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Method for enhancing stability of nano gold and biological detection method adopting the same

A biological detection and nano-gold technology, which is applied in the direction of analyzing materials through chemical reactions and material analysis through observing the impact on chemical indicators, etc., can solve the cumbersome experimental steps and the stability of double-stranded DNA and single-stranded DNA The difference is not very large, the stability effect and other issues, to achieve the effect of improving the range and sensitivity

Active Publication Date: 2010-06-30
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The experimental steps are relatively cumbersome, and the amount of DNA, DNase, RNase or nucleic acid aptamer is large
The second type of approach is the non-crosslinked nanoparticle aggregation method, which takes advantage of the reduced stability of the nanoparticles upon addition of the analyte.
[0009] 1. The limitations of the types of detectable substances are limited to the presence of double-stranded DNA, or / and single-stranded DNA, or / and dNTP, or / and ADP and uridine, or / and ATP in the detection system
[0010] 2. When using double-stranded DNA and single-stranded DNA to detect the difference in the stability of gold nanoparticles, the difference in the stability of gold nanoparticles between double-stranded DNA and single-stranded DNA is not very large, especially the stability of gold nanoparticles by single-stranded DNA. The stabilization effect is significantly affected by its composition. For example, single-stranded DNA containing continuous T cannot stabilize gold nanoparticles well. The mole amount of the detected DNA needs to be as close as possible to the mole amount of the DNA probe before it can be directly observed by the naked eye. Significant difference in

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  • Method for enhancing stability of nano gold and biological detection method adopting the same
  • Method for enhancing stability of nano gold and biological detection method adopting the same
  • Method for enhancing stability of nano gold and biological detection method adopting the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Preparation of unmodified gold nanoparticles and improvement of the stability of gold nanoparticles by dNMP.

[0055] An aqueous solution of trisodium citrate (25ml, 38.8mM) was quickly added to a boiling solution of auric acid HAuCI4 (250ml, 1mM). After a few minutes, the color of the solution changed from light yellow to dark red. The solution was stirred at reflux for 15 minutes to complete the reaction. Then cool slowly to room temperature. 4 degrees save. According to the ultraviolet absorption intensity of nano gold at 520nm (Fig. 1(C)), the concentration of prepared nano gold is 12.7nM, and the size is 13nm (see Fig. 1(A) and (B).

[0056] In the stability experiment of nano-gold, the concentration of nano-gold is 3nM, 100 μL. dNMP is a mixed solution of dAMP, dTMP, dGMP and dCMP (100μM or / 1mM), in which the ratio of each single nucleotide is the same as that of single-stranded DNA (5'CCGCAAATTGTTC). The amount of 0.6M NaCl phosphate buffer in Ta...

Embodiment 2

[0059] Example 2: Differences in the stabilizing effects of dNMP, single-stranded DNA and double-stranded DNA on unmodified gold nanoparticles

[0060] Double-stranded DNA sample PM / A: 0.25 μl 100 μM 5-terminal phosphorylated single-stranded DNA probe A (5'CCGCAAGACCGCTAGC), 0.25 μl 100 μM target DNA PM (GCTAGCGGTCTTGCGG) fully complementary to the single-stranded DNA probe, 1 microliter of buffer (0.6M NaCl, 10 mM phosphate, pH 7.4), and 0.5 microliter of water were kept at room temperature for 10 minutes.

[0061] Single-stranded DNA sample PM: 0.25 μl of 100 μM target DNA PM (GCTAGCGGTCTTGCGG) that is fully complementary to the single-stranded DNA probe, 1 μl of buffer (0.6M NaCl, 10 mM phosphate, pH 7.4), and 0.75 μl of The water was kept at room temperature for 10 minutes.

[0062] ssDNA sample A: 0.25 µl 100 µM 5-terminal phosphorylated ssDNA probe A (5'CCGCAAGACCGCTAGC), 1 µl buffer (0.6M NaCl, 10 mM phosphate, pH 7.4), and 0.75 µl of water at room temperature for 10 ...

Embodiment 3

[0065] Example 3: Detection of the activity of lambda exonuclease by enhancing the stability of gold nanoparticles

[0066] 10 microliters of enzyme reaction solution contains 5-terminal phosphorylated single-stranded DNA probe A (100pmole, 10μM), (5'PO 4 -CCGCAAGACCGCTAGC) target DNA PM (100pmole, 10μM) fully complementary to the single-stranded DNA probe (GCTAGCGGTCTTGCGG), reaction buffer (67mM Glycine-KOH, 2.5mMMgCI2, 50μg / ml BSA, pH 9.4), and different amounts of λ Exonuclease (from 1unit to 0.1unit). The reaction was carried out at 37°C for 1 hour, followed by heating at 75°C for 10 minutes to inactivate the enzyme. Then the reaction solution was mixed with 200 μl of gold nanoparticles (12.7 nM), and 100 μl of 0.2 M NaCl phosphate solution was added. UV-Vis absorbance measurements were performed and peak shifts were used to quantify color changes and enzyme activity.

[0067] The selectivity of the lambda exonuclease to the substrate can also be performed according to...

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Abstract

The invention provides a method for enhancing the stability of nano gold and a biological detection method adopting the same. Mononucleotide dNMP can better stabilize the nano gold than single-stranded DNA with the same composition and the same base concentration. Various nucleases with matrix selectivity can digest nucleic acid to form the dNMP. The property of the dNMP, which enables the dNMP to better stabilize the nano gold, is integrated with specific enzyme reaction, and the change of the color of the nano gold can be used to simply and conveniently detect various analytes including enzyme and DNA. The method greatly broadens the range of detected substances biologically detected by the unmodified nano gold, and increases the sensitivity of detection. Taking DNA detection for an example, the mole ratio range of a detecting probe and a target is broadened by three orders of magnitude. The method can be applied to the detection of various types of substances including micromolecules and heavy metal ions.

Description

technical field [0001] The invention relates to a method for enhancing the stability of nano-gold and a biological detection method using the same, belonging to the field of nano-biotechnology. Background technique [0002] Nano-gold particles have unique physical properties, especially optical properties, such as nano-gold has a high extinction coefficient. And these properties vary with the size, shape and surface modification of the nanoparticles. Therefore, nanomaterials, especially gold nanoparticles, are very suitable for biological detection and environmental detection as signals to mark the presence or absence of detection substances. [0003] Nanogold has been used in recent years to detect various substances, such as DNA, proteins and metal ions. [0004] In the prior art, in order to use nanogold for biological detection and environmental detection, the unique surface plasmon resonance optical properties of nanogold must be used. Independently existing nanogold ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78
Inventor 娄新徽赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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