A mediator-less immunodetection method for microorganisms is realized by incorporating the newly developed field-effect enzymatic detection (FEED) technique with the conventional electrochemical immunosensing approach. The gating voltage of FEED facilitates the transduction of electrical signal through the bulky immune complex so that the detection does not rely on the use of mediators or other diffusional substances. The voltage-controlled intrinsic amplification provided by the detection system allows detection in low-concentration samples without target pre-enrichment, leading to ultrasensitive and rapid detection. The detection approach is demonstrated with E. coliO157:H7, a model microorganism, in milk with an estimated detection limit of 20 CFU mL−1 (where CFU is a colony-forming unit) without performing sample pre-enrichment and centrifugation of sample followed by the resuspension of the pellet in a buffer solution, resulting in a significantly shortened assay time of 67 min. Optimizing the gating voltage resulted in the detection of 12 CFU mL−1 of the bacterium in milk. The novel detection approach can be used as a detection platform for ultrasensitive, specific and rapid detection of microorganism pathogens

Bonastre

Choi

Daly

Ferri

Giese

Gray

Ivnitski

Lovley

Mathebe

Medina

Page

Rinaldi

Skládal

Tans

Tavernier

Tokarskyy

Turano

Zhang

English

Wang, Jiapeng

Xu, Yan

Yau, Siu-Tung

EngagedScholarship @ Cleveland State University

Cleveland State University
EngagedScholarship@CSU
Electrical Engineering & Computer Science Faculty
Publications
Electrical Engineering & Computer Science
Department
4-15-2014
Mediator-less immunodetection with voltage-
controlled intrinsic amplification for ultrasensitive
and rapid detection of microorganism pathogens
Jiapeng Wang
Cleveland State University
Yan Xu
Cleveland State University
Siu-Tung Yau
Cleveland State University, s.yau@csuohio.edu
Follow this and additional works at: https://engagedscholarship.csuohio.edu/enece_facpub
How does access to this work benefit you? Let us know!
Publisher's Statement
This is the accepted version of the following article: J. Wang and S. Yau, "Detection of alpha-
Methylacyl-CoA Racemase in Serum and Urine Using a Highly Sensitive Electrochemical
Immunodetector," Electroanalysis, vol. 26, pp. 1441-1444, 2014. , which has been published in final
form at http://onlinelibrary.wiley.com/doi/10.1002/celc.201300180/abstract
Repository Citation
Wang, Jiapeng; Xu, Yan; and Yau, Siu-Tung, "Mediator-less immunodetection with voltage-controlled intrinsic amplification for ultrasensitive and rapid
detection of microorganism pathogens" (2014). Electrical Engineering & Computer Science Faculty Publications. 311.
https://engagedscholarship.csuohio.edu/enece_facpub/311
This Article is brought to you for free and open access by the Electrical Engineering & Computer Science Department at EngagedScholarship@CSU. It
has been accepted for inclusion in Electrical Engineering & Computer Science Faculty Publications by an authorized administrator of
EngagedScholarship@CSU. For more information, please contact library.es@csuohio.edu.
Original Citation
J. Wang and S. Yau, "Detection of alpha-Methylacyl-CoA Racemase in Serum and Urine Using a Highly Sensitive Electrochemical
Immunodetector," Electroanalysis, vol. 26, pp. 1441-1444, 2014.
Detection of a-Methylacyl-CoA Racemase in Serum and
Urine Using a Highly Sensitive Electrochemical
Immunodetector
Jiapeng Wang[a] and Siu-Tung Yau*[a, b]
Because of its lack of specificity in the diagnosis of pros-
tate cancer, prostate specific antigen (PSA) is deemed un-
satisfactory as a biomarker for the diagnosis of the dis-
ease [1]. New biomarkers with improved performance
factors are needed for the early diagnosis of prostate
cancer [2]. One potential biomarker is a-methylacyl-CoA
racemase (AMACR), an enzyme that is involved in per-
oxisomal-oxidation of dietary branched-chain fatty acids.
Recent studies have shown that, compared with expres-
sion in normal or benign prostate epithelium, AMACR is
consistently overexpressed in prostate cancer epithelium,
making it a specific marker for cancer cells within the
prostate gland [3–5]. The fact that AMACR is present in
serum and urine of prostate cancer patients allows it to
be used as a convenient biomarker for the diagnosis of
prostate cancer.
Previously, AMACR in the urine of prostate cancer pa-
tients has been detected in a fluidic system using a macro-
cantilever-based piezoelectric sensor, whose surface was
immobilized with anti-AMACR [6]. Detection of
AMACR on the femto-gram/mL level was achieved di-
rectly in patient urine without a sample preparation step
and without the use of labeled reagents. Another sensor-
based detection of AMACR was made using a iridium
nanoparticle-based H2O2 electrochemical sensor [7]. The
sensor was used to detect H2O2 that was released in the
AMACR-catalyzed conversion of (2R)-pristanoyl-CoA to
(2S)-pristanoyl-CoA. The sample preparation required in-
cubation for 72 hours. The sensor provided detection on
the mg/mL level and showed 100% accuracy, which was
demonstrated using blood samples of patients.
This paper reports the detection of AMACR on the
pico-gram(pg)/mL level, using a novel immunosensing
method, which consists of a three-electrode system modi-
fied with a gating electrode for applying a gating voltage
VG to the immune complex immobilized on an electrode.
The detection was performed in serum and urine samples
with a limit of 100 pg/mL. The detection system was real-
ized by integrating gating electrodes with screen-printed
electrodes (SPEs). This emerging detection technique can
be potentially used to form a disposable platform for the
ultrasensitive and low-cost detection of AMACR for
point-of-care applications.
The detection of AMACR was achieved using the de-
tection system described in Supporting Information, the
operation of which requires the sandwich structure, Ab-
AMACR-Ab(HRP), to be formed on the AMACR-de-
tecting electrode. The detection signal was the measured
peak current of the reduction peak of HRP without using
H2O2, the substrate of HRP. A similar reagentless detec-
tion approach was also demonstrated previously [8].
Control experiments were performed in order to avoid
misinterpretation of experimental results due to non-spe-
cific binding. Figure 1a shows a set of cyclic voltammo-
grams (CVs) obtained with a SPE modified with PANI as
described above. The modified electrode was first immo-
Abstract : The detection of a-methylacyl-CoA racemase
(AMACR), a novel biomarker for prostate cancer, is
demonstrated in serum and urine using a novel immuno-
detection method. The detection system consists of
a three-electrode conventional electrochemical cell modi-
fied with a gating electrode for applying a gating voltage
VG to the immune complex immobilized on the working
electrode to provide signal amplification. The detection
system is realized by integrating gating electrodes with
screen-printed electrodes. This detection method does not
require involved sample preparation procedures. The de-
tection was demonstrated in serum and urine samples on
the nanogram/mL level with VG equal to 0.6 V. Detection
in serum was also performed on the picogram/mL level
with a limit of 100 picogram/mL with VG=0.6 V being
a necessary condition.
Keywords: Biomarker detection · Prostate cancer biomarker · Immunoassay · Screen printed electrode
[a] J. Wang, S.-T. Yau
Department of Electrical and Computer Engineering,
Cleveland State University, Cleveland, Ohio 44115, USA
[b] S.-T. Yau
The Applied Bioengineering Program, Cleveland State
University, Cleveland, Ohio 44115, USA
*e-mail: s.yau@csuohio.edu
bilized with the primary Ab as described below and sub-
sequently incubated with the labeled secondary
Ab(HRP) for 30 min. The SPE was then washed with de-
ionized water. CV1 was obtained with this electrode in
serum. CV2 was obtained with the same electrode in
serum when a VG of 0.6 V was applied. The two CVs
appear to be featureless and have similar magnitudes.
Thus, the secondary Ab does not create non-specific bind-
ing.
Figure 1b shows a set of CVs obtained with a PANI-
modified SPE, which had the Ab-AMACR-Ab(HRP)
structure formed on its surface. The AMACR concentra-
tion used to prepare this electrode was 100 ng/mL. CV1
was obtained in serum, showing a pair of redox peaks
with a formal potential of 0.32 V vs. Ag/AgCl. The re-
duction peak is at 0.42 V and the weak oxidation peak
is at 0.32 V as indicated by the arrow. Previous works
on the direct electron transfer associated with immobi-
lized HRP show that the formal potential of this redox
process is between 0.5 V and 0.3 V vs. Ag/AgCl [9,10].
Thus, the redox peaks in CV1 indicate the formation of
Ab-AMACR-Ab(HRP) immuno-complex. CV2 was ob-
tained when VG of 0.6 V was applied. The reduction peak
at 0.42 V appears to be enhanced, illustrating the am-
plification provided by FEED. This effect was essential
for measuring the signals at low AMACR concentrations.
Detection of AMACR was previously carried out in
serum with the lowest concentration at mg/mL [7]. In the
present work, detection of AMACR in serum was first
demonstrated on the ng/mL level. Figure 2a shows the
calibration curve of AMACR detection in serum obtained
with VG=0.6 V. The detection covers a range from100 ng/
mL to 1 ng/mL. The data points show a nearly linear de-
pendence on the concentration. The straight line is the re-
gression line for the data points, having a correlation co-
efficient of 0.9730. The sensitivity derived using the
straight line is 0.0048710 mAmL/ng. The small error
bars indicate reproducible device performance. Figure 2b
shows the calibration curve of AMACR detection in
urine obtained with VG=0.6 V. The general features of
the calibration curve are similar to those for the serum
case except that the current level is reduced.
Detection of AMACR in serum was also achieved on
the pg/mL level, where the application of VG is indispen-
sable since the redox peaks of HRP are extremely weak
on CVs. Figure 2c shows the detection calibration curve
of AMACR in serum on the pg/mL level. The curve was
obtained with VG=0.6 V. The first date point shows the
zero concentration signal. The data points indicate
a nearly linear dependence of the signal on AMACR con-
centration. The straight line is the regression line for the
data points, having a correlation coefficient of 0.9890.
The sensitivity derived using the straight line is
12.9 mAmL/ng. The detection limit of this concentration
range is estimated to be 204 pg/mL, using the signal/
noise=3 method. The small error bars indicate reproduci-
ble device performance.
The results presented above show that sensitive detec-
tion of AMACR can be performed in serum and urine
without sample preparation. The detection was made pos-
sible by the amplification provided by the novel detection
system. The specificity provided by the immuno-detection
approach allowed the detection to be carried out in
serum and urine samples. The detection was demonstrat-
ed in serum and urine samples on the nanogram/mL
level. Detection in serum was also performed on the pg/
mL level with a limit of 100 pg/mL.
Experimental
The basic principle of the detection is explained and the
detection mechanism is elucidated in previous publica-
tions [11,12]. Briefly, the detection system consists of
a conventional three-electrode electrochemical cell modi-
fied with additional gating electrodes for applying
a gating voltage VG to the detecting (working) electrode,
Fig. 1. (a) Control experiment with primary Ab and HRP-la-
beled secondary Ab. (b) CVs obtained with 100 ng/mL SPE,
showing the amplification effect. The scan rate of 20 mV/s was
used to obtain the CVs.
on which a redox enzyme, the sensing element, is immo-
bilized. VG rearranges ions in the sample solution at the
electrode-solution interface, inducing an electric field that
penetrates the immobilized enzyme. The field reduces the
effective height of the tunnel barrier between the active
site of the enzyme and the electrode, therefore increasing
the tunnel rate of electrons and resulting in intrinsic am-
plification of the signal current. In the present work, the
enzyme was conjugated to the detecting antibody and im-
mobilized on the electrode via the immune-complex. A
detailed description of the immunosensing system used in
this work is provided in Supporting Information.
Recombinant human AMACR was purchased
(Genway Biotech; Cat. No. GWB-CFE200) and used as
the positive control. Monoclonal anti-AMACR and poly-
clonal anti-AMACR (Genway Biotech; Cat. Nos. GWB-
8770CE, GWB-AMACRA) were used as the primary/
capture antibody and the secondary/detection antibody,
respectively. Horseradish peroxidase (HRP) was conju-
gated to the detection antibody using a conjugation kit.
Human serum (Thermo Scientific Pierce; Product #
31876) and synthetic urine (Quick Fix) were purchased.
SPEs were purchased from DropSens. The working
electrode of the SPE is a circular carbon electrode with
a 4 mm diameter. The working electrode, silver reference
electrode and the carbon counter electrode are fabricated
on the top side of the SPE. The dimensions of the SPE
are 3.4 cm1.0 cm0.05 cm. The working electrode of
the purchased SPE was covered with a layer of polyani-
line (PANI). The PANI layer was used to host the capture
antibody and therefore the immune complex on electro-
des [13] and as electron mediator, shuttling electrons be-
tween HRP and the electrode [14]. The PANI layer was
synthesized on the electrode using electrochemical poly-
merization of aniline in a solution of 0.1 M aniline and
1 M HCl at a potential of 1.2 V for 100 s [15]. The PANI
prepared using this method was used previously in
a HRP-based biosensor to detect H2O2 at pH 6.8 [16] as
well as in a pH sensor [17] because the conductivity of
PANI persists up to pH 9. Alternatively, sulfonated PANI
was prepared by adding poly(vinylsulfonic acid) (Aldrich,
cat. # 27,842–4) to aniline and HCl during electrochemi-
cal polymerization as described previously [18], to in-
crease the redox activity of PANI at pH 7. The results
shown in this work were obtained with PANI prepared
using HCl only while the two kinds of PANI led to similar
results.
AMACR was spiked into serum and urine samples.
The AMACR sample solution did not require treatment.
The PANI layer was modified by depositing 2 mL of gluta-
raldehyde (25% with water, diluted 100 times with water)
on PANI until dry. Glutaraldehyde was used as a cross-
linker, coupling the capture antibody to the highly porous
PANI so that the immune complex was firmly entrapped
in PANI. Previous XPS analysis [13] shows that glutaral-
dehyde binds to the amine groups on PANI to form an
imine bond. AMACR-detecting electrodes were formed
by incubating the modified SPE with 10 mL of 1 mg/mL
Fig. 2. AMACR detection calibration curve on the ng/mL level
in (a) serum and (b) urine with VG=0.6 V. (c) AMACR detec-
tion calibration curve on the pg/mL level in serum with VG=
0.6 V. The scan rate of 20 mV/s was used to obtain the CVs.
primary antibody dissolved in PBS for 1 h at room tem-
perature. To detect AMACR, an AMACR-detecting elec-
trode was incubated with 5–10 mL of an AMACR sample
(a AMACR standard serum solution) for 1 h at 37 8C.
After rinsing and blocking possible open areas on the
electrode with bovine serum albumin (40mg/mL diluted
to 10% in Dulbecco’s phosphate-buffered saline), the
same electrode was incubated with 5–10 mL of 0.5 mg/mL
HRP-labeled secondary antibody for 30 min at 37 8C and
then rinsed with de-ionized water. The Ab-AMACR-
Ab(HRP) sandwich structure now has formed on the
electrode. Finally, the electrode was used as the working
electrode for electrochemical measurements. A piece of
0.5 mm-diameter copper wire coated with a thin layer of
insulator (enamel) was used as the gating electrode for
applying VG. The wire was bent to form a U-shaped struc-
ture and was attached on the sensing electrode using non-
conductive epoxy. All AMACR samples were prepared
with undiluted serum sample as received. The measure-
ments of the reduction peak currents of three electrodes
were used to determine each data point on the calibration
curves.
Electrochemical measurements were made by immers-
ing the detecting electrode in a phosphate saline solution
(PBS) contained in a glass beaker. The electrode was
driven by a commercial electrochemical controller (CHI
660C Work Station). A potential scan rate of 20 mV/
s was used in recording cyclic voltammograms (CV). PBS
(0.1 M at pH 7) was prepared using de-ionized water
(18.2 W-cm). Commercially available hydrogen peroxide
(Fisher scientific, 30% concentration) was diluted to the
concentrations used in the experiment. All measurements
were made with deaerated PBS at room temperature. Re-
producible results were obtained by repeating each mea-
surement multiple times.
Acknowledgement
This work was supported by Cleveland State University.
There is no potential conflicts of interest and/or sources
of funding for all authors involved in the writing of this
manuscript.
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Mediator-less immunodetection with voltage-controlled intrinsic amplification for ultrasensitive and rapid detection of microorganism pathogens

Cleveland-Marshall College of Law

Cleveland State UniversityEngagedScholarship@CSUElectrical Engineering & Computer Science FacultyPublicationsElectrical Engineering & Computer ScienceDepartment4-15-2014Mediator-less immunodetection with voltage-controlled intrinsic amplification for ultrasensitiveand rapid detection of microorganism pathogensJiapeng WangCleveland State UniversityYan XuCleveland State UniversitySiu-Tung YauCleveland State University, s.yau@csuohio.eduFollow this and additional works at: https://engagedscholarship.csuohio.edu/enece_facpubHow does access to this work benefit you? Let us know!Publisher's StatementThis is the accepted version of the following article: J. Wang and S. Yau, "Detection of alpha-Methylacyl-CoA Racemase in Serum and Urine Using a Highly Sensitive ElectrochemicalImmunodetector," Electroanalysis, vol. 26, pp. 1441-1444, 2014. , which has been published in finalform at http://onlinelibrary.wiley.com/doi/10.1002/celc.201300180/abstractRepository CitationWang, Jiapeng; Xu, Yan; and Yau, Siu-Tung, "Mediator-less immunodetection with voltage-controlled intrinsic amplification for ultrasensitive and rapiddetection of microorganism pathogens" (2014). Electrical Engineering & Computer Science Faculty Publications. 311.https://engagedscholarship.csuohio.edu/enece_facpub/311This Article is brought to you for free and open access by the Electrical Engineering & Computer Science Department at EngagedScholarship@CSU. Ithas been accepted for inclusion in Electrical Engineering & Computer Science Faculty Publications by an authorized administrator ofEngagedScholarship@CSU. For more information, please contact library.es@csuohio.edu.Original CitationJ. Wang and S. Yau, "Detection of alpha-Methylacyl-CoA Racemase in Serum and Urine Using a Highly Sensitive ElectrochemicalImmunodetector," Electroanalysis, vol. 26, pp. 1441-1444, 2014.Detection of a-Methylacyl-CoA Racemase in Serum andUrine Using a Highly Sensitive ElectrochemicalImmunodetectorJiapeng Wang[a] and Siu-Tung Yau*[a, b]Because of its lack of specificity in the diagnosis of pros-tate cancer, prostate specific antigen (PSA) is deemed un-satisfactory as a biomarker for the diagnosis of the dis-ease [1]. New biomarkers with improved performancefactors are needed for the early diagnosis of prostatecancer [2]. One potential biomarker is a-methylacyl-CoAracemase (AMACR), an enzyme that is involved in per-oxisomal-oxidation of dietary branched-chain fatty acids.Recent studies have shown that, compared with expres-sion in normal or benign prostate epithelium, AMACR isconsistently overexpressed in prostate cancer epithelium,making it a specific marker for cancer cells within theprostate gland [3–5]. The fact that AMACR is present inserum and urine of prostate cancer patients allows it tobe used as a convenient biomarker for the diagnosis ofprostate cancer.Previously, AMACR in the urine of prostate cancer pa-tients has been detected in a fluidic system using a macro-cantilever-based piezoelectric sensor, whose surface wasimmobilized with anti-AMACR [6]. Detection ofAMACR on the femto-gram/mL level was achieved di-rectly in patient urine without a sample preparation stepand without the use of labeled reagents. Another sensor-based detection of AMACR was made using a iridiumnanoparticle-based H2O2 electrochemical sensor [7]. Thesensor was used to detect H2O2 that was released in theAMACR-catalyzed conversion of (2R)-pristanoyl-CoA to(2S)-pristanoyl-CoA. The sample preparation required in-cubation for 72 hours. The sensor provided detection onthe mg/mL level and showed 100% accuracy, which wasdemonstrated using blood samples of patients.This paper reports the detection of AMACR on thepico-gram(pg)/mL level, using a novel immunosensingmethod, which consists of a three-electrode system modi-fied with a gating electrode for applying a gating voltageVG to the immune complex immobilized on an electrode.The detection was performed in serum and urine sampleswith a limit of 100 pg/mL. The detection system was real-ized by integrating gating electrodes with screen-printedelectrodes (SPEs). This emerging detection technique canbe potentially used to form a disposable platform for theultrasensitive and low-cost detection of AMACR forpoint-of-care applications.The detection of AMACR was achieved using the de-tection system described in Supporting Information, theoperation of which requires the sandwich structure, Ab-AMACR-Ab(HRP), to be formed on the AMACR-de-tecting electrode. The detection signal was the measuredpeak current of the reduction peak of HRP without usingH2O2, the substrate of HRP. A similar reagentless detec-tion approach was also demonstrated previously [8].Control experiments were performed in order to avoidmisinterpretation of experimental results due to non-spe-cific binding. Figure 1a shows a set of cyclic voltammo-grams (CVs) obtained with a SPE modified with PANI asdescribed above. The modified electrode was first immo-Abstract : The detection of a-methylacyl-CoA racemase(AMACR), a novel biomarker for prostate cancer, isdemonstrated in serum and urine using a novel immuno-detection method. The detection system consists ofa three-electrode conventional electrochemical cell modi-fied with a gating electrode for applying a gating voltageVG to the immune complex immobilized on the workingelectrode to provide signal amplification. The detectionsystem is realized by integrating gating electrodes withscreen-printed electrodes. This detection method does notrequire involved sample preparation procedures. The de-tection was demonstrated in serum and urine samples onthe nanogram/mL level with VG equal to 0.6 V. Detectionin serum was also performed on the picogram/mL levelwith a limit of 100 picogram/mL with VG=0.6 V beinga necessary condition.Keywords: Biomarker detection · Prostate cancer biomarker · Immunoassay · Screen printed electrode[a] J. Wang, S.-T. YauDepartment of Electrical and Computer Engineering,Cleveland State University, Cleveland, Ohio 44115, USA[b] S.-T. YauThe Applied Bioengineering Program, Cleveland StateUniversity, Cleveland, Ohio 44115, USA*e-mail: s.yau@csuohio.edubilized with the primary Ab as described below and sub-sequently incubated with the labeled secondaryAb(HRP) for 30 min. The SPE was then washed with de-ionized water. CV1 was obtained with this electrode inserum. CV2 was obtained with the same electrode inserum when a VG of 0.6 V was applied. The two CVsappear to be featureless and have similar magnitudes.Thus, the secondary Ab does not create non-specific bind-ing.Figure 1b shows a set of CVs obtained with a PANI-modified SPE, which had the Ab-AMACR-Ab(HRP)structure formed on its surface. The AMACR concentra-tion used to prepare this electrode was 100 ng/mL. CV1was obtained in serum, showing a pair of redox peakswith a formal potential of 0.32 V vs. Ag/AgCl. The re-duction peak is at 0.42 V and the weak oxidation peakis at 0.32 V as indicated by the arrow. Previous workson the direct electron transfer associated with immobi-lized HRP show that the formal potential of this redoxprocess is between 0.5 V and 0.3 V vs. Ag/AgCl [9,10].Thus, the redox peaks in CV1 indicate the formation ofAb-AMACR-Ab(HRP) immuno-complex. CV2 was ob-tained when VG of 0.6 V was applied. The reduction peakat 0.42 V appears to be enhanced, illustrating the am-plification provided by FEED. This effect was essentialfor measuring the signals at low AMACR concentrations.Detection of AMACR was previously carried out inserum with the lowest concentration at mg/mL [7]. In thepresent work, detection of AMACR in serum was firstdemonstrated on the ng/mL level. Figure 2a shows thecalibration curve of AMACR detection in serum obtainedwith VG=0.6 V. The detection covers a range from100 ng/mL to 1 ng/mL. The data points show a nearly linear de-pendence on the concentration. The straight line is the re-gression line for the data points, having a correlation co-efficient of 0.9730. The sensitivity derived using thestraight line is 0.0048710 mAmL/ng. The small errorbars indicate reproducible device performance. Figure 2bshows the calibration curve of AMACR detection inurine obtained with VG=0.6 V. The general features ofthe calibration curve are similar to those for the serumcase except that the current level is reduced.Detection of AMACR in serum was also achieved onthe pg/mL level, where the application of VG is indispen-sable since the redox peaks of HRP are extremely weakon CVs. Figure 2c shows the detection calibration curveof AMACR in serum on the pg/mL level. The curve wasobtained with VG=0.6 V. The first date point shows thezero concentration signal. The data points indicatea nearly linear dependence of the signal on AMACR con-centration. The straight line is the regression line for thedata points, having a correlation coefficient of 0.9890.The sensitivity derived using the straight line is12.9 mAmL/ng. The detection limit of this concentrationrange is estimated to be 204 pg/mL, using the signal/noise=3 method. The small error bars indicate reproduci-ble device performance.The results presented above show that sensitive detec-tion of AMACR can be performed in serum and urinewithout sample preparation. The detection was made pos-sible by the amplification provided by the novel detectionsystem. The specificity provided by the immuno-detectionapproach allowed the detection to be carried out inserum and urine samples. The detection was demonstrat-ed in serum and urine samples on the nanogram/mLlevel. Detection in serum was also performed on the pg/mL level with a limit of 100 pg/mL.ExperimentalThe basic principle of the detection is explained and thedetection mechanism is elucidated in previous publica-tions [11,12]. Briefly, the detection system consists ofa conventional three-electrode electrochemical cell modi-fied with additional gating electrodes for applyinga gating voltage VG to the detecting (working) electrode,Fig. 1. (a) Control experiment with primary Ab and HRP-la-beled secondary Ab. (b) CVs obtained with 100 ng/mL SPE,showing the amplification effect. The scan rate of 20 mV/s wasused to obtain the CVs.on which a redox enzyme, the sensing element, is immo-bilized. VG rearranges ions in the sample solution at theelectrode-solution interface, inducing an electric field thatpenetrates the immobilized enzyme. The field reduces theeffective height of the tunnel barrier between the activesite of the enzyme and the electrode, therefore increasingthe tunnel rate of electrons and resulting in intrinsic am-plification of the signal current. In the present work, theenzyme was conjugated to the detecting antibody and im-mobilized on the electrode via the immune-complex. Adetailed description of the immunosensing system used inthis work is provided in Supporting Information.Recombinant human AMACR was purchased(Genway Biotech; Cat. No. GWB-CFE200) and used asthe positive control. Monoclonal anti-AMACR and poly-clonal anti-AMACR (Genway Biotech; Cat. Nos. GWB-8770CE, GWB-AMACRA) were used as the primary/capture antibody and the secondary/detection antibody,respectively. Horseradish peroxidase (HRP) was conju-gated to the detection antibody using a conjugation kit.Human serum (Thermo Scientific Pierce; Product #31876) and synthetic urine (Quick Fix) were purchased.SPEs were purchased from DropSens. The workingelectrode of the SPE is a circular carbon electrode witha 4 mm diameter. The working electrode, silver referenceelectrode and the carbon counter electrode are fabricatedon the top side of the SPE. The dimensions of the SPEare 3.4 cm1.0 cm0.05 cm. The working electrode ofthe purchased SPE was covered with a layer of polyani-line (PANI). The PANI layer was used to host the captureantibody and therefore the immune complex on electro-des [13] and as electron mediator, shuttling electrons be-tween HRP and the electrode [14]. The PANI layer wassynthesized on the electrode using electrochemical poly-merization of aniline in a solution of 0.1 M aniline and1 M HCl at a potential of 1.2 V for 100 s [15]. The PANIprepared using this method was used previously ina HRP-based biosensor to detect H2O2 at pH 6.8 [16] aswell as in a pH sensor [17] because the conductivity ofPANI persists up to pH 9. Alternatively, sulfonated PANIwas prepared by adding poly(vinylsulfonic acid) (Aldrich,cat. # 27,842–4) to aniline and HCl during electrochemi-cal polymerization as described previously [18], to in-crease the redox activity of PANI at pH 7. The resultsshown in this work were obtained with PANI preparedusing HCl only while the two kinds of PANI led to similarresults.AMACR was spiked into serum and urine samples.The AMACR sample solution did not require treatment.The PANI layer was modified by depositing 2 mL of gluta-raldehyde (25% with water, diluted 100 times with water)on PANI until dry. Glutaraldehyde was used as a cross-linker, coupling the capture antibody to the highly porousPANI so that the immune complex was firmly entrappedin PANI. Previous XPS analysis [13] shows that glutaral-dehyde binds to the amine groups on PANI to form animine bond. AMACR-detecting electrodes were formedby incubating the modified SPE with 10 mL of 1 mg/mLFig. 2. AMACR detection calibration curve on the ng/mL levelin (a) serum and (b) urine with VG=0.6 V. (c) AMACR detec-tion calibration curve on the pg/mL level in serum with VG=0.6 V. The scan rate of 20 mV/s was used to obtain the CVs.primary antibody dissolved in PBS for 1 h at room tem-perature. To detect AMACR, an AMACR-detecting elec-trode was incubated with 5–10 mL of an AMACR sample(a AMACR standard serum solution) for 1 h at 37 8C.After rinsing and blocking possible open areas on theelectrode with bovine serum albumin (40mg/mL dilutedto 10% in Dulbecco’s phosphate-buffered saline), thesame electrode was incubated with 5–10 mL of 0.5 mg/mLHRP-labeled secondary antibody for 30 min at 37 8C andthen rinsed with de-ionized water. The Ab-AMACR-Ab(HRP) sandwich structure now has formed on theelectrode. Finally, the electrode was used as the workingelectrode for electrochemical measurements. A piece of0.5 mm-diameter copper wire coated with a thin layer ofinsulator (enamel) was used as the gating electrode forapplying VG. The wire was bent to form a U-shaped struc-ture and was attached on the sensing electrode using non-conductive epoxy. All AMACR samples were preparedwith undiluted serum sample as received. The measure-ments of the reduction peak currents of three electrodeswere used to determine each data point on the calibrationcurves.Electrochemical measurements were made by immers-ing the detecting electrode in a phosphate saline solution(PBS) contained in a glass beaker. The electrode wasdriven by a commercial electrochemical controller (CHI660C Work Station). A potential scan rate of 20 mV/s was used in recording cyclic voltammograms (CV). PBS(0.1 M at pH 7) was prepared using de-ionized water(18.2 W-cm). Commercially available hydrogen peroxide(Fisher scientific, 30% concentration) was diluted to theconcentrations used in the experiment. All measurementswere made with deaerated PBS at room temperature. Re-producible results were obtained by repeating each mea-surement multiple times.AcknowledgementThis work was supported by Cleveland State University.There is no potential conflicts of interest and/or sourcesof funding for all authors involved in the writing of thismanuscript.References[1] J. R. Prensner, M. A. Rubin, J. T. Wei, A. M. Chinnaiyan,Sci. Transl. Med. 2012, 127, doi: 10.1126/sci-translmed.3003180.[2] G. Sardana, B. Dowell, E. P. Diamandis, Clin. Chem. 2008,54, 1951–1960.[3] Z. Jiang, B. A. Woda, K. L. Rock, Y. Xu, L. Savas, A. Khan,Am. J. Surg. Patho.l 2001, 25, 1397–1404.[4] M. A. Rubin, M. Zhou, S. M. Dhanasekaran, S. Varambally,T. R. Barrette, M. G. Sanda, JAMA 2002, 287, 1662–1670.[5] J. Luo, S. Zha, W. R. Gage, T. A. Dunn, J. L. Hicks, C. J.Bennett, Cancer Res. 2002, 62, 2220–2226.[6] D. Maraldo, F. U. Garcia, R. Mutharasan, Anal. Chem.2007, 79, 7683–7690.[7] P.-Y. Lin, K.-L. Cheng, J. D. McGuffin-Cawley, F.-S. Shieu,A. C. Samia, S. Gupta, M. Cooney, C. L. 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A mediator-less immunodetection method for microorganisms is realized by incorporating the newly developed field-effect enzymatic detection (FEED) technique with the conventional electrochemical immunosensing approach. The gating voltage of FEED facilitates the transduction of electrical signal through the bulky immune complex so that the detection does not rely on the use of mediators or other diffusional substances. The voltage-controlled intrinsic amplification provided by the detection system allows detection in low-concentration samples without target pre-enrichment, leading to ultrasensitive and rapid detection. The detection approach is demonstrated with E. coli O157:H7, a model microorganism, in milk with an estimated detection limit of 20 CFU mL−1 (where CFU is a colony-forming unit) without performing sample pre-enrichment and centrifugation of sample followed by the resuspension of the pellet in a buffer solution, resulting in a significantly shortened assay time of 67 min. Optimizing the gating voltage resulted in the detection of 12 CFU mL−1 of the bacterium in milk. The novel detection approach can be used as a detection platform for ultrasensitive, specific and rapid detection of microorganism pathogens

Yau, Siu Tang

Mediatorless Immunoassay with Voltage-Controlled Intrinsic Amplification for Ultrasensitive and Rapid Detection of Microorganism Pathogens

Jiapeng Wang

Yan Xu

Siu-Tung Yau

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ChemElectroChem

https://engagedscholarship.csuohio.edu/cgi/viewcontent.cgi?article=1319&amp;context=enece_facpub

Mediator-less immunodetection with voltage-controlled intrinsic amplification for ultrasensitive and rapid detection of microorganism pathogens

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EngagedScholarship @ Cleveland State University

Cleveland-Marshall College of Law

EngagedScholarship @ Cleveland State University

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Cleveland-Marshall College of Law