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Method for improving detection sensitivity of cocaine

A technology for detecting sensitivity and cocaine, which is applied in the direction of biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of affecting sensitivity, large experimental error, and insufficient sensitivity, so as to reduce interaction, improve color gradient, and increase sensitivity Effect

Inactive Publication Date: 2011-01-19
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • 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.
The sensitivity of the detection is not high enough, the gradient of the color of the nano-gold with the concentration of cocaine is not large enough, and the standard deviation of the detection is relatively large
These deficiencies are mainly due to the weak ability of relatively long single-stranded nucleic acid aptamers to stabilize unmodified gold nanoparticles, which is not very different from the ability of nucleic acid aptamers and cocaine to form double-stranded DNA structures to stabilize gold nanoparticles.
The degree of aggregation at a specific salt concentration is relatively large, resulting in a large experimental error, which directly affects the sensitivity of detection

Method used

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  • Method for improving detection sensitivity of cocaine
  • Method for improving detection sensitivity of cocaine
  • Method for improving detection sensitivity of cocaine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of unmodified gold nanoparticles.

[0036] 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 ± 1nM, and the size is 13nm (see Fig. 1(A) and (B)).

Embodiment 2

[0037] Embodiment 2: one-stage nucleic acid probe (probe 1), two-stage nucleic acid probe (probe 2 and probe 3) and three-stage nucleic acid probe of the present invention (probe 3, probe 3) that are used for cocaine detection Differences in the influence of probe 4 and probe 5) on the stability of unmodified gold nanoparticles.

[0038] First prepare the following nucleic acid probe samples with the same molar concentration:

[0039] One-piece nucleic acid probe (probe 1) sample: 1 μl of 100 μM probe 1, 3 μl of cocaine detection buffer (0.3M NaCl, 50 mM Tris-HCl, pH 8.0), and 2 μl of water in Mix well at room temperature.

[0040] Two-stage nucleic acid probe (probe 2 and probe 3) sample: 1 microliter of 100 μM probe 2, 1 microliter of 100 μM probe 3, 3 microliters of cocaine detection buffer (0.3M NaCl, 50 mM Tris -HCl, pH 8.0), and 1 μl of water were mixed well at room temperature.

[0041] Three-part nucleic acid probe (probes 3, 4, and 5) sample: 1 µl of 100 µM probe 3...

Embodiment 3

[0044] Example 3: Detection of cocaine using a three-segment nucleic acid probe.

[0045] Preparation of cocaine standard samples: Cocaine solutions with concentrations of 500 μM, 200 μM, 100 μM, 50 μM, 20 μM, and 0 μM were prepared in a buffer solution (0.15M NaCl, 25 mM Tris-HCl, pH 8.0).

[0046] 0.5 μl each of 200 μM nucleic acid probe 3, 200 μM probe 4, and 200 μM probe 5, 1.5 μl of 50 mM Tris-HCl buffer containing 0.3 M NaCl, and mix well. Take 0.5 microliters of the above mixed solution, add 4.5 microliters of cocaine solutions of different concentrations, and store at room temperature for 30 minutes. Take 2 microliters from each of the above samples, add 100 microliters of 12±1nM nano-gold solution, store at room temperature for 5 minutes, and then add 15 microliters of 10mM phosphate buffer solution containing 0.2M NaCl.

[0047] Perform UV-vis absorption measurements ( Figure 4A ), and taking pictures ( Figure 4C ). Draw the standard curve of cocaine detection ...

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Abstract

The invention relates to a method for improving the detection sensitivity of cocaine. The method is characterized in that a nucleic acid aptamer for cocaine detection is broken into three sections. The method comprises the following steps of: (1) mixing the cocaine and three sections of nucleic acid aptamer probes to ensure that the detected cocaine and the aptamer probes form a compound; and (2)performing fluorescence detection, electrochemical detection or color detection. As the length of the three sections of nucleic acid probes is obviously shorter than that of one or two sections of nucleic acid probes, the interaction among probes and between the probes and other components in a detection system capable of causing background noise is greatly reduced. Thus the detection sensitivityof the cocaine is greatly improved. Taking the nanogold color detection of the cocaine for example, the sensitivity of the color detection of the cocaine is improved by one magnitude order by the three sections of nucleic acid probes. The method can be popularized to the detection of various materials comprising small molecules and heavy metal ions.

Description

technical field [0001] The invention relates to a method for improving cocaine detection sensitivity by using a three-segment nucleic acid probe, and belongs to the field of nano-biotechnology. Background technique [0002] Drug detection technology represented by cocaine has made great progress in recent years, especially the detection technology using nucleic acid aptamers has attracted great attention. Nucleic acid aptamers are from ~10 14 The DNA or RNA library of random sequences is screened by the method of systematic molecular evolution of exponential enrichment (Systematic Evolution of Ligands by EXponential enrichment, referred to as SELEX), which can specifically bind to the target molecule, thereby specifically recognizing the target molecule (Ellington AD, Szostak JW (1990) Nature 346:818-822. Tuerk C, Gold L (1990) Science 249:505-510.). Nucleic acid aptamers can be used for the detection of various biomolecules including proteins, small molecules, heavy metal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 娄新徽袁敏赵建龙
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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