Biosensor for detecting tobramycin and detection method

A biosensor, tobramycin technology, applied in biochemical equipment and methods, biological testing, microbial determination/inspection, etc., can solve the problems of low specificity and sensitivity, fast detection speed, long detection period, etc. Simple method, good practicability and high stability

Active Publication Date: 2021-09-03
JIANGSU INSTITUTE OF EDUCATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problems of relatively low specificity and sensitivity, high cost and long detection period in the detection method of tobramycin in the prior art, and provide a biosensor for detecting tobramycin with low cost , when used to detect tobramycin, the specificity and sensitivity are relatively high, and the detection speed is fast

Method used

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  • Biosensor for detecting tobramycin and detection method
  • Biosensor for detecting tobramycin and detection method
  • Biosensor for detecting tobramycin and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A biosensor for detecting tobramycin, comprising an aptamer probe, hairpin probe H1, hairpin probe H2, Klenow Fragment polymerase, Nt.BbvCI endonuclease, buffer and graphene oxide;

[0038]The sequence of the aptamer probe is: 5'-GAG GAA CTG GAG TCA CAA GCT GAG GAT GTG ACTCCA GGC ACT TAGTCACA-3' (SEQ ID No: 1), the aptamer probe is the aptamer Aptamer and The amplified template T is hybridized, and the nucleotide sequence of the aptamer Aptamer is: 5'-TG ACT CCA GGC ACT TAG TCA-3' (SEQ ID No: 2), the nucleotide sequence of the amplified template T The acid sequence is: 5'-GAG GAA CTG GAG TCA CAA GCT GAG GA-3' (SEQ ID No: 3).

[0039] The nucleotide sequence of the hairpin probe H1 is: 5'-GAG GAA CTG GAG TCA CAA GCT GAC ATATCT CAG CTT GTG ACT CCA G-3' (SEQ ID No: 4).

[0040] The nucleotide sequence of the hairpin probe H2 is: 5'-TCA GCT TGT GAC TCC AGT TCC TCC TGGAGT CAC AAG CTG AGA TATG-3' (SEQ ID No: 5).

[0041] The aptamer Aptamer and the amplified template T hybr...

Embodiment 2

[0050] The schematic diagram of the biosensor detecting tobramycin of the present invention is shown in figure 1 .

[0051] The method utilizing the biosensor of embodiment 1 to detect tobramycin comprises the steps:

[0052] (1) Hybridize the aptamer Aptamer and the template T into an aptamer probe, 1uL (100μM) aptamer probe, 61.5uL water, 62.5uL 2x T-100Na (25mM Tris, 100mM NaCl, pH 7.3), Incubate in a 95°C metal bath for 3-5 minutes, then slowly cool for 1-2 hours to obtain aptamer hairpin structure products;

[0053] (2) Mix 1uL (100μM) of H1 and H2 modified with fluorescent groups, 79uL of water and 20uL of 5x T-500Na (25 mMTris, 500mM NaCl, pH7.3), and incubate in a 95℃ metal bath for 3- 5min, after slow cooling for 1h, a hairpin structure product was obtained; the whole reaction process should be protected from light;

[0054] (3) Add 1.5uL (800nM) aptamer hairpin structure product, 3uL tobramycin detection substance, 3uL 10xsmartcut buffer, 1uL (10mM) dNTP, 2U Nt.Bb...

Embodiment 3

[0058] figure 2 It is the feasibility verification result of the biosensor of Example 1. In the figure, the --- line represents the addition of fluorescent hairpin H1; the ... line represents the addition of fluorescent hairpin H1 / H2; the - - - - line represents the addition of Aptamer probes and fluorescent hairpins H1 / H2, the target tobramycin was not added to the above three samples. -··-··· represents the addition of tobramycin, aptamer probe and fluorescent hairpin H1; -·-·-·-· line represents the addition of tobramycin and fluorescent hairpin H1 / H2;—— It represents the addition of tobramycin, aptamer probes and fluorescent hairpins H1 / H2, and the target substance tobramycin is added to the above three samples. All samples were spiked with excess graphene oxide prior to testing. The experimental results show that only when tobramycin, aptamer probe and H1 / H2 exist at the same time, a strong fluorescent signal can be detected, which is consistent with the detection prin...

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Abstract

The invention discloses a biosensor for detecting tobramycin. The biosensor comprises an aptamer probe, a hairpin probe H1, a hairpin probe H2, Klenow Fragment polymerase, Nt.BbvCI endonuclease, a buffer solution and graphene oxide. The sequence of the aptamer probe is as shown in SEQ ID No: 1, the aptamer probe is formed by hybridizing an aptamer Aptamer with the sequence as shown in SEQ ID No: 2 and an amplification template T with the sequence as shown in SEQ ID No: 3. The sequence of the hairpin probe H1 is as shown in SEQ ID No: 4, and the sequence of the hairpin probe H2 is as shown in SEQ ID No: 5. When the biosensor is used for detecting tobramycin, the method is simple, the practicability is good, the stability is high, the detection lower limit reaches 0.06 nM and is lower than that of the existing similar sensors, and the biosensor has wide application prospects in the fields of environmental monitoring and food safety.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a biosensor and a detection method for detecting tobramycin. Background technique [0002] Tobramycin is an aminoglycoside antibiotic mainly used to treat bacterial infections caused by aerobic Gram-negative bacteria and some Gram-positive bacteria. However, the abuse of tobramycin may cause irreversible side effects on human life and health, including nephrotoxicity, neuromuscular blockade and allergic reactions. Due to the low price of tobramycin, tobramycin is still widely used in animal husbandry so far, resulting in potential residues in the food chain, such as lake water, milk, eggs and meat. [0003] Many routine analytical techniques have been used to detect antibiotics, including gas chromatography, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis, and enzyme-linked immunos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C12Q1/6825C12Q1/682
CPCG01N33/5308C12Q1/6825C12Q1/682C12Q2525/205C12Q2525/301C12Q2531/119C12Q2521/301C12Q2563/107Y02A50/30
Inventor 吕蓓李大为周兵
Owner JIANGSU INSTITUTE OF EDUCATION
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