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Microfluidic fluorescence immunoassay chip for rapidly and quantitatively detecting cTnI in whole blood

A quantitative detection, fluorescence immunological technology, applied in the field of immunoassay, can solve the problems of poor sensitivity, linearity, repeatability and quantitative accuracy, not suitable for acute diagnosis and small sample detection, poor sensitivity and linear range, etc., to shorten the detection time. , to meet the effect of detection anytime, anywhere, high sensitivity

Pending Publication Date: 2020-06-19
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ELISA method has mature technology and low detection cost, but the sensitivity and linear range are poor, the operation is complicated, the repeatability is poor, and the detection time is long
Colloidal gold immunochromatography is simple to operate, but poor in sensitivity, linearity, repeatability and quantitative accuracy
Immunoturbidimetry and chemiluminescence are highly sensitive and accurate, but they need to be equipped with expensive large-scale instruments, and the detection time is long, so they are not suitable for acute diagnosis and small sample detection
It also has the characteristics of poor sensitivity and linear range

Method used

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  • Microfluidic fluorescence immunoassay chip for rapidly and quantitatively detecting cTnI in whole blood
  • Microfluidic fluorescence immunoassay chip for rapidly and quantitatively detecting cTnI in whole blood
  • Microfluidic fluorescence immunoassay chip for rapidly and quantitatively detecting cTnI in whole blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Microfluidic Fluorescent Immunochip for Rapid Quantitative Detection of cTnI

[0064] In this implementation example, the microfluidic fluorescent immune chip for rapid quantitative detection of cTnI includes a central plate 1 and a bottom plate 9, the central plate 1 is an optically transparent material, and the bottom plate 9 is located adjacent to the lower side of the central plate 1, so The region where the center plate and the bottom plate overlap each other around this recess are joined to each other directly and in a fluid-tight manner by laser welding.

[0065] A sample inlet 3 for introducing samples into the chip is provided on one side of the surface of the center plate. The sample inlet is closed in a pressure-tight manner, and enters the first sample mixing area sequentially from the sample inlet 3 through the flow channel. 4. The tracer area and the second sample mixing area, the two sample mixing areas extend in a serpentine shape, the tracer a...

Embodiment 2

[0088] Example 2 Rapid Quantitative Detection of cTnI Microfluidic Fluorescence Immunochip Drawing Standard Curve Determination and Information Storage

[0089] Take the chip out of storage conditions and equilibrate to room temperature before testing;

[0090] Step 1. Prepare cTnI calibrator with diluent, concentration: 0, 0.1, 1, 5, 15, 25ng / mL

[0091] Step 2. Take 50 μL of the calibrator and add it to the sample inlet of the microfluidic chip. Before each sampling, the pipette head needs to be replaced to avoid cross-contamination. After 10 minutes, read the system detection signal through Response IQ. Each standard The concentration of the calibrator was detected twice, and the signal value measurement results of the calibrator solution series are shown in Table 1. The regression curve of the calibrator dose-signal value was obtained by four-parameter logistic fitting, as shown in Table 1. image 3 shown.

[0092] Table 1

[0093]

Embodiment 3

[0094] Example 3 Methodological Verification of Microfluidic Fluorescent Immunochip for Rapid Quantitative Detection of cTnI

[0095] The chip in embodiment 1 is verified according to the conventional manufacturing and verification procedures in the art, and the results are as follows:

[0096] 1. Chip precision measurement

[0097] 1.1 Intra-batch precision analysis

[0098] A batch of chips in Example 1 were used to measure high and low concentration quality control solution series respectively, and 10 chips were measured in parallel, and the intra-assay coefficients of variation were 3.45% and 1.97%, respectively. The results are shown in Table 2.

[0099] Table 2

[0100] Target value (ng / mL) Measurement times Intra-analytical CV(%) 1 10 3.45 10 10 1.97

[0101] 1.2 Batch-to-batch precision analysis

[0102] The chips in Example 1 are taken in three batches, and each batch of chips is measured for high and low concentration quality control...

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Abstract

The invention discloses a microfluidic fluorescence immunoassay chip for rapidly and quantitatively detecting cTnI in whole blood, and belongs to the field of immunoassay. The chip comprises a tracingreagent and a capturing reagent, wherein the tracing reagent comprises a cyanine dye Cy5 labeled cTnI monoclonal antibody and a quality control material; wherein the capturing reagent contains a cTnImonoclonal antibody and a quality control substance monoclonal antibody. The chip marks a cTnI antigen in blood through a tracing antibody to form an immune complex, and the immune complex is captured by a capture antibody and emits light under excitation of exciting light. The invention has high sensitivity and specificity and a wide detection range, and can be used for evaluating the cTnI levelof a patient and prompting acute myocardial infarction.

Description

technical field [0001] The invention relates to the field of immune analysis, in particular to a microfluidic fluorescent immune chip for rapidly detecting cTnI. Background technique [0002] Cardiac troponin is a key protein that regulates heart contraction. Trimeric complex composed of three subunits of cardiac troponin I (cTnI), cardiac troponin C (cTnC) and cardiac troponin T (cTnT). The physiological role of cTnI is to inhibit the activity of ATPase in the actin-myosin complex in the absence of calcium to prevent muscle contraction. The molecular weight of cTnI is about 24kDa, composed of 209 amino acids, and it is a protein rich in α-helix. There are three subtypes of TnI (Troponin I): There are fast skeletal muscle type and slow skeletal muscle type in skeletal muscle troponin I (sTnI), which have similar molecular weight (20KD), but the amino acid between the two There is about 40% difference in the sequence; the third type is the cardiac type. There is also a 40...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N21/64
CPCG01N33/6893G01N33/577G01N21/6428G01N2333/4712G01N2800/324
Inventor 王鹏郭闻轩任传路
Owner BEIJING LEADMAN BIOCHEM
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