Inkjet-Printed Microtiter Plates for Portable Electrochemical Immunoassays.
––––––––––––––––––––SUPPORTING INFORMATION–––––––––––––––––––
Milica Jović, Yingdi Zhu, Andreas Lesch, Alexandra Bondarenko, Fernando Cortés-Salazar,
Frédéric Gumy, Hubert H. Girault*
Laboratoire d'Electrochimie Physique et Analytique, École Polytechnique Fédérale de Lausanne
— Valais Wallis, CH-1951 Sion, Switzerland.
TABLE OF CONTENT
SI.I Optimization of the atrazine immunoassay protocol for electrochemical detection. ...............2
SI.II Scanning electrochemical microscopy of different magnetic beads. ......................................5
SI.III Reproducibility of IJP microtiter plates for amperometric detection of PAP and TMB. ......5
SI.IV Evaluation of long-term stability of IJP microtiter plates……………………….……...…..9
1
SI.I Optimization of the atrazine immunoassay protocol for electrochemical detection
The commercial kit for ATR was designed for colorimetric detection and the assay protocol
needed to be adjusted and optimized for the electrochemical detection. The first step was the
investigation of the sensitivity of the electrochemical signal for different concentrations of HRP
enzyme (Figure S2a). Briefly, 50 μL of the MB suspension was loaded into the vials, sedimented
and separated from the supernatant. Thereafter, 50 μL of Ab-HRP solutions with various enzyme
concentrations were added and incubated for 30 min at room temperature (RT) under stirring
(1000 rpm). Next, the formed immunocomplex was washed 3 times with 100 µL of the washing
solution, using a magnetic separation stand to precipitate the magnetic beads bound with the AbHRP and to separate them from the supernatant. Finally, 50 μL of the TMB substrate solution
was added and the suspension was transferred into a respective microchip well. The potential of
– 0.1 V was applied for the amperometric detection of enzymatically produced TMB DI. The
detected currents were in a good agreement with the HRP dilution (Figure S2a) proposing that
the electrochemical detection step can successfully be coupled with the atrazine immunoassay
protocol.
Afterwards, the immunoassay reagent volumes were optimized in order to reach the highest
sensitivity for the electrochemical detection. For that purpose the volume of the immunoreagents
were adjusted by the proportional increase of all component volumes by factors 2.5, 5 and 10 so
that the sensitivity of the competitive immunoassay remained intact (Procedures A, B, C, D,
respectively; Table S1). Firstly, ATR, diluent and ATR-HRP (conjugate) solutions were mixed
with a MB-Ab suspension (see below the employed volumes), placed into vials and incubated
during 30 min at RT under stirring (1000 rpm). Thereafter, the system was rinsed 3 times with
the washing solution (volume according to the applied procedure) using a magnetic separation
rack to precipitate the MBs with the immunocomplex and to separate them from the supernatant.
The volumes of the washing solution were adjusted based on the initial volumes of the
immunoreagents. Finally, 50 μL of the TMB substrate solution was added into each vial, the
2
suspension was transferred into the microchip and the according optimal potential was applied
for both detections. As it can be seen from the Figure S2b, increasing the volumes of the
immunoreagents increases the electrochemical signal. Consequently, for procedure D the
signal/blank ratio was the highest, thus the procedure was chosen as the optimal (compromise
between the reagents consumption and sensitivity) and such was applied for the further
experiments.
Table S1: Optimization of the reagent volumes and washing steps for the atrazine immunoassay
protocol.
PROCEDURE A
PROCEDURE B
PROCEDURE C
PROCEDURE D
25 µL diluent
50 µL diluent
100 µL diluent
250 µL diluent
25 µL ATR-HRP
50 µL ATR-HRP
100 µL ATR-HRP
250 µL ATR-HRP
50 µL MB-Ab
100 µL MB-Ab
200 µL MB-Ab
500 µL MB-Ab
WASHING
WASHING
WASHING
WASHING
1. step 100 µL
1. step 200 µL
1. step 400 µL
1. step 1000 µL
2. step 100 µL
2. step 100 µL
2. step 200 µL
2. step 500 µL
3. step 50 µL
3. step 50 µL
3. step 100 µL
3. step 100 µL
The ATR commercial kit involves the addition of 500 µL of TMB solution for the detection.
However, the maximum volume of the microchip wells is equal to 50 µL, hence it was necessary
to adjust the volume of substrate solution. The experiment was done using the optimized
reagents volumes by Procedure D, with a difference in the last step where various volumes of
TMB substrate solution were introduced: 50 µL, 100 µL, 200 µL and 500 µL (Figure S2c).
However, after the enzymatic reaction only 40 µL of the final suspension was transferred into the
wells for the detection due to the volume capacity of 50 µL. When 500 μL of the TMB substrate
solution was used the amperometric signal was quite low (~ 50 nA). However, decreasing the
volumes of the TMB solution is causing smaller dilution and thus increases the electrochemical
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signal. In conclusion, the addition of TMB in the volume equal to the maximum well volume (50
µL) resulted in four times higher signal value than the background. The TMB volume of 50 µL
was chosen as the optimal and implemented in the further experiments.
Figure S1. a) Sensitivity of the electrochemical signal for the detection of different HRP enzyme
concentrations: Ab-HRP dilutions were 1/1000, 1/5000, 1/10 000 and 1/50 000 in PBS. b)
Optimization of the immunoreagents volumes for the amperometric detection of ATR. c)
Optimization of the TMB volume for the amperometric detection of ATR. TMB DI produced in
the reaction between TMB and H2O2 catalyzed by HRP was electrochemically detected in all
three experiments at – 0.1 V during 60 s.
The ELISA kit for the TSH quantification was designed for the amperometric detection of PAP
and used according to the provided protocol.
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SI.II Scanning electrochemical microscopy of different magnetic beads
MBs have been widely used because of their chemical and physical stability and high surface to
volume ratio in comparison with a planar solid phase, which can be easy modified with
biomolecules. The small size of MBs ensures a fast equilibrium between antigen and antibody,
and thereby fast immunoassays. Moreover, MBs are compatible with miniaturized systems due
to their high degree of freedom and easy manipulation with an external magnetic field. MBs can
be easily modified with Abs or other biomolecules that provides their wide application as a solid
phase for immunoassays through different surface functionalizations: protein A, protein G,
amine, carboxylic acid, epoxy, tosylactivated, silane, streptavidine, and used to be coated by
hydrophilic polymers to prevent aggregation. In terms of electrochemical assays, the quality of
the coating is of high importance due to possible interference of Fe3O4 core on the detected
signal. In order to investigate the electrochemical properties of different MBs, scanning
electrochemical microscopy (SECM) was performed. SECM is an analytical technique where the
electrochemical signal is collected on the ultramicroelectrode (UME) in a solution of a RedOx
mediator when it is positioned or moved in a proximity to a substrate. Obtained signal depends
on the topography and electrochemical properties of an investigated substrate.
Experimentally, 2.5 μL of the suspensions of MBs-ProteinA/G (dilution 1 to 20 in PBS), MBsAbTSH1 and MBs-AbATR were transferred into an electrochemical well filled with 2 mM
FcMeOH solution in 10 mM PBS and installed above a magnet so that the MBs formed small
plugs positioned in one line. SECM experiments were provided by a custom-built SECM setup
running under SECMx software combined with IVIUM potentiostat (IVIUM Technologies,
Netherlands) operating in a three-electrode mode. An Ag wire was used as a quasi-reference
electrode (QRE) and a Pt wire – as a counter (CE). All the reported further potentials are given
with respect to the Ag-QRE. The surface of the electrochemical well was levelled based on the
negative feedback approach curves performed at the potential equal to 0.25 V on a glassy Pt
UME (diameter of Pt 10 μm, RG 7). Thereafter, the UME was positioned on 200 µm distance
5
over the substrate and the current was recorded on the UME during scanning above the surface at
the constant height with the step 10 µm and the translation speed 10 µm/s.
In terms of SECM, scanning above the plug of well-coated and therefore isolated plugs of MBs
should provide a negative feedback due to the decrease in the distance between the UME and the
substrate and blocking the diffusion of FcMeOH to the electrode. MBs-AbTSH1 and MBsProteinAG presented the expected decrease in the detected current, however MBs-AbATR showed
positive feedback: increase in the current on the UME when it was passing above the MBs plug
(Figure S). It indicates accessibility of the Fe3O4 core to the redox mediator and recycling
FcMeOH+ into FcMeOH on the MBs.
1.05
1.00
I/I0
0.95
0.90
MB-AbATR
MB-AbTSH1
MB-ProteinA/G
0.85
0.80
0.75
0
500
1000
1500
2000
Electrode latteral position / m
Figure S2. SECM signal obtained during lateral movement of the electrode above the MBs plugs
at a constant distance. E = 0.250 V, QRE = Ag, CE = Pt, working electrode = Pt UME, 25 μm of
diameter, RG = 7.
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SI.III Reproducibility of IJP microtiter plates for amperometric detection of PAP and
TMB
The reproducibility of IJP microtiter plates was determined by the amperometric detection of
para-aminophenol (PAP) and 3,3′,5,5′-Tetramethylbenzidine diimine (TMB DI). For this
purpose, 1 mL of PAPP was incubated with 0.5 μL of Ab-ALP stock solution and 50 µL of TMB
+ H2O2 was incubated with 25 µL ATR-HRP overnight to complete the enzymatic production of
PAP and TMB DI, respectively. Thereafter, the obtained solution was transferred into each well
of the microchip and the electrochemical signal was read out simultaneously in all 8 wells at the
corresponding optimal potential during 60 s (Figure S3). Current values obtained at 60 s were
used for plots in Figure S4.
Figure S3. Original measurement plots for amperometric detection of enzymatically produced:
a) PAP from the reaction between commercial PAPP substrate (24 mM, pH 7.6) and Ab-ALP
(applied potential 0.05 V); b) TMB DI from the reaction between commercial TMB substrate
(TMB and H2O2 in an organic base, pH 6) and Ab-HRP (applied potential - 0.1 V).
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Figure S4. Reproducibility of the electrochemical signal for the IJP microtiter plates based on
the amperometric detection of enzymatically produced: a) PAP from the reaction between
commercial PAPP substrate (24 mM, pH 7.6) and Ab-ALP (applied potential 0.05 V); b) TMB
DI from the reaction between commercial TMB substrate (TMB and H2O2 in an organic base,
pH 6) and Ab-HRP (applied potential - 0.1 V).
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SI.IV Evaluation of long-term stability of IJP microtiter plates
Long-term stability of IJP plates was evaluated by recording the consecutive immunoassay
experiments in the same microchip well using 30 µIU/ml of TSH and protocol described in the
Section 2.4. After each measurement microchip well was intensively, but gently washed with DI
water and dry with N2. Figure S5 is showing a high stability of IJP microtiter plates, allowing
multiple uses of the microchips up to 20 times while keeping the electrode responses within a
variability of 2%.
Figure S5. Evaluation of long-term stability and multiple usage of IJP microtiter plates using the
repeated immunoassay protocol with 30 µIU/ml of TSH.
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