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SPR-SERS dual-mode sensor for detecting disease nucleic acid markers as well as preparation method and application of SPR-SERS dual-mode sensor

A marker and sensor technology, applied in the fields of biological detection and spectroscopic detection, can solve the problems of missed diagnosis or diagnosis error, nucleic acid amplification, etc., and achieve the effects of reliable detection, good specificity and simple preparation process

Active Publication Date: 2021-08-24
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Purpose of the invention: In order to overcome the shortcomings of the existing single detection mode that are prone to missed diagnosis or diagnostic errors, and to solve the problem of nucleic acid amplification in the previously disclosed patent documents, the present invention proposes a method based on colloidal gold nanoparticles (AuNPs) SPR-SERS dual-mode sensor, and designed a catalytic hairpin self-assembly (CHA)-induced AuNP network sensor specifically for the detection of gastric cancer nucleic acid marker miRNA-652

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  • SPR-SERS dual-mode sensor for detecting disease nucleic acid markers as well as preparation method and application of SPR-SERS dual-mode sensor
  • SPR-SERS dual-mode sensor for detecting disease nucleic acid markers as well as preparation method and application of SPR-SERS dual-mode sensor
  • SPR-SERS dual-mode sensor for detecting disease nucleic acid markers as well as preparation method and application of SPR-SERS dual-mode sensor

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Experimental program
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Effect test

Embodiment 1

[0060] Example 1 Preparation of SPR-SERS dual-mode sensor

[0061] 1. Preparation of the first reagent

[0062] (1) Place the hairpin DNA single strands H1 and H2 in a mixer for annealing at 95°C for 5 minutes to keep the hairpin structure;

[0063] (2) Mix 10 μL of 100 μM captured single-stranded C, 10 μL of 100 μM hairpin DNA single-stranded H1 with 10 μL of 100 mM TCEP solution (disulfide bond reducing agent) and place them in a constant temperature mixer at 25°C for overnight reaction To reduce disulfide bonds formed between sulfhydryl groups;

[0064] (3) Mix 100 μL 2.3nM AuNPs and 10 μL 10 μM captured single-stranded C in a 0.5×TBE solution, and incubate overnight at 25°C at 300 rpm to obtain a mixture 1;

[0065] (4) Add salt to the mixed solution 1 obtained in step (3) for aging: slowly add 10 μL of 2M NaCl solution to the above-mentioned mixture 1 in 4 times at intervals of 30 minutes, the final NaCl concentration is 0.3M, and then at 25°C 300rpm Shake overnight un...

Embodiment 2

[0076] Working curve and detection limit of embodiment 2SPR-SERS dual-mode sensor

[0077] The target miRNA-652 was diluted with 10% normal human serum to different concentrations of 0 (blank), 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM and 10 nM. Take 10 μL of the first reagent, 10 μL of the third reagent, and 5 μL of 10 μM of the second reagent and mix them in 45 μL of PBS buffer, add 5 μL of different concentrations of miRNA-652, shake in a mixer at 25°C for 3 hours, and centrifuge and wash 3 times. The final product was redispersed in 40 μL of PBS buffer for SPR-SERS dual-mode detection. SPR was detected by dark-field microscopy (DFM), and 20 μL of the final product after miRNA-652 detection was directly dropped on the center of clean indium tin oxide (ITO) glass (25 × 5 mm), adsorbed at 25 ° C for 1 min, and then washed with N 2 Blow dry, drop PBS buffer solution on the ITO glass and take DFM pictures. After the shooting was completed, Image Pro Plus software was used for...

Embodiment 3

[0079] Example 3 The specificity of SPR-SERS dual-mode sensor

[0080] The target miRNA-652 was diluted to 1pM with PBS buffer, and the complete mismatch sequence (UM), three-base mismatch sequence (TM) and single-base mismatch sequence (SM) were all diluted to 100pM with PBS buffer. Take 10 μL of the first reagent, 10 μL of the third reagent, and 5 μL of 10 μM of the second reagent and mix them in 45 μL of PBS buffer, add 5 μL of 1 pM miRNA-652, 100 pM complete mismatch sequence (UM), and 100 pM three-base mismatch sequence ( TM), 100pM single-base mismatch sequence (SM) and blank sample group (PBS buffer), after shaking in a mixer at 25°C for 3h, centrifugation and washing, SPR-SERS dual-mode detection was performed. Get the integral of the dark field colors corresponding to the target miRNA-652, complete mismatch sequence (UM), three-base mismatch sequence (TM), single-base mismatch sequence (SM) and blank sample group (PBS buffer) Optical density, SERS spectrum and the ra...

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Abstract

The invention discloses an SPR-SERS dual-mode sensor for detecting a disease nucleic acid marker as well as a preparation method and the application of the SPR-SERS dual-mode sensor. According to the SPR-SERS dual-mode sensor, an SPR sensing mode is realized by extracting integral optical density of dark field color in a dark field microscopic image, and a ratio type SERS sensing mode is realized by acquiring an intensity ratio of SERS characteristic peaks of ROX molecules and 4-mercaptobenzoic acid (4-MBA) molecules. For miRNA-652 detection, nucleic acid single chains C, H1 and H2 in the first reagent, the second reagent and the third reagent are DNA chains which are designed for miRNA-652 and have different nucleotide sequences. The SPR-SERS dual-mode sensor disclosed by the invention is simple to prepare, high in detection sensitivity and good in reliability, the detection limits of an SPR sensing mode and an SERS sensing mode on nucleic acid detection in serum can reach the level of flying mole per liter, and nucleic acid markers related to diseases can be detected in complex biological samples.

Description

technical field [0001] The invention belongs to the fields of biological detection and spectroscopy detection, and in particular relates to an SPR-SERS dual-mode sensor for detection of gastric cancer nucleic acid marker miRNA-652, a preparation method and application thereof. Background technique [0002] Accurate detection of trace nucleic acids is essential for reliable analysis of early disease-related biomarkers, which play an important role in disease diagnosis, treatment, and pathogenesis. However, genetic variation due to certain subtle changes in nucleic acid sequence can produce significant biological effects. Therefore, the development of ultrasensitive and reliable methods to detect biomarkers associated with low-abundance diseases is crucial and is becoming an irresistible trend. Traditional analytical methods such as Northern blot hybridization, microarray technology, and real-time quantitative polymerase chain reaction (PCR) have been widely used in nucleic a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825C12Q1/6886G01N21/65G01N21/59G01N21/49
CPCC12Q1/6825C12Q1/6886G01N21/658G01N21/59G01N21/5907G01N21/49C12Q2600/158C12Q2600/178C12Q2525/207C12Q2563/137C12Q2563/155C12Q2565/607C12Q2525/301C12Q2565/628C12Q2565/632Y02A50/30
Inventor 张晶晶蒋新宇李雪宋春元汪联辉
Owner NANJING UNIV OF POSTS & TELECOMM
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