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Universal aptamer colloidal gold lateral chromatography test paper for detecting small molecular substances

A technology of small molecular substances and lateral flow chromatography, which is applied in the fields of medicine, environment, food safety detection, nanobiological sensing, and analytical chemistry. It can solve problems such as detection sensitivity needs to be improved, is not conducive to OTA competition, and poor product stability. Achieve the effects of increasing binding efficiency, high repeatability, and reducing dosage

Pending Publication Date: 2021-09-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The test strip couples the aptamer to AuNPs, which is not conducive to the competition of OTA in the sample for the aptamer, the product stability is poor, and the design is more complicated and the versatility is poor
[0007] At present, for the nucleic acid aptamer colloidal gold lateral flow test paper that has been developed, for a specific target, the probes on the test strip need to be repeatedly optimized and designed, the versatility is poor, and the detection sensitivity needs to be improved

Method used

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  • Universal aptamer colloidal gold lateral chromatography test paper for detecting small molecular substances
  • Universal aptamer colloidal gold lateral chromatography test paper for detecting small molecular substances
  • Universal aptamer colloidal gold lateral chromatography test paper for detecting small molecular substances

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Preparation of Kanamycin Rapid Detection Test Strips Based on Nucleic Aptamers

[0057] The specific steps are:

[0058] 1. Design of aptamer sequences and probes

[0059] In order to ensure that the biotinylated aptamer can bind to the nucleic acid on the probe and be captured by SA in the T-line region, nucleic acid strands of different sequences (polyA-DNA (20) , polyA-DNA (15) , polyA-DNA (5+10) , polyA-DNA (5+5) , polyA-DNA (10+5) ) coupled with AuNPs (as shown in Table 1). The selected corresponding aptamer concentration was 0.5 μM, and the same concentration of kanamycin (150 ng / mL) was added. The result is as Figure 8 As shown, when multiple T bases are connected at the 3' end of the aptamer sequence, the signal intensity can be significantly increased, and the signal can be enhanced by up to 6 times. When a nucleic acid probe with a nucleotide sequence such as SEQ ID NO.2 is used strand polyA-DNA (10+5) , the change of the corresponding sig...

Embodiment 2

[0091] Embodiment two: with test strip to the mensuration of kanamycin standard solution

[0092] (1) Preparation of Kanamycin Standard Solution

[0093] Kanamycin standard solution was diluted with Runningbuffer (4×SSC, pH 7) to a final concentration of 0.5, 5, 15, 25, 50, 150, 250 and 400 ng / mL. The kanamycin aptamer whose nucleotide sequence is shown in SEQ ID NO.1 was diluted to 0.5 μM with ultrapure water.

[0094] (2) Establishment of standard curve for detection of kanamycin nucleic acid test strips:

[0095] In step (1), 99 μL of kanamycin standard solution of different concentrations and 1 μL of kanamycin aptamer solution were mixed and incubated for 20 min. After the mixed reaction, the mixed liquid was added to the sample pad for detection. After 3 min of reaction, the (T / C) relative signal intensity, and establish a standard curve of the corresponding relationship between (T / C) relative optical signal intensity and different kanamycin concentrations.

[0096] T...

Embodiment 3

[0098] Embodiment three: use test strip to the mensuration of OTA standard solution

[0099] (1) Preparation of OTA standard solution

[0100] The OTA standard solution was diluted with Running buffer (4×SSC, pH7) to a final concentration of 1, 10, 50, 100, 250 and 500 ng / mL. Dilute the OTA aptamer whose nucleotide sequence is shown in SEQ ID NO.1 to 0.5 μM with ultrapure water.

[0101] (2) Establishment of OTA nucleic acid test strip detection standard curve:

[0102] Mix and incubate 99 μL of OTA standard solutions with different concentrations and 1 μL OTA aptamer solution in step (1) for 20 minutes, add the mixed droplets to the sample pad for detection after the mixed reaction, and measure the (T / C) relative signal intensity after 3 minutes of reaction , to establish a standard curve of the corresponding relationship between (T / C) relative optical signal intensity and different OTA concentrations.

[0103] The result is as Figure 5 As shown, when the OTA concentrati...

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Abstract

The invention discloses universal aptamer colloidal gold lateral chromatography test paper for detecting small molecular substances, and belongs to the fields of analytical chemistry, medicine, environment, food safety detection, nano biosensing and the like. PolyA-DNA is used as an anchoring block to be anchored on gold nanoparticles to serve as a probe, AuNPs@poly-DNA is used for rapidly and sensitively capturing an aptamer, part of complementary nucleic acid chains of SA and DNA sprayed in a detection area and a control area do not need to be changed, and the other substance can be detected only by changing the nucleic acid chains on the probe. And the rapid, sensitive and low-cost universal colloidal gold lateral chromatography test paper is developed. The test strip method is simple, convenient and rapid to detect small molecular substances, detection can be carried out at any time, an experiment result can be observed only by adding a test solution into a sample port and completely developing the test strip after 5 minutes, and the detection efficiency can be greatly improved. Qualitative analysis is carried out by naked eyes, and quantitative analysis is carried out by using a colloidal gold test paper quantitative analyzer.

Description

technical field [0001] The invention relates to a general-purpose aptamer colloidal gold lateral flow chromatography test paper for detecting small molecular substances, and belongs to the fields of analytical chemistry, medicine, environment, food safety detection, nanobiological sensing and the like. Background technique [0002] Lateral flow chromatography, a paper-based assay platform for detecting targets, is of great interest to researchers due to its potential to provide results within minutes. Because of its low cost and ease of development and production, lateral flow chromatography has been identified for on-site testing applications and is widely used in various fields, including biomedicine, food safety, quality control, and environmental hygiene. Lateral flow chromatography can be applied to a range of biological samples, including urine, saliva, sweat, serum, plasma, and blood. Therefore, the application value of the lateral flow chromatography analysis techni...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/558G01N33/53G01N33/52
CPCG01N33/587G01N33/558G01N33/5308G01N33/52G01N33/54388B01L3/5023B01L2300/0825C12Q1/6834
Inventor 彭池方常芮李秀萍
Owner JIANGNAN UNIV
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