Electrochemical biosensor for detecting EBV related gene and preparation method thereof
An electrochemical and genetic technology is applied in the field of electrochemical biosensors for detecting Epstein-Barr virus-related genes and their preparation, which can solve the problems of temperature-controlled complex sequence design, time-consuming sensitivity, and difficulty in detection.
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Embodiment 1
[0085] Example 1 Preparation of electrochemical EBV-related gene detection sensor
[0086] 1. Materials and methods
[0087] 1.1 Materials
[0088] Lambda exonuclease (hereinafter referred to as λexo) and 10×λexo reaction buffer (670mM glycine-KOH, 25mM MgCl 2 , 500 μg mL-1BSA, pH 9.4) were purchased from New England Biolabs, USA, 6-mercaptohexanol (MCH), amino-terminated PAMAM dendrimer (G5.0) and tris(2-carboxyethyl)phosphine Hydrochloride (TCEP) was purchased from Sigma-Aldrich, USA, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 1×TE buffer (pH 8.0) Purchased from Sangon Bioengineering (Shanghai) Co., Ltd., AgNO 3 Purchased from Guangzhou Jinhuada Chemical Reagent Co., Ltd., NaBH 4 Purchased from Chengdu Kelong Chemical Reagent Factory, ethanol precipitation kit and DNA Marker (20bp) were purchased from Bao Biological Engineering (Dalian) Co., Ltd. (Dalian TaKaRa Company), and the nucleic acid dye GoldView I was purchased from Beijing Suolaibao T...
Embodiment 2
[0133] Example 2 Characterization and inspection of electrochemical EBV-related gene sensors
[0134] The high resolution transmission electron microscopy (HRTEM) characterization of the nanomaterials prepared in Example 1 is as follows:
[0135] (1) if figure 2 As shown in A, AgDNCs exhibits a uniform, monomer-dispersed spherical structure under HRTEM, with a particle size of about 3.81 nm;
[0136] (2) if figure 2 As shown in B, DNA / AgNCs exhibits a spherical structure under HRTEM with a particle size of less than 2nm;
[0137] (3) if figure 2 As shown in C, the AgDNCs@DNA / AgNCs-LP copolymer exhibits a relatively uniform size under HRTEM, with a particle size of about 7.19 nm.
[0138] The electrochemical EBV-related gene sensor obtained in Example 1 is characterized as follows:
[0139] 1. Electrochemical characterization of the assembly process of electrochemical EBV-related gene sensors
[0140] Such as image 3 Shown are the AC impedance curves of working elect...
Embodiment 3
[0177] Example 3 Research on Electrochemical EBV-related Gene Sensor and Its Conditions of Use
[0178] In order to obtain the best experimental performance, we investigated the important conditional parameters during the experiment, such as the ratio of LP to DNA / AgNCs, λexo concentration and time for λexo-assisted non-phosphorylated substrate preference reaction. For each experimental condition, at least 5 points were taken from low to high concentrations for a series of experiments.
[0179] 1. In order to investigate the effect of the concentration ratio of LP and DNA / AgNCs on the electrochemical EBV-related gene sensor, this experiment used different molar ratios of LP and DNA / AgNCs (the molar ratios of LP and DNA / AgNCs were 3:1, 2, respectively. :1, 1:1, 1:2, 1:3), other experimental methods are the same as in Example 1, such as Figure 7 As shown, when the molar ratio of LP to DNA / AgNCs is 2:1, the sensing system shows the highest signal-to-noise ratio, which is the op...
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