CN110736736B

CN110736736B - Homogeneous phase chemiluminescence POCT detection method and device using same

Info

Publication number: CN110736736B
Application number: CN201910652109.6A
Authority: CN
Inventors: 杨阳; 赵卫国; 刘宇卉; 李临
Original assignee: Shanghai Suoxin Biological Technology Co ltd
Current assignee: Kemei Diagnostic Technology Suzhou Co ltd
Priority date: 2018-07-18
Filing date: 2019-07-18
Publication date: 2022-10-14
Anticipated expiration: 2039-07-18
Also published as: CN110736736A

Abstract

The invention relates to a homogeneous phase chemiluminescence POCT detection method and a device using the same. The method comprises the following steps: s1, mixing a sample to be detected with an acceptor reagent and a donor reagent to form a mixture to be detected; s2, exciting the mixture to be detected to perform chemiluminescence by using energy or active compounds, and measuring the signal intensity of the chemiluminescence in real time; 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 microspheres is equal to the particle size of the acceptor microspheres. The method has the characteristics of high sensitivity, high precision and wide range of the chemiluminescence analysis technology, and the POCT detection technology is rapid and portable.

Description

Homogeneous phase chemiluminescence POCT detection method and device using same

Technical Field

The invention belongs to the technical field of homogeneous chemiluminescence, and particularly relates to a homogeneous immunoassay POCT detection method and a system using the same.

Background

Homogeneous chemiluminescence analysis refers to a method for performing chemiluminescence detection without the need to separate the complex formed after binding and the remaining free reactants.

The existing homogeneous phase chemiluminescence analysis has the following defects:

A. the instrument system is large in size and large in occupied area, and meanwhile, because the test flux is large, the reagent card adopts 100 tests as a whole unit, so that the requirement on the sample scale of a laboratory is met;

B. instrument systems and reagents are expensive, maintenance cost is high, and the method is not suitable for basic medical institutions;

C. the instrument has large volume and cannot be carried about to enter a diagnosis and treatment site;

D. the chemiluminescence system mainly adopts serum and plasma as samples, and generally can not adopt whole blood, thereby limiting the application range of the chemiluminescence system.

Meanwhile, in recent years, a point-of-care testing (POCT) technology for clinical testing (bedside detection) near a patient, which is abbreviated as POCT technology, is emerging, wherein the POCT technology mainly adopts fluorescent quantitative chromatography or colloidal gold, and mainly adopts a rapid diagnosis technology for immunoassay by a membrane chromatography method through fluorescent microspheres or colloidal gold wrapped by fluorescent materials. However, since these two techniques mainly perform release detection on NC membranes, the CV of the membrane itself is 5% or more, so that the POCT detection CV by a solid-phase membrane method is generally 10% or more, the detection precision is poor, and it is extremely difficult to quantify items requiring high sensitivity, such as cTnI. In addition, a novel technology such as a microfluidic chip type POCT detection technology is adopted, so that the method has the advantages of high reaction speed, small sample demand and the like, and the problem of low detection sensitivity is caused due to insufficient reaction.

Therefore, it is highly desirable to provide a homogeneous chemiluminescence POCT detection method with high sensitivity, high precision, and wide range, and also with the characteristics of rapidness and portability.

Disclosure of Invention

The invention aims to solve the technical problem of providing a homogeneous chemiluminescence POCT detection method, which has the characteristics of high sensitivity, high precision, wide range, rapidness, portability and the like of a chemiluminescence analysis technology.

The invention also provides a system utilizing the homogeneous phase chemiluminescence POCT detection method, the system separately and independently designs the reagent cup strip and the POCT analyzer for integrated use, and the reagent cup strip is used for collecting a sample to be detected, so that the system is convenient to carry.

Therefore, the first aspect of the present invention provides a homogeneous chemiluminescence POCT detection method, which comprises the following steps:

s1, mixing a sample to be detected with an acceptor reagent and a donor reagent to form a mixture to be detected;

s2, exciting the mixture to be detected to perform chemiluminescence by using energy or active compounds, and immediately 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 microspheres is equal to the particle size of the acceptor microspheres.

In some embodiments of the present invention, in step S1, the sample to be tested is first mixed with the acceptor reagent to form a first mixture, and then the first mixture is mixed with the donor reagent to form the mixture to be tested.

In other embodiments of the present invention, in step S2, the mixture to be measured is irradiated with 600 to 700nm red excitation light to excite the mixture to be measured to generate chemiluminescence.

In some embodiments of the invention, the average particle size of the donor microspheres and the average particle size of the acceptor microspheres are both 20nm to 350nm, preferably 50nm to 300nm, more preferably 100nm to 250nm, and most preferably 180nm to 220nm.

In some embodiments of the invention, the donor microsphere comprises a first support, the interior of the first support being filled with a sensitizer, the surface of the first support being chemically bonded to the label.

In some embodiments of the invention, the surface of the first support is not coated or linked with a polysaccharide substance, which is directly chemically bonded to the label.

In some embodiments of the invention, the label is avidin; preferably, the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidin, and further preferably selected from the group consisting of neutravidin or streptavidin.

In some embodiments of the present invention, the avidin is chemically bonded to the surface of the first support by reacting an amino group with an aldehyde group on the surface of the first support to form a schiff base.

In some embodiments of the invention, the surface of the first support carries a bonding functional group for chemically bonding a label to the surface of the first support.

In some embodiments of the invention, the bonding functional group is selected from the group consisting of amine, amide, hydroxyl, aldehyde, carboxyl, maleimide, and thiol; preferably selected from aldehyde groups and/or carboxyl groups.

In some embodiments of the invention, the bonding functional group content of the first support surface is 100 to 500nmol/mg, preferably 200 to 400nmol/mg.

In some embodiments of the present invention, the surface of the first carrier is coated with hydrophilic aldehyde dextran, and the aldehyde group of the aldehyde dextran is chemically bonded with the label.

In some embodiments of the present invention, the photosensitizer is selected from one of methylene blue, rose bengal, a porphyrin and phthalocyanine.

In some embodiments of the present invention, the acceptor microsphere includes a second support, the second support is filled with the luminescent composition, the surface of the second support is coated with at least one polysaccharide layer, and the surface of the polysaccharide layer is connected with a biomolecule.

In some embodiments of the invention, the surface of the second carrier is coated with hydrophilic carboxydextran.

In some embodiments of the present invention, the luminescent composition comprises a europium complex; further preferably, the europium complex is MTTA-EU ³⁺ 。

In some embodiments of the present invention, the material of the first carrier and/or the second carrier is selected from agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate or polyacrylate; preferably selected from polystyrene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate or polyacrylate.

In some embodiments of the invention, the donor microspheres have a coefficient of variation of particle size distribution in the donor agent C.V.value of 5% or more; and/or the presence of a gas in the gas,

the particle size distribution variation coefficient C.V value of the receptor microsphere in the receptor reagent is more than or equal to 5%.

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

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

In a second aspect, the invention provides a homogeneous chemiluminescent POCT detection device, which can detect the antibody or antigen to be detected in the sample to be detected in real time by using the method of the first aspect.

In some 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 tested for containing a sample to be tested containing target molecules to be tested;

a first 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 acceptor microspheres having a particle size equal to the particle size of the donor microspheres;

b. the sample adding mechanism is used for mutually moving the reagents contained in the hole sites among the hole sites;

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

In other embodiments of the present invention, the sample well site to be tested, the donor reagent well site, and the acceptor reagent well site are all covered with a membrane to close the aperture.

In some embodiments of the invention, the reagent cup strips are provided with barcode zones at the sides in the width direction, which barcode zones contain information of the reagent cup strips.

In some embodiments of the invention, the well site further comprises a diluent well site for holding a diluent.

In some preferred embodiments of the present invention, the sample adding 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 some 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 some embodiments of the invention, the detection assembly comprises an exciter capable of emitting red excitation light between 600 and 700 nm.

In some embodiments of the invention, the detection wavelength of the chemiluminescent signal produced by the reaction of the acceptor microsphere with reactive oxygen species is 450-650nm.

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

In some embodiments of the invention, the device further comprises an incubation module for providing a suitable ambient temperature for the chemiluminescent reaction.

In other embodiments of the present invention, the cross-sections of the sample well site to be tested, the donor reagent well site and the acceptor reagent well site are different from each other in shape.

In a third aspect, the present invention provides a use of the homogeneous chemiluminescent POCT detection method according to the first aspect or the homogeneous chemiluminescent POCT detection device according to the second aspect of the invention in chemiluminescent assay.

The invention has the beneficial effects that:

1. the homogeneous chemiluminescence POCT detection method has the characteristics of high sensitivity, high precision, wide range, rapidness, portability and the like of a chemiluminescence analysis technology; in addition, the homogeneous chemiluminescence POCT detection method overcomes the problem of high CV brought by an NC membrane due to pure liquid phase detection, so that the detection precision is high, the CV can be controlled within 5 percent generally, and the POCT detection technology can reach or even exceed the level of chemiluminescence analysis technology in the precision aspect.

2. In the invention, hydrophilic carboxyl glucan is coated on the surface of the acceptor microsphere, and hydrophilic aldehyde glucan is coated on the surface of the donor microsphere, so that 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, thereby greatly improving the detection accuracy.

3. Compared with the traditional solid phase such as a membrane, a microporous plate, plastic beads and the like, the reaction area can be greatly increased, the detection range can be effectively expanded, and the Hook effect is reduced. Meanwhile, the donor microspheres and the acceptor microspheres used in the method have the same particle size, so that the precision and the sensitivity of the method are further improved.

4. The homogeneous chemiluminescence POCT detection method requires a small amount of sample, so that the amount of antibody consumed is also small. Therefore, the prepared detection reagent has lower cost and higher market competitiveness.

5. According to the device utilizing the homogeneous phase chemiluminescence POCT detection method, the reagent cup strip and the POCT analyzer are separately and independently designed, and a reagent card is used for collecting a sample to be detected, so that the device is convenient to carry; the POCT analyzer is simple to operate, realizes full automation, avoids interference caused by cleaning, and has the advantage of high detection precision.

6. The system has no cleaning mechanism, so that the instrument design is more miniaturized, more stable and more portable, and the application field of the instrument is expanded.

Drawings

The present invention will be described in detail below with reference to the accompanying drawings. In the figure:

FIG. 1 is a schematic structural diagram of a homogeneous chemiluminescent POCT detection device according to the present invention.

Fig. 2 is a first schematic view of the structure of the reagent cup strip.

Fig. 3 is a schematic view of the structure of the reagent cup strip II.

Fig. 4 is a schematic view of the pipetting assembly of fig. 1.

Reference numerals: 1-reagent cup strip; 11-the hole site of the sample to be detected; 12-first reagent well site; 13-second reagent well site; 131-a detection cup; 15-barcode region; 2-a sample adding mechanism; 21-a pipetting assembly; 211-a piston mechanism; 212-a connector; 213-pipette; 22-a vertical movement assembly; 23-a horizontal movement assembly; 3-a detection mechanism; 31-a base; 311-grooves.

FIG. 5 is a Gaussian distribution diagram of aldehyde-based polystyrene latex microspheres prepared in example 12.

Fig. 6 is a Nicomp distribution plot of aldehydic polystyrene latex microspheres prepared in example 12.

FIG. 7 is a Gaussian distribution plot of donor microspheres prepared in example 12.

FIG. 8 is a Gaussian distribution plot of dextran-coated microspheres prepared in example 13

FIG. 9 is a Gaussian distribution plot of donor microspheres prepared in example 13.

FIG. 10 is a graph showing the Gaussian distribution of aldehyde-based polystyrene latex microspheres prepared in example 14.

FIG. 11 is a graph showing a Gaussian distribution of aldehyde-based polystyrene latex microspheres with a luminescent composition embedded therein prepared in example 14.

FIG. 12 is a Gaussian distribution plot of dextran-coated aldehyde polystyrene latex microspheres embedded with a luminescent composition prepared in example 14.

FIG. 13 is a Gaussian distribution plot of acceptor microspheres prepared in example 14 with an average particle size around 250nm.

In the drawings, like parts are provided with like reference numerals. The figures are not drawn to scale.

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 no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in 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 of

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 a mixture that may contain a target molecule to be tested, including but not limited to a protein, hormone, antibody or antigen. Typical test samples that can be used in the disclosed methods include body fluids such as whole blood, serum, plasma, saliva, urine, and the like. The sample to be tested can be diluted with a diluent as required before use. For example, to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before the on-line detection and then detected on the detection instrument.

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 in the present invention, the detection method of the specific binding reaction includes but is not limited to: double antibody sandwich, competition, neutralization competition, indirect or capture.

In the present invention, the "donor microsphere" may be a polymer microparticle filled with a photosensitizer and formed by coating a functional group on a carrier, and may generate active oxygen (e.g., singlet oxygen) under light excitation, in which case the donor microsphere may also be referred to as a photosensitive microsphere or a photosensitive microparticle. The inside of the donor microsphere is filled with a photosensitizer. The photosensitizer may be one known in the art, preferably a compound that is relatively photostable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, and phthalocyanines, as well as derivatives of these compounds having 1-50 atom substituents that are used to render these compounds more lipophilic or more hydrophilic and/or as linkers to specific binding pair members. The donor microspheres may also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, etc., which release singlet oxygen upon heating or upon direct absorption of light by these compounds.

The "acceptor microsphere" may be a polymer particle formed by coating a functional group on a carrier 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 hydrophilic carboxyl dextran, and a chemical composition capable of reacting with active oxygen (such as singlet oxygen) is filled in the luminescent microsphere acceptor microsphere. In some embodiments of the invention, the chemical composition undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that can decompose with or subsequently emit light. In some preferred embodiments of the present invention, the luminescent composition comprises a europium complex; further preferably, the europium complex is MTTA-EU ³⁺ 。

The "carrier" according to the present invention is selected from the group consisting of strips, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres, which may be microspheres or microparticles known to those skilled in the art, which may be of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which may be magnetic or non-magnetic, which has any density, but preferably has a density close to that of water, preferably capable of floating in water, and which are composed of transparent, partially transparent or opaque materials.

The term "antibody" as used herein is used in the broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to antigen, including but not limited to Fab, fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any case desired, the antibody may be further conjugated with other moieties, such as a specific binding pair member, e.g., biotin or streptavidin (a member of a biotin-streptavidin specific binding pair member), and the like.

The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and that binds to the immune response product antibodies and sensitized lymphocytes in vitro and in vivo to produce an immune effect.

The term "biotin" is widely found in animal and plant tissues, and two cyclic structures are present on the molecule, namely, an imidazolone ring and a thiophene ring, wherein the imidazolone ring is the main site for binding with streptavidin. Activated biotin can be conjugated to almost any biological macromolecule known, including proteins, nucleic acids, polysaccharides, lipids, and the like, mediated by a protein cross-linking agent; "streptavidin" is a protein secreted by Streptomyces and has a molecular weight of 65kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which is capable of binding one biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby creating a "tentacle effect" that increases assay sensitivity. Any reagent used in the present invention, including antigens, antibodies, oxygen-accepting microspheres or oxygen-donating microspheres, may be conjugated to any member of the biotin-streptavidin specific binding pair as desired.

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

The 'coefficient of variation of particle size distribution c.v value' described in the present invention refers to the coefficient of variation of particle size in Gaussian distribution in the detection result of the nano-particle size analyzer. The coefficient of variation is calculated as: c.v value = (standard deviation SD/Mean) × 100%.

The term "Nicomp distribution" as used herein refers to an algorithmic distribution in the US PSS nanometer particle sizer, NICOMP. Compared with a Gaussian single-peak algorithm, the Nicomp multi-peak algorithm has unique advantages in the analysis of multi-component liquid dispersion systems with nonuniform particle size distribution and the stability analysis of colloidal systems.

Detailed description of the preferred embodiments

The principle of the homogeneous phase chemiluminescence POCT detection method is as follows: target molecules to be detected in a sample to be detected react with the donor microspheres and the acceptor microspheres to form immune complexes, the donor microspheres and the acceptor microspheres are drawn close by the interaction, and under the irradiation of laser (the wavelength is 680 nm), oxygen in the surrounding environment is converted into more active singlet oxygen by the photosensitizer on the donor microspheres. The singlet oxygen diffuses into the acceptor microsphere to react with the luminescent compound on the acceptor microsphere, so that the luminescent group on the acceptor microsphere is further activated to emit light with the wavelength of 520-620nm. The half-life of singlet oxygen is 4 mus, and the diffusion distance in solution is about 200 nm. If the biomolecules do not have interaction, the singlet oxygen cannot diffuse to the receptor microsphere, and no optical signal is generated. Therefore, the concentration of the target molecule to be detected in the sample to be detected can be calculated by measuring the light intensity emitted by the mixture to be detected.

FIG. 1 shows a homogeneous chemiluminescence POCT detection device according to the invention, which comprises a reagent cup strip 1 (shown in FIG. 2), a sample adding mechanism 2 and a detection mechanism 3 (arranged on the left side of the sample adding mechanism 2, which is shielded by the sample adding mechanism 2 in FIG. 1). The reagent cup strip 1 is provided with a plurality of hole sites for containing reagents. The sample adding mechanism 2 moves the reagents contained in the hole sites among the hole sites. The detection mechanism 3 is electrically connected with the sample adding mechanism 2 and is used for detecting a chemiluminescent signal generated by the reaction of the receptor microsphere and the singlet oxygen.

The hole sites include, but are not limited to, a sample hole site to be detected, a first reagent hole site and a second reagent hole site, wherein the sample hole site to be detected is used for containing a sample to be detected containing target molecules to be detected. The first reagent well site is for holding a donor reagent comprising donor microspheres capable of generating singlet oxygen in an excited state. The second reagent hole site is used for containing an acceptor reagent containing acceptor microspheres, the acceptor microspheres can react with singlet oxygen to generate chemiluminescent signals, and the particle size of the acceptor microspheres is equal to that of the donor microspheres. The hole site can be selected and functionally expanded according to actual needs, and for example, the hole site can comprise a third reagent site, a fourth reagent site and the like. Furthermore, the method is simple. The position of each hole site can also be adjusted according to the need, and is not necessarily arranged according to a fixed sequence. For example, the first reagent well site and the second reagent well site are not necessarily located immediately adjacent to each other, and there may be a third reagent well site in between.

The homogeneous chemiluminescence POCT detection method comprises the following steps:

In the invention, the advantages that the acceptor microspheres and the donor microspheres have the same diameter are as follows:

1. the same batch of polystyrene microsphere filling dye can be used when the diameter of the acceptor microsphere is the same as that of the donor microsphere, and the density of carboxyl or aldehyde functional groups on the surface of the microsphere can be ensured to be the same.

2. Chemical reaction collision theory indicates that reactant molecules must collide with each other to be possible to react, and the collision between the acceptor microsphere coated with the antibody and the donor microsphere in immunoassay is random collision generated by brownian motion. Brownian motion is related to particle size, and the collision probability of microspheres with the same particle size is the same.

3. The principle of detecting the luminescent signal is that the photosensitizer in the donor microsphere is irradiated by 680nm laser to release singlet oxygen, the existence time of the singlet oxygen is microsecond level, the propagation distance is only 200nm, and the luminescent efficiency of the microsphere with smaller particle size is high.

In some embodiments of the invention, the average particle size of the donor microspheres and the average particle size of the acceptor microspheres are both 20nm to 350nm, preferably 50nm to 300nm, more preferably 100nm to 250nm, and most preferably 180nm to 220nm. For example, in some embodiments of the invention, the average particle size of the donor and acceptor microspheres may be 20nm, 50nm, 70nm, 90nm, 100nm, 120nm, 140nm, 160nm, 180nm, 200nm, 220nm, 240nm, or 250nm.

In some particularly preferred embodiments of the present invention, the average particle size of the donor microspheres and the average particle size of the acceptor microspheres are both 200nm, and the luminescence signal value of the detection is optimal, and the sensitivity of the detection is optimal.

In other embodiments of the invention, the surface of the first support is not coated or linked with a polysaccharide substance, which is directly chemically bonded to the label.

In some embodiments of the invention, the label is avidin.

In other embodiments of the invention, the avidin is selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin and avidin-like, preferably selected from the group consisting of neutravidin or streptavidin.

In other embodiments of the present invention, the surface of the first support carries a bonding functional group for chemically bonding a label to the surface of the first support.

In other embodiments of the present invention, the bonded functional group content of the first support surface is 100 to 500nmol/mg, preferably 200 to 400nmol/mg.

In some embodiments of the invention, the surface of the first support is coated with hydrophilic aldehyde dextran, the aldehyde group of which is chemically bonded to the label.

In some preferred embodiments of the present invention, the photosensitizer is selected from one of methylene blue, rose bengal, a porphyrin and a phthalocyanine. The carrying amount of the photosensitizer is not particularly limited, and it may be an amount commonly used in the art.

In other embodiments of the present invention, the acceptor microsphere includes a second carrier, the second carrier is filled with the luminescent composition, the surface of the second carrier is coated with at least one polysaccharide layer, and the surface of the polysaccharide layer is connected with a biomolecule.

When the microspheres containing the carrier are 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 other embodiments of the present invention, the luminescent composition comprises a europium complex; further preferably, the europium complex is MTTA-EU ³⁺ . Europium complex filled in polystyrene microsphere interacts with polystyrene microsphere, and luminous efficiency of europium complex is further improved. In a further preferred embodiment of the present invention, the europium complex is MTTA-EU ³⁺ 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, the structural formula of which is shown in formula I, and the synthesis reference is CN200510130851.9.

Europium complex MTTA-EU ³⁺ The synthesis of the (europium (III) complex) is as follows:

(1) A500 mL three-necked flask was charged with 732mg of MTTA (1 mmoL) and 366mg of EuCl ₃ ·6H ₂ O (1 mmoL) 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-EU ³⁺ 。

In some embodiments of the invention, the donor microspheres and the acceptor microspheres are polystyrene microspheres.

In some embodiments of the invention, the biomolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule, and a lipid molecule; preferably a protein molecule. Of course, the biomacromolecule is not limited to protein molecules, nucleic acid molecules, polysaccharide molecules and lipid molecules, and any substance that can be designed to satisfy the above conditions can be used as the biomolecular in the present invention, as long as it is combined with the prior art under the technical idea disclosed in the present invention, and will not be described herein again.

In some preferred embodiments of the invention, the proteinaceous molecule is an antigen and/or an antibody; wherein, the antigen refers to a substance with immunogenicity; the antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters.

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

In addition, it is generally accepted by those skilled in the art that the more uniform the particle size of the microspheres, the better the performance of homogeneous chemiluminescent detection using the microspheres. Current research on microspheres employed in homogeneous chemiluminescence therefore tends to result in microspheres of more uniform particle size. The inventor of the application finds that, after research, when the microsphere with uniform particle size is used for homogeneous chemiluminescence detection, the sensitivity and the detection range of the detection result are difficult to guarantee at the same time. However, by adopting the microspheres with proper uniformity of particle size (for example, the variation coefficient of the particle size distribution of the microspheres is more than 5%), the sensitivity of the light-activated chemiluminescence detection can be ensured, and the detection range can be widened.

Thus, in some embodiments of the invention, the donor microspheres have a coefficient of variation of particle size distribution in the donor agent, C.V., value, of 5% or more.

In other embodiments of the present invention, the donor microspheres have a coefficient of variation of particle size distribution, C.V, value of 8% or more in the donor agent; preferably, the variation coefficient C.V value of the particle size distribution of the donor microsphere in the reagent is more than or equal to 10%.

In some embodiments of the invention, the donor microspheres have a coefficient of variation of particle size distribution, C.V, value in the donor agent of 40% or less; still more preferably, the donor microspheres have a coefficient of variation in particle size distribution, C.V, in the donor agent of 20% or less.

In some embodiments of the invention, the donor microsphere may have a coefficient of variation in particle size distribution, c.v, in the recipient agent of 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 25%, 30%, 35%, or 40%, etc.

It should be noted that the c.v value of the variation coefficient of the particle size distribution of the donor microsphere refers to the c.v value of the variation coefficient of the particle size distribution of the donor microsphere coated with the desired substance.

In some embodiments of the invention, the acceptor microspheres have a coefficient of variation of particle size distribution in the acceptor reagent having a C.V value of 5% or more.

In some embodiments of the invention, the acceptor microsphere has a coefficient of variation of particle size distribution in the acceptor reagent, C.V, value of 8% or more; preferably, the variation coefficient C.V value of the particle size distribution of the acceptor microsphere in the acceptor reagent is more than or equal to 10%.

In other embodiments of the present invention, the acceptor microsphere has a coefficient of variation of particle size distribution, C.V, in the acceptor reagent of 40% or less; even more preferably, the receptor microsphere has a particle size distribution variation coefficient C.V value of less than or equal to 20% in the receptor reagent.

In some embodiments of the invention, the receptor microsphere may have a coefficient of variation in particle size distribution, c.v, in the receptor reagent of 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 25%, 30%, 35%, 40%, or the like.

It should be noted that the c.v value of the variation coefficient of particle size distribution of the acceptor microsphere described in the present invention refers to the c.v value of the variation coefficient of particle size distribution after the acceptor microsphere is coated with a desired substance.

In some embodiments of the invention, the c.v value of the coefficient of variation of particle size distribution is calculated from a Gaussian distribution. In some embodiments of the invention, the reactive oxygen species is singlet oxygen.

In some preferred embodiments of the present invention, in step S1, the sample to be tested is diluted with a diluent, and then mixed with the acceptor reagent and the donor reagent to form a mixture to be tested. The diluent comprises a buffer, a protein, a stabilizer, a preservative and the like. The diluent has the functions of dilution and buffering, and the accuracy of a final detection result and the stability of a sample to be detected are improved.

When some specific detection methods are used, besides the sample to be detected, the donor reagent and the acceptor reagent, additional reagents are needed to be successfully or optimally performed, so the preferable technical scheme is as follows: in step S1, the sample to be tested is mixed with an additional reagent, and then mixed with the donor reagent. It should be noted that the additional reagent described in the present invention does not refer to a certain kind of reagent, and the additional reagent is added to ensure the successful or optimized performance of certain detection methods based on specific reactions.

Under the technical idea disclosed by the invention, the detection method of the specific reaction includes but is not limited to: double antibody sandwich, competition, neutralization competition, indirect or capture. Taking the sandwich method as an example: the immune complex pattern was: donor microsphere-streptavidin-biotin-antibody 1-antigen-antibody 2-acceptor microsphere, in which case the additional reagent is biotinylated antigen or antibody; the donor reagent is a donor microsphere coated by streptavidin, and the acceptor reagent is an acceptor microsphere coated by antigen or antibody.

The second aspect of the present invention relates to a homogeneous chemiluminescent POCT detection device, which uses the method according to the first aspect of the present invention to detect the antibody or antigen to be detected in the sample to be detected in real time.

In some embodiments of the invention, the apparatus comprises:

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

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

In other embodiments of the present invention, the sample well site to be tested, the donor reagent well site and the acceptor reagent well site are coated to close the openings, so as to ensure that the substances therein are not polluted. The covering film can be a disposable sealing film or a reusable sealing film.

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. The barcode may be a one-dimensional or two-dimensional code.

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, and the mode can be infrared or radio frequency; 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 some 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 diluent. In some embodiments of the invention, the reagent cup strip is further provided with additional reagent holes for holding additional reagents, the additional reagent holes being covered to close the opening.

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

a pipetting assembly for aspirating or discharging a liquid;

a base for carrying the reagent cup strips;

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

In some embodiments of the invention, the detection wavelength of the chemiluminescent signal produced by the reaction of the acceptor microsphere and reactive oxygen species is 450-650nm.

In some preferred embodiments of the present invention, the pipetting assembly includes a piston mechanism, a connector and a pipette, which are arranged in sequence from top to bottom, the piston mechanism is connected to the connector, and the pipette is arranged at the edge of the end face of the base; when liquid transfer is needed, the connecting piece descends and is connected with the pipette, and the piston mechanism can move up and down to drive the pipette to suck or discharge liquid.

In some embodiments of the invention, the device further comprises an incubation module for providing a suitable ambient temperature for the chemiluminescent reaction. When in detection, the incubation module adopts a metal bath, a water bath or an oil bath and the like, so that the temperature of the reagent cup strip and the substances in the reagent cup strip is 20-50 ℃.

In other embodiments of the present invention, the cross-sections of the sample well site to be tested, the donor reagent well site and the acceptor reagent well site are different in shape from each other.

The using process of the device comprises the following steps: after the sample to be detected, the donor reagent hole site and the acceptor reagent hole site are respectively filled with the sample to be detected, the donor reagent and the acceptor reagent, the reagent card is placed in the POCT analyzer, a sample to be detected with a corresponding volume is taken by using a sample adding mechanism, a first reagent hole site is added, after a certain time reaction, a certain volume of mixed liquid is continuously taken and added into a second reagent hole site, an exciter in the detection assembly is used for emitting laser to irradiate towards the second reagent hole site, after a certain time reaction, the detection mechanism detects a chemiluminescence signal generated by the reaction of the acceptor microspheres and active oxygen, and the concentration of target molecules to be detected in the sample to be detected is calculated.

Example III

In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.

Example 1: preparation of acceptor microspheres

1. A25 mL round-bottom flask was prepared, 0.1g of europium (III) complex and 10mL of 95% ethanol were added, magnetic stirring was performed, and the temperature in a water bath was raised to 70 ℃ to obtain a europium (III) complex solution.

2. A100 mL three-necked flask was prepared, and then 10mL of 95% ethanol, 10mL of water and 10mL of 10% concentration polystyrene microspheres coated with a carboxyl dextran hydrogel having a particle size of 200nm were added thereto, and the mixture was magnetically stirred and heated in a water bath to 70 ℃.

3. Slowly and dropwise adding the europium (III) complex solution in the step 1 into the three-neck flask in the step 2, reacting for 2 hours at 70 ℃, stopping stirring, and naturally cooling to obtain the emulsion.

4. The emulsion was centrifuged for 1 hour at 30000g, and the supernatant was discarded after centrifugation and then resuspended in 50% ethanol. After repeating the centrifugal washing 3 times, the mixture was resuspended in 50mM CB buffer solution having a pH =10 to a final concentration of 20mg/mL to obtain a receptor microsphere solution having a particle size of 200 nm.

5. The same method is used for preparing acceptor microsphere solutions with the grain diameters of 100nm, 150nm, 250nm and 350nm respectively.

Example 2: receptor microsphere coated antibody

1. Weighing 10mg of receptor microsphere coated with carboxyl dextran hydrogel according to the preparation amount, placing the receptor microsphere in a centrifuge tube, centrifuging at 10000rpm for 60min.

2. The supernatant was discarded, and 2mg of Anti-PCT antibody I (which may be the antibody examples for any other analytical item (Anti-cTnI antibody I and Anti-PCT antibody I)), 50. Mu.L of Tween-20 (50 mg/mL), and a volume of 0.05M MES pH =6.0 was added to the pellet to give a final concentration of 10mg/mL of receptor microspheres.

3. And (5) quickly mixing by ultrasound.

4. Add 50. Mu.L of NaBH to the centrifuge tube ₃ CN (50 mg/mL,0.05M MES pH =6.0 preparation) is mixed evenly and placed in a rotary mixer for reaction for 36-48h at 37 ℃.

5. And (3) sealing: 1mL of BSA (50 mg/mL,0.05M MES pH =6.0 formulation) was added and placed in a rotary mixer at 37 ℃ for 12-16h.

6. Cleaning: wash 3 times with 0.05M MES buffer.

7. And sampling to determine the concentration, the grain diameter and the signal value of the washed receptor microsphere coated with the antibody.

Example 3: preparation of Donor microspheres

1. A25 mL round-bottomed flask was prepared, 0.1g of copper (II) phthalocyanine and 10mL of DMF were added, and the mixture was magnetically stirred, and then heated in a water bath to 70 ℃ to obtain a copper (II) phthalocyanine solution.

2. Preparing a 100mL three-neck flask, adding 10mL95% ethanol, 10mL water and 10mL polystyrene microspheres which are 10% in concentration and have the particle size of 200nm and are coated with aldehyde dextran hydrogel, magnetically stirring, and heating in a water bath to 70 ℃.

3. And (3) slowly dropwise adding the copper (II) phthalocyanine solution obtained in the step (1) into the three-neck flask obtained in the step (2), reacting for 2 hours at 70 ℃, stopping stirring, and naturally cooling to obtain the emulsion.

4. The emulsion was centrifuged for 1 hour at 30000g, the supernatant discarded after centrifugation and resuspended in 50% ethanol. After 3 times of repeated centrifugal washing, the microspheres were resuspended in 50mM CB buffer solution with pH =10 to a final concentration of 20mg/mL to obtain a donor microsphere solution with a particle size of 200 nm.

5. The same method is used to prepare donor microsphere solutions with the particle sizes of 80nm, 100nm, 150nm, 250nm and 350nm respectively.

Example 4: donor microsphere coated avidin

1. Donor microsphere suspension treatment: and (3) absorbing a certain amount of donor microspheres to centrifuge in a high-speed refrigerated centrifuge, removing supernatant, adding a certain amount of MES buffer solution, performing ultrasonic treatment on an ultrasonic cell disruption instrument until the particles are resuspended, and adding the MES buffer solution to adjust the concentration of the donor microspheres to 100mg/mL.

2. Preparing an avidin solution: an amount of avidin (which may also be streptavidin or neutravidin) was weighed and dissolved to 8mg/mL with MES buffer.

3. Mixing: the treated donor microsphere suspension, avidin at 8mg/mL, and MES buffer were mixed at a volume ratio of 2.

4. Reaction: preparing 25mg/mL NaBH by MES buffer solution ₃ The CN solution was added in a volume ratio to the reaction solution 1. The reaction was rotated at 37 ℃ for 48 hours.

5. And (3) sealing: preparing 75mg/mL Gly solution and 25mg/mL NaBH by MES buffer solution ₃ The CN solution was added to the above solution in a volume ratio to the reaction solution 2. Then 200mg/mL BSA solution (MES buffer) with the volume ratio of 5 to 8 is added, and the mixture is rapidly mixed,the reaction was rotated at 37 ℃ for 16 hours.

6. Cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, resuspending by an ultrasonic method, centrifuging again, cleaning for 3 times, finally suspending by a small amount of buffer solution, measuring the solid content, and adjusting the concentration to 10mg/mL by the buffer solution.

Example 5: preparation of Biotin-labeled antibody (additional reagent)

1. Antibody treatment: anti-PCT antibody II (which may be the antibody example corresponding to any other assay item (Anti-cTnI antibody II and Anti-PCT antibody II)) was dialyzed against 0.1M NaHCO ₃ Solution, antibody concentration was determined and adjusted to 1mg/mL.

2. A16.17 mg/mL biotin solution was prepared in DMSO.

3. Marking: the prepared biotin solution and the treated Anti-PCT antibody II (which can be antibody examples (Anti-cTnI antibody II and Anti-PCT antibody II) corresponding to any other analysis items) with the concentration of 1mg/mL are mixed according to the volume ratio of 10000. Standing and reacting for 12-16 hours at the temperature of 2-8 ℃.

4. And (3) dialysis: the reacted biotin-labeled antibody was dialyzed against biotin-labeled dialysis buffer (pH = 8.00).

5. Dialyzed biotinylated antibody was aspirated and transferred to a clean centrifuge tube, and samples were taken to determine antibody concentration. The concentration of the biotin labeled antibody which is qualified for quality inspection is adjusted to 0.5mg/mL.

Example 6: homogeneous phase chemiluminescence POCT detection device

In this embodiment, the hole site of the sample to be tested, the first reagent hole site and the second reagent hole site are coated with a sealing film to seal the hole opening, and the coating film may be a disposable sealing film or a reusable sealing film.

In this embodiment, the particle size of the donor microsphere in the first reagent hole for containing the donor reagent is selected from 50-500nm, and the particle size of the donor microsphere in the donor reagent is 0.1-100 μ g/mL. The second reagent hole is used for containing the acceptor microspheres in the acceptor reagent, the particle size of the acceptor microspheres is 50-500nm, and the concentration of the acceptor microspheres in the acceptor reagent is 10-400 mu g/mL.

In this embodiment, as shown in fig. 2, the cross-sectional shapes of the sample hole site to be tested 11, the first reagent hole site 12 and the second reagent hole site 13 are different from each other, so as to distinguish different liquids contained in different hole sites. In the reagent cup strip shown in fig. 2, the cross section of the sample well site 11 to be detected is oval, the cross section of the first reagent well site 12 is square, the cross section of the second reagent well site 13 is circular, and the detection cup 131 is arranged in the second reagent well site 13.

In this embodiment, as shown in fig. 2, the hole sites further include a dilution hole site 14, and the dilution hole site 14 is used for containing a dilution liquid. In one embodiment, dilution hole sites 14 are configured with a rectangular cross-sectional shape, as shown in FIG. 2.

In this embodiment, as shown in fig. 3, the cross section of the sample hole site 11A to be detected is circular, the cross section of the first reagent hole site 12A is elliptical, the second reagent hole site 13A is located at the detection position, and the detection cup 131A is disposed in the second reagent hole site 13A. Preferably, the well locations further comprise dilution well locations 14A. More preferably, the well site further comprises an additional reagent well site 15A, the additional reagent well site 15A being adapted to contain an additional reagent. In case the well sites of the reagent cup strip 1 comprise dilution well site 14A and additional well site 15A, the dilution and additional reagents are added in the following order: adding a sample to be tested to a diluent hole site 14A, mixing the sample with diluent for dilution, adding the diluted sample to be tested into an additional reagent hole site 15A after dilution, continuing to add the mixed liquid into a first reagent hole site 12A after mixing the diluted sample with the additional reagent, reacting for a certain time, continuing to add the mixed liquid into a detection cup 131A of a second reagent hole site 13A, and performing subsequent processes.

In order to facilitate the identification and reading of the information of the sample to be tested, the reagent cup strip 1 is further provided with a barcode area 15 along the side in the width direction, as shown in fig. 3, the barcode area 15 containing the information of the reagent cup strip 1. The bar code area 15 is provided with a bar code, and the bar code is a one-dimensional or two-dimensional code.

In this embodiment, as shown in fig. 1, the sample adding mechanism 2 includes a pipetting assembly 21, a vertical movement assembly 22, and a horizontal movement assembly 23. Pipetting assembly 21 is used to aspirate or discharge liquids. The pipetting assembly 21 is arranged on the vertical moving assembly 22, and the vertical moving assembly 22 is used for driving the pipetting assembly 21 to vertically move. The vertical moving assembly 22 is arranged on the horizontal moving assembly 23, and the horizontal moving assembly 23 is used for driving the pipetting assembly 21 to move horizontally. The vertical moving assembly 22 is arranged on the horizontal moving assembly 23, and the horizontal moving assembly 23 is used for driving the pipetting assembly 21 to move horizontally. Regarding the structure of the vertical moving assembly 22 and the horizontal moving assembly 23, the prior art is adopted, and the structure of the vertical moving assembly 22 and the structure of the horizontal moving assembly 23 can be the same or similar, and both structures comprise a guide rail, a slide block and a driving device, wherein the guide rail of the vertical moving assembly 22 and the guide rail of the horizontal moving assembly 23 are perpendicular to each other. As shown in fig. 1, the detecting mechanism 3 includes a base 31, a driving assembly (not shown), and a detecting assembly. The base 31 is intended to carry a reagent cup strip 1. The drive assembly is adapted to drive the base 31 in rotation around its centre and to bring the reagent cup strips 1 in rotation. The detection assembly is used for detecting a chemiluminescent reaction in the reagent cup strip 1. As shown in FIG. 1, the cross-section of the base 31 is circular, a plurality of grooves 311 for accommodating the reagent cup strips 1 are formed in the base 31, and the grooves 311 are uniformly distributed on the base 31. When the reagent cup strips 1 in the recesses 311 move to a position where the length direction thereof is parallel to the horizontal moving guide, the sample adding mechanism 2 moves the reagents contained in the wells between the wells by the cooperation of the vertical moving component 22 and the horizontal moving component 23. Wherein, the driving assembly is arranged below the base 31, and can drive the base 31 to rotate around the center thereof, so that the detection hole sites of the reagent cup strips 1 in the grooves 311 can sequentially pass through the lower part of the detecting assembly.

In this embodiment, the detection assembly includes an exciter capable of emitting red excitation light of 600-700 nm and a detector capable of detecting chemiluminescence with a wavelength of 450-650nm. During detection, the exciter emits red exciting light to irradiate the reaction system, the reaction system produces light-excited chemiluminescence reaction, and the detector reads signals. The detector is selected from a single photon counter, a photomultiplier tube, a silicon photocell or a photometric integrating sphere. It will be appreciated that the detection mechanism 3 further comprises a motion control module for controlling the vertical movement of the activator and the detector for completing the detection of the detection hole locations in cooperation with the reagent cup strips on the base 31.

In this embodiment, as shown in fig. 4, the pipetting assembly 21 comprises a piston mechanism 211, a connector 212 and a pipette 213 arranged in sequence from top to bottom, the piston mechanism 211 is connected with the connector 212, and the pipette 213 is arranged at the edge of the end face of the base 31 (as shown in fig. 1). When the connecting piece 212 moves to the position right above the pipette 213, the vertical moving assembly 22 drives the connecting piece 212 to descend, the connecting piece 212 is connected with the pipette 213, and the connecting piece 212 is a rigid connector which is tightly matched with the top end of the pipette, so that the air tightness of the structure is ensured. Piston mechanism 211 comprises piston rod, cavity and piston control module, and the piston rod setting is in the cavity, and the piston rod can be driven by the motor and move up and down, inflation or compressed air to cooperate the pipette to aspirate or discharge liquid. After the liquid is sucked by the pipette 213, the vertical moving component 22 and the horizontal moving component 23 drive the pipette 213 to move to the position above the hole to be discharged, and then the piston mechanism 211 is controlled to discharge the liquid in the pipette 213 to the hole.

In this embodiment, the homogeneous chemiluminescence POCT detection apparatus further comprises an incubation module, and the incubation module is used for providing a suitable ambient temperature for a chemiluminescence reaction of the reagent in the reagent cup strip.

Example 7: the use process of the homogeneous phase chemiluminescence POCT detection device.

The using process comprises the following steps: respectively adding 25 mu L of sample to be detected, 25 mu L of biotin-labeled antibody, 175 mu L of mixed solution of avidin-coated donor microspheres and 25 mu L of receptor reagent into a sample hole site to be detected, an additional reagent hole site, a first reagent hole site and a second reagent hole site of a reagent cup strip, then placing the reagent cup strip into a POCT analyzer developed by Boyang biotechnology (Shanghai) Limited, adding the sample with the corresponding volume to be detected into the additional reagent hole site by a sample adding mechanism, vibrating and incubating for 10 minutes at 37 ℃; adding the incubated liquid in the additional reagent hole site into the first reagent hole site, vibrating, and incubating at 37 ℃ for 10 minutes to form mixed liquid; the mixed liquid is added to the second reagent hole site, vibrated and incubated at 37 ℃ for 10 minutes to form a mixture to be tested. And irradiating the second reagent hole site by using laser emitted by an exciter in the detection assembly, reacting for a certain time, detecting a chemiluminescent signal generated by the reaction of the receptor microsphere and active oxygen by using a detection mechanism, and calculating the concentration of target molecules to be detected in a sample to be detected.

Example 8: homogeneous chemiluminescence POCT detection

The device described in example 6, the method described in example 7, and the microsphere composition (taking the Anti-PCT antibody i coated receptor microsphere as an example) with different particle sizes are used to detect PCT in a sample to be detected, and the detection results are shown in table 1.

TABLE 1

As can be seen from table 1, under the condition that the particle size of the donor microsphere is fixed, the detected luminescence signal value gradually decreases as the particle size of the acceptor microsphere increases, and when the particle size of the acceptor microsphere is equal to the particle size of the donor microsphere, the detected luminescence signal value is the largest. And when the particle size of the donor microsphere and the particle size of the acceptor microsphere are both 200nm, the detected luminescence signal value is optimal, and the detection sensitivity is best.

Example 9: preparation of quality control product and calibrator

1. Preparation of quality control product

And (3) taking the newborn bovine serum as a diluent, and respectively diluting the pure antigen products into 2 working solutions with different concentrations, wherein the working solutions are quality control products Q1 and Q2. And taking the quality control products Q1 and Q2 to be detected to repeatedly calibrate on the instrument system of the company for three times. And measuring 10 holes each time, and calculating the overall mean value and SD, wherein the mean value +/-3 SD is the allowable range of the concentration measurement of the quality control substance.

2. Preparation of calibrator

The pure antigen is diluted into a series of concentrations by calf serum (containing preservative), and is frozen and stored for later use after being calibrated by national standard for immunoassay. The shelf life is 2 years when the tea is stored at-20 ℃.

Simultaneously analyzing and measuring the calibrator and the national standard substance with corresponding concentration, fitting by using 4 parameters or other models, and requiring that the absolute value of the correlation coefficient (r) of the calibrator dose-response curve is not lower than 0.9900; while the two dose-response curves did not deviate significantly from parallelism (t-test); the ratio of the measured titer of the calibrator to the calibrated titer is 0.90-1.10 by taking the national standard as a standard.

Example 10: detection of batch-to-batch precision of homogeneous chemiluminescence POCT detection method

A sample to be tested: the quality control product Q1 prepared in example 9;

the process is as follows: repeating the detection 20 times to obtain a light intensity value (RLU)

Criteria for outlier determination: not less than 3SD

Reagents used in the detection:

(1) Receptor microspheres prepared in example 2 (the particle size of the oxygen-receiving microspheres is 200nm, and the oxygen-receiving microspheres are respectively matched with anti-cTnI antibody I and anti-PCT antibody I);

(2) Additional reagents prepared in example 5 (biotin-labeled antibody, anti-cTnI antibody ii and anti-PCT antibody ii, respectively));

(3) Example 4 preparation of donor microspheres (different particle size (80 nm, 200 nm) coated avidin) donor microspheres).

The reagents (1) - (3) were combined to form a reagent set 1 and a reagent set 2 shown in table 2, the cTnI and the PCT antigen were detected separately (after dilution to appropriate concentrations, the same samples were detected by the reagent sets 1 and 2 at the same time), and the detection was repeated for 20 wells, as described in example 7, and the detection results are shown in table 3.

Table 2:

reagent grouping	Reagent set 1	Reagent set 2
			Particle size/nm of acceptor microspheres	200nm	200nm
Donor microsphere particle size/nm	80nm	200nm

Table 3:

as can be seen from Table 3, when the light intensity of reagent set 2 is increased as compared with reagent set 1, that is, when the particle size of the acceptor microsphere is equal to that of the donor microsphere, the light intensity detected by this method is increased. Meanwhile, compared with the reagent group 1, the reagent group 2 has a smaller Coefficient of Variation (CV), i.e., when the particle size of the acceptor microspheres is equal to that of the donor microspheres, the precision is higher.

Example 11: detection of analytical sensitivity of homogeneous chemiluminescence POCT detection method

A sample to be detected: a zero value calibrator;

Sensitivity: RLU substitution calibration curve

The reagents and procedures used in the detection were the same as those in example 10, and the results are shown in Table 4.

Table 4:

as can be seen from table 4, the sensitivity of detection in reagent set 2 is better than that in reagent set 1, i.e., when the particle size of the acceptor microsphere is equal to that of the donor microsphere, the sensitivity of the reagent is advantageously improved.

Comparative example 1: preparation of comparative donor and acceptor microspheres

1. Receptor microsphere coated antibody

1. anti-PCT antibody I was dialyzed into 50mM CB buffer at pH =10 and found to be 1mg/mL.

2. 0.5mL of the receptor microsphere prepared in example 1 and 0.5mL of anti-PCT antibody I were added to a 2mL centrifuge tube, mixed and added with 100. Mu.L of 10mg/mL NaBH ₄ The solution (50 mM CB buffer) was reacted at 2-8 ℃ for 4 hours.

3. After the reaction, 0.5mL of a 100mg/mLBSA solution (50 mM CB buffer) was added, and the reaction was carried out at 2 to 8 ℃ for 2 hours.

4. After completion of the reaction, the reaction mixture was centrifuged at 45min,30000g, and the supernatant was discarded after centrifugation, followed by resuspension in 50mM MES buffer. And repeating the centrifugal washing for 4 times, and diluting to a final concentration of 100 mu g/mL to obtain the anti-PCT antibody I-coated receptor microspheres with the particle sizes of 100nm, 150nm, 200nm, 250nm and 350nm respectively.

2. Donor microsphere coated antibody

1. The anti-PCT antibody II was dialyzed into 50mM CB buffer with pH =10, and the concentration was measured to be 1mg/mL.

2. Adding 0.5mL of photosensitive microspheres and 0.5mL of paired antibody II into a 2mL centrifuge tube, uniformly mixing, and adding 100 mu L of 10mg/mL NaBH ₄ The solution (50 mM CB buffer) was reacted at 2-8 ℃ for 4 hours.

3. After the reaction was completed, 0.5mL of 100mg/mL BSA solution (50 mM CB buffer) was added and the reaction was carried out at 2-8 ℃ for 2 hours.

4. After completion of the reaction, the reaction mixture was centrifuged at 45min and 30000g, and after centrifugation, the supernatant was discarded and resuspended in 50mM MES buffer. The centrifugal washing was repeated 4 times and diluted to a final concentration of 100. Mu.g/mL. Obtaining the donor microspheres coated with the anti-PCT antibody II with the grain sizes of 100nm, 150nm, 200nm, 250nm and 350nm respectively.

Comparative example 2: homogeneous chemiluminescence POCT detection by adopting contrast donor microspheres and acceptor microspheres

The procedure of the assay was the same as in example 8 except that the donor microspheres and the acceptor microspheres used were replaced with the comparative donor microspheres and acceptor microspheres prepared in comparative example 1, and the assay results are shown in table 5.

TABLE 5

As can be seen from Table 5, the amount of luminescence signal detected by the donor microsphere and the acceptor microsphere is significantly reduced, and the detection sensitivity is significantly reduced. And when the particle size of the acceptor microsphere is equal to that of the donor microsphere in the donor microsphere and the acceptor microsphere, the detected light-emitting signal value is not optimal.

Example 12: donor microsphere with average particle size of 250nm and surface not coated or connected with polysaccharide and preparation of donor reagent

Preparation of aldehyde group polystyrene latex microsphere

a) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10min, N was introduced thereinto ₂ 30min。

b) 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system of the step a), and continuously introducing N ₂ 30min。

c) The reaction system was heated to 70 ℃ and reacted for 15 hours.

d) The emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. And washing the obtained emulsion by using deionized water through centrifugal sedimentation for a plurality of times until the conductivity of the supernatant at the beginning of centrifugation is close to that of the deionized water, then diluting the supernatant with water, and storing the diluted supernatant in an emulsion form.

e) The latex microspheres had a Gaussian distribution of mean particle size 201.3nm, coefficient of variation (c.v.) =8.0%, gaussian distribution as shown in fig. 1, and Nicomp distribution as shown in fig. 2, as measured by a nano-particle sizer. The aldehyde group content of the latex microsphere is 260nmol/mg measured by an electric conductivity titration method.

Filling of (II) sensitizers

a) A25 ml round-bottomed flask was prepared, 0.11g of copper phthalocyanine and 10ml of N, N-dimethylformamide were added thereto, and magnetically stirred, and the temperature was raised to 75 ℃ in a water bath to obtain a photosensitizer solution.

b) Preparing a 100ml three-neck flask, adding 10ml95% ethanol, 10ml water and 10ml aldehyde polystyrene latex microspheres obtained in the step (I) with the concentration of 10%, magnetically stirring, and heating in a water bath to 70 ℃.

c) Slowly dropwise adding the solution obtained in the step a) into the three-neck flask obtained in the step b), reacting at 70 ℃ for 2 hours, stopping stirring, and naturally cooling to obtain an emulsion.

d) The emulsion was centrifuged for 1 hour at 30000G, the supernatant discarded after centrifugation and resuspended in 50% ethanol. After three repeated centrifugal washes, the suspension was resuspended in 50mM CB buffer, pH =10, to a final concentration of 20mg/ml.

(III) preparation of Donor reagent by modifying avidin on the surface of microsphere

a) Treating microsphere suspension: and (3) sucking a certain amount of the microspheres prepared in the step (II) to centrifuge in a high-speed refrigerated centrifuge, removing the supernatant, adding a certain amount of MES buffer solution, performing ultrasonic treatment on an ultrasonic cell disruption instrument until the microspheres are resuspended, and adding the MES buffer solution to adjust the concentration of the microspheres to 100mg/ml.

b) Preparing an avidin solution: a certain amount of streptavidin was weighed and dissolved in MES buffer to 8mg/ml.

c) Mixing: the treated microsphere suspension, avidin at 8mg/ml and MES buffer were mixed at a volume ratio of 2.

d) Reaction: preparing 25mg/ml NaBH by MES buffer solution ₃ CN solution is added according to the volume ratio of the CN solution to the reaction solution 1. The reaction was rotated at 37 ℃ for 48 hours.

e) And (3) sealing: MES buffer solution is prepared into 75mg/ml Gly solution and 25mg/ml NaBH ₃ The CN solution was added to the above solution in a volume ratio to the reaction solution 2. Then 200mg/ml BSA solution (MES buffer) was added thereto, and the volume ratio of the BSA solution to the reaction solution was adjusted5, rapidly mixing, and rotating at 37 ℃ for 16 hours.

f) Cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, resuspending by an ultrasonic method, centrifuging again, washing for 3 times, finally suspending by a small amount of donor microsphere buffer solution, measuring the solid content, and adjusting the concentration to 150 mu g/ml by the donor microsphere buffer solution to obtain the donor reagent containing the donor microspheres.

The average gaussian distribution particle size of the donor microspheres measured by a nano-particle sizer was 227.7nm, and the coefficient of variation (c.v.) =6.5%, as shown in fig. 3.

Example 13: preparation of polysaccharide-coated donor microspheres with average particle size of 250nm and donor reagent

The preparation of aldehyde-based polystyrene latex microspheres and the filling process of the sensitizer were the same as the preparation steps of (i) and (ii) in example 12.

Preparation of (mono) aminodextran

a) A500 mL four-neck flask was placed in an oil bath pan, equipped with a condenser tube, and charged with nitrogen.

b) 10g of dextran with the average molecular weight distribution of 500000KDa, 100ml of deionized water, 2g of NaOH and 10g of N- (2, 3-epoxypropyl) phthalimide are sequentially added and mechanically stirred.

c) After the oil bath is carried out for 2 hours at the temperature of 90 ℃, the heating is closed, and the stirring is maintained for natural cooling.

d) The reaction mixture separated out the main mixture in 2L of methanol, the solid was collected and dried.

e) A200 mL four-necked flask was placed in an oil bath pan, equipped with a condenser tube, and purged with nitrogen.

f) The dried solid, 100mL of deionized water, 1.8g of sodium acetate, and 5mL of 50% hydrazine hydrate were sequentially added, the pH was adjusted to 4, and the mixture was mechanically stirred.

g) After being subjected to oil bath at 85 ℃ for 1 hour, the heating is closed, and the stirring is maintained for natural cooling.

h) The pH of the reaction solution is adjusted to be neutral and then filtered, and the filtrate is collected.

i) The filtrate is put into a dialysis bag, and is dialyzed for 2 days at the temperature of 4 ℃ by deionized water, and the water is changed for 3 to 4 times every day.

j) After dialysis, the resulting solution was lyophilized to obtain 9.0g of an aminodextran solid.

k) The amino group content was found to be 0.83mmol/g by TNBSA method.

Preparation of (di) aldehyde dextran

a) 10g of dextran with a mean molecular weight distribution of 500000kDa was weighed into a 250 beaker, and 100mL of 0.1M/pH =6.0 phosphate buffer was added and dissolved with stirring at room temperature.

b) 1.8g of sodium metaperiodate was weighed into a 50mL beaker, 10mL of 0.1M/pH =6.0 phosphate buffer was added, and dissolved with stirring at room temperature.

c) Slowly and dropwise adding the sodium metaperiodate solution into the dextran solution, reacting until no bubbles are generated, and continuing stirring for 1 hour.

d) The reaction mixture is put into a dialysis bag, and is dialyzed for 2 days at the temperature of 4 ℃ by deionized water, and the water is changed for 3 to 4 times every day.

e) After dialysis, the mixture was freeze-dried to obtain 9.6g of aldehyde dextran solid.

f) The aldehyde group content was measured by using the BCA Kit to be 0.94mmol/g.

(III) microsphere coating dextran

a) 50mg of aminodextran solid was placed in a 20mL round bottom flask, 5mL of 50mM/pH =10 carbonate buffer was added, and dissolved by stirring in the dark at 30 ℃.

b) 100mg of donor microspheres were added to the aminodextran solution and stirred for 2 hours.

c) 10mg of sodium borohydride was dissolved in 0.5mL of 50mM/pH =10 carbonate buffer solution, and the solution was added dropwise to the reaction solution and reacted overnight at 30 ℃ in the dark.

d) The mixture after the reaction was centrifuged at 30000G, and the supernatant was discarded, and then added with 50mM/pH =10 carbonate buffer for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume was adjusted to 20mg/ml by using 50mM/pH =10 carbonate buffer.

e) 100mg of aldehyde dextran solid was placed in a 20mL round bottom flask, and 5mL of 50mM/pH =10 carbonate buffer was added and dissolved with stirring at 30 ℃ in the dark.

f) The microspheres are added into an aldehyde dextran solution and stirred for 2 hours.

g) After 15mg of sodium borohydride was dissolved in 0.5mL of 50mM/pH =10 carbonate buffer solution, the solution was added dropwise to the reaction solution, and the reaction was carried out overnight at 30 ℃ in the dark.

h) After the reaction, the mixture 30000G was centrifuged, the supernatant was discarded, and 50mM/pH =10 carbonate buffer was added for ultrasonic dispersion. After three repeated centrifugal washes, the volume was determined to be 20mg/ml using 50mM/pH =10 carbonate buffer.

i) The gaussian distribution average particle size of the microspheres measured by a nano-particle sizer was 235.6nm, and the coefficient of variation (c.v.) =8.1%, as shown in fig. 4.

(IV) modifying avidin on the surface of microsphere and preparing donor reagent

g) Treating microsphere suspension: and (3) sucking a certain amount of microspheres prepared in the step (three) into a high-speed freezing centrifugal machine for centrifugation, removing a supernatant, adding a certain amount of MES buffer solution, performing ultrasound on an ultrasonic cell disruption instrument until the microspheres are resuspended, and adding the MES buffer solution to adjust the concentration of the donor microspheres to 100mg/ml.

h) Preparing an avidin solution: a certain amount of neutravidin was weighed and dissolved to 8mg/ml by adding MES buffer.

i) Mixing: mixing the treated microsphere suspension, 8mg/ml avidin and MES buffer solution according to the volume ratio of 2.

j) Reaction: preparing 25mg/ml NaBH by MES buffer solution ₃ CN solution is added according to the volume ratio of the CN solution to the reaction solution 1. The reaction was rotated at 37 ℃ for 48 hours.

k) And (3) sealing: preparing 75mg/ml Gly solution and 25mg/ml NaBH by using MES buffer solution ₃ The CN solution was added to the above solution in a volume ratio to the reaction solution 2. Then 200mg/ml BSA solution (MES buffer) was added at a volume ratio of 5:8 to the reaction solution, and the mixture was rapidly mixed and subjected to a rotary reaction at 37 ℃ for 16 hours.

l) cleaning: adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, resuspending by an ultrasonic method, centrifuging again, cleaning for 3 times, finally suspending by a small amount of donor microsphere buffer solution, measuring the solid content, and adjusting the concentration to 150 mu g/ml by using the donor microsphere buffer solution to obtain the donor reagent containing the donor microspheres.

The average gaussian distribution particle size of the donor microspheres measured by a nano-particle sizer was 249.9nm, and the coefficient of variation (c.v.) =11.6%, as shown in fig. 5.

Example 14: preparation of acceptor microspheres with average particle size of 250nm

1. Preparation and characterization process of aldehyde polystyrene latex microspheres

1) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10min, N was introduced thereinto ₂ 30min；

2) 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system in the step 1, and continuously introducing N ₂ 30min；

3) Heating the reaction system to 70 ℃ and reacting for 15 hours;

4) The emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. Washing the obtained emulsion with deionized water by secondary centrifugal sedimentation until the conductivity of the supernatant at the beginning of centrifugation is close to that of the deionized water, then diluting with water, and storing in an emulsion form;

5) The mean particle size of Gaussian distribution of the particle size of the latex microspheres measured by a nano-particle sizer was 202.2nm, the coefficient of variation (c.v.) =4.60%, and the Gaussian distribution curve is shown in fig. 6. The aldehyde group content of the latex microsphere is 280nmol/mg measured by an electric conductivity titration method.

2. Process and characterization of embedding luminescent compositions inside microspheres

1) A25 ml round-bottom flask was prepared, and 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (MTTA-EU) were added ³⁺ ) 10ml of 95% ethanol, magnetically stirring, heating in a water bath to 70 ℃ to obtain a complex solution;

2) Preparing a 100ml three-neck flask, adding 10ml of 95% ethanol, 10ml of water and 10ml of aldehyde polystyrene latex microspheres with the concentration of 10% obtained in the step (1), magnetically stirring, and heating in a water bath to 70 ℃;

3) Slowly dropwise adding the complex solution obtained in the step 1) into the three-neck flask obtained in the step 2), reacting at 70 ℃ for 2 hours, stopping stirring, and naturally cooling;

4) And centrifuging the emulsion for 1 hour at 30000G, and removing supernatant after centrifugation to obtain the aldehyde polystyrene microspheres embedded with the luminescent composition.

5) The average particle size of the microspheres in Gaussian distribution measured by a nanometer particle sizer was 204.9nm, and the coefficient of variation (C.V.) =5.00% (as shown in FIG. 7)

3. Process and characterization for coating polysaccharide coating on microsphere surface

1) Taking 50mg of aminodextran solid, putting the aminodextran solid in a 20mL round-bottom flask, adding 5mL of 50mM/pH =10 carbonate buffer solution, and stirring and dissolving the aminodextran solid at 30 ℃ in the dark;

2) Adding 100mg of aldehyde polystyrene microspheres which are prepared in the step 2 and are filled with the luminescent composition into the aminodextran solution, and stirring for 2 hours;

3) Dissolving 10mg of sodium borohydride in 0.5mL of 50mM/pH =10 carbonate buffer solution, dropwise adding the solution into the reaction solution, and reacting overnight at 30 ℃ in the dark;

4) After the reaction, the mixture 30000G was centrifuged, the supernatant was discarded, and 50mM/pH =10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugal washing for three times, fixing the volume by using 50mM/pH =10 carbonate buffer solution to ensure that the final concentration is 20mg/ml;

5) Adding 100mg aldehyde dextran solid into a 20mL round-bottom flask, adding 5mL 50mM/pH =10 carbonate buffer solution, and stirring and dissolving at 30 ℃ in the dark;

6) Adding the microspheres into an aldehyde dextran solution and stirring for 2 hours;

7) Dissolving 15mg of sodium borohydride in 0.5mL of 50mM/pH =10 carbonate buffer solution, dropwise adding the solution into the reaction solution, and reacting overnight at 30 ℃ in the dark;

8) After the reaction, the mixture 30000G was centrifuged, the supernatant was discarded, and 50mM/pH =10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume was adjusted to 20mg/ml by using 50mM/pH =10 carbonate buffer.

9) The Gaussian distribution mean particle size of the microspheres at this time was 241.6nm, and the coefficient of variation (c.v.) =12.90%, as measured by a nano-particle sizer (see fig. 8).

Conjugation procedure for PCT antibody

1) Paired PCT antibody was dialyzed into 50mM CB buffer at PH =10, and the concentration was determined to be 1mg/ml.

2) Adding 0.5ml of microspheres obtained in the step 3 and 0.5ml of paired antibody I obtained in the step 1) into a 2ml centrifuge tube, uniformly mixing, and adding 100 mu l of 10mg/ml NaBH ₄ The solution (50 mM CB buffer) was reacted at 2 to 8 ℃ for 4 hours.

3) After completion of the reaction, 0.5ml of a 100mg/ml BSA solution (50 mM CB buffer) was added thereto, and the reaction was carried out at 2 to 8 ℃ for 2 hours.

4) After completion of the reaction, the reaction mixture was centrifuged for 45min,30000G, and the supernatant was discarded after centrifugation, followed by resuspension in 50mM MES buffer. The centrifugal washing was repeated four times, and diluted with a buffer solution to a final concentration of 50. Mu.g/ml to obtain a PCT antibody-coupled receptor microsphere solution.

The mean particle size of Gaussian distribution of the particle size of the receptor microsphere measured by a nano-particle sizer was 253.5nm, and the coefficient of variation (c.v value) =9.60% (as shown in fig. 9).

Example 15: test results and analysis on computer (test substance: PCT antigen)

The PCT homogeneous chemiluminescent assay kit (photo-activated chemiluminescent assay) used in this example consisted of reagent 1 (R1 ') containing a first anti-PCT antibody coated acceptor microsphere, reagent 2 (R2 ') containing a biotin-labeled second anti-PCT antibody, and additionally contained a universal solution (R3 ') containing donor microspheres. Wherein, R1' is the receptor reagent prepared by using the receptor microsphere (coefficient of variation of particle size distribution c.v = 9.6%) in example 14; r3' is the donor reagent prepared using the donor microspheres of examples 12 and 13.

The detection process is completed on the homogeneous chemiluminescence POCT detection device described in example 6, and the detection results are output, and the specific detection results are shown in table 6 below.

TABLE 6

From the results in Table 6, it is understood that the sensitivity and the upper limit of detection of the detection by the method described in the present application are excellent, and the sensitivity and the upper limit of detection of the detection by the donor reagent in example 12 are superior to those of the donor reagent in example 13. Therefore, the performance of the donor microsphere without the polysaccharide coating on the surface is more excellent.

It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described in relation to an exemplary embodiment, and it is understood that the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Claims

1. A homogeneous phase chemiluminescence POCT detection method comprises the following steps:

2. The method of claim 1, wherein in step S1, the sample to be tested is first mixed with the receptor reagent to form a first mixture, and then the first mixture is mixed with the donor reagent to form the mixture to be tested.

3. The method according to claim 1 or 2, wherein in step S2, the mixture to be tested is excited to generate chemiluminescence by irradiating the mixture with 600nm to 700nm red excitation light.

4. The method of claim 3, wherein the donor microspheres and the acceptor microspheres each have an average particle size of 20nm to 350nm.

5. The method of claim 3, wherein the donor microspheres and the acceptor microspheres each have an average particle size of 50nm to 300nm.

6. The method of claim 3, wherein the donor microspheres and the acceptor microspheres each have an average particle size of 100nm to 250nm.

7. The method of claim 3, wherein the donor microspheres and the acceptor microspheres each have an average particle size of 180nm to 220nm.

8. The method of any one of claims 1-2, 4-7, wherein the donor microsphere comprises a first support, wherein the interior of the first support is filled with a photosensitizer, and wherein the surface of the first support is chemically bonded to a label.

9. The method of claim 8, wherein the label is avidin, selected from the group consisting of ovalbumin, streptavidin, vitellin, neutravidin, and avidin-like.

10. The method of claim 9, wherein the avidin is selected from neutravidin and streptavidin.

11. The method according to claim 9 or 10, wherein the surface of the first support is free of a coating or attached polysaccharide substance, which is directly chemically bound to the label.

12. The method of claim 11, wherein the avidin is chemically bonded to the surface of the first support by reacting an amino group with an aldehyde group on the surface of the first support to form a schiff base.

13. The method according to claim 12, wherein the surface of the first support carries a bonding functional group for chemically bonding a label to the surface of the first support.

14. The method of claim 13, wherein the bonding functional group is selected from the group consisting of an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, a maleimide group, and a thiol group.

15. The method of claim 13, wherein the bonding functional group is selected from an aldehyde group or a carboxyl group.

16. The method according to any one of claims 13 to 15, wherein the bonded functional group content of the first support surface is 100 to 500nmol/mg.

17. The method according to any one of claims 13 to 15, wherein the bonded functional group content of the first support surface is from 200 nmol/mg to 400nmol/mg.

18. The method according to claim 9 or 10, wherein the surface of the first carrier is coated with hydrophilic aldehyde dextran, the aldehyde group of which is chemically bonded with a label.

19. The method of claim 8, wherein said photosensitizer is selected from one of methylene blue, rose bengal, a porphyrin and a phthalocyanine.

20. The method of any one of claims 9-10, 13-15, and 19, wherein the acceptor microsphere comprises a second support, the interior of the second support is filled with the luminescent composition, the surface of the second support is coated with at least one polysaccharide layer, and the surface of the polysaccharide layer is connected with the biomolecule.

21. The method of claim 20, wherein the surface of the second carrier is coated with hydrophilic carboxydextran.

22. The method of claim 20, wherein the luminescent composition comprises a europium complex.

23. The method of claim 22, wherein said europium complex is MTTA-EU3+.

24. The method according to any one of claims 21 to 23, wherein the first carrier and/or the second carrier is selected from agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate or polyacrylate.

25. The method according to any one of claims 21 to 23, wherein the first carrier and/or the second carrier is made of a material selected from the group consisting of polystyrene, polypropylene, poly (4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, and polyacrylate.

26. The method of any one of claims 1-2, 4-7, 9-10, 13-15, 19, 21-23, wherein the donor microspheres have a coefficient of variation of particle size distribution (cv) in the donor agent of at least 5%; and/or the presence of a gas in the gas,

the particle size distribution variation coefficient C.V value of the receptor microsphere in the receptor reagent is more than or equal to 5 percent.

27. The method of any one of claims 1-2, 4-7, 9-10, 13-15, 19, 21-23, wherein the donor microspheres have a coefficient of variation of particle size distribution, c.v., in the donor agent of 8% or more; and/or the presence of a gas in the gas,

the particle size distribution variation coefficient C.V value of the receptor microsphere in the receptor reagent is more than or equal to 8 percent.

28. The method of any one of claims 1-2, 4-7, 9-10, 13-15, 19, 21-23, wherein the donor microspheres have a coefficient of variation of particle size distribution (cv) in the donor agent of 10% or more; and/or the presence of a gas in the atmosphere,

the variation coefficient C.V value of the particle size distribution of the receptor microsphere in the receptor reagent is more than or equal to 10%.

29. The method of claim 26, wherein the donor microspheres have a coefficient of variation of particle size distribution, c.v., in the donor agent of 40% or less; and/or the presence of a gas in the gas,

the variation coefficient C.V value of the particle size distribution of the receptor microsphere in the receptor reagent is less than or equal to 40 percent.

30. The method of claim 26, wherein the donor microspheres have a coefficient of variation in particle size distribution (C.V) value of 20% or less in the donor reagent; and/or the presence of a gas in the atmosphere,

the variation coefficient C.V value of the particle size distribution of the receptor microsphere in the receptor reagent is less than or equal to 20 percent.

31. The method of any one of claims 1-2, 4-7, 9-10, 13-15, 21-23, 29-30, wherein the reactive oxygen species is singlet oxygen.

32. The method according to any one of claims 1 to 2, 4 to 7, 9 to 10, 13 to 15, 21 to 23, and 29 to 30, wherein in step S1, the sample to be tested is diluted with a diluent and then mixed with the acceptor reagent and the donor reagent to form a mixture to be tested.

33. A homogeneous chemiluminescence POCT assay device for in-situ detection of an antibody or antigen to be detected in a sample to be detected using the method of any one of claims 1 to 32; the device comprises:

a second reagent well site for holding a recipient reagent comprising recipient microspheres, said recipient microspheres capable of reacting with reactive oxygen species to produce a chemiluminescent signal, and said recipient microspheres having a particle size equal to the particle size of said donor microspheres;

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.

34. The apparatus of claim 33, wherein the sample well site to be tested, the donor reagent well site, and the acceptor reagent well site are each coated to close an aperture.

35. The device as claimed in claim 33, wherein the reagent cup strip is provided with a barcode area at a side in the width direction, said barcode area containing information of said reagent cup strip.

36. The apparatus of claim 33, wherein the well site further comprises a diluent well site for holding diluent.

37. The apparatus of any one of claims 33-36, wherein the sample application mechanism comprises:

a pipetting assembly for aspirating or discharging a liquid;

38. The apparatus of claim 37, wherein the detection mechanism comprises:

a base for carrying the reagent cup strips;

39. The device of claim 38, wherein the detection assembly comprises an exciter capable of emitting red excitation light between 600nm and 700 nm.

40. The device of any one of claims 33-36, 38-39, wherein the detection wavelength of the chemiluminescent signal produced by the reaction of the acceptor microspheres with reactive oxygen species is between 450nm and 650nm.

41. The apparatus of any one of claims 38-39, wherein the pipetting assembly comprises, in order from top to bottom, a piston mechanism, a connector, and a pipette, the piston mechanism being connected to the connector, the pipette being disposed at an end face edge of the base; when liquid transfer is needed, the connecting piece descends and is connected with the pipette, and the piston mechanism can move up and down to drive the pipette to suck or discharge liquid.

42. The device of claim 41, further comprising an incubation module for providing a suitable ambient temperature for the chemiluminescent reaction.

43. The device of any one of claims 33-36, 38-39, 42, wherein the cross-sectional shapes of the sample well site to be tested, the donor reagent well site and the acceptor reagent well site are different from each other.

44. Use of a homogeneous chemiluminescent POCT assay according to any one of claims 1 to 32 or a homogeneous chemiluminescent POCT assay device according to any one of claims 33 to 43 in a chemiluminescent assay.