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Colorimetric and fluorescent double-signal biosensor for detecting Hg<2+>, and detection method of biosensor

A biosensor and detection method technology, applied in the field of biosensing, can solve problems such as low sensitivity and narrow applicability, achieve high amplification efficiency, wide application range, and overcome false positive effects

Active Publication Date: 2017-02-01
MATERIAL INST OF CHINA ACADEMY OF ENG PHYSICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to overcome the shortcoming of narrow applicability and low sensitivity of colorimetric method, and utilize the advantage of convenient colorimetric method and high sensitivity of fluorescence method to provide a kind of colorimetric method based on target-catalyzed hairpin assembly enzyme-free amplification technology. Dual-signal ultrasensitive Hg and fluorescent signal 2+ Biosensors and detection methods

Method used

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  • Colorimetric and fluorescent double-signal biosensor for detecting Hg&lt;2+&gt;, and detection method of biosensor
  • Colorimetric and fluorescent double-signal biosensor for detecting Hg&lt;2+&gt;, and detection method of biosensor
  • Colorimetric and fluorescent double-signal biosensor for detecting Hg&lt;2+&gt;, and detection method of biosensor

Examples

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Embodiment

[0036] The following examples will further illustrate the present invention, but do not limit the present invention thereby.

[0037] In the following examples:

[0038] Chlorauric acid, sodium citrate, Tris-HCl buffer solution, mercuric nitrate, FAM, and concentrated sulfuric acid are analytically pure, and ultrapure water is self-made in the laboratory. The nucleotide sequences used in Examples 1-4 are shown in Table 1. The number of T mismatches between the helper DNA and H1 in Examples 1-4 is 6.

[0039] Auxiliary DNA and nucleotide sequences used in table 1 embodiment 1-4

[0040]

Embodiment 5

[0041] The nucleotide sequences used in Example 5 are shown in Table 2. The number of T mismatches between the helper DNA and H1 in Example 5 is 8.

[0042] Auxiliary DNA and nucleotide sequence used in table 2 embodiment 5

[0043]

[0044] The sequence listings of HI, H2, and H3 in Tables 1 and 2 above are shown in SEQUENCE LISTING.

[0045] instrument

[0046] Fluorescence photometer, the detection conditions are: the excitation wavelength and emission wavelength of FAM are set to 492nm and 517nm respectively, the slit width is 10nm, the fluorescence emission spectrum of the sample is excited by 492nm light, and the scanning range is: 505-600nm, scanning step The length is 1nm.

[0047] UV-visible spectrophotometer.

[0048] Since the sensitivity of fluorescence detection is higher than that of colorimetric detection, the following examples are all detected with a fluorescence photometer, and only a brief description is given for colorimetric detection.

Embodiment 1

[0050] Preparation of AuNPs

[0051] Weigh 0.01g of chloroauric acid and dissolve it in 100mL of ultrapure water, heat and boil under reflux; add 2.5mL of 1wt% sodium citrate solution under rapid stirring, heat and react for 30min and then cool to room temperature naturally; the prepared AuNPs are kept at 4°C±2°C save in .

[0052] Dual signal Hg with colorimetric and fluorescent signals 2+ Preparation and detection process of biosensor

[0053] Mix the Tris-HCl buffer solution of Hg(NO3)2, molecular hairpin probes H1, H2 and H3 and the Tris-HCl buffer solution of the auxiliary DNA, wherein the concentration of the Tris-HCl buffer solution is 50mmol / L, containing 50mmol / L MgCl2 and 0.5mol / L NaCl, pH 8.0, the volume after mixing is about 100μL, the concentration of molecular hairpin probes H1, H2 and H3 is 30-300nmol / L, the concentration of auxiliary DNA is 20-500nmol / L, Hg2+ The concentration of AuNPs is 0.2-100nmol / L, react at room temperature for 1-5h, then add 900μL AuNP...

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Abstract

The invention discloses a colorimetric and fluorescent double-signal biosensor for detecting Hg<2+>, and a detection method of the biosensor. The colorimetric and fluorescent double-signal biosensor comprises molecular hairpin probes (H1, H2 and H3), auxiliary deoxyribonucleic acid (DNA) and gold nanoparticles. The molecular hairpin probes are fluorescein (FAM)-labeled molecular hairpins. The AuNPs is synthesized by using a method of reducing chloroauric acid by means of sodium citrate. The detection method comprises the steps of dissolving 0.01g of the chloroauric acid in 100mL of ultrapure water, and boiling by reflux heating; adding 2.5mL of a 1wt% sodium citrate solution under the condition of rapidly stirring, carrying out a reaction for 30min, and then cooling to the room temperature to prepare the AuNPs; mixing mercury nitrate with H1, H2 and H3 (30-300 nmol / L) and a Tris-HCl buffer solution of the auxiliary DNA (20-500 nmol / L), carrying out a reaction for 1-5h at the room temperature, then adding the AuNPs, and carrying out a reaction for 5min; detecting respectively by using an ultraviolet-visible spectrophotometer and a fluorimeter. The double-signal biosensor based on the AuNPs and a catalytic hairpin assembly non-enzyme amplification technology is cheap in preparation and high in detection sensitivity, and overcomes the defect of narrow applicability of a single detection mode.

Description

technical field [0001] The present invention relates to the field of biosensing, in particular to a method for Hg 2+ Dual-signal biosensors and detection methods with both colorimetric and fluorescent signals for detection. Background technique [0002] Mercury is one of the most toxic and dangerous heavy metal elements, and its accumulation in living organisms has brought serious health hazards to people, such as slow growth and development, organ damage, and even death. The U.S. Environmental Protection Agency (USEPA) stipulates that mercury ions (Hg 2+ ) content is not higher than 10nmol / L. It is necessary to develop a highly selective and sensitive analytical method for Hg in the environment 2+ detection. Traditional detection methods include atomic emission spectroscopy (AES) (Moreton J A, Delves HT. Simple direct method for the determination of total mercury levels in blood and urine and nitric acid digests of fish by inductively coupled plasma massspectrometry[J]....

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N21/31
CPCG01N21/31G01N21/6486
Inventor 姜交来蔡定州杜云峰云雯廖俊生
Owner MATERIAL INST OF CHINA ACADEMY OF ENG PHYSICS
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