CN116381229A - Centrifugal microfluidic chip and photochemiluminescence multi-target detection method - Google Patents

Abstract

The invention discloses a centrifugal microfluidic chip, which relates to the technical field of light-induced chemiluminescence, and comprises a chip main body, and a microfluidic control unit provided on the chip main body includes: a first sample adding unit, a reaction unit and a second sample adding unit , the first sample loading unit includes a first sample loading chamber, the first sample loading chamber is used to add the sample to be tested; the reaction unit includes a plurality of reaction chambers, the reaction chambers are connected with the first sample loading chamber, and different The first lyophilized reaction reagent containing acceptor microspheres of the target; the second sample adding unit includes a reagent chamber and a second sample adding chamber, and the second lyophilized reaction reagent containing donor microspheres is preset in the reagent chamber. The second sample adding chamber is used for adding a reconstitution solution, which can redissolve the second freeze-dried reaction reagent, and the reagent chamber communicates with the reaction unit. The invention also discloses a multi-target detection method of photo-chemiluminescence based on the centrifugal microfluidic chip. The detection structure of the invention is simple, the volume is small, and it can realize multi-target detection of photo-excited chemiluminescence.

Description

Centrifugal microfluidic chip and photochemiluminescence multi-target detection method

technical field

The invention relates to the field of photochemiluminescence technology, in particular to a centrifugal microfluidic chip and a photochemiluminescence multi-target detection method.

Background technique

Light initiated chemiluminescence assay (LICA) proposed by American scientist Ullman et al. in the 1990s is a new type of homogeneous and wash-free chemiluminescence technology, which mainly relies on the interaction between donor microspheres and acceptor microspheres. As a result, the two microspheres will approach each other due to the specific binding of the antibody antigen, thereby connecting the energy transfer chain. After being excited by the 680nm laser, the photosensitizer loaded inside the donor microspheres will convert the oxygen in the surrounding environment into more active monomeric oxygen, and the monomeric oxygen diffuses to the surface of the acceptor microspheres within 200nm, triggering a series of The chemiluminescent reaction of cascade amplification finally makes the luminescent material on the acceptor microspheres generate a 615nm fluorescent signal.

Compared with traditional immunoassay techniques, LICA technology has obvious advantages: uniform and sufficient reaction, good repeatability of results; easy operation, fast detection speed; high sensitivity and wide detection range; rarely affected by hemoglobin , bilirubin and other impurities interfere with the detection signal of LICA, almost no non-specific fluorescence is generated, and it has good detection specificity.

At present, the existing LICA detection usually uses the LICA fully automated detection instrument for detection, but the LICA fully automated detection instrument can only realize single target detection, and needs to be driven by a mechanical arm to add samples and pipette. The instrument is bulky and can only be used in large-scale detection experiments. room to operate.

Therefore, it is urgent to provide a photochemiluminescence detection instrument with a simple detection structure and capable of realizing multi-target detection.

Contents of the invention

The purpose of the present invention is to provide a centrifugal microfluidic chip and photochemiluminescence multi-target detection method to solve the problems in the prior art. The detection structure is simple, the volume is small, and it can realize photochemiluminescence multi-target detection.

To achieve the above object, the present invention provides the following scheme:

The present invention provides a centrifugal microfluidic chip, which is used for multi-target detection of photo-induced chemiluminescence, including a chip body, and a microfluidic unit is arranged on the chip body, and the microfluidic unit includes:

a first sample adding unit, the first sample adding unit includes a first sample adding chamber, and the first sample adding chamber is used to add a sample to be tested;

A reaction unit, the reaction unit includes a plurality of reaction chambers, the reaction chamber communicates with the first sample loading chamber, the sample to be tested in the first sample loading chamber can enter the reaction chamber, the A first freeze-dried reaction reagent is preset in the reaction chamber, and at least two first freeze-dried reaction reagents of different targets are preset in the reaction chamber; wherein, the first freeze-dried reaction reagent contains receptor microbes ball;

The second sample adding unit, the second sample adding unit includes a connected reagent chamber and a second sample adding chamber, the second freeze-dried reaction reagent is preset in the reagent chamber, and the second sample adding chamber is used for Adding the reconstitution solution, the reconstitution solution in the second sample loading chamber can enter the reagent chamber, so that the second freeze-dried reaction reagent in the reagent chamber is redissolved to form a second reaction solution, and the reagent chamber is also combined with The reaction units are connected; wherein, the second freeze-dried reaction reagent contains donor microspheres.

Preferably, the microfluidic unit further includes a distribution unit, the inlet of the distribution unit communicates with the first sample loading chamber and the reagent chamber, the outlet of the distribution unit communicates with the reaction chamber, the The distribution unit can quantitatively distribute the sample to be tested and the second reaction solution into the reaction chamber.

Preferably, the distribution unit includes a distribution pipe and a distribution chamber, the inlet of the distribution pipe communicates with the first sample chamber and the reagent chamber, and the distribution pipe is sequentially connected with a plurality of The distribution chamber; wherein, the distribution chamber corresponds to the reaction chamber one by one, and any of the distribution chambers is in communication with one of the reaction chambers;

The outlet of the distribution pipeline is also connected with a waste liquid chamber.

Preferably, the microfluidic unit further includes a buffer chamber, the inlet of the buffer chamber communicates with the first sample loading chamber and the reagent chamber, and the outlet of the buffer chamber communicates with the inlet of the distribution pipeline.

Preferably, the reagent chamber communicates with the inlet of the buffer chamber through a siphon pipe.

Preferably, the siphon channel is modified hydrophilically.

Preferably, the microfluidic unit further includes an exhaust unit, the exhaust unit includes an exhaust pipe and an exhaust hole, and the first sample loading chamber and the second sample loading chamber are provided with a an exhaust hole, the reagent chamber is connected to one of the exhaust holes through the exhaust pipeline, and the distribution pipeline is connected to one of the exhaust holes;

The buffer cavity is connected with a positioning hole to realize exhaust, and the positioning hole can be used for positioning and installing the chip main body.

Preferably, the first lyophilized reaction reagent is a reaction reagent formed by mixing and lyophilizing antibody-coupled receptor microspheres and biotinylated antibodies, and the second lyophilized reaction reagent is a streptavidin-coupled donor reagent. Reagents formed by lyophilization of microspheres.

Preferably, there are multiple chip bodies, each of the chip bodies is provided with a microfluidic unit, and all the chip bodies can be evenly distributed on a rotating tray along the circumference;

Alternatively, a plurality of microfluidic units are evenly distributed along the circumference of the chip body, and the chip body can be installed on a rotating tray.

The present invention also provides a multi-target photo-chemiluminescent detection method, which is implemented based on the above-mentioned centrifugal microfluidic chip, including steps:

S1. Add the sample to be tested into the first sample adding chamber, and distribute it to each of the reaction chambers, and perform a first reaction with the first freeze-dried reaction reagent in each of the reaction chambers to form an intermediate reaction solution; wherein, at least The first freeze-dried reaction reagents of different targets are preset in the two reaction chambers to form intermediate reaction solutions of different targets;

S2. Distributing the second reaction solution to each of the reaction chambers, and performing a second reaction with the intermediate reaction solution of different targets;

S3. After the reaction is completed, a detection instrument is used to detect the reaction solution that has completed the second reaction in each of the reaction chambers, so as to realize multi-target detection.

Compared with the prior art, the present invention has achieved the following technical effects:

The present invention adopts the centrifugal microfluidic chip for multi-target detection of photo-excited chemiluminescence, and sets multiple reaction chambers to realize simultaneous detection of various targets. The independent reaction chamber also avoids contamination between reagents and reduces non-specific reactions. , can better realize the high sensitivity and high specificity detection of photo-induced chemiluminescence multi-target; and the centrifugal microfluidic chip is used for multi-target detection of photo-induced chemiluminescence, only one motor can be used to manipulate the movement of the fluid, and then realize The mixing of samples and multi-step reaction greatly simplifies the structure and hardware system, and can take into account portability while realizing automation.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.

FIG. 1 is a schematic structural view of a chip body in an embodiment of the present invention;

Fig. 2 is a schematic structural view of a rotating tray in an embodiment of the present invention;

3 is a flow chart of the multi-target detection method of photochemiluminescence in Example 2 of the present invention;

Fig. 4 is a schematic diagram of the fluid drive of the centrifugal microfluidic chip in Example 2 of the present invention;

Fig. 5 is a standard curve diagram for the detection of five kinds of enterotoxin LICA in Example 3 of the present invention;

Figure 6 is a schematic diagram of the specificity of the detection of five enterotoxins LICA in Example 3 of the present invention;

Fig. 7 is the standard curve diagram of 5 kinds of diarrhea virus LICA detection in the embodiment four of the present invention;

Fig. 8 is a schematic diagram of the specificity of detection of five diarrhea viruses LICA in Example 4 of the present invention.

In the figure: 100-chip main body, 1-first sample loading chamber, 2-second sample feeding chamber, 3-reagent chamber, 4-liquid filling hole, 5-vent hole, 6-exhaust pipe, 7-siphon tube Road, 8-buffer chamber, 9-distribution chamber, 10-reaction chamber, 11-distribution pipe, 12-positioning hole, 13-waste liquid chamber, 200-rotary tray, 201-installation groove, 202-positioning column.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The purpose of the present invention is to provide a centrifugal microfluidic chip and photochemiluminescence multi-target detection method to solve the problems in the prior art. The detection structure is simple, the volume is small, and it can realize photochemiluminescence multi-target detection.

In order to make the above objects, features and advantages of the present invention more comprehensible, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Embodiment one

As shown in Fig. 1-Fig. 2, this embodiment provides a centrifugal microfluidic chip for multi-target photochemiluminescence detection, including a chip body 100, on which a microfluidic unit is arranged, the The microfluidic unit mainly includes: a first sample loading unit, a reaction unit, and a second sample loading unit; wherein, the first sample loading unit includes a first sample loading chamber 1, and the first sample loading chamber 1 is used for Add the sample to be tested; the reaction unit includes a plurality of reaction chambers 10, the reaction chamber 10 communicates with the first sample loading chamber 1, and the sample to be tested in the first sample loading chamber 1 can enter the In the reaction chamber 10, a first freeze-dried reaction reagent is preset in the reaction chamber 10, and at least two first freeze-dried reaction reagents of different targets are preset in the reaction chamber 10; wherein, the first The freeze-dried reaction reagent contains acceptor microspheres; the second sample loading unit includes a connected reagent chamber 3 and a second sample loading chamber 2, and the second freeze-dried reaction reagent is preset in the reagent chamber 3, so The second sample adding chamber 2 is used to add a complex solution, and the complex solution in the second sample adding chamber 2 can enter the reagent chamber 3 to redissolve the second freeze-dried reaction reagent in the reagent chamber 3, A second reaction solution is formed, and the reagent chamber 3 is also communicated with the reaction unit; wherein, the second freeze-dried reaction reagent contains donor microspheres.

In this embodiment, since the first lyophilized reaction reagent contains acceptor microspheres, and the second lyophilized reaction reagent contains donor microspheres, when the second lyophilized reaction reagent is added to the reaction chamber 10, the donor microspheres The interaction with the acceptor microspheres can generate light-induced chemiluminescent reaction, and the reaction solution in the reaction chamber 10 generates a fluorescent signal, and the reaction is detected by acquiring the fluorescent signal value in each reaction chamber 10 in real time.

At the same time, in this embodiment, a centrifugal microfluidic chip is used for multi-target detection of photo-induced chemiluminescence, and multiple reaction chambers 10 are set to realize simultaneous detection of various targets, and independent reaction chambers 10 also avoid contamination between reagents. , which reduces non-specific reactions, and can better achieve high-sensitivity and high-specificity detection of multiple targets of photo-induced chemiluminescence; and the centrifugal microfluidic chip is used for multi-target detection of photo-chemiluminescence, which can be operated with only one motor The movement of the fluid, and then the mixing of samples and multi-step reactions, greatly simplifies the structure and hardware system, and can take into account portability while realizing automation.

As a preferred implementation, in this embodiment, as shown in Figures 1-2, there are multiple chip bodies 100, and each of the chip bodies 100 is a separate detection chip, each The detection chip is equipped with a microfluidic unit, and all the chip bodies 100 can be evenly distributed on a rotating tray 200 along the circumference, and the rotating tray 200 is driven by a motor to rotate, thereby driving a plurality of chip bodies 100 on it. Rotate to realize fluid control in the centrifugal microfluidic chip. Wherein, as shown in FIG. 2 , the rotating tray 200 is provided with a plurality of installation slots 201 , and the installation slots 201 are provided in one-to-one correspondence with the chip main bodies 100 , and the chip main bodies 100 can be clipped into the corresponding installation slots 201 .

It needs to be further explained that the control of the fluid in the centrifugal microfluidic chip is achieved by driving the rotating tray 200 and the plurality of chip bodies 100 on it to rotate through the motor, which is a mature prior art in this field, and will not be discussed in this embodiment. Let me repeat. In this embodiment, the chambers are sequentially arranged along the liquid flow sequence from inside to outside (from the center of the rotating tray 200 to the outer edge). Specifically, the reaction chamber 10 is located between the first sample loading chamber 1 and the second sample loading chamber. Outside the chamber 2, the liquid in the first sample loading chamber 1 and the second sample loading chamber 2 can flow into the reaction chamber 10 under the centrifugal force generated when the chip rotates, and the reagent chamber 3 is located outside the second sample loading chamber 2 , the liquid in the second sample loading chamber 2 can first enter the reagent chamber 3 and then enter the reaction chamber 10 under the action of centrifugal force.

Alternatively, the chip body 100 may be a circular chip on which a plurality of microfluidic units are evenly distributed along the circumference, and the circular chip is mounted on a rotating tray 200 .

In this embodiment, the chip body 100 can be made of white polymethyl methacrylate (PMMA) or polycarbonate/polystyrene (PC/PS) through high-precision precision machining-opening mold injection molding.

In this embodiment, when the detection is not performed, the detection reagent required for the two-step reaction of LICA detection is pre-lyophilized into a bead shape to form the first lyophilized reaction reagent and the second lyophilized reaction reagent, which are stored in the reaction chamber 10 respectively. In the reagent chamber 3, a single microfluidic unit is sealed with an encapsulation film; after step-by-step centrifugation and incubation, the fluorescent signal detection is carried out immediately. Wherein, the first lyophilized reaction reagent is preferably a reaction reagent formed by lyophilizing antibody-coupled acceptor microspheres and biotinylated antibodies, and the second lyophilized reaction reagent is preferably a streptavidin-coupled donor Reagents formed by lyophilization of microspheres.

In this embodiment, the microfluidic unit further includes a distribution unit, the inlet of the distribution unit communicates with the first sample loading chamber 1 and the reagent chamber 3, and the outlet of the distribution unit communicates with the reaction chamber 3. The cavity 10 is connected, and the distribution unit can quantitatively distribute the sample to be tested and the second reaction solution into the reaction cavity 10 . Specifically, the distribution unit includes a distribution pipe 11 and a distribution chamber 9, the inlet of the distribution pipe 11 communicates with the first sample chamber 1 and the reagent chamber 3, and the distribution pipe 11 is in the direction of liquid flow. A plurality of the distribution chambers 9 are connected in sequence; wherein, the distribution chambers 9 correspond to the reaction chambers 10 one by one, any of the distribution chambers 9 communicates with one of the reaction chambers 10, and each distribution chamber 9 have the same volume.

In this embodiment, the outlet of the distribution pipe 11 is also connected with a waste liquid chamber 13 for storing the waste liquid of the remaining sample to be tested and the second reaction solution after quantitative distribution by the distribution unit.

Specifically, when the distribution unit performs quantitative distribution of the sample to be tested and the second reaction solution, the sample to be tested or the second reaction solution is filled with all the distribution chambers 9, so that the volume of the solution in each distribution chamber 9 is the same to achieve quantitative distribution. After filling all the distribution chambers 9, the remaining waste liquid flows into the waste liquid chamber 13 for collection.

In this embodiment, the microfluidic unit further includes a buffer chamber 8, the inlet of the buffer chamber 8 communicates with the first sample loading chamber 1 and the reagent chamber 3, and the outlet of the buffer chamber 8 communicates with the reagent chamber 3. The inlet of the distribution pipe 11 is connected; a buffer chamber 8 is provided to realize the buffering of the sample to be tested and the second reaction solution.

In this embodiment, the reagent chamber 3 is communicated with the inlet of the buffer chamber 8 through a siphon pipe 7, and the siphon pipe 7 is hydrophilically modified, specifically, absolute ethanol (containing 0.1% Triton-100 ) solution to modify the siphon pipe 7 hydrophilically; setting the siphon pipe 7 in this embodiment can realize the precise control of the first step reaction and the second step reaction sequence of LICA, and centrifuge in the first step (the sample to be tested is added to the distribution chamber 9), the siphon pipe 7 is filled with no liquid, and the second reaction solution will remain in the reagent chamber 3. After the first step of reaction is completed, the siphon pipe 7 is filled with liquid, and the second step of centrifugation and reaction begins.

In this embodiment, the microfluidic unit further includes an exhaust unit, the exhaust unit includes an exhaust pipe 6 and an exhaust hole 5, the first sample loading chamber 1 and the second sample loading chamber 2 are provided with a vent hole 5 to realize exhaust, and the first sample chamber 1 and the second chamber 2 are provided with a liquid port 4 for sample addition; The reagent chamber 3 is connected to a vent hole 5 through a vent pipe 6 for exhausting, and the distribution pipe 11 is also connected to a vent hole 5; the buffer chamber 8 is connected with a positioning hole 12 , to achieve exhaust, and the positioning hole 12 can be used for the positioning and installation of the chip main body 100, specifically, when the chip main body 100 is installed in the corresponding installation groove 201 on the rotary tray 200, the chip main body 100 can be positioned by The hole 12 is sleeved on the positioning column 202 to realize positioning and fixing.

In this embodiment, it should be noted that the liquid filling hole 4, the exhaust hole 5 or the positioning hole 12 of each cavity are all located inside the corresponding cavity, which can effectively prevent the liquid from flowing out under the action of centrifugal force.

Embodiment two

As shown in Figures 3-4, this embodiment provides a multi-target detection method for photo-induced chemiluminescence, which is implemented based on the centrifugal microfluidic chip as described in Example 1, including steps:

S1, adding the sample to be tested into the first sample loading chamber 1, and distributing it to each of the reaction chambers 10, and performing a first reaction with the first freeze-dried reaction reagent in the reaction chamber 10 to form an intermediate reaction solution; wherein , at least two of the reaction chambers 10 are preset with first freeze-dried reaction reagents of different targets, so as to form intermediate reaction solutions of different targets;

S2. Distributing the second reaction solution to each of the reaction chambers 10, and performing a second reaction with the intermediate reaction solution of different targets in each of the reaction chambers 10;

S3. After the reaction is completed, the reaction solution that completed the second reaction in each of the reaction chambers 10 is detected by a detection instrument to realize multi-target detection; wherein, the detection instrument is a mature technology in the field and can be selected according to needs, preferably Multifunctional microplate reader.

Specifically, the specific workflow of the photo-chemiluminescence multi-target detection method in this embodiment is as follows:

First, use a pipette gun to add 60 μL of the sample to be tested into the first sample loading chamber 1, and 60 μL of the complex solution into the second sample loading chamber 2, place the chip main body 100 on the rotating tray 200, and centrifuge at a low speed of 1300 rpm to make the sample to be tested. The test sample flows through the buffer chamber 8 and enters the distribution pipeline 11 and is distributed to each distribution chamber 9. The volume of the distribution chamber 9 is 10 μL, and the excess liquid enters the waste liquid chamber 13; Reagent dissolves. The chip main body 100 keeps rotating, and the rotating speed is increased to 3000rpm, so that the sample solution in the distribution chamber 9 breaks through the thin tube at the bottom of the distribution chamber 9 and enters the reaction chamber 10, and is mixed with the first freeze-dried reaction reagent (antibody-coupled receptor microspheres and biological Antigenated antibody) was mixed, and after reacting at 37°C for 15 minutes, the antigen to be detected in the sample to be tested was captured and formed a double antibody sandwich. After incubation, the hydrophilically modified siphon pipe 7 is filled with the second reaction solution due to the action of capillary force; then, the low-speed centrifugation at 1300 rpm is repeated, and the second reaction solution in the reagent chamber 3 flows through the siphon pipe 7 through the buffer chamber 8 and enters each chamber. Distributing chamber 9; increase the rotation speed to 3000 rpm, so that the second reaction solution enters the reaction chamber 10, and the volume of the reaction chamber 10 is 20 μL. After reacting at 37° C. for 10 minutes, the chip main body 100 is detected in a multi-functional microplate reader, and the fluorescence signal value of the reaction solution in each reaction chamber 10 is read immediately.

Embodiment three

In this example, taking type 5 Staphylococcus aureus enterotoxin as an example, the specific steps of LICA detection are described:

Mice were immunized with recombinantly expressed type 5 Staphylococcus aureus enterotoxin antigen, splenocytes were taken from the mice for cell fusion to prepare hybridoma cells, and cell lines with high titers were obtained by screening, and antibody purification was performed to obtain monoclonal antibodies A3#, B1#, C4#, D5#, E7#, respectively biotinylated, used for Staphylococcus aureus enterotoxin A (SEA), Staphylococcus aureus enterotoxin B (SEB), Staphylococcus aureus enterotoxin C (SEC), specific detection of S. aureus enterotoxin D (SED) and S. aureus enterotoxin E (SEE).

Goats were co-immunized with the recombinantly expressed type 5 enterotoxin antigen of Staphylococcus aureus, the serum titer was determined, and the goat serum was purified to obtain goat polyclones that could be used for the detection of SEA, SEB, SEC, SED and SEE type 5 enterotoxins at the same time Antibodies, for conjugation to receptor microspheres. For the preparation of biotinylated antibodies, refer to the instruction manual of the biotin labeling kit from Thermo Company. The acceptor microspheres and streptavidin-coupled donor microspheres were purchased from PerkinElmer. Ball marking instructions.

Mix biotinylated antibodies (1 μg/mL) of 5 kinds of enterotoxins and goat polyclonal antibody-coupled receptor microspheres (50 μg/mL) respectively, freeze-dry them into beads, the size of which is 10 mm ³ , freeze-dry The last five kinds of beads were respectively placed in the five reaction chambers 10 of the chip main body 100; the donor microspheres (40 μg/mL) were also freeze-dried in the form of beads, and the size of the beads was 10 mm ³ , and two pieces were taken after freeze-drying. The droplet is placed in the reagent chamber 3 of the chip main body 100 . After being sealed with packaging film, store at 4°C.

Add different concentrations of Staphylococcus aureus enterotoxin antigens into the first sample chamber 1, add complex solution into the second sample chamber 2, and perform two-step centrifugation and signal detection; repeat 3 times for each antigen concentration. Taking the antigen concentration as the abscissa and the measured fluorescence signal intensity as the ordinate, draw a standard curve to obtain a four-parameter fitting equation for the detection of 5 kinds of enterotoxins. The critical value is 0 ng/mL of the LICA signal average plus three times the standard deviation , the lowest detection limit is the lowest antigen concentration greater than the critical value; as shown in Figure 5, the ^R2 values of the standard curves for the detection of five enterotoxins are all above 0.99, which has a good fit. The lowest detection limits of SEA, SEB, SEC, SED and SEE were 35.27pg/mL, 15.55pg/mL, 9.58pg/mL, 11.91pg/mL and 26.59pg/mL, respectively.

Prepare SEA, SEB, SEC, SED and SEE antigen solutions with a concentration of 100ng/mL for the specific detection of five Staphylococcus aureus enterotoxins; each antigen concentration was repeated 3 times. As shown in Figure 6, the results showed that there was no cross-reaction among the five enterotoxins, indicating that the detection of the five enterotoxins by the centrifugal microfluidic chip had good specificity.

After diluting the liquid pure milk purchased in the supermarket by 2 times, 5 kinds of enterotoxin antigens of different concentrations were added to prepare simulated samples of 5 kinds of enterotoxin antigens, and 60 μL of simulated samples of different concentrations were added to the pre-prepared chip main body 100 solution and 60 μL complex solution. Each simulated sample was repeated 3 times, the centrifugal microfluidic chip was centrifuged, incubated and detected, the fluorescence detection signal was read, the detection concentration of the simulated sample was calculated through the standard curve, and the recovery rate of the simulated sample was calculated through the concentration of the added standard; As shown in Table 1, the results show that the recovery rates of the five enterotoxins are between 89.52% and 109.09%, and the CV (coefficient of variation, Coefficient ofVariation) is all lower than 10%, indicating that the centrifugal microfluidic chip can be accurately and reliably 5 kinds of Staphylococcus aureus enterotoxins in samples were detected accurately.

Table 1 The detection recoveries of 5 kinds of enterotoxins in simulated samples

Embodiment four

In this embodiment, five kinds of diarrhea viruses are taken as examples to illustrate the specific steps of LICA detection:

The five types of diarrhea viruses are Rotavirus, Astrovirus, Adenovirus, Norovirus GI and Norovirus GII; among them, Rotavirus (Rotavirus) antibody pair was purchased from American Fitzgerald Company, inactivated virus antigen was purchased from Canada Microbix Company; Astrovirus (Astrovirus) antibody pair and recombinant protein antigen were purchased from American EastCoast Bio Company; intestinal adenovirus (Adenovirus) The antibody pair was purchased from Fitzgerald Company in the United States, the inactivated adenovirus type 40 virus antigen was purchased from Meridian Company in the United States; the antibody pair of Norovirus GI (Norovirus GⅠ) and Norovirus GII (Norovirus GⅡ) antibody pair, and the recombinant protein antigen was purchased from Spain Certest Biotec Corporation.

The detection antibodies of 5 kinds of diarrhea viruses were labeled with biotin. For the method, please refer to the instruction manual of the biotin labeling kit of Thermo Company; Spheres and streptavidin-coupled donor microspheres were purchased from PerkinElmer.

Mix biotinylated antibodies (0.5 μg/mL) of 5 kinds of diarrhea viruses and antibody-coupled receptor microspheres (12.5 μg/mL) of 5 kinds of diarrhea viruses respectively, freeze-dry and form droplet beads with a size of 10 mm ^3. The five kinds of beads after freeze-drying were placed in the five reaction chambers 10 of the chip main body 100; The size is 10mm ³ , and after freeze-drying, take 2 beads and place them in the reagent chamber 3 of the chip main body 100 . After being sealed with packaging film, store at 4°C.

Add different concentrations of diarrhea virus antigens into the first sample chamber 1 of the chip body 100 after packaging, and add a complex solution into the second sample chamber 2 to perform two-step centrifugation and signal detection; each antigen concentration is repeated 3 times. Taking the antigen concentration as the abscissa and the measured fluorescence signal intensity as the ordinate, draw a standard curve to obtain a four-parameter fitting equation for the detection of five diarrhea viruses. The critical value is 0 ng/mL of the LICA signal average plus three times the standard deviation , the lowest detection limit is the lowest antigen concentration greater than the cut-off value. As shown in Figure 7, the R ^values of the standard curves for the detection of five diarrhea viruses are all above 0.99, which has a good fit; detection of rotavirus, astrovirus, enteric adenovirus, and norovirus GI The minimum detection limits of norovirus type GII were 839.46pg/mL, 29.96pg/mL, 4.43pg/mL, 9.35pg/mL and 8.22pg/mL, respectively.

Prepare rotavirus, astrovirus, enteric adenovirus, norovirus GI and norovirus GII antigen solutions at a concentration of 100ng/mL for the specific detection of 5 diarrhea viruses; repeat for each antigen concentration 3 times. As shown in Figure 8, the results showed that there was no cross-reaction among the five diarrhea viruses, indicating that the detection of the five diarrhea viruses by the centrifugal microfluidic chip had good specificity.

Antigens of rotavirus, astrovirus, enteric adenovirus, norovirus type GI, and norovirus type GII were diluted to different concentrations using PBS (phosphate buffer saline). Take 1.0mL of different concentrations of diarrhea virus antigen solutions and add them to 5mg of healthy human feces respectively, oscillate to mix the diarrhea virus antigen and feces thoroughly, and prepare a 0.5% (w/v) suspension, which is the preparation of 5 kinds of diarrhea virus simulated stool samples. 60 μL of simulated sample solutions of different concentrations and 60 μL of complex solution were added to the pre-prepared chip main body 100 . Each simulated sample was repeated 3 times, the centrifugal microfluidic chip was centrifuged, incubated and detected, the fluorescence detection signal was read, the detection concentration of the simulated sample was calculated through the standard curve, and the recovery rate of the simulated sample was calculated through the concentration of the added standard; As shown in Table 2, the results show that the recovery rates of the five diarrhea viruses are between 86.69% and 114.63%, and the CV (coefficient of variation, Coefficient ofVariation) is all lower than 10%, indicating that the chip can accurately and reliably detect the 5 diarrhea viruses.

The detection recoveries of 5 kinds of diarrhea viruses in table 2 simulated samples

In the present invention, specific examples have been used to illustrate the principle and implementation of the present invention. The description of the above embodiments is only used to help understand the method and core idea of the present invention; meanwhile, for those of ordinary skill in the art, according to the present invention The idea of the invention will have changes in the specific implementation and scope of application. In summary, the contents of this specification should not be construed as limiting the present invention.

Claims (10)

1. A centrifugal microfluidic chip, characterized in that: the device is used for photo-excitation chemiluminescence multi-target detection and comprises a chip main body, wherein a microfluidic unit is arranged on the chip main body and comprises:

the first sample adding unit comprises a first sample adding cavity, and the first sample adding cavity is used for adding a sample to be tested;

the reaction unit comprises a plurality of reaction chambers, wherein the reaction chambers are communicated with the first sample adding chamber, a sample to be detected in the first sample adding chamber can enter the reaction chambers, first freeze-drying reaction reagents are preset in the reaction chambers, and at least two first freeze-drying reaction reagents with different targets are preset in the reaction chambers; wherein the first freeze-drying reaction reagent contains receptor microspheres;

the second sample adding unit comprises a reagent cavity and a second sample adding cavity which are communicated, a second freeze-drying reaction reagent is preset in the reagent cavity, the second sample adding cavity is used for adding a compound solution, the compound solution in the second sample adding cavity can enter the reagent cavity to enable the second freeze-drying reaction reagent in the reagent cavity to be dissolved again, so that a second reaction solution is formed, and the reagent cavity is also communicated with the reaction unit; wherein the second lyophilized reactant comprises donor microspheres.

2. The centrifugal microfluidic chip according to claim 1, wherein: the microfluidic unit further comprises a distribution unit, an inlet of the distribution unit is communicated with the first sample adding cavity and the reagent cavity, an outlet of the distribution unit is communicated with the reaction cavity, and the distribution unit can quantitatively distribute the sample to be detected and the second reaction solution into the reaction cavity.

3. The centrifugal microfluidic chip according to claim 2, wherein: the distribution unit comprises a distribution pipeline and a distribution cavity, wherein an inlet of the distribution pipeline is communicated with the first sample adding cavity and the reagent cavity, and a plurality of distribution cavities are sequentially connected to the distribution pipeline along the liquid flow direction; wherein the distribution cavities are in one-to-one correspondence with the reaction cavities, and any distribution cavity is communicated with one reaction cavity;

and the outlet of the distribution pipeline is also connected with a waste liquid cavity.

4. A centrifugal microfluidic chip according to claim 3, wherein: the microfluidic unit further comprises a buffer cavity, wherein an inlet of the buffer cavity is communicated with the first sample adding cavity and the reagent cavity, and an outlet of the buffer cavity is communicated with an inlet of the distribution pipeline.

5. The centrifugal microfluidic chip according to claim 4, wherein: the reagent cavity is communicated with the inlet of the buffer cavity through a siphon pipeline.

6. The centrifugal microfluidic chip according to claim 5, wherein: the siphon pipeline is subjected to hydrophilic modification.

7. The centrifugal microfluidic chip according to claim 4, wherein: the microfluidic unit further comprises an exhaust unit, the exhaust unit comprises an exhaust pipeline and an exhaust hole, the exhaust hole is formed in each of the first sample adding cavity and the second sample adding cavity, the reagent cavity is connected with one exhaust hole through the exhaust pipeline, and the distribution pipeline is connected with one exhaust hole;

the buffer cavity is connected with a positioning hole for realizing exhaust, and the positioning hole can be used for positioning and mounting the chip main body.

8. The centrifugal microfluidic chip according to any one of claims 1 to 7, wherein: the first freeze-drying reaction reagent is a reaction reagent formed by mixing and freeze-drying antibody coupled receptor microspheres and biotinylated antibodies, and the second freeze-drying reaction reagent is a reaction reagent formed by freeze-drying streptavidin coupled donor microspheres.

9. The centrifugal microfluidic chip according to any one of claims 1 to 7, wherein: the chip main bodies are provided with a plurality of micro-fluidic units, and each chip main body is provided with a plurality of micro-fluidic units which can be uniformly distributed on a rotary tray along the circumference;

or, a plurality of microfluidic units are uniformly distributed on the chip main body along the circumference, and the chip main body can be mounted on a rotary tray.

10. A photo-excitation chemiluminescence multi-target detection method is characterized by comprising the following steps of: implemented on the basis of a centrifugal microfluidic chip according to any one of claims 1-9, comprising the steps of:

s1, adding a sample to be detected into a first sample adding cavity, distributing the sample to each reaction cavity, and carrying out a first reaction with a first freeze-drying reaction reagent in each reaction cavity to form an intermediate reaction solution; the reaction chambers are internally preset with first freeze-drying reaction reagents of different targets to form intermediate reaction solutions of the different targets;

s2, distributing a second reaction solution to each reaction cavity, and carrying out a second reaction with the intermediate reaction solutions of different targets;

and S3, after the reaction is finished, detecting the reaction liquid in which the second reaction is finished in each reaction cavity through a detection instrument, so as to realize multi-target detection.

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