A microfluidic chip for detecting entamoeba histolytica infection

Abstract

This invention relates to a method for preparing a microfluidic chip for detecting Entamoeba histolytica infection. The preparation method includes the following steps: S1, providing a microfluidic chip, the microfluidic chip including channels; S2, modifying the channels so that the surface of the channels can bind Entamoeba histolytica recombinant antigens; S3, binding the Entamoeba histolytica recombinant antigens to the surface of the channels, such that each Entamoeba histolytica recombinant antigen bound to the surface of the channels can bind at most one Entamoeba histolytica-specific antibody, and each Entamoeba histolytica-specific antibody can be bound by at most one gold nanosphere bound to anti-human IgG antibody; S4, blocking the sites on the surface of the channels that are not bound to the Entamoeba histolytica recombinant antigens. The method for detecting Entamoeba histolytica infection of this invention has relatively high sensitivity and significantly improves detection efficiency.

Description

Technical Field

[0001] This invention relates to the field of immunodiagnostic technology, and in particular to a microfluidic chip for detecting Entamoeba histolytica infection. Background Technology

[0002] Entamoeba histolytica is a pathogenic protozoan that causes amoebic dysentery or amoebic liver abscess after infection. It can cause severe diarrhea, abdominal pain, fever, and abdominal discomfort. If left untreated, it can be life-threatening. Studies have shown that when a person is infected with Entamoeba histolytica, specific antibodies against Entamoeba histolytica are produced in the serum. These specific antibodies can bind to certain structural proteins of Entamoeba histolytica to form immune complexes. By detecting these immune complexes and combining them with other symptoms, such as amoebic liver abscess, it is possible to diagnose Entamoeba histolytica infection, acute amoebic dysentery, and chronic amoebic dysentery. At the same time, it can also be used to monitor the effectiveness of treatment and predict recurrence.

[0003] Currently, the main techniques used to detect specific antibodies against Entamoeba histolytica in serum include indirect hemagglutination assay (IHA), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay (IFA). These techniques are all based on the principle of antigen-antibody binding. However, they have certain limitations in practical applications. For example, IHA has low sensitivity and specificity and may produce false positives due to cross-reactions with other parasites, making it unsuitable for detecting early infections. While ELISA and IFA methods are relatively sensitive, they are expensive, require specialized equipment and well-trained personnel, and involve long testing times, labor-intensive operations, and large consumption of reagents and samples.

[0004] Microfluidic chips are a technology that combines miniaturization, portability, high sensitivity, and speed. They achieve microprocessing and precise quantitative control of samples through microchannels and microreaction devices at the micrometer level, showing broad application prospects in the field of biodetection and analysis. Digital immunoassay, on the other hand, uses a high-sensitivity imaging system to image the chip, and then employs digital signal processing algorithms to count and quantify target molecules, achieving high sensitivity and other advantages. It can be applied in fields such as bioanalysis, molecular diagnostics, and drug screening.

[0005] Therefore, there is an urgent need for a microfluidic chip for detecting Entamoeba histolytica infection. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a microfluidic chip for detecting Entamoeba histolytica infection.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A first aspect of the present invention is to provide a method for fabricating a microfluidic chip, the microfluidic chip being used to detect Entamoeba histolytica infection, the fabrication method comprising the following steps:

[0009] S1. A microfluidic chip is provided, the microfluidic chip comprising: channels;

[0010] S2. Modify the channel so that the surface of the channel can bind recombinant Entamoeba histolytica antigen;

[0011] S3. The recombinant antigen of Entamoeba histolytica is bound to the surface of the channel, so that each recombinant antigen of Entamoeba histolytica bound to the surface of the channel can bind to at most one Entamoeba histolytica-specific antibody, and each Entamoeba histolytica-specific antibody can be bound to at most one gold nanosphere bound to anti-human IgG antibody.

[0012] S4. Block the sites on the surface of the channel that are not bound to the recombinant antigen of Entamoeba histolytica.

[0013] Preferably, in step S2, the modification includes: passing a modifying reagent through the channel and washing the channel with deionized water, wherein...

[0014] The modifying agent includes at least one of polylysine, epoxy resin, or polyethylene glycol.

[0015] Preferably, in step S3, the binding of the recombinant Entamoeba histolytica antigen includes: allowing a solution of the recombinant Entamoeba histolytica antigen to flow through the channel, wherein,

[0016] The concentration of the solution of the recombinant Entamoeba histolytica antigen is 40 pg / μL, and the flow rate of the solution is 3 mm / s. Thus, the distance between adjacent recombinant Entamoeba histolytica antigens bound to the surface of the channel is greater than 60 nm. Therefore, each recombinant Entamoeba histolytica antigen bound to the surface of the channel can bind to at most one Entamoeba histolytica-specific antibody, and each Entamoeba histolytica-specific antibody can be bound to at most one gold nanosphere conjugated with anti-human IgG antibody. Other combinations of concentration and flow rate that result in a distance between adjacent recombinant Entamoeba histolytica antigens bound to the surface of the channel greater than 60 nm are also included within the scope of this patent.

[0017] Preferably, in step S4, the sealing includes: allowing phosphate buffer containing skim milk to flow through the channel, and washing the channel with PBST washing solution.

[0018] A second aspect of the present invention is to provide a microfluidic chip, fabricated using the method described above, the microfluidic chip comprising: an inlet, an outlet, and a channel for connecting the inlet and the outlet; wherein,

[0019] The channel includes: an inlet channel connected to the inlet pipe, a first bend section connected to the inlet channel pipe, an S-shaped channel connected to the first bend section pipe, a second bend section connected to the S-shaped channel pipe, and an outlet channel connected to the second bend section pipe.

[0020] The first curved segment and the second curved segment are located in the imaging region;

[0021] The sample outlet channel is connected to the sample outlet pipeline.

[0022] A third aspect of the present invention is to provide a method for detecting Entamoeba histolytica infection, employing a microfluidic chip as described above, the method comprising the steps of:

[0023] A1. Inject the biological sample into the channel through the inlet, and after the biological sample flows through the channel, it flows out of the channel through the outlet.

[0024] A2. After washing the channel with PBST washing solution, inject the solution of gold nanospheres bound with anti-human IgG antibody into the channel through the injection port, so that the solution of gold nanospheres bound with anti-human IgG antibody flows through the channel.

[0025] A3. Take an image of the imaging area to identify the number of gold nanospheres bound with anti-human IgG antibodies within the imaging area.

[0026] Preferably, the biological sample is at least one of plasma or serum.

[0027] Preferably, the determination result is obtained by subtracting the number of gold nanospheres bound to anti-human IgG antibodies in the second curved segment of the imaging region from the number of gold nanospheres bound to anti-human IgG antibodies in the first curved segment of the imaging region.

[0028] Preferably, if the judgment result of the biological sample is higher than that of the negative control, then the biological sample is infected with Entamoeba histolytica; if the judgment result of the biological sample is not higher than that of the negative control, then the biological sample is not infected with Entamoeba histolytica.

[0029] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0030] The method for detecting Entamoeba histolytica infection of the present invention has relatively high sensitivity and can obtain a high signal-to-noise ratio in a short time, thereby greatly improving detection efficiency. At the same time, the detection cost is low and less experimental waste is generated. It is a rapid qualitative detection method for Entamoeba histolytica. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the microfluidic chip in Embodiment 1 of the present invention;

[0032] The attached figures are labeled as follows: 1 for the inlet, 2 for the outlet, and 3 for the imaging area;

[0033] Figure 2 The image shows the detection results of 25 serum samples in Example 3 of this invention;

[0034] Figure 3 This is a diagram showing the number of gold nanospheres bound to anti-human IgG antibodies in the first curved segment of imaging region 3 in the standard positive serum of Embodiment 3 of the present invention.

[0035] Figure 4 This is a diagram showing the number of gold nanospheres bound to anti-human IgG antibodies in the second curved segment of imaging region 3 in Example 3 of the present invention, based on the standard positive serum.

[0036] Figure 5 This is a diagram showing the number of gold nanospheres bound to anti-human IgG antibodies in the first curved segment of imaging region 3 in the standard negative serum of Embodiment 3 of the present invention.

[0037] Figure 6 This is a diagram showing the number of gold nanospheres bound to anti-human IgG antibodies in the second curved segment of imaging region 3 in Example 3 of the present invention, representing the standard negative serum.

[0038] Figure 7 This is a graph showing the detection results of 25 serum samples in the comparative examples of this invention;

[0039] Figure 8 This is a comparison chart of the detection results of Embodiment 3 and the comparative example of the present invention. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0043] Example 1

[0044] like Figure 1 As shown, this embodiment provides a microfluidic chip, including: an inlet 1, an outlet 2, and a channel for connecting the inlet 1 and the outlet 2; wherein,

[0045] The channel is 16cm long, 200μm wide, and 25μm high; the channel includes: an injection channel connected to the injection port 1, a first bend connected to the injection channel, an S-shaped channel connected to the first bend, a second bend connected to the S-shaped channel, and an outlet channel connected to the second bend.

[0046] The first curved segment and the second curved segment are located in imaging region 3, and the cross-sectional area of ​​imaging region 3 is 1750 pixels × 1750 pixels.

[0047] The sample outlet channel is connected to the sample outlet 2 pipeline.

[0048] This embodiment also provides a method for fabricating the microfluidic chip, the steps of which include:

[0049] After drying a 3-inch silicon wafer with a 95°C hot plate, SU-8 photoresist is applied to the center of the wafer. The SU-8 photoresist is then evenly distributed using a spin coater, increasing from 100 rpm to 500 rpm and maintaining this speed for 10 seconds, then increasing from 300 rpm to 3000 rpm and maintaining this speed for 40 seconds. The wafer is then sequentially heated with a 65°C hot plate for 2 minutes, a 95°C hot plate for 5 minutes, and a 65°C hot plate for 6 minutes. Afterward, the wafer is exposed to UV light for 30 seconds. This process is repeated, followed by further heating with a 65°C hot plate for 2 minutes, a 95°C hot plate for 5 minutes, and a 65°C hot plate for 6 minutes. The wafer is then developed for 1-3 minutes with a negative developer containing propylene glycol methyl ether acetate. After rinsing with isopropanol and drying with nitrogen, the wafer is heated with a 100°C hot plate for 2 minutes. To ensure a stronger bond between the photoresist and the silicon wafer surface, a drop of trimethylchlorosilane is placed on the etched channel side of the silicon wafer and transferred to a plastic petri dish, with the etched channel side facing upwards. PDMS A and B components are mixed at a 10:1 mass ratio and poured into the plastic petri dish, then vacuumed to remove air bubbles. The petri dish is then placed in an 80°C oven for 1 hour to cure the PDMS. The PDMS layer is then completely peeled off the silicon wafer and cut along the edge of the microfluidic chip. A through-hole is drilled at both the inlet (1) and outlet (2) using a 0.75mm punch. A 22cm × 60cm × 0.17mm (thickness) optical slide is taken, cleaned sequentially with anhydrous ethanol and ultrapure water, and dried with nitrogen. The PDMS layer and the optical slide are treated with oxygen plasma for 5 seconds each. The channel side of the PDMS layer faces the optical slide, and the PDMS layer is then bonded to the optical slide.

[0050] Example 2

[0051] This embodiment provides a microfluidic chip for detecting Entamoeba histolytica infection and its preparation method, wherein the preparation method includes the following steps:

[0052] S1. Provide a microfluidic chip as described in Embodiment 1, transfer the microfluidic chip to a 40x objective lens (Olympus, UPLFLN40X, air lens NA0.95), and adjust the focal plane to the lower surface of the channel in the microfluidic chip;

[0053] S2. 5 μL of poly-L-lysine (Sigma, RNBJ6951) is injected into the channel at 3 mm / s through the injection port 1. After incubation for 5 min, it is collected through the outlet port 2 so that the surface of the channel can bind the recombinant antigen of Entamoeba histolytica.

[0054] 10 μL of ultrapure water is injected into the channel at 3 mm / s through the inlet 1 and collected through the outlet 2.

[0055] S3. A solution of 10 μL of 40 pg / μL Entamoeba histolytica recombinant antigen is injected into the channel at 3 mm / s through the injection port 1. After incubation for 10 min, the solution is collected through the outlet port 2 so that the surface of the channel is bound to the Entamoeba histolytica recombinant antigen.

[0056] S4. Inject 10 μL of 3% skim milk (BD Difco) through the injection port 1. TM Phosphate buffer (Sketch Milk, BD, 1053907) was injected into the channel at 3 mm / s, and after incubation for 10 min, it was collected through the outlet 2 to block the unbound sites of the recombinant Entamoeba histolytica antigen on the surface of the channel.

[0057] 20 μL of PBST washing solution is injected into the channel at 3 mm / s through the inlet 1 and collected through the outlet 2;

[0058] The nucleotide sequence corresponding to the recombinant histolytica antigen is shown in SEQ ID NO:1, TCAAATGGTGAATGTAAGCCATGTACCAATCATTGTAGTGAATGTAGT.

[0059] Example 3

[0060] This embodiment provides a method for detecting Entamoeba histolytica infection, using a microfluidic chip as described in Embodiment 2. The steps of the method include:

[0061] A1. After diluting the serum 400 times, inject 2 μL of the diluted serum into the channel at 3 mm / s through the injection port 1, and collect it through the outlet port 2;

[0062] A2. Inject 20 μL of PBST washing solution into the channel at 3 mm / s through the inlet 1 and collect it through the outlet 2.

[0063] 5 μL of 10 μL was injected through the injection port 1. 8A solution of gold nanospheres (60 nm in diameter) conjugated with anti-human IgG antibodies per μL was injected into the channel at a rate of 3 mm / s and reacted for 10 min.

[0064] A3. Take an image of the imaging area 3, remove the background of the microscopic imaging using ImageJ, and identify the number of gold nanospheres bound to anti-human IgG antibodies in the first curved segment of the imaging area 3 and the number of gold nanospheres bound to anti-human IgG antibodies in the second curved segment of the imaging area 3 respectively. The difference between the two is the judgment result.

[0065] Using standard positive serum as a positive control and standard negative serum as a negative control, the experiment was repeated 20 times. The sum of the average of the 20 negative control judgment results and the standard deviation between the 20 negative control judgment results was used as the judgment criterion.

[0066] If the serum's judgment result is higher than the judgment standard, then the serum is infected with Entamoeba histolytica; if the serum's judgment result is not higher than the judgment standard, then the serum is not infected with Entamoeba histolytica.

[0067] The method for preparing the solution of gold nanospheres conjugated with anti-human IgG antibodies includes:

[0068] Take 1 mL of 60 nm PEG-modified gold nanoparticles (Xianfeng Nano, 101710), centrifuge at 13000 rpm for 10 min, and remove the supernatant; add 1 mL of 10 mmol / L marin ethanesulfonic acid buffer to an EP tube; add 1000 μL of 1 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (Aladdin) and 200 μL of 1 mg / mL N-hydroxythiosuccinimide sodium salt (Aladdin); redisperse the PEG-modified gold nanoparticles in MES buffer, add 50 μg of HRP-labeled anti-human IgG antibody (Momentive Specialty Chemicals (NY), RTV-615-044), and incubate at 37 °C in the dark for 2 h; centrifuge and wash to remove unbound anti-human IgG antibody, redisperse the EG-modified gold nanoparticles in 1 mL of water, and store at 4 °C in the dark.

[0069] Specifically, the test results of 25 known serum samples are as follows: Figure 2 As shown, the test results for organisms (healthy individuals) corresponding to samples 1-15 and organisms (cryptospora-infected individuals) corresponding to samples 23-25 ​​were negative, while the test results for organisms (patients) corresponding to samples 16-22 were positive.

[0070] Specifically, such as Figure 3As shown, the number of gold nanospheres bound to anti-human IgG antibodies in the first curved segment of imaging region 3, as indicated, is 12028. Figure 4 As shown, the number of gold nanospheres bound to anti-human IgG antibodies in the second curved segment of imaging region 3 of the standard positive serum is 1395, and the judgment result is 10633; Figure 5 As shown, the number of gold nanospheres bound to anti-human IgG antibodies in the first curved segment of imaging region 3 of the standard negative serum is 308. Figure 6 As shown, the number of gold nanospheres bound to anti-human IgG antibodies in the second curved segment of the standard negative serum in imaging region 3 is 55, and the judgment result is 253. The standard negative serum was repeated 20 times in parallel. The average of the judgment results of the 20 negative controls (641) and the standard deviation between the judgment results of the 20 negative controls (321) were summed to obtain a judgment standard of 1604. That is, if the judgment result of the serum is higher than 1604, the serum is infected with Entamoeba histolytica (positive); if the judgment result of the serum is not higher than 1604, the serum is not infected with Entamoeba histolytica (negative).

[0071] Comparative Example

[0072] This comparative example provides another method for detecting Entamoeba histolytica, the steps of which include:

[0073] Add 100 μL of coating buffer containing 0.5 μg of the recombinant Entamoeba histolytica antigen to each well of a 96-well plate and incubate overnight at 4°C in a humidified chamber. Wash the plate sequentially with 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, and 400 μL of PBST buffer. Add 400 μL of phosphate buffer containing 3% skim milk to each well and incubate for 1 hour in a humidified chamber. After aspirating the blocking buffer, add 100 μL of serum diluted 400 times with PBS to each well and incubate for 1 hour in a humidified chamber. Wash the plate with 0 μL, 250 μL, 300 μL, 350 μL, and 400 μL of PBST washing buffer. Add 100 μL of the HRP-labeled anti-human IgG antibody diluted 1000 times with 3% skim milk to each well and incubate in a humidified chamber for 1 h. Wash the plate sequentially with 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, and 400 μL of PBST washing buffer. Add 200 μL of chromogenic solution to each well and incubate at room temperature in the dark for 30 min. Read the absorbance at 450 nm using a microplate reader. Add 50 μL of 2 mol / L H2SO4 to each well to stop the chromogenic reaction and read the absorbance at 490 nm using a microplate reader. The results of the same 25 serum samples are as follows: Figure 7-8As shown, the detection results of Example 3 are clearly in good agreement with those of the comparative example (R0). 2 The accuracy of the detection results in Example 3 for judging negative and positive results is 100%, and there is no cross-reaction with other intestinal infections, that is, the specificity of the detection results in Example 3 is 100%; in addition, the total duration of Examples 1-3 is only 30 min, while the duration of the comparative example is about 24 h, that is, the method of the present invention for detecting Entamoeba histolytica infection has relatively high sensitivity.

[0074] In summary, enzyme-linked immunosorbent assay (ELISA) has low sensitivity and requires a long time to separate the target signal from background noise, resulting in low detection efficiency. In contrast, the method for detecting Entamoeba histolytica infection of the present invention has relatively high sensitivity, thus requiring only a short time to obtain a high signal-to-noise ratio, thereby significantly improving detection efficiency. At the same time, the detection cost is low and less experimental waste is generated, making it a rapid qualitative detection method for Entamoeba histolytica.

[0075] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a microfluidic chip for detecting Entamoeba histolytica infection, characterized by, The preparation method includes the following steps: S1. A microfluidic chip is provided, the microfluidic chip comprising: channels; S2. Modify the channel so that its surface can bind recombinant Entamoeba histolytica antigen; the modification includes: passing a modifying reagent through the channel and washing the channel with deionized water, wherein... The modifying agent includes at least one of polylysine, epoxy resin, or polyethylene glycol. S3. The recombinant antigen of Entamoeba histolytica is bound to the surface of the channel, so that each recombinant antigen of Entamoeba histolytica bound to the surface of the channel can bind to at most one Entamoeba histolytica-specific antibody, and each Entamoeba histolytica-specific antibody can be bound to at most one gold nanosphere bound to anti-human IgG antibody. The binding of the recombinant Entamoeba histolytica antigen comprises: allowing a solution of the recombinant Entamoeba histolytica antigen to flow through the channel, wherein, The concentration of the solution of the recombinant Entamoeba histolytica antigen is 40 pg / μL, and the flow rate of the solution of the recombinant Entamoeba histolytica antigen is 3 mm / s. S4. Blocking the sites on the surface of the channel that are not bound to the recombinant antigen of Entamoeba histolytica; the blocking includes: passing a phosphate buffer containing skim milk through the channel and washing the channel with PBST washing solution.

2. A microfluidic chip prepared by the method of claim 1, wherein, The microfluidic chip includes: an inlet (1), an outlet (2), and a channel for connecting the inlet (1) and the outlet (2); wherein, The channel includes: an injection channel connected to the injection port (1) pipeline, a first bend section connected to the injection channel pipeline, an S-shaped channel connected to the first bend section pipeline, a second bend section connected to the S-shaped channel pipeline, and an outlet channel connected to the second bend section pipeline; The first curved segment and the second curved segment are located in the imaging region (3); The sample outlet channel is connected to the sample outlet (2) pipeline.

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