Online monitoring of reporter cell biosensors in a multiplexed array format

摘要

Reporter cell biosensors utilize reporter cells that produce a measureable signal with respect to changes in the chemical or physical environment. Pseudomonas aeruginosa produces the electroactive blue-green compound pyocyanin that is ideal as a reporter compound because it is detectable electrochemically, spectroscopically, and visually. The FAD dependant monooxygenase PhzS affects the final step in pyocyanin biosynthesis. To construct a reporter cell, a deletion mutant of phzS was generated in P. aeruginosa PAO1, and this strain was complemented by plasmid pDOH54 which contains IPTG inducible Ptrc promoter upstream of phzS. A low cost (<$3) sterilizable electrochemical cell with carbon paste working and auxiliary electrodes and Ag/AgCl quasi-reference electrode was developed for electrochemical monitoring of the reporter cells. Using this electrochemical cell, pyocyanin standards were quantified in the concentration range 571 nM-38.1 muM (R2>0.99) with cyclic voltammetry at 0.5 V/s, and square wave voltammetry at 400 Hz was used to detect pyocyanin down to 114 nM. Electrochemical monitoring of live cells revealed that reporter strain P. aeruginosa (delta)phzS pDOH54 had eightfold higher electrochemical activity than the P. aeruginosa (delta)phzS strain, indicating that electrochemical activity of the reporter cells is primarily affected by PhzS mediated pyocyanin production. P. aeruginosa (delta)phzS pDOH54 cells exposed to 0.0 - 75. muM IPTG did not show significantly different electrochemical activity (p>0.05) over a 19.3 hour monitoring period which implicates non-selective low level induction of phzS of the Ptrc promoter in MOPS buffered glucose medium. To enable reporter cell biosensors to operate remotely, a biosensor network was constructed using the Rabbit 4200 embedded, Ethernet-enabled microprocessor coupled to a PMT and arsenic responsive reporter Pseudomonas fluorescens arsR::lux strain 18HR. Arsenic was detected remotely in real time at concentrations of 1 ppm and 10 ppm in less than 60 minutes after exposure. Also, web services technology was integrated into biosensor and humidity control instrumentation for remote web browser monitoring. Web pages using client-side AJAX controls were found to use less than 2 kb per screen update compared to greater than 20 kb per screen update for an equivalent server-side solution based on ASP.NET web forms controls. This research demonstrates the feasibility of distributing reporter cell biosensors in a networked array format with detection over the Internet. Future work will modify the regulation of phzS for development of a reporter cell biosensor that utilizes the pyocyanin biosynthetic pathway of P. aeruginosa..