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A tetrahedral frame nucleic acid sERS probe, sensor and preparation method and application thereof

A tetrahedron and sensor technology, applied in biochemical equipment and methods, instruments, scientific instruments, etc., can solve the problems of poor enhancement effect of metal nanoparticles, expensive detection instruments, unstable detection signals, etc., to improve the spatial positioning range and accessibility, improve biorecognition efficiency, and reduce the effect of non-specific adsorption

Active Publication Date: 2022-07-12
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, there are some techniques for detecting ampicillin, such as high performance liquid chromatography, electrochemical analysis, mass spectrometry, capillary electrophoresis, microbiological methods, etc. These methods have some shortcomings, such as expensive detection instruments, long time-consuming, and difficult to operate. Complicated, large error and other disadvantages
Although there are existing SERS methods for detecting ampicillin, those methods have a small linear range; the detection limit is high; and direct detection requires pretreatment of the sample; especially the metal nanoparticles have poor enhancement effect and are prone to agglomeration, resulting in the detection signal. Unstable, unable to obtain Raman spectra with good enhancement effect and good reproducibility
Tetrahedral framework nucleic acid probes (tFNA probes) have the characteristics of uniform distribution and uniform orientation on the substrate surface, which can provide highly reproducible SERS signals, but the size of tFNA probes is very large, and it is hardly used in the field of SERS

Method used

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  • A tetrahedral frame nucleic acid sERS probe, sensor and preparation method and application thereof
  • A tetrahedral frame nucleic acid sERS probe, sensor and preparation method and application thereof
  • A tetrahedral frame nucleic acid sERS probe, sensor and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Preparation of tFNA probes

[0049] Take 1 μL each of 100 μM Tetra-A, Tetra-B, Tetra-C and Tetra-D, 10 μL of 30 mM tris(2-carboxyethyl)phosphine (TCEP) and 86 μL of TM buffer solution (made up of 20 mM Tris-HCl, 50 mM MgCl 2 •6H 2 O composition, pH 8.0) and mix well. After heating at 95 °C for 10 minutes, the temperature was rapidly lowered to 4 °C for more than 30 seconds, and the temperature was controlled with a PCR instrument to obtain a tFNA probe containing functionalized nucleic acid with a final concentration of 1 μM. Characterized by gel electrophoresis images, figure 1 From left to right are Tetra-A single-strand solution, Tetra-B single-strand solution, Tetra-C single-strand solution, Tetra-D single-strand solution, Tetra-A and Tetra-B two strands hybridized solution, Tetra -A, Tetra-B and Tetra-C three-chain hybridization solution, Tetra-A, Tetra-B, Tetra-C and Tetra-D four-chain hybridization solution (ie tFNA), DNA Marker solution. Because the molecu...

Embodiment 2

[0051] Preparation of long-range substrates

[0052] A gold film with a thickness of 100 nm was deposited on a glass substrate with a length and width of 1 cm × 1 cm by a thermal evaporator at 2 × 10 -6 The thickness of the Au film was controlled at a deposition rate of 0.02 nm / s at a pressure of mbar. Then MgF with a thickness of 200 nm was 2 Thermal evaporation coating onto gold surface to form MgF 2 / Au mirror / glass multilayer structure (for its synthesis reference ACS Appl. Mater. Interfaces 2021, 13(15), 18301–18313). Take 6 mL of prepared gold nano-hexagonal plate particles with an optical density of 2 (the synthetic reference is Adv. Optical Mater. 2016, 4, 76–85), add it to a 10 mL beaker, and add 2 mL of n-hexane to form water / n-hexane interface, drop 1-2 mL of ethanol into the above solution at a rate of 0.1 mL / min. Discontinuous golden luster hexagonal plate islands appear at the water / n-hexane interface at this time, and then these hexagonal plate islands rap...

Embodiment 3

[0054] Assembly of tFNA probes on long-range SERS substrates

[0055] 100 μL of 1 μM tFNA probe prepared in Example 1 was dropped on the LR-SERS substrate prepared in Example 2, and incubated at 37°C for 2 hours, so that the three vertices of the tFNA probe were self-assembled and connected by Au-S bonds. On the long-range SERS substrate, rinse with TM buffer to remove the unbound tFNA probe, and record the Raman response signal, because the Raman signal of Cy5 on the tFNA probe molecule is recorded. At this time, Cy5 is closer to the substrate surface, The peak intensity is about 10795 cps and the Raman signal is strong.

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Abstract

The invention discloses a tetrahedral frame nucleic acid SERS probe, a sensor and a preparation method and application thereof. The probe comprises a single-stranded probe Tetra-A modified with a cyanine dye 5, a single-stranded probe Tetra-B, a Tetra- C and Tetra‑D; The sensor is formed by the assembly of probes on long-range SERS substrates and hybridization to ampicillin aptamers. The probe of the invention is evenly distributed and oriented on the surface of the substrate, and can provide a highly reproducible SERS signal with good specificity; the long-range surface-enhanced Raman scattering characteristic of the long-range substrate overcomes the problem of the large size of the tFNA probe. This leads to its limitation in SERS detection, so that the probe can still obtain a strong Raman signal on the surface of the SERS substrate. The invention combines the tFNA probe and the long-range surface-enhanced Raman substrate for the first time to develop a novel ampicillin detection sensor with high sensitivity, high selectivity and high repeatability.

Description

technical field [0001] The invention belongs to biological Raman detection, in particular to a tetrahedral frame nucleic acid SERS probe, a sensor and a preparation method and application thereof. Background technique [0002] Ampicillin, also known as ampicillin, is a β-lactam antibiotic, which has been widely used due to its wide applicability, good activity, good tolerance, low toxicity, low cost and good clinical effect on bacterial infections. For the treatment of various bacterial infections in humans, livestock, poultry and aquaculture. In recent years, although ampicillin as a growth regulator or anti-infective has promoted the rapid development of animal husbandry and feed processing industries, the illegal and excessive use of ampicillin in animals and livestock has directly led to the use of antifungal drugs in the production of human and animal-derived foods. The health and safety problems caused by the pollution and residues are becoming more and more prominent...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6816G01N21/65
CPCC12N15/11C12Q1/6816G01N21/658C12N2310/531C12Q2525/205C12Q2565/519C12Q2525/30C12Q2565/607C12Q2565/632Y02A40/70
Inventor 吴萍祝晶甜郏雯雨方宁宁蔡称心
Owner NANJING NORMAL UNIVERSITY
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