Biosensors and Bioelectronics 86 (2016) 690–696

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Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

Detection of Hepatitis C core antibody by dual-affinity yeast chimera

and smartphone-based electrochemical sensing
Eliah Aronoff-Spencer a,n,1, A.G. Venkatesh b,1, Alex Sun b, Howard Brickner a,
David Looney a,c, Drew A. Hall b
a
School of Medicine, University of California, San Diego, La Jolla, CA, 92093 USA
b
Department of Electrical and Computer Engineering, University of California, San Diego, La Jolla, CA, 92093 USA
c
VA San Diego Healthcare System, San Diego, CA, 92161 USA

art ic l e i nf o a b s t r a c t

Article history: Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold
Received 21 April 2016 binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the
Received in revised form gold sensor surface while simultaneously acting as a “renewable” capture reagent for anti-HCV core
17 June 2016
antibody. This streamlined functionalization and detection strategy removes the need for traditional
Accepted 8 July 2016
Available online 9 July 2016
purification and immobilization techniques. With this biobrick construct, both optical and electro-
chemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV
Keywords: core antibody down to 12.3 pM concentrations while the electrochemical assay demonstrated higher
mHealth binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-
Point-of-care diagnostics
based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art
Hepatitis C virus
electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-
Yeast biobrick
Whole cell biosensor care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a
Electrochemical biosensor variety of settings.
& 2016 Elsevier B.V. All rights reserved.

1. Introduction point-of-care (POC) diagnostic devices are key to effectively screen

and monitor the treatment of hundreds of millions, especially
Hepatitis C Viral (HCV) infection is a global healthcare concern those living in resource poor settings, where both testing and
due to the high morbidity and mortality associated with chronic treatment costs remain significant constraints (Edlin and Win-
infection (Chen and Morgan, 2006; Saito et al., 1990). HCV is pri- kelstein, 2014; Graham and Swan, 2015).
marily acquired through blood transfusion or unsterilized medical Anti-HCV antibody and HCV RNA load testing is expected to
needles/devices and causes chronic liver disease with strong epi- cost $20 billion USD by the end of this decade (Uliana et al., 2014).
demiological linkage to subsequent hepatocellular carcinoma Three generations of anti-HCV antibody tests have relied on en-
(Saito et al., 1990). The virus undergoes a high rate of RNA muta- zyme-linked immunosorbent assay (ELISA) technology to detect
tion leading to significant genetic variations impeding vaccine viral proteins: NS4 alone in the first generation (Kuo et al., 1989);
development (Stoll-Keller et al., 2009) and confounding control the HCV core, NS3, and NS4 in the second (Gretch, 1997); and the
efforts. New, highly-effective and well-tolerated direct-acting an- core, NS3, NS4, and NS5 in the third (Barrera et al., 1995; Kao et al.,
tiviral drugs (Edlin and Winkelstein, 2014; Graham and Swan, 1996; Uyttendaele et al., 1994). The increasing assay sensitivity of
2015) are now driving disease control efforts globally (Hagan and each successive generation has reduced the time to detect anti-
Schinazi, 2013), making eradication of HCV infection within a HCV antibody from 16 to 5 weeks after initial infection (Uliana
generation achievable. However, this will require identifying and et al., 2014). The HCV core antigen remains crucial, as it highly
treating an estimated population of 130 – 150 million mono-in- conserved (Ribeiro et al., 2012) and the serum concentration of the
fected (Suthar and Harries, 2015) and 5.5 million HIV co-infected protein correlates with the viral RNA concentration, allowing it to
individuals (Rouet et al., 2015) worldwide. Accurate, low-cost be used in as an indirect marker for viral replication (Bouvier-Alias
et al., 2002; Gaudy et al., 2005; Park et al., 2010; Zanetti et al.,
n
2003).
Correspondence to: University of California, San Diego, 9500 Gilman Drive, La
Jolla, CA 92093, USA.
Recombinant HCV core proteins have been successfully ex-
E-mail address: earonoffspencer@ucsd.edu (E. Aronoff-Spencer). pressed in a variety of host systems including bacteria (Majeau
1
These authors contributed equally to this work. et al., 2004), yeast (Acosta-Rivero et al., 2002), mammalian cells

http://dx.doi.org/10.1016/j.bios.2016.07.023
0956-5663/& 2016 Elsevier B.V. All rights reserved.

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(Harada et al., 1991), insect cells (Baumert et al., 1998), and plants respectively were added to facilitate insertion of HCV-core and
(Madesis et al., 2010). While production of HCV core protein in GBP genes. The complete HCV-core gene was cloned by poly-
large quantities has been realized, cultivation must be tightly merase chain reaction (PCR) with forward and reverse primers
controlled, purification often requires multiple steps, and im- consisting of NheI and BamHI, respectively, from pCMV3010-HCV1,
mobilization on a testing substrate all combine to increase the an HCV1 genome library constructed from a clinical sample. The
costs and complexity of assay production. An alternative approach PCR product was then restriction digested (NheI and BamHI),
is illustrated by one-step purification and surface binding of hu- cleaned, and ligated into pYD and pCTCON2 vectors. The DNA
man papilloma virus (HPV) L1 capsid glutathione s-transferase sequence corresponding to GBP (sequence: MHGKTQATSGTIQS)
fusion proteins to a glutathione-casein coated ELISA plate for de- was cloned from template (Stanley, 1997) by PCR with both for-
tection of anti-HPV antibody in human serum (Sehr et al., 2002). A ward and reverse primers consisting of BamHI site. The PCR pro-
chimera of viral antigen and a high affinity substrate binding duct was then digested with BamHI and inserted adjacent to HCV-
moiety simplifies the production of immunologic tests. Other low- core gene by ligation. The sequence of all vectors was confirmed by
cost genetically-engineered fusion proteins have been produced in population sequencing.
non-pathogenic E. coli (van Bloois et al., 2011), S. cerevisiae, Lac-
tobacillus sp. (Qin et al., 2014), and Bacillus sp. (Du et al., 2005;
2.2. Transformation and induction of yeast display vectors
Iwanicki et al., 2014) for a variety of diagnostic applications (Cherf
and Cochran, 2015). Recently, Jaun Franco et al. (2013) reported
The constructed vectors were transformed into S. cerevisiae
site-directed antibody deposition for surface plasmon resonance
EBY100 strain from ATCC using DSY yeast transformation kit
(SPR) using protein A-gold binding domain (PAG) antibody fusion
(Dualsystems Biotech AG, #P01003). The transformed HCV-core/
proteins. This strategy led to proteins that bound avidly and in the
GBP expressing yeasts were then freshly streaked on SD þCAA
correct orientation to gold monolayers, and is the foundation of
selective media plates at 30 °C overnight. A fresh single colony was
the current work.
subsequently inoculated in 5 mL of SR þCAA media and incubated
Here we present a platform to detect anti-HCV core antibody
for 2 days at 30 °C, diluted to achieve 1 OD600, and aliquoted in
combining yeast biobricks and scalable sensing methods. Yeast
1 mL batches. The expression was induced by inoculating 0.5 mL
cells were genetically engineered to display HCV core protein
from 1 mL aliquots in 5 mL of S/Galactose and Raffinose þCAA
concatenated to gold binding peptide (GBP) repeats, creating af-
media and incubated for 48 h at 20 °C. Preparation of media is
finity reagents capable of self-assembly on gold surfaces. This
described in supplementary of reference (Venkatesh et al., 2015).
technique enables single-step purification, surface preparation and
deposition, and is compatible with a host of technologies ranging
from optical imaging to electrochemical sensing. We developed 2.3. Immuno-fluorescent assay for yeast display validation
and compared both optical and electrochemical assays to explore
the avidity and efficiency of yeast-based reagents. Ultimately, 100 mL galactose induced yeast cells were centrifuged in 96
functionalized gold screen printed electrodes (SPE) were used to well plates and re-suspended in 50 mL 1  PBS containing 1% bo-
detect HCV antibodies by a low-cost potentiostat yielding direct vine serum albumin (BSA) and incubated at room temperature for
transduction of immune-complex formation into a signal that is 30 min to pre-block the surface. Mouse monoclonal anti-HCV core
sensed, processed, and transmitted by a smartphone (Fig. 1). IgG (Santa Cruz Biotech, #sc-57800) of desired concentration was
added to the yeast cells and the plate was gently rocked for 2 h at
room temperature. The yeast cells were then washed 3  with
2. Materials and methods 50 mL 1  PBS by centrifugation and resuspension. The Alexa Fluor
488 conjugated donkey anti-mouse IgG (Thermo Fischer, #A-
2.1. Construction of HCV-core/GBP yeast display vectors 21202) was subsequently added incubated for 1 h at room tem-
perature followed by 3  wash with 50 mL 1  PBS. The pellets
Yeast display vectors pYD and pCTCON2 were kindly provided were then re-suspended in 20 mL 1  PBS and measured for
by K. Dane Wittrup, MIT, MA (Wang et al., 2005) and canonical fluorescence using flow cytometer with suitable excitation and
restriction sites such as NheI and BamHI at N- and C-terminal emission filters (BD Accuri C6).

Fig. 1. Illustration of yeast biobrick chimera enabled detection of anti-HCV core antibody with POC smartphone potentiostat platform. (a) Detection of anti-HCV core
antibody by fluorescence and electrochemical methods. (b) Illustration of smartphone-based potentiostat that connects to a host device through the audio jack.

Please cite this article as: Aronoff-Spencer, E., et al., Biosensors and Bioelectronics (2016), http://dx.doi.org/10.1016/j.bios.2016.07.023i
692 E. Aronoff-Spencer et al. / Biosensors and Bioelectronics 86 (2016) 690–696

2.4. Procedure for cleaning electrode where the calibration curve saturates, minus 10s.

Gold SPEs from Dropsens (DRP-250AT) were cleaned with a

mixture of 50 mM KOH and H2O2 (3:1) for 10 min to remove or- 2.7. Smartphone-based potentiostat
ganics from the gold surface. The gold surface was further elec-
trochemically cleaned in the presence of 50 mM sulfuric acid by A portable potentiostat was designed as a peripheral module
sweeping a potential between  0.4 V to þ0.4 V at a rate of that plugs directly into the headphone jack of the user's smart-
100 mV/s for 12 cycles, using a benchtop potentiostat (CH Instru- phone (Sun et al., 2014, 2016). The block diagram in Fig. 2a illus-
ments 750E). The electrode was finally rinsed with deionized trates how the module harvests power through the left audio
water and air-blow dried. channel, communicates bi-directionally with its smartphone host
via the right channel and the microphone, and makes electro-
2.5. Functionalization of gold surface with displayed HCV-core/GBP
chemical measurements. The smartphone provided energy to the
chimera
device by sending a sinusoidal tone (5–20 kHz) that was rectified
50 mL 1 OD600 cells grown in induction media was dropped on and then regulated to a 4-volt DC supply. The harvesting circuit
the clean gold surface of the electrode and incubated overnight at also includes a tunable impedance matching network automated
4 °C. The electrodes were then rinsed with 1 mL 1  Tris buffered by the smartphone and completely hidden from the operator (Yao
saline (TBS) pH 7.4, and treated with 50 mL 1% sodium azide in 1  et al., 2015) to ensure optimum power transfer. For digital com-
TBS for 90 min at room temperature and rinsed with 1 mL TBS. The munication, the microcontroller in the module sent and received
electrode chip was blocked with 1  TBS containing 2% BSA and serial RS-232 packets across the right audio output and the mi-
0.05% TWEEN-20 for 1 h at room temperature and rinsed with crophone channels at a baud rate of 2.4 kHz. Via this commu-
1 mL TBS. nication protocol, the smartphone, once given user input, sent
commands to the microcontroller to set different test parameters
2.6. Electrochemical sandwich ELISA
for cyclic voltammetry (CV) measurements, such as scan-rate (25–
100 mV/s) and scan range (72 V) as well as to start and stop tests.
The functionalized electrode was incubated with 20 mL of
Finally, the module's low-power potentiostat were interfaced with
mouse anti-HCV core monoclonal IgG (Santa Cruz Biotech, #sc-
57800) with desired concentration for 2 h at room temperature disposable screen printed electrodes plugged into the device to
and rinsed with 1 mL TBS. 20 mL of alkaline phosphatase (ALP) make CV measurements (Fig. 2b). CV operates by applying a tri-
conjugated secondary IgG's from rabbit (Abcam, # ab6729) tar- angular voltage waveform between the working and reference
geted towards mouse IgG was added and incubated for 1 h at room electrodes in an electrochemical cell and recording the current
temperature. The electrode was then rinsed 3x with 1 mL TBS and generated at the working electrode. In this module, the micro-
air-blow dried. The immune complex formation was detected by controller generated the voltage ramps for the potentiostat to use
adding 50 mL substrate solution (100 mM glycine pH 10.4, 6 mM p- and the resulting measured current signal was amplified and then
aminophenyl phosphate (pAPP) (Santa Cruz Biotech, #sc-281392), transformed into a voltage by the potentiostat. Using a voltage
1 mM ZnCl2, and 1 mM MgCl2) to the electrode and incubated at controlled oscillator (VCO), this voltage data output by the po-
37 °C for 10 min. The conversion of pAPP to p-aminophenol (pAP)
tentiostat was first converted to a frequency signal within the
by ALP was detected by cyclic voltammetry by a potential sweep
audio band and then sent to the phone to be recorded, demodu-
between þ 0.3 V to 0.25 V at a rate of 25 mV/s for 10 cycles using
lated, and reconstructed (Fig. 2c). To make reconstructing the
CHI 750E or smartphone-based potentiostat. The measured cur-
rent vs antibody concentration was fitted with 4-parameter lo- voltammogram, which is the current plotted against the input
gistic regression. The limit of detection (LOD) and limit of quan- voltage of a CV measurement, easier when the smartphone is
tification (LOQ) were calculated as the amplitude of the control processing the data, marker tones, which signify the voltage
sample (0 nM) plus 3s and 10s, respectively. The linear dynamic transition points of the applied potential, were generated by the
range (DR) was calculated from the LOQ to the upper boundary, microcontroller and inserted into the output of the VCO (Fig. 2c).

Fig. 2. POC Smartphone-based potentiostat platform: (a) Block diagram of potentiostat circuit interfaced through 3.5 mm audio jack, (b) Photograph of smartphone po-
tentiostat measuring anti-HCV core antibody by cyclic voltammetry, and (c) Demodulation and reconstruction of cyclic voltammogram.

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3. Results and discussion protein only biobricks, methods that will be the subject of up-
coming reports. The specificity of HCV core to selectively capture
3.1. Expression of yeast biobricks anti-HCV core IgG was measured for several off-target antibodies
and found to have signal comparable to that when no anti-HCV
Yeast display has advantages of high surface expression and core antibody is present (Supplementary S2).
eukaryotic protein processing mechanisms (Rosano and Ceccarelli,
2014) compared to traditional bacterial platforms. Likewise, yeast 3.3. Smartphone-based potentiostat
display offers advantages in the cost of reagent production as well
as improved assay stability and shelf life of lyophilized cells (Gray In order to demonstrate the POC utility of the electrochemical
et al., 2012). “Dual–affinity biobricks” were engineered by cloning assay, a smartphone-based potentiostat was developed that can be
the full length HCV core gene spanning 572 bp from HCV genotype controlled by a mobile app and interfaces with yeast biobrick
1 into the pYD and pCTCON2 vectors (Boder and Wittrup, 1997) functionalized SPEs for portable and accurate electrochemical
with gold binding peptide repeats (6–9  ), to yield the HCV core measurements (Sun et al., 2014, 2016). The potentiostat interfaces
and GBP fusion protein linked by glycine-serine repeats to Aga1–2 with the phone through the audio jack making it universally
and the yeast surface (Fig. 3). compatible with any smartphone, regardless of make or model –
These constructs create a cell-based reagent capable of con- unlike devices that use proprietary ports such as Lightening or USB
comitant surface immobilization and antibody capture. We en- that are only found on a subset of smartphones. This also obviates
gineered pYD6, pYD8, and pCTCON2 vectors to compare C- and N- the need for replacing or recharging an external battery since the
terminal Aga-2 linkage and the effect of order (Core/GBP vs. GBP/ phone can provide the power. Most importantly, the audio port
Core) of motif expression. Gal-raffinose induction showed greater enables the device to take advantage of the many capabilities of
than 80% surface expression for pCTCON2 while lower and variable the smartphone, including processing power, battery, network
expression was observed for pYD6 and pYD8. Fig. 3b shows the connectivity, and easy-to-develop software applications. This also
expression efficiency of the two vector systems as measured by avoids adding redundant components that are already available on
flow cytometry, where the control corresponds to yeast cells the phone allowing it to be more portable and cost-effective as
without the HCV core/GBP gene inserted in the transformed vec- compared to stand-alone variants (Cruz et al., 2014; Ionescu et al.,
tor. Immuno-precipitation and fluorescent imaging of pCTCON2 2010; Laksanasopin et al., 2015; Lillehoj et al., 2013; Nemiroski
yeast confirmed high surface expression and avid binding of core et al., 2014; Rowe et al., 2011; Wang et al., 2015; Zhang et al.,
directed antibodies to the yeast biobrick surface (Fig. 4). 2016).
The main challenge in using the headphone jack is that the
3.2. Immuno-fluorescent and electrochemical assays audio channels are AC coupled meaning that no DC signal or
power can be directly transmitted between the phone and the
Standard optical calibration assays were performed to assess potentiostat. Hence, in order to power to the device, a sinusoidal
the limit of detection (LOD), dynamic range (DR) and dissociation AC signal with a frequency in the audio band (20 Hz–20 kHz) is
constant (Kd), of surface displayed chimera to monoclonal anti- sent through the left channel to be harvested by the device. For
HCV core antibodies (Fig. 5a). A LOD of 12.3 pM, DR of 30 pM–3 nM bidirectional digital communication, RS-232 is used with a baud
and Kd of 277737 pM were observed in optimized optical ex- rate of 2.4 kHz, which is within the audio bandwidth, allowing
periments. Subsequently, cyclic voltammetry was employed with these packets of data to pass through the AC coupling and be
yeast functionalized gold SPEs for electrochemical testing. Pre- decoded without additional conditioning or processing. Since CV
liminary assays in this format provided consistent, but elevated measurements result in slow varying waveforms close to DC, the
binding parameters with a 2 nM LOD, 4–250 nM DR, and output of the potentiostat cannot be directly passed to the phone
49.94 76.69 nM Kd (Fig. 4b). Individual voltammograms are either. One solution would be to digitize this signal and send it
shown in the Supplemental (S1). The observed differences in assay digitally. However, this requires adding an analog to digital con-
parameters between the immuno-fluorescent and electrochemical verter (ADC) to the device, which would increase the power con-
assay are attributed to signal leakage from the electrode surface, sumption and cost. Instead, we push this burden back onto the
which is unmeasured in the electrochemical method but captured phone with its far superior ADC and processing power by mod-
in bulk optical measurements. This phenomenon, attributed to the ulating the signal using a VCO. The analog signal from the po-
distance between the yeast surface and the sensing electrode, may tentiostat is translated into a frequency modulated signal within
be addressed by creating yeast surface monolayers or secreting the audio band allowing it to be sent directly to the phone to be

Fig. 3. Selection of expression vector for yeast cells to display HCV core protein on its surface: (a) Position of HCV core protein and gold binding peptide in the Aga1-Aga2
display system using in the pYD6, pYD8, and pCTCON2 vector systems. (b) Comparison of vector efficiency to display HCV core protein on yeast cell's surface, assayed with
mouse anti-HCV core antibody.

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694 E. Aronoff-Spencer et al. / Biosensors and Bioelectronics 86 (2016) 690–696

Fig. 4. Validation of HCV-core expression by immuno-fluorescent assay: (a) Bright field microscopic image of yeast cell expressing HCV core protein, (b) Fluorescent image of
yeast cells expressing HCV core protein detected by HCV core directed antibody (mouse anti-HCV core and Alexa Fluor 488-anti-mouse).

Fig. 5. Detection of mouse monoclonal anti-HCV core antibody: (a) immuno-fluorescent assay and (b) electrochemical assay with CHI electrochemical workstation. The
mean and standard deviation of all concentrations were from triplicates and fitted with 4 – parameter logistic regression.

processed. This device with the power harvesting network and SPE immobilized immune complex, with parallel comparison to a
communication schemes can measure bidirectional currents as CHI reference platform (Fig. 6) (Sun et al., 2014). Fig. 6a shows
low as 300 pA, consumes a peak power of 6.9 mW during mea- voltammograms of the same electrodes measured consecutively
surements, which is within the power budget set by the typical by the CHI and smartphone-based potentiostat. Although the
output of mobile devices, and harvests power with up to 79% voltage scanning windows are slightly different, both data sets
efficiency. cover the range required to detect pAP concentration,þ100 mV to
The functionality of the smartphone-based potentiostat was þ200 mV with respect to reference electrode (highlighted in
verified by electrochemical measurement of pAP concentration, gray). Shortening this range minimizes the runtime of the CV scan
formed as a result of enzymatic conversion by ALP present in the allowing the smartphone-based potentiostat to conserve power

Fig. 6. Performance of smartphone-based potentiostat (SP): (a) Cyclic voltammograms and (b) Extracted peak current of CHI and SP for Blank (substrate (pAPP) on a 2% BSA
blocked SPE), Control (0 nM anti-HCV antibody) and Test (100 nM anti-HCV antibody). All measurements taken in triplicate and error bars represent 71 standard deviation.

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and reduces the measurement time. The tests labelled “Blank” Special thanks to Dr. Chip Schooley for ongoing laboratory support.
represent the absolute background noise, in which only electro-
chemically inactive substrate (pAPP) was added to SPEs blocked
only with BSA and measured by cyclic voltammetry. In both the Appendix A. Supporting information
positive (100 nM anti-HCV antibody) and control experiments, the
SPEs contained all the assay components with the exception that Supplementary data associated with this article can be found in
no anti-HCV core antibody was added to the control SPEs. Direct the online version at doi:10.1016/j.bios.2016.07.023.
comparison of CHI and smartphone-based potentiostat based on
Bland-Altman plot (not shown) revealed 95% limits of agreement
between the two instruments of þ 0.24 μA and  0.39 μA and an References
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