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Probe set for detecting SARS-CoV-2 RNA, ECL biosensor and preparation method and application thereof

A biosensor, DNA probe technology, applied in biochemical equipment and methods, microbial determination/inspection, chemiluminescence/bioluminescence, etc., can solve problems such as increasing electron transfer distance, reagent consumption, affecting ECL intensity, etc. High electron transfer efficiency, good specificity and reproducibility, the effect of enhancing RuECL intensity

Pending Publication Date: 2021-09-17
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the separation of ECL reagents and co-reactants from each other, the electron transfer distance is increased, which brings about serious consumption of reagents and affects the strength of ECL.

Method used

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  • Probe set for detecting SARS-CoV-2 RNA, ECL biosensor and preparation method and application thereof
  • Probe set for detecting SARS-CoV-2 RNA, ECL biosensor and preparation method and application thereof
  • Probe set for detecting SARS-CoV-2 RNA, ECL biosensor and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] 1.1 Synthesis of Ru-PEI / Au@ZIF-8

[0083] 1.1.1 Synthesis of ZIF-8

[0084] 1) Zinc acetate dehydrating agent (Zn(AC) 2 -2H 2 O) heated to 55°C and kept for 45 minutes to remove moisture from the crystals to give Zn(AC) 2 powder;

[0085] 2) Add Zn(AC) 2 The powder (200mg) and 2-methylimidazole (295mg) were dissolved in methanol (22ml). The mixture was then diluted with water under continuous stirring to obtain a final concentration of 1 mmol Zn(AC) 2 and 3.3 mmol of 2-methylimidazole. When the mixture turns milky, age with stirring at room temperature for a day.

[0086] 3) The mixture was centrifuged, washed repeatedly with methanol and water, and then dried in an incubator to obtain a white powder, namely ZIF-8, which was used for the next step of modification.

[0087] Carry out amino modification to ZIF-8 surface, comprise the following steps:

[0088] 1) Sonicate 1.5 mg of ZIF-8 into 20 ml of N,N-dimethylformamide (DMF) to obtain a uniformly dispersed sus...

Embodiment 2

[0103] 2.1 The preparation of ECL biosensor, including the following steps:

[0104] S1: Before building the sensor, the GCE needs to be cleaned. A GCE with a diameter of 3 mm was immersed in a piranha solution (98% (v / v) H 2 SO 4 and 30% (v / v) H 2 o 2 The molar ratio is 4 to 1) to remove non-specific adsorbed substances on the electrode surface, and then repeatedly polished with 0.05 μm alumina powder and ultrasonically cleaned in ethanol and water until a bright and clean interface appeared. Then activate the electrode in sulfuric acid with a concentration of 0.1M, the voltage is -0.8-0.8V, and the scan rate is 0.1V / s until a continuous and stable CV peak appears. After rinsing with ultrapure water, the electrode was dried in N 2 Dry under the hood for the next step of biosensor construction.

[0105] S2: Drop 0.5% (mass percentage) of Nafion (N169478-5ml, Aladdin) and 8 μL of Ru-PEI / Au@ZIF-8 on the spot-free surface of GCE to obtain Nafion / GCE and Ru-PEI / Au in turn @...

Embodiment 3

[0125] Example 3 Detection of SARS-CoV-2 RNA using CRISPR-Cas 12a trans-cleavage properties and ECL biosensor

[0126] In order to prove the use of CRISPR-Cas 12a trans-cleavage characteristics in Example 2 and the detection level of SARS-CoV-2 RNA by ECL biosensors, this example analyzed the relationship between ECL intensity and target RNA concentration, and obtained their The mathematical relationship between them enables the possibility of inferring the target concentration from the signal intensity. Such as Figure 4 As shown in (A), when the concentration of SARS-CoV-2 RNA (SEQ ID: NO.5) in the sample increases, the ECL intensity gradually becomes stronger, which further confirms that the target RNA can drive the DSN enzyme to cycle with the CHA reaction process, Activate the activity of the CRISPR-Cas12a system to achieve the hydrolysis of Fc-labeled DNA probes. This embodiment also explored the linear relationship between ECL intensity and target RNA concentration, a...

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Abstract

The invention provides a system for detecting SARS-CoV-2RNA. The system comprises a probe group, a CRISPR / Cas12a system and an ECL biosensor. A self-enhanced ruthenium compound is linked to the surface of ZIF-8, and high intramolecular electron transfer efficiency is obtained under the condition that a co-reactant is not added, so that a strong and stable ECL signal is obtained. The DSN participates in target circulation and CHA signal amplification, so that cascade amplification of signals is realized, and unstable target RNA is converted into stable dsDNA to be output. The dsDNA in the strategy can activate the trans-cleavage characteristic of Cas12a, and the Fc-labeled DNA probe which shows affinity with C3N4 is cleaved. Therefore, the concentration of the target RNA can determine the number of the Fc-labeled DNA probes adsorbed on the surface of C3N4, so that the ECL intensity is directly influenced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a probe set for detecting SARS-CoV-2 RNA, an electrochemical sensor and a preparation method and application thereof. Background technique [0002] Coronavirus disease 2019 (COVID-19), caused by a novel coronavirus (severe acute respiratory syndrome-associated coronavirus 2, SARS-CoV-2), has posed major threats and challenges worldwide (Lukas et al., 2020 ). Due to its high infectivity, high morbidity and high mortality, it has caused huge damage and loss to global public health and world economic activities, and has claimed the lives of more than one million people around the world. Currently, early diagnosis of this virus and the development of antiviral vaccines and drugs provide the main means to suppress the SARS-CoV-2 pandemic. Especially with the emergence of virus mutations, the need for early and rapid diagnosis of viruses appears to be continuous and urgent. SARS-CoV-2 d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682C12Q1/6825C12Q1/70G01N21/76
CPCC12Q1/682C12Q1/6825C12Q1/701G01N21/76Y02A50/30
Inventor 张凯范振强丁月娣谢敏浩
Owner JIANGSU INST OF NUCLEAR MEDICINE
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