The project design

  BPA was detected by tyrosinase. In the presence of dissolved oxygen, tyrosinase catalyzes the conversion of BPA into o-quinones, which can be reduced and release electrons, which can be detected in an external band circuit through a galvanic cell.

build

1.Synthesis of tyrosinase gene by direct DNA synthesis.

2.The tyrosinase gene was inserted into pSB1A3 plasmid by molecular cloning.

3.Prepare competent cells for transformation.

4.Molecular cloning and screening were carried out to obtain positive clones, and the labeled DH5α/pSB1A3 target fragment colonies were selected for colony PCR with Tap enzyme.

detection

1. The successful transformation of E. coli to Tyr was confirmed by Sanger sequencing. E. coli BL21 containing pET23b was used as control

2. Determination of tyrosinase activity According to a previous spectrophotometric method, this method measures the conversion of L-dopa to dopa red, a red oxidation product. The initial rate of reaction is proportional to the concentration of enzyme. The enzyme activity was determined by centrifugation at 8000rpm for 10 min

3. Electrochemical measurements were performed using a Chi660C Electrochemical workstation (ChenhuaCo, Shanghai., China) with a conventional single chamber and three electrode cells with an internal volume of 5 mL. CV reactions were obtained in a 3 mL reaction mixture of 0.1MPBS, pH=7.0 and BPA at a scanning rate of 100mV/s. The immobilized electrode was used as the working electrode, and the Ag/ silver chloride and planar platinum electrodes were used as the reference and counter electrodes, respectively.

Learning

  The detection reaction is not obvious, because these chemical molecules need to be transported across the membrane, which may hinder the efficiency of the reaction. When the concentration of BPA is low, the corresponding concentration is not well detected. Consider using surface display technology to increase the chances of BPA molecules coming into contact with the bio detector, thereby increasing sensitivity.

design

  Based on tyrosinase, an electrochemical biosensor was developed to detect bisphenol A by cell surface display technology. The BPA-recognizing Tyrosinase was fused with the carrier protein INP, and the INP was used to anchor Tyrosinase to the surface of E.coli cells.

Construction

1. Obtain the target gene - Tyrosinase

2. The target gene was implanted into E.coli DH5α/ pSB1A3-MRFP

3. The plasmid was extracted, DNA was amplified by PCR, and double digestion and gel recovery experiments were performed

4. Competent cells were prepared, and the engineered vector pSB1A3-target fragment was transformed into the expression host E.coli BL21

5. Molecular cloning and screening were performed to obtain positive clones, and the labeled DH5α/pSB1A3 target fragment colonies were selected for colony PCR with Tap enzyme.

Test

1. Electrochemical detection Electrochemical measurements were performed using a Chi660C Electrochemical workstation (ChenhuaCo, Shanghai., China) with a conventional single chamber and three electrode cells with an internal volume of 5 mL. CV reactions were obtained in a 3 mL reaction mixture of 0.1MPBS, pH=7.0 and BPA at a scanning rate of 100mV/s. The stationary electrode was used as the working electrode, and the Ag/ silver chloride and planar platinum electrodes were used as the reference electrode and counter electrode, respectively. In the amperometric study, the optimized potential was applied to the enzyme electrode in 50mmPBS solution (pH 7.0) to obtain a stable current response

2. Electrochemical detection of BPA in real samples The performance of the biosensor was investigated by selecting three commercial canned tea and fruit juice as samples containing BPA to detect BPA concentration. HPLC assays were performed in parallel as described above to test the accuracy of the biosensor. The recovery rate of BPA was calculated by randomly adding different final concentrations of pure BPA solution to the sample. All tests were performed in triplicate and their mean values were considered. Use OriginPro8.0 software

Learning

  A functional biosensor with LOD, accuracy, and sensitivity compared to other biosensors was constructed by simple adsorption of cells on GC electrodes. These results indicate that adsorption is a feasible method to immobilize living cells with highly active and surface-displayed tyrosinase. Further development of this biosensor should focus on improving its capability and durability