阅读说明：本技术 一种降钙素原均相化学发光检测试剂盒、检测方法与装置 (procalcitonin homogeneous phase chemiluminescence detection kit, detection method and device ) 是由 杨阳 赵卫国 刘宇卉 李临 于 2018-07-18 设计创作，主要内容包括：本发明涉及均相化学发光检测技术领域的一种降钙素原均相化学发光检测试剂盒、检测方法与装置。所述试剂盒包括：供体试剂,其包括供体微球以及与之结合的第一标记物,所述供体微球能够在激发状态产生活性氧；和,受体试剂,其包括受体微球以及与之结合的第一结合单元,所述受体微球能够与活性氧反应生成可检测的化学发光信号,所述第一结合单元能够与降钙素原的第一表位特异性结合；其中,所述供体微球的粒径不小于受体微球的粒径。试剂盒中的供体微球的粒径不小于受体微球的粒径,提高了该试剂盒的精密性和灵敏度。(The invention relates to a homogeneous chemiluminescence detection kit, a detection method and a device for procalcitonin, belonging to the technical field of homogeneous chemiluminescence detection, wherein the kit comprises a donor reagent and an acceptor reagent, wherein the donor reagent comprises donor microspheres and markers combined with the donor microspheres, the donor microspheres can generate active oxygen in an excited state, the acceptor reagent comprises acceptor microspheres and combination units combined with the acceptor microspheres, the acceptor microspheres can react with the active oxygen to generate detectable chemiluminescence signals, the combination units can be specifically combined with epitope of procalcitonin, the particle size of the donor microspheres is not smaller than that of the acceptor microspheres, the particle size of the donor microspheres in the kit is not smaller than that of the acceptor microspheres, and the precision and the sensitivity of the kit are improved.)

1, A procalcitonin homogeneous phase chemiluminescent assay kit, comprising:

a donor reagent comprising a donor microsphere capable of generating a reactive oxygen species in an excited state and an th label bound thereto,

a receptor reagent comprising a receptor microsphere and th binding unit bound thereto, wherein the receptor microsphere is capable of reacting with active oxygen to generate a detectable chemiluminescent signal, and the th binding unit is capable of specifically binding to th epitope of procalcitonin;

wherein the particle size of the donor microsphere is not smaller than that of the acceptor microsphere.

2. The kit according to claim 1, wherein the particle size of the donor and acceptor microspheres is selected from the group consisting of 20nm to 400nm, preferably from 50nm to 350nm, more preferably from 100nm to 300nm, and most preferably from 150nm to 250 nm.

3. The kit of claim 2, wherein the donor microspheres have a particle size equal to the particle size of the acceptor microspheres.

4. The kit of claim 3, wherein the donor microspheres and the acceptor microspheres each have a particle size of 200 nm.

5. The kit of claim 2, wherein the donor microspheres have a particle size larger than the particle size of the acceptor microspheres.

6. The kit according to claim 5, wherein the ratio of the particle sizes of the donor microspheres to the acceptor microspheres is 1.06-8.60, preferably 1.2-4.0, more preferably 1.5-2.01.

7. The kit of any of , wherein the donor microspheres are surface-coated with hydrophilic aldehyde dextran.

8. The kit of any of of claims 1-7, wherein the receptor microspheres are surface-coated with hydrophilic carboxyglucose.

9. The kit of any of claims 1-8, wherein the donor microspheres are filled with a photosensitizer selected from the group consisting of of methylene blue, rose bengal, porphyrin and phthalocyanine.

10. The kit of any of claims 1-9, wherein the luminescent microsphere is filled with a luminescent compound, preferably the luminescent compound is an europium complex, and further preferably the europium complex is MTTA-EU³⁺。

11. The kit of any of , wherein the donor and acceptor microspheres are polystyrene microspheres.

12. The kit of any of claims 1-11, wherein the reactive oxygen species is singlet oxygen.

13. The kit of any of claims 1-12, further comprising a reagent comprising a specific binder for a second binding unit capable of specifically binding to a second epitope of procalcitonin that is an epitope of different binding properties or of the same binding properties at different positions to the epitope and a marker bound thereto.

14. The kit of claim 13, wherein:

said th binding unit and said second binding unit are each independently selected from the group consisting of a polyclonal antibody, a monoclonal antibody, an antibody binding fragment, an artificial antibody, a modified antibody with binding specificity for procalcitonin, preferably from the group consisting of a polyclonal antibody and/or a monoclonal antibody.

15. The kit of claim 14, wherein:

said th binding unit and/or said second binding unit independently comprise at least two different monoclonal antibodies, antibody binding fragments, artificial antibodies or modified antibodies capable of having binding specificity for an epitope of procalcitonin with different binding properties or an epitope of the same binding properties at different positions.

16. The kit of any of , wherein the marker is selected from the group consisting of avidin and/or streptavidin.

17. The kit of any of of claims 1-16, wherein the concentration of the acceptor microspheres in the acceptor reagent is selected from the group consisting of 50-300 μ g/mL, preferably 80-250 μ g/mL, and more preferably 100-200 μ g/mL.

18. The kit of any of claims 1-17, wherein the concentration of the donor microspheres in the donor reagent is selected from 1-15 μ g/mL, preferably 2-10 μ g/mL, and more preferably 4-8 μ g/mL.

19, homogeneous chemiluminescent assay for procalcitonin using the kit of any of claims 1-18 for chemiluminescent assay.

20. The method according to claim 19, characterized in that it comprises the steps of:

s1, mixing the sample to be tested with the receptor reagent and the donor reagent to form a mixture to be tested;

s2, exciting the mixture to be detected to generate chemiluminescence by using energy or active compounds, and measuring the signal intensity of the chemiluminescence;

wherein the donor reagent comprises donor microspheres capable of generating reactive oxygen species in an excited state; the receptor reagent comprises a receptor microsphere capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal; the particle size of the donor microsphere is not smaller than that of the acceptor microsphere.

chemiluminescent assay device for detecting procalcitonin in a test sample using the kit of any of claims 1-18 or the method of claim 19 or 20.

22. The apparatus of claim 21, wherein the apparatus is a POCT point-of-care testing apparatus.

Technical Field

The invention belongs to the technical field of homogeneous phase chemiluminescence detection, and particularly relates to an procalcitonin homogeneous phase chemiluminescence detection kit, a detection method and a detection device.

Background

Procalcitonin (PCT), a procalcitonin, was found in 1990, it has been shown that an increase in serum concentration is closely related to the occurrence of infection, and in normal individuals, the serum concentration is very low, and is only 10-50pg/ml, which is undetectable by the method.

At present, ELISA method, colloidal gold method and homogeneous phase chemiluminescence analysis are commonly used for detecting biological macromolecule procalcitonin, wherein the chemiluminescence analysis method is methods for detecting light waves emitted by chemiluminescence substances, the chemiluminescence substances are used as labels in nucleic acid detection and immunoassay, for example, one molecule in a specific binding pair can be combined with the chemiluminescence substances through various ways to form a luminescence compound, the compound can react with a detected object (the other molecule in the specific binding pair) in a sample and is distributed in a solid phase and a liquid phase, the distribution ratio is related to the amount of the detected object, and the corresponding concentration of the detected object in the sample can be obtained by measuring the luminescence amount in the solid phase or the liquid phase.

To increase the efficiency of the emission of the donor and/or acceptor microspheres, methods to increase the efficiency of the light exposure of the dye in the donor microsphere and/or the efficiency and efficiency of the emission of the light-emitting compound in the acceptor microsphere are commonly used in the art .

Although the detection sensitivity of the chemiluminescence detection method can be greatly improved by using the method in the field, methods which can improve the luminous efficiency by steps on the basis of the prior art to detect procalcitonin still need to be developed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides procalcitonin homogeneous phase chemiluminescence detection kits, and the method for detecting procalcitonin by adopting the kits has high detection sensitivity.

To this end, the invention according to the provides a homogeneous chemiluminescent assay kit for procalcitonin, comprising:

a donor reagent comprising a donor microsphere capable of generating a reactive oxygen species in an excited state and an th label bound thereto,

a receptor reagent comprising a receptor microsphere and th binding unit bound thereto, wherein the receptor microsphere is capable of reacting with active oxygen to generate a detectable chemiluminescent signal, and the th binding unit is capable of specifically binding to th epitope of procalcitonin;

wherein the particle size of the donor microsphere is not smaller than that of the acceptor microsphere.

In embodiments of the present invention, the donor and acceptor microspheres have a particle size selected from the group consisting of 20nm to 400nm, preferably from 50nm to 350nm, more preferably from 100nm to 300nm, and most preferably from 150nm to 250 nm.

In preferred embodiments of the present invention, the donor microspheres have a particle size equal to the particle size of the acceptor microspheres.

In preferred embodiments of the present invention, the donor and acceptor microspheres each have a particle size of 200 nm.

In preferred embodiments of the present invention, the donor microspheres have a larger particle size than the acceptor microspheres.

In preferred embodiments of the present invention, the ratio of the particle size of the donor microspheres to that of the acceptor microspheres is 1.06 to 8.60, preferably 1.2 to 4.0, and more preferably 1.5 to 2.01.

In , the donor microspheres are surface coated with hydrophilic aldehyde dextran.

In still other embodiments of the invention, the acceptor microspheres are surface coated with hydrophilic carboxyglucose.

In embodiments of the invention, the donor microspheres are filled with a photosensitizer selected from of methylene blue, rose bengal, porphyrin and phthalocyanine.

In other embodiments of the present invention, the luminescent microsphere is filled with a luminescent compound, preferably the luminescent compound is a europium complex, and further preferably the europium complex is MTTA-EU³⁺。

In , the donor and acceptor microspheres are polystyrene microspheres.

In still other embodiments of the invention, the reactive oxygen species is singlet oxygen.

In embodiments of the invention, the kit further comprises a reagent comprising a specific binder of a second binding unit capable of specifically binding to a second epitope of procalcitonin which is an epitope of different binding properties or of the same binding properties at different positions as the epitope, and a label bound thereto.

In preferred embodiments of the present invention, the th binding unit and the second binding unit are each independently selected from the group consisting of a polyclonal antibody, a monoclonal antibody, an antibody binding fragment, an artificial antibody, a modified antibody, preferably selected from the group consisting of a polyclonal antibody and/or a monoclonal antibody, having binding specificity for procalcitonin.

In preferred embodiments of the invention, the th binding unit and/or the second binding unit independently comprise at least two different monoclonal antibodies, antibody binding fragments, artificial antibodies or modified antibodies capable of binding specificity to an epitope of procalcitonin having different binding characteristics or an epitope of the same binding characteristics at different positions.

In embodiments of the invention, the marker is selected from avidin and/or streptavidin.

In preferred embodiments of the present invention, the concentration of the acceptor microsphere in the acceptor reagent is selected from 50-300 μ g/mL, preferably 80-250 μ g/mL, and more preferably 100-200 μ g/mL.

In another preferred embodiments of the present invention, the concentration of the donor microsphere in the donor reagent is selected from 1-15 μ g/mL, preferably 2-10 μ g/mL, and more preferably 4-8 μ g/mL.

In a second aspect, the invention provides homogeneous-phase chemiluminescent assay methods for procalcitonin using a kit according to aspect for chemiluminescent assay.

In embodiments of the present invention, the method includes the steps of:

s1, mixing the sample to be tested with the receptor reagent and the donor reagent to form a mixture to be tested;

s2, exciting the mixture to be detected to generate chemiluminescence by using energy or active compounds, and measuring the signal intensity of the chemiluminescence;

wherein the donor reagent comprises donor microspheres capable of generating reactive oxygen species in an excited state; the receptor reagent comprises a receptor microsphere capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal; the particle size of the donor microsphere is not smaller than that of the acceptor microsphere.

In a third aspect, the present invention provides chemiluminescent detection devices for detecting procalcitonin in a test sample using the kit according to aspect of the invention or the method according to the second aspect of the invention.

In preferred embodiments of the present invention, the device is a POCT point of care device.

The invention has the beneficial effects that: by controlling the matrixes and the grain sizes of the acceptor microspheres and the donor microspheres, the kit improves the luminous efficiency of detection when being used for detecting procalcitonin by chemiluminescence, and has good detection sensitivity. In addition, the acceptor microspheres and the donor microspheres in the kit are polystyrene microspheres, hydrophilic carboxyl glucose is coated on the surfaces of the acceptor microspheres, and hydrophilic aldehyde dextran is coated on the surfaces of the donor microspheres, so that nonspecific adsorption is greatly reduced, the influence of other environmental factors outside the system such as pH value, electrolyte and the like is reduced, and the detection accuracy can be improved.

Detailed Description

In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that each intervening value, to the extent that there is a stated range of upper and lower limits and any other stated or intervening value in that stated range is encompassed within the invention, that the upper and lower limits of such smaller ranges may independently be included in the smaller ranges, and that there is also included in the invention, subject to any specifically excluded limit in the stated range, in the event that a stated range includes or two limits, any range or both excluding those included limits is also encompassed within the invention.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Term (I)

The term "homogeneous" as used herein is defined in english as "homogeneous" and means that the bound antigen-antibody complex and the remaining free antigen or antibody are detected without separation.

The term "test sample" as used herein refers to mixtures that may contain analytes including, but not limited to, proteins, hormones, antibodies or antigens, typical test samples that may be used in the disclosed methods include body fluids such as whole blood, serum, plasma, saliva, urine, etc. the test sample may be diluted with a diluent as necessary prior to use.

The term "binding" as used herein refers to direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.

The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure. Under the technical idea disclosed by the invention, the detection method of the specific binding reaction comprises but is not limited to the following steps: double antibody sandwich, competition, neutralization competition, indirect or capture.

In the present invention, the "donor microsphere" can be a polymeric microparticle coated on a substrate with a functional group to form a photosensitizer-filled polymer microparticle capable of generating reactive oxygen species (e.g., singlet oxygen) upon photoexcitation, in which case the donor microsphere can also be referred to as a photosensitive microsphere or photosensitive microparticle, the surface of the donor microsphere has hydrophilic aldehyde dextran and the interior of the donor microsphere is filled with a photosensitizer, which can be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrin, and phthalocyanine, and derivatives of these compounds having 1-50 atomic substituents that are used to render these compounds more lipophilic or hydrophilic and/or as a linking group to a specific binding partner.

The "acceptor microsphere" may be a polymer particle formed by coating a functional group on a substrate to be filled with a luminescent compound, and in this case, may be referred to as a luminescent microsphere or a luminescent particle. The surface of the luminescent microsphere acceptor microsphere is provided with an affinity embodiments of the invention, the chemiluminescent agent undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that can decompose with or subsequently emit light, preferred embodiments of the invention, the luminescent compound is a europium complex, and further preferably the europium complex is MTTA-EU³⁺。

The term "monoclonal antibody" as used herein refers to an immunoglobulin secreted from a monoclonal B lymphocyte, which can be prepared by methods known to those skilled in the art.

The term "polyclonal antibody" as used herein refers to a collection of immunoglobulins produced from or more clones of B lymphocytes, which can be prepared by methods known to those skilled in the art.

The term "biotin" as used herein is broadly applicable to animal and plant tissues, which have two cyclic structures on the molecule, namely, an imidazolone ring and a thiophene ring, wherein the imidazolone ring is the main site for binding with streptavidin, the activated biotin can be coupled with almost all known biological macromolecules including proteins, nucleic acids, polysaccharides, lipids, etc., under the mediation of a protein cross-linking agent, and "streptavidin" is a protein secreted by streptomyces, and the "streptavidin" molecule with a molecular weight of 65 kD. consists of 4 identical peptide chains, each of which can bind biotin.

The term "particle size" as used herein refers to the average particle size of the microspheres, as measured by conventional particle sizers.

Detailed description of the preferred embodiments

The present invention will be described in more detail below.

The homogeneous chemiluminescent assay kit for procalcitonin according to aspect of the present invention comprises:

a donor reagent comprising a donor microsphere capable of generating a reactive oxygen species in an excited state and an th label bound thereto,

a receptor reagent comprising a receptor microsphere and th binding unit bound thereto, wherein the receptor microsphere is capable of reacting with active oxygen to generate a detectable chemiluminescent signal, and the th binding unit is capable of specifically binding to th epitope of procalcitonin;

wherein the particle size of the donor microsphere is not smaller than that of the acceptor microsphere.

According to the invention, by controlling the particle size relationship between the donor microsphere and the acceptor microsphere in the reagent kit, when the reagent kit is used for chemiluminescence detection of procalcitonin, the detection luminous efficiency is improved, and the detection sensitivity is good.

In embodiments of the invention, the donor and acceptor microspheres have a particle size selected from 20nm to 400nm, preferably from 50nm to 350nm, more preferably from 100nm to 300nm, and most preferably from 150nm to 250nm, e.g., in embodiments of the invention, the donor and acceptor microspheres may have a particle size of 20nm, 50nm, 70nm, 90nm, 100nm, 120nm, 140nm, 160nm, 180nm, 200nm, 220nm, 240nm, and 250 nm.

In preferred embodiments of the present invention, the donor microspheres have a particle size equal to the particle size of the acceptor microspheres.

In preferred embodiments of the present invention, the donor and acceptor microspheres each have a particle size of 200 nm.

In preferred embodiments of the present invention, the donor microspheres have a larger particle size than the acceptor microspheres.

In preferred embodiments of the present invention, the ratio of the particle size of the donor microspheres to the particle size of the acceptor microspheres is 1.06-8.60, preferably 1.2-4.0, more preferably 1.5-2.01. for example, in preferred embodiments of the present invention, the donor microspheres have a particle size of 150nm, the particle size of the acceptor microspheres is 100nm, and the ratio of the particle size of the donor microspheres to the particle size of the acceptor microspheres is 1.5.

In , the donor microspheres are surface coated with hydrophilic aldehyde dextran.

In still other embodiments of the invention, the acceptor microspheres are surface coated with hydrophilic carboxyglucose.

When the microsphere is used for detection, nonspecific adsorption can be greatly reduced, and the influence of other environmental factors outside a system such as pH value, electrolyte and the like is reduced, so that the detection accuracy is improved.

In the embodiments of the invention, the donor microspheres are filled with a photosensitizer selected from of methylene blue, rose bengal, porphyrin and phthalocyanine.

In other embodiments of the present invention, the acceptor microspheres are filled with a luminescent compound, in other preferred embodiments of the present invention, the luminescent compound is an europium complex, the europium complex filled in the polystyrene microspheres interacts with the polystyrene microspheres to further increase the luminescence efficiency of the polystyrene microspheres at , and in other preferred embodiments of the present invention, the europium complex is MTTA-EU³⁺The complex can directly capture singlet oxygen generated by phthalocyanine dye in the photosensitive microsphere and then emit red light with europium ion characteristic wavelength of 614-615 nm.

MTTA: [4 ' - (10-methyl-9-anthryl) -2,2 ': 6 ' 2 ' -bipyridine-6, 6 ' -dimethylamine ] tetraacetic acid has a structural formula shown in a formula I, and the synthesis is referred to CN 200510130851.9.

Europium complex MTTA-EU³⁺The synthesis of the (europium (III) complex) is as follows:

(1) a500 mL three-necked flask was charged with 732mg of MTTA (1mmoL) and 366mg of EuCl₃·6H₂O (1mmoL) was dissolved in 100mL of methanol and refluxed at 70 ℃ for 2 hours with stirring.

(2) The solvent was distilled off under reduced pressure.

(3) To the resultant was added 50mL of diethyl ether, and the cake was collected by filtration and washed three times with acetone.

(4) Vacuum drying to obtain 830mg MTTA-EU³⁺。

In , the donor and acceptor microspheres are polystyrene microspheres.

In still other embodiments of the invention, the reactive oxygen species is singlet oxygen.

In embodiments of the invention, the kit further includes a reagent comprising a specific binder of a second binding unit capable of specifically binding to a second epitope of procalcitonin that is an epitope of different binding characteristics or of the same binding characteristics at different locations to the epitope and a marker bound thereto, the concentration of the reagent may be selected from 60 μ g/mL, 70 μ g/mL, 80 μ g/mL or 90 μ g/mL.

In preferred embodiments of the present invention, the th binding unit and the second binding unit are each independently selected from the group consisting of a polyclonal antibody, a monoclonal antibody, an antibody binding fragment, an artificial antibody, a modified antibody, preferably selected from the group consisting of a polyclonal antibody and/or a monoclonal antibody, having binding specificity for procalcitonin.

In preferred embodiments of the invention, the th binding unit and/or the second binding unit independently comprise at least two different monoclonal antibodies, antibody binding fragments, artificial antibodies or modified antibodies capable of binding specificity to an epitope of procalcitonin having different binding characteristics or an epitope of the same binding characteristics at different positions.

In embodiments of the invention, the marker is selected from avidin and/or streptavidin.

In preferred embodiments of the present invention, the concentration of the acceptor microspheres in the acceptor reagent is selected from 50-300 μ g/mL, preferably 80-250 μ g/mL, and more preferably 100-200 μ g/mL. in embodiments of the present invention, the concentration of the acceptor microspheres in the acceptor reagent may include, but is not limited to, one selected from 120 μ g/mL, 140 μ g/mL, 160 μ g/mL, or 80 μ g/mL.

In other preferred embodiments of the present invention, the concentration of the donor microspheres in the donor reagent is selected from 1-15 μ g/mL, preferably 2-10 μ g/mL, and more preferably 4-8 μ g/mL. in embodiments of the present invention, the concentration of the donor microspheres in the donor reagent can include, but is not limited to, a concentration selected from 5 μ g/mL, 6 μ g/mL, or 7 μ g/mL.

According to , the kit further comprises a diluent for diluting the sample to be tested, wherein the diluent comprises a buffer solution, a protein, a stabilizer, a preservative and the like, and the diluent has the functions of dilution and buffering, so that the accuracy of the final test result and the stability of the sample to be tested are improved.

In a second aspect, the invention provides homogeneous-phase chemiluminescent assay methods for procalcitonin using a kit according to aspect for chemiluminescent assay.

In embodiments of the present invention, the method includes the steps of:

s1, mixing the sample to be tested with the receptor reagent and the donor reagent to form a mixture to be tested;

s2, exciting the mixture to be detected to generate chemiluminescence by using energy or active compounds, and measuring the signal intensity of the chemiluminescence;

wherein the donor reagent comprises donor microspheres capable of generating reactive oxygen species in an excited state; the receptor reagent comprises a receptor microsphere capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal; the particle size of the donor microsphere is not smaller than that of the acceptor microsphere.

In , in step S1, the test sample is first mixed with the acceptor reagent to form a th mixture, and then the th mixture is mixed with the donor reagent to form a test mixture.

In another embodiments of the present invention, in step S2, the mixture to be tested is irradiated by 600-700 nm red excitation light to excite the mixture to be tested to generate chemiluminescence.

In the preferred embodiments of the present invention, in step S1, the sample to be tested is diluted with a diluent, and then mixed with the receptor reagent and the donor reagent to form a mixture to be tested.

In a third aspect, the present invention provides chemiluminescent detection devices for detecting procalcitonin in a test sample using the kit according to aspect of the invention or the method according to the second aspect of the invention.

In preferred embodiments of the present invention, the device is a POCT point of care device.

In embodiments of the invention, the apparatus comprises:

a. the reagent cup strip is provided with a plurality of hole sites for containing reagents, and the hole sites at least comprise:

a sample hole site to be detected for containing a sample to be detected containing target molecules to be detected;

a reagent well site for holding a donor reagent comprising donor microspheres capable of generating reactive oxygen species in an excited state;

a second reagent well site for holding an acceptor reagent comprising acceptor microspheres capable of reacting with reactive oxygen species to produce a chemiluminescent signal, the donor microspheres having a particle size not smaller than the acceptor microspheres;

b. the sample adding mechanism is used for mutually moving the reagents contained in the hole sites among the hole sites; the mass transferred by the sample adding mechanism is 1-500 mu L each time;

c. and the detection mechanism is electrically connected with the sample adding mechanism and is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere and the active oxygen.

In other embodiments of the present invention, the sample well to be tested, the donor reagent well and the acceptor reagent well are coated to seal the opening of the well, so as to prevent the contamination of the substances therein.

In order to conveniently identify and read the information of the sample to be tested, the preferable technical scheme is that the side surface of the reagent cup strip along the width direction is provided with a bar code area, and the bar code area contains the information of the reagent cup strip, wherein the bar code can be -dimensional or two-dimensional.

Correspondingly, the POCT device also comprises a bar code scanning module, and the bar code scanning module is used for identifying and reading information in the bar code.

The bar code scanning module supports IC card scanning and bar code medium (paper or reagent card) printing scanning, and the information reading adopts contact scanning or non-contact scanning in a mode of infrared or radio frequency and the like; the information includes, but is not limited to, assay project name, standard curve, reagent composition, lot number, expiration date, manufacturer information.

In order to improve the accuracy of the final detection result and the stability of the sample to be detected, in embodiments of the present invention, the reagent cup strip is further provided with a diluent hole site, and the diluent hole site is used for containing a diluent.

In , the reagent cup strip is further provided with an additional reagent hole for containing an additional reagent, and the additional reagent hole is covered with a film to close the opening.

In preferred embodiments of the present invention, the sample application mechanism comprises:

a pipetting assembly for aspirating or discharging a liquid;

the liquid transfer assembly is arranged on the vertical moving assembly, and the vertical moving assembly is used for driving the liquid transfer assembly to vertically move;

the vertical moving assembly is arranged on the horizontal moving assembly, and the horizontal moving assembly is used for driving the liquid transfer assembly to move horizontally.

In preferred embodiments of the present invention, the detection mechanism comprises:

a base for carrying the reagent cup strips;

the driving assembly is used for driving the base to rotate around the center of the base and driving the reagent cup strips to rotate;

the detection component is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere in the reagent cup strip and active oxygen.

In the specific embodiments of the invention, the detection assembly comprises an exciter capable of emitting 600-700 nm red excitation light.

In , the detection wavelength of the chemiluminescent signal generated by the reaction of the acceptor microsphere and active oxygen is 450-650 nm.

In preferred embodiments of the present invention, the liquid transfer assembly includes a piston mechanism, a connector and a pipette sequentially arranged from top to bottom, the piston mechanism is connected to the connector, the pipette is arranged at the edge of the end face of the base, when liquid transfer is required, the connector descends and is connected to the pipette, and the piston mechanism can move up and down to drive the pipette to suck or discharge liquid.

In , the device further comprises an incubation module for providing a suitable ambient temperature for the chemiluminescent reaction, wherein the temperature of the reagent cup strip and the contents of the reagent cup strip is 20-50 ℃ by means of a metal bath, water bath or oil bath.

In still other embodiments of the present invention, the sample well site to be tested, the donor reagent well site, and the acceptor reagent well site have cross-sections with different shapes.

The using process of the device comprises the steps of respectively containing a sample to be detected, a donor reagent hole and an acceptor reagent hole in the sample hole to be detected, placing the reagent card in the POCT analyzer, taking the sample to be detected with the corresponding volume by using a sample adding mechanism, adding the sample to be detected into an reagent hole, reacting for time, continuously taking mixed liquid with the fixed volume, adding the mixed liquid into a second reagent hole, irradiating laser to the second reagent hole by using an exciter in the detection assembly, reacting for time, detecting a chemiluminescence signal generated by the reaction of acceptor microspheres and active oxygen by using the detection mechanism, and calculating the concentration of procalcitonin in the sample to be detected.

Example III

In order that the invention may be more readily understood, the invention is now described in further detail at with reference to the following examples, which are intended to be illustrative only and are not intended to limit the scope of the invention.