Experimental section
Chemicals.
([Os(bpy)2DPPZ]Cl2) was prepared
according the procedures reported in the work of Petralia, Sciuto, Di
Pietro et al (2017). The engineered E. coli strains, specific for
AsIII and HgII, the ampicillin antibiotic and the 4-aminophenyl
β-D-galactopyranoside powder were provided by the EEM-Lab of the
University of Lausanne and used as described in the section below.
Filtered sterile 10X (1 mM) phosphate-buffered saline solution, or PBS,
(137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4and 1.47 mM KH2PO4, pH=7, Fisher
Scientific) was diluted 10-fold for usage. Sterile Luria Bertani (LB)
broth and chloride potassium (KCl) were purchased from Sigma. Sodium
arsenite (NaAsO2), mercury chloride
(HgCl2) and cadmium sulphate (CdSO4)
powders, from Sigma, were dissolved in filtered sterile 1 mM PBS and
used for the analysis.
Whole-cell-based
module. The whole-cell-based module is represented by the sensing
element for the AsIII obtained by the molecular cloning experiment
described in the work of Cortés-Salazar et al. (2013).
The sensing element is a genetically modified E. coli strain
containing the recombinant plasmid pMV132-arsR-ABS in which thears operon promoter sequence (Pars) is fused,
downstream, to the ars R and lac Z gene.
Electrochemical
module. The electrochemical (EC) module of the biosensing system
consists of (a) a miniaturized device and (b) a portable electronic
system to perform the analysis. The EC device reported in Fig. 1, was
manufactured using the VLSI technology on a 6” silicon wafer substrate
according to the procedure described in Petralia, Sciuto and Conoci et
al., 2017. It is a silicon chip contains 4 EC-cells for multiplexing
analysis, each contains three planar microelectrodes so structured: a
working electrode (WE) made in platinum, counter and reference electrode
(CE, RE) made in gold through sputtering process. Table 1 contains the
size of each EC cell. The electrode-to-electrode distances are 100 μm.
The silicon chip is integrated with a polycarbonate ring creating 4
microchambers (20 μL total volume) and electrically isolated from the
substrate by a thermally grown silicon oxide layer (Fig. 1c). The whole
device is fixed on a plastic holder for an easily handling. The EC
device is connected with a customised board developed by
STMicroelectronics acting as miniaturised potentiostat and used to
perform the electrical driving of CV measurements on samples (Fig 1b-c).
Experimental procedure for analysis. To prepare the
samples for the analysis, the E. coli sensing element was plated
24 h in LB-agar added with ampicillin 100 µg/ml. The day after, few
colonies of the plate were suspended in 20 mL of fresh LB broth and
incubated overnight at 21°C – 150 rpm. The last 3 h cells were
incubated at 37°C – 150 rpm until the optical density
(OD600nm) was around 0.6. Subsequently, the culture was
diluted 2-fold in tap water (final volume of 10 mL) and used to prepare
the following mixes: 800 µL of diluted E. coli culture + 100 µL
of 2.5 – 5 – 10 – 50 – 100 ppb NaAsO2 (water as
zero) + 100 µL of 10 mM PAPG. Once induced by incubation at 30°C – 350
rpm – 2 h, 20 µL of the induced mixes have been immediately spotted
into the microchambers of the EC device and analyzed by CV. For each
concentration, samples have been prepared and measured in triplicate.
The experimental procedure was repeated by substituting the
NaAsO2 with HgCl2 (HgII) and
CdSO4 (CdII) to test the cross-reactivity.
Electrochemical measurements. CV measurements were
carried out by using the portable electronic board linked to a laptop
via USB connection (Fig. 1C). A RealTerm and MATLAB software were used
to manage, respectively, the electronic settings for the CV experiments
and the collection and post-processing of data coming from the board.
The experiment was set using a scan rate of 10 mV/s and a voltage range
of −1.5 V / +1.5 V, and focusing on the anodic peak of PAP at around
+1.2 V. Before each test, the platinum surface of the WE was activated
by 50 sweeps of CV in KCl 0.1 M. The LoD was obtained by measuring 3
replicas of a blank sample and calculating the mean result and the
standard deviation (SD) according to the following equation LoD = 3 x
SDRegression / Slope, while the Limit of Quantification
was calculated by the follows equation LoQ = (LoD/3) x 10. The delta
current signal (I delta) was calculated as the difference between the
current recorded in whole-cell biosensor in presence of the metal
analyte and the current signal with 0 ppm of analyte (blank).