Experiment purpose
To create an electrochemical biosensor based on tyrosinase to detect bisphenol A by displaying tyrosinase on the surface of Escherichia coli cells.
1. Extraction of target gene tyrosinase and INP (Ice Nucleation Protein)
2. Activated strain E. coli BL21/ psb1a3
3. Extract plasmid and then amplify its DNA and the target gene (tyrosinase and INP) with PCR
4. Carry out double enzyme digestion to the fusion fragment
5. Perform nucleic acid gel electrophoresis and recover the gell
6. Prepare the receptor cells and transform the engineered fusion fragment to the plasmid psb1a3 and then transform plasmid into the E.coli
7. Carry out molecular cloning, screening and obtain positive clones
8. Culture the positive clones
Experimental results and verification
Verification of the transformed plasmid
Figure. 1. Carrying out nucleic acid gel electrophoresis
To verify that the gene fragment (INP and tyrosinase) is successfully expressed in the plasmid, our team completed nucleic acid gel electrophoresis to verify its presence.
Figure. 2. Nucleic acid gel electrophoresis results
As can seen by figure. 2., the marker is on the left of the gel. The second nucleic acid stain (from top to bottom) two samples to the left demonstrated the same kDa value with the marker. Thus it is verified that the modified gene expression is successful.
Construction of electrode
Functional electrodes for the biosensor were produced using the recombinant E. coliTyr cells directly adsorbed on the GC electrode. But, to confirm the construction of a functional electrode, we completed a reference experiment with the GFP gene fused with InaK-Tyr and transferred into E. coli for expression.
Figure. 3. Microscopy images I and III was taken under bright field miscroscope, III and IV under fluorescence microscope
After the functional electrode was prepared, it was observed using fluorescence microscopy. The resulting images indicated that part of the engineered E. coli (III and IV) emitted fluorescence which was not emitted by the controlled E. coli (I and II), indicating that the engineering strains had adhered to the GC electrode to form functional E. coli-Tyr GC electrode.
Electrode response to BPA
After confirming the functional electrodes, the E. coli-Tyr GC electrode was used to investigate the presence of BPA.
Figure. 4. Voltammogram of electrodes in detection of BPA
We obtained the CV graph in PBS (phosphate-buffered saline) concentration of 0.1 M (and pH 7.0) with 0.1 nM of BPA, at the scan rate of 100 mV/s. This modified electrode exhibited a typical reduction current profile toward BPA (as can be seen by the yellow line on figure. 4.). On the other hand, the E. coli-psb 1a3 GC control electrode did not sense the BPA (black line on figure. 4.). These results demonstrate that the functional electrode based on the engineered strain E. coli-Tyr was successfully constructed and that it could respond to BPA.
To verify that the sensor can operate in a wide range of environments and to find the optimum response conditions in order to optimize the operating parameters to draw the calibration curve, we investigated the effects of cell loading, applied potential, pH and temperature on the performance of the biosensor.
The investigation was conducted using 50 nM of BPA.
Figure. 5. Effects of loaded cells on biosensor performance
Figure. 6. Effects of applied potential on biosensor performance
Figure. 7. Effects of pH on biosensor performance
Figure. 8. Effects of pH on biosensor performance
From figures 5-8 above, the optimum working environment for the biosensor was proven to be temperature at 3 x loaded cells, -100mV, pH 7.0, and 35 °C. This environment was later utlilized in out calibration curve. Moreover, this experiment demonstrated that our biosensor is usable in a variable environment.
Calibration curve of the biosensor
Figure. 9. Voltammogram of electrodes in detection of BPA
After determining the optimum operating parameters for the biosensor to function, we contructed amperometry and measured amperometric response curves for successive additions of BPA standard solution to the PBS under stirring.
As shown on Figure. 5, reduction current increased rapidly within the first 20 s and stabilized in 100 s. A strong linear relationship was observed between the current values and the concentration of BPA within the range of – M.
Figure. 10. Voltammogram of electrodes in detection of BPA
A calibration curve is contructed with calculated to be 0. 9967. The equation for the whole curve is I = − 0.04633c + 0.05368. This linear relationship demonstrated that the proposed biosensor is successful, as to give electrosignal responses to different BPA concentration.
Analysis of the tyrosinase cell-surface display system in biosensors
Table 1. Linear range and LOD values for different configuration of biosensors (The reference for this may be found at the bottom of this page)
Tyrosinase has been widely used for BPA detection in various biosensors due to its low-cost and high activity. In these biosensors, the enzyme is usually immobilized on different chemically modified electrodes.
Although chemical modification can enhance the stability of the enzyme, the decrease in cell viability and the consequent loss in enzyme activity should be considered during the process. When compared with chemical modification, bio-modification such as microbial cell-surface display systems (as shown on Table 1), has demonstrated astonishingly low LOD values and a wide linear range.
As can be seen on table. 1., the nanographene-based tyrosinase biosensor displayed superior analytical performance over a linear range of 100-2000 , with an LOD of 33 . The gold nanoparticle modified graphene used for BPA detection also showed a good linear relationship in the concentration range of 2.5 × –3.0 μM, with an LOD of 0.001 μM.
The detection ranges of these systems were narrower than those of our biosensor, which suggests the superiority of our proposed biosensor in real environmental monitoring. The LOD of our biosensor for BPA detection was M, which is lower than that of several sensors based on chemically modified electrodes, for instance the LOD of 0.005 μM for a biosensor based on nanoflower–chitosanAuNPs/GCE, and the LOD of 0.02 mM for a biosensor based on polyglutamic acid–MWCNT– / GCE. Thus, the biosensor developed in our group is both sensitive and technically competent.
Application of the biosensor （detection in real samples)
Figure. 7. The three tea samples we used
Figure. 8. The three juice samples we used
Table 2. BPA measurement using the biosensor in real samples of teas and juices
The practical performance of the constructed biosensor in detecting the concentration of BPA in tea and juice samples was examined. To make sure our biosensor is applicable in real life, we applied our biosensor in real samples to which were added different BPA standard solutions were analyzed (as in Table 2). The BPA content in three different tea and juice samples was successfully measured using the biosensor.
The recovery rates of BPA in the tea samples were in the range of 97.78%− 98.29%, while the values in the juice samples were in the range of 98.20%− 100.32%. These results indicate that the proposed biosensor is a promising and reliable tool for accurate and quick detection of BPA in natural samples.
Conclusion
In conclusion, our experiments have demonstrated that our project is successful as to contructing a cell-surface display system of bisphenol A and that sends electric signals if BPA is detected. In our ultimate attempt is to use the biosensor to detect BPA, we received high recovery rate that demonstrates our model is able to predict BPA values with high accuracy. Moreover, proven by our experiment, biosensors the surface display system is more sensitive to BPA in the environment and thus more prefered, not to be mention its wide range compared to other biosenser configurations. Thus it is proven that our design matches our aim.
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9 From our exeriment
