Nano probe based method for detecting trace proteins by using microfluidic chip
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A technology of microfluidic chips and nanoprobes, applied in measuring devices, processes for producing decorative surface effects, biological testing, etc., can solve problems such as false positives and pollution, and achieve high sensitivity and specificity
Inactive Publication Date: 2011-08-10
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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There have been reports, such as nano-gold combined with horseradish peroxidase to detect tumor proteins, and nano-gold combined with fluorescent dyes to detect nucleoproteins, etc., but these methods have certain defects. The former requires a specific microplate reader, and the latter Those who need fluorescent reagents and expensive fluorescent detectors are likely to cause pollution and false positives
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Embodiment 1
[0064] Embodiment 1: the detection of standard substance hepatitis B surface antigen
[0065] (1) Preparation of microfluidic chip
[0066] 1. Silicon wafers are used as the base material, and SU-8 photoresist is used for exposure and development to make pipe molds and valve molds;
[0067] 2. Mix PDMS and curing agent at a ratio of 20:1 (weight ratio), remove air bubbles, pour on the pipe mold, heat and cure at 90°C for 1 hour, and make the pipe microstructure;
[0068] 3. Mix PDMS and curing agent at a ratio of 10:1 (weight ratio), remove air bubbles, pour on the valve mold, heat and cure at 90°C for 1 hour, and make the valve microstructure;
[0069] 4. Peel off the PDMS on the valve mold, and use a punching needle to punch the air hole ( figure 2 A, B, and C three holes); and then fit together with the pipeline microstructure;
[0070] 5. Then peel off the pipe-valve bonding body and punch holes ( figure 2 Among them, 1 and 3 are sample injection holes, and 2 and 4 a...
Embodiment 2
[0096] Embodiment 2: clinical serum sample detection (1ng / ml hepatitis B serum specimen)
[0097] (1) Preparation of microfluidic chip
[0098] It is the same as (1) in Example 1.
[0099] (2) Preparation of detection reagents and DNA
[0100] It is the same as (2) in Example 1.
[0101] (3) Labeling of monoclonal antibodies and DNA
[0102] It is the same as (3) in Example 1.
[0103] (4) Testing and interpretation of results
[0104] 1. Sample processing
[0105] Dilute the clinical serum samples with calf serum to a concentration of 1pg / ml, 10pg / ml, and 100pg / ml, and take 10ul into the injection port 1.
[0106] 2. The method is the same as (4) in the implementation case 1, see the result Figure 4 .
[0107] The present invention dilutes the clinical serum samples by 1000, 100, and 10 times for sensitivity test, because the MMP antibody can capture the hepatitis B surface antigen in the clinical serum samples, and form a sandwich structure with the NP probe, so tha...
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Abstract
The invention relates to a nano probe based method for detecting trace proteins by using a microfluidic chip, which is characterized by comprising the steps: manufacturing a microstructure by using a standard photoetching process, and sealing a glass sheet (spotted with a DNA (deoxyribonucleic acid) probe) and the microstructure to prepare a required microfluidic chip; simultaneously labeling a monoclonal secondary antibody and a Barcode DNA with a signal amplification function on a nano gold particle, and labeling a monoclonal primary antibody on a magnetic bead; and detecting the trace target proteins in a microfluidic chip channel by means of immunoreaction of antigens and antibodies as well as gradual amplification and silver staining development of signals. The nano probe based method provided by the invention integrates the procedures of enrichment, separation and detection of biological samples, has the characteristics of specificity, rapidness and high sensitivity, and is expected to be applied to the diagnosis and detection of trace proteins (the antigens or antibodies) in clinical laboratory medicine. The sensitivity can reach a pg/ml level and is improved by 1000 times compared with the common ELISA (enzyme-linked immunosorbent assay) method in clinical application.
Description
technical field [0001] The invention belongs to the detection field of trace proteins, in particular to a method for detecting target proteins based on nano-probe combined with microfluidic chip technology. Background technique [0002] my country is a country with a high incidence of viral hepatitis, and the incidence of hepatitis B (HBV) ranks first in the world. Chronic infection can lead to chronic inflammation, necrosis and fibrosis of the liver, and some patients may develop liver cirrhosis or even hepatocellular carcinoma (HCC), which is extremely harmful to the health and life of patients and has become a serious social and public health problem. Therefore, early and timely detection of hepatitis virus in patients is particularly important. At present, the main methods of clinical diagnosis of HBV are ELISA and polymerase chain reaction, but there are still some limitations. The missed detection in the window period of the former is the current bottleneck affecting...
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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68B81C1/00G01N33/543G01N33/532
Inventor 张宏莲陈强毛红菊郭慧金庆辉赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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