CN110579603A - A virus detection sensor, a device and method for detecting virus concentration - Google Patents

Abstract

The invention discloses a virus detection sensor, a device and method for detecting virus concentration. The sensor includes a bracket, a detection probe and a capillary glass tube, and the inner wall of the capillary glass tube is modified with antibodies that specifically bind to the virus to be detected. , the detection probe includes a detection body and a photoelectric conversion circuit, the photoelectric conversion circuit includes a photosensitive element and a light source, the detection body is fixed on a bracket, a circular detection channel is arranged in the center of the detection body, a capillary glass tube passes through the detection channel, and the capillary glass tube is fixed On the bracket, the first groove and the second groove are symmetrical on the side wall of the detection channel, the light source is fixed in the first groove, and the photosensitive element is fixed in the second groove. The sensor has a simple structure and an ingenious design. The device has a simple structure, can realize automatic virus detection, and improves the detection efficiency and accuracy. The method is simple, and the standard curve method is used for detection, which is convenient and quick.

Description

A virus detection sensor, a device and method for detecting virus concentration

technical field

The invention relates to the technical field of biological detection, in particular to a virus detection sensor, a device and method for detecting virus concentration.

Background technique

At present, there are many kinds of human diseases caused by viruses. Different viruses have different pathogenic mechanisms. At the cellular level, the main destructive effect of viruses is to cause cell lysis, thereby causing cell death. In multicellular organisms, once enough cells in the body die, there are consequences for the body's health. Some viruses are capable of causing chronic infections and can replicate continuously in the body without being compromised by the host defense system. People who are chronically infected are carriers of the virus because they act as a reservoir of virus that remains infectious, and when there is a high proportion of carriers in the population, the disease can develop into an epidemic. Therefore, virus detection technology is of great significance in the field of biomedicine and is an important part of human health assurance.

At present, the commonly used clinical detection methods are mostly nucleic acid detection and protein detection, and the detection process has high technical and equipment requirements. Currently commonly used virus technologies include: virus detection method and device (patent CN201510144530), a virus detection device and its use method (CN201711486707), a virus trait detection device (CN201820145057) and the like. However, the implementation process of the above device is too cumbersome, with high technical requirements, low efficiency, high cost, and increased inspection costs, which is not conducive to popularization.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the above-mentioned prior art, the present invention provides a virus detection sensor, a device and method for detecting virus concentration, the sensor is simple in structure, ingenious in design, and ingeniously combines the light-shielding property of the magnetic ball with photoelectric conversion , the detection is convenient and fast, and the cost is low.

The device has a simple structure, can realize automatic virus detection, and improves the detection efficiency and detection accuracy.

The method is simple, and the standard curve method is used for detection, which is convenient and quick.

The technical scheme adopted to realize the above-mentioned purpose of the present invention is:

A virus detection sensor includes a bracket, a detection probe and a capillary glass tube, the inner wall of the capillary glass tube is modified with an antibody that specifically binds to the virus to be detected, the detection probe includes a detection body and a photoelectric conversion circuit, and the photoelectric conversion circuit includes a photosensitive element and light source, the detection body is fixed on the bracket, the center of the detection body is provided with a circular detection channel, the capillary glass tube passes through the detection channel, the capillary glass tube and the detection channel are gap-fitted, the capillary glass tube is fixed on the bracket, and the side wall of the detection channel The first groove and the second groove are symmetrical, the light source is fixed in the first groove, the photosensitive element is fixed in the second groove, and the light source is facing the photosensitive element.

The bracket includes a support plate and a clamp, there are two clamps, the clamp includes a support body and an elastic C-type clamp, the support body is provided with a semicircular third groove, and the diameter of the third groove is the same as the capillary. The outer diameter of the glass tube is equal, the support body is symmetrically fixed on the support plate, the detection body is located between the two support bodies, the two sides of the capillary glass tube are respectively placed in two third grooves, and the two sides of the capillary glass tube pass through respectively The C-type clamp is fixed on the two supports.

Including the virus detection sensor, power mechanism, valve control mechanism, reagent supply mechanism, magnetic separation mechanism and detection mechanism of claim 1;

The power mechanism includes a suction pump, a first suction branch pipe and a first valve, one end of the first suction branch pipe is communicated with the outlet of the suction pump, and the first valve is installed on the first suction branch pipe;

The reagent supply mechanism includes N reagent bottles, and the other ends of the first air suction branch pipes are respectively connected with the N reagent bottle air paths;

The magnetic separation mechanism includes a microfluidic chip, a sample discharge pipe and a waste discharge pipe. Each reagent bottle is controlled by a valve control mechanism to communicate with the inlet liquid path of the microfluidic chip. The microfluidic chip includes a buffer moving channel and a sample moving channel. The buffer moving channel is communicated with the inlet of the capillary glass tube through the sample discharge pipe, and the sample moving channel is communicated with the waste discharge pipe.

The valve control mechanism includes a multi-way pneumatic solenoid valve, a controller and a valve control chip, the controller is connected with the multi-way pneumatic solenoid valve, and the multi-way pneumatic solenoid valve has one air inlet and N air outlets;

The valve control chip includes a clamp and a glass chip. The glass chip includes a cover sheet, an organic silicon film and a substrate. The glass chip is a sandwich structure. The silicone film is located between the cover sheet and the substrate, and the cover sheet and the substrate are fixed by the clamp. connect;

The cover sheet is respectively provided with N first liquid inlet holes, two first liquid discharge holes and N gas passage grooves, the N gas passage grooves are all sealed by clamps, and N second liquid inlet holes are respectively provided on the organic silicon film. hole and two second drain holes;

A first liquid channel and a second liquid channel are respectively provided on the substrate. The first liquid channel consists of a first main channel, a first liquid outlet channel and Q first liquid inlet sub-channels, and each first liquid inlet sub-channel. One end of the channel is communicated with the first main channel respectively, and one of the first liquid inlet channel is communicated with one end of the first main channel, and one end of the first liquid outlet channel is communicated with the other end of the first main channel , the second liquid channel consists of a second main channel, a second liquid outlet channel and P second liquid inlet sub-channels, one end of each second liquid inlet sub-channel is respectively connected with the second main channel, and wherein A second liquid inlet channel is communicated with one end of the second main channel, and one end of the second liquid outlet channel is communicated with the other end of the second main channel, Q+P=N;

The N reagent bottles are respectively connected with the N first liquid inlet holes, the N first liquid inlet holes are respectively connected with the N second liquid inlet holes, and the Q second liquid inlet holes are respectively connected with the Q first liquid inlet holes. The other ends of the channels are connected, and the remaining P second liquid inlet holes are respectively connected with the other ends of the P second liquid inlet channels;

The N gas outlets are respectively communicated with the N gas passages, and the projection of each gas passage on the substrate separates the corresponding first liquid inlet channel or second liquid inlet channel;

The power mechanism also includes a second suction branch pipe and a second valve, one end of the second suction branch pipe is connected to the outlet of the suction pump, the second valve is installed on the second suction branch pipe, and the other end of the second suction branch pipe is connected to the inlet. air connection;

The microfluidic chip further comprises a first injection port, a second injection port, a first discharge port and a second discharge port, the first injection port and the first discharge port are respectively connected with both ends of the buffer moving channel, and the second injection port and the second discharge port are respectively connected with the two ends of the sample moving channel, the other ends of the first liquid outlet channel and the second liquid outlet channel are respectively connected with the two second liquid discharge holes, and the two second liquid discharge holes are respectively connected with the two second liquid discharge holes. The first liquid discharge holes are in communication, one of the first liquid discharge holes is in communication with the first injection port, the first discharge port is in communication with one end of the sample discharge pipe, the other end of the sample discharge pipe is in communication with the capillary glass tube, and the other first row The liquid hole is communicated with the second injection port, and the second discharge port is communicated with the waste discharge pipe.

The clamp includes a first splint and a second splint, the glass chip is located between the first splint and the second splint, the first splint is sealed with the cover sheet, the second splint is sealed with the substrate, the first splint and the The second splint is fixedly connected by bolts, and the first splint is connected with N liquid inlet pipes, two discharge pipes and N inlet pipes, N reagent bottles are respectively connected with one end of the N liquid inlet pipes, and N liquid inlet pipes are respectively connected. The other end of the pipe is connected with N first liquid inlet holes respectively, one end of the two liquid discharge pipes is connected with the two first liquid discharge holes respectively, and the other ends of the two liquid discharge pipes are respectively connected with the first injection port and the second injection port. , one end of the N intake pipes is respectively connected with the N gas outlets, and the other ends of the N intake pipes are respectively communicated with the N gas passage grooves.

The reagent supply mechanism further includes N liquid injection pipes and N-1 communication pipes, one end of the N liquid injection pipes is respectively connected with the N reagent bottles, and the other ends of the N liquid injection pipes are respectively connected with the N liquid injection pipes. One end of the pipe is communicated, the other end of the first air suction branch is communicated with the bottle mouth of the first reagent bottle, and the bottle mouths of two adjacent reagent bottles are communicated through a communication pipe.

It also includes Diwen screen, the virus detection sensor is connected with the controller, and the controller is connected with the Diwen screen.

A method for detecting virus concentration, comprising the steps of:

1. Modify the magnetic microspheres in the magnetic microsphere suspension to an antibody that can specifically bind to the virus to be tested to obtain an antibody-modified magnetic sphere suspension;

2. Add antibody-modified magnetic sphere suspension to the virus stock solution containing the virus to be tested for reaction, so that the virus to be tested can be completely specifically bound to the antibody, and at the same time, dilute the virus stock solution to the virus concentration of C ₁ , C ₂ ...... C _m virus standard solution, C ₁ , C ₂ ...... C _m increases in turn, C ₁ ≤ 5ng/ml, C _m ≥ 155ng/ml;

3. Add blocking reagent to one pair of reagent bottles, add deionized water and PBS buffer to one pair of reagent bottles, and add virus standard solution of volume V and concentration C ₁ to one of the reagent bottles. The reagent bottle containing the virus standard solution with a concentration of _C1 is marked as the sample bottle;

4. Control the valve control mechanism to connect the two reagent bottles containing the closed reagents with the inlet of the microfluidic chip, so that the two reagent bottles containing the closed reagents are connected to the buffer moving channel and the sample moving channel respectively. The reagents are respectively passed into the communication between the buffer moving channel and the sample moving channel to reduce non-specific adsorption. After the sealing is completed, the valve control mechanism is controlled to separate the two reagent bottles containing the sealing reagent from the inlet of the microfluidic chip;

5. Control the valve control mechanism to connect the two reagent bottles filled with deionized water and PBS buffer with the inlet of the microfluidic chip, so that the reagent bottle filled with PBS buffer is connected with the buffer moving channel, filled with deionized water The reagent bottle is connected with the sample moving channel, the PBS buffer is passed into the buffer moving channel, the ionized water sample is passed into the moving channel, and the cleaning buffer moving channel is connected with the sample moving channel. After cleaning, control the valve to control The mechanism separates the two reagent bottles containing deionized water and PBS buffer from the inlet of the microfluidic chip;

6. Control the valve control mechanism to connect the two reagent bottles containing the virus standard solution and the PBS buffer with the inlet of the microfluidic chip, so that the reagent bottle containing the virus standard solution is connected to the sample moving channel, so that the reagent bottle containing the PBS buffer is connected. The reagent bottle is connected with the buffer moving channel, the PBS buffer is passed into the buffer moving channel, the virus standard solution is passed into the sample moving channel, and the magnetic microspheres in the virus standard solution are enriched in the buffer moving channel, and the Flowing into the capillary glass tube, the magnetic microspheres are captured at the capillary glass tube. When the virus standard solution is fully introduced, the valve control mechanism is controlled to isolate the reagent bottle containing the virus standard solution from the inlet of the microfluidic chip. The voltage at both ends does not change, and the valve control mechanism is controlled to isolate the reagent bottle containing the PBS buffer from the inlet of the microfluidic chip, and simultaneously record the voltage V ₁ at both ends of the photosensitive element;

7. After cleaning the sample bottle, add virus standard solution with volume V and concentration _C2 into the sample bottle; or directly add virus standard solution with volume V and concentration _C2 to one of the reagent bottles;

8. Repeat steps 8.4-8.6, the voltage across the photosensitive element measured in this step is V ₂ ;

9. Repeat steps 8.7-8.8 until all virus standard solutions are detected, and the voltages at both ends of the photosensitive element are respectively measured as V ₃ ...... V _m ;

10. Take the concentration of the virus standard solution as the abscissa and the resistance at both ends of the photosensitive element as the ordinate, draw according to the detected data, get the standard curve after fitting, and obtain the functional relationship of the standard curve according to the standard curve y=k ₁ x+b ₁ ;

11. Detect the virus liquid to be tested with the method of steps 8.3-8.6, measure the voltage at both ends of the photosensitive element as V', and substitute V' into the functional relationship to obtain the virus concentration in the virus liquid to be tested.

Compared with the prior art, the beneficial effects and advantages of the present invention are:

1. The present invention realizes the automation of virus detection, can accurately inject reagents, reduces waste of reagents in manual operations, and can also greatly reduce errors or erroneous diagnoses caused by manual errors.

2. The present invention utilizes the magnetic properties of the magnetic microspheres to realize the separation of viral proteins, and utilizes the black properties of the magnetic spheres, combined with the voltammetric characteristics of the photoresistor, to realize the judgment of the virus concentration in the solution, and to enrich the viral proteins. It is integrated with the content detection on the same device, which simplifies the tedious operation process in the traditional detection process.

3. The present invention is simple in implementation, low in cost, high in efficiency, does not require high-end technical requirements, and effectively simplifies the complex technological process of traditional virus detection.

4. The present invention can be used in preliminary diagnosis in the early stage, can reduce the cost of inspection of precision instruments, can reduce unnecessary waste, and save the cost of detection.

Description of drawings

Figure 1 is a schematic diagram of the structure of a virus detection sensor.

FIG. 2 is a schematic diagram of the structure of the detection body.

FIG. 3 is a circuit diagram of a photoelectric conversion circuit.

FIG. 4 is a schematic structural diagram of a device for detecting virus concentration.

FIG. 5 is a schematic structural diagram of a valve control chip.

FIG. 6 is an exploded view of the structure of the valve control chip.

FIG. 7 is a schematic structural diagram of a microfluidic chip.

FIG. 8 is a graph showing the relationship between the virus concentration in the virus standard solution and the voltage across the photoresistor.

Figure 9 is a standard curve graph.

Among them, 1-capillary glass tube, 2-detection body, 3-C-type clamp, 4-support body, 5-support plate, 6-first groove, 7-second groove, 8-light emitting diode, 9- -Photoresistor, 10-Third groove, 11-Air pump, 12-First pump branch, 13-First valve, 14-First reagent bottle, 15-Second reagent bottle, 16-Third reagent bottle , 17- fourth reagent bottle, 18- fifth reagent bottle, 19- microfluidic chip, 20- multiple pneumatic solenoid valve, 21- controller, 22- valve control chip, 23- Diwen screen, 24- sample Discharge pipe, 25-discharge pipe, 26-second suction branch pipe, 27-second valve, 28-liquid injection pipe, 29-connecting pipe, 30-buffer moving channel, 31-sample moving channel, 32-first An injection port, 33-second injection port, 34-first discharge port, 35-second discharge port, 36-first splint, 37-second splint, 38-liquid inlet pipe, 39-liquid discharge pipe, 40 -Inlet pipe, 41-cover sheet, 42-organic silicon film, 43-substrate, 44-first liquid inlet hole, 45-first liquid discharge hole, 46-gas channel, 47-second liquid inlet hole, 48- The second drain hole, 49- The first main channel, 50- The first liquid inlet channel, 51- The first liquid outlet channel, 52- The second main channel, 53- The second liquid outlet channel channel, 54 - the second liquid inlet channel, 55 - the first waste liquid bottle, 56 - the second waste liquid bottle.

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

Example 1

The structure of the virus detection sensor provided in this embodiment is shown in FIG. 1 , including a bracket, a detection probe, and a capillary glass tube 1 .

The bracket includes a support plate and a clamp, and the clamp has two. The fixture includes a support body 4 and an elastic C-shaped clamp 3. Both the support body 4 and the support plate 5 are square. The support body 4 is symmetrically fixed on one surface of the support plate 5. The plates are in close contact with each other, and a semicircular third groove 10 is symmetrically arranged on the other side of the two support bodies 4 that is parallel to and not in contact with the support plate 5 .

The detection probe includes a detection body 2 and a photoelectric conversion circuit. The photoelectric conversion circuit includes a photoresistor 9 and a light emitting diode 8 . The detection body 2 is in a square shape, the detection body 2 is opaque to light, and a circular detection channel is provided in the center of the detection body 2 . As shown in FIG. 2, the first groove 6 and the second groove 7 are symmetrical on the side wall of the detection channel, the light-emitting diode 8 is fixed in the first groove 6, the photoresistor 9 is fixed in the second groove 7, and the light-emitting diode 8 is fixed in the second groove 7. The stage tube is opposite to the photoresistor, and the photoresistor can receive the light received by the light-emitting diode.

The capillary glass tube 1 is transparent, and the inner wall of the thin glass tube 1 is modified with an antibody that specifically binds to the virus sample to be tested. The capillary glass tube 1 passes through the detection channel, and the capillary glass tube 1 is gap-fitted with the detection channel. The two sides of the capillary glass tube 1 are respectively placed in the two third grooves 10 , and the two sides of the capillary glass tube 1 are respectively fixed on the two supports 4 by the C-shaped clamps 3 .

The method of modifying the antibody on the inner wall of the thin glass tube is as follows:

1. Hydrophilic treatment of capillary glass tube:

1) Take a 5mL reagent tube, add 1ml of hydrogen peroxide, and then take 3ml of concentrated sulfuric acid and slowly add it to the hydrogen peroxide along the wall of the cup;

2) Soak the capillary glass tube in the solution for 1 hour;

3) Clean the capillary glass tube with absolute ethanol;

2. Modification of capillary glass tube:

1) Take 1 mL of absolute ethanol into a centrifuge tube, inject 25 μL APTES into absolute ethanol, and mix evenly (note: after taking APTES, quickly seal the APTES reagent bottle, APTES is easy to hydrolyze) to obtain a mixed solution;

2) Use the injection to take the prepared mixed solution and inject it into the capillary glass tube, use a syringe pump to inject, the speed is 2 μL/min, and the time is about 5 hours. Pay attention to observe whether there are air bubbles during the injection process, and manually adjust if there are air bubbles;

3) Clean the capillary glass tube with absolute ethanol to remove unreacted APTES;

3. Antibody modification

1) Place the EP tube in a METTLER TOLEDO AT20 microbalance and set it to zero, and use the EP tube to weigh 10 μg/ml of EDC4 mg and 2 μL of antibody respectively;

2) Take 98 μL of PBS buffer (PH=7.2) into the EP tube, add EDC and antibody, and mix well to obtain the antibody backup solution;

3) Use a syringe pump to inject the antibody stock solution into the capillary glass tube at a speed of 0.5 μL/min and keep it for half an hour, and the antibody modification on the inner wall of the thin glass tube is completed.

The circuit diagram of the photoelectric conversion circuit is shown in Figure 3. When the light received by the photoresistor changes, the resistance value of the photoresistor changes. According to the principle of voltage division, it can be known that the output voltage will also change accordingly. If the initial value is too large and exceeds the full voltage, you can change the resistor divider value by connecting R1, R2 and R3 with a jumper cap to change the initial value. After changing the resistor value, connect the pin CALI of the PIC12F683 to the ground GND briefly. Afterwards, the circuit will automatically calibrate the voltage value, so that the initial voltage value is about 1/2 of V _CC , so that the changing voltage can be guaranteed to be within a limited range and has reference value.

Example 2

The structure of the device for detecting virus concentration provided in this embodiment is shown in FIG. 4 , including the virus detection sensor, power mechanism, valve control mechanism, reagent supply mechanism, magnetic separation mechanism, detection mechanism, and first waste liquid bottle of Example 1 55 and the second waste liquid bottle 56.

The power mechanism includes a suction pump 11, a first suction branch pipe 12, a first valve 13, a second suction branch pipe 26 and a second valve 27. The first valve 13 and the second valve 27 are both electrical proportional valves. One end of the first suction branch pipe 12 and the second suction branch pipe 26 are both connected to the outlet of the suction pump 11 , the first valve 13 is installed on the first suction branch pipe 12 , and the second valve 27 is installed on the second suction branch pipe 26 .

The reagent supply mechanism includes a first reagent bottle 14 , a second reagent bottle 15 , a third reagent bottle 16 , a fourth reagent bottle 17 , a fifth reagent bottle 18 , a liquid injection pipe 28 and a communication pipe 29 . The other end is communicated with the mouth of the first reagent bottle 14, the mouth of the first reagent bottle 14 is communicated with the mouth of the second reagent bottle 15 through a communication pipe, and the mouth of the second reagent bottle 15 and the mouth of the third reagent bottle 16 are communicated through a communication pipe The mouth of the third reagent bottle 16 is communicated with the mouth of the fourth reagent bottle 17 through a communication pipe, and the mouth of the fourth reagent bottle 17 is communicated with the mouth of the fifth reagent bottle 18 through a communication pipe.

The valve control mechanism includes a multi-channel pneumatic solenoid valve 20, a valve control chip 22 and a controller. Multi-way pneumatic solenoid valve 20 The multi-way pneumatic solenoid valve 20 adopts ITV-1030 electro-pneumatic proportional valve, and the multi-way pneumatic solenoid valve 20 has one air inlet and five air outlets.

The valve control chip includes a clamp and a glass chip. The glass chip includes a cover sheet 41, an organic silicon film 42 and a substrate 43. The organic silicon film adopts PDMS film, the glass chip is a sandwich structure, and the organic silicon film 42 is located between the cover sheet 41 and the substrate. between 43. The fixture includes a first splint 36 and a second splint 37, the glass chip is located between the first splint 36 and the second splint 37, the first splint 36 is sealed with the cover sheet 41, and the second splint 37 is sealed with the substrate 43. , the first clamping plate 36 and the second clamping plate 37 are fixedly connected by bolts, and the cover sheet 41 and the base sheet 43 are fixedly connected by the clamping force between the first clamping plate 36 and the second clamping plate 37 .

The first splint 36 is connected with five liquid inlet pipes 38, two liquid discharge pipes 39 and five air inlet pipes 40, and the cover sheet is respectively provided with five first liquid inlet holes 44, two first liquid discharge holes 45 and There are five gas passage grooves 46 , the gas passage grooves 46 are L-shaped, and the organic silicon film 42 is respectively provided with five second liquid inlet holes 47 and two second liquid discharge holes 48 .

The substrate is respectively provided with a first liquid channel and a second liquid channel. The first liquid channel consists of a first main channel 49, a first liquid outlet channel 51 and two first liquid inlet sub-channels 50. One end of each first liquid inlet sub-channel 50 is connected to the first main channel 49 respectively. One end of the first liquid inlet channel 50 is communicated with one end of the first main channel 49 , and one end of the first liquid outlet channel 50 is communicated with the other end of the first main channel 49 . The second liquid channel consists of a second main channel 52 , a second liquid outlet channel 53 and three second liquid inlet sub-channels 54 . One end of each second liquid inlet sub-channel 54 is connected to the second main channel 52 respectively. One end of the second liquid inlet channel 54 is communicated with one end of the first main channel 52 , and one end of the second liquid outlet channel 53 is communicated with the other end of the second main channel 52 .

The five air outlets are respectively communicated with one end of the five air inlet pipes 40, and the other ends of the five air inlet pipes 40 are respectively communicated with the five gas passage grooves 46, and one of the flat parts of each gas passage groove 46 is on the substrate 43. The projection of is perpendicular to the corresponding first liquid inlet channel 50 or second liquid inlet channel 54 .

One ends of the five liquid injection pipes 28 are respectively connected with the first reagent bottle 14 , the second reagent bottle 15 , the third reagent bottle 16 , the fourth reagent bottle 17 and the fifth reagent bottle 18 , and the other ends of the five liquid injection pipes 28 They are respectively connected with one end of the five liquid inlet pipes 38, the other ends of the five liquid inlet pipes 38 are respectively connected with the five first liquid inlet holes 44, and the five first liquid inlet holes 44 are respectively connected with the five second liquid inlet holes 44 therein. The liquid holes 47 are connected, wherein the two second liquid inlet holes 47 are respectively connected with the other ends of the two first liquid inlet channels 50, and the remaining three second liquid inlet holes 47 are respectively connected with the three second liquid inlet channels. The other end of the channel 54 is connected. One end of the two liquid discharge pipes 39 is connected with the two first liquid discharge holes 45 respectively, the two second liquid discharge holes 48 are respectively connected with the two first liquid discharge holes 45, the first liquid discharge channel 51 and the second liquid discharge groove The other ends of the channel 53 are respectively communicated with the two second liquid discharge holes 48 .

The magnetic separation mechanism includes a microfluidic chip 19 , a sample discharge pipe 24 and a waste discharge pipe 25 . The microfluidic chip adopts the microfluidic chip disclosed in the Chinese invention patent "A Microfluidic Chip for Three-Dimensional Magnetophoretic Separation" (Patent Application No. 201910081980.5). The microfluidic chip includes a first injection port 32, a second The injection port 33 , the first discharge port 34 , the second discharge port 35 , the buffer moving channel 30 and the sample moving channel 31 . The other ends of the two liquid discharge pipes 39 are respectively communicated with the first injection port 32 and the second injection port 33, the first injection port 32 and the first discharge port 34 are respectively communicated with both ends of the buffer moving channel 30, and the second injection port 33 and the second discharge port 35 communicate with both ends of the sample moving channel 31, respectively. The first discharge port 34 communicates with one end of the sample discharge pipe 24 , the other end of the sample discharge pipe 24 communicates with the capillary glass tube 1 , and the second discharge port 35 communicates with the waste discharge pipe 25 . The first waste liquid bottle 55 is located directly below the outlet of the capillary glass tube 1 , and the second waste liquid bottle 56 is located directly below the outlet of the waste discharge pipe 25 .

The controller adopts STM32f107 chip, the photoelectric conversion circuit and the multi-channel pneumatic solenoid valve 20 are respectively connected with the controller 21 , and the controller 21 is connected with the Diwen screen 23 . The Diwen bottle is equipped with a multi-way pneumatic solenoid valve working program. When using it, you only need to click the button to open the detection on the operation interface of the Diwen screen.

Example 3

1. Preparation of antibody-modified magnetic sphere suspension:

1) Place the EP tube in a METTLER TOLEDO AT20 microbalance to zero, and use the EP tube to weigh 4 mg of 10 μg/ml EDC and 2 mg of 5 μg/ml NHS;

2) Take 20 μL of magnetic nanospheres into the EP tube, then inject PBS buffer (PH=6.8) with a pipette, spin and wash on the magnetic stand, suck out the PBS solution with a pipette, and wash the magnetic nanospheres according to this method After washing three times, add 400 μL of PBS solution to the EP tube;

3) Add EDC and NHS to the EP tube, EDC is relatively insoluble, so it should be added first, and then the EP tube is placed in a constant temperature oscillator to shake for 30 minutes;

4) After shaking, wash three times on a magnetic stand with PBS buffer (PH=7.2) to wash off excess EDC and NHS powder;

5) Add H7N9 antibody to the EP tube, shake in a constant temperature oscillator for 4 hours to obtain an antibody-modified magnetic sphere suspension;

2. Take five H7N9 virus stock solutions, add antibody-modified magnetic sphere suspensions to the five virus stock solutions, and carry out a specific reaction, so that the virus to be tested can be completely specifically bound to the antibody. At the same time, the antibody-modified magnetic sphere suspension will Five virus stock solutions were diluted to virus standard solutions with virus concentrations of 5ng/ml, 12.5ng/ml, 16.7ng/ml, 25ng/ml, 50ng/ml and 125ng/ml;

3. Add BSA solution to the first reagent bottle 14 and the third reagent bottle 16 respectively, add PBS buffer to the second reagent bottle 15, add deionized water to the fourth reagent bottle 17, and add the fifth reagent bottle 18 Add 100 μL of virus standard solution with a virus concentration of 5ng/ml;

4. Open the air pump 11 and the second valve 27, click the button to start detection in the Diwen screen 23, and automatically control the multi-way pneumatic solenoid valve 20 to work through the Diwen screen 23, and the multi-way pneumatic solenoid valve 20 automatically opens the corresponding outlet. The gas port is introduced into the corresponding gas channel 46, and the gas presses the corresponding position on the organic silicon film 42 to the first liquid inlet channel 50 connected with the second reagent bottle 15 and the fourth reagent bottle 17 and 17. The second liquid inlet channel 54 connected to the fifth reagent bottle 18 separates the second reagent bottle 15 from the first main channel 49 and the fourth reagent bottle 17 and the fifth reagent bottle 18 from the second main channel 52 , and the first reagent bottle 14 is communicated with the first main channel 49, the third reagent bottle 16 is communicated with the second main channel 52, the first valve 13 is opened, and the BSA solution is passed into the buffer moving channel 30 and the sample moving channel respectively. During the communication of the channel 31, the closed buffer moving channel 30 and the sample moving channel 31 are connected to reduce the non-specific adsorption of the buffer moving channel and the sample moving channel. The first reagent bottle is separated from the first main channel, and the third reagent bottle is separated from the second main channel;

5. The multi-way pneumatic solenoid valve 20 automatically closes the corresponding air outlet, so that the second reagent bottle 15 is communicated with the first main channel 49, and the fourth reagent bottle 17 is communicated with the second main channel 52, so that the PBS buffer is communicated. into the buffer mobile channel 30, the ionized water sample is passed into the mobile channel communication 31, after cleaning, the multi-way pneumatic solenoid valve automatically opens the corresponding outlet, so that the second reagent bottle 15 is isolated from the first main channel 49, The fourth reagent bottle 17 is isolated from the second main channel 52;

6. The multi-way pneumatic solenoid valve 20 automatically closes the corresponding air outlet, so that the second reagent bottle 15 is communicated with the first main channel 49, and the fifth reagent bottle 18 is communicated with the second main channel 52, so that the PBS buffer is communicated. Into the buffer moving channel 30, the virus standard solution is passed into the moving channel communication 31, the magnetic nanospheres in the virus standard solution are enriched in the buffer moving channel, and flow to the capillary glass tube 1, and the magnetic nanospheres are in the capillary. The glass tube is captured at 1, and the magnetic nanospheres will block the light emitted by the light-emitting diode, which will cause the resistance value of the photoresistor 9 to change, and then affect the voltage value across the photoresistor 9. The solenoid valve 20 automatically opens the corresponding air outlet to isolate the fourth reagent bottle from the second main channel. When the Divin screen shows that the voltage at both ends of the photosensitive element no longer changes, the multi-way pneumatic solenoid valve 20 automatically opens the corresponding air outlet. , isolate the second reagent bottle 15 from the first main channel, record the voltage across the photosensitive element, and close the button to start detection on the Divin screen;

7. After cleaning the fifth reagent bottle, put 100 μL of virus standard solution with a virus concentration of 12.5ng/ml into the fifth reagent bottle;

8. Repeat steps 4-6;

9. Repeat steps 7-8 until all virus standard solutions are tested. The test results are shown in the following table:

Virus concentration (ng/ml) 5 12.5 16.7 25 50 125 Voltage value (V) 2.4 2.3 2.13 1.45 0.86 0.53

10. Take the concentration of the virus standard solution as the abscissa and the resistance at both ends of the photosensitive element as the ordinate, and draw according to the detected data, as shown in Figure 8, and obtain the standard curve after fitting, as shown in Figure 9, according to the standard The curve obtains the functional relationship of the standard curve y=-0.037x+2.68;

11. Detect the virus liquid to be tested with the method of steps 3-8, measure the voltage at both ends of the photosensitive element as V', and substitute V' into the functional relationship to obtain the concentration of the virus in the virus liquid to be tested.

Claims (8)

1. a virus detection sensor, is characterized in that: comprise support, detection probe and capillary glass tube, on the capillary glass tube inwall, the antibody that carries out specific binding with virus to be tested is modified, and detection probe comprises detection body and photoelectric conversion circuit, The photoelectric conversion circuit includes a photosensitive element and a light source. The detection body is fixed on the bracket. The center of the detection body is provided with a circular detection channel. The capillary glass tube passes through the detection channel. The capillary glass tube is gap-fitted with the detection channel. The capillary glass tube is fixed on the bracket The first groove and the second groove are symmetrical on the side wall of the detection channel, the light source is fixed in the first groove, the photosensitive element is fixed in the second groove, and the light source is facing the photosensitive element. 2. The virus detection sensor according to claim 1, characterized in that: the bracket comprises a support plate and a clamp, the clamps have two, the clamp comprises a support body and an elastic C-type clamp, and the support body is provided with A semicircular third groove, the diameter of the third groove is equal to the outer diameter of the capillary glass tube, the support body is symmetrically fixed on the support plate, the detection body is located between the two support bodies, and the two sides of the capillary glass tube are placed respectively. in the two third grooves, and the two sides of the capillary glass tube are respectively fixed on the two supports through C-shaped clamps. 3. A device for detecting virus concentration, characterized in that: comprising the virus detection sensor described in claim 1, a power mechanism, a valve control mechanism, a reagent supply mechanism, a magnetic separation mechanism and a detection mechanism; The power mechanism includes a suction pump, a first suction branch pipe and a first valve, one end of the first suction branch pipe is communicated with the outlet of the suction pump, and the first valve is installed on the first suction branch pipe; The reagent supply mechanism includes N reagent bottles, and the other ends of the first air suction branch pipes are respectively connected with the N reagent bottle air paths; The magnetic separation mechanism includes a microfluidic chip, a sample discharge pipe and a waste discharge pipe. Each reagent bottle is controlled by a valve control mechanism to communicate with the inlet liquid path of the microfluidic chip. The microfluidic chip includes a buffer moving channel and a sample moving channel. The buffer moving channel is communicated with the inlet of the capillary glass tube through the sample discharge pipe, and the sample moving channel is communicated with the waste discharge pipe. 4. the device of detecting virus concentration according to claim 1 is characterized in that: described valve control mechanism comprises multi-way pneumatic solenoid valve, controller and valve control chip, and controller is connected with multi-way pneumatic solenoid valve, The pneumatic solenoid valve has one air inlet and N air outlets; The valve control chip includes a clamp and a glass chip. The glass chip includes a cover sheet, an organic silicon film and a substrate. The glass chip is a sandwich structure. The silicone film is located between the cover sheet and the substrate, and the cover sheet and the substrate are fixed by the clamp. connect; The cover sheet is respectively provided with N first liquid inlet holes, two first liquid discharge holes and N gas passage grooves, the N gas passage grooves are all sealed by clamps, and N second liquid inlet holes are respectively provided on the organic silicon film hole and two second drain holes; A first liquid channel and a second liquid channel are respectively provided on the substrate. The first liquid channel consists of a first main channel, a first liquid outlet channel and Q first liquid inlet sub-channels, and each first liquid inlet sub-channel. One end of the channel is communicated with the first main channel respectively, and one of the first liquid inlet channel is communicated with one end of the first main channel, and one end of the first liquid outlet channel is communicated with the other end of the first main channel , the second liquid channel consists of a second main channel, a second liquid outlet channel and P second liquid inlet sub-channels, one end of each second liquid inlet sub-channel is respectively connected with the second main channel, and wherein A second liquid inlet channel is communicated with one end of the second main channel, and one end of the second liquid outlet channel is communicated with the other end of the second main channel, Q+P=N; The N reagent bottles are respectively connected with the N first liquid inlet holes, the N first liquid inlet holes are respectively connected with the N second liquid inlet holes, and the Q second liquid inlet holes are respectively connected with the Q first liquid inlet holes. The other ends of the channels are connected, and the remaining P second liquid inlet holes are respectively connected with the other ends of the P second liquid inlet channels; The N gas outlets are respectively communicated with the N gas passages, and the projection of each gas passage on the substrate separates the corresponding first liquid inlet sub-channel or second liquid inlet sub-channel; The power mechanism also includes a second suction branch pipe and a second valve, one end of the second suction branch pipe is connected to the outlet of the suction pump, the second valve is installed on the second suction branch pipe, and the other end of the second suction branch pipe is connected to the inlet. air connection; The microfluidic chip further comprises a first injection port, a second injection port, a first discharge port and a second discharge port, the first injection port and the first discharge port are respectively connected with both ends of the buffer moving channel, and the second injection port and the second discharge port are respectively connected with the two ends of the sample moving channel, the other ends of the first liquid outlet channel and the second liquid outlet channel are respectively connected with the two second liquid discharge holes, and the two second liquid discharge holes are respectively connected with the two second liquid discharge holes. The first liquid discharge holes are connected, one of the first liquid discharge holes is connected with the first injection port, the first discharge port is connected with one end of the sample discharge pipe, the other end of the sample discharge pipe is connected with the capillary glass tube, and the other first row The liquid hole is communicated with the second injection port, and the second discharge port is communicated with the waste discharge pipe. 5. The device for detecting virus concentration according to claim 4, wherein the clamp comprises a first splint and a second splint, the glass chip is located between the first splint and the second splint, and the first splint and the cover The sheet is sealed and fitted, the second splint is sealed and fitted with the substrate, the first splint and the second splint are fixedly connected by bolts, and N liquid inlet pipes, two liquid discharge pipes and N air inlet pipes are connected on the first splint. The N reagent bottles are respectively connected to one end of the N liquid inlet pipes, the other ends of the N liquid inlet pipes are respectively connected to the N first liquid inlet holes, and one end of the two liquid discharge pipes is respectively connected to the two first liquid discharge holes. The other ends of the two liquid discharge pipes are respectively connected with the first injection port and the second injection port. 6. The device for detecting virus concentration according to claim 4, wherein the reagent supply mechanism further comprises N liquid injection pipes and N-1 communication pipes, and one end of the N liquid injection pipes is respectively connected with N liquid injection pipes. The other ends of the N liquid injection pipes are connected with one end of the N liquid inlet pipes respectively, the other end of the first air suction branch pipe is connected with the bottle mouth of the first reagent bottle, and the bottle mouths of the two adjacent reagent bottles pass through The connecting pipe is connected. 7 . The device for detecting virus concentration according to claim 4 , further comprising a Diwen screen, the virus detection sensor is connected to the controller, and the controller is connected to the Diwen screen. 8 . 8. a method for detecting virus concentration, is characterized in that comprising the steps: 8.1. Modify the magnetic microspheres in the magnetic microsphere suspension to an antibody that can specifically bind to the virus to be tested to obtain an antibody-modified magnetic sphere suspension; 8.2. Add the antibody-modified magnetic sphere suspension to the virus stock solution containing the virus to be tested for reaction, so that the virus to be tested can be completely specifically bound to the antibody, and at the same time, dilute the virus stock solution to the virus concentration of C ₁ , C ₂ ...... C _m virus standard solution, C ₁ , C ₂ ...... C _m increases in turn, C ₁ ≤ 5ng/ml, C _m ≥ 155ng/ml; 8.3. Add blocking reagent to one pair of reagent bottles, add deionized water and PBS buffer to one pair of reagent bottles, add virus standard solution of volume V and concentration C ₁ to one of the reagent bottles, The reagent bottle containing the virus standard solution with a concentration of _C1 is marked as the sample bottle; 8.4. Control the valve control mechanism to connect the two reagent bottles containing the closed reagents with the inlet of the microfluidic chip, so that the two reagent bottles containing the closed reagents are connected to the buffer moving channel and the sample moving channel respectively. The reagents are respectively passed into the communication between the buffer moving channel and the sample moving channel to reduce non-specific adsorption. After the sealing is completed, the valve control mechanism is controlled to separate the two reagent bottles containing the sealing reagent from the inlet of the microfluidic chip; 8.5. Control the valve control mechanism to connect the two reagent bottles containing deionized water and PBS buffer with the inlet of the microfluidic chip, so that the reagent bottle containing PBS buffer is connected to the buffer moving channel, containing deionized water. The reagent bottle is connected with the sample moving channel, the PBS buffer is passed into the buffer moving channel, the ionized water sample is passed into the moving channel, and the cleaning buffer moving channel is connected with the sample moving channel. After cleaning, control the valve to control The mechanism separates the two reagent bottles containing deionized water and PBS buffer from the inlet of the microfluidic chip; 8.6. Control the valve control mechanism to connect the two reagent bottles containing the virus standard solution and the PBS buffer with the inlet of the microfluidic chip, so that the reagent bottle containing the virus standard solution is connected to the sample moving channel, so that the reagent bottle containing the PBS buffer is connected. The reagent bottle is connected with the buffer moving channel, the PBS buffer is passed into the buffer moving channel, the virus standard solution is passed into the sample moving channel, and the magnetic microspheres in the virus standard solution are enriched in the buffer moving channel, and the Flowing into the capillary glass tube, the magnetic microspheres are captured at the capillary glass tube. When the virus standard solution is fully introduced, the valve control mechanism is controlled to isolate the reagent bottle containing the virus standard solution from the inlet of the microfluidic chip. The voltage at both ends does not change, and the valve control mechanism is controlled to isolate the reagent bottle containing the PBS buffer from the inlet of the microfluidic chip, and simultaneously record the voltage V ₁ at both ends of the photosensitive element; 8.7. After cleaning the sample bottle, add virus standard solution with volume V and concentration _C2 to the sample bottle; or directly add virus standard solution with volume V and concentration _C2 to one of the reagent bottles; 8.8. Repeat steps 8.4-8.6. The voltage across the photosensitive element measured in this step is V ₂ ; 8.9. Repeat steps 8.7-8.8 until all virus standard solutions are detected, and the voltages at both ends of the photosensitive element are respectively measured as V ₃ ...... V _m ; 8.10. Take the concentration of the virus standard solution as the abscissa and the resistance at both ends of the photosensitive element as the ordinate, draw according to the detected data, get the standard curve after fitting, and obtain the functional relationship of the standard curve according to the standard curve y=k ₁ x+b ₁ ; 8.11. Detect the virus liquid to be tested with the method of steps 8.3-8.6, measure the voltage at both ends of the photosensitive element as V', and substitute V' into the functional relationship to obtain the virus concentration in the virus liquid to be tested.