Part:BBa_I732005:Experience

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Introduction

In order to be able to utilize the electrochemical detection system, various response levels must be correlated to cell concentration. Because our output signal is the product of a reaction sequence, it would not be sufficient to only characterize the performance of one particular part operating alone. This novel approach to characterizing lacZ through the oxidation of chlorophenol red was done in conditions allowing all the necessary reactions to occur simultaneously - just as if used in a field-ready biosensor.

Results

The final results of our data processing is shown in Figure 1 below. The calculations done to arrive at this final curve follow.

Figure 1 Maximum rate of current change per voltage versus cell concentration for a standard electrochemical cell system.

By repeating this procedure for the various tested cell levels, we can construct one of our standard curves - the maximum rate of current change versus number of cells.

We determined the concentration of cells in our culture to be 7.35*10⁸ cells/mL. For the purposes of making a biosensor, we can see that having 10⁵ to 10⁷ cells in our electrochemical system provides a strong signal, while not risking saturating our detection mechanism. This is completely feasible with a small amount of our cell culture.

Appendix

In order to construct our standard curve for current response vs. number of cells, several experiments were conducted with varying numbers of cells, with a constant amount of CPRG (100uL at 0.5M), NaH₂PO₄ buffer (1mL at 0.5M), and nanopure water (up to 20mL). The cell was left for 5 minutes before the experiment was run.

Figure 2 below shows experimental data for a cyclic voltammetry experiment done with 7.5*10⁵ cells.

Figure 2 Current plotted against voltage during a cyclic voltammetry run.

We observe the characteristic dip indicative of CPR oxidation at approximately -0.71V, showing the presence of CPR in solution. Next, the time derivative of this curve will be taken, shown below.

Figure 3 Rate of current change versus input voltage for a cyclic voltammetry assay.

Using Figure 3, we determine the maximum rate of change of current to occur at -0.755V, with a maximum of 258.45uA/V.

By repeating this procedure for the various tested cell levels, we can construct one of our standard curves - the maximum rate of current change versus number of cells.

Figure 4 Maximum rate of current change per voltage versus the number of cells.

Finally, by varying the amount of CPR in solution, and running cyclic voltammetry experiments in a simple system of only CPR, buffer, and nanopure water, we can construct a standard curve of CPR concentration versus maximum rate of current change. This graph is shown below.

Figure 5 Maximum rate of current change versus concentration of CPR in our electrochemical cell.

Currently, work is being done to refine our ability to differentiate between varying levels of CPR concentration. Our preliminary data is shown below.

Figure 6 Current drop versus concentration of CPR. This data was calculated based on the drop of each individual peak observed at the ionization voltage of -0.7V.

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••••• Lri

This part was used as a reporter. It performed as expected. β-gal assays showed that this part was expressed in the expected pattern and was useful as a reporter.

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