High-flux rapid malaria serum detection method based on micro-fluidic chip

A microfluidic chip and high-throughput technology, applied in the field of biomedicine, can solve the problems of low degree of automation, inability to distinguish Plasmodium species, and easy cross-contamination, so as to shorten the inspection time, improve the detection accuracy, The effect of reducing inspection costs

Active Publication Date: 2012-10-31
FUDAN UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above method can generally only detect a single antigen such as HRP2 or LDH in the patient's serum. Although the operation is simple, it cannot distinguish the Plasmodium species, and has many problems such as low degree of automation, often accompanied by non-specific adsorption, and prone to cross-contamination. shortcoming

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Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Preparation of microfluidic chip

[0037]Put the silicon wafer into Piranha solution (98% concentrated sulfuric acid: 30% hydrogen peroxide = 7:3), boil and clean for 15 minutes; rinse it with deionized water for 5 times, dry it with nitrogen, and bake it at 200 °C for 30 minutes; Pour the SU-8 glue on the center of the silicon wafer and rotate it slowly so that the SU-8 covers most of the silicon wafer; use a spin coater at 3000 rpm for 60s to make the glue evenly distributed, and let it stand for 10 minutes to relieve edge protrusions effect; followed by soft baking, the purpose of soft baking is to volatilize the solvent in the SU-8 photoresist, the key to process control is to make the solvent volatilize at a controllable rate; keep the temperature at 65°C, 95°C and 65°C, respectively 3min, 6min and 3min. After that, the temperature was slowly lowered to room temperature at a rate of 0.5°C / min; a contact exposure machine (wavelength 365nm) was used; pos...

Embodiment 2

[0038] Example 2 Detection of serum from malaria patients by enzyme-linked immunosorbent assay (ELISA)

[0039] 100 μl of Coating Buffer containing 100 ng of Plasmodium protein merozoite surface antigen 1 (MSP1) and erythrocyte-binding antigen 175 (EBA175) were added to each well of a 96-well microtiter plate, and the cells were placed in a humidified box at 4°C overnight. After washing the plate with PBS (containing 0.05% Tween20), 400 μl of PBS containing 3% skim milk was added to each well, and the plate was blocked at room temperature for 1 h in a humidified box. 100 μl of healthy human or patient serum (1:400 dilution) was added to each well, and the negative control was healthy human serum (1:400 dilution) and incubated at room temperature for 1 h in a humidified box. After washing the plate with PBS (containing 0.05% Tween20), add 100 μl horseradish peroxidase-labeled goat anti-human IgG (1:1000 dilution) 100 μl to each well, and incubate in a humid box for 1 h at roo...

Embodiment 3

[0041] Example 3 Serological detection of malaria patients with microfluidic chip

[0042] Fill the column with polymeric fillers through the injection channel, close the valve at the bottom of the column, control the air pressure of the valve not to exceed 20 psi, and control the speed of the column. After the analysis chamber of the chip, after several times of PBS washing, several filler volumes of recombinant Plasmodium MSP1 and EBA175 antigen solutions were pumped into different analysis chambers, and through specific binding, the Plasmodium protein antigen and the polymerized microspheres were formed. Filler binding. After several PBS washes, PBS containing 5% bovine albumin was pumped into the microfluidic chip analysis chamber to block the polymerized microspheres bound to recombinant Plasmodium antigens. The serum of malaria patients was then diluted and pumped into the analysis chamber of the microfluidic chip, and the anti-plasma parasite MSP1 and EBA175 antibodi...

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Abstract

The invention belongs to the fields of biomedical research and clinical applications, and relates to a serum immunological detection method. According to the invention, based on a micro-fluidic chip, a method specially used for high-flux rapid malaria serum detection is established. With the method provided by the invention, immunological determinations of two plasmodium antigens are simultaneously carried out on a micro-fluidic chip. Analysis capacity and antigen-antibody reaction specificity of the micro-fluidic chip are combined, such that defects of conventional immunoassay can be effectively overcome, reaction efficiency can be improved, operation steps can be simplified, detection time can be shortened, patient serum sample usage amount can be greatly reduced, and consumptions of reagents and energy are greatly reduced. Thereby, the entire detection cost is reduced. Also, the method is advantaged in high automation degree. With the method, specimen room contamination can be prevented, and detection accuracy can be improved.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a serum immunological detection method, in particular to a method for high-throughput rapid detection of malaria serum based on a microfluidic chip. Background technique [0002] Malaria is an important parasitic disease caused by Plasmodium and transmitted by the bite of Anopheles mosquito. According to the latest WHO estimates, 41% of the world's people live in malaria-endemic areas, distributed in more than 100 countries, with 300 to 500 million clinical cases and about 2.7 million deaths every year. On average, one person dies from malaria every 15 seconds , mostly children and pregnant women. At present, malaria, tuberculosis, and AIDS are listed as the three most serious infectious diseases in the world. For the prevention and treatment of malaria, the World Health Organization regards the development of accurate and rapid malaria diagnosis technology as one of the priority counter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N35/00
CPCY02A50/30
Inventor 程训佳隋国栋刘思秀蒋建国张晶玲付永锋
Owner FUDAN UNIV
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