working electrodes. The method has been explained in a previ-
ously published protocol.[22] The StDT-coated Au/Si electrodes
were mounted on the vacuum holder of a spin coater, and a solu-
tion of CdS/ZnS QDs (dissolved in toluene at a QD concentration of
12.6 µM) was added while rotating the electrode at 6000 rpm for
1 minute. This type of electrode was used for the studies with the
enzymes in solution, or as the basis for the enzyme immobilization
during sensor development.
Supporting Information
Supporting Information is available from the Wiley Online Library
or from the author.
Enzyme Immobilization: Enzymes were immobilized on the sur-
face of a QDs-modified Au electrode (surface area of 1 cm2) using
a layer-by-layer (LbL) assembly technique.[41] For this, 18 nM of
human guanylate kinase GMPK (purified as highly pure recombi-
nant fusion protein with His6-SUMO tag,[1] 8 µL of pyruvate kinase
PK (12 units mL−1 solution; Sigma-Aldrich), and 10 µL of lactate
dehydrogenase LDH (15 units mL−1 solution; Sigma-Aldrich) were
adsorbed on the surface of each electrode, followed by applica-
tion of a 20 to 30 µL solution containing the strong poly-anion
poly(sodium-4-styrene sulfonate) (PSS, MW = 70 kDa, 59 µM;
Sigma-Aldrich) on top of the enzyme solution. After incubation for
15 minutes, without letting the solution dry, the electrodes were
spun for 1 minute at 6000 rpm in order to remove the non-attached
macromolecules. The electrodes were rinsed with water, and once
they were dried, 20 to 30 µL of a solution containing the weak
poly-cation poly(allylamine) hydrochloride (PAH, MW = 56 kDa,
33 µM; Sigma-Aldrich) was added to the PSS layer. After rinsing,
another PSS/PAH bilayer was deposited following the same proce-
dure. This resulted in two PSS/PAH bilayers, with PAH forming the
outermost layer. The overall structure of the assembled electrode
can be expressed as (PSS/PAH)2/LDH;PK;GMPK/QD/StDT/Au/Si.
Photocurrent Measurements: The homebuilt set-up for
recording the photocurrents has been described in previous
publications.[22,25,26] A Xe arc lamp (emission spectrum λ =
300 – 700 nm) controlled by a lamp power supply LPS 220
(Photon Technology International) was used as the light source.
An optical chopper (Scitec instruments) was introduced in the light
path ahead of the lens, which focused the light onto the working
electrode, thus allowing us to modulate the incident light at a
desired frequency. A plano-convex lens and a 45° mirror (Linos
Germany) were used to focus light from the Xe arc lamp on the
working electrode in the electrochemical cell. The illumination was
periodically modulated with a chopper. The electrochemical cell
(1 mL solution volume) with the three-electrode arrangement was
connected to a potentiostat. In this arrangement, the working elec-
trode (WE, i.e., the CdS@ZnS/StDT/Au/Si chip) was set to ground
potential. The fixed potential Ubias = +50 mV was controlled via
the reference electrode (RE, i.e., the Ag/AgCl electrode) using an
operational amplifier (OP), which supplied the required voltage
through the counter electrode (CE, i.e., a platinum wire). In order
to improve the signal-to-noise ratio for the photocurrent measure-
ments I’(t), a lock-in amplifier (EG&G Model # 5210) was used. All
measurements were performed at room temperature in 100 mM
Acknowledgements
N. Sabir and N. Khan contributed equally to this work. This work was
supported by the German Research Foundation (grant PA794/15–1
to WJP, grant WI 1361/6–1 to GW, and grant Li 706/8–1 to FL). NS
acknowledges the Higher Education Commission (HEC) of Pakistan
and GC University Faisalabad (GCUF) of Pakistan. NK's doctoral
thesis work has been funded by a DAAD scholarship. MK and NK
acknowledge continuous support by the Max Planck Society.
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modulation frequency of 17.3 Hz was applied for the lock-in of the
chopper. The time constant was set to 300 ms during the meas-
urements. The modulated illumination was switched on and off for
fixed periods of 30 s with a shutter (15 s on, 15 s off). The output
of the lock-in amplifier, namely, the rectified mean amplitude I =
<|I’|> of the photocurrent, was recorded through the serial port
interface using a personal computer (PC).
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2015, 11, No. 43, 5844–5850