阅读说明：本技术 用于过敏原检测的系统和方法 (Systems and methods for allergen detection ) 是由 A·吉尔博-格芬 A·L·威克斯 V·维拉里尔 P·墨菲 E·A·罗伯森 D·卡彭特 D 于 2019-02-21 设计创作，主要内容包括：本发明涉及用于食物样品中的过敏原检测的装置和系统。过敏原检测系统包括一次性分析盒和具有优化的光学系统的检测装置。(The present invention relates to a device and a system for allergen detection in a food sample. The allergen detection system comprises a disposable cartridge and a detection device with an optimized optical system.)

1. An assembly for detecting a molecule of interest in a sample, the assembly comprising:

a sample processing cartridge configured to receive the sample for processing to a state that allows the molecule of interest to interact with a detection agent; and

a detector unit configured to receive the sample processing cartridge in a configuration that allows a detection mechanism housed by the detector unit to detect an interaction of the molecule of interest with the detection agent, wherein the interaction triggers a visual indication on the detector unit that the molecule of interest is detected.

2. The assembly of claim 1, wherein the molecule of interest is an allergen.

3. The assembly of claim 1 or 2, wherein the detection agent is an antibody or variant thereof, a nucleic acid molecule, or a small molecule.

4. The assembly of claim 3, wherein the detection agent is a nucleic acid molecule comprising a nucleic acid sequence that binds to the molecule of interest.

5. The assembly of claim 4, wherein the nucleic acid molecule is a Signal Polynucleotide (SPN) derived from an aptamer comprising a nucleic acid sequence that binds to the molecule of interest.

6. The assembly of any one of claims 1 to 5, wherein the sample processing cartridge comprises:

a homogenizer configured to produce a homogenized sample, thereby releasing the molecule of interest from the matrix of the sample into an extraction buffer in the presence of the detection agent;

a first conduit for transferring the homogenized sample and detection agent through a filtration system to provide a filtrate comprising the molecule of interest and the detection agent;

a second conduit for transferring the filtrate to a detection chamber having a window, wherein the detection mechanism of the detector unit analyzes the detection chamber through the window to identify interactions of the molecule of interest with the detection agent in the detection chamber.

7. The assembly of claim 6, wherein the homogenizer is powered by a motor located in the detector unit, wherein the motor is functionally coupled to the homogenizer when the sample processing cartridge is received by the detector unit.

8. The assembly of claim 6 or 7, wherein the sample processing cartridge further comprises: a chamber containing a wash buffer for washing the detection chamber; and a waste chamber for receiving the effluent contents of the detection chamber.

9. The assembly of claim 8, wherein the sample processing cartridge further comprises a rotary valve switching system for providing a plurality of fluid flow paths for transferring the homogenized sample to the filtration system, for transferring the filtrate to the detection chamber, for transferring the wash buffer to the detection chamber, and for transferring the contents of the detection chamber to the waste chamber.

10. The assembly of claim 9, wherein the rotary valve switching system is further configured to provide a closed position to prevent fluid movement in the sample processing cartridge.

11. The assembly of any one of claims 6 to 10, wherein the detection chamber comprises a transparent substrate having detection probe molecules immobilized thereon, the detection probes configured to probe interact with the detection agent, wherein interaction of the molecule of interest with the detection agent prevents probe interaction of the detection agent with the detection probes.

12. The assembly of claim 11, wherein the transparent substrate further comprises optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

13. The assembly of claim 11, wherein the transparent substrate further comprises two different optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

14. The assembly of claim 12 or 13, wherein the detection agent comprises an optically detectable group that is activated upon the probe interaction.

15. The assembly of claim 14, wherein the optically detectable group is a fluorophore.

16. The assembly of any one of claims 13 to 15, wherein the detection mechanism housed by the detector unit is a fluorescence detection system having laser light for exciting fluorescence, the fluorescence detection system configured to detect a fluorescence emission signal and/or a fluorescence scatter signal when the probe interaction is performed and subjected to laser excitation.

17. The assembly of claim 16, wherein the detection mechanism comprises a plurality of optical elements placed in a linear or folded arrangement within a stepped bore in the detector unit.

18. The assembly of claim 16 or 17, wherein the detector unit further comprises a signal processor for analyzing fluorescence emission signals and/or fluorescence scatter signals to identify the probe interactions and to transmit the identification of the molecules of interest or the molecular source of interest as the visual indication in order to inform an operator of the assembly whether the molecules of interest or the molecular source of interest are present in the sample.

19. The assembly of any one of claims 11 to 18, wherein the transparent substrate comprises a plurality of different detection probes for detecting a plurality of different detection agents configured to provide a plurality of different interactions with different molecules of interest in the sample.

20. The assembly of any one of claims 6 to 19, wherein the sample processing cartridge further comprises a sample concentrator for concentrating the filtrate prior to transferring the filtrate to the detection chamber.

21. The assembly of any one of claims 1 to 20, further comprising a sampler comprising: a hollow tube having a cutting edge for cutting a source to create and hold the sample within the hollow tube; and a plunger for pushing the sample out of the hollow tube and into a port in the sample processing cartridge.

22. A detection system for detecting allergens in a food sample, comprising:

a sampler for collecting a test sample;

a disposable cartridge configured to receive the sample and process the sample to a state that allows an allergen of interest in the sample to interact with a detection agent, the cartridge comprising:

(i) a sample receiving chamber having a homogenizer configured to homogenize the sample with an extraction buffer in the presence of the detection agent, thereby allowing the allergen of interest in the sample to interact with the detection agent,

(ii) a filtration system configured to provide a filtrate comprising the allergen of interest and the detection agent,

(iii) a detection chamber having a window, wherein the detection chamber comprises a separate substrate having detection probe molecules immobilized thereon,

(iv) a chamber containing a wash buffer for washing the detection chamber,

(v) a waste chamber for receiving and storing the effluent contents of the detection chamber,

(vi) a rotary valve switching system and conduits configured to transfer the homogenized sample and detection agent through the filtration system, transfer the filtrate to the detection chamber, transfer the wash buffer to the detection chamber, and transfer effluent contents from the detection chamber to the waste chamber, an

(vii) An air flow system configured to regulate air pressure and flow rate in the cartridge; and

a detector unit configured to receive the disposable cartridge and operate sample processing to detect an interaction between the allergen of interest and the detection agent within the disposable cartridge, the detector unit comprising:

(i) a homogenizing motor configured to drive the homogenizer of the cartridge,

(ii) a valve motor configured to drive the rotary valve switching system of the cartridge,

(iii) a pump configured to drive a flow of fluid in the cartridge,

(iv) a detection mechanism that detects an interaction between the allergen of interest and the detection agent, wherein the interaction triggers a visual indication on a display of the detector unit as to whether the allergen of interest is present, and

(v) a power supply for supplying power to the electronic device,

wherein the detection unit comprises an outer housing having a receptacle for the disposable cartridge and an execution button for executing a procedure.

23. The detection system of claim 22, wherein the filtration system is a filter assembly comprising: a body filter having a cotton body for filtering coarse debris from the homogenized sample; and a membrane filter having a pore size of about 1 μm.

24. The detection system of claim 22, wherein the detection agent is a nucleic acid molecule comprising a nucleic acid sequence that binds to the allergen of interest and a fluorophore.

25. The detection system of claim 24, wherein the detection agent is preloaded into the extraction buffer.

26. The detection system according to any one of claims 22 to 25, wherein the detection probe molecule is configured to interact with the detection agent, wherein interaction of the allergen of interest with the detection agent prevents the detection agent from probing with the detection probe.

27. The detection system of claim 26, wherein the detection probe is a nucleic acid molecule comprising a nucleic acid sequence complementary to a nucleic acid sequence of the detection agent.

28. The detection system according to any one of claims 22 to 27, wherein the substrate further comprises optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

29. The detection system according to claim 28, wherein the substrate further comprises two different optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

30. The detection system according to claim 28 or 29, wherein the detection probes are immobilized in a local area of the substrate, referred to as the action area, and wherein the control probes are immobilized in a separate local area of the substrate, referred to as the control panel.

31. The detection system of any one of claims 22 to 30, wherein the detection mechanism housed by the detector unit is a fluorescence detection system configured to detect fluorescence emission signals and/or fluorescence scatter signals from the detection chamber.

32. The detection system of claim 31, wherein the fluorescence detection system comprises: (i) a laser for exciting fluorescence; (ii) a plurality of optical elements for directing laser excitation to the substrate within the detection chamber; (iii) a plurality of collecting lenses for collecting the fluorescent light emitted from the substrate; (iv) a fluorescence detector for measuring light emitted from the substrate; and (v) a signal processor for analyzing the fluorescence emission signal and/or the fluorescence scattering signal to identify the probe interaction and to transmit the identification of the allergen of interest as the visual indication to inform an operator whether the allergen of interest is present in the sample.

33. The detection system of claim 32, wherein the optical elements of the fluorescence detection system are placed in a linear or folded arrangement within a stepped bore in the detector unit.

34. The detection system of any of claims 22 to 33, wherein the rotary valve motor comprises: a DC gear motor having two optical sensors, namely an output optical sensor and a straight-axis optical sensor; and a microcontroller including an output coupling and an encoder wheel, a direct motor shaft, and a direct shaft encoder wheel.

35. A sample processing cartridge for processing a sample to detect a molecule of interest in the sample, comprising:

(i) a sample receiving chamber having a homogenizer configured to homogenize the sample with an extraction buffer in the presence of a detection agent, thereby allowing a protein of interest in the sample to interact with the detection agent,

(ii) a filtration system configured to provide a filtrate comprising the molecule of interest and the detection agent,

(iii) a detection chamber having a window, wherein the detection chamber comprises a separate substrate having detection probe molecules immobilized thereon,

(iv) a chamber containing a wash buffer for washing the detection chamber,

(v) a waste chamber for receiving and storing the effluent contents of the detection chamber,

(vi) a rotary valve switching system and conduits configured to transfer the homogenized sample and detection agent through the filtration system, transfer the filtrate to the detection chamber, transfer the wash buffer to the detection chamber, and transfer effluent contents from the detection chamber to the waste chamber, and

(vii) an air flow system configured to regulate air pressure and flow rate in the cartridge.

36. The sample processing cartridge of claim 35, wherein the detection agent is a nucleic acid molecule comprising a nucleic acid sequence that binds to the allergen of interest and a fluorophore.

37. The sample processing cartridge of claim 36, wherein the detection agent is preloaded into the extraction buffer.

38. The sample processing cartridge of claim 37, wherein the detection probe molecules immobilized on the substrate are configured to interact with the detection agent, wherein interaction of the allergen of interest with the detection agent prevents the detection agent from probing with the detection probe.

39. The sample processing cartridge of claim 38, wherein the detection probe is a nucleic acid molecule comprising a nucleic acid sequence complementary to a nucleic acid sequence of the detection agent.

40. The sample processing cartridge of any one of claims 35 to 39, wherein the substrate further comprises optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

41. The sample processing cartridge of any one of claims 35 to 39, wherein the substrate further comprises two different optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism.

42. The sample processing cartridge of any one of claims 35 to 41, wherein the substrate is a glass chip, or a plastic chip, or a film chip.

43. The sample processing cartridge of any one of claims 35 to 42, wherein the filtration system consists of a bulk filter and a membrane filter.

44. The sample processing cartridge of claim 43, wherein the body filter comprises cotton, and wherein the film filter is a PET film.

45. The sample processing cartridge of claim 44, wherein the membrane filter has a pore size of 1 μm.

46. The sample processing cartridge of any one of claims 35 to 45, wherein the rotary valve switching system is further configured to provide a closed position to prevent fluid movement in the cartridge.

47. The sample processing cartridge of any one of claims 35 to 46, wherein the molecule of interest is an allergen.

48. The sample processing cartridge of any one of claims 35 to 47, wherein the cartridge is made of a polymer with minimal autofluorescence.

49. A sample processing cup or cup for processing a sample to a state that allows detection of a molecule of interest in the sample, comprising:

a top cover configured to receive the sample and seal the cup or cup-shaped container, wherein the top cover comprises a port for receiving the sample and at least one breather filter to allow air to enter,

a body portion configured to process the sample to a state that allows interaction of the molecule of interest with a detection agent, wherein the body portion comprises:

(i) a chamber having a homogenizer for homogenizing the sample in an extraction buffer, thereby releasing the molecule of interest from the matrix of the sample into the extraction buffer and interacting with a detection agent present in the extraction buffer,

(ii) a conduit for transferring the homogenized sample through a filtration system contained in the body portion to provide a filtrate comprising the molecule of interest and the detection agent,

(iii) a separate chamber for containing a wash buffer for washing the molecule of interest and the detection agent,

(iv) a separate chamber for receiving and storing results from washing the molecule of interest and the detection agent,

(v) a conduit for transferring the filtrate to a detection chamber, an

(vi) A rotary valve switching system for providing a plurality of fluid flow paths for transferring homogenized sample to the filtration system, for transferring the filtrate to a separate detection chamber, for transferring the wash buffer to the detection chamber, and for transferring the contents of the detection chamber to a waste chamber; and

a bottom cover configured to be connected to the cup body portion so as to form a detection chamber at a bottom of the test cup, and configured to provide a connection surface with the detector unit, wherein the detection chamber inside the bottom cover includes: (i) a separate substrate comprising optically detectable detection probe molecules immobilized thereon, the optically detectable detection probe molecules interacting with the molecules of interest, (ii) a plurality of fluidic pathways, and (iii) a window, wherein a detection mechanism of the detector unit analyzes the interaction between the homogenized sample and the detection probes and identifies the molecules of interest in the sample,

wherein an exterior of the bottom cover comprises a plurality of ports for connecting a plurality of motors housed in the detector unit for operating the homogenizer, the rotary valve system and the flow of fluid.

50. The sample processing cup or cup-like container of claim 49, wherein the bottom lid further comprises a data chip.

51. The sample processing cup or cup-like container of claim 49 or 50, wherein said detection agent is a nucleic acid molecule comprising a nucleic acid sequence and a fluorophore that binds to said molecule of interest in said sample.

52. The sample processing cup or cup-shaped container of claim 51, wherein the detection probe molecule undergoes a probe interaction with the detection agent, wherein the interaction of the molecule of interest with the detection agent prevents the detection agent from undergoing the probe interaction, and wherein the detection probe molecule is a short nucleic acid molecule comprising a nucleic acid sequence complementary to a nucleic acid sequence of the detection agent.

53. The sample processing cup or cup-like container of claim 52, wherein the base further comprises one or more optically detectable control probe molecules immobilized thereon for normalizing the signal output detected by the detection mechanism.

54. The sample processing cup or cup-like container of claim 53, wherein the substrate is a glass chip comprising a local area having detection probe molecules immobilized thereon, and two local areas having two optically detectable control probes immobilized thereon, each control area being positioned on one side of the local area having the detection probes.

55. An optical system for detecting a fluorescent signal, comprising:

a laser source configured to provide optical excitation energy;

a plurality of optical components configured to direct a laser excitation source to an action area of a substrate to form a spot covering the action area, wherein detectable probe molecules are immobilized on the action area, and configured to direct the laser excitation source to a control area of the same substrate, wherein control probes are immobilized on the control area, thereby exciting detection probes and the control probes immobilized thereon;

a plurality of light collecting elements configured to collect light energy emitted from the active area and the control area of the substrate, respectively;

a fluorescence detector for measuring light emitted from the region of action of the substrate; and

a processor for processing the measurements from the fluorescence detector.

56. The optical system of claim 55, wherein the optical elements for directing the laser excitation source comprise a collimating lens, a band pass filter, and a cylindrical lens; and wherein the optical components for collecting the emitted light include a collection lens shaped with a concave first surface to optimize imaging and minimize stray light, a band pass filter, a long pass filter, and a focusing lens.

57. The optical system of claim 56, wherein the fluorescence detector comprises two photodiode lenses, two control array photodiodes, a signal array photodiode, and a collection printed circuit board.

58. A method for detecting the presence or absence of a molecule of interest in a sample, the method comprising the steps of:

(a) collecting a sample;

(b) homogenizing the sample in an extraction buffer in the presence of a detection agent, thereby releasing the molecule of interest from the sample to interact with the detection agent comprising a fluorophore;

(c) filtering the homogenized sample comprising the molecule of interest and the detection agent;

(d) contacting the filtrate comprising the molecule of interest and the detection agent with a detection probe molecule that undergoes a probe interaction with the detection agent, wherein the interaction of the molecule of interest with the detection agent prevents the detection agent from undergoing a probe interaction with the detection probe;

(e) washing the mixture of step (d) with a wash buffer;

(f) measuring a signal output from the probe interaction of the detection probe molecule and the detection agent; and

(g) detecting the presence or absence of the molecule of interest in the sample.

59. The method of claim 58, wherein the detection agent is an antibody or variant thereof, a nucleic acid molecule, or a small molecule.

60. The method of claim 59, wherein the detection agent is a nucleic acid molecule comprising a nucleic acid sequence that binds to the molecule of interest and a fluorophore attached to one end of the sequence.

61. The method of claim 60, wherein the detection agent is stored comprising MgCl₂In said buffer of (1).

62. The method of claim 60 or 61, wherein the detection probe molecule is a nucleic acid molecule comprising a short nucleic acid sequence complementary to a sequence of the detection agent, wherein the probe molecule performs a probe interaction with the detection agent and interaction of the molecule of interest with the detection agent prevents the detection agent from performing the probe interaction.

63. The method of claim 62, wherein the detection probe molecules are immobilized on the surface in specific localized regions of the substrate.

64. The method of claim 63, wherein the substrate further comprises at least one control region, wherein optically detectable control probe molecules are immobilized on the at least one control region for normalizing signal output from the detection probe molecules.

65. The method of any one of claims 58 to 64, wherein the homogenized sample comprising the molecule of interest and the detection agent is filtered through a filter assembly comprising a coarse filter, a depth filter, and a membrane filter.

66. The method of claim 65, wherein the depth filter is a cotton depth filter.

67. The method according to any one of claims 58 to 66, wherein the molecule of interest is an allergen.

68. A system for detecting the presence or absence of an allergen in a sample, the system comprising:

an apparatus comprising an optical system configured to measure a fluorescent signal output, thereby detecting the presence or absence of the allergen; and

a disposable cartridge configured to process the sample, the cartridge interfacing with a receiver of the device, the cartridge comprising:

(i) an upper module comprising a plurality of chambers isolated from one another, each chamber of the plurality of chambers comprising a lower port to allow entry and/or exit of a fluid, the plurality of chambers comprising:

(1) a homogenization chamber comprising a homogenizer for homogenizing the sample in an extraction buffer and extracting the allergen;

(2) a washing buffer chamber;

(3) a waste chamber configured to receive liquid waste; and

(4) a detection chamber in optical communication with the optical system for detecting the allergen; and

(ii) a base configured to be connected to the upper module, the base comprising:

(1) a plurality of fluid paths connecting the lower port of each chamber when the cartridge is inserted into the receptacle;

(2) a valve configured to form a plurality of bridging fluid connections between respective fluid paths of the plurality of fluid paths, thereby allowing selective fluid movement into and/or out of the plurality of chambers.

69. The system of claim 68, wherein the valve is a rotary valve driven by a motor positioned in the device, the motor including one or more optical sensors for determining a position of the rotary valve.

70. The system of claim 68 or 69, wherein the plurality of bridging fluid connections comprises:

(a) a first fluid connection between the wash buffer chamber and the reaction chamber; and

(b) a second fluid connection between the homogenization chamber and the detection chamber.

71. The system of any one of claims 68-70, wherein the cartridge further comprises:

(iii) a filter assembly and a filter fluid path between the homogenization chamber and the filter assembly to obtain a filtered sample after homogenization of the sample in the homogenization chamber; and

(iv) a filtrate chamber for receiving the filtered sample.

72. The system of claim 71, wherein the second fluid connection comprises the filtrate chamber between the homogenization chamber and the detection chamber, wherein the rotary valve is configured to make the second fluid connection between the filtrate chamber and the detection chamber.

73. The system of any one of claims 68-72, wherein the rotary valve comprises a position in which all bridging fluid connections are closed.

74. The system of any one of claims 68 to 73, wherein said upper module further comprises an extraction buffer reservoir and a fluid channel extending from said extraction buffer reservoir to said homogenization chamber.

75. The system of any one of claims 68 to 74, wherein the detection chamber comprises a substrate on which detection probe molecules are immobilized; the substrate is configured to detect the allergen.

76. The system of claim 75, wherein the substrate is a glass chip having anchored nucleic acid detection probes that hybridize to free Signal Polynucleotides (SPNs) having fluorescent probes attached thereto, the SPNs comprising nucleic acid sequences that specifically bind to the allergen, wherein the SPNs do not bind to the nucleic acid probes when bound to the allergen.

77. The system according to claim 76, wherein the glass chip comprises at least two control panels printed with oligonucleotide sequences that do not bind to the SPN or the allergen of interest in the sample.

78. The system of any one of claims 68-77, wherein the cartridge is disposable.

79. A kit comprising a sample processing cartridge according to any one of claims 35 to 48, or a test cup according to any one of claims 49 to 54, and instructions for testing for the presence of an allergen in a sample using the cartridge or the cup.

80. The kit according to claim 79, further comprising a sampler for collecting a sample.

Technical Field

The present invention relates to a portable device and system for detecting allergens in a sample, such as a food sample. The invention also provides methods for detecting the presence or absence of an allergen in a sample.

Background

Allergies (e.g., food allergies) are common medical conditions. It is estimated that up to 2% of adults and up to 8% of children (especially children under three years of age) suffer from food allergies (approximately 1500 million people) in the united states, and it is believed that the prevalence is increasing. A portable device that enables a person with food allergy to test their food and accurately and immediately determine the allergen content would be of great benefit to provide a informed decision as to whether to eat or not.

Researchers have attempted to develop suitable devices and methods to meet this need, such as those disclosed in the following applications: U.S. patent nos. 5,824,554 to McKay; U.S. patent application publication nos. 2008/0182339 and 8,617,903 to Jung et al; U.S. patent application publication nos. 2010/0210033 to Scott et al; U.S. Pat. Nos. 7,527,765 to Royds; U.S. patent nos. 9,201,068 to Suni et al; and U.S. patent No. 9,034,168 to Khattak and Sever. There remains a need for improved molecular detection techniques. There is also a need for devices and systems that can detect allergens of interest in less time, with higher sensitivity and specificity and with less technical expertise than devices used today.

The present invention provides a portable detection device for rapidly and accurately detecting allergens in a sample by using aptamer-based Signaling Polynucleotides (SPNs). SPN as a detection agent specifically binds to an allergen of interest to form an SPN-protein complex. The sensor that captures SPN can include a chip (e.g., a DNA chip) printed with nucleic acid molecules that hybridize to SPN. The detection system may include a separate sampler, a disposable cartridge/vessel for processing the sample and performing the detection assay, and a detector including an optical system for operating to detect and detect the reaction signal. The detection agent (e.g., SPN) and the sensor (e.g., DNA chip) can be integrated into the disposable cartridge of the present invention. The cartridge, detection agent and detection sensor may also be used in other detection systems. Other capture agents, such as antibodies specific for allergen proteins, may also be used in the detection system of the present invention. The consumer may use the device in a non-clinical setting, such as in a home, a restaurant, and any other facility that provides food.

Disclosure of Invention

The present invention provides systems, devices, disposable vessels/cartridges, optical systems and methods for molecular detection in various types of samples, in particular allergens in food samples. The allergen detection device and system is portable and handheld.

One aspect of the invention is an assembly for detecting a molecule of interest in a sample. The assembly includes a sample processing cartridge configured for receiving a sample for processing to a state that allows interaction of a molecule of interest with a detection agent. The assembly includes a detector unit configured to receive a sample processing cartridge in a configuration that allows a detection mechanism housed by the detector unit to detect interaction of a molecule of interest with a detection agent. The interaction triggers a visual indication on the detector unit as to whether the molecule of interest is present in the sample.

The molecule of interest may be a protein or a functional fragment thereof, a nucleic acid molecule, or a polysaccharide or a functional fragment thereof. In some embodiments, the molecule of interest may be an allergen (such as a food allergen). Allergens are antigens (parts or functional fragments of molecules such as proteins and polysaccharides) that elicit an immune response that leads to allergic conditions.

In some embodiments, the detection agent is an antibody or variant thereof, a nucleic acid molecule, or a small molecule. In some embodiments, wherein the detection agent is a nucleic acid molecule comprising a nucleic acid sequence that binds to the molecule of interest. The nucleic acid-based detection agent may be a signal polynucleotide derived from an aptamer that includes a nucleic acid sequence that binds to the molecule to be detected.

In some embodiments, the sample processing cartridge comprises a homogenizer configured to produce a homogenized sample, thereby releasing the molecule of interest from the matrix of the sample into the extraction buffer in the presence of the detection agent. The sample processing cartridge further comprises: a first conduit for transferring the homogenized sample and detection agent through a filtration system to provide a filtrate comprising the molecule of interest and detection agent; a second conduit to transfer the filtrate to a detection chamber having a window. The detection mechanism of the detector unit analyzes the detection chamber through the window to identify interactions of the molecule of interest in the detection chamber with the detection agent.

The homogenizer may be powered by a motor located in the detector unit, the motor being functionally coupled to the homogenizer when the sample processing cartridge is received by the detector unit.

The sample processing cartridge may further include: a chamber containing a wash buffer for washing the detection chamber; and a waste chamber for receiving the effluent contents of the detection chamber after washing.

In some embodiments, the sample processing cartridge further comprises a rotary valve switching system providing a plurality of fluid flow paths and channels for transferring the homogenized sample to the filtration system, for transferring the filtrate to the detection chamber, for transferring the wash buffer to the detection chamber, and for transferring the contents of the detection chamber to the waste chamber. The rotary valve switching system may also be configured to provide a closed position to prevent fluid movement in the sample processing cartridge. In some embodiments, the rotary valve switching system may be powered by a motor located in the detector unit, the motor being functionally coupled to the rotary valve system when the sample processing cartridge is received by the detector unit.

In some embodiments, the detection chamber comprises a transparent substrate having detection probe molecules immobilized thereon. The detection probe is configured to perform a probe interaction with the detection agent. Interaction of the molecule of interest with the detection agent prevents the detection agent from performing a probe interaction with the detection probe. The transparent substrate may further comprise optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism. In some embodiments, the transparent substrate includes two different optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism. Control probe molecules are nucleic acid molecules that bind neither to the molecule of interest nor to the detection agent. In some embodiments, the substrate may be a glass chip.

In some embodiments, the detection agent comprises an optically detectable group that is activated upon probe interaction. The optically detectable group may be a fluorophore.

In some embodiments, the detection mechanism housed by the detector unit is a fluorescence detection system having a laser for exciting fluorescence, the fluorescence detection system configured to detect a fluorescence emission signal and/or a fluorescence scatter signal when the probe interaction is performed and excited by the laser. The detection mechanism may include a plurality of optical elements placed in a straight or folded arrangement within a stepped bore (stepped bore) in the detector unit.

In some embodiments, the detector unit further comprises a signal processor for analyzing the fluorescence emission signal and/or the fluorescence scatter signal to identify probe interactions and to transmit an identification of the molecule of interest or the source of the molecule of interest to the visual indication in order to inform an operator of the assembly whether the molecule of interest or the source of the molecule of interest is present in the sample.

In some embodiments, the transparent substrate comprises a plurality of different detection probes for detecting a plurality of different detection agents configured to provide a plurality of different interactions with a plurality of different molecules of interest.

In some embodiments, the sample processing cartridge further comprises a sample concentrator for concentrating the filtrate prior to transferring the filtrate to the detection chamber.

In some embodiments, the assembly further comprises a sampler. The sampler comprises a hollow tube having a cutting edge for cutting the source to produce and hold a sample as a sample (core) within the hollow tube. This embodiment of the sampler also has a plunger for pushing the sample out of the hollow tube and into a port in the sample processing cartridge.

Another aspect of the invention relates to a test system and device for detecting the presence or absence of one or more allergens of interest in a sample. In various embodiments, a detection system comprises: at least one disposable processing cartridge configured to receive a test sample and process the sample to a state that allows an allergen of interest in the sample to interact with a detection agent; and an integrally formed detector unit configured to receive the disposable cartridge and operate the sample processing to detect an interaction between the allergen of interest and the detection agent within the disposable cartridge. The detector unit may be removably connected to the disposable cartridge. In some embodiments, the system may further comprise a sampler for collecting the test sample and transferring the collected sample to the sample processing cartridge.

In some embodiments, the sampler for collecting the test sample is a food sampler comprising: a hollow tube having a cutting edge for cutting the source to produce and hold a sample as a sample within the hollow tube; and a plunger for pushing the sample out of the hollow tube and into the port in the sample processing cartridge. The sampler may be operatively connected to the disposable cartridge for transferring the collected test sample to the cartridge.

In some embodiments, the disposable process cartridge may include: (i) a sample receiving chamber having a homogenizer configured to homogenize a sample with an extraction buffer in the presence of a detection agent, thereby allowing an allergen of interest in the sample to interact with the detection agent, (ii) a filtration system configured to provide a filtrate comprising the allergen of interest and the detection agent, (iii) a detection chamber having a window, wherein the detection chamber comprises a separate substrate having detection probe molecules immobilized thereon, (iv) a chamber containing a washing buffer for washing the detection chamber, (v) a waste chamber for receiving and storing effluent contents of the detection chamber after washing, (vi) a rotary valve switching system and a conduit configured to transfer the homogenized sample and detection agent through the filtration system, transfer the filtrate to the detection chamber, and transfer the washing buffer to and from the detection chamber to the waste chamber, and (vii) an air flow system configured to regulate air pressure and flow rate in the cartridge.

In some examples, the disposable processing cartridge is configured to detect a particular allergen. In other examples, the sample processing cartridge is configured to detect more than one allergen.

In some embodiments, the detector unit may include an external housing having a receiver for the disposable process cartridge and an execution button for executing the process. The detector unit is thereby configured to drive the detection process. In some embodiments, the detector unit may comprise: (i) a motor configured to drive a homogenizer of the cartridge, (ii) a motor configured to drive a rotary valve switching system of the cartridge, (iii) a pump configured to drive a flow of fluid in the cartridge, (iv) a detection mechanism for detecting an interaction between an allergen of interest and a detection agent, wherein the interaction triggers a visual indication on a display of the detector unit as to whether the allergen of interest is present, and (v) a display window allowing an operator to view the detection result.

In some embodiments, the filtration system of the sample processing cartridge is a filter assembly comprising a body filter (bulk filter) and a membrane filter. The bulk filter may include a coarse filter and a depth filter having a cotton (cotton roll) for filtering coarse debris from the processed sample. The film has a pore size of 1 μm to 2 μm. In some embodiments, the filter assembly may further comprise a filter cap that may lock the rotary valve.

In some embodiments, the sample processing cartridge comprises a detection agent that specifically binds to the allergen of interest. In some embodiments, the detection agent is pre-storedExtracting in buffer solution. The detection agent is a nucleic acid molecule comprising a nucleic acid sequence that binds to the allergen of interest, and a fluorophore attached to one end of the nucleic acid sequence. Nucleic acid-based detection agents can be stored including MgCl₂In the buffer of (1). In some examples, the detection agent is a Signaling Polynucleotide (SPN) derived from an aptamer that specifically binds to the allergen of interest and has high affinity.

In some embodiments, the detection chambers in the cartridge comprise a separate substrate on which the detection probe molecules are immobilized. The detection probe molecule is configured to interact with a detection agent, wherein interaction of the allergen of interest with the detection agent prevents interaction of the detection agent with the probe molecule. In some embodiments, the detection probe is a nucleic acid molecule comprising a short nucleic acid sequence that is complementary to the nucleic acid sequence of the detection agent. In some embodiments, the detection probe molecules are immobilized in a specific local area of the substrate, referred to as a reaction panel.

In some embodiments, the substrate further comprises optically detectable control probe molecules immobilized thereon for normalizing the signal output measured by the detection mechanism. In some examples, the control probes are immobilized in a specific local area of the substrate, referred to as a control panel. In some embodiments, the substrate comprises at least one reaction panel and at least two control panels. In a preferred embodiment, the substrate is a glass chip. The detection chamber may include at least one optical window aligned with the substrate. In one embodiment, the optical window is configured for measuring a signal output from the interaction of the detection probe with the detection agent by a detection mechanism of the detector unit. In other embodiments, the detection chamber may comprise a separate window configured for measuring scattered light from the substrate by the detection mechanism.

In some embodiments, the disposable processing cartridge may include a data chip configured to provide cartridge information.

In some embodiments, the detection mechanism is a fluorescence detection system configured to detect a fluorescence emission signal and/or a fluorescence scatter signal from the detection chamber. In some embodiments, the fluorescence detection system comprises: (i) a laser for exciting fluorescence, (ii) a plurality of optical components for directing laser excitation to a substrate within the detection chamber, (iii) a plurality of collection lenses configured to collect fluorescence emitted from the substrate, (iv) a fluorescence detector for measuring light emitted from the substrate, (v) a signal processor for analyzing the fluorescence emission signal and/or the fluorescence scattering signal to identify probe interactions and to transmit an identification of the allergen of interest to a visual indication to inform an operator whether the allergen of interest is present in the sample.

In some embodiments, the optical elements of the fluorescence detection system are placed in a linear or folded arrangement within a stepped bore in the detector unit.

In some embodiments, a Printed Circuit Board (PCB) may be connected directly or indirectly to the fluorescence detection system for displaying the test readings. The results may be displayed as numbers, icons, colors, and/or letters or other equivalent forms.

In one aspect of the invention, the sample processing cartridge is configured as a disposable test cup or cup-like container. The disposable test cup or cup-shaped receptacle may be configured as an analysis module in which a sample is processed and allergens of interest in the test sample are detected by interaction with a detection agent. In some embodiments, a disposable test cup or cup-like container comprises: (i) a top cover configured to receive a sample and seal the cup or cup-shaped container, wherein the top cover comprises a port for receiving the sample and at least one vent filter allowing air to enter, (ii) a body portion configured to process the sample to a state allowing interaction of the allergen of interest with a detection agent, (iii) a bottom cover configured to be connected to the cup body portion, thereby forming a detection chamber with a window at the bottom of the assembled test cup, and configured to provide a connection surface with the detector unit. The exterior of the bottom cover comprises a plurality of ports for connecting a plurality of motors located in the detection unit to operate the homogenizer, the rotary valve system and the flow of fluid. The window of the detection chamber is connected to a detection mechanism in the detector unit.

In some embodiments, the detection chamber in the bottom lid interior comprises: (i) a separate substrate comprising optically detectable detection probe molecules immobilized thereon, the optically detectable detection probe molecules interacting with a detection agent, (ii) a plurality of fluidic pathways, and (iii) a window, wherein a detection mechanism of the detector unit analyzes the interaction between the homogenized sample and the detection probe molecules and identifies an allergen of interest in the sample.

In some embodiments, the cup body portion may be divided into multiple compartments (e.g., chambers) dedicated to various functions, including sample collection and homogenization, buffer and reagent storage, filtrate collection, washing, and waste collection. In one embodiment, the cup body portion may comprise: (i) a chamber with a homogenizer for homogenizing the sample in an extraction buffer, thereby releasing the molecule of interest from the matrix of the sample into the extraction buffer and interacting with the detection agent present in the extraction buffer, (ii) a catheter, (ii) for transferring the homogenized sample through a filtration system contained in the body portion to provide a filtrate containing the molecule of interest and the detection agent, (iii) a separate chamber, for containing a wash buffer for washing the molecule of interest and the detection agent, (iv) a separate chamber, for receiving and storing results from washing the molecule of interest and the detection agent, (v) a catheter, (vii) rotary valve switching system, fluid path and vent required for transfer of filtrate to detection chamber, and (vi) fluid flow in compartment within cartridge.

In one aspect of the present invention, a fluorescence detection system for detecting a fluorescence signal includes: (i) a laser source configured to provide optical excitation energy; (ii) a plurality of optical components configured to guide a laser excitation source to an active region of a substrate on which detectable probe molecules are immobilized to form a spot covering the active region, and to a control region of the same substrate on which control probes are immobilized, thereby exciting detection probe molecules and control probes immobilized thereon, (iii) a plurality of light collection components configured to collect light energy emitted from the active region and the control region of the substrate, respectively; (iv) a fluorescence detector for measuring light emitted from the action area of the substrate and/or from the control area of the substrate; and (v) a processor for processing the measurements from the fluorescence detector.

Another aspect of the invention relates to a system for detecting the presence or absence of an allergen in a sample, the system comprising: (a) a detector unit comprising an optical system configured to measure a fluorescence signal output, thereby detecting the presence or absence of an allergen; and (b) a disposable cartridge configured to process a sample, the disposable cartridge interfacing with a receiver of a detector unit, the cartridge comprising: (1) an upper module comprising a plurality of chambers isolated from one another, each chamber of the plurality of chambers comprising a lower port to allow entry and/or exit of a fluid, the plurality of chambers comprising: (i) a homogenization chamber comprising a homogenizer for homogenizing the sample and extracting the allergen; (ii) a washing buffer chamber; (iii) a waste chamber configured to receive liquid waste; and (iv) a reaction chamber in optical communication with the optical system for detecting the allergen; and (2) a base configured to be connected to the upper module, the base comprising: (i) a plurality of fluid paths connecting the lower port of each chamber when the cartridge is inserted into the receptacle; and (ii) a valve configured to form a plurality of bridging fluid connections between respective ones of the plurality of fluid paths, thereby allowing selective fluid movement into and/or out of the plurality of chambers.

In some embodiments of the system, the plurality of bridging fluid connections comprises: (a) a first fluid connection between the wash buffer chamber and the reaction chamber; and (b) a second fluid connection between the homogenization chamber and the reaction chamber.

In some embodiments of the system, the cartridge further comprises: (3) a filter assembly and a filter fluid path between the homogenization chamber and the filter assembly to obtain a filtered sample after homogenizing the sample in the homogenization chamber; (4) and the filtrate chamber is used for accommodating the filtered sample.

Another aspect of the invention relates to a method for detecting the presence or absence of a molecule of interest in a sample, the method comprising the steps of: (a) collecting a sample suspected of containing the allergen of interest, (b) homogenizing the sample in an extraction buffer in the presence of a detection agent, thereby releasing the molecule of interest from the sample to interact with the detection agent comprising a fluorophore, (c) filtering the homogenized sample containing the molecule of interest and the detection agent; (d) contacting the filtrate comprising the molecule of interest and the detection agent with a detection probe molecule that undergoes a probe interaction with the detection agent, wherein the interaction of the molecule of interest with the detection agent prevents the detection agent from undergoing a probe interaction with the detection probe; (e) washing the contact of step (d) with a wash buffer; (f) measuring a signal output from probe interaction of the detection probe molecule with the detection agent; and (g) processing and digitizing the detection signal and visualizing the interaction between the detection probe and the detection agent.

In some embodiments, the detection agent is an antibody or variant thereof, a nucleic acid molecule, or a small molecule. In a preferred embodiment, the detection agent is a nucleic acid molecule comprising a nucleic acid sequence that binds to the molecule of interest and a fluorescent group attached to one end of the sequence. In some embodiments, the nucleic acid-based detection agent can be stored to comprise MgCl₂In the buffer of (1).

In some embodiments, the detection probe molecule is a nucleic acid molecule comprising a short nucleic acid sequence complementary to a sequence of a detection agent, wherein the probe molecule undergoes probe interaction with the detection agent, and interaction of the molecule of interest with the detection agent prevents the detection agent from undergoing probe interaction.

In another aspect the invention relates to a kit comprising: a sample processing cartridge (e.g., a test cup as described herein), and instructions for use of the cartridge in testing for the presence of an allergen in a sample. In some embodiments, the kit may further comprise a sampler for collecting the sample.

In some embodiments, the detection system may include a user interface that is accessible and controllable by the software application. The software may be run by a software application on a personal device such as a smart phone, tablet computer, personal computer, laptop computer, smart watch, and/or other device. In some cases, the software may be run by an internet browser. In some embodiments, the software may be connected to remote and localized servers called "clouds".

Drawings

Fig. 1 is a perspective view of an embodiment of a detection system according to the invention, comprising: a detection device 100 having an outer housing 101 and a port or receptacle 102 configured for receiving a disposable cartridge 300; a separate food sampler 200, as an example of a sampler; and a disposable test cup 300 as an example of the cartridge. Optionally, a cover 103 covers the receptacle 102. This embodiment of the system 100 has an execute/action button 104 that allows the user to perform an allergen detection test and may include a USB port 105.

Fig. 2A is an exploded perspective view of one embodiment of a food sampler 200 as an example of a sampler.

Fig. 2B is a perspective view of food sampler 200.

Figure 3A is a perspective view of an embodiment of a disposable test cup 300 comprising a cup top 310, a cup body 320, and a cup bottom 330.

Figure 3B is a cross-sectional view of the test cup 300 showing features inside the cup 300.

Figure 3C is an exploded view of an embodiment of the disposable test cup 300.

Fig. 3D is a top perspective view (left view) and a bottom perspective view (right view) of the top cover 312.

Fig. 3E is a top perspective view (left view) and a bottom perspective view (right view) of the cup body 320.

Fig. 3F is a top perspective view (top view) of the bottom of the upper housing 320a shown in fig. 3C and a bottom perspective view (bottom view) of the interior of the outer housing 320b shown in fig. 3C.

Fig. 3G is a bottom perspective view (left view) and a top perspective view (right view) of the cup bottom cover 337 shown in fig. 3C.

Fig. 3H is a bottom perspective view of the cup bottom surface after assembly of the bottom 330 and cup body 320.

Fig. 3I is an exploded view of the cup top cover 311.

Fig. 4A is an exploded view of one embodiment of a filter assembly 325.

Fig. 4B is a cross-sectional perspective view of one embodiment of filtrate chamber 322, which includes a filter bed chamber 431 for housing a filter assembly 325, a collection tank 432, and a filtrate collection chamber 433.

Figure 5A is a perspective view of an alternative embodiment of a test cup 300.

Fig. 5B is an exploded view of the disposable test cup 300 of fig. 5A (filter 325 not shown).

Fig. 5C is a cross-sectional elevation view of the cup 300 of fig. 5A.

Fig. 5D is an exploded perspective view of an alternative embodiment of test cup 300.

Fig. 5E is a bottom perspective view (upper view) and a top perspective view (lower view) of the cup body 320 shown in fig. 5D.

Fig. 5F is a bottom perspective view of the cup bottom 337 and the bottom of the cup body 320 shown in fig. 5D.

Fig. 5G is an alternative embodiment of a filter assembly 525.

Fig. 5H is a cross-sectional view of the filter cap 541 of the filter assembly 525 when assembled with the valve 350.

Fig. 5I includes a perspective view of the rotary valve 350 (top view), a side view of the rotary valve 350 (bottom left view), and a bottom view of the bottom of the rotary valve 350 (bottom right view).

Fig. 5J is a bottom view (top view) of the cup bottom cover 337 and a top view (bottom view) of the cup bottom cover 337 shown in fig. 5D.

Fig. 5K is a top view of the chip panel 532 shown in fig. 5D.

Fig. 6A is a top view of the upper cup body 510 showing features related to homogenization, filtration (F), washing (W1 and W2), and waste.

Fig. 6B is a schematic diagram showing the position of the rotary valve 350 during sample preparation and sample washing.

Fig. 6C is a diagram showing the flow of fluid inside the cup 300.

Fig. 7A is a perspective view of the device 100.

Fig. 7B is a top view of device 100 without cover 103.

Fig. 8A is a longitudinal cross-sectional perspective view of device 100.

Fig. 8B is a side (lateral) cross-sectional perspective view of the device 100.

Fig. 9A is a valve motor 820 and associated components for controlling the operation of the rotary valve 350.

Fig. 9B is a top perspective view of an output coupling 920 associated with a motor.

FIG. 10A is a top perspective view of one embodiment of an optical system 830.

Fig. 10B is a side view of the optical system 830 of fig. 10A.

Fig. 11A is a diagram of the chip sensor 333 showing a test area and a control area.

Fig. 11B is a top view of optical system 830 and chip 333, showing the reflectance that provides fluorescence measurements of chip 333.

Fig. 12A shows the optical assembly 830 in a straight mode.

Fig. 12B shows the optical assembly 830 in a folded mode.

Fig. 12C is a cut-away perspective view of one end of the device 100 (the right side of fig. 8B), showing the emission optics 1210, which includes lenses 1221, 1223 and filters 1222a and 1222B placed in a stepped bore 1224 in the device 100.

FIG. 13A shows the reaction without MgCl₂And MgCl₂Buffer comparison of solutions, MgCl₂Histogram of SPN intensity in lyophilized formulation.

FIG. 13B shows Slave MgCl₂The percentage of magnesium recovered in the formulation that was deposited on a cotton filter supported on a 1 μm screen.

Detailed Description

The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

The use of analytical devices ensures that food safety has not yet reached the point of achieving its novels. In particular, no portable device based on a simple yet accurate, sensitive and fast detection scheme has been developed to detect a variety of known allergens. One of the latest reviews on aptamer-based analysis in food safety control shows that although a number of commercial analytical tools have been developed to detect allergens, most of them rely on immunoassays. Further indicating that selection of aptamers for this composition is occurring (Amaya-Gonz a lez et al, Sensors 2013,13,16292-16311, which is incorporated herein by reference in its entirety).

The present invention provides a detection system and device that can specifically detect low concentrations of allergens in a variety of food samples. The detection system and/or device of the present invention is a small, portable and hand-held product that is intended to be of compact size, thereby enhancing its portability and discreet operation. A user may carry the detection system and apparatus of the present invention and perform a rapid and real-time test for the presence of one or more allergens in a food sample prior to eating the food. The detection system and apparatus according to the present invention may be used by a user at any location, such as in a home or restaurant. The test system and/or device displays the test results as standard readings and any user can conduct the test in accordance with the brief instructions on how to operate the test system and device.

In some embodiments, the detection systems and devices are configured for simple, fast, and sensitive one-step performance. The system may complete the allergen detection test in less than 5 minutes, or less than 4 minutes, or less than 3 minutes, or less than 2 minutes, or less than 1 minute. In some examples, allergen detection may be completed within approximately 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, or 15 seconds.

The construction process for producing the detection system and device according to the invention may be an electro-mechanical engineering construction process, which integrates electrical engineering, mechanical engineering and computer engineering to carry out and control the process of allergen detection testing. Embodiments of the detection system and apparatus have the following features, including but not limited to: a rechargeable or replaceable battery, a motor drive for processing the test sample, a pump for controlling the flow of the processed sample solution and buffer in the cartridge, a printed circuit board and a connector that couples and integrates the different components for rapid allergen testing. An embodiment of the detection apparatus of the present invention further comprises: an optical system configured for detecting the presence and concentration of an allergen of interest in a test sample and converting the detection signal into a readable signal; and the shell provides support for other parts of the detection device and integrates different parts together into a functional product.

In some embodiments, the detection system and/or device is configured such that a disposable cartridge (e.g., a disposable test cup or cup-like container) dedicated to one or more specific allergens is configured for receiving and processing a test sample and performing a detection test, with all solutions contained therein. Thus, all solutions can be confined in a disposable cup or cup-like container. As a non-limiting example, a user may use a disposable peanut test cup to detect peanuts in any food sample and discard the test. This prevents cross-contamination when different allergen tests are performed using the same device.

In some embodiments, a separate sampler is provided that can measure and size the test sample. In one version, the sampler may further pre-treat the test sample, such as cutting the sample into small pieces, mixing, scraping and/or grinding to render the sample suitable for allergen protein extraction.

According to the present invention, a nucleic acid molecule (i.e., an aptamer) that specifically binds to an allergen of interest in a sample is used as a detection agent. The nucleic acid agent may be an aptamer capable of recognizing the allergen of interest and a Signaling Polynucleotide (SPN) derived from the aptamer. In some embodiments, the SPN captures allergen proteins in the sample to form SPN protein complexes. Another detection probe, such as a short nucleic acid sequence complementary to the SPN sequence, can be used to anchor the SPN to a solid substrate for signal detection. In other embodiments, the detection agent can be attached to a solid substrate, such as a magnetic particle, silica, agarose particle, polystyrene bead, glass surface, microwell, surface of a chip (e.g., microchip), and the like. Within the scope of the present invention, such detection agents and sensors may also be incorporated into any suitable detection systems and instruments for similar purposes.

Aptamers and SPNs that specifically bind to the allergen of interest may be those disclosed in the commonly owned applications: U.S. provisional application serial No. 62/418,984 filed on 8/11/2016; U.S. provisional application serial No. 62/435,106 filed on 12, 16, 2016; U.S. provisional application serial No. 62/512,299 filed on 30/5/2017; and PCT patent application publication No. WO/2018/089391 filed on 8/11/2017; the contents of each of the applications are incorporated herein by reference in their entirety.

Detection system

According to the present invention, the allergen detection system of the present invention may comprise: at least one disposable cartridge for performing an allergen detection test; and the detection device is used for detecting and visualizing the result of the detection test. Optionally, the detection system may further comprise at least one sampler for collecting the test sample. The sampler may be any tool that can be used to collect a portion of the test sample, such as a spoon or a chopstick. In some aspects, as discussed below, specially designed samplers may be incorporated into the present detection system.

As shown in fig. 1, an embodiment of the detection system of the present invention comprises: a detection device 100 configured for processing a test sample, performing an allergen detection test, and detecting the result of the detection test; a separate food sampler 200, as an example of a sampler; and a disposable test cup 300 as an example of the cartridge. The test device 100 includes an outer housing 101 that provides support for the components of the test device 100. The port or receptacle 102 of the testing device 100 is configured for docking the disposable test cup 300 and also includes a lid 103 to open and close the instrument. The outer housing 101 also provides surface space for buttons by which a user can operate the device. An execute/action button 104 and a USB port 105 may be included that allow the user to perform the allergen detection test. Optionally, a power plug (not shown) may also be included. During the course of performing the allergen detection test, the food sampler 200 with the sample contained therein is inserted into the disposable test cup 300, and the disposable test cup 300 is inserted into the port 102 of the detection device 100 for detection.

Sampling device

Collecting a sample of appropriate size is an important step in conducting an allergen detection test. In some embodiments of the present invention, a separate sampler for picking up and collecting a test sample (e.g., a food sample) is provided. In one aspect, a sample-pack-plunger concept for picking and collecting food samples is disclosed herein. Such a mechanism may measure and collect one or several size fractions of the test sample and provide pre-treatment steps such as cutting, grinding, scraping and/or mixing to facilitate homogenization and extraction or release of allergen proteins from the test sample. In accordance with the present invention, individual food samplers 200 are configured to take different types of food samples and collect appropriately sized portions of the test sample.

As shown in fig. 2A, food sampler 200 may include three parts: a plunger 210 at the distal end; a handle 220 configured for coupling with the sampler; and the sampler 230 at the proximal end. The plunger 210 has: a distal portion at the distal end provided with a sampler top grip 211 (fig. 2A) that facilitates manipulation of plunger 210 up and down; a plunger stop 212 in the middle of the plunger body; and a seal 213 at the proximal end of the plunger body. Handle 220 may include a snap fit 221 at the distal end and a skirt 222 at the proximal end that connects to sampler 230. Sampler 230 may include a proximal portion provided with a cutting edge 231 at the proximal-most end (fig. 2A). The sampler 230 is configured for cutting and holding a collected sample to be discharged into the disposable test cup 300.

In one embodiment, the plunger 210 may be inserted into the sampler 230, wherein the proximal end of the plunger 210 may protrude from the sampler 230 for direct contact with the test sample and, together with the cutting edge 231 of the sampler 230, pick up a specifically sized portion of the test sample (fig. 2B). According to the present invention, the plunger 210 is used to expel sampled food from the sampler 230 into the disposable test cup 300, and also to pull specific food (such as liquids and creamy food) into the sampler 230. By interacting with the snap-fit 221, the features of the plunger stop 212 may prevent the plunger 210 from being pulled back too far or out of the sampler body 230 during sampling. To draw liquid into sampler 230 by pulling back plunger 210, seal 213 at the proximal-most end of plunger 210 may maintain an airtight seal. In some embodiments, plunger 210 may be provided with other types of seals, including molded features or mechanical seals. Handle 220 is configured for a user to hold the sampling components of sampler 200. For example, skirt 222 provides a means for a user to operate food sampler 200, push sampler 230 downward, and drive sampler 230 into a food sample to be collected.

In some embodiments, the cutting edge 231 may be configured for pre-processing the collected sample, thereby allowing the sampled food to be sampled in a pushing, twisting, and/or cutting manner. As some non-limiting examples, the cutting edge 231 may be a flat edge, a sharp edge, a serrated edge with various numbers of teeth, a sharp serrated edge, and a thin-walled edge. In other versions, the inner diameter of sampler 230 varies in the range of approximately 5.5mm to 7.5 mm. Preferably, the inner diameter of sampler 230 may vary in the range of about 6.0mm to about 6.5 mm. The inner diameter of the sampler 230 may be 6.0mm, 6.1mm, 6.2mm, 6.3mm, 6.4mm, 6.5mm, 6.6mm, 6.7mm, 6.8mm, 6.9mm, or 7.0 mm. The dimensions of the sampler 230 are optimized to allow the user to collect an appropriate amount of the test sample (e.g., 1.0g to 0.5 g).

The components of food sampler 200 may be configured in any shape that is easy to handle, such as triangular, square, octagonal, circular, elliptical, and the like.

In other embodiments, food sampler 200 may also be provided with a means for weighing the test sample being picked up, such as a spring, scale, or equivalent thereof. As a non-limiting example, food sampler 200 may be provided with a weighing tension module.

Food sampler 200 may be made of a plastic material including, but not limited to, Polycarbonate (PC), Polystyrene (PS), Polymethylmethacrylate (PMMA), Polyester (PET), polypropylene (PP), High Density Polyethylene (HDPE), polyvinyl chloride (PVC), thermoplastic elastomer (TPE), Thermoplastic Polyurethane (TPU), acetal (POM), Polytetrafluoroethylene (PTFE), or any polymer, and combinations thereof.

A sampler (e.g., sampler 200) may be operatively associated with an analysis cartridge (e.g., disposable cup 300) and/or a detection device (e.g., device 100). Optionally, the sampler may comprise an interface for connecting to the cartridge. Optionally, a cap may be positioned at the proximal end of the sampler. Sampler 200 may also include a sensor positioned with sampler 200 to detect the presence of a sample in the sampler.

Disposable processing box

In some embodiments, the present invention provides a test cartridge or vessel. As used herein, the terms "cassette" and "vessel" are used interchangeably. The cartridge is configured for performing a detection test. The test kit is disposable and is intended for use with a particular allergen. The disposable cartridge is configured for: dissociation of food samples and allergen protein extraction, filtration of food particles, storage of reaction solutions/reagents and detection agents, such as antibodies and nucleic acid molecules that specifically bind to allergen proteins, and capture of allergens of interest using detection agents. In one approach, the detection agent is a nucleic acid molecule, such as an aptamer and/or an SPN derived from an aptamer. In other embodiments, the detection agent can be an antibody specific for an allergen protein, such as an antibody specific for the peanut allergen protein Ara H1. According to the present invention, at least one individual cartridge is provided as part of the detection system. In other embodiments, the cartridge can be configured for use in any other detection system.

In some embodiments, the cartridge may be configured to include one or more separate chambers, each configured for separate functions, such as for sample reception, protein extraction, filtration, and storage of buffers, reagents, and waste fluids. The cartridge may further include: means for processing the sample (e.g., a homogenizer); a filter for filtering out large particles; and channels and ports for controlling fluid flow within the cassette.

In some embodiments, the disposable cartridge is intended to be used only once for allergen testing in a sample and may therefore be made of low cost plastic materials, such as Acrylonitrile Butadiene Styrene (ABS), COC (cyclic olefin copolymer), COP (cyclic olefin polymer), transparent High Density Polyethylene (HDPE), Polycarbonate (PC), Polymethylmethacrylate (PMMA), polypropylene (PP), Polyvinylchloride (PVC), Polystyrene (PS), Polyester (PET), or other thermoplastics. Thus, the disposable cartridge may be configured for any particular allergen of interest. In some embodiments, the disposable cartridges may be configured for only one specific allergen, which may avoid cross-contamination with other allergen reactions.

In some embodiments, the disposable cartridge is made of polypropylene (PP), COC (cyclic olefin copolymer), COP (cyclic olefin polymer), PMMA (polymethyl methacrylate), or Acrylonitrile Butadiene Styrene (ABS).

In other embodiments, the disposable cartridges may be configured to detect two or more different allergens in a test sample in parallel. In some aspects, the disposable cartridge may be configured for parallel detection of two, three, four, five, six, seven, or eight different allergens. In one approach, detecting the presence of multiple (e.g., two, three, four, five, or more) allergens simultaneously may produce a positive signal indicating which allergen is present. In another aspect, a system is provided to detect the presence of an allergen, such as a peanut or tree nut, and generate a signal to indicate the presence of such allergen.

In some embodiments, the disposable cartridge may be a disposable test cup or cup-like container. According to one embodiment of the test cup, as shown in FIG. 3A, the assembled disposable test cup 300 comprises three parts: a cup top 310, a cup body 320, and a cup bottom 330. The cup 300 further comprises: a homogenizing rotor 340 that rotates in two directions to homogenize the sample; and a rotary valve 350 for fluid flow within the cup (fig. 3B).

In some embodiments, the test cup body 320 may include multiple chambers. In one embodiment, as shown in fig. 3B, the test cup body 320 includes: a homogenizing chamber 321 comprising a food processing reservoir 601 (shown in fig. 6C); a filtrate chamber 322 for collecting the sample solution after filtration through a filter (e.g., a 2-state filter 325); a waste chamber 323 comprising a waste reservoir 603 (shown in fig. 6C); and optionally, a wash buffer storage chamber 324, including a wash buffer storage reservoir 602 (shown in fig. 6C). The reaction chamber 331 (also referred to herein as a signal detection chamber) at the cup bottom 320 is shown in fig. 3E and 3H. All of the analytical reactions take place in the reaction chamber 331 and produce a detectable signal (e.g., a fluorescent signal) therein. In some embodiments, for example, a detector agent (e.g., SPN) pre-stored in the homogenization chamber 321 can be pre-mixed with a test sample in the homogenization chamber 321, the test sample is homogenized in the homogenization chamber and the extracted allergen protein reacts with the detector agent. The mixed reaction complex may be transferred to the filter 325 before being transferred to the reaction chamber 331, wherein a detection signal is measured.

In alternative embodiments, more than one buffer and reagent storage reservoir may be included in the buffer and reagent storage chamber 324. As a non-limiting example, the extraction buffer and the wash buffer may be stored separately in reservoirs within the buffer storage chamber 324.

Fig. 3C shows an exploded view of a disposable test cup 300 configured to contain three major components, a top 310, a housing or body 320, and a bottom 330. In one embodiment, the cup top 310 may include: a cup cover 311; a top cap 312 having a food sampler port 313 (in fig. 3B and 3D) for receiving food sampler 200; two or more breather filters 314 are included to ensure that only air is introduced and fluid does not escape from the test cup 300. The top portion may have two covers 311. As shown in fig. 3I, the second cover 311b at the bottom is configured to reseal and retain liquid during operation. Top cover 311a may be peeled open to insert a test sample collected by sampler 200 and then reclosed after the assay is complete. The top cover 312 may also include at least one small hole for drawing air to flow the fluid (fig. 3C). The cup body 320 is made up of two separate parts: an upper housing 320a and an outer housing 320 b. A filter or filter assembly 325 is included in the cup body for processing the sample. The filter 325 may be attached to the cup body by a gasket 326. The cup base assembly 330 includes a bottom cover 337 that holds other components including the reaction chamber 331 (in fig. 3E and 3G), the detection sensor (i.e., the glass chip 333), and a chip gasket 334 that facilitates attachment of the glass chip 333 to the bottom of the reaction chamber 331. The bottom cap 337 also includes ports/bores (bit)340a for holding the homogenizing rotor 340 and ports/bores 350a for holding the rotary valve 350 (as shown in figure 3G). These bores provide means for connecting the homogenizing rotor 340 and the rotary valve 350 to the motor of the inspection apparatus 100. For example, a rotor gasket may be configured to the upper housing 320a to seal the rotor 340 to the housing 320 to avoid leakage of fluid.

In some embodiments, the cup may also be configured to include a bar code that can store lot-specific parameters. In one example, the barcode can be a data chip 335 that stores specific parameters of the cup 300, including information of the SPN (e.g., fluorophore label, target allergen, and intensity of the SPN, etc.), expiration date, manufacturing information, and the like.

Figure 3D also illustrates features of the top cover 312 of the cup shown in figure 3A. Sampler port 313 is included to receive a sampler and transfer a collected test sample to sample processing chamber 321. By way of non-limiting example, port 313 may be configured to receive food sampler 200 shown in fig. 2B. Fig. 3E is a top perspective view of the cup housing body 320. The upper housing 320a and the outer housing 320b shown in fig. 3C are assembled together in this view. The upper housing 320a may include one or more chambers operatively connected. In this embodiment, the homogenization chamber 321, the filtration chamber 322 and the waste chamber 323 (left figure) can be seen. The bottom of the cup body 320 includes a reaction chamber 331 (right view) having an inlet and an outlet 336 for fluid flow. The rotor 340 and the rotary valve 350 may be assembled in the cup 300 to form a functional test cartridge (right drawing).

Fig. 3F also shows the bottom external interface (top) of the upper housing (320 a shown in fig. 3C) and the bottom internal interface (bottom) of the outer housing 320b shown in fig. 3C. The two energy director faces 361 (face 1) and 362 (face 2) at the exterior interface of the upper housing 320a interact with the two welded mating faces (i.e., faces 363 (face 1) and 364 (face 2)) at the interior interface of the bottom of the outer housing 320b to hold the housing components 320a and 320b together to form the cup body 320. A fluid path 370 is also included to allow liquid to flow in the cup bottom 330. The rotor 340 and the rotary valve 350 are assembled into the cup 300 through the rotor port 340a and the rotary valve port 350a, respectively.

Fig. 3G also shows the bottom cover 337 of the cup 300 shown in fig. 3A and 3C. After the components are assembled together to form the functional test cup 300, a specialized region 332 within the reaction chamber 331 can include a detection sensor that includes a detection agent, such as SPN, specific to the allergen to be detected. In one embodiment, the detection sensor is a glass chip 333 positioned to the active region 332 by a glass gasket 334 (shown in FIG. 3C). A glass gasket 334 may be included to seal the glass chip 333 in place at the bottom of the active area 332 of the reaction chamber 331 and prevent fluid leakage. Alternatively, the layers may be mated together using adhesive or ultrasonic bonding. In some aspects of the present invention, the glass chip 333 may be directly disposed at the bottom of the reaction chamber 331 (e.g., the bottom surface of the sensor region 332) as a part of the cup bottom cover 337 and integrated into the cup body 320 as a whole. The entire unit may be made of PMMA (polymethylmethacrylate), also known as acrylic or acrylic glass. The transparent PMMA acrylic glass can be used as an optical window for signal detection.

As shown in fig. 3H, the base 330 is assembled with the cup body 320. From this bottom perspective view, the bottom surface of the cup includes a plurality of ports for fluid paths (e.g., fluid inlet/outlet 336) and pump ports 380 as well as ports to connect the rotor 340 and rotary valve 350 shown in fig. 3C to the detection device 100.

Means for blocking fluid flow between components of the cup 300 may be included in the cup 300. In one embodiment, a dump valve 315 (in fig. 3C) is included in the cup housing 320a to block the flow of fluid in the homogenization chamber 321 to a rotary valve 350 disposed at the bottom of the cup 300. The dump valve 315 is held in place by the rotary valve 350 during shipping and at the end of life. The rotary valve 350 locks the dump valve 315 over the filter (e.g., filter assembly 325) during transport and prevents fluid flow after completion of the detection assay. In some embodiments, the rotary valve 350 may include a valve shaft operatively connected to the dump valve 315 and locking the dump valve (as shown in fig. 3C). The rotary valve 350 may be attached to the cup 300 by any available means known in the art. In one embodiment, a valve gasket (e.g., gasket 504 shown in FIG. 5A) may be used. Alternatively, the rotary valve 350 may be attached to the cup by a wave coil spring. The rotary valve 350 may be actuated in several steps to direct the flow to the appropriate chamber within the cup 300. By way of non-limiting example, the position of the rotary valve 350 during the test of detection is shown in FIG. 6B.

In some embodiments, a filter assembly (e.g., filter 325 shown in fig. 3C and 4A) is included in the cartridge for removing large particles and other interfering components, such as fat from the food matrix, from the sample before the processed sample is transferred into the reaction chamber 331.

In some embodiments, the filter mechanism may be a filter assembly. The filter assembly may be a simple membrane filter 420. The film 420 may be nylon, PE, PET, PES (polyethersulfone), Porex^TMGlass fibers or film polymers such as Mixed Cellulose Esters (MCE), cellulose acetate, PTFE, polycarbonate, PCTE (polycarbonate) or PVDF (polyvinylidene fluoride), and the like. It may be a thin film (e.g., 150 μm thick) with high porosity. In some embodiments, the pore size of the filter membrane 420 may be in the range of 0.01 μm to 600 μm, or in the range of 0.1 μm to 100 μm, or in the range of 0.1 μm to 50 μm, or in the range of 1 μm to 20 μm, or in the range of 20 μm to 100 μm, or in the range of 20 μm to 300 μm, or in the range of 100 μm to 600 μm, or any size in between. For example, the pore size can be about 0.02 μm, about 0.05 μm, about 0.1 μm, about 0.2 μm, about 0.5 μm, about 1.0 μm, about 1.5 μm, about 2.0 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4.0 μm, about 4.5 μm, about 5.0 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, or about 600 μm.

In some alternative embodiments, the filter assembly may be a complex filter assembly 325 (shown in fig. 4A) that includes multiple layers of filter material. In one example, filter assembly 325 may include a body filter 410 (fig. 4A) comprised of a coarse filter 411, a depth filter 412, and a membrane filter 420. In one embodiment, coarse filter 411 and depth filter 412 may be secured by retainer ring 413 to form body filter 410 on membrane filter 420. In other embodiments, the body filter 410 may also include a powder located inside or on top of the filter. The powder may be selected from cellulose, PVPP, resins, etc. In some examples, the powder does not bind to nucleic acids and proteins.

In some embodiments, filter assembly 325 may be optimized to remove oil from high fat samples without removing proteins and nucleic acids, thereby achieving excellent sample cleaning. In other embodiments, the ratio of the depth and width of the filter assembly 325 may be optimized to maximize filtration efficiency.

In some embodiments, the filter assembly 325 can be placed within the filter bed chamber 431 (FIG. 4B) in the disposable cup body 320. The filter bed chamber 431 may be connected to the homogenizing chamber 321. The homogenized product may be supplied to a filter assembly 325 within a filter bed chamber 431. The filtrate is collected by a collection tank 432 (also referred to herein as a filtrate chamber). The collected filtrate may then exit the jet to flow to the reaction chamber 331 (fig. 3B). In one example, the collected filtrate may be transported directly from the collection tank 432 to the reaction chamber 331. In another example, the filtrate may be first delivered to the filtrate collection chamber 433 before being delivered to the reaction chamber 331 through the inlet/outlet 336 (fig. 3G). Fluid can be delivered to the reaction chamber 331 through a fluid path 370 (shown in fig. 3F) at the bottom of the cup 320.

In some embodiments, the filtrate collection chamber 433 can also include a filtrate concentrator configured to concentrate the sample filtrate before the sample filtrate flows to the reaction chamber 331 for signal detection. The concentrator may be a hemispherical, or conical concentrator, or tall tube.

According to this embodiment, the processed sample (e.g., homogenized product from chamber 321) is filtered sequentially through a coarse filter 411, a depth filter 412, and a membrane filter 420. The coarse filter 411 may filter large particle suspensions, e.g. particles larger than 1mm, from the sample. The depth filter 412 can remove small particle collections and oil components from a sample, such as a food sample. The pore size of the depth filter 412 may be in the range of about 1 μm to about 500 μm, or in the range of about 1 μm to about 100 μm, or in the range of about 1 μm to about 50 μm, or in the range of about 1 μm to about 20 μm, or in the range of about 4 μm to about 15 μm. For example, the pore size of the depth filter 412 may be about 2 μm, or about 3 μm, or about 4 μm, or about 5 μm, or about 6 μm, or about 7 μm, or about 8 μm, or about 9 μm, or about 10 μm, or about 11 μm, or about 12 μm, or about 13 μm, or about 14 μm, or about 15 μm, or about 16 μm, or about 17 μm, or about 18 μm, or about 19 μm, or about 20 μm, or about 25 μm, or about 30 μm, or about 35 μm, or about 40 μm, or about 45 μm, or about 50 μm.

Depth filter 412 may be composed of, for example, cotton (including but not limited to raw and bleached cotton), polyester mesh (monofilament polyester fiber), or sand (silica). In some embodiments, the filter material may be hydrophobic, hydrophilic, or oleophobic. In some examples, the material does not bind to nucleic acids and proteins. In one embodiment, the depth filter is a cotton depth filter. The dimensions of the cotton depth filter may vary. For example, the cotton depth filter may have a width to height ratio in the range of about 1:5 to about 1: 20. The cotton depth filter 412 may be configured to correlate total filter volume and amount of food filtered.

The membrane filter 420 may remove small particles of a size less than 10 μm, or a size less than 5 μm, or a size less than 1 μm. The pore size of the film may be in the range of about 0.001 μm to about 20 μm, or 0.01 μm to about 10 μm. Preferably, the pore size of the filter membrane may be about 0.001 μm, or about 0.01, or about 0.015 μm, or about 0.02 μm, or about 0.025 μm, or about 0.03 μm, or about 0.035 μm, or about 0.04 μm, or about 0.045 μm, or about 0.05 μm, or about 0.055 μm, or about 0.06 μm, or about 0.065 μm, or about 0.07 μm, or about 0.075 μm, or about 0.08 μm, or about 0.085 μm, or about 0.09 μm, or about 0.095 μm, or about 0.1 μm, or about 0.15 μm, or about 0.2 μm, or about 0.25 μm, or about 0.3 μm, or about 0.35 μm, or about 0.15 μm, or about 0.2 μm, or about 0.65 μm, or about 0.55 μm, or about 0.5 μm, or about 0.75 μm, or about 0.5 μm, Or about 0.85 μm, or about 0.9 μm, or about 1.0 μm, or about 1.5 μm, or about 2.0 μm, or about 3.0 μm, or about 3.5 μm, or about 4.0 μm, or about 4.5 μm, or about 5.0 μm, or about 6.0 μm, or about 7.0 μm, or about 8.0 μm, or about 9.0 μm, or about 10 μm. As discussed, the film may be a nylon film, PE, PET, PEs (polyethersulfone) film, glass fiber film, polymer film such as Mixed Cellulose Ester (MCE) film, cellulose acetate film, cellulose nitrate film, PTFE film, polycarbonate film, track etched polycarbonate film, PCTE (polycarbonate) film, polypropylene film, PVDF (polyvinylidene fluoride) film, or nylon and polyamide film.

In one embodiment, the membrane filter is a PET membrane filter with a pore size of 1 μm. The small pore size prevents particles larger than 1 μm from entering the reaction chamber. In another embodiment, the filter assembly may comprise a cotton filter combined with a PET mesh with a pore size of 1 μm.

In some embodiments, the filtration mechanism has low protein binding, low nucleic acid binding, or no nucleic acid binding. The filter may be used as a bulk filter to remove fat and emulsifiers as well as large particles to obtain a filtrate with a viscosity comparable to the viscosity of the buffer.

In some embodiments, filter assembly 325, including coarse filter 411, depth filter 412, and membrane filter 420, can provide maximum recovery of Signal Polynucleotides (SPNs) and other detection agents.

In some embodiments, the filtration mechanism may complete the filtration process in less than 1 minute, preferably in about 30 seconds. In one example, the filter mechanism may be capable of collecting a sample at a pressure of less than 10psi in 35 seconds, or 30 seconds, or 25 seconds, or 20 seconds. In some embodiments, the pressure may be less than 9psi, or less than 8psi, or less than 7psi, or less than 6psi, or less than 5 psi.

In some alternative embodiments, filter chamber 322 may include one or more additional chambers configured for filtering processed samples. As shown in fig. 4B, filter chamber 322 may also include a separate filter bed chamber 431 in which a filter assembly 325 (shown in fig. 4A) is inserted and connected to a collection tank 432. A collection tank 432 is configured to collect filtrate that flows through the filter assembly 325, and the tank 432 may be directly connected to the flow cell jet to flow the filtrate to the reaction chamber 331 for signal detection. Optionally, a further collection/concentration chamber 433 may be included in the filter chamber 322 configured for collecting and/or concentrating the filtrate collected by the collection tank 432 before it is conveyed to the reaction chamber 331 for signal detection. The collection/concentration compartment 433 is collected to the filter bed compartment 431 by a collection trough 432.

Fig. 5A-5C illustrate an alternative embodiment of a disposable cartridge 300 (fig. 5A). Similarly, as shown in fig. 5B, the cup includes three portions: a cup top cover 310, a cup can 320, and a cup bottom cover 330, which are operatively connected to form an analysis module. At the top of the cup is a top cap 310, from which a test sample is placed in the cup for testing. A top gasket 501 may be included to seal the top 310 to the cup body. The upper cup body 510 includes a homogenization chamber, a waste chamber, a chamber for washing (e.g., wash 1 chamber (W1), wash 2 chamber (W2) shown in fig. 6A), and an air vent set for controlling air and thus fluid flow. A rotor 340 is arranged in the homogenization chamber for homogenizing the test sample in the homogenization buffer. The shape of the rotor may be adjusted during assembly to fit the cup. An intermediate gasket 502 is positioned at the bottom of the upper cup body 510 to seal the body 510 to a manifold 520 having apertures for fluid flow. The manifold 520 is configured to hold the filter 325 and the fluid path 370 for fluid flow. Another middle gasket 503 is added to seal the manifold 520 to the bottom cover 330, where the reaction chamber, glass chip, glass gasket, and memory chip (e.g., EPROM) are positioned. The rotor 340 is sealed to the bottom by an O-ring 505 (shown in fig. 5C). The rotary valve 350 is configured to the bottom 330 by a valve gasket 504. The configuration of each component of the cup shown in FIG. 5B is also shown in the cross-sectional view of FIG. 5C.

In accordance with the present invention, a further alternative embodiment of the disposable cup 300 is shown in FIG. 5D. Fig. 5E-5K also show the components of the disposable cup 300 of fig. 5D. As shown in fig. 5D, the cartridge includes a top portion 310, a body portion 320, and a bottom portion 330. The rotor 340 is sealed to the cup body by a gasket 533. The rotary valve 350 is assembled to the cartridge by a coil spring 535. When performing a test assay, the rotary valve 350 may rotate and move the seal 533 to release the rotor 340, thereby homogenizing the test sample. In this embodiment, a separate jet panel 532 is disposed between the bottom of the cup body 320 and the bottom cover 337, including the jet channel therein. When the components of the test cup are assembled together, a reaction chamber 331 is formed between the fluidic panel 532 and the bottom cover 337. The DNA chip 333 may be operatively connected to the fluidic panel 532 and the sensor region 332 of the reaction chamber 331 through the chip PSA 534. The fluidic path of plate 532 directs the processed sample to reaction chamber 331 for signal detection.

The cup top 310 may include a top cover 311 with two tabs 311a and 311b as shown in fig. 3I. The cup body 320 may be configured to provide several separate chambers including a homogenization chamber 321, a filtration chamber 322, a waste chamber 323, two or more washing spaces (W1 and W2), as shown in fig. 5E (upper drawing). In some examples, filter chamber 322 has vent 531. The wetting of vent 531 may signal the pressure sensor of the electronics that chamber 322 is full (fig. 5D). At the bottom of the cup body 320, several ports are designed, similar to other designs, including a port for the rotor 340 and a port for the rotary valve 350 (e.g., the rotary valve 350 shown in fig. 5I) for assembling the functional cartridge. These ports align with the ports (e.g., 340a and 350a shown in fig. 5J) of the bottom cover 337 when sealing the cup bottom cover 337 to the cup body 320 and sealing the cup.

In this embodiment, the fluidic panel 532 is inserted into the bottom of the cup body 320; the panel is configured to hold the DNA chip 333 by the chip PSA 534 and provide the necessary fluidic paths (e.g., 370) to flow the processed sample to the DNA chip 333. Fig. 5K shows an exemplary configuration of a fluidic panel 532, where a DNA chip 333 may be attached to the reaction chamber 331 and the inlet and outlet channels 336 will flow the sample to the DNA chip for detection reactions.

In some examples, the filter assembly 325 is inserted into the homogenization chamber 321 to filter the processed sample. In one example, the filter component 325 may be the filter shown in FIG. 4A. In another example, an alternative filter assembly 525 may be configured to include a filter 544 (e.g., a mesh filter) inserted into a filter pad 543, a body filter 542, and a filter cap 541 (fig. 5G). The filter assembly 525 may be secured by the rotary valve 350 and controlled by the valve 350 (fig. 5H).

In some embodiments, the reaction chamber 331 may include a dedicated sensing region 332 configured for holding a detection sensor for signal detection. In some aspects of the invention, the detection sensor may be a solid substrate (e.g., glass surface, chip, and microwell) whose surface is covered with capture probes, such as short nucleic acid sequences complementary to SPNs that bind to the target allergen. In some embodiments, the sensing region 332 within the reaction chamber 331 can be a glass chip 333 (fig. 3C and 5D).

In some embodiments, the reaction chamber 331 includes at least one optical window. In one embodiment, the chamber includes two optical windows, namely, a primary optical window and a secondary optical window. In some embodiments, the primary optical window serves as an interface for the reaction chamber 331 to the detection device 100 (and in particular to the optical system 830 (shown in FIGS. 10A, 10B, and 12A-12C) of the detection device 100. A detection sensor (e.g., a glass chip 333) may be positioned between the optical window and the interface of the optical system.

In some embodiments, the glass chip 333 (i.e., DNA chip) printed with nucleic acid molecules is aligned with the optical window. In some embodiments, the DNA chip comprises at least one reaction panel and at least two control panels. In some versions of the invention, the reaction panels of the chips face the reaction chambers 331 flanked by the inlet and outlet channels 336 of the cartridge 300. In some embodiments, the reaction panel of the glass chip 333 may be coated/printed with short nucleic acid probes that hybridize to SPNs with high specificity and binding affinity for the allergen of interest. Then, when the SPN is hybridized with the nucleic acid probe, it can be anchored to the chip.

In a preferred embodiment, the sensor DNA chip (e.g., 333 in fig. 3C) may include: a reaction panel printed with short complementary sequences that hybridize to SPNs specific for the allergen of interest; and two or more control regions (control panels) covalently linked to nucleic acid molecules (as control nucleic acid molecules) that do not react with SPNs or allergens. The complementary probe sequence can bind to SPN only when SPN is not bound to the target allergen protein. In some aspects of the invention, the nucleic acid molecules printed in the control panel are labeled with probes (e.g., fluorophores). The control panel provides a mechanism for the optical device to normalize the signal output with respect to the reaction panel and confirm proper operation procedures. An exemplary configuration of the chip 333 is shown in fig. 11A.

In some embodiments, the DNA coated chip 333 can be prepackaged into the reaction chamber 331 of the cartridge. In other embodiments, the DNA coated chip 333 can be packaged separately from a disposable cartridge (e.g., the cup 300 in fig. 1).

In some embodiments, the solid substrate used to fabricate the sensor chip may be glass with high optical transparency, such as borosilicate glass and soda glass.

In some embodiments, the solid substrate for printing DNA may be made of a plastic material with high optical transparency. As non-limiting examples, the substrate may be selected from the group consisting of: polydimethylsiloxane (PDMS), Cyclic Olefin Copolymer (COC), Polymethylmethacrylate (PMMA), Polycarbonate (PC), Cyclic Olefin Polymer (COP), Polyamide (PA), Polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), Polystyrene (PS), Polyoxymethylene (POM), Polyetheretherketone (PEEK), Polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (pvdf)Vinylidene fluoride (PVDF), polyvinyl alcohol, polyimides, polybutylene terephthalate (PBT), Fluorinated Ethylene Propylene (FEP), perfluoroalkoxy alkanes (PFA), polypropylene carbonate (PPC), Polyethersulfone (PES), polyethylene terephthalate (PET), cellulose, poly (4-vinylbenzyl chloride) (PVBC),Hydrogels, Polyimides (PI), 1, 2-Polybutadiene (PB), fluoropolymers and copolymers (e.g., Polytetrafluoroethylene (PTFE), perfluoroethylene propylene copolymer (FEP), Ethylene Tetrafluoroethylene (ETFE)), norbornene group containing polymers, polymethylmethacrylate, acrylic polymers or copolymers, polystyrene, substituted polystyrene, polyimide, silicone elastomers, fluoropolymers, polyolefins, epoxy resins, polyurethanes, polyesters, polyethylene terephthalate, polysulfone (polyperfone), and polyetherketone, or combinations thereof.

The cup bottom 330 is configured to close the disposable test cup 300 and provide a means for coupling the test cup 300 to the test device 100. In some embodiments, the bottom side of the bottom component 330 of the cup 300 shown in fig. 3G includes a plurality of interfaces for connecting the cup 300 to the detection apparatus 100 for operation, including: a homogenizing rotor interface 340a, which may couple the homogenizing rotor 340 to a motor in the apparatus 100 for controlling homogenization; a valve interface 350a that may couple the rotary valve 350 to a motor in the device 100 for controlling the rotation of the valve; a pump interface 380 for connection to a pump in the test device 100.

In some embodiments, a valve system is provided to control the fluid flow of samples, detection agents, buffers, and other reagents through different portions of the cartridge. In addition to the flexible membranes, foil seals, and pinch valves discussed herein, other valves may be included to control fluid flow during the detection assay process, including swing check valves, gate valves, ball valves, globe valves (globe valves), rotary valves, custom valves, or other commercially available valves. For example, a gland seal or rotary valve 350 may be used to control the flow of the processed sample solution within the cup 300. In some examples, pinch valves or rotary valves are used to completely isolate the fluid from other internal valve components. In other examples, a pneumatic valve (e.g., a pneumatic pinch valve) may be used to control fluid flow, which is operated by a pressurized air supply.

In one embodiment, the means for controlling fluid flow within the cup chamber may be incorporated, for example, in the cup base assembly 330. The device may include flow channels, tunnels, valves, gaskets, vents, and air connections. In one embodiment, fluidic channels may be configured in the fluidic panel 532, as shown in fig. 5D.

In other embodiments, the valve system of the present invention may include an additional air vent included in the test cup 300 to control air flow when the DNA-coated glass chip is used as a detection sensor. The DNA chip may be purged with air during the allergen detection assay process. The single air inlet port may be opened based on the requirements of the system. The valve system discussed herein may be used to keep the air vent unit inactive until use. The air ports allow air to enter the cartridge (e.g., cup 300) and the air vents allow air to enter the various chambers when fluid is added to or removed from the chambers. The air vent may also have a membrane incorporated therein to prevent spillage and act as a mechanism to control the amount of fluid filled by occluding the vent membrane to prevent further flow and filling functions.

In a preferred embodiment, a rotary valve 350 (shown in FIGS. 3C and 5B) may be used to control and regulate the fluid flow and rate in test cup 300. The rotary valve 350 may include a valve shaft and a valve disc operable by an associated detection device (e.g., device 100). In some embodiments, the rotary valve 350 may be positioned at a particular angle by rotating the valve member counterclockwise (CCW) or Clockwise (CW) during the course of a test assay in each step of a repeated wash and air purge cycle. The air holes allow air to enter. Air is drawn into the system via vacuum pressure to perform an air purge function. The angle may be in the range of about 2 ° to about 75 °.

As a non-limiting example, the valve may be positioned at about 38.5 ° relative to the gas vent, wherein the pump 840 is closed and the reaction chamber 331 is dry (referred to as the home position). After processing and homogenization of the test sample, the pump is turned on and the valve 350CCW is rotated and parked at an angle of about 68.5 ° allowing the processed sample to be transported to the filter chamber 322. Next, the valve member can be rotated again in different directions to park at different angles, such as at about 57 deg., to flow the wash buffer to the reaction chamber 331, and at about 72 deg. to purge the DNA chip with air. After the pre-washing of the DNA chip, the valve member may be rotated to a home position at about 38.5 deg.. The treated sample solution is pulled through the filter assembly 325. After filtration, the valve member can be rotated and parked at an angle of about 2 °, allowing the collected filtrate to flow into the reaction chamber 331 where the chemical reaction takes place. The valve 350 will rotate and park at about 57 ° to allow the wash buffer to flow to the reaction chamber 331 and at about 72 ° to purge the DNA chip with air. The washing and air purging steps may be repeated one or more times until the optical measurements indicate a clean background.

In one embodiment, the valve system may be a rotary valve operating as shown in FIG. 6B. In this embodiment, the rotary valve 350 is positioned to control air and fluid flow in the system. Rotary valve 350 drives homogenization in homogenization chamber 321, filtration and collection of filtrate (F), sample washing (e.g., wash 1(W1) and wash 2(W2)), and waste collection (fig. 6A). In step 1 of fig. 6B, the rotary valve 350 is in the closed position, no connection is made between any of the chambers. In step 2 of fig. 6B, the rotary valve 350 connects the wash 1 chamber W1 to the reaction chamber 331 to flush the reaction chamber 331 with the wash buffer, and then pushes the wash buffer out to the waste chamber 323. In step 3 of fig. 6B, the rotary valve 350 connects the homogenization chamber 321 to the filtrate chamber F to perform the filtration step. In step 4 of fig. 6B, a rotary valve 350 connects the filtrate chamber F to the reaction chamber 331 to send the filtrate to the reaction chamber 331 for reaction and analysis. In step 5 of fig. 6B, the rotary valve 350 connects the wash 2 chamber W2 to the reaction chamber to flush the reaction chamber 331 again.

In some embodiments, the extraction buffer may be pre-stored in the homogenization chamber 321, for example, in a foil-sealed reservoir, such as the food processing reservoir 601 (fig. 6C). Alternatively, the extraction buffer may be stored separately in a separate buffer reservoir in the cup body 320, similar to the reservoir of the wash buffer storage reservoir 602 (buffer storage chamber 324 (optional) shown in fig. 6C). The extraction buffer and wash waste after sample homogenization may be stored in separate waste reservoirs 603 within the waste chamber 323. The waste chamber 323 has a sufficient volume to store a volume greater than the amount of fluid used during the detection assay.

According to the present invention, the homogenizing rotor 340 may be configured small enough to fit into the disposable test cup 300, and in particular into the homogenizing chamber 321, where the homogenizer processes the sample to be tested. In addition, the homogenizing rotor 340 may be optimized to increase the efficacy of sample homogenization and protein extraction. In one embodiment, the homogenizing rotor 340 may comprise one or more blades or their equivalents at the proximal end. In some examples, the rotor 340 may include one, two, three, or more blades. The homogenizing rotor 340 is configured to pull the test sample from the food sampler 200 into the bottom of the homogenizing chamber 321.

Alternatively, the homogenizing rotor 340 may also include a central rod extending through the rotor, which is connected to the second interface bore by the cup body 320. The central rod may be used as an additional bearing surface, or to transfer rotational motion to the rotor 340. When the rotor 340 is mounted to the cup body 320 through a port (e.g., 340a) at the bottom of the cup, the blade tips may remain submerged in the extraction buffer during operation. In another alternative embodiment, the homogenizing rotor 340 may have an extension to provide a channel through the bottom of the cup; the channel may serve as a secondary support and/or additional location for power transfer. In this embodiment, the lower portion of the rotor is tapered to fit to the shaft, thereby forming a one-piece rotor. According to the invention, the depth level of the blades of the homogenizing rotor 340, with or without a central rod, is positioned to ensure that the blade tips remain in the fluid during sample processing.

In contrast to other homogenizers (e.g., U.S. patent No. 6,398,402; incorporated herein by reference in its entirety), the custom blade core of the present invention takes food and forces the food into the toothed surface of the custom cap as the blade rotates. The homogenizer rotor may be made of any thermoplastic material, including but not limited to Polyamide (PA), Acrylonitrile Butadiene Styrene (ABS), Polycarbonate (PC), High Impact Polystyrene (HIPS), and acetal (POM).

The disposable cartridge may be of any shape, for example, circular, elliptical, rectangular or oval. Any of these shapes may be provided with finger cuts or indentations. The disposable cartridge may be asymmetric or symmetric.

Optionally, a label or foil seal may be included on the top of the cap 311 to provide a final fluid seal and identification of the test cup 300. For example, the peanut indicia indicates that the disposable test cup 300 is used to detect peanut allergens in a food sample.

Detection device

In some embodiments, the detection apparatus 100 may be configured to have: an outer housing 101 providing a support surface for the components of the detection device 100; a lid 103 that opens the detection device 100 for insertion of the disposable test cup 300 and covers the cup during operation. The small cover 103 may be located on one side of the device (as shown in fig. 1 and 7A), or may be located in the center (not shown). In some aspects of the invention, the cover may be transparent, allowing all operations to be visible through the cover 103. The device may also include a USB port 105 for transferring data.

One embodiment of an allergen detection device 100 according to the present invention is depicted in fig. 1 and 7A. As shown in fig. 1, a test device 100 including an outer housing 101 provides a support for holding the components of the test device 100 together. The outer housing 101 may be formed of plastic or other suitable supporting material. The device also has a port or receptacle 102 for docking with a test cup 300 (fig. 1 and 7A).

In order to perform the allergen detection test, the detection device 100 is provided with means for operating the homogenizing assembly (e.g. a motor) and the necessary connectors to connect the motor to the homogenizing assembly; means for controlling the rotary valve (e.g., a motor); means for driving and controlling the flow of the treated sample solution during the course of the allergen detection test; an optical system; means for detecting a fluorescent signal from a detection reaction between the allergen in the test sample and the detection agent; means for visualizing the detected signals, including converting and digitizing the detected signals; a user interface for displaying the test results; and a power source.

As seen from the transparent cover 103 (fig. 7A), the device 100 has an interface comprising a region for coupling components of the cartridge 300 (when inserted) for handling the reaction (fig. 7B). These areas include: a homogenizing bore 710 for coupling the rotor 340 to the motor; a vacuum bore 720 for coupling the cup with a vacuum pump; a rotary valve drive bore 730 for coupling the rotary valve 350 to a valve motor; and a cover glass 740 aligned with the glass chip 333 through an optical window of the reaction chamber 331. A data chip reader 750 is also included to read the data chip 335. The pins 760 are used to help place the cup 300 in the receptacle of the device 100.

In one embodiment of the present invention, as shown in figure 8A, the components of the inspection device 100 that are integrated to provide all the motions and actuations for operating the inspection tests include a motor 810 that may be connected to the homogenizing rotor 340 within the homogenizing chamber 321 within the cup body 320. The motor 810 may be connected by a multi-part coupling assembly comprising a gear train/drive platen for driving the rotor during homogenization in the allergen detection test; a valve motor 820 for driving the rotary valve 350; an optical system 830 connected to the reaction chamber 331 of the disposable test cup 300 (not shown); a vacuum pump 840 for controlling and regulating air and fluid flow (not shown in FIG. 8A); a PCB display 850; and a power supply 860 (fig. 8B). A means for holding a test cup (i.e., a cup holder 870) is included for holding the test cup 300. Each component is described in detail below.

1. Homogenizing assembly

In one embodiment, the motor 810 may be connected to the homogenizing rotor 340 within the test cup 300 by a multi-part rotor coupling assembly. The rotor coupling assembly may include: a coupler directly connected with the distal end cap of the rotor 340; and a gear head as part of a gear train or drive (not shown) for connection to the motor 810. In some embodiments, the couplers may have different dimensions at each end, or the same dimensions at each end of the couplers. The distal end of the coupling assembly may be connected to the rotor 340 through a rotor port 340a at the cup bottom 330. It is also within the scope of the invention that other alternative means for connecting the motor 810 to the homogenizing rotor 340 may be used to form the functional homogenizing assembly.

In some embodiments, Motor 810 can be a commercially available Motor, such as, for example, Maxon RE-max and/or Maxon A-max (Maxon Motor ag, san Mateo, Calif., USA).

Optionally, a heating system (e.g., resistive heating or Peltier heater) may be provided to increase the temperature of homogenization, thus increasing the efficiency of sample dissociation and shortening the processing time. The temperature may be increased to between 60 ℃ and 95 ℃ but should be kept at 95 ℃ or less. The increase in temperature may also facilitate binding between the detector molecule and the allergen being detected. Optionally, a fan or Peltier cooler may be provided to rapidly reduce the temperature after the test is performed.

The motor 810 drives the homogenization assembly to homogenize the test sample in the extraction buffer and dissociate/extract the allergen proteins. The treated sample solution can be pumped or pressed through the flow tube to the next chamber for analysis, e.g., to reaction chamber 331 where it is mixed with the preloaded detection molecule (e.g., signal polynucleotide) for detection testing. Alternatively, the treated sample solution may first be pumped or pressed through a flow tube to the filter assembly 325 and then to the filtrate chamber 322 before being delivered to the reaction chamber 331 for analysis.

2. Filtration

In some embodiments, a device for controlling filtration of the treated test sample may be included in the detection device. The food sample will be pressed through a filter membrane or filter assembly before the extraction solution is delivered to the reaction chamber 331 and/or other chambers for further processing, such as washing. One example is a filter membrane. The membrane provides the function of filtering specific particles from the treated protein solution. For example, the filter membrane may filter particles from about 0.1 μm to about 1000 μm, or from about 1 μm to about 600 μm, or from about 1 μm to about 100 μm, or from about 1 μm to about 20 μm. In some examples, the filter membrane can remove particles up to about 20 μm, or about 19 μm, or about 18 μm, or about 17 μm, or about 16 μm, or about 15 μm, or about 14 μm, or about 13 μm, or about 12 μm, or about 11 μm, or about 10 μm, or about 9 μm, or about 8 μm, or about 7 μm, or about 6 μm, or about 5 μm, or about 4 μm, or about 3 μm, or about 2 μm, or about 1 μm, or about 0.5 μm, or about 0.1 μm. In one example, the filter membrane may remove particles up to about 1 μm from the treated sample. In some aspects, filter membranes may be used in combination to filter specific particles from an assay for analysis. The filter membrane may comprise a multi-stage filter. The filter membrane and/or filter assembly may be in any configuration relative to the flow valve. For example, the flow valve may be above, below, or between any stage of filtration.

In some embodiments, the filter assembly may be a complex filter assembly 325 as shown in fig. 4A, where the processed sample is filtered sequentially through a coarse filter 411, a depth filter 412, and a membrane filter 420.

3. Pump and fluid movement

According to the present invention, there is provided a device for driving and controlling the flow of a processed sample solution. In some embodiments, the device may be a vacuum system or an external pressure. By way of non-limiting example, the device may be a platen configured for multiple functions (e.g., a welded plastic flip cover) because it may support the axis of the gear train and may provide pumping (sealed air passage) for vacuum to be applied from the pump 840 to the test cup 300. The pump 840 can be connected to the test cup 300 through a pump port 720 (fig. 7B) located at the bottom that connects to a pump interface 380 (fig. 3G) on the bottom 330 of the test cup 300 when the cup is inserted into the device.

The pump 840 may be a piezoelectric micropump (e.g., Takasago Electric, inc., ancient house, japan) or a peristaltic pump that can be used to control the flow and automatically adjust it to a target flow rate. The flow rate of the pump can be adjusted by varying the driver voltage or driving frequency. As a non-limiting example, the pump 840 may be a peristaltic pump. In another embodiment, pump 840 may be a piezoelectric pump currently on the market with specifications suitable for the aliquoting functions required for the filtered sample solution to flow to the different chambers within test cup 300. The pump 840 may be a vacuum pump or other small pump configured for laboratory use, such as a KBF pump (e.g., KNF Neuberger, trenden, new jersey, usa).

Alternatively, a syringe pump, a diaphragm, and/or a micro-peristaltic pump may be used to control fluid movement during the course of operation of the detection assay and/or the support jet. In one example, an air operated diaphragm pump may be used.

4. Rotary valve control

In some embodiments, the rotary valve 350 (e.g., as shown in FIG. 5I) used to control the fluid flow needs to be in a precise position. Means are provided for controlling the rotary valve and the control mechanism enables the valve to be rotated in both directions and stopped at the desired position accurately. In some embodiments, the device 100 includes a valve motor 820 (in fig. 7B). As shown in fig. 9A, the valve motor 820 may be a low cost DC gear motor 910 with two low cost optical sensors (931, 932) and a microcontroller. The output coupling 920 is engaged with the rotary valve 350. In some embodiments, output coupler 920 has a half-moon shaped shelf 970 as shown in fig. 9B that interrupts output optical sensor 931 with a protruding half. The output optical sensor signal switches between high and low depending on whether the protruding shelf interrupts the sensor or not. A Microcontroller (MCU) detects these transitions and derives the absolute position of the output from the signal. The location of these transitions is important and application specific because these transitions are used to account for gear lash during direction changes.

The direct motor shaft 940 has a paddle wheel that interrupts the direct shaft optical sensor 932, allowing the direct shaft optical sensor 932 to output a series of pulses, the number of pulses per revolution being determined by the number of paddles on the wheel 950. The MCU reads the series of pulses and determines the angular movement of the output coupling. The resolution depends on the number of paddles of the direct shaft encoder wheel 950, and the gear reduction ratio of the gear box 960.

The MCU interprets the outputs of the two optical sensors and can drive the outputs to the desired positions as long as the output coupler shelf position transitions, the number of paddles on the direct encoder wheel 920, and the gear ratio are known. During the change of direction, the motor must rotate a fixed amount before seeing the output transition. The fixed amount is selected to overcome backlash of the gears. Once the fixed amount is overcome, the MCU can start counting direct signal pulses at the next output signal transition and be confident that they correspond to an accurate output of position and movement.

5. Optical system

The detection device 100 of the present invention includes an optical system that detects an optical signal (e.g., a fluorescent signal) generated by the interaction between the allergen in the sample and the detection agent (e.g., aptamer and SPN). Depending on the type of fluorescence signal to be detected, the optical system may comprise different components and variable configurations. The optical system is adjacent to and aligned with the cartridge, e.g., the primary optical window and the optional secondary optical window of the reaction chamber 331 of the test cup 300 as described above.

In some embodiments, the optical system 830 can include excitation optics 1010 and emission optics 1020 (fig. 10A and 10B). In one embodiment, as shown in FIG. 10A, excitation optics 1010 may include: a laser diode 1011 configured to transmit the excitation optical signal to a sensing region (e.g., 332) in the reaction chamber 331; a collimating lens 1012 configured to focus light from the light source; a filter 1013 (e.g., a band pass filter); a focusing lens 1014; and an optional LED power monitor photodiode. The emission optics 1020 may include: a focusing lens 1015 configured to focus at least a portion of the allergen-related optical signal onto a detector (photodiode); two filters, including a long pass filter 1016 and a band pass filter 1017; a collection lens 1018 configured to collect light emitted from the reaction chamber; and an orifice 1019. The emission optics collect light emitted from a solid surface (e.g., a DNA chip) in the detection chamber 331 and this signal is detected by a detector 1030 configured to detect allergen-related optical signals emitted from the sensing region 332. In some aspects, the excitation power monitoring may be integrated into the laser diode 1011 (not shown in fig. 10A).

The light source 1011 is arranged to transmit excitation light in an excitation wavelength range. Suitable light sources include, but are not limited to, lasers, semiconductor lasers, Light Emitting Diodes (LEDs), and organic LEDs.

An optical lens 1012 may be used with the light source 1011 to provide the excitation source light to the fluorophores. Optical lens 1014 may be used to limit the range of excitation light wavelengths. In some aspects, the filter may be a band pass filter.

Fluorophore-labeled SPNs specific for the target allergen are capable of emitting an allergen binding-related optical signal (e.g., fluorescence) in at least one emission wavelength range in response to excitation light in at least one excitation wavelength range.

In some embodiments, the emission optics 1020 are operable to collect emissions upon interaction between a target allergen in a test sample from the reaction chamber 331 and a detection agent. Optionally, a mirror may be interposed between the emission optics 1020 and the detector 1030. The mirror can be rotated over a large angular range (e.g., 1 ° to 90 °), which can facilitate forming a compact optical unit within a small portable detection device.

In some embodiments, more than one emission optical system 1020 may be included in the detection apparatus. As a non-limiting example, three photodiode optical systems may be provided to measure fluorescence signals from an unknown test region and two control regions on a glass chip (see, e.g., fig. 11B). In further aspects, an additional collection lens 1018 may also be included in emission optics 1020. The collection lens may be configured to detect several different signals from the chip 333. For example, when performing detection assays using DNA glass chips, more than two control regions can be constructed on a solid surface in addition to the detection region for allergen detection. When allergen-derived signals are measured, the internal control signals from each control region can be detected simultaneously. In this case, more than two collection lenses 1018 may be included in the optical system 830, one lens 1018 for signals from the detection area and the remaining collection lenses 1018 for signals from the control area.

A detector (e.g., photodiode) 1030 is arranged to detect light emitted from the fluidic chip in the emission wavelength range. Suitable detectors include, but are not limited to, photodiodes, Complementary Metal Oxide Semiconductor (CMOS) detectors, photomultiplier tubes (PMTs), microchannel plate detectors, quantum dot photoconductors, phototransistors, photoresistors, Active Pixel Sensors (APS), gaseous ionization detectors, or Charge Coupled Device (CCD) detectors. In some aspects, a single and/or universal detector may be used.

In some embodiments, the optical system 830 may be configured to detect fluorescent signals from a solid substrate (e.g., the DNA chip 333 shown in fig. 11A). The DNA chip can be configured to contain a central reaction panel labeled as an "unknown" signal region on the chip (fig. 11A), and at least two control regions at various locations on the chip (fig. 11A). In this case, the optical system 830 is configured to simultaneously measure the detection signal and the internal control signal (fig. 11B).

In one example, the optical system 830 includes two collection lenses 1018 and corresponding optical components, such as a control array photodiode for each lens 1018. Fig. 10B shows a side view of the optical system 830 shown in fig. 10A within the detection apparatus 100. In this embodiment, two collection lenses 1018 are included in the optical system, one for collecting control array signals from the DNA chip (e.g., two signals 1101 and 1102 shown in FIG. 11B), and one dedicated to unknown detection signals from the DNA chip (e.g., detection signal 1102 shown in FIG. 11B). Including a signal array diode 1021 (e.g., laser diode 1011 shown in fig. 10A) and two control sensing photodiodes 1022 for each optical path. Additionally, two prisms 1023 may be added to two collection lenses configured to collect signals from two control areas (1018). The prism 1023 may bend the control array light to the photodiode sensor area.

In some embodiments, optical system 830 may be configured in a straight mode, as shown in fig. 12A. Excitation optics 1210 is configured to transmit excitation optical signals to a glass chip 333 (e.g., a DNA-coated chip) in reaction chamber 331, which may include a laser diode 1211, a collimating lens 1212, a bandpass filter 1213, and a cylindrical lens 1214. The cylindrical lens 1214 may make the excitation light form a line to cover the reaction panel and the control panel on the glass chip (for example, fig. 11B). The emission optics 1220 aligned with the glass chip 333 may include: a collecting lens 1221 configured to collect light emitted from the glass chip 333; a band-pass filter 1222 a; a long pass filter 1222 b; and a focusing lens 1223 configured to focus at least a portion of the allergen-related optical signal onto the chip reader 1230. The chip reader 1230 includes: three photodiode lenses 1231, two control array photodiodes 1232, a signal array photodiode 1233, and a collection PCB 1234 (fig. 12A). In some embodiments, the collection lens 1221 can be shaped to include a concave first surface to optimize imaging and minimize stray light.

As a non-limiting example, the excitation optics 1210 and emission optics 1220 may be folded and configured into a stepped bore 1224 in the device 100 (see fig. 12C). Excitation fold mirror 1240 and collection fold mirror 1250 can be configured to minimize the light path from excitation optics 1210 and emission optics 1220, respectively (in fig. 12B). The minimized volume may modulate the laser at a frequency that minimizes interference from ambient light sources. A photodiode shield 1260 can be added to cover and protect the chip reader 1230 (fig. 12B). The reader 1230 is then positioned close to the collection lens 1221 to minimize scattered light. Fig. 12C shows an example of a stepped bore 1224 for holding emission optics 1220 in the device. Fig. 12C shows the aperture 1270 of the collection lens 1221.

The laser source (e.g., 1211) may be modulated and/or polarized and oriented to minimize reflection from the glass chip. Thus, the chip readers may be synchronized to measure the modulated light.

The optical system 830 described above is an illustrative example of some embodiments. Alternate embodiments may have different configurations and/or different components.

In other embodiments, a computer or other digital control system may be used to communicate with the filters, fluorescence detector, absorption detector, and scatter detector. A computer or other digital control system controls the optical filter to subsequently illuminate the sample with each of a plurality of wavelengths while measuring the absorption and fluorescence of the sample based on signals received from the fluorescence and absorption detectors.

6. Display device

As shown in the cut-away side view in fig. 8B, a Printed Circuit Board (PCB)850 is connected to the optical system 830. The PCB 850 may be configured to be compact with respect to the size of the test device 100 and, at the same time, may provide sufficient space to display the test results.

Thus, the test results may be displayed with a backlit icon, LED or LCD screen, OLED, segmented display, or on an attached mobile phone application. The user can see that the sample is being processed, that the sample is fully processed (total protein indicator) and an indication of the test result. The user is also able to view the status of the battery and which type of cartridge (barcode on cartridge or LED assembly) is placed in the device. For example, the test results will show that (1) the actual number ppm or mg; or (2) binary result yes/no; or (3) risk analysis-high/medium/low or high/low, risk present; or (4) a ppm range of less than 1ppm/1 to 10 ppm/greater than 10 ppm; or (5) a mg range of less than 1mg/1-10 mg/greater than 10 mg. The results may also be displayed as numbers, colors, icons, and/or letters.

In accordance with the present invention, the detection device 100 may also include other features, such as a device for providing power and a device for providing process control. In some embodiments, one or more switches are provided to connect the motor, micropump and/or gear train or drive to the power source. These switches may be simple micro switches that can turn the detection means on and off by connecting and disconnecting the battery.

The power supply 860 may be a lithium ion AA format battery or any commercially available battery suitable for supporting small medical devices, such as a Rhino 610 battery, a turkiggy Nanotech highly rechargeable Li Po battery, or a Pentax D-L163 battery.

In the description herein, it is to be understood that all enumerated connections between components may be either direct or indirect operative connections. Other components may also include those disclosed in applicants' PCT patent publication No. wo/2018/156535; the contents of which are incorporated herein by reference in their entirety.

Detection assay

In another aspect of the present invention, there is provided an allergen detection test carried out using the present detection system and device.

In some embodiments, the allergen detection test comprises the steps of: (a) collecting a specific amount of a test sample suspected of containing the allergen of interest; (b) homogenizing the sample using an extraction/homogenization buffer and extracting the allergen protein; (c) contacting the treated sample with a detection agent that specifically binds to the allergen of interest; (d) contacting the mixture in (c) with a detection sensor comprising a solid substrate printed with nucleic acid probes; (e) measuring a fluorescent signal from the reaction; and (f) processing and digitizing the detected signals and visualizing the interaction between the detection agent and the allergen.

In some aspects of the invention, the method further comprises the steps of: unbound compounds are washed from the detection sensor to remove any non-specific binding interactions.

In some aspects of the invention, the method further comprises the steps of: the treated sample is filtered before contacting the treated sample with a detection sensor (e.g., a DNA chip).

In some embodiments, test samples of appropriate size are collected for detection assays to provide reliable and sensitive results from the assay. In some examples, a sampling mechanism is used that can efficiently and non-destructively collect test samples to quickly and efficiently extract allergen proteins for detection.

A sized portion of the test sample may be collected using a food sampler 200 such as that shown in fig. 2B. Food sampler 200 may collect a sample of appropriate size from which sufficient protein may be extracted for detection testing. The mass of the size fraction may range from 0.1g to 1g, preferably 0.5 g. In addition, food sampler 200 may pre-process the collected test sample by cutting, grinding, mixing, scraping, and/or filtering. The pre-treated test sample will be introduced into the homogenization chamber 321 for processing and allergen protein extraction.

The collected test samples were processed in extraction/homogenization buffer. In some aspects, the extraction buffer is stored in the homogenization chamber 321 and can be mixed with the test sample by the homogenization rotor 340. In other versions, the extraction buffer may be released into the homogenization chamber 321 from another separate storage chamber. The test sample and extraction buffer will be mixed together by the homogenizing rotor 340 and the sample is homogenized.

The extraction buffer may be a universal target extraction buffer that can take enough target protein from any test sample and is optimized to maximize protein extraction. In some embodiments, the formulation of the universal protein extraction buffer can extract proteins at room temperature and in a minimal time (less than 1 minute). The same buffer may be used during food sampling, homogenization and filtration. The extraction buffer may be a PBS-based buffer comprising 10%, 20% or 40% ethanol, or a Tris-based buffer comprising Tris base pH 8.0, 5mM MEDTA and 20% ethanol, or a modified PBS or Tris buffer. In some examples, the buffer may be a HEPES-based buffer. Some examples of modified PBS buffers may include: p + buffer and K buffer. Some examples of Tris-based buffers may include buffer a +, buffer A, B, C, D, E, and buffer T. In some embodiments, the extraction buffer may be optimized for increased protein extraction. A detailed description of each modified buffer is disclosed in PCT patent publication No. WO/2015/066027; the contents of which are incorporated herein by reference in their entirety.

According to the invention, MgCl is added after homogenization of the sample₂. In some embodiments, after homogenizing the sample, MgCl is added₂Solutions (e.g., 30. mu.L of 1M MgCl₂Solution) is added to the homogenization chamber (e.g., 321 in fig. 3).

In other embodiments, solid MgCl may be used during the reaction₂Preparation to replace MgCl₂And (3) solution. The solid formulation may be provided as: MgCl in the homogenization chamber (e.g., 321 in FIG. 3)₂Lyophilized pellets, which are dissolved by homogenization products after filtration; or a filtration component deposited or layered in a filter (e.g., filter membrane 420 in fig. 4A and filter assembly 325 in fig. 4A, or filter assembly 525 in fig. 5G) that is dissolved by the homogenized product during filtration; or MgCl deposited on the inner surface of the homogenization chamber 321 or on a separate support₂A film. Regardless of the formulation, MgCl₂Will dissolve in less than 1 minute (preferably less than 30 seconds) to contact the treated sample homogenization product. MgCl₂Can dissolve in about 10 seconds, or about 15 seconds, or about 20 seconds, or about 25 seconds, or about 30 seconds. The solid formulation will release MgCl within this short period of time₂To reach a final concentration of 30 mM. In some embodiments, the solid MgCl₂The preparation can be made into powder without breaking.

The volume of extraction buffer may be 0.5mL to 3.0 mL. In some embodiments, the volume of extraction buffer can be 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL, or 3.0 mL. Over time and in different food matrices, the volumes have been determined to be effective and repeatable.

According to the present invention, test samples are homogenized and processed using a homogenizing assembly that has been optimized by high speed homogenization to maximize processing of the test samples.

In some aspects of the invention, a filtration mechanism may be connected to the homogenizer. The homogenized sample solution is then driven through a filter in the process to further extract allergen proteins and remove particles that may interfere with flow and optical measurements during testing, thereby reducing the amount of other molecules extracted from the test sample. The filtering step may further achieve a uniform viscosity of the sample to control the jet during the assay. In the case of using a DNA glass chip as a detection sensor, filtration can remove fats and emulsifiers that may adhere to the chip and interfere with optical measurements during testing. In some embodiments, a filtration membrane (such as a cell filter from CORNING (CORNING, new york, usa) or similar custom embodiments) may be connected to the homogenizer. The filtration process may be a multi-stage arrangement with different pore sizes from the first filter to the second filter or to the third filter. The filtration process can be adjusted and optimized according to the food substrate to be tested. By way of non-limiting example, a filter assembly having a small pore size may be used to capture particles and absorb large amounts of liquid when processing dry food, and therefore, may be used for longer periods of time and higher pressures during filtration. In another example, when treating greasy foods, coarse filtration (bulk filtration) may be performed to absorb fat and emulsifiers. Filtering may further facilitate the removal of fluorescent mist or particles from fluorescent food, which would interfere with the optical measurement.

The filter may be a simple membrane filter or an assembly of combinations of filter materials such as PET, cotton, and sand. In some embodiments, the homogenized sample may be filtered through a filter membrane, or filter assembly (e.g., filter assembly 325 in fig. 4A).

In some aspects of the invention, the sampling process can achieve effective protein extraction in less than 1 minute. In one version, the rate of digestion may be less than 2 minutes, including food pick-up, digestion, and reading. Approximately, the process may last 15 seconds, 30 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, or 2 minutes.

The extracted allergen proteins may be mixed with one or more detection agents specific for one or more allergens of interest. The interaction between the allergen protein extraction and the detection agent will produce a detectable signal indicative of the presence or amount of one or more allergens in the test sample. As used herein, the term "detection agent" or "allergen detection agent" refers to any molecule that interacts and/or binds with one or more allergens to allow detection of the allergen in a sample. The detection agent may be a protein-based agent (such as an antibody), a nucleic acid-based agent, or a small molecule.

In some embodiments, the detection agent is a Signal Polynucleotide (SPN) based nucleic acid molecule. SPNs comprise a core nucleic acid sequence that binds with high specificity and affinity to a target allergen protein. The core nucleic acid sequence may be 5-100 nucleic acids in length, or 10-80 nucleic acids in length, or 10-50 nucleic acids in length. SPN may be derived from aptamers selected by the SELEX method. As used herein, the term "aptamer" refers to a species of nucleic acid engineered to bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms, by iterative rounds of in vitro selection or equivalently SELEX (exponential enrichment of ligand system evolution). The binding specificity and high affinity to the target molecule, the sensitivity and reproducibility at ambient temperature, the relatively low production cost, and the possibility of developing aptamer core sequences capable of recognizing any protein, ensure an efficient but simple detection assay.

SPNs useful as detection agents according to the present invention may be aptamers specific to common allergens such as peanuts, tree nuts, fish, gluten, milk and eggs. For example, the detection agent may be an aptamer or SPN as described in applicants' relevant literature: 62/418,984, 62/435,106, 2016, 12, 16, 2016, 62/512,299, 2017, 5, 30, 8, 2016; and PCT publication No. WO/2018/089391, filed on 8/11/2017; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the detection agent (e.g., SPN) can be labeled with a fluorescent label. The fluorescent label may be a fluorophore with a suitable excitation maximum in the range of 200nm to 700nm, while the emission maximum may be in the range of 300nm to 800 nm. The fluorophore may further have a fluorescence relaxation time in the range of 1 nanosecond to 7 nanoseconds, preferably 3 nanoseconds to 5 nanoseconds. As non-limiting examples, fluorophores that can be probed at one end of the SPN may include derivatives of boron-dipyrromethene (BODIPY, e.g., BODIPY TMR dye; BODIPY FL dye), fluorescein (fluoroscein) and its derivatives, rhodamine (rhodamine) and its derivatives, dansyl (dansyl) and its derivatives (e.g., dansylcadam), Texas red (Texas red), eosin, cyanine dyes, indocyanine, oxonol, thiacarbocyanine (thiacarbocyanine), merocyanine (merocyanine), squaraine and its derivatives Seta, Setau and squar dyes (Square dyes), naphthalene and its derivatives, coumarin and its derivatives, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazoles, anthraquinone, pyrene and its derivatives, oxazines and derivatives, Nile red (Nile red), Nile blue (Nile blue), cresol purple, oxazine 170, xanthine, acridine yellow, auramine, Crystal violet, malachite green, porphine, phthalocyanine, bilirubin, tetramethylrhodamine, hydroxycoumarin, aminocoumarin; methoxycoumarin, cascade Blue (cascade Blue), pacific Blue (pacific Blue), pacific orange (pacific orange), NBD, R-Phycoerythrin (PE), Red613 (Red 613); PerCP, trured; FluorX, Cy2, Cy3, Cy5 and Cy7, TRITC, X-Rhodamine (X-Rhodamine), lissamine Rhodamine B (Lissamine Rhodamine B), Allophycocyanin (APC), and AlexaDyes (e.g. Alexa)488、Alexa500、Alexa514、Alexa

532、Alexa

546、Alexa555、Alexa568、Alexa594、Alexa

610、Alexa

633、Alexa

637、Alexa647、Alexa660、Alexa680 and Alexa

700)。

In one example, SPNs are labeled with Cy5 at the 5' end of the SPN nucleic acid sequence.In another example, the SPN is treated with Alexa at one end of the SPN nucleic acid sequence647 and marking.

In some embodiments, SPNs specific for the allergen of interest can be pre-stored in the extraction/homogenization buffer in the homogenization chamber 321 (fig. 3B and 3E). The extracted allergen proteins (if present in the test sample) will bind to SPN, thereby forming protein: SPN complexes. SPN complexes can be detected by the detection sensor during the testing process.

In some embodiments, the detection agents for the eight major food allergens (i.e., wheat, egg, milk, peanut, tree nut, fish, crustaceans, and soybean) may be provided as disposables. In one embodiment, the structure of the detection agent can be associated with MgCl₂Or a buffer doped with KCl. MgCl₂The structures are tightly closed and KCl slightly opens them for binding.

In some embodiments, the detection sensor is a solid substrate printed with nucleic acids. As used herein, the term "detection sensor" refers to an instrument that can capture a reaction signal (i.e., a reaction signal derived from the binding of an allergen protein to a detection agent), measure the quantity and/or quality of a target, and convert the measurement into a signal that can be digitally measured.

In some embodiments, the detection sensor is a solid substrate, such as a glass chip (as referred to herein as a nucleic acid chip or DNA chip), coated with nucleic acid molecules. For example, the detection sensor may be a glass chip 333 inserted into the reaction chamber 331 of the present invention. The detection sensor may also be a separate glass chip, for example, made of: a glass wafer and soda glass, or a microwell, or acrylic glass, or a microchip, or a plastic chip made of COC (cyclic olefin copolymer) and COP (cyclic olefin polymer), or a film-like substrate (for example, nitrocellulose), the surface of which is coated with nucleic acid molecules.

In some embodiments, the nucleic acid coated chip can include at least one reaction panel and at least two control panels. The reaction panel is printed with nucleic acid probes that hybridize to SPNs. As used herein, the term "nucleic acid probe" refers to a short oligonucleotide comprising a nucleic acid sequence complementary to a nucleic acid sequence of an SPN. The short complementary sequence of the probe can hybridize to free SPN. When SPN is not bound to the target allergen, SPN can be anchored to the probe by hybridization. SPN complexes prevent hybridization between SPN and its nucleic acid probes when SPN binds to a target allergen to form SPN complexes.

In some examples, the probe includes a short nucleic acid sequence complementary to a sequence at the 3' end of the SPN that specifically binds to the target allergen protein. In this case, SPNs specific for the allergen protein of interest are provided in the extraction/homogenization buffer. When the sample is processed in the homogenization chamber 321, the allergen of interest (if present in the test sample) will bind to the SPN and form a protein SPN complex. When the sample solution flows to the detection sensor (e.g., DNA chip 333 in reaction chamber 331 (FIG. 3B)), the bound allergen proteins prevent the SPN from hybridizing to the complementary SPN probes on the chip surface. SPN complexes were washed away and no fluorescent signal was detected. In the absence of the target allergen protein in the test sample, free SPNs will bind to complementary SPN probes on the chip surface. A fluorescent signal will be detected from the reaction panel (as shown in fig. 11A and 11B).

In some embodiments, the detection sensor (e.g., a chip printed with nucleic acids) further comprises at least two control panels. The control panel is printed with nucleic acid molecules that do not bind to SPNs or proteins (referred to herein as "control nucleic acid molecules"). In some examples, the control nucleic acid molecule is labeled with a fluorescent label.

In some embodiments, nucleic acid probes can be printed to the reaction panel at the center of the glass chip ("unknown"), and control nucleic acid molecules can be printed to both control panels at each side of the reaction panel on the glass chip, as shown in fig. 11A.

In some embodiments, the nucleic acid chip (DNA chip) may be prepared by any known DNA printing technique known in the art. In some embodiments, the DNA chip may be prepared by pipetting the nucleic acid solution onto a glass chip using single-point pipetting, or by stamping a stamp with wet PDMS comprising the nucleic acid probe solution followed by stamping the stamp onto a glass slide, or by flowing with a microfluidic culture chamber.

By way of non-limiting example, a glass wafer may be laser diced to produce 10x 10mm glass "chips". Each chip contains three panels: one reaction panel (i.e., the "unknown" region in the chip shown in FIG. 11A) is flanked by two control panels (FIG. 11A). The reaction panel comprises covalently bound short complementary nucleic acid probes that bind to SPNs specific for allergen proteins. The SPN is derived from an aptamer and is modified to include CY5 fluorophore. In the absence of the target allergen protein, SPNs can bind freely to the probes in the reaction panel, resulting in a high fluorescence signal. In the presence of the target allergen protein, the SPN probe hybridization interface is blocked by the binding of the target protein to the SPN, resulting in a decrease in the fluorescence signal on the reaction panel. In the detection assay, the reaction panel of the chip faces a small reaction chamber (e.g., reaction chamber 331) flanked by inlet and outlet channels (e.g., 336 in fig. 3G) of the cartridge (e.g., cup 300). During food homogenization, SPN in the extraction buffer binds to the target allergen, if present in the sample, forming a protein-SPN complex. The treated sample solution comprising the protein SPN complex is moved by a jet driven by a vacuum pump into the reaction chamber 331 via an inlet. The solution is then discharged via an outlet channel into a waste chamber 323. After exposure to the sample, the reaction panel is then washed to reveal a fluorescent signal having an intensity correlated with the target allergen concentration.

According to the present invention, the two control panels are constantly bright areas on the chip sensor that produce constant signals as background signals 1101 and 1102 (FIG. 11B). In addition, the two control panels compensate for laser illumination and/or disposable cartridge misalignment. If the cartridge is properly aligned, the fluorescent background signals 1101 and 1102 will be equal (as shown in FIG. 11B). If the measured control signals are not equal, a correction factor lookup table will be used to correct the unknown signal for cartridge/laser misalignment. The final measurement is a comparison of the signal level of the signal 1103 of the unknown test area with the signal level of the control area. The comparison level may be one of the lot specific parameters used for the test.

Food samples with high background fluorescence measurements from the region of action may produce false negative results. A verification method may be provided to adjust the process.

The final fluorescence measurements of the reaction panel can be analyzed after comparison with the control panel and any batch-specific parameters, and a result report can be provided.

Thus, the light absorption and light scattering signals can also be measured at baseline levels before and/or after injection of the treated food sample. These measurements will provide additional parameters to adjust the detection assay. For example, such a signal can be used to look for residual food in the reaction chamber 331 after a washing step.

In addition to the parameters discussed above, one or more other batch-specific parameters may also be measured. Optimization of the parameters may, for example, minimize differences in control and unknown signal levels of the chip.

In some embodiments, the monitoring process may be automated and may be controlled by a software application. The evaluation of the DNA chip and test sample, the washing process and the final signal measurement can be monitored during the detection assay.

The family of allergens that can be detected using the detection systems and devices described herein include allergens from food, the environment, or from non-human proteins such as house pet dander. Food allergens include, but are not limited to, proteins in the following: legume crops such as peanuts, peas, lentils and beans, and the plant lupins associated with legume crops; tree nuts such as almonds, cashews, walnuts, brazil nuts, hazelnuts/hazelnuts, pecans, pistachios, beech nuts, butternuts, chestnuts, chinquapin, coconuts, ginkgo nuts, lychee nuts, macadamia nuts, java olive seeds (nangai nuts) and pine nuts; eggs, fish, crustaceans (such as crabs, crayfish, lobsters, shrimps, and prawns), mollusks (such as clams, oysters, mussels, and scallops); milk, soy, wheat, gluten, corn, meat (such as beef, pork, mutton, and chicken); gelatin; a sulfite; seeds (such as sesame, sunflower and poppy seeds); for example, seeds from plants (such as lupins, sunflowers or poppy) may be used in food products (such as seeded bread) or may be ground to make flour for making bread or pastry.

Applications of

The detection systems, devices, and methods described herein contemplate the use of nucleic acid-based detector molecules (such as aptamers) to detect allergens in food samples. The portable device allows a user to test a food sample for the presence or absence of one or more allergens. The family of allergens that can be detected using the devices described herein include those from: allergens of leguminous crops (such as peanuts), tree nuts, eggs, milk, soybeans, spices, seeds, fish, crustaceans, wheat gluten, rice, fruits and vegetables. Allergens may be present in flour or grains. The device is capable of confirming the presence or absence of these allergens and quantifying the amount of these allergens.

In a broad concept, the detection systems, apparatus and methods described herein can be used to detect any protein content in a sample in a variety of different applications, such as, for example, civilian disease medical diagnosis and battlefield background, environmental monitoring/control and military applications for detecting biological weapons, in addition to food safety. In even a wide variety of applications, the detection systems, devices and methods of the invention can be used to detect any biological molecule that binds to a nucleic acid-based detection molecule. As non-limiting examples, the detection systems, devices, and methods may be used for in-situ detection of malignant tumor markers, in-situ diagnostics (exposure to chemical agents, traumatic head injury, etc.), third world applications (TB, HTV testing, etc.), emergency care (stroke markers, head injury, etc.), and many other applications.

As another non-limiting example, the detection systems, devices, and methods of the present invention can detect and identify pathogenic microorganisms in a sample. Pathogens that can be detected include bacteria, yeast, fungi, viruses and virus-like organisms. Pathogens cause diseases in animals and plants; contaminated food, water, soil or other sources; or as a biological agent in the military field. The device is capable of detecting and identifying pathogens.

Another important application includes the use of the detection system, apparatus and method of the present invention in medical care, such as diagnosing a disease, staging the progression of a disease and monitoring the response to a treatment. As a non-limiting example, the detection apparatus of the present invention may be used to test the presence, absence or amount of biomarkers associated with a disease (e.g., a malignancy) to predict the disease or disease progression. The detection systems, apparatus and methods of the present invention are configured for analyzing small test samples and can be implemented by a user without requiring extensive laboratory training.

Other extended applications outside the field of food safety include field use by military organizations, testing of antibiotics and biopharmaceuticals, environmental testing of products such as pesticides and fertilizers, testing of dietary supplements and various food ingredients and additives prepared in bulk, such as caffeine and nicotine, and testing of clinical specimens (saliva, skin, blood, etc.) to determine whether an individual is exposed to significant levels of an individual's allergens.

Equivalents and ranges

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited by the above description but rather is as set forth in the appended claims.

Many possible alternative features are introduced during the course of this description. It is understood that such alternative features may be substituted in various combinations to yield different embodiments of the invention, as determined by those skilled in the art.

To the extent that any patent, publication, internet site, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated herein only: the materials incorporated do not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. Thus, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein is incorporated herein only to the extent that: no conflict arises between the incorporated material and the prior art disclosed material.

In the claims, articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Unless indicated to the contrary or otherwise evident from the context, claims or descriptions including an "or" between one or more members are deemed to be satisfied if one or more members of a group are present, used, or associated in a given product or process. The invention includes embodiments in which exactly one member of the set is present in, used in, or associated with a given product or process. The invention includes embodiments in which more than one or all of the components are present in, used in, or associated with a given product or process.

It should also be noted that the term "comprising" is intended to be open-ended and allows, but does not require, the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, up to one tenth of the unit of the lower limit of the stated range, unless the context clearly dictates otherwise.

In addition, it should be understood that any particular embodiment of the present invention falling within the prior art may be explicitly excluded from any one or more claims. Since these embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if the exclusion is not explicitly set forth herein. Any particular embodiment of the compositions of the present invention (e.g., any antibiotic, therapeutic or active ingredient; any method of manufacture; any method of use, etc.) may be excluded from any one or more claims for any reason, whether or not related to the presence of prior art.

It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the scope of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

While the present invention has been described with some specificity and length with respect to the several embodiments described, it is not intended to limit the invention to any such detail or embodiment or any particular embodiment, but rather reference should be made thereto in order to provide the broadest possible interpretation of such claims in connection with the appended claims of the prior art and, therefore, to effectively encompass the intended scope of the invention.

Examples of the invention

Example 1: testing filter materials and filtration efficiency

Various filter materials and combinations thereof are tested for filtration efficiency and effect on signal measurement, e.g., loss of detection agent (SPN). Commercially available filter materials such as film (PES, glass fiber, PET, PVDF, etc.), cotton, sand, mesh and silica were tested.

A filter comprising a combination of different filter materials is assembled. In one example, the filter assembly consists of a cotton and glass filter with a pore size of 1 μm. The cotton depth filter and the paper filter are configured to sequentially filter the sample. The filter assembly was tested for filtering different food substrates. Recovery of protein and SPN during the filtration process was measured. Various cotton bodies are used to construct the depth filter, and the cotton depth filter is combined with a membrane filter. These filter assemblies were tested for filtration efficiency and SPN recovery. In one study, 0.5g of food samples were collected and homogenized in 5mL of EPPS buffer (pH 8.4) (Tween 0.1%) and the homogenized food samples were incubated with 5nM SPN (signal polynucleotide) labeled Cy5 specific for allergen proteins. After incubation, a portion of the mixture was passed through a filter assembly and the recovery of protein and SPN was measured and compared to the measurement before filtration.

The filters were further tested and optimized to ensure filtration efficiency and avoid significant SPN loss. In addition to testing different filter materials and combinations thereof, other parameters such as pore size, filtration area (e.g., surface area/diameter of depth filter, height), filtration volume, filtration time and pressure required to drive the filtration process were tested and optimized for various food substrates.

In one study, depth filters with different filter volumes were assembled using bleached cotton balls. Constructing cotton filters having different width (i.e., diameter) to height ratios; the width to height ratio of each mold is in the range of about 1:30 to about 1: 5. The cotton depth filter was then tested for filtration efficiency at different food qualities and buffer volumes. In another study, these models of cotton filters had a pore size of 1 μm and a filtration area of about 20mm²The PET film filter of (1) is assembled together. Various food samples were homogenized and filtered through each filter assembly using different volumes of buffer. The filtrates were collected and compared for recovery for each condition.

In another study, food samples were either supplemented with or without 50ppm peanuts. The dosed sample is homogenized, for example using a rotor 340 (e.g., as shown in fig. 3B and 3C), and the extract is mixed with SPN that specifically binds to peanut allergens. SPN comprises a Cy5 label at the 5' end of the sequence. The mixture is filtered through a depth filter (for example, a depth filter made of cotton) and a membrane filter (pore size: 1 μm). The fluorescence signal is measured and compared to the measurement of the mixture before filtration.

In a separate study, several parameters of each filter assembly were tested and measured, including pressure and time required for filtration, protein and nucleic acid binding, washing efficiency, and assay compatibility and sensitivity. Compatibility was determined as a baseline strength measurement.

₂Example 2: MgCl formulation

After homogenization of the sample in the extraction buffer, several solid mgcls were tested₂Preparation instead of adding MgCl₂And (3) solution. Each formulation tested was evaluated for the following characteristics: (1) dissolving time; (2) dissolved MgCl₂(ii) a final concentration of (d); (3) the effect of additives in the formulation on the detection assay; (4) can be dissolved without stirring; (5) it does not break into powder and does not block the outlet of the homogenization chamber.

Lyophilized MgCl₂Preparation

Freeze-drying of a total of 34 MgCl in 1.5mL Edward (Eppendorf) tubes₂Formulations, and tested for dissolution time, mechanical stability, and other characteristics when exposed to extraction buffer for 10 seconds without agitation. Of these formulations, 2 dissolve rapidly and do not form a powder. Mixing a plurality of MgCl₂The formulation was exposed to the extraction buffer without stirring for 10 seconds and the magnesium content in the recovered buffer was determined by the BioVision magnesium assay and the assays described herein. The assay results showed that lyophilized MgCl comprised maltodextrin and hydroxyethyl cellulose (HEC)₂The highest intensity signal of SPN in buffer is given by the formulation (table 1) as shown in fig. 13A.

MgCl₂As a filter component

Mixing MgCl₂The formulation (table 1) was deposited on a cotton filter and dried at 60 ℃. The extraction buffer was pulled through the cotton filter at 1psi vacuum. The percentage of magnesium recovered in the filtrate was measured by the BioVision colorimetric magnesium assay. MgCl comprising maltodextrin and hydroxyethyl cellulose (HEC)₂Formulations (Table 1) with MgCl₂MgCl on solution and filter₂The results of (1) were compared (FIG. 13B).

MgCl₂As a film

Will alwaysIn total 10 different MgCl₂The formulation was deposited on a polystyrene support and cured. Dissolution time was measured and all formulations dissolved within 10 seconds. The results show that none of the formulations have strong adhesion to polystyrene supports.

Table 1: MgCl₂Components of the preparation

Based on the test results, several fast dissolving solid MgCl were selected₂Formulations (as shown in table 2). The dissolution time of the filter deposit depends on the flow rate. When the fastest flow rate was tested, the solid formulation dissolved within 10 seconds (as shown in table 2).

TABLE 2 fast dissolving and mechanically robust solid MgCl₂Preparation