阅读说明：本技术 一种基于智能移动终端的酶联免疫层析传感器及使用方法 (Enzyme-linked immunosorbent assay sensor based on intelligent mobile terminal and use method ) 是由 吴泽 陆锦辉 刘博超 于 2020-06-30 设计创作，主要内容包括：本发明公开了一种基于智能移动终端的酶联免疫层析传感器及使用方法。一种基于智能移动终端的酶联免疫层析传感器,其包括：结果阅读装置,其包括提供光源的暗室、导光基座和带有光传感器的智能移动终端,其中所述暗室底部设有便于光束通过的通孔,所述导光基座上设置有导光孔,所述光传感器、导光孔及通孔相对应设置且位于同一光路径上；层析装置,其包括依次连接的进样层、结合层、具有层析膜的层析桶及吸水层,所述层析桶与结合层、吸水层为可拆卸连接,便于将层析反应后的层析桶置于所述导光基座上。本发明基于智能手机的即时检测装置,将免疫检测过程与智能移动终端相结合,借助智能移动终端的光传感器实现检测结果的判读。(The invention discloses an enzyme-linked immunosorbent assay sensor based on an intelligent mobile terminal and a using method thereof. An enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal, comprising: the result reading device comprises a darkroom for providing a light source, a light guide base and an intelligent mobile terminal with a light sensor, wherein the bottom of the darkroom is provided with a through hole for light beams to pass through, the light guide base is provided with a light guide hole, and the light sensor, the light guide hole and the through hole are correspondingly arranged and are positioned on the same light path; the chromatography device comprises a sample injection layer, a binding layer, a chromatography barrel with a chromatography film and a water absorption layer which are sequentially connected, wherein the chromatography barrel is detachably connected with the binding layer and the water absorption layer, and the chromatography barrel after chromatography reaction is conveniently arranged on the light guide base. The instant detection device based on the smart phone combines the immunodetection process with the smart mobile terminal, and realizes the interpretation of the detection result by means of the optical sensor of the smart mobile terminal.)

1. An enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal is characterized by comprising:

the result reading device comprises a darkroom for providing a light source, a light guide base and an intelligent mobile terminal with a light sensor, wherein the bottom of the darkroom is provided with a through hole for light beams to pass through, the light guide base is provided with a light guide hole, and the light sensor, the light guide hole and the through hole are correspondingly arranged and are positioned on the same light path;

the chromatographic device comprises a sample introduction layer, a binding layer, a chromatographic barrel with a chromatographic membrane and a water absorption layer which are sequentially connected, wherein the chromatographic barrel is detachably connected with the binding layer and the water absorption layer, so that the chromatographic barrel after chromatographic reaction is conveniently placed on the light guide base;

the intelligent mobile terminal is used for detecting the light intensity of the color development liquid passing through the chromatography barrel through the optical sensor.

2. The ELISA sensor based on the intelligent mobile terminal of claim 1, wherein the result reading device further comprises a base stand, a clamping groove for limiting the light guide base is arranged on the base stand, an accommodating cavity for accommodating the side part of the intelligent mobile terminal is arranged below the base stand, and a through hole or a through groove is formed in the base stand to facilitate light to pass through; the darkroom is arranged above the clamping groove.

3. The ELISA sensor based on the smart mobile terminal of claim 2, wherein the light guide base moves laterally relative to the card slot or the light guide base moves vertically relative to the card slot.

4. The ELISA sensor based on the intelligent mobile terminal of claim 1, wherein a light source component is arranged in the darkroom and used for providing a light source; the light source assembly comprises a light-emitting light source and a light filter arranged in the light-emitting direction.

5. The ELISA sensor based on the intelligent mobile terminal of claim 1, wherein an optical fiber is arranged in the light guide hole.

6. The ELISA sensor based on the intelligent mobile terminal of claim 2, wherein the light guide base is provided with a plurality of barrel fixing grooves, and each barrel fixing groove is provided with the light guide hole.

7. The ELISA sensor based on the smart mobile terminal of claim 6, wherein the light guide base moves laterally along the card slot.

8. The ELISA sensor based on the intelligent mobile terminal of claim 1, wherein the surface of the binding layer is sprayed with platinum nanoparticles decorated by antibody.

9. The ELISA sensor based on the intelligent mobile terminal of claim 8, wherein the sample introduction layer, the binding layer, the chromatography barrel with the chromatography membrane and the water absorption layer are sequentially arranged along a vertical direction, and a sample injection port is arranged at the side of the sample introduction layer.

10. The use method of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal according to claim 1, characterized in that the use method is as follows:

dropwise adding the sample to the sample introduction layer, and waiting for the completion of chromatography; the chromatography barrel is detached and placed on the light guide base, color development liquid is added into the chromatography barrel, and the chromatography barrel is kept standing in a dark place; then, the light guide base is placed below a darkroom, and the optical sensor of the intelligent mobile terminal is placed below the light guide hole; and starting the light source, and detecting the light intensity of the color development liquid passing through the chromatography barrel by the intelligent mobile terminal through the light sensor to finally obtain the concentration of the detection object.

Technical Field

The invention relates to the technical field of rapid detection, in particular to an enzyme-linked immunosorbent assay sensor based on an intelligent mobile terminal and a using method thereof.

Background

In the detection field, including clinical detection, food safety detection, biological detection and the like, Enzyme linked immunosorbent assay (ELISA) is one of the recognized gold standards, and the principle is that known antigens or antibodies are adsorbed on the surface of a solid phase carrier (polystyrene microplate), the antigens or antibodies labeled by Enzyme are combined on the carrier through immunoreaction, finally, the Enzyme substrate is catalyzed to develop color, and the OD value of specific wavelength light penetrating through the substrate solution is measured by an instrument to perform result interpretation. Has high sensitivity, relatively low cost and good stability, but requires special instruments and professionals for operation, and the detection time is longer, generally 2-4 hours. An Immunochromatographic Test Strip (ICS) detection technology is a rapid detection method which is widely used in recent years, and the principle is that Gold nanoparticles (Gold nanoparticles, AuNPs) with surfaces modified with antibodies 1 are sprayed on solid fibers (generally glass fibers), paired antibodies or antigens are fixed on a Test line (T line) of a Nitrocellulose (NC) membrane, secondary antibodies are fixed on a Control line (C line) of the NC membrane, a sample is firstly combined with the Gold-labeled antibodies 1 after being added, and then chromatography is respectively combined with the paired antibodies and the secondary antibodies through the NC membrane, so that the AuNPs are left on the NC membrane in a specific area to present a red line. Compared with ELISA, the detection time only needs 5-15 minutes, no special operator is needed, the price is lower, the sensitivity is much lower, the result is judged by naked eyes, and the error is larger. In many cases, especially in the face of public safety issues with local outbreaks, not only is the sensitivity and specificity of detection required, but also the rapidity, accuracy, cost and portability of detection are required, and immediate feedback of the detection results is required, so that conventional ELISA and ICS alone are often not satisfactory.

Disclosure of Invention

In order to solve the problems, the invention provides an enzyme-linked immunosorbent assay sensor based on an intelligent mobile terminal and a use method thereof, the enzyme-linked immunosorbent assay sensor (NLICA) is an instant detection device based on the intelligent mobile phone, combines an immunodetection process with the intelligent mobile terminal, can realize interpretation of a detection result by virtue of an ambient light sensor of the intelligent mobile terminal, does not need to depend on other detection equipment, does not need professional technical personnel for operation, and can realize the integration of simplification, portability and detection analysis of detection.

In order to achieve the purpose, the invention adopts the following technical scheme:

an enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal, comprising:

the result reading device comprises a darkroom for providing a light source, a light guide base and an intelligent mobile terminal with a light sensor, wherein the bottom of the darkroom is provided with a through hole for light beams to pass through, the light guide base is provided with a light guide hole, and the light sensor, the light guide hole and the through hole are correspondingly arranged and are positioned on the same light path;

the chromatographic device comprises a sample introduction layer, a binding layer, a chromatographic barrel with a chromatographic membrane and a water absorption layer which are sequentially connected, wherein the chromatographic barrel is detachably connected with the binding layer and the water absorption layer, so that the chromatographic barrel after chromatographic reaction is conveniently placed on the light guide base;

the intelligent mobile terminal is used for detecting the light intensity of the color development liquid passing through the chromatography barrel through the optical sensor.

As a preferred embodiment of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal provided by the present invention, a light source assembly is disposed in the darkroom and is used for providing a light source; the light source assembly comprises a light-emitting light source and a light filter arranged in the light-emitting direction.

In the present invention, the light source is preferably a laser light source, and more preferably, an LED lamp bead, such as an LED lamp bead with an excitation light wavelength of 450nm, but not limited thereto.

In the present invention, the filter is a narrow-band filter with a bandwidth of 20nm and filters light of 450nm, but is not limited thereto.

In the present invention, the result reading device further comprises a control circuit, specifically, it comprises a battery, a charging interface, a switch, a resistor and a wire; wherein the battery is preferably but not limited to a lithium battery, more preferably a lithium battery with a specification of 32mm 26mm 3mm and a capacity of 300 mAh; the specification of the charging interface is Micro-usb; the resistor specification is preferably in the range of 0-10K omega, which functions to adjust the light intensity of the light source.

Further, a radiator is arranged above the light-emitting source, preferably but not limited to an aluminum-based multi-fin radiator, so that the working heat of the light-emitting source can be led out in time.

As a preferred embodiment of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal provided by the present invention, the result reading device further comprises a base table, on which a slot for limiting the light guide base is provided and an accommodating cavity for accommodating a side portion of the intelligent mobile terminal is provided below the slot, the base table is provided with a through hole or a through groove for allowing light to pass through; the darkroom is arranged above the clamping groove.

As a preferred embodiment of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal provided by the present invention, the light guide base moves laterally relative to the card slot, for example, the light guide base is slidably disposed in the card slot; or, the light guide base moves vertically relative to the card slot, for example, the light guide base is directly clamped in the card slot.

As a preferred embodiment of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal provided by the present invention, an optical fiber is disposed in the light guide hole.

As a preferred embodiment of the enzyme-linked immunochromatographic sensor based on an intelligent mobile terminal provided by the present invention, the light guide base is provided with a plurality of barrel fixing grooves, and each barrel fixing groove is provided with the light guide hole. The light guide base moves transversely along the clamping groove.

So design can once bear a plurality of chromatography buckets through setting up a plurality of solid bucket grooves, realizes carrying out quick quantitative determination on the scene to a plurality of samples in succession. The chromatography barrel can be connected in the barrel fixing groove in a threaded mode, can also be connected in the barrel fixing groove in an interference fit mode, can also be connected in the barrel fixing groove in a concave-convex structure or a buckling mode, and is not limited to the above.

In the invention, the light guide base is preferably but not limited to a trapezoid strip, the light guide holes are distributed in the upper part of the light guide base in an array mode, a silica gel pad and the optical fiber which passes through the silica gel pad are arranged in each light guide hole, and when the positioning line on the light guide base moves to the indication scale, the optical fiber is superposed with the center point of the through hole below the darkroom.

Further, the light guide holes are preferably 9mm in diameter and 4mm deep, the interval between the two light guide holes is 15mm, and shearing threads are arranged in the holes; the silica gel pad is preferably 9mm in diameter and 2mm in thickness and is used for sealing to prevent the color development liquid from seeping out; the optical fiber is preferably made of PMMA (polymethyl methacrylate), the diameter of the optical fiber is 1.2mm, the cutting length of the optical fiber is 4mm, and the optical fiber extends out of the bottom of the base by 1 mm.

Correspondingly, the clamping groove is provided with a long strip-shaped clamping groove, the groove structure of the clamping groove corresponds to that of the light guide base to be a trapezoid structure, and the light guide base can conveniently slide into the clamping groove in the corresponding and transverse directions.

In the invention, the alignment part is arranged on the side edge of the base table and/or the outer side of the darkroom, and the optical sensor of the intelligent mobile terminal is inserted into the accommodating cavity opposite to the alignment part, so that the optical sensor corresponds to the bottom end of the optical fiber, namely, the darkroom through hole, the optical fiber and the optical sensor are positioned on the same optical path.

As a preferred embodiment of the enzyme-linked immunosorbent assay sensor based on the intelligent mobile terminal, the surface of the binding layer is sprayed with platinum nanoparticles modified by antibodies.

As a preferred embodiment of the enzyme-linked immunosorbent assay sensor based on the intelligent mobile terminal, the sample introduction layer, the binding layer, the chromatography barrel with the chromatography membrane and the water absorption layer are sequentially arranged along the vertical direction, and the sample introduction layer side is provided with a sample injection port.

In the invention, the sample introduction layer and the sample adding port are used as sample introduction layers and are positioned at the bottom of the chromatography device, the sample introduction layer is arranged at the bottom of the chromatography device, the sample adding port is arranged at the side edge of the bottom of the chromatography device, and the sample adding port is communicated with a sample pad of the sample introduction layer; a bonding pad is obliquely arranged in the bonding layer, and the bottom of the bonding pad is communicated with the sample pad; the chromatographic membrane is fixedly arranged on the barrel wall of the chromatographic barrel, and the bottom of the chromatographic membrane is communicated with the top of the combination pad; and the water absorption layer is internally provided with water absorption paper which is communicated with the top of the chromatographic membrane.

It is worth noting that a plane barrel wall can be arranged in the chromatography barrel, and a plane vertical plate can also be inserted into the chromatography barrel, so that the chromatography membrane can be conveniently and fixedly arranged. The structure of the chromatography barrel is not particularly limited, and the chromatography membrane can be fixedly arranged. In the invention, the inside of the chromatography barrel preferably adopts a D-shaped cross section, and one side of the plane of the chromatography barrel is used for fixedly arranging the chromatographic membrane.

Before the chromatographic device is assembled, the sample pad and the combined pad are soaked in the sample pad treatment solution and the combined pad treatment solution respectively in advance and are dried; the antibody for detection is covalently coupled to the surface of platinum nanoparticles (PtNPs) through an intermediate compound; after the treatment of the bonding pad, the surface of the pad is sprayed with antibody-modified PtNPs through a three-position spraying platform. After a sample solution is dripped into the sample adding port, the solution enters the sample pad from the sample adding hole and then enters the combination pad through chromatography, so that the PtNPs on the combination pad enter the solution and then flow upwards together with the solution through chromatography, meanwhile, a target object in the solution and antibodies on the surfaces of the PtNPs generate immunoreaction to form PtNPs-antibody complexes, and then flow upwards through chromatography to enter the chromatographic membrane and generate immunoreaction with paired antibodies or antigens on the membrane, so that the PtNPs are left in a specific area on the NC membrane.

Further, the sample pad treatment fluid formulation is preferably: 10mM PBS, 0.5% -2% Triton, 0.05% NaN₃And a pH of 7.0 to 8.5, but is not limited thereto. The combined pad treatment fluid formula is preferably: 10mM PBS, 1% -10% sucrose, 1% -10% trehalose, 0.5% BSA, 0.5% Tween-20, 0.05% NaN₃And a pH of 7.0 to 8.0, but is not limited thereto.

It should be noted that, in the present invention, the base platform, the light guide base, the chromatography barrel, the sample injection layer, the bonding layer, and the water absorption layer are all preferably obtained by 3D printing, and the materials used in the present invention are photosensitive resin, polylactic acid, and the like, but are not limited thereto.

A use method of the enzyme-linked immunochromatographic sensor based on the intelligent mobile terminal comprises the following steps: dropwise adding the sample to the sample introduction layer, and waiting for the completion of chromatography; the chromatography barrel is detached and placed on the light guide base, color development liquid is added into the chromatography barrel, and the chromatography barrel is kept standing in a dark place; then, the light guide base is placed below a darkroom, and the optical sensor of the intelligent mobile terminal is placed below the light guide hole; and starting the light source, and detecting the light intensity of the color development liquid passing through the chromatography barrel by the intelligent mobile terminal through the light sensor to finally obtain the concentration of the detection object.

The intelligent mobile terminal is an intelligent terminal with an ambient light sensor, and preferably an Android operating system is an intelligent mobile phone or a tablet; more preferably, the operating system is a smartphone or tablet with Android 8.0.0 or more.

The detection principle of the invention is as follows: the final result of the nano enzyme-linked immunosorbent assay sensor is interpreted according to the intensity of monochromatic light after passing through the chromatography barrel, PtNPs left on a chromatography film in the chromatography barrel after immunochromatographic reaction can catalyze TMB substrate liquid (namely color development liquid) to develop color, the color development liquid has strong absorption to 450nm light, a switch of a reading device is turned on, the light emitted by an LED lamp bead (namely a light emitting source) is transmitted to the chromatography barrel through light transmission, the light is transmitted to an environment light sensor at the top of the intelligent mobile terminal through optical fibers in a light guide hole, the amount of the PtNPs is in direct proportion to the depth of the color and is in inverse proportion to the finally passed light intensity.

Compared with the prior art, the invention has the following advantages:

1. the invention is an instant detection device based on a smart phone, which combines an immunodetection process with the smart phone, can realize result interpretation by an ambient light sensor of the smart phone, and can also upload the result to a network in real time, and accurately analyze and feed back the detection result by big data;

2. the complicated washing and reaction steps of ELISA are completed by utilizing the chromatography process, and the complexity of ELISA operation is greatly reduced under the condition of little loss of detection sensitivity.

3. The invention utilizes an enzyme-catalyzed chromogenic system after chromatography, greatly improves the sensitivity compared with the common lateral chromatography, and has convenient operation and shorter time consumption compared with ELISA;

4. the invention uses Au @ PtNPs with peroxidase-like activity as the nanoenzyme, and has the advantages of good thermal stability, convenient and fast synthesis, low preparation cost and the like;

5. all the supporting structures are formed by 3D printing, and the manufacturing cost is extremely low;

6. the LED lamp beads and the optical fibers are matched for use, so that the stability of signal transmission is improved, and the complexity of a system is reduced;

7. the invention has simple operation, low use cost, small and exquisite appearance, convenient carrying and short detection time, and is more suitable for families, basic medical institutions and detection sites compared with the traditional detection analysis method;

8. the nano enzyme-linked immunosorbent assay sensor based on the smart phone has wide application range, can be used for various detection items, such as food safety detection, environmental sanitation detection, clinical target detection and the like, is basically suitable for detection items of an immunochromatography method and an enzyme-linked immunosorbent assay, is suitable for different items, only needs to replace corresponding antibodies, and has strong applicability and plasticity.

Drawings

Fig. 1 is a real object diagram of a nano enzyme-linked immunochromatographic sensor device based on a smart phone according to the present invention.

Fig. 2 is an exploded view of the sensor device according to the present invention.

Fig. 3 is a side cross-sectional view of the sensor device structure of the present invention, wherein the lamp bead, the optical filter, the optical fiber, and the optical sensor of the mobile phone are located on the same center line.

FIG. 4 is a side cross-sectional view of a structure of a chromatographic portion of the sensor of the present invention.

FIG. 5 is a schematic view showing the combination of the chromatography cartridge and the base in the sensor of the present invention.

Fig. 6 is a flow chart of the use of the sensor of the present invention.

FIG. 7 is a standard curve for detecting HCV core antigens in the use of the sensor of the present invention.

In the figure: 1. the top cap, 2, the battery, 3, the varistor, 4, the interface that charges, 5, the switch, 6, the radiator, 7, the excitation light source, 8, the light filter, 9, the chromatography bucket, 10, the leaded light base, 11, the draw-in groove, 12, hold the chamber, 13, the light sensor, 14, the smart mobile phone, 15, the optic fibre, 16, the upper cover, 17, the layer that absorbs water, 18, the bonding pad, 19, the bonding layer, 20, the sample pad, 21, advance the kind layer, 22, the pad that absorbs water, 23, the sample loading mouth, 24, reaction zone, 25, chromatographic film, 26, the location line, 27, the silica gel pad.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.