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Method for measuring materials inside human single blood erythrocyte by mocro-fluidic chip chemiluminescence

A microfluidic chip, chemiluminescence technology, applied in chemiluminescence/bioluminescence, analysis by chemical reaction of materials, etc., can solve the problems of inability to perform single-cell analysis, time-consuming, and complicated operation of the analyzer. Simple injection, fast analysis, and high separation efficiency

Inactive Publication Date: 2010-03-17
GUANGXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to overcome the shortcomings of the current traditional method, which is cumbersome to operate, takes a long time, and cannot perform single-cell analysis, the present invention proposes a method for the determination of substances in a single human red blood cell by chemiluminescence with a microfluidic chip, and analyzes glutathione in a single red blood cell. Peptide (GSH) and cysteine ​​(Cys) content

Method used

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  • Method for measuring materials inside human single blood erythrocyte by mocro-fluidic chip chemiluminescence
  • Method for measuring materials inside human single blood erythrocyte by mocro-fluidic chip chemiluminescence
  • Method for measuring materials inside human single blood erythrocyte by mocro-fluidic chip chemiluminescence

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Embodiment 1

[0028] (1) Preparation of red blood cell suspension:

[0029] Take 3mL of fresh venous blood samples from normal people in a microcentrifuge tube, then add 3 times the volume of PBS solution, mix it gently, and then centrifuge at 4°C for 10min (1000r / min). Repeat the above steps four times to clarify the upper cell solution, and finally add 3mL PBS solution into the centrifuge tube, and gently blow the cells into a suspension. This suspension is used for single cell analysis.

[0030] (2) Microfluidic chip electrophoretic chemiluminescent detection of mixed standard solutions of hemoglobin (Hb), glutathione (GSH) and cysteine ​​(Cys)

[0031] Before starting, the chip channel was washed with 0.1mol / L sodium hydroxide, ultrapure water and electrophoresis buffer (20mmol / L borax solution containing 3.0mmol / L luminol, pH 10.2) for 10min each, and then observed under a microscope. The chip channel is filled with buffer solution without fine particles and air bubbles. The sample ...

Embodiment 2

[0036] Determination of single red blood cells in patients with benign tumors.

[0037] Fresh venous blood samples from patients with benign tumors were used instead of normal human blood samples, and the separation and detection were carried out according to the steps in Example 1, and the experimental conditions were the same as in Example 1. Figure 5 Shown are: (A) the electrophoresis spectrum of a single red blood cell in a patient with benign thyroid tumor; (B) the electrophoresis spectrum of a single red blood cell in a patient with benign breast tumor; (C) the electrophoresis spectrum of a single red blood cell in a patient with benign gastric tumor. The corresponding peaks in the figure are: peak 1 is glutathione (GSH), and peak 2 is hemoglobin (Hb).

Embodiment 3

[0039] Determination of single red blood cells in patients with malignancy.

[0040] Fresh venous blood samples from patients with malignant tumors were used instead of normal human blood samples, and the separation and detection were carried out according to the steps in Example 1, and the experimental conditions were the same as in Example 1. Figure 6 Shown are: (A) the electrophoresis spectrum of a single red blood cell of a liver cancer patient; (B) the electrophoresis spectrum of a single red blood cell of a patient with thyroid cancer; (C) the electrophoresis spectrum of a single red blood cell of a breast cancer patient. The corresponding peaks in the figure are: peak 1 is cysteine ​​(Cys), peak 2 is glutathione (GSH), and peak 3 is hemoglobin (Hb).

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Abstract

The invention provides a method for measuring materials inside human single blood erythrocyte by mocro-fluidic chip chemiluminescence, which can analyze the contents of cysteine, glutathione and hemoglobin in a single blood erythrocyte. An electrophoresis chemiluminescence detection system of a mocro-fluidic chip is adopted; five power supply output ends of a high voltage power supply are respectively connected with a buffer solution pool B, a sample pool S, a waste liquie pool SW, a chemiluminescence reagent pool R and a buffer waste liquie pool BW of the mocro-fluidic chip; the mocro-fluidicchip is arranged on an objective lens of a chemiluminescence detector; the chemiluminescence detector is an inverted microscope combined with a photomultiplier; the photomultiplier is electrically connected with a data collector; and the data collector is electrically connected with a computer. The method can carry out early diagnosis on occurrence of lesions by measuring the increased degree ofthe concentration of the glutathione and the cysteine in the single blood erythrocyte, has high analysis speed, high sensitivity and high resolution, is suitable for early diagnosis to health examination and the lesions of a human body, and can be used as an effective means of preventive medicine.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for measuring the contents of hemoglobin (Hb), glutathione (GSH) and cysteine ​​(Cys) in human single red blood cells by chemiluminescence of a microfluidic chip. Background technique [0002] Single-cell analysis is of great significance for understanding many physiological and pathological processes, as well as early diagnosis and treatment of diseases. Because the diameter of cells is generally micron, the body is very small, and the content of intracellular components is very low (fmol-zmol) and extremely complex, so it is very difficult to detect ultra-trace components in cells. [0003] Microfluidic chip electrophoresis (MCE) is a new separation technology developed after capillary electrophoresis, and is widely used in the fields of chemistry, biology, medicine and environmental monitoring due to its unique advantages. The microfluidic chip has a multi-channel...

Claims

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Application Information

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IPC IPC(8): G01N21/76
Inventor 赵书林黄勇叶芳贵石明李向堂
Owner GUANGXI NORMAL UNIV
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