CN103792354B - A kind of micro-fluidic paper substrate chip detecting antibody of HCV and preparation method thereof - Google Patents

Abstract

The present invention relates to a kind of micro-fluidic paper substrate chip detecting antibody of HCV and preparation method thereof.Micro-fluidic paper substrate chip according to the present invention comprises multiple miniature sense channel, and each passage is coated with HCV-cAg, HCVNS5 antigen, HCVNS4 antigen, HCVNS3 antigen and combination thereof respectively.Multichannel detectability makes micro-fluidic paper substrate chip can carry out the detection of multiple immunization program simultaneously.By the examination of integrated micro on micro-fluidic paper substrate chip with make a definite diagnosis test, significantly simplify complicated HCV diagnostic procedure, thus significantly improve diagnosis efficiency.

Description

A microfluidic paper-based chip for detecting hepatitis C virus antibody and its preparation method

technical field

The invention relates to the field of disease diagnosis, in particular to a microfluidic paper-based chip for detecting hepatitis C virus antibody, a preparation method thereof, and a detection kit sample.

Background technique

Hepatitis C is an infectious disease caused by the hepatitis C virus (HCV), which spreads globally and has great harm. HCV kills more people than AIDS each year in the United States and is the leading cause of cirrhosis, liver cancer, and the need for liver transplantation [1]. WHO estimates that at least 150 million people worldwide are infected with HCV [2], and in the next ten years, the harm of HCV to global health will continue to increase. The cumulative number of HCV patients in my country has exceeded 43 million. A major reason for the prevalence of HCV is the low diagnostic rate of HCV. Most people with hepatitis C are unaware of their infection. By the time HCV infection is discovered, patients are often at an advanced stage of the disease. Advanced symptoms are not only difficult to cure and reverse, but also consume a lot of medical and social resources. The US Centers for Disease Control and Prevention recommends that people born between 1946 and 1965 (baby boomers, approximately 76 million people) should be diagnosed with HCV [3]. Because of the wide spread and low diagnosis rate of HCV, the disease has been called "the silent pandemic" [4].

One of the main reasons for the low diagnosis rate of HCV is that the current detection and diagnosis methods for HCV have some shortcomings. First, the diagnostic algorithm for HCV is a step-by-step process [5]. The screening results obtained in the first step require a second step of confirmatory testing. The screening diagnostic method is mainly enzyme-linked immunosorbent assay (ELISA), although it has high sensitivity, but its specificity is low, and it is easy to produce false positive results. Therefore, it is necessary to use more specific methods to confirm the diagnosis results. One of the diagnostic methods is recombinant immunoblotting (RIBA). It uses an independent HCV antigen for detection, which can ensure high specificity of detection results. Because the step-by-step diagnosis process is relatively complicated, it will limit the improvement of diagnosis efficiency. Statistics show that the missed diagnosis rate of hepatitis C in my country is as high as 52%. Even in developed countries such as the United Kingdom and the United States, only 25% to 50% of patients can get confirmed results. More than half of the patients did not get a confirmed diagnosis after the screening results were obtained [6]. Therefore, it is not known whether these patients were actually infected with HCV. Unwitting HCV-infected patients will inevitably accelerate the spread of the disease. In addition, the HCV diagnostic techniques themselves (such as ELISA and RIBA tests) are expensive, take a long time (6-7 hours), and consume a large amount of sample (more than 20 microliters) [7]. These all limit the feasibility of HCV diagnosis in large-scale populations, affect the coverage of HCV diagnosis, and eventually lead to the widespread spread and serious harm of HCV.

Microfluidic paper-based chip is a new technology developed in recent years, which is characterized by the use of a special paper base with a certain geometric size and microstructure, so that the liquid can flow in a certain direction within a certain boundary on the paper relying on capillary force [8-11]. Paper base refers to a porous and hydrophilic layered material. The capillary force of the paper base can replace power sources and drive systems such as power supplies and water pumps. In addition, the paper-based chip has the potential and advantages of small size, large specific surface area, and relatively cheap price, with less sample consumption, faster reaction speed, and multiplex reaction (or detection). Microfluidic paper-based chips have great potential to build an integrated, cheap and rapid clinical diagnosis platform.

Contents of the invention

According to one aspect of the present invention, there is provided a microfluidic paper-based chip for detecting hepatitis C virus antibody, which includes more than two detection channels, each of which includes a sample loading area and a detection area; its characteristics in:

Each detection area is coated with one or more selected from the following: HCV core antigen, HCVNS5 antigen, HCVNS4 antigen, HCVNS3 antigen.

In some embodiments, each detection channel may or may not share the same sample loading area. In a preferred embodiment, each detection channel shares the same sample loading area, which makes it possible to realize multi-channel simultaneous sample application with only one drop.

In some embodiments, the distances from the detection area to the sample loading area may be equal or unequal. In a preferred embodiment, the distances from the detection area of each detection channel to the sample loading area are equal. It can be understood that once the sample is loaded, the sample can reach the detection area at the same time, which is convenient for time monitoring of subsequent operations and also helps to shorten the experimental process. On the microfluidic paper-based chip, the area outside the detection area is coated with an inert component selected from the following: BSA, hemocyanin, skimmed milk powder, casein, and ovalbumin.

In some embodiments, more than two detection channels are divided on the paper base; a sample loading area and a detection area are further divided on the detection channel; each detection area is physically connected to the sample loading area through a paper base channel. The liquid can flow from the sample loading area of the paper base to the detection area of the paper base along the paper base channel.

In some embodiments, the microfluidic paper-based chip of the present invention is suitable for chemiluminescent or chromogenic immunodetection.

In some embodiments, 2 to 20 detection channels are included, preferably 3-18, 3-12, 5-12, 5-10 detection channels; most preferably 7 or 8 detection channels.

The term "paper base" as used herein refers to a porous, hydrophilic layered material that allows liquid to flow thereon; the material has a certain geometry that allows control of the flow direction and boundaries of the liquid; the material Part of the region can adsorb or immobilize proteins such as antigens or antibodies.

When each detection channel shares the same sample loading area and the distance from the detection area to the sample loading area of each detection channel is equal, the paper base of the present invention can be embodied as any appropriate shape, including but not limited to the one shown in Figure 1A to Figure 1D shape. Although the specific example of the present invention is based on the specific shape and size shown in FIG. 1A , those skilled in the art will understand that changes made on this basis are also included within the scope of the present invention.

In some embodiments, the paper base is a hydrophilic porous layered material; the paper base is selected from polyvinylidene fluoride, polycarbonate, methyl ethyl cellulose, nitrocellulose paper; wherein methyl ethyl cellulose is preferred Base cellulose, nitrocellulose paper. In a preferred embodiment, the paper base is nitrocellulose paper; nitrocellulose is a nitrated cellulose with nitric acid groups on the backbone of the cellulose that can interact non-covalently with proteins Force, so that the protein is stably adsorbed on the nitrocellulose.

In some specific embodiments, the paper base is Amersham Protran NC nitrocellulose paper. Available nitrocellulose papers have pore sizes of 0.45 microns, 0.2 microns or 0.1 microns. In some embodiments, the paper base is Amersham Protran N 0.45 nitrocellulose paper.

In some embodiments, the microfluidic paper-based chip for detecting antibodies to hepatitis C virus may further include a blank detection channel. The detection area of the blank detection channel is coated with inert components, such as BSA, hemocyanin, skimmed milk powder, casein, and ovalbumin.

In some embodiments, the microfluidic paper-based chip for detecting antibodies to hepatitis C virus may further include a positive control detection channel. The detection area of the positive control detection channel is coated with anti-human IgG antibody. In some embodiments, the microfluidic paper-based chip for detecting antibodies to hepatitis C virus may further include a negative control detection channel.

In some embodiments, the antigen can be a natural antigen or a recombinant antigen.

In some embodiments, the detection zone is 5-6 square millimeters.

In some embodiments, on the microfluidic paper-based chip, other areas except the detection area are coated with an inert protein; preferably bovine serum albumin.

In some embodiments, the length of the detection channel is between 1-15 mm, preferably 7-8 mm; the width is between 1-4 mm, preferably 2 mm.

In a preferred embodiment, the microfluidic paper-based chip for detecting hepatitis C virus antibody includes 8 detection channels, wherein the detection areas of the 5 detection channels are respectively coated with: HCV core antigen, HCVNS5 antigen, HCVNS4 Combination of antigens, HCVNS3 antigens and HCV antigens, the combination of HCV antigens refers to the combination of HCV core antigens, HCVNS5 antigens, HCVNS4 antigens and HCVNS3 antigens; wherein the detection area of one detection channel is coated with anti-human IgG antibodies as Positive control detection channel; wherein the detection area of one detection channel is coated with BSA as a blank detection channel; the detection area of one detection channel is coated with a combination of HCV antigen as a negative control detection channel; the paper base is a pore size of 0.45 micron nitrocellulose paper; the detection area is 5-6 square millimeters, the detection channel length is 5-8 mm, and the detection channel width is 1-4 mm.

In another preferred embodiment, the microfluidic paper-based chip for detecting hepatitis C virus antibody includes 7 detection channels, wherein the detection areas of 4 detection channels are respectively coated with: HCV core antigen, HCVNS5 antigen, The combination of HCV NS4 antigen and HCV antigen, the combination of HCV antigen refers to the combination of HCV core antigen, HCV NS5 antigen and HCV NS4 antigen; wherein the detection area of one detection channel is coated with anti-human IgG antibody as a positive control detection channel; The detection area of one of the detection channels is coated with BSA as a blank detection channel; the detection area of one of the detection channels is coated with a combination of HCV antigens as a negative control detection channel; the paper base is nitrocellulose with a pore size of 0.45 microns plain paper; the detection area is 5-6 square millimeters; the detection channel length is 5-8 mm; the detection channel width is 1-4 mm.

According to another aspect of the present invention, there is provided a test kit for detecting hepatitis C virus antibody, which includes:

The microfluidic paper-based chip for detecting hepatitis C virus antibody of the present invention;

sample diluent;

detergent;

Enzyme-labeled mouse or enzyme-labeled rabbit anti-human IgG; and

Chemiluminescent or chromogenic substrates.

In some embodiments, the kit may also optionally include positive control sera and negative control sera.

In some embodiments, the kit for detecting antibodies against hepatitis C virus may also optionally include one or more of instructions for use, packaging, and a dropper as required.

In some embodiments, the sample diluent is a PBS solution containing 10% (w/w) BSA, pH 7.4.

In some embodiments, the washing solution is a PBS solution containing 0.05% (V/V) Tween-20, pH 7.4.

In some embodiments, the enzyme-labeled mouse or enzyme-labeled rabbit anti-human IgG is mouse or rabbit anti-human IgG labeled with horseradish peroxidase, alkaline phosphatase or β-galactosidase.

In some embodiments, the chemiluminescent substrate or chromogenic substrate is a substrate of horseradish peroxidase, alkaline phosphatase and β-galactosidase; the substrate is selected from the group consisting of: luminol, tetra Methylbenzidine and p-nitrophenyl phosphate. In some embodiments, the substrate is a mixture of luminol and hydrogen peroxide, such as Immobilon Western Chemiluminescent Substrate for HRP (WBKLS0500).

According to another aspect of the present invention, there is provided a method for preparing a microfluidic paper-based chip for detecting hepatitis C virus antibody, comprising the steps of:

1) Provide paper base;

2) Emboss the paper base with a mold to obtain a microfluidic paper base chip;

3) The detection area of the microfluidic paper-based chip is coated with one or more of the following: HCV core antigen, HCVNS5 antigen, HCVNS4 antigen, HCVNS3 antigen, BSA and anti-human IgG antibody, at room temperature dry naturally;

4) Put the microfluidic paper-based chip into BSA and seal it for 20-40 minutes;

In some embodiments, the microfluidic paper-based chip includes 2-20, preferably 3-12, preferably 5-12, most preferably 7 or 8 detection channels; in some embodiments, each detection channel includes one A sample application area and a detection area; in some embodiments, the paper base is methyl ethyl cellulose or nitrocellulose paper; in preferred embodiments, the paper base is nitrocellulose paper. In some embodiments, the detection area is 5-6 square millimeters; the length of the detection channel is 5-8mm; the width of the detection channel is 1-4mm; in some embodiments, the detection channels share the same loading area; in some embodiments , the distance from the detection area of each detection channel to the sample loading area is equal; in a preferred embodiment, in step 2), a metal mold is used to emboss the paper base in the shape shown in Figure 1A to obtain a microfluidic Paper-based chip; in the most preferred embodiment, in step 2), the microfluidic paper-based chip is obtained by embossing the paper base with a metal mold according to the shape and size shown in Figure 1A.

Those skilled in the art understand that after the shape and size of the paper base are determined, a matching metal mold can be made according to the specific shape and size for cutting out the paper base chip of the present invention.

In the prior art, the production of paper-based chips is often carried out at relatively high temperatures, but the manufacturing method of the present application has no special requirements on temperature. In some embodiments, step 2) is carried out at 0-40°C; Preferably 4-35°C, 10-30°C; 15-25°C; most preferably room temperature 18-25°C.

In a specific embodiment, the preparation method of the microfluidic paper-based chip for detecting hepatitis C virus antibody of the present invention comprises the steps of:

1) Provide paper base, said paper base is Amersham ProtranNC0.45 nitrocellulose paper;

2) According to the shape and size shown in Figure 1A, use a mold to imprint on the paper base to obtain a microfluidic paper base chip;

3) The detection area of the microfluidic paper-based chip is respectively coated with: HCV core antigen, HCVNS5 antigen, HCVNS4 antigen, HCVNS3 antigen, a combination of HCV antigens, an anti-human IgG antibody, and BSA. The combination of HCV antigens is Refers to the combination of HCV core antigen, HCVNS5 antigen, HCVNS4 antigen and HCVNS3 antigen; naturally dry at room temperature;

4) Put the microfluidic paper-based chip into BSA and block for 30 minutes to obtain the microfluidic paper-based chip for detecting hepatitis C virus antibody;

The microfluidic paper-based chip includes 8 detection channels, each of which includes a sample loading area and a detection area, the detection channels share the same sample loading area, and the distance between the detection area of each detection channel and the sample loading area is equal.

According to another aspect of the present invention, there is provided a method for detecting hepatitis C virus antibody, comprising the steps of:

1) The biological sample to be tested is obtained from the subject, and the biological sample is selected from whole blood, serum, and plasma;

2) Load the biological samples to be tested, negative and positive control samples onto the microfluidic paper-based chip of the present invention;

3) Add enzyme-labeled mouse or rabbit anti-human IgG;

4) Add chemiluminescence substrate or chromogenic substrate;

5) Read the light intensity.

Description of drawings

Fig. 1A is a microfluidic paper-based chip of the present invention. 1 is nitrocellulose paper, 16 is a detection area, and samples can also be directly added dropwise, 17 is a sample loading area, and 18 is a paper-based channel for liquid flow.

Figures 1B to 1D, microfluidic paper-based chips with 8 detection channels, 20 detection channels and 5 detection channels respectively; the detection areas are marked with dyes.

Figure 2 is a microfluidic paper-based chip for HCV detection.

1 for nitrocellulose paper,

2 combinations coated with HCV antigens,

3 combinations coated with HCV antigens,

4 coated with HCV core antigen,

5 coated with HCVNS5 antigen,

6 coated with HCVNS4 antigen,

7 coated with anti-human IgG antibody,

8 is a blank detection area,

9 is the two detection areas 2 and 3 that make up the ELISA,

10 is the four detection areas 4-7 that make up RIBA,

0 is reserved, and can be coated with HCVNS3 antigen or other antigens or antibodies as needed.

Fig. 3 is a schematic diagram of the reaction principle of the indirect immune reaction detection performed on the paper-based detection area. 1 is nitrocellulose paper, 11 is HCV antigen, 12 is bovine serum albumin, 13 is anti-HCV antibody in human sample, 14 is enzyme-labeled secondary antibody against human IgG, and 15 is enzyme.

Fig. 4A is a chemiluminescent image of HCV patient serum detected using the strategy in Fig. 2 . 1 is nitrocellulose paper; the dotted line is the edge of the microfluidic paper-based chip. The scale is 5 mm.

Fig. 4B is a chemiluminescent image of non-HCV patient serum detected using the strategy in Fig. 2 . 1 is nitrocellulose paper. The dotted line is the edge of the microfluidic paper-based chip. The scale is 5 mm.

5A to 5C are schematic diagrams and characterization experiments of sample injection in the center of the microfluidic paper-based chip. Different volumes of dye liquid (2, 3.5 and 6 microliters) were dropped in the center of the microfluidic paper-based chip (marked by the dotted line). Under the capillary force of the paper base, the liquid can be automatically distributed into the surrounding detection area. Dashed arrows indicate the flow direction of liquid samples.

Fig. 6A is a chemiluminescent image of the results of detecting different volumes of mouse IgG. 1 is nitrocellulose paper. The scale is 5 mm.

Figure 6B is the linearity result of detecting different volumes of mouse IgG. 1 is nitrocellulose paper. The scale is 5 mm.

Fig. 7A is the chemiluminescence images of detecting different concentrations of mouse IgG (668fmol-2.67amol). 1 is nitrocellulose paper. The scale is 5 mm.

Fig. 7B is the non-linear Hill equation relationship for detection of different concentrations of mouse IgG (668fmol-2.67amol). 1 is nitrocellulose paper. The scale is 5 mm.

Fig. 8A is the chemiluminescence images of detecting different concentrations of mouse IgG (26.7 fmol-267 amol). 1 is nitrocellulose paper. The scale is 5 mm.

Fig. 8B is a linear relationship for detection of different concentrations of mouse IgG (26.7fmol-267amol). 1 is nitrocellulose paper. The scale is 5 mm.

Figures 9A, 9B show a pair of intermeshing, sharp-edged metal molds.

Detailed ways

Specific materials and their sources used in specific embodiments of the invention are provided below. However, it should be understood that these are merely exemplary and not intended to limit the present invention, and materials with the same or similar type, model, quality, property or function as the following reagents and instruments can be used to implement the present invention.

Example

Example 1: Preparation method of microfluidic paper-based chip for detection of hepatitis C virus antibody

Step 1: Provide microfluidic substrate

The paper base is Amersham Protran NC0.45 (purchased from GE healthcare).

Step 2: Making Microfluidic Paper-Based Chips

Using a pair of intermeshing metal molds with sharp edges (as shown in Figures 9A-9B ), the microfluidic paper-based chip was fabricated at room temperature by embossing according to the dimensions shown in Figure 1A.

Step 3: Add 0.5 μl of HCV antigen mixture (mixed by ab7978, ab68616 and ab43027, channels 2 and 3), HCV core antigen (purchased from abcam, ab7978, lane 4), HCVNS5 antigen (purchased from abcam, ab68616, lane 5), recombinant HCVNS4 antigen (purchased from abcam, ab43027, lane 6), anti-human IgG antibody (purchased from Sigma-Aldrich, SAB3701331, lane 7); Add BSA dropwise on the detection area of channel 8 as a blank detection area; detection areas 2 and 3 are ELISA detection areas; detection areas 4-7 constitute the RIBA detection area (see Figure 2). Let dry at room temperature.

Step 4: Immerse the microfluidic paper-based chip obtained in Step 3 into a 5% (w/w) BSA solution for sealing, and take it out after 30 minutes.

Step 5: Add desiccant as needed. After packaging, store in a refrigerator at 2-8 degrees for later use.

Example 2. Assembly of the kit

Assemble into a kit according to the following composition:

Microfluidic chip prepared by the method described in Example 1; horseradish peroxidase-labeled secondary antibody, sample diluent, washing solution and substrate. According to needs, the kit can also be equipped with accompanying supplies such as instructions for use, packaging, and droppers.

test case

Test Example 1: Detection of Human Serum Samples

Step 1: Acquisition of Human Serum Samples

The positive and negative sera came from discarded serum from the Laboratory Department of Peking Union Medical College Hospital, which complied with the corresponding ethical principles and norms.

Step 2: Sample loading

On the chip prepared in Example 1, a 50-fold diluted serum sample (0.5 μl) was added dropwise to each detection area. Serum diluent was PBS solution (pH 7.4) containing 10% w/w BSA. After one minute, the chip was placed in PBS solution (pH 7.4) containing 0.05% (v/v) Tween-20, and excess serum samples were washed away. Repeat the washing step two more times. Add negative control serum (0.5 μl) dropwise to the negative control detection channel.

Step 3: Soak the microfluidic paper-based chip in HRP-labeled mouse anti-human IgG solution (purchased from CST, 7076) (1:5000) for 3 minutes to form the immune complex shown in Figure 3.

Step 4: Put the microfluidic paper-based chip into 0.05% v/v Tween-20, PBS solution, wash for 2 minutes, and repeat twice.

Step 5: Mix the chemiluminescent substrate (Milipore, Immobilon Western Chemiluminescent Substrate for HRP, WBKLS0500), take 250 microliters on the microfluidic paper-based chip, and suck it out after 3 minutes.

Step 6: Put the microfluidic paper-based chip in a chemiluminescence detector (Las4000) for detection, with an exposure time of 15 minutes to obtain a chemiluminescence image, as shown in Figure 4A and Figure 4B.

Step 7: Process the image to obtain the data shown in Table 1.

result:

(1) The results of ELISA can be obtained in detection areas 2 and 3, expressed as the ratio of signal value to critical value (S/Co); the critical value is taken from the signal value of negative serum samples; detection areas 4 to 7 can be obtained RIBA results, expressed as the ratio of the signal value to the signal value of the positive control. The HCV patient sera all obtained positive results in detection areas 3, 4, 5, 6, and 7. Serum from non-HCV patients only obtained positive control results in the No. 7 detection area.

(2) Detection time: If the ELISA detection method is used, the incubation time for each step needs at least 30 minutes to 1 hour. If you use the RIBA method, it will be slower, such as the one produced by Novartis kit, the incubation step alone takes 4-4.5 hours. However, the chip of the present application makes it possible to shorten the incubation process to a few minutes; even the entire detection process is less than 30 minutes (from sample loading to chemiluminescent color development).

(3) The amount of detection reagents: the sample volume required for using a microfluidic chip is 0.5 microliters, while the ELISA experiment is 50-100 microliters, and RIBA uses 20-50 microliters, so it can only be used for some precious samples. Small amounts get results. The amount of reagents used in microfluidic chips (such as enzyme-labeled secondary antibodies) is about one-twentieth of the amount of conventional reagents, which can reduce the amount of reagents and thus reduce costs.

Table 1 shows the detection results of positive sera and three patient sera using the microfluidic paper-based chip of the present invention and the comparison with the ELISA kit. The diagnostic test results of the paper-based chip were consistent with ELISA, and the S/Co was slightly higher. The test results of a single antigen test are counted with 0.4 as the threshold, which can well distinguish positive and negative results. If the sample has more than two single antigen signal values greater than 0.4, it is judged as positive, while only one or less single antigen signal value is greater than 0.4, it is judged as negative.

Table 1. Comparison between the microfluidic paper-based chip of the present invention and the ELISA kit

Test Example 2: Linearity Test

Step 1: Make a microfluidic paper-based chip according to steps 1-2 of Example 1.

Step 2: Add different volumes of mouse IgG solution (1 μM) dropwise to the detection area, and let it dry at room temperature.

Step 3: Immerse the entire microfluidic paper-based chip in 5% BSA solution for sealing, and take it out after 30 minutes.

Step 4: Soak the microfluidic paper-based chip in HRP-labeled anti-mouse IgG solution (1:5000) for 15 minutes.

Step 5: Put the microfluidic paper-based chip into 0.05% Tween-20 PBS pH7.4 solution, wash for 5 minutes, and repeat three times.

Step 6: Mix the chemiluminescent substrate, take 250 microliters onto the microfluidic paper-based chip, and aspirate after 3 minutes.

Step 7: Put the microfluidic paper-based chip in a chemiluminescence detector (Las4000) for detection, with an exposure time of 15 minutes to obtain a chemiluminescence image, as shown in Figure 6A.

Step 8: Perform data processing and analysis on the chemiluminescence image to obtain the relationship between the luminescence intensity and the volume of the dripping solution, as shown in Figure 6B, and the correlation coefficient is 0.988.

Test Example 3: Linearity Test

Step 1: Make a microfluidic paper-based chip according to steps 1-2 of Example 1.

Step 2: Drop different concentrations of mouse IgG solution (0.5μl) on the detection area and let it dry at room temperature.

Step 3: Immerse the entire microfluidic paper-based chip in 5% BSA solution for sealing, and take it out after 30 minutes.

Step 4: Soak the microfluidic paper-based chip in HRP-labeled anti-mouse IgG solution (1:5000) for 15 minutes.

Step 5: Put the microfluidic paper-based chip into 0.05% Tween-20 PBS pH7.4 solution, wash for 5 minutes, and repeat three times.

Step 6: Mix the chemiluminescent substrate, take 250 microliters onto the microfluidic paper-based chip, and aspirate after 3 minutes.

Step 7: Put the microfluidic paper-based chip in a chemiluminescence detector (Las4000) for detection, with an exposure time of 15 minutes to obtain a chemiluminescence image, as shown in Figures 7A-7B and 8A-8B.

Step 8: Perform data processing and analysis on the chemiluminescence image to obtain the relationship between the luminescence intensity and the volume of the dripping solution. In the range from 667fmol to 267amol, the luminous intensity has a nonlinear relationship with the concentration, which fits well with the Hill equation, and the correlation coefficient is 0.999 (Figure 7A-7B). In the range from 26.7 fmol to 267 amol, the luminous intensity has a linear relationship with the concentration. The correlation coefficient was 0.995 and the detection limit was 267 amol (Fig. 8A-8B).

summary:

The invention builds a microfluidic paper-based chip with multiple channels on the nitrocellulose film, which can make each detection channel independent of each other and minimize cross-contamination. Each channel has a detection area. The detection area is small, about 5-6 square millimeters. Different HCV antigens or their mixtures were dropped on the detection area respectively, and allowed to dry naturally at room temperature. Because the nitrocellulose membrane has good protein adsorption capacity, the protein in the dripping liquid is immobilized in the corresponding detection area. Subsequently, the paper-based chip was placed in 3%-5% BSA for sealing. The serum sample is added dropwise to the sample loading area, and is transported to the detection area by capillary force or directly dropped to the detection area. The types (or quantities) of proteins immobilized in each detection area are different, so each detection area can perform different immunoassays. After the HCV antigen captures the human HCV antibodies in the serum, excess samples are washed away by three washes. After adding an anti-human IgG enzyme-labeled secondary antibody and corresponding washing, chemiluminescence or colorimetric detection can be realized by adding an enzyme substrate. Immobilizing the detection area of the HCV antigen mixture can serve the purpose of ELISA detection, while the immunoassay of immobilizing a single HCV antigen can serve the purpose of RIBA detection. Other detection areas of the microfluidic paper-based chip can also be used to detect IgG in serum as a positive control. The multiplex detection capability enables the microfluidic paper-based chip to perform multiple serum immunoassays at the same time. Improving the efficiency of HCV detection and diagnosis by integrating screening and confirmatory tests on a microfluidic paper-based chip.

By integrating the traditional multi-step HCV detection method on the microfluidic paper-based chip, the HCV diagnosis process is greatly simplified and the diagnostic efficiency is greatly improved. Main beneficial effects of the present invention are as follows:

1) Integrating screening tests and confirmatory tests into one-step testing simplifies the diagnostic process.

2) Since the area of each detection area on the paper-based chip is small, the sample loading volume can be very small, such as 300 nanoliters, and the reagent consumption of serum can even be as low as 6 nanoliters. Such low serum consumption will allow fingertip blood to replace venous blood draws. This not only causes less harm to patients, but also is easy to operate and detect, and is suitable for large-scale blood collection, thereby further improving diagnostic efficiency.

3) Because the specific surface area of the paper-based detection area is large, the diffusion distance is small. Incubation and washing times for samples can be significantly shortened. For example, the sample incubation time is only 1 minute, and it only takes about 30 minutes to complete a complete detection process.

4) Using cheap paper base instead of plastic orifice plate, the consumption of reagents is also greatly reduced due to the reduction in size, so the cost of the entire detection platform is also greatly reduced accordingly. The cost of a paper-based detection unit is roughly 1/25 of the cost of a well in a 96-well plate ELISA.

In conclusion, improving the diagnostic strategy and technology of HCV through the microfluidic paper-based chip technology can improve the diagnostic efficiency and coverage, and ultimately can control the spread and harm of HCV more effectively.

references:

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Claims (13)

1. A microfluidic paper-based chip for detecting hepatitis C virus antibody, which includes 8 detection channels, each of which includes a sample loading area and a detection area; The detection channels share the same sample loading area; The distance from the detection area of each detection channel to the sample loading area is equal; The detection areas of the five detection channels are respectively coated with: a combination of HCV core antigen, HCVNS5 antigen, HCVNS4 antigen, HCVNS3 antigen and HCV antigen, and the combination of HCV antigens refers to HCV core antigen, HCVNS5 antigen, HCVNS4 antigen and HCVNS3 antigen in combination; The detection area of one of the detection channels is coated with an anti-human IgG antibody as a positive control detection channel; The detection area of one of the detection channels is covered with BSA as a blank detection channel; The detection area of one of the detection channels is coated with a combination of HCV antigens as a negative control detection channel; The paper base is a nitrocellulose paper with a pore size of 0.45 micron; The detection area is 5-6 square millimeters; The detection channel length is 5-8mm; The detection channel width is 1-4mm; The HCV core antigen is ab7978 of abcam, the HCVNS5 antigen is ab68616 of abcam, and the recombinant HCVNS4 antigen is ab43027 of abcam. 2. A microfluidic paper-based chip for detecting hepatitis C virus antibody, which includes 7 detection channels, each of which includes a sample loading area and a detection area; The detection channels share the same sample loading area; The distance from the detection area of each detection channel to the sample loading area is equal; Wherein the detection areas of the four detection channels are coated with: HCV core antigen, HCVNS5 antigen, HCVNS4 antigen, and HCV antigen combination, the HCV antigen combination refers to the HCV core antigen, HCVNS5 antigen and HCVNS4 antigen combination ; The detection area of one of the detection channels is coated with an anti-human IgG antibody as a positive control detection channel; The detection area of one of the detection channels is covered with BSA as a blank detection channel; The detection area of one of the detection channels is coated with a combination of HCV antigens as a negative control detection channel; The paper base is nitrocellulose paper with a pore size of 0.45 micron; The detection area is 5-6 square millimeters; The detection channel length is 5-8mm; The detection channel width is 1-4mm; The HCV core antigen is ab7978 of abcam, the HCVNS5 antigen is ab68616 of abcam, and the recombinant HCVNS4 antigen is ab43027 of abcam. 3. The microfluidic paper-based chip for detecting hepatitis C virus antibody according to claim 1 or 2, wherein the paper base is AmershamProtranNC0.45 type nitrocellulose paper. 4. A test kit for detecting hepatitis C virus antibody, comprising: The microfluidic paper-based chip for detecting hepatitis C virus antibody described in any one of claims 1-3; sample diluent; detergent; Enzyme-labeled mouse or enzyme-labeled rabbit anti-human IgG; and Chemiluminescent or chromogenic substrates. 5. The test kit for detecting hepatitis C virus antibody according to claim 4, further comprising positive control serum and negative control serum. 6. The test kit for detecting hepatitis C virus antibody according to claim 4, wherein: The sample diluent is a PBS solution containing 10% (w/w) BSA, pH7.4; The washing solution is a PBS solution containing 0.05% (v/v) Tween-20, pH 7.4. 7. The test kit for detecting hepatitis C virus antibody according to claim 4, wherein: The enzyme-labeled mouse or enzyme-labeled rabbit anti-human IgG is mouse or rabbit anti-human IgG labeled with horseradish peroxidase, alkaline phosphatase or β-galactosidase; The chemiluminescent substrate or chromogenic substrate is the substrate of horseradish peroxidase, alkaline phosphatase and β-galactosidase. 8. The test kit for detecting hepatitis C virus antibody according to claim 7, wherein said chemiluminescent substrate or chromogenic substrate is selected from: luminol, tetramethylbenzidine and p-nitrophenyl phosphate . 9. The kit for detecting hepatitis C virus antibody according to claim 4, further comprising one or more selected from the following: instructions, packaging, dropper. 10. A method for preparing a microfluidic paper-based chip for detecting hepatitis C virus antibody, comprising the steps of: 1) Provide paper base; 2) Using a pair of intermeshing metal molds with sharp edges to imprint on the paper base to obtain a microfluidic paper base chip; 3) The detection area of the microfluidic paper-based chip is coated with one selected from the following: HCV core antigen, HCVNS5 antigen, HCVNS4 antigen, HCVNS3 antigen, HCV antigen combination, BSA and anti-human IgG antibody. Dry naturally at room temperature; 4) Blocking the microfluidic paper-based chip with BSA for 20-40 minutes to obtain a microfluidic paper-based chip for detecting hepatitis C virus antibody; Wherein the combination of HCV antigens refers to: a combination of HCV core antigens, HCVNS5 antigens, HCVNS4 antigens and HCVNS3 antigens, or a combination of HCV core antigens, HCVNS5 antigens and HCVNS4 antigens; Wherein the microfluidic paper-based chip includes 5-12 detection channels, each of which includes a sample loading area and a detection area, and each detection channel shares the same sample loading area; The paper base is methyl ethyl cellulose or nitrocellulose paper; The detection area is 5-6 square millimeters; The detection channel length is 5-8mm; The detection channel width is 1-4mm. 11. The method according to claim 10, wherein the HCV core antigen is ab7978 of abcam, the HCVNS5 antigen is ab68616 of abcam, and the recombinant HCV NS4 antigen is ab43027 of abcam. 12. The method according to claim 10, wherein the microfluidic paper-based chip comprises 7 or 8 detection channels. 13. The method according to claim 10, wherein the distances from the detection area of each detection channel to the sample loading area are equal.