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Nucleic acid detection method based on surface plasma resonance technology

A technology of surface plasma and detection method, applied in the field of nucleic acid analysis, can solve the problems of insufficient detection sensitivity, low probe density, poor binding stability, etc., and achieve the effects of simple operation, low cost and stable assembly structure

Inactive Publication Date: 2016-10-12
NANJING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems of low probe density, poor binding stability and insufficient detection sensitivity in the existing SPR detection method for nucleic acid, the present invention provides a simple sensitivity with double sensitization signal, stable structure and good repeatability. High nucleic acid detection method based on surface plasmon resonance technology

Method used

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  • Nucleic acid detection method based on surface plasma resonance technology
  • Nucleic acid detection method based on surface plasma resonance technology
  • Nucleic acid detection method based on surface plasma resonance technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Electrodeposition of reduced graphene oxide nanosheets on the surface of the SPR chip, the specific process is as follows:

[0048] (1) The bare SPR chip was prepared in piranha acid (concentrated H 2 SO 4 : Concentrated H 2 o 2 =1:3) after soaking for 10min, then in boiling H 2 o 2 Mixed solution with ammonia water (H 2 o 2 The mass concentration of ammonia and ammonia water is 5%) and soaked for 10min, and finally rinsed twice with ultrapure water and ethanol. 2 Blow dry for later use;

[0049] (2) Add 0.5mg·mL -1 GO solution in 0.1M Na 2 SO 4 Ultrasonic for 2h, get 0.5mg·mL -1 GO dispersion;

[0050] (3) Cyclic voltammetry scanning on-line electrical reduction of GO, the cycle voltage is -1.5 ~ +0.5V, and the cycle speed is 50mVs -1 , for 1 to 600 cycles, to obtain an SPR chip with electrodeposited reduced graphene oxide nanosheets on the surface.

[0051] figure 2 A is the cyclic voltammetry curve, where (a) is the 1st time, (b) is the 500th time, an...

Embodiment 2

[0053] The underlying construction of cDNA / NTPy / rGO and its hybridization performance, the synthesis steps are:

[0054] (1) Micropipette 0.2 mL of dimethylformamide containing 2 mM aminotriacetic acid (NTA)-grafted perylene derivative (NTPy) into the container and spread it on the electrodeposited surface after 500 cycles. On the SPR chip of graphite oxide, and incubate for 1h to lay the foundation of the π-stacking scaffold, and repeatedly pump in / out DMF to wash away the weak adsorbate;

[0055] (2) Continuously, 0.5mL, 10mM Ni(ClO 4 ) 2 The acetic acid buffer solution was drop-coated on the above-mentioned treated chip, and left to stand for 2-6 hours to complex Ni 2+ and rinse with acetic acid buffer;

[0056] (3) The divisible mixed eluent of 1 μM cDNA and 5 μM ethanolamine blocking agent (blocking non-specific binding sites) is introduced onto the chip obtained in step 2, and the construction of the bottom layer is completed through layer-by-layer assembly to form cD...

Embodiment 3

[0060] The non-covalent assembly of dsDNA / NTPy / rGO, the reaction process is as follows:

[0061] Inject 100 μL, 1 pM tDNA into the detection cuvette, and perform initial biometric recognition with the cDNA / NTPy / rGO chip synthesized in Example 2, to realize binding and capture of target DNA, form dsDNA / NTPy / rGO, and complete the detection step.

[0062] Figure 5 Atomic force microscopy (AFM) images of rGO / Au (A), NTPy / rGO / Au (B) and dsDNA / NTPy / rGO / Au (C). Figure 5 A shows that within the scanned area of ​​2 × 2 μm, rGO maintains a broken morphology, which is consistent with figure 2 D is equivalent, rGO fragments occupy the entire area, and have a broad size distribution, with an average height of about 2nm in the plane (Z-offset=0nm). Figure 5 B shows that NTPy is in situ adsorbed on rGO, given the van der Waals radius of NTA, the average height of the sampling interval is increased by 6 nm, indicating that rGO and NTPy are tightly connected through π conjugation. Because...

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Abstract

The invention discloses a nucleic acid detection method based on surface plasmon resonance technology. First, graphene oxide is electrodeposited on the surface of a bare SPR chip, and then the activated molecular layer of perylene derivatives grafted with aminotriacetic acid is adsorbed through conjugation. Assemble the 5′ end biotinylated capture cDNA probe as the sensing interface to detect the target tDNA, and after realizing the binding and capture of tDNA, add the 3′ end biotinylated response rDNA to form a long chain dsDNA and expose its biotin molecules , combined with avidinated horseradish peroxidase to complete the assembly, and finally add aniline and hydrogen peroxide mixed solution, use horseradish peroxidase to catalyze the aniline polymerization deposition reaction to form polyaniline, and realize the detection of target nucleic acid according to the SPR signal. The present invention uses two-dimensional nanomaterials for "bottom-up" construction, and forms an SPR sensing platform with double-sensitized signals with a top-mounted "top-down" gravimetric reactor, which can achieve specificity and sensitivity to target DNA. Sensitive detection, the detection limit can reach femtomolar level.

Description

technical field [0001] The invention belongs to the field of nucleic acid analysis, and relates to a nucleic acid detection method based on surface plasmon resonance technology, in particular to a nucleic acid detection method using a high-density probe assembly method and an enzyme-catalyzed polymerization reaction to improve the quality response surface plasmon resonance signal method. Background technique [0002] The analysis method of specific DNA / RNA sequence at low physiological level is particularly important for early detection of cancer and screening of infectious viruses. Surface plasmon resonance (SPR) technology is a charge layer formed by surface plasmons on the interface between metal and dielectric. Under the excitation of electromagnetic waves, surface plasmons resonate to generate SPR signals based on mass changes, which can be conveniently and flexibly applied in nucleic acid detection. [0003] Nucleic acid sensors based on mass changes usually detect t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/552
CPCG01N21/554
Inventor 邓盛元袁培新郑晨昱宋宏鑫姚传广崔宏达
Owner NANJING UNIV OF SCI & TECH
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