CN114113583A

CN114113583A - A microfilament detection reaction device and its application

Info

Publication number: CN114113583A
Application number: CN202111568946.4A
Authority: CN
Inventors: 王天奇; 王济赫; 王一
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-12-21
Filing date: 2021-12-21
Publication date: 2022-03-01

Abstract

The invention relates to a microwire type detection reaction device which comprises a microwire carrier. The microwire type detection reaction device can be used for enriching and detecting target components, can reduce the space of a reaction system, reduces the dimensionality of the reaction system, saves reagent and equipment consumption, improves the reaction efficiency, improves the operation convenience and reduces the requirements on auxiliary equipment and environment.

Description

Microwire type detection reaction device and application thereof

Technical Field

The invention belongs to the technical field of target component detection, and relates to a microwire type detection reaction device and application thereof.

Background

The detection of target components such as bioactive substances or microorganisms is one of the most common works in the fields of biology, medicine, agriculture, environmental protection, and the like, and among the numerous detection methods, the most used method is to make the target components be affinity-enriched from a sample by utilizing affinity action. For example, antigens and antibodies with specific adsorption are commonly used affinity substances for biomedical assays. Other common affinity substances are: paired nucleotide fragments, enzymes and their substrates, receptors and their ligands, etc.

In the application of utilizing the specific adsorption effect of the antigen and the antibody, detection methods such as an antigen-antibody neutralization experiment, an antigen-antibody precipitation experiment, a rosette experiment and the like appear. Among the immunolabeling detection techniques, the enzyme-linked immunosorbent assay (ELISA) is a widely used detection method based on the principle that an antigen and an antibody can perform a specific affinity reaction. The main principle of the sandwich method is as follows: for a certain target component to be detected, usually a target antigen, a substance having an affinity effect with the target component, usually a specific antibody, called a first antibody, is coated on a well plate, usually a 96 well plate, that is, a certain amount of antibody is adsorbed on the surface of the well, a base plate for detection is made, after the detection is started, a sample to be detected, for example, a certain amount of serum with different dilutions, is added into the well, after a period of time, the target component to be detected, namely the target antigen in the serum, is combined with the first antibody coated on the inner surface of the well through diffusion, then the liquid in the well is poured off, the residual substance in the well which is not combined with the first antibody is washed away by using a washing solution, a second antibody which is combined with horseradish peroxidase in advance and can be specifically combined with the antigen is added, the second antibody carrying the horseradish peroxidase meets the combined complex of the target antigen and the first antibody, and (3) combining the complex, washing away the surplus unbound second antibody, adding an indicating solution containing a horseradish peroxidase catalytic substrate, carrying out reactions such as color development, luminescence and the like on the horseradish peroxidase catalytic substrate, and judging the existence of the target antigen in the serum or calculating the concentration titer by visual inspection, specific wavelength light absorption or fluorescence analysis and the like. Based on this principle, reagent companies have widely produced a wide variety of ELISA detection kits based on 96-well plates.

The reaction system of these ELISA kits is present in the wells of a 96-well plate, and usually the well diameter of the 96-well plate is about 0.64cm, and the bottom area is about 0.32cm²Volume 365. mu.l, coating antibody on the surface of the well, and adding the sample into the wellThe product or reagent usually has a volume of not less than two-thirds of the pore volume, wherein the detected substance and the reaction substance can only react with the coating antibody or the conjugate when reaching the inner surface of the pore through diffusion, and the efficiency is not high; the detection is completed by special techniques and personnel, tools and instruments with higher requirements, such as a sample adding gun, a sample adding gun head, a microplate reader, an incubation incubator and the like, and the detection activity also has higher requirements on the laboratory environment.

Therefore, at present, a novel target component detection device and a novel target component detection technology which are convenient to operate, high in reaction efficiency, small in space and dimension of a reaction system and low in requirements on auxiliary equipment and environment need to be researched and developed.

Disclosure of Invention

The present invention has been made in view of the problems of the prior art, and an object of the present invention is to provide a microwire-type detection reaction apparatus capable of loading a probe having specific affinity for a target component, enriching the target component with the probe, and further detecting the target component. The detection reaction device is used for enriching and detecting target components, the space of a reaction system can be reduced, the dimensionality of the reaction system is reduced, reagent and equipment consumption is saved, the cost is reduced, the reaction efficiency is improved, the operation convenience is improved, and the requirements on auxiliary equipment and the environment are reduced.

To this end, the present invention provides, in a first aspect, a microwire type assay reaction device comprising a microwire carrier.

In some embodiments of the invention, the microwire carriers are filaments having a circular or round-like cross-section; the base material of the microwire carrier comprises stainless steel microwires, alloy microwires, metal microwires, glass fibers, quartz fibers, high polymer fibers, natural fibers and carbon fibers; the metal comprises one or more of molybdenum, tungsten, copper and silver; preferably, the diameter of the microwire carrier is 0.1 μm-0.5 mm.

According to the invention, the detection reaction device also comprises a micro-tube cavity which can enable the micro-wire carrier to pass through, and a micro-wire carrier fixing structure and/or a component which are arranged at two ends of the micro-tube cavity; preferably, the inner surface of the micro-tube cavity and the surface of the micro-wire carrier are arranged at equal intervals; further preferably, the pitch is 0.2 μm to 5 mm.

According to some embodiments of the present invention, the microtube cavity is formed by a capillary or by two substrates with grooves and capable of sealing the microtube cavity; preferably, the capillary and the substrate with grooves and capable of synthesizing the substrate of the cavity of the capillary comprise one or more of glass, quartz, mica, silicon compound and high polymer; further preferably, the inner surfaces of the capillary and the groove are smooth surfaces or rough surfaces.

In some embodiments of the invention, the microtube cavity is formed by a capillary tube; preferably, the capillary is a thick walled capillary.

In some embodiments of the invention, the holding structure and/or member of the microwire carrier is an M-shaped frame disposed at both ends of the capillary tube; still further preferably, the two ends of the capillary cavity are arranged to be reduced in diameter, and one or more through-going channels are arranged on the side wall of the capillary near the edge of the reduced diameter portion to serve as fluid channels.

In some embodiments of the present invention, the fixing structure and/or the component of the microwire carrier is a capillary cap disposed at two ends of the capillary, a through hole is disposed at a top center of the capillary cap for fixing the microwire carrier, and one or more through holes are disposed between the top center and the edge of the capillary cap for serving as a fluid channel.

In some embodiments of the present invention, the fixing structure and/or member of the microwire carrier is a capillary cap disposed at two ends of the capillary tube, the top center of the capillary cap is provided with a through hole for fixing the microwire carrier, and the side wall of the capillary tube near the edge of the capillary cap is provided with one or more through holes for serving as a fluid channel.

In some embodiments of the present invention, the fixing structure and/or member of the microwire carrier is formed by changing the shape of the end of the microwire carrier at two ends of the micro-tube cavity to form an expanded, flat, wing-like or multi-wing arrow-tail shape, and the deformed width of the microwire carrier exceeds the inner diameter of the micro-tube cavity, so that the deformed portion is clamped at two ends of the micro-tube cavity, and the microwire carrier in the extended state is fixed.

According to other embodiments of the present invention, the microtube cavity is formed by two substrates which are provided with grooves and can be sealed in a matching way to the synthesized microtube cavity.

In some embodiments of the present invention, the groove is a through linear groove, and the fixing structure and/or the member of the microwire carrier is a fastener arranged at the outer side of two ends of the groove;

in some embodiments of the present invention, the grooves are right-angled zigzag or pi-shaped grooves, and after two substrates with grooves and capable of sealing the synthesized micro-tube cavity are closed, two through holes are formed on the side walls of the two ends of the micro-tube cavity formed along the length direction, respectively, for serving as fluid channels; the fixing structure and/or the component of the microfilament carrier are reducing clamping grooves or outer side buckles arranged at two ends of the groove.

According to the invention, the microwire carrier is a pretreated microwire carrier, and the pretreatment comprises one or more of polishing, cleaning by a cleaning agent, acid treatment, alkali treatment and surface roughening of the surface of the microwire carrier; preferably, the microwire carrier is a coating film with an affinity microstructure on the surface and/or a microwire carrier with an activating group on the surface; further preferably, the activating group comprises one or more of hydroxyl, carboxyl, amino and amide.

In a second aspect, the present invention provides the use of a microwire-type detection reaction apparatus according to the first aspect of the invention for detecting a target component.

In the present invention, the application includes:

a, loading a probe with specific affinity to a target component on a microwire carrier to obtain the microwire carrier loaded with the probe;

and step B, enriching the target component by using the microwire carrier loaded with the probe, and detecting or otherwise applying the target component.

According to some embodiments of the invention, the step B comprises:

step I, fixing two ends of the microwire carrier loaded with the probes on fixing devices of the microwire carrier respectively to obtain an extended microwire carrier loaded with the probes;

step J, fully contacting the stretched microwire carrier loaded with the probe with a sample to be detected to enrich the target component on the microwire carrier, so as to obtain the microwire carrier with the target component enriched on the surface;

and step K, detecting or otherwise applying the target components enriched on the microfilament carrier in the step J.

In some embodiments of the invention, step K comprises: sequentially contacting the microfilament carrier with the target components enriched on the surface with a washing liquid, an indicator and an auxiliary reagent, and directly or indirectly detecting the target components in a visual inspection or instrument measurement mode;

in other embodiments of the present invention, step K comprises: eluting, recovering, detecting or otherwise applying the enriched target components on the microwire carrier.

According to some further embodiments of the invention, the step B comprises:

step L, enabling the microwire carrier loaded with the probe to penetrate through the micro-tube cavity, and fixing two ends of the microwire carrier on the fixed structures or the components of the microwire carrier at two ends of the micro-tube cavity respectively to obtain the micro-tube cavity with the microwire carrier loaded with the probe in the tube cavity;

step M, enabling a sample to be detected to flow through a micro-tube cavity containing a micro-wire carrier loaded with a probe, and fully contacting the micro-wire carrier loaded with the probe, so that a target component is enriched on the micro-wire carrier, and obtaining the micro-tube cavity enriched with the target component on the surface of the micro-wire carrier;

and step N, detecting or otherwise applying the target components enriched on the microfilament carrier in the step M.

In some embodiments of the invention, the step N comprises: sequentially flowing the washing liquid, the indicator and the auxiliary reagent into a micro-tube cavity containing a micro-wire carrier with the surface enriched with target components, sequentially contacting the micro-wire carrier, and directly or indirectly detecting the target components in a visual or instrument measurement mode;

in other embodiments of the present invention, the step N comprises: and taking the microwire out of the cavity of the microtube, and eluting, recovering, detecting or otherwise applying the target component enriched on the microwire.

In the invention, the probe comprises one or more of an affinity substrate, a group of the affinity substrate and an affinity microstructure; the affinity substrate comprises one or more of antibodies, receptors, ligands, biotin, avidin, lectin, antigen, hapten, polynucleotide fragments, peptide chains, complements, cytokines, oligosaccharides, polysaccharides, enzymes and hormones.

In some embodiments of the invention, the affinity microstructures include carbon chain structures and/or hole structures, spatial conformations with specific binding capacity.

In the invention, the sample to be detected comprises one or more of blood, plasma, serum, tissue fluid, cerebrospinal fluid, milk, saliva, urine, secretion extracting solution, tissue extract, biochemical analysis sample, culture solution, fermentation liquor, water body sample, industrial fluid and food, feed, medicine, veterinary medicine, pesticide and extract to be detected for target components.

The invention has the following beneficial effects:

the microwire type detection reaction device provided by the invention can load the probe with specific affinity to the target component, enrich the target component through the probe and further detect the target component. The detection reaction device is used for enriching and detecting target components, the space of a reaction system can be reduced, the dimensionality of the reaction system is reduced, reagent and equipment consumption is saved, the cost is reduced, the reaction efficiency is improved, the operation convenience is improved, and the requirements on auxiliary equipment and the environment are reduced.

Drawings

For the present invention to be readily understood, the following description is made with reference to the accompanying drawings.

Fig. 1 is a schematic view of a micro-tube cavity containing a probe-loaded microwire carrier, the micro-tube cavity being formed by a capillary.

Fig. 2 is a schematic view of a capillary cap secured to the end of a capillary tube.

Figure 3 is a schematic illustration of a grooved substrate that can be mated to seal a synthetic microtube chamber.

FIG. 4 is a schematic cross-sectional view of a microtube chamber containing a microwire carrier loaded with probes, said chamber being formed by sealing substrates with grooves in apposition.

Fig. 5 is a schematic diagram of the deformation fixation of the microwire carrier ends at the two ends of the microtube cavity.

FIG. 6 is a schematic representation of a microwire carrier.

Fig. 7 is a schematic view of batch assembly of the microwire carriers.

FIG. 8 is a schematic view of a continuous assembly of microwire carriers.

The above schematic drawings are for reference only, wherein like reference numerals or numerals indicate like or similar meanings or functions, and wherein the reference numerals are as follows: 1 a microfilament carrier; 10 microwire carrier base stock; 11 finished product of microfilament carrier; 14, buckling; 2, a capillary tube; 20 micro-tube cavities; 21 capillary outer wall; 22 capillary inner wall (inner wall of micro-tube cavity); a 24M-shaped frame; 25 a capillary cap; 252 capillary cap outer edges; 253 a second through-channel (disposed between the top center and the rim of the capillary cap); 254 a first through-channel (arranged in the center of the top of the capillary cap); 3 a substrate; 30 microtube cavities; 31 grooves; 312 the first pore canal communicated with the micro-tube cavity; 313 a second pore canal communicated with the micro-tube cavity; the inner wall of the cavity of the 32 micro-tube; 33, buckles (arranged on the outer sides of the two ends of the groove); 34 reducing neck (arranged at two ends of the groove); 26 a deformed portion of the microwire carrier at the end; 4, hollowing out the line roller; 41 hollowing out the central shaft of the line roller; 42 a radial cross-shaped stent; 43 supporting the edge; 44 a rectangular bracket; 5 a microfilament carrier preparation device; 51 an I base line roller; 52 a second baseroll; 53 assembling the rollers; 54 a drying roller; 55, a take-up roll; 57 a drying device; 6, a container; 61 treating the cell.

Detailed Description

In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to the appended drawings. 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.

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

Term (I)

The term "knot" as used herein refers to a button or knot that is fastened or secured for fastening or securing.

Embodiments II

As described above, the reaction system of the conventional ELISA well plate kit is present in the wells of a 96 well plate, and the well diameter of the 96 well plate is usually about 0.64cm, and the bottom area is about 0.32cm²The volume is 365 microliter, the antibody is coated on the inner surface of the hole, the sample or reagent added into the hole is usually not less than two thirds of the hole volume, and the detected substance can react with the coated antibody or the conjugate only when reaching the inner surface of the hole through diffusion; the detection is completed by special techniques and personnel, tools and instruments with higher requirements, such as a sample adding gun, a sample adding gun head, a microplate reader, an incubation incubator and the like, and the detection activity also has higher requirements on the laboratory environment. In view of this, the present inventors have conducted extensive studies on the structure and operation technique of the well plate kit.

The inventor researches and designs and finds that a microfilament is used as a carrier for loading a probe with specific affinity to a target component, and the probe is used for specifically selecting and enriching the target component and further displaying and detecting the target component or desorbing the target component for detection; the present invention was thus obtained.

Accordingly, in a first aspect, the present invention provides a microwire-type assay reaction device. Although the microwire type detection reaction device is invented for overcoming the defects of an ELISA (enzyme-linked immunosorbent assay) pore plate kit at first, the microwire type detection reaction device is not limited to the conventional detection items for detecting the traditional ELISA pore plate kit, and can also be applied to all the devices capable of carrying out specific selective enrichment and detection on target components by probes with specific affinity on the target components; wherein, the target component comprises one or more of antibodies, receptors, ligands, biotin, avidin, agglutinin, antigens, haptens, polynucleotide fragments, peptide chains, complements, cytokines, oligosaccharides, polysaccharides, enzymes and hormones which may exist in a sample to be detected.

The microwire type detection reaction apparatus of the present invention is mainly composed of a microwire carrier, and it is understood from the above that the microwire carrier, which is a main component of the microwire type detection reaction apparatus of the present invention, can be used as a reaction substrate, and probes having specific affinity for a target component can be loaded on the surface of the reaction substrate, and the target component can be further enriched and detected by the probes loaded on the surface.

Specifically, the microwire carrier with the surface loaded with the probe with specific affinity for the target component has the function of separating or adsorbing the affinity target component from the fluid flowing through or on or near the surface of the microwire carrier, when a sample to be detected is filled or flows through the surface of the microwire carrier, the target component is adsorbed or attracted to the surface of the microwire carrier or near the microwire carrier by the probe on the surface of the microwire carrier to realize enrichment or separation, and then detection can be realized in one or more modes of color development, luminescence, recombination of a marking substance, enzymatic reaction, spectrum, mass spectrum, magnetic spectrum, ray, photoelectric signal and the like.

According to some embodiments of the present invention, the microwire type detection reaction apparatus comprises a microwire carrier which is a filament having a circular or quasi-circular cross-section.

In the invention, the substrate of the microwire carrier comprises stainless steel microwires, alloy microwires, metal microwires, glass fibers, quartz fibers, high polymer fibers, natural fibers and carbon fibers; wherein the metal comprises one or more of molybdenum, tungsten, copper and silver.

In some embodiments of the invention, the microwire carrier has a diameter of between 0.1 micron and 0.5 mm.

In some embodiments of the present invention, the reaction device for detecting a target component is used by loading a probe on the above microwire carrier, and then placing the microwire carrier loaded with the probe in a stretched state so that it becomes a stretched filament.

The fixing device for fixing the microwire carrier in the present invention is not particularly limited as long as the microwire carrier can be placed in a stretched state and suspended in the middle of the cavity of the micro-tube, for example, the fixing device for fixing the microwire carrier can adopt a bracket with a clamping device, the bracket and the clamping device can be a bracket and a clamping device which are conventional in the art, and the microwire carrier can be fixed on the fixing device of the microwire carrier by one or more of sintering, clamping, bonding, knotting and binding.

According to some further embodiments of the present invention, the detection reaction device further comprises a micro-tube cavity capable of allowing the micro-wire carrier to pass through, and a micro-wire carrier fixing structure and/or member disposed at two ends of the micro-tube cavity.

The micro-tube cavity can ensure that a sample to be detected, washing liquor, an indicator, an auxiliary reagent and the like are sucked between the inner wall of the micro-tube cavity and the micro-wire carrier through negative pressure suction, positive pressure push or a capillary mechanism. In the present invention, there is no particular limitation on the channels for the inflow and outflow of the sample and the reagent at both ends of the microtube chamber, as long as the sample and the reagent can flow in from one end and flow out from the other end.

In the invention, the inner surface of the micro-tube cavity and the surface of the micro-wire carrier are arranged at equal intervals (namely, are arranged in the middle); preferably, the spacing is between 0.2 microns and 5 millimeters.

Specifically, the diameter of the inner cavity of the micro-tube cavity is larger than that of the micro-wire carrier, and the fixing structures and/or members of the micro-wire carrier are arranged at two ends of the micro-tube cavity, so that the micro-wire carrier can be centrally suspended in the center of the micro-tube cavity (namely, centrally arranged), a gap is formed between the inner wall of the micro-tube cavity and the surface of the micro-wire carrier, namely, the inner surface of the micro-tube cavity and the surface of the micro-wire carrier are arranged at equal intervals, and the fluid of a sample to be detected and a reagent can be filled or pass through the gap.

The microwire carrier with the surface loaded with the probe with specific affinity for the target component has the function of separating or adsorbing the affinity target component from fluid flowing through or on the surface of or nearby the microwire carrier, when a sample to be detected is filled or flows through a micro-tube cavity containing the microwire carrier, the target component is adsorbed or attracted to the surface of the microwire carrier or nearby the microwire carrier by the probe on the surface of the microwire carrier to realize enrichment or separation, and detection can be realized through one or more modes of color development, luminescence, recombination of a marking substance, enzymatic reaction, spectrum, mass spectrum, magnetic spectrum, ray, photoelectric signal and the like.

According to some embodiments of the present invention, the outer surface of the detection reaction device of the present invention may be additionally provided with a prism-shaped edge, a scale and an opposite background substrate, so as to facilitate visual reading of the reaction result.

In some specific embodiments of the present invention, the detection reaction device is shown in fig. 1, fig. 2 and fig. 5, and the microtube cavity 20 is formed by a capillary 2. It is understood that the microtube cavity 20 is directly provided by the capillary 2, i.e. the cavity of the capillary 2 is the microtube cavity 20.

Specifically, the microwire carrier is fixed and extended by a fixing structure and/or a component of the microwire carrier 1 connected to two ends of the capillary 2, the microwire carrier is centrally suspended in the center of the micro-tube cavity 20 of the capillary, an annular fluid channel is formed between the microwire carrier 1 and the inner wall 22 of the micro-tube cavity 20 of the capillary, and the fluid of the sample to be detected and the reagent fills or passes through the gap.

In other embodiments of the present invention, the detection reaction device is shown in fig. 3 and 4, and the microtube cavity 30 is formed by two substrates 3 with grooves and can be sealed to form the microtube cavity.

In the invention, the base materials of the capillary tube and the substrate with the groove and capable of synthesizing the cavity of the capillary tube comprise one or more of glass, quartz, mica, silicon compound and high polymer.

The inventors have studied and found that when the inner surfaces of the capillary and the groove (of the substrate) are treated with a smooth surface or a rough surface, the loading amount of the probe and the enrichment rate and the enrichment amount of the target component can be increased, and the sensitivity of detection can be improved.

In some particularly preferred embodiments of the invention, the microtube cavity is formed by a capillary tube; preferably, the capillary is a thick-walled capillary, and the fixing structure and/or the component of the microwire carrier is arranged at two ends of the capillary.

In the invention, the microwire carrier is fixed on the fixed structure and/or the component of the microwire carrier in one or more modes of sintering, clamping, bonding, buckling and binding.

In some preferred embodiments of the present invention, the detection reaction device is shown in fig. 1, and the fixing structure and/or member of the microwire carrier is an M-shaped frame 24 fixed at both ends of the capillary; preferably, the two ends of the capillary cavity are arranged to be reduced in diameter, and one or more through-holes (not shown) are arranged on the side wall of the capillary near the edge of the reduced diameter part and used as a fluid channel.

In the invention, the inner diameter formed by necking is slightly larger than or equal to the diameter of the microwire carrier, and the length is not particularly required.

In some particularly preferred examples, the detection reaction device is shown in fig. 1, and as can be seen from fig. 1, the detection reaction device of the present invention comprises a microwire carrier 1 and a microtube cavity 20; the micro-tube cavity 20 is directly provided by the capillary tube 2, that is, the cavity of the capillary tube 2 is the micro-tube cavity 20; the fixing structure and/or the component of the microwire carrier is an M-shaped frame 24 connected with the two ends of the capillary tube 2; the microwire carrier 1 is tensioned and fixed by the buckles 14 on the M-shaped frame 24 connected with the two ends of the capillary 2, is suspended in the center of the micro-tube cavity 20 of the capillary, and forms an annular fluid channel between the microwire carrier 1 and the inner wall 22 of the micro-tube cavity 20 of the capillary, and fluid of a sample to be detected and a reagent is filled in or passes through the channel.

In other preferred embodiments of the present invention, the holding structure and/or member of the microwire carrier is a capillary cap 25 disposed at both ends of the capillary tube.

In some specific embodiments of the present invention, the detection reaction device is shown in fig. 2, the fixing structure and/or component of the microwire carrier is a capillary cap 25 disposed at two ends of the capillary, a first through hole 254 is disposed at the top center of the capillary cap 25 for fixing the microwire carrier 1, and one or more second through holes 253 disposed between the top center and the edge of the capillary cap for fluid passage.

In some particularly preferred examples, the detection reaction device is shown in fig. 2, and as can be seen from fig. 2, the detection reaction device of the present invention comprises a microwire carrier 1, a microtube cavity 20; the micro-tube cavity 20 is directly provided by the capillary tube 2, that is, the cavity of the capillary tube 2 is the micro-tube cavity 20; the fixing structure and/or the component of the microwire carrier is a capillary cap 25, the top center of the capillary cap 25 is provided with a first through hole 254 for fixing the microwire carrier 1, and one or more second through holes 253 for serving as fluid channels are also arranged between the top center of the capillary cap and the edge 252; the microwire carrier 1 is fixed through the I-th through hole 254 at the top center of the capillary cap 25 arranged at two ends of the capillary 2, is tensioned and fixed through the knot 14 at the outlet position of the I-th through hole 254 at the top center of the capillary cap 25, is suspended in the center of the micro-tube cavity 20 of the capillary, forms an annular fluid channel between the microwire carrier 1 and the inner wall 22 of the micro-tube cavity 20 of the capillary, and is filled with or passes through fluid of a sample to be detected and a reagent.

In other specific embodiments of the present invention, the fixing structure and/or member of the microwire carrier is a capillary cap disposed at two ends of the capillary tube, the top center of the capillary cap is provided with a through hole for fixing the microwire carrier, and the side wall of the capillary tube near the edge of the capillary cap is provided with one or more through holes for fluid passage (not shown in the figure).

In some particularly preferred examples, the detection reaction device of the present invention comprises a microwire carrier 1, a microtube cavity 20; the micro-tube cavity 20 is directly provided by the capillary tube 2, that is, the cavity of the capillary tube 2 is the micro-tube cavity 20; the fixing structure and/or the component of the microwire carrier is a capillary cap 25, the center of the top of the capillary cap 25 is provided with a through hole channel for fixing the microwire carrier 1, and the side wall of the capillary close to the edge of the capillary cap is provided with one or more through hole channels for serving as a fluid channel; the microwire carrier 1 is fixed through a through hole channel arranged at the top center of a capillary cap 25 at two ends of a capillary 2, is tensioned and fixed through buckling at an outlet position of the through hole channel at the top center of the capillary cap 25, is centrally suspended at the center of a micro-tube cavity 20 of the capillary, forms an annular fluid channel between the microwire carrier 1 and an inner wall 22 of the micro-tube cavity 20 of the capillary, and is filled with or passes through fluid of a sample to be detected and a reagent.

In other specific preferred embodiments according to the present invention, the detection reaction device is shown in fig. 3 and 4, and the microtube cavity 20 is formed by two substrates 3 with grooves and can be sealed to form the microtube cavity.

In some embodiments of the present invention, the groove is a through-going in-line groove 31, and the fixing structure and/or member of the microwire carrier 1 is a snap 33 disposed at the outer side of the two ends of the groove.

In some embodiments of the present invention, the grooves are right-angled zigzag or pi-shaped grooves, and after two substrates with grooves and capable of sealing the synthesized micro-tube cavity are closed, two through holes are formed on the side walls of the two ends of the micro-tube cavity formed along the length direction, respectively, for serving as fluid channels; the fixing structure and/or the component of the microfilament carrier are reducing clamping grooves 34 or outer side buckles 33 arranged at two ends of the groove.

In some particularly preferred examples, the detection reaction device is shown in fig. 3 and 4, and as can be seen from fig. 3 and 4, the detection reaction device of the present invention comprises a microwire carrier 1, a microtube cavity 20; the micro-tube cavity 20 is formed by sealing two substrates 3 which are provided with grooves 31 and can be combined into a micro-tube cavity. The groove 31 is a right-angle Z-shaped groove, two pieces of substrates which are provided with grooves and can be combined and sealed with the synthesized micro-tube cavity respectively form two pore channels (312 and 313) which are communicated with the micro-tube cavity on the side walls of two ends of the micro-tube cavity 30 formed along the length direction and are used as fluid channels; the fixing structure and/or the component of the microfilament carrier are reducing clamping grooves 34 or outer side buckles 33 arranged at two ends of the groove; the microwire carrier 1 is tensioned and fixed through reducing clamping grooves 34 or outer side buckles 33 arranged at two ends of a micro-tube cavity 30 formed along the length direction, and is centrally suspended in the center of the micro-tube cavity 30, an annular fluid channel is formed between the microwire carrier 1 and the inner wall 32 of the micro-tube cavity 30, and fluid of a sample to be detected and a reagent fills or passes through the channel.

In some specific embodiments of the present invention, the detection reaction device is shown in fig. 5, and as can be seen from fig. 5, the detection reaction device of the present invention comprises a microwire carrier 1, a microtube cavity 20; the micro-tube cavity 20 is directly provided by the capillary tube 2, that is, the cavity of the capillary tube 2 is the micro-tube cavity 20; the fixing structure and/or the component of the microwire carrier is that the shape of the two ends of the microtube cavity 20 is changed, the deformed width exceeds the inner diameter of the microtube cavity through the deformation part 26 at the end part, so that the deformation part is clamped at the two ends of the microtube cavity, the microwire carrier in the stretching state is centrally suspended in the center of the microtube cavity 20 of the capillary, an annular fluid channel is formed between the microwire carrier 1 and the inner wall 22 of the microtube cavity 20 of the capillary, and the fluid of the sample to be detected and the reagent is filled in or passes through the channel.

Based on the above, it can be understood that the microwire carrier, which is the main component of the detection reaction device according to the first aspect of the present invention, is actually a reaction substrate, and therefore, a probe with specific affinity for a target component can be loaded on the surface of the reaction substrate layer by layer, and the target component can be enriched, detected or used for other reaction applications, for example, as a reaction substrate for performing a micro-assembly reaction.

Accordingly, the second aspect of the present invention provides the use of a microwire-type detection reaction apparatus according to the first aspect of the present invention for detecting a target component, which can also be understood as a method for detecting a target component using the microwire-type detection reaction apparatus according to the first aspect of the present invention.

In the present invention, the application includes:

In the invention, the probe comprises one or more of a specific affinity substrate, an affinity group fragment of the affinity substrate and an affinity microstructure; the affinity substrate comprises one or more of antibodies, receptors, ligands, biotin, avidin, lectin, antigen, hapten, polynucleotide fragments, peptide chains, complements, cytokines, oligosaccharides, polysaccharides, enzymes and hormones. The fragments of affinity groups of the affinity substrate include fragments comprising an affinity group in parts of the fragments of the above-mentioned substances, wherein the fragments comprising an affinity group are to be understood as affinity domains in the affinity substrate which make the substrate an affinity substrate.

It will be appreciated by those skilled in the art that the probe and the target component have specific affinity for each other, and thus the probe and the target component are referred to as being relative, for example, an antigen and an antibody having specific affinity for each other, and if the target component to be detected is an antibody, the microwire carrier is loaded with an anti-antibody having specific affinity for the antibody, and so on.

According to the invention, the probe can be directly loaded on the microwire carrier in a physical adsorption mode by depending on the surface energy, can be connected to the surface of the microwire carrier in a bridging mode, and can be loaded on the surface of the microwire carrier in a mode of combining physical adsorption and bridging. In order to realize the loading of the probe, in the step a, the microwire carrier needs to be pretreated to obtain an activated microwire carrier, and then the probe is loaded on the microwire carrier to obtain the microwire carrier loaded with the probe.

The method of pretreatment in the present invention is not particularly limited as long as the surface of the microwire carrier can be cleaned and activated; for example, the pretreatment includes one or more of polishing, cleaning with a cleaning agent, acid treatment, alkali treatment, surface roughening, and the like of the surface of the microwire carrier.

The method for polishing the surface of the microwire carrier in the present invention is not particularly limited, and the surface of the microwire carrier can be polished by a method conventional in the art.

In some embodiments, the cleaning agent comprises one or more of water, methanol, ethanol, acetone, toluene, xylene, surfactants, and the like.

The acid treatment method in the invention is not particularly limited as long as the surface of the microfilament carrier can be cleaned and activated; for example, acid treatment methods conventional in the art may be employed.

Likewise, the alkali treatment method in the present invention is not particularly limited as long as the surface of the microwire carrier can be cleaned and activated; for example, an alkali treatment method which is conventional in the art may be employed.

The method for roughening the surface in the present invention is not particularly limited as long as the surface roughness of the microwire carrier can be increased, and for example, the surface may be treated with blasting and/or roughening agent including one or more of alkaline microetching agent, blasting-effect sand surface agent, acidic white matte agent, acidic matte additive, and the like.

In some embodiments of the present invention, the probe is loaded directly on the microwire carrier by physical adsorption by means of surface coating and surface spraying. Probes loaded in this manner are primarily referred to as affinity microstructures, which include carbon chain structures and/or hole structures, spatial conformations with specific binding capacity.

The surface coating and spraying in the present invention are not particularly limited as long as a coating film having an affinity microstructure can be formed on the surface of the microwire support.

In other embodiments of the present invention, the probes are loaded on the surface of the microwire carrier by combining physical adsorption with bridging.

Specifically, for example, the active groups are first loaded on the surface of the microwire carrier by means of chemical activator treatment, surface coating and surface spraying, and then the probes are directly connected to the active groups by chemical reaction, or the probes are connected to the active groups by means of a cross-linking agent, so that the probes are connected to the surface of the microwire carrier.

In the process of loading the probe on the surface of the microwire carrier by combining physical adsorption and bridging, a chemical activating agent is adopted to directly adsorb the surfactant with active groups on the surface of the microwire carrier through sulfonation, nitration, amination and azide treatment, so that the active groups are connected to the surface of the microwire carrier; the probe is connected with the active group through chemical reaction, so as to be loaded on the surface of the microwire carrier.

Similarly, in the process of loading the probe on the surface of the microwire carrier by adopting the mode of combining physical adsorption and bridging, the surface coating and surface spraying enable the coating film with the active group to be coated on the surface of the metal microwire, so that the active group is connected to the surface of the microwire carrier; the probe is connected with the active group through chemical reaction, so as to be loaded on the surface of the microwire carrier.

The cross-linking agent in the present invention is not particularly limited as long as it can be loaded on the surface of the microwire carrier and further attach the probe to the surface of the microwire carrier through a cross-linking bond, and for example, one or more cross-linking agents conventional in the art, such as glutaraldehyde, polylysine, polysaccharide, and a cross-linking agent containing a thiol group, may be used.

In some preferred embodiments of the present invention, in step a, different probes are loaded on different segments of a microwire carrier, thereby achieving simultaneous enrichment and detection of different target components; of course, a plurality of microwire carriers each carrying one probe can also be used simultaneously for simultaneously enriching and detecting a plurality of target substances.

In other embodiments of the present invention, the sample to be tested comprises one or more of blood, plasma, serum, interstitial fluid, cerebrospinal fluid, milk, saliva, urine, secretion extract, tissue extract, biochemical analysis sample, culture fluid, fermentation fluid, water sample, industrial fluid and food, feed, drug, veterinary drug, pesticide and extract sample in which a target component is to be determined.

According to some embodiments of the present invention, the target component is enriched and detected using a target component detection reaction apparatus mainly composed of a microwire carrier, and the step B includes:

In some specific embodiments of the present invention, the step K includes: the microfilament carrier with the surface enriched with the target components is sequentially contacted with a washing liquid, an indicator and an auxiliary reagent, and the target components are directly or indirectly detected in a visual or instrumental measurement mode.

The indicator in the present invention refers to a reagent which generates a change in a signal such as light, electricity, magnetism, etc. by itself or by acting on other substances after binding to a target component.

In other specific embodiments of the present invention, the step K includes: eluting and recovering the enriched target components on the microfilament carrier, and detecting by using some detection instruments or applying the detection instruments for other applications.

The auxiliary reagent can assist in completing detection, and specifically comprises an anticoagulant, an antagonist, a substrate of an enzyme, a cosolvent, a chelating agent, a blocking agent, a masking agent, a buffer solution, a washing solution and the like.

The detection apparatus in the present invention is not particularly limited as long as it can separate, purify, and detect the target component; for example, the detection instrument may include a luminometer, liquid chromatography, thin layer chromatography, and the like.

In some particularly preferred examples of the invention, the microwire carrier 1 may be wound on a patterned wire roll 4, as shown in fig. 6. Fig. 7 shows the use state of the thread roller 4, and as can be seen from fig. 7, the thread roller 4 is a hollow regular octagonal prism, and is composed of a central shaft 41, radial cross-shaped brackets 42 arranged at both ends of the prism, a supporting edge 43, and a rectangular bracket 44 connected with the central shaft 41 of the thread roller 4 and having an opening at the lower part. In use, the carrier 44 is manipulated to immerse the wire roll with the carrier 1 wound thereon in the container 6 for batch assembly.

In other specific preferred examples of the present invention, the microwire carrier 1 may be prepared using a microwire carrier preparing apparatus 5 shown in fig. 8. As can be seen from fig. 8, the microwire carrier preparation device 5 comprises an i base line roller 51, an ii base line roller 52, one or more assembly rollers 53, one or more drying rollers 54, one or more processing pools 61, one or more drying devices 57, and a take-up roller 55, which are arranged in sequence, wherein the assembly rollers 53 are arranged at the bottom of the processing pools 61, and the drying devices 57 are arranged before the drying rollers 54 and close to the drying rollers 54; the first base line roller 51 is wound with the microwire carrier raw material 10, the microwire carrier raw material 10 passes through the first base line roller 52 to the assembly roller 53, is dried by the drying device 57 after reaction and assembly in the treatment tank 61, and then enters the next treatment tank … … through the drying roller, passes through the treatment tanks one by one, and is dried by the drying device 57 after assembly of the microwire carrier is completed, and the obtained microwire carrier finished product 11 is rolled up through the take-up device. Thereby, the assembling process of the microfilament carrier can be continuously carried out.

According to other embodiments of the present invention, a target component detection reaction apparatus mainly composed of a microwire carrier and a micro cavity is adopted to enrich and detect a target component, and the step B includes:

step L, enabling the microwire carrier loaded with the probes to penetrate through the micro-tube cavity, and fixing two ends of the microwire carrier on the fixed structures or the components of the microwire carrier at two ends of the micro-tube cavity respectively to obtain the micro-tube cavity containing the microwire carrier loaded with the probes;

step M, enabling a sample to be detected to flow through a micro-tube cavity containing a micro-wire carrier loaded with a probe, and fully contacting the micro-wire carrier loaded with the probe, so that a target component is enriched on the micro-wire carrier, and obtaining the micro-tube cavity containing the micro-wire carrier with the surface enriched with the target component;

In some specific embodiments of the present invention, the step N includes: the washing liquid, the indicator and the auxiliary reagent sequentially flow into a micro-tube cavity containing the microwire carrier with the surface enriched with the target component, and the micro-tube cavity is sequentially contacted with the microwire carrier, so that the target component is directly or indirectly detected in a visual or instrumental measurement mode.

In other specific embodiments of the present invention, the step N includes: and taking the microwire out of the cavity of the microtube, and eluting, recovering, detecting or otherwise applying the target component enriched on the microwire.

In some specific embodiments of the present invention, the target component is enriched and detected by using a target component detection reaction apparatus mainly composed of a microwire carrier and a microtube cavity formed by a capillary, and the step B includes:

step S1, the microwire carrier loaded with the probe passes through the cavity of the micro-tube, and the two ends of the microwire carrier are respectively fixed on the fixed structure and/or the component of the microwire carrier at the two ends of the capillary tube, so as to obtain the capillary tube containing the microwire carrier loaded with the probe in the cavity;

step S2, adding the capillary containing the microwire carrier loaded with the probe into a sample to be detected, enabling the sample to enter a micro-tube cavity of the capillary containing the microwire carrier loaded with the probe and fully contacting the microwire carrier loaded with the probe, so that the target component is enriched on the microwire carrier, and obtaining the capillary with the target component enriched on the surface of the microwire carrier;

and step S3, detecting or otherwise applying the target components enriched on the microwire carrier in the step S2.

In some specific embodiments of the present invention, the step N includes: and sequentially adding washing liquor, an indicator, an auxiliary reagent and the like into the capillary containing the microwire carrier with the surface enriched with the target component to enable the capillary to be in contact with the microwire carrier, and detecting the target component in a visual inspection or instrument detection mode after the surface of the microwire carrier develops color or emits light.

In other specific embodiments of the present invention, the step N includes: and taking the microfilament out of the capillary, and eluting, recovering, detecting or otherwise applying the target component enriched on the microfilament.

In some specific embodiments of the present invention, a target component detection reaction apparatus mainly composed of a microwire carrier and a micro-cavity formed by sealing two substrates with involutory microtube grooves in an involutory manner is used to enrich and detect a target component, and the step B includes:

step T1, passing or placing the microwire carrier loaded with the probe into the micro-tube groove of one substrate, closing the other substrate, and fixing the two ends of the substrate on the fixing structures and/or the components (such as buckles) of the microwire carrier at the two ends of the micro-tube cavity respectively to obtain the micro-tube cavity containing the microwire carrier loaded with the probe;

step T2, adding a sample to be detected into the micro-tube cavity containing the micro-wire carrier loaded with the probe, enabling the sample to enter the micro-tube cavity containing the micro-wire carrier loaded with the probe and fully contacting the micro-wire carrier loaded with the probe, so that the target component is enriched on the micro-wire carrier, and obtaining the micro-tube cavity containing the micro-wire carrier with the surface enriched with the target component;

and step T3, detecting or otherwise applying the target components enriched on the microwire carrier in the step T2.

In some specific embodiments of the present invention, the step N includes: sequentially adding washing liquid, indicator, auxiliary reagent and the like into a micro-tube cavity containing the micro-wire carrier with the surface enriched with the target component to enable the micro-tube cavity to be in contact with the micro-wire carrier, and detecting the target component in a visual inspection or instrument detection mode after the surface of the micro-wire carrier develops color or emits light.

The invention adopts the microfilament carrier to load the affinity reagent, such as the first antibody, because the diameter of the microfilament carrier is usually micron-sized, the specific surface area is large, which is beneficial to loading the affinity substance with high efficiency, the microfilament carrier can be continuously manufactured in a whole strip, the batch production is stable, the raw material is saved, and the quality is improved; the reaction system is in the capillary cavity near the surface of the microwire carrier, the spatial dimension is greatly reduced, the reaction system can be similar to a one-dimensional space, the mass transfer efficiency is accelerated while the reagent consumption is reduced, the reaction efficiency is improved, the detection timeliness is improved, and the detection sensitivity is improved; the sample volume can be adjusted to increase, thereby reducing the detection limit; and the convenience is improved, the requirements of the environment and the matched device instrument are properly reduced, and the detection result can be visually displayed like a mercury (alcohol) thermometer.

Example (b):

the present invention will be specifically described below with reference to specific examples. The experimental methods described below are, unless otherwise specified, all routine laboratory procedures. The experimental materials described below, unless otherwise specified, are commercially available.

Example 1:

the invention adopts a batch production mode to prepare the detection tube for detecting the HIV antibody in the serum and the detection method.

Firstly, preparing the microfilament carrier for loading the antigen by adopting a batch production method. 316L stainless steel microwire (S) was selected, 50 microns in diameter. As shown in fig. 6 and 7, the polished S is wound on the microwire carrier winding stent (B) while maintaining the interval between the wires not less than 1 mm. Immersing the B wound with the S into a treatment solution mixed by a hydrochloric acid pool of 20 percent and an equal proportion of 15 percent, and keeping the S suspended in the solution for acid etching for 2 hours at 45 ℃; taking out, respectively cleaning in pure water and 75% ethanol pools for 15min under a 100khz ultrasonic machine, taking out, and blow-drying with nitrogen gas to form a fine-pore rough surface on the S, so that the S can be used for loading the antigen.

And secondly, loading the acquired HIV antigen produced by a commercial genetic engineering method onto a microfilament carrier. Spraying HIV antigen protein (200 μ g. mL) diluted with 5mmol/L PBS onto the surface of the substrate B^-1) The surface was wetted and incubated at 37 ℃ for 30min in a humid environment. Then, the mixture was placed in a 10 mmol.L apparatus containing 0.2% Tween-20^-1Washing in phosphate buffered saline (PBS, pH7.4) bath is repeated three times, then soaking B in 0.5% NaCl solution containing 100 μ g/mL bovine serum albumin, sealing the gap without HIV antigen adsorbed thereon at 37 deg.C for 30min, and taking out and drying.

And thirdly, manufacturing a 10 cm detection tube. The capillaries were pretreated for use, each capillary was perfused with a 0.5% NaCl solution containing 100. mu.g/mL BSA to block potential active sites, and 20. mu.L of ELISA general wash solution was injected into each tube to wash away excess BSA after incubation at 37 ℃ for 30 min. Cutting S wound on the B and adsorbed with the HIV antigen protein into a straight section of 11cm according to the required length, vertically suspending and hanging the straight section on a cantilever of a 3D operation table, vertically fixing a capillary tube with the inner diameter of 300 microns and the length of 10 cm beside the operation table, adjusting and calibrating to enable the tail end of the microwire to be opposite to the opening of the capillary tube, descending the cantilever, and sliding the microwire carrier into the capillary tube. Fixing the microwire carrier at two ends of the capillary tube by using a buckle device, and suspending the microwire carrier at the center of the capillary tube to obtain the detection tube for detecting the HIV antibody in serum.

The detection method is explained next. Anticoagulating a blood sample to be detected, centrifugally separating serum of the sample to be detected, and preparing a standard sample series by using a human anti-HIV antibody standard substance with known concentration. The detection tubes are numbered in sequence according to the needs of the experiment, 10 mu L of serum sample and standard series sample are respectively injected from the inlets of the detection tubes by using a micro-injector, then 20 mu L of ELISA universal washing liquid is injected, then fluorescence labeled antibody is injected, 10 mu L of FITC (fluorescein isothiocyanate) labeled rabbit anti-human IgG antibody with the concentration of 0.2 mu g/mL is injected, 20 mu L of ELISA universal washing liquid is injected again, the redundant fluorescein labeled antibody is washed off, the detection tubes are placed in a cassette, and the detection tubes are observed under the ultraviolet irradiation. Fitting a regression equation according to the length of the color development section of the standard series detection tube, drawing a standard curve, and calculating the titer of the anti-AIDS virus antibody in the blood sample according to the color development length of the sample detection tube.

Example 2:

the invention adopts a continuous production mode to prepare the whole-axis microwire carrier (no-load wire) without loading the probe, and is used for manufacturing the detection tube for detecting the hepatitis B virus surface antigen in the serum.

Firstly, the method adopts a continuous production mode to prepare the unloaded yarn of the whole shaft. As shown in FIG. 8, 316L stainless steel microwires (S) having a diameter of 50 μm were selected. And mounting the polished whole shaft S (weighing about 100g and having a length of about 6km) on a rotatable shaft lever R1, passing the microfilament through the rotating shafts of the processing tanks V through leads, adjusting the rotating speed, keeping the advance speed of the microfilament at 1m/min, and finishing the surface treatment of each link in a hot air drying mode during the period to form continuous processing production. The first step is a pretreatment link, pure water is injected into a V1 pool, ultrasonic waves are loaded on the liquid surface with the depth of 30cm, and S is cleaned; then the mixture enters a V2 pool filled with 75% ethanol, the liquid level is kept at 30cm, ultrasonic waves are loaded, the mixture is cleaned and dried, and the dried mixture enters a V3 pool, wherein the mixed titanium dioxide-based material (tetraethyl orthotitanate (TEOT) and ethanol (C) are filled₂H₅OH) preparing TEOT, wherein the molar ratio of C2H5OH is 1:44, adding 36% hydrochloric acid, adjusting the pH value to 2, performing acid hydrolysis for 2 hours at 45 ℃, keeping the liquid level of 40cm deep, performing blow-drying after liquid level is reached, forming a titanium dioxide-based coating on the surface of the microwire carrier, then entering a V4 pool, filling 0.1M ethanol solution of (3-aminopropyl) triethoxysilane (APTS) into the V4 pool, keeping the liquid level at 75 ℃, keeping the liquid level height at 40cm, introducing amino groups on the surface of the microwire carrier, performing blow-drying after liquid level is reached, entering a V5 pool, performing immersion-washing with 30cm deep ethanol, entering a V6 pool, performing immersion-washing with 30cm deep pure water, performing blow-drying after liquid level is reached, taking a scroll, performing vacuum sealing packaging after the whole scroll is completed, performing low-temperature cold storage, and preparing the microwire for loading a probeWire carrier (unloaded wire).

Next, a test tube for detecting hepatitis B virus surface antigen in serum was prepared by pretreating the capillary tube according to the method of example 1, and then loading the microwire carrier (idle wire) into the pretreated capillary tube. Then, a probe is loaded on a microwire carrier (idle wire), a single-chain fragment of a commercially available antibody of anti-hepatitis B virus surface antigen expressed by escherichia coli is diluted to 60 mu g/mL by 5mmol/L PBS, 20 mu L of diluent is injected into each capillary, incubation is carried out at 37 ℃ for 30min, so that the antibody is fully combined with amino groups on the surface of the microwire carrier, then 20 mu L of ELISA universal washing liquid is injected into each tube to wash off redundant antibody, then 20 mu L of 0.5% NaCl solution containing 100 mu g/mL bovine serum albumin is injected into each tube, incubation is carried out at 37 ℃ for 30min, potential active sites which are not combined with the antibody are blocked, and 20 mu L of ELISA universal washing liquid is injected into each tube to wash off redundant bovine serum albumin, thereby completing the preparation of the detection tube.

The detection method is explained next. Similarly as described in example 1, the test blood sample was anticoagulated, and the test sample serum was centrifuged while preparing a standard sample series with a standard of hepatitis B surface antigen protein at a known concentration. The detection tubes are numbered in sequence according to the needs of the experiment, 10 mu L of serum sample and standard series sample are respectively injected from the inlets of the detection tubes by microinjectors, then 20 mu L of ELISA universal lotion is respectively injected, then fluorescence labeled antibody is respectively injected, 10 mu L of FITC (fluorescein isothiocyanate) labeled rabbit anti-hepatitis B virus surface antigen antibody with the concentration of 0.2 mu g/mL is injected again, 20 mu L of ELISA universal lotion is injected again, the redundant fluorescein labeled antibody is washed off, the detection tubes are placed in a cassette, and the detection tubes are observed under the irradiation of ultraviolet light. Fitting a regression equation according to the length of the color development segment of the standard series detection tube, drawing a standard curve, and calculating the titer of the hepatitis B virus surface antigen in the blood sample according to the color development length of the sample detection tube.

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 with reference to exemplary embodiments, but 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 microwire type detection reaction device comprises a microwire carrier.

2. The microwire-type detection reaction device according to claim 1, wherein said microwire carrier is a filament having a circular or round-like cross-section; the base material of the microwire carrier comprises stainless steel microwires, alloy microwires, metal microwires, glass fibers, quartz fibers, high polymer fibers, natural fibers and carbon fibers; the metal comprises one or more of molybdenum, tungsten, copper and silver; preferably, the diameter of the microwire carrier is 0.1 μm-0.5 mm.

3. The microwire type detection reaction device according to claim 1 or 2, further comprising a microtube chamber capable of passing the microwire carrier therethrough, and fixing structures and/or members of the microwire carrier disposed at both ends of the microtube chamber; preferably, the inner surface of the micro-tube cavity and the surface of the micro-wire carrier are arranged at equal intervals; further preferably, the pitch is 0.2 μm to 5 mm.

4. The microwire type detection reaction device according to claim 3, wherein said micro-tube cavity is formed by a capillary or by two substrates having grooves and being sealed to each other to form a micro-tube cavity; preferably, the capillary and the substrate with grooves and capable of synthesizing the substrate of the cavity of the capillary comprise one or more of glass, quartz, mica, silicon compound and high polymer; further preferably, the inner surfaces of the capillary and the groove are smooth surfaces or rough surfaces.

5. The microwire type detection reaction apparatus according to claim 4,

the micro-tube cavity is formed by a capillary tube; preferably, the capillary is a thick-walled capillary;

further preferably, the fixing structure and/or member of the microwire carrier is an M-shaped frame disposed at both ends of the capillary tube; still further preferably, both ends of the capillary cavity are provided with a reduced diameter, and one or more through-holes are arranged on the side wall of the capillary close to the edge of the reduced diameter part and used as a fluid channel;

and/or the fixing structure and/or the component of the microwire carrier is a capillary cap arranged at two ends of the capillary, the center of the top of the capillary cap is provided with a through hole channel for fixing the microwire carrier, and one or more through hole channels are also arranged between the center of the top of the capillary cap and the edge and are used as fluid channels;

and/or the fixing structure and/or the component of the microwire carrier is a capillary cap arranged at two ends of the capillary, the center of the top of the capillary cap is provided with a through hole channel for fixing the microwire carrier, and the side wall of the capillary close to the edge of the capillary cap is provided with one or more through hole channels for serving as a fluid channel;

and/or the fixing structure and/or the component of the microwire carrier is that the shape of the end parts of the microwire carrier at the two ends of the micro-tube cavity is changed to form an expanded, flat sheet shape, wing shape or multi-wing arrow tail shape, and the width of the deformed microwire carrier exceeds the inner diameter of the micro-tube cavity, so that the deformed part is clamped at the two ends of the micro-tube cavity, and the stretched microwire carrier is fixed.

6. The microwire type detection reaction apparatus according to claim 4,

the micro-tube cavity is formed by sealing two substrates which are provided with grooves and can be combined into a micro-tube cavity;

the groove is a through straight-line-shaped groove, and the fixing structure and/or the component of the microfilament carrier are/is a buckle arranged at the outer sides of two ends of the groove;

and/or the grooves are right-angled Z-shaped or II-shaped grooves, two substrates which are provided with the grooves and can be combined and sealed to form the micro-tube cavity are oppositely sealed, and two through pore channels are respectively formed on the side walls of two ends of the micro-tube cavity formed along the length direction and are used as fluid channels; the fixing structure and/or the component of the microfilament carrier are reducing clamping grooves or outer side buckles arranged at two ends of the groove.

7. The microwire type assay reaction device according to any of claims 1-6, wherein said microwire carrier is a pretreated microwire carrier, said pretreatment comprising one or more of polishing, cleaning with a detergent, acid treatment, alkali treatment, surface roughening of the surface of the microwire carrier; preferably, the microwire carrier is a coating film with an affinity microstructure on the surface and/or a microwire carrier with an activating group on the surface; further preferably, the activating group comprises one or more of hydroxyl, carboxyl, amino and amide.

8. Use of the microwire-type assay reaction device according to any one of claims 1 to 7 for the detection of target components; preferably, the application comprises:

9. The use according to claim 8, wherein said step B comprises:

step K, detecting or otherwise applying the target components enriched on the microfilament carrier in the step J;

preferably, the step K includes: sequentially contacting the microfilament carrier with the target components enriched on the surface with a washing liquid, an indicator and an auxiliary reagent, and directly or indirectly detecting the target components in a visual inspection or instrument measurement mode;

or, the step K includes: eluting, recovering, detecting or otherwise applying the enriched target components on the microwire carrier.

10. The use according to claim 8, wherein said step B comprises:

step N, detecting or otherwise applying the target components enriched on the microfilament carrier in the step M;

preferably, the step N includes: sequentially flowing the washing liquid, the indicator and the auxiliary reagent into a micro-tube cavity containing a micro-wire carrier with the surface enriched with target components, sequentially contacting the micro-wire carrier, and directly or indirectly detecting the target components in a visual or instrument measurement mode;

or, the step N includes: and taking the microwire out of the cavity of the microtube, and eluting, recovering, detecting or otherwise applying the target component enriched on the microwire.

11. Use according to any one of claims 8 to 10,

the probe comprises one or more of an affinity substrate, a group of the affinity substrate and an affinity microstructure; wherein, the affinity substrate comprises one or more of antibody, receptor, ligand, biotin, avidin, agglutinin, antigen, hapten, polynucleotide fragment, peptide chain, complement, cytokine, oligosaccharide, polysaccharide, enzyme and hormone;

and/or the affinity microstructure comprises a carbon chain structure and/or a hole structure with specific binding capacity, a spatial conformation;

and/or the sample to be detected comprises one or more of blood, plasma, serum, tissue fluid, cerebrospinal fluid, milk, saliva, urine, secretion extracting solution, tissue extract, biochemical analysis sample, culture solution, fermentation liquor, water body sample, industrial fluid and food, feed, medicine, veterinary medicine, pesticide and extract sample to be detected.