Thiram on-site detection sensor based on polypeptide nano laccase and preparation method of thiram on-site detection sensor
An on-site detection and thiram technology, applied in the field of biosensors, can solve the problems of further improvement in sensitivity and limited signal amplification ability, and achieve the effects of low cost, high selectivity, and high sensitivity
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Embodiment 1
[0021] Embodiment 1: Preparation of polypeptide nano-laccase material
[0022] Copper chloride aqueous solution (0.24mol L -1 , 2.5mL), glutathione aqueous solution (0.12mol L -1, 5 mL), N,N-dimethylformamide solution (≥99.9%) (10.0 mL) were mixed in a centrifuge tube at a volume ratio of 1:2:4. The mixture was shaken evenly with an adjustable mixer, and then the mixture was transferred to a 20mL stainless steel polytetrafluoroethylene-lined autoclave. The reaction kettle was reacted at a constant temperature of 140° C. for 4.5 hours in an oven, and the polypeptide nano-laccase was prepared by a hydrothermal method. After the reaction was complete, the autoclave was cooled to room temperature and transferred to a centrifuge tube. The polypeptide nano-laccase was collected by multiple centrifugation (15000 rpm, 5 min), and washed three times with N,N-dimethylformamide solution (≥99.9%) and deionized water respectively. Finally, the precipitate was collected and freeze-dried...
Embodiment 2
[0023] Example 2: Construction of thiram double field detection sensor based on polypeptide nano-laccase
[0024] 25 μL of polypeptide nano-laccase aqueous solution (0.05 mg mL -1 ) and 50μL different concentrations (2.5, 12.5, 25, 50, 125 and 250μg L -1 ) of thiram solution (dissolved in 5% acetone aqueous solution) was mixed and incubated at 37°C for 25min to realize the inhibition of thiram on polypeptide nano-laccase activity; then, after the reaction was sufficient, 2,4- Dichlorophenol aqueous solution (100μL, 1mg mL -1 ), 4-aminoantipyrine aqueous solution (100 μ L, 1 mg mL -1 ) and MES aqueous solution (pH=7.4, 30mM, 100μL), the volume ratio of the three mixed solutions was always kept 1:1:1. Incubate at 60° C. for 1 h after adding the chromogenic substrate. Cool it and put it into a glass cuvette, measure the absorption spectrum of the solution generated by the color reaction between different concentrations of thiram and polypeptide nano-laccase by ultraviolet spe...
Embodiment 3
[0026] Example 3: Construction of a paper-based colorimetric sensor for thiram on-site detection based on polypeptide nano-laccase
[0027] 20 μL polypeptide nano-laccase aqueous solution (concentration 0.15mg mL -1 ) were dropped on 7 pieces of ordinary water-absorbing test paper (circular in shape and 6mm in diameter), and dried at room temperature; then, 20 μL of thiram solution (dissolved in 5% acetone aqueous solution) (0.0125 , 0.025, 0.125, 0.25, 0.5, 1.25 and 2.5mg L -1 ), incubate at 37°C for 10min, then drop 1mg mL -1 2,4-dichlorophenol aqueous solution (2,4-DP), 1mg mL -1 4-Aminoantipyrine aqueous solution (4-AP) and MES (morpholineethanesulfonic acid monohydrate) aqueous solution (pH=7.4, 30mM) are used as the mixed chromogenic substrate solution (three kinds of mixed chromogenic substrate solutions The volume ratio of the solution is 1:1:1, and the total volume of the mixed chromogenic substrate solution is 20 μL) and incubate at 60°C for 20 min. By observing...
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