for the entire population [8]. PA also utilizes QS for cell-to-cell
communication to enable biofilm formation, and regulate the
production of virulence factors which challenge the host defense
systems and provoke acute and eventually chronic infections [4, 9,
10 ]. Among>50 different quinolones produced by PA, the most
predominant are 2-heptyl-3-hydroxy-4(1H)-quinolone, classified
asPseudomonasquinolone signal (PQS), and its biosynthetic pre-
cursor 2-heptyl-4-hydroxyquinoline (HHQ). PQS and HHQ func-
tion as signaling molecules and boost pathogenicity, for instance, in
the production of pyocyanin (PYO) and biofilm formation [11].
Other potential biomarkers such as a thiazolyl-indole alkaloid have
also been isolated from PA, but have been far less studied [12].
Typically, PA infections are diagnosed in the clinic using plate
culturing and polymerase chain reaction (PCR) which are not
quantitative and require time-consuming sample preparation,
respectively. Despite sensitive, PCR is open to significant interfer-
ence by contamination, while mispriming and false negatives can
present a challenge to the clinical implementation of this technol-
ogy [13]. Rapid, sensitive, and selective electrochemical strategies
would offer advantages over these and other techniques.
The protocols described here involve the detection of PYO,
HHQ, and PQS by simple electrochemical methods using a boron-
doped diamond (BDD) electrode [14, 15]. We have also reported
on the detection of barakacin, derived from a ruminal PA strain
[16]. However, this compound is less documented and therefore is
not discussed further here.
This chapter describes the detection of PA signaling molecules
PYO, HHQ, and PQS as standards (Fig.1) using the BDD elec-
trode. Detection of PYO, HHQ, and PQS in bacterial cultures of
PA14 is then described (Fig. 2) using liquid-liquid extraction
(LLE) followed by solid-phase extraction (SPE) [14] as a precon-
centration step for the improvement of detection sensitivity. Finally,
an improved procedure, involving rapid and direct detection of
PYO, HHQ, and PQS, is described using cationic surfactant hex-
adecyltrimethylammonium bromide (CTAB) to disrupt the bacte-
rial membrane, aiding the release of biomarkers (Fig.3)[15].2 Materials
Prepare all buffer solutions in deionized water (DW) using an
appropriate water purification system and analytical grade reagents.
Store all buffer solutions at room temperature (unless otherwise
stated). Prepare 2 mM of the stock solutions of HHQ, PQS, or
PYO in acetonitrile (ACN) (seeNote 1) and store them in a
refrigerator at 4C. Follow all regulations when disposing of
waste materials.108 Alyah Buzid et al.