Quorum Sensing

7. Elute PYO, HHQ, and PQS from the SPE cartridge using
   0.5 ml of 5 % of 4.5 M ammonium formate in methanol
   (95 % CH 3 OH:5 % DW, v/v).


8. Add 0.3 ml of the eluent (step 7) to the 0.3 ml of electrolyte,
   50 mM acetate buffer at pH 5.0 consisting of 20 % ACN, and
   perform the DPV detection on the BDD electrode vs. Ag/
   AgCl.


9. Repeat the same procedure (step 1– 8 ) at each different time
   interval (5, 6, 7, and 8 h) (Fig.2).

3.2 Direct Detection
of PYO, HHQ, and PQS
in PA14 Bacterial
Cultures Based on In
Situ Cationic
Surfactant-Assisted
Membrane Disruption

1. Add a 0.25 ml sample of the bacterial culture to 0.75 ml of
   1 mM CTAB prepared in 50 mM phosphate buffer, pH 7.0
   (1:4, culture:CTAB, v/v).


2. Equilibrate this bacterial culture, CTAB, and buffer mixture for
   5 min at room temperature.


3. Add 0.2 ml of the bacterial culture, CTAB, and buffer mixture
   to 0.8 ml of electrolyte, 50 mM acetate buffer (pH 5.0) con-
   sisting of 20 % ACN, and perform the DPV detection at the
   BDD electrode vs. Ag/AgCl (Fig.3).

4 Notes

1. Stock solutions of PYO, HHQ, and PQS: 2 mM each in ACN.
   Weight 2.1 mg of PYO (C 13 H 10 N 2 O) and then add 5 ml of
   ACN. Weight 2.4 mg of HHQ (C 16 H 21 NO) and then add
   5 ml of ACN. Weight 2.6 mg of PQS (C 16 H 21 NO 2 ) and then
   add 5 ml of ACN.


2. The BDD electrode was polished with wet polishing (nylon
   and MasterTex) pads. Always add some DW to polishing pads
   before the addition of alumina. First, use wet nylon paper with
   0.3μm alumina slurries and make figure-8 motions on the
   polishing pad to clean the surface. Then, use wet MasterTex
   paper with 0.05μM alumina and make figure-8 motions on the
   polishing pad to further polish the rough surface of the BDD
   electrode. After rinsing the electrode with DW, ethanol or
   propanol can be used to sonicate the electrode for 5 min to
   remove any residual organic species. Subsequently, sonicate the
   electrode in DW for 10 min to remove any residual alumina
   particles.


3. The DPV potential is swept between – 0.6 Vand +2.0 V for the
   electrochemical detection of PYO, HHQ, and PQS.


4. Insert the BDD electrode into the electrochemical cell, connect
   the Pt and Ag/AgCl electrodes. Then, use CV between1.0
   and +2.0 V vs. Ag/AgCl (3 M KCl) at a scan rate of 100 mV
   s^1 in a 50 mM acetate buffer (pH 5.0) (seeNote 5) until a
   steady CV profile is obtained.

Rapid Detection ofP. aeruginosaSignal 113