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IEEE SENSORS JOURNAL, VOL. 19, NO.

14, JULY 15, 2019 5769

Multi-Panel, On-Single-Chip Memristive Biosensing


Ioulia Tzouvadaki, Abuduwaili Tuoheti, Séverine Lorrain, Manfredo Quadroni, Marie-Agnès Doucey,
Giovanni De Micheli , Fellow, IEEE, Danilo Demarchi , Senior Member, IEEE,
and Sandro Carrara , Fellow, IEEE

Abstract— Memristive biosensors have demonstrated excellent TABLE I


capabilities for ultrasensitive bio-detection. In this paper, memris- D EVELOPING M EMRISTIVE B IOSENSORS
tive biosensing chips are designed, fabricated, and implemented
in a for the first time presented multi-panel on-chip detection for
discrete sensing of a target molecule through separate function-
alization of individual devices on the same chip. The biosensing
scheme is validated by means of labeled (i.e. fluorescence) and
label-free (i.e. electrical) characterization methods. This novel
memristive multi-panel sensing paradigm paves the way for fast
and ultrasensitive point-of-care (PoC) devices allowing the detec-
tion of specific targets in complex matrixes where non-specific
molecules are present as well, and opening the great potential
for the application of memristive phenomena in multiplexed
ultrasensitive bio-detection and theranostics.
Index Terms— Memristive biosensors, bio-functionalization,
multi-panel detection.
I. I NTRODUCTION

S EMICONDUCTOR nanowires may provide low-cost


microchips and therefore are considered as effective and
highly promising building blocks for miniaturized bioas-
introduction of charged biological species on the nanodevices’
surface (bio-functionalization procedures and target molecule
uptake) [6]–[8].
says dedicated to medical applications, in both diagnostics
Moreover, the design and realization of memristive biosens-
and therapeutics. In this framework, silicon nanowire-arrays
ing electronic platforms was realized and validated [9], [10]
that exhibit memristive electrical properties [1]–[3] are bio-
providing a fast, fully-automatized and simultaneous sensing
functionalized with receptor molecules i.e. antibodies or DNA
output of multiple individual memristive biosensors on a
aptamers, giving rise to the so-called memristive biosensors.
single chip. Highlights of the development steps of memristive
These memristive sensors have already successfully achieved
biosensors are summarized in Table I.
ultrasensitive detection for cancer biomarkers, demonstrating
Meanwhile, the importance of multi-panel detection in
atto-molar concentration sensing performance [4] as well as
a lab-on-a-chip framework is even more pronounced when
effective screening of therapeutic compounds, along with
considering the aspect of theranostics applications where both
the possibility for continuous drug monitoring [5] therefore
disease biomarkers and therapeutic compounds are simulta-
showing immense potential for ultrasensitive and precise
neously monitored in order to reflect the effect of the treat-
biosensing. The bio-detection is based on the hysteresis mod-
ment [16]–[19]. This scheme may involve target molecules
ification appearing in the electrical characteristics upon the
that are competing or complementary to each other. The first
Manuscript received February 6, 2019; accepted March 7, 2019. Date of case concerns for instance a disease marker and the specific
publication March 12, 2019; date of current version June 19, 2019. This therapeutic drug or its metabolites, evaluating the efficiency of
work was supported by the multidisciplinary FNS under Grant CR32I3
156915. The associate editor coordinating the review of this paper and the medical treatment in the form of a feedback loop, allowing
approving it for publication was Dr. Cheng-Ta Chiang. (Corresponding appropriate adjustments for enhancing the therapy [20]–[22].
author: Ioulia Tzouvadaki.) The case of complementary markers refers to simultaneous
I. Tzouvadaki, G. D. Micheli, and S. Carrara are with the
Integrated System Laboratory, EPFL, CH-1015 Lausanne, Switzerland detection of multiple biomarkers indicating the same disease
(e-mail: ioulia.tzouvadaki@alumni.epfl.ch; giovanni.demicheli@epfl.ch; or disorder targeting at a more accurate evaluation of the
sandro.carrara@epfl.ch). disease evolution and providing enhanced diagnosis [23].
A. Tuoheti and D. Demarchi are with Department of Electronics and
Telecommunications, Politecnico di Torino, 10129 Torino, Italy (e-mail: Furthermore, the discrete sensing of specific biomarkers
abuduwaili.tuoheti@polito.it; danilo.demarchi@polito.it). when included in a more complex matrix or cocktail of
S. Lorrain and M. Quadroni are with the University of Lausanne multiple substances consists another significant challenge in
(UNIL), CH-1015 Lausanne, Switzerland (e-mail: severine.lorrain@unil.ch;
manfredo.quadroni@unil.ch). biosensing.
M.-A. Doucey is with the University of Lausanne (UNIL), In this work, a proof-of-concept study demonstrating multi-
CH-1015 Lausanne, Switzerland, and also with the Ludwig Institute panel detection paradigm involving memristive biosensors and
for Cancer Research, CH-1066 Lausanne, Switzerland (e-mail:
marie-agnes.doucey@unil.ch). individualized sensing is for the first time presented and
Digital Object Identifier 10.1109/JSEN.2019.2904393 validated through electrical and fluorescence characterization
1558-1748 © 2019 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission.
See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.

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5770 IEEE SENSORS JOURNAL, VOL. 19, NO. 14, JULY 15, 2019

surface to antibody against Prostate Specific Antigen (PSA),


i.e. anti-PSA antibody (Abcam-ab10185), solution in Phos-
phate Buffered Saline (PBS) (pH 7.4 Sigma-Aldrich) for 4 h
at room temperature, thoroughly rinsed with the same buffer
and gently dried with N2 flow. Then, the antigen uptake
was performed through successive 45 min incubations of
the sensing chip in solutions of PSA (Millipore Angebot
R-1939458.1; 539834 purchased from Merck) in PBS. The
measurements were performed with the prober needles placed
on the Pt pads instead of the NiSi pads.
Fig. 1. Optical microscopy image depicting the selective spotting with The electrical monitoring of the memristive nano-bio-
receptor molecules. A closer view of the surface of the multi-sensing chip is sensors was performed utilizing the configuration consisting
shown, depicting the twelve sensors locations, that are integrated with metal of a Cascade Microtech Probe Station in combination to a
lines. The discrete functionalization scheme is visible for each individual
sensor and shown as a droplet located exactly at the sensor’s regions. The Hewlett-Packard 4165A Precision Semiconductor Parameter
memristive devices are integrated with the extension Pt electrodes in order Analyzer. Fluorescence characterization is carried out using
to enable the electrical characterization as well as the integration with the scanner Typhoon Trio (GE Healthcare).
electronic readout platform as reported in [9] and [10].

techniques. This biosensing scheme paves the way for indi- B. Multiplexing Approach for Sensing
vidualized biomarker sensing in a complex matrix, as well
A single-chip, detection in a multi-panel mode is for
as for multiple biomarkers/drugs detection on the same chip
the first time attempted implementing memristive biosen-
and application in theranostics. Sensing chips of memristive
sors. The memristive devices included in the same 1 cm ×
biosensors conjugated with metallic extension electrodes are
1 cm chip are first subjected to a silanization step with
validated for successful sensing performance and then imple-
GPTES (440167, Sigma-Aldrich). Then the devices are sep-
mented for multi-panel sensing.
arately bio-functionalized with different antibody solutions.
Antibodies either from mouse, goat or rabbit were diluted
II. M ATERIALS AND M ETHODS
within PBS to get a final concentration of 0.5 mg/ml with
A. Memristive Biosensing Chips 0.5% trehalose (T9449, D-(+)-Trehalose dihydrate from corn
Two-terminal silicon (Si) nanofabricated wire-arrays starch = 99%, Sigma). Multiplexed biosensing requires post-
exhibiting memristive electrical properties are acquired fabrication nanowire functionalization with probe molecules,
through a top-down fabrication process using Silicon-on- (though robotic spotting [24], ink-jet printing [25], selec-
Insulator (SOI) wafers as described in [7], by implementing tive heating [26], electrochemically [27], or via dip-pen
electron beam lithography and Deep Reactive Ion Etching nanolithography [28]). In this work, a micro-spotter (GeSim
(DRIE). The wires are suspended and anchored between NanoPlotter 2.1) is used for specific and separate bio-
Nickel Silicide (NiSi) pads forming Schottky-barriers and functionalization of each nanodevice with the different anti-
serving for the electrical characterization of the nanodevices. bodies under consideration, with a drop of 400 pL antibody-
Metal lines are integrated on top of the already fabricated solution of an initial diameter set at 150 µm. A rabbit antibody
nanostructures and serve as extension line electrodes to the is used as a positive-control bio-functionalization reagent and
NiSi pads of the devices, as described in [9]. The metal is spotted to various devices of the sensing chip. Meanwhile,
lines are designed taking into consideration the fact that three different negative controls are taken into consideration:
the hysteretic properties of the memristive biosensors are a mouse and a goat antibody both applied at the same
sensitive to additional resistive elements introduced in series concentrations as the positive control as well as plain buffer
to the nanofabricated devices, eliminating or even completing solution (PBS).
masking the memristive properties of the nanodevices. For The micro-spotting procedure finally results in discrete
this reason, the extension lines design follows the outcomes of bio-functionalization of different devices (Fig. 1). Following
a relevant computational study and calculations [9], [13] for the bio-functionalization the chip remains in a humid
respecting the resistance tolerance of the system. A common chamber for 1 h in order to ensure the attachment of the
source and separated drains are considered for individual bio-functionalization regents on the nanodevice surface. The
measurement of twelve integrated sensors (as seen in Fig. 1) additional humidity introduced, aims at the conservation of
that allow possibility for adequate statistical analysis to the liquid antibody-solution drops which are very sensitive to
determine average value and standard deviation. This evaporation issues. In order to prevent non-specific binding
statistical analysis is required for obtaining reliable results of proteins during the detection measurement, the remaining
and overcome the variability of the sensors originating from active GPTES-derived groups are passivated by applying
the DRIE process. In addition, such realization of multiple 10 mM ethanolamine in PBS solution at room temperature.
sensors allows the possibility for simultaneous sensing (multi- Washing process is further performed to remove unreacted
panel sensing), that consists the main aim of the present study. molecules.
To confirm the sound functionality of the metal lines the An additional blocking step is carried out with PBS
memristive devices were functionalized by exposure of the containing 3% gelatin from cold water fish skin. This further

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TZOUVADAKI et al.: MULTI-PANEL, ON-SINGLE-CHIP MEMRISTIVE BIOSENSING 5771

Fig. 3. Fluorescence results demonstrate the successful multi-panel bio-


functionalization.

Fig. 2. Indicative semi-logarithmic current to voltage characteristics obtained


for the same device measured at the Pt pads using the Probe Station and
Keithley configuration. The different graphs correspond to the bare device,
after the bio-functionalization with Ab and with increasing antigen uptake
(from 1 fM to 10 nM).

blocking forms a stable and specific receptor layer, due to the


passivation property of neutrally charged gelatin molecules
that aims to prevent the empty sites of the device active area
from interacting with charged non-specific species from the
analyte solution. Then, what follows is the antigen uptake
through simultaneous incubation of all the nanodevices under Fig. 4. Electrical characterization results bringing proof of the successful
consideration for 45 min in humid environment. A pipette multi-panel bio-functionalization. The average value of the voltage gap
acquired for increasing concentrations of four different molecules which are
of 2 µL is used to efficiently transfer a sufficient amount the target molecule and three negative controls. No significant signal difference
of antigen solution directly and restricted at the region of is depicted for the case of the negative-control bio-functionalized sensors in
interest i.e. nanowires’ region. Two different aspects are contrast to the decreasing voltage gap trend with increasing concentration
shown for the case of the target molecule.
considered through the antigen uptake. A selected region
of the chip is incubated in a high concentration of antigen-
tagged with fluorescent substance, destined for a fluorescence 1) Fluorescence Sensing: The results obtained from fluores-
study and another region is incubated with successive 45 min cence characterization are illustrated in Fig. 3. It is depicted
incubations increasing antigen concentrations belonging to the that selected bio-functionalization is successfully achieved
range close and below the clinical range of most biomarkers, and only the devices functionalized with the positive control
like for example PSA. Each incubation is followed by a antibody finally demonstrate the capability to bind the target
washing step with gelatin, two washing steps with PBS and antigens. Despite the fact that accurate bio-functionalization is
a final washing step with PBS 10% in order to remove any achieved strictly at the region of each suspended nanowire, due
salts originated from the buffer. to adhesion phenomena occurring during the incubation time
the bio-functionalization solution is slightly spread outside
III. R ESULTS AND D ISCUSSION the target area and finally stabilized to a slightly larger
A. Performance of Memristive Biosensing Chips droplet, after established hydrodynamic and hydrostatic forces
equilibrium, as well as evaporation aspects.
The electrical response using the fabricated chips is acquired
2) Label-Free Sensing: The nanodevices, bio-functionalized
before and after the bio-functionalization process and antigen
with the different antibodies and the PBS, are electrically char-
uptake with increasing antigen concentration (Fig. 2). The
acterized (baseline measurement), followed by the electrical
obtained results clearly show that the hysteresis is successfully
monitoring of the nanodevices with increasing antigen concen-
maintained and the voltage gap is acquired as expected upon
trations. The introduction of charged residues on the surface
bio-functionalization and decreases with the increasing antigen
of the nanodevices, as it is expected, induces the appearance
uptake as shown in Fig. 2.
of a voltage gap in the semi-logarithmic current to voltage
characteristics. Meanwhile, the binding of antigen (through the
B. Multi-Panel Sensing With Memristive Biosensors receptor-target molecule relation) introduces a masking contri-
For the multi-panel detection two separate methods bution to the effect already brought by antibodies, resulting in
(fluorescence detection and label-free sensing through a decreasing voltage gap. Indeed, the voltage gap presented at
electrical characterization) are independently implemented to the electrical characteristics of the nanodevices functionalized
verify the multi-panel sensing capabilities of the memristive with the positive control antibody demonstrates smaller values
biosensors. after the antigen uptake, verifying successful antibody-antigen

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5772 IEEE SENSORS JOURNAL, VOL. 19, NO. 14, JULY 15, 2019

Fig. 5. Illustration of the memristive bio-sensing platform, implemented for theranostics in combination with specially designed microfluidic circuit.

binding (Fig. 4). However, no significant difference is recorder designed memristive sensing chips offering disposable sens-
for the case of the negative-control bio-functionalized devices ing modules by integrating memristive wires and extension
(Fig. 4). Further study of the analytical performance of the metal electrodes are validated for successfully providing the
nanodevices at the different antigen steps, demonstrates a electrical characteristics of the devices before and after the
decreasing behavior only for the nanodevices functionalized bio-functionalization and the target uptake steps. Most impor-
with the positive control antibody while the voltage gap for the tantly, this novel sensing paradigm demonstrates successful
other nanodevices remains constant at the levels of the values implementation of the memristive biosensors in a multi-panel
after the bio-functionalization. This finding brings proof of sensing scheme in both labeled and label-free sensing. The
the possibility for efficient individualized bio-functionalization system presented in this work paves the way for advanced
of different nanodevices on the same chip. Moreover, these bio-detection with memristive biosensors, allowing individu-
results demonstrate the capability of the memristive biosensors alized detection of specific targets in complex matrixes in the
to provide discrete sensing of a target molecule against other presence of non-specific molecules or in even more complex
non-specific molecules through a tailored functionalization of schemes i.e. cells, tumor extracts, holding also great promise
individual devices. Therefore, this study consists a very impor- for fast and ultrasensitive sensing in theranostics.
tant step towards future implementation of the memristive
biosensors for the detection of a specific biomarker or drug
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TZOUVADAKI et al.: MULTI-PANEL, ON-SINGLE-CHIP MEMRISTIVE BIOSENSING 5773

[8] F. Puppo, F. L. Traversa, M. Di Ventra, G. De Micheli, and S. Carrara, Ioulia Tzouvadaki received the B.Sc. degree in
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pp. 2120–2127, Dec. 2016. in 2008 and the M.Sc. degree from the Politecnico
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no. 5, pp. 1205–1209, 2012. Microelectronics in 2014. Then, he was a Software
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drugs detection in human serum for personalized therapy,” Biosensors Italy from 2015 to 2016. He is currently a Doc-
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[18] M. Lee, K. Y. Baik, M. Noah, Y.-K. Kwon, J.-O. Lee, S. Hong, and Telecommunications, Politecnico di Torino, under the supervision of
“Nanowire and nanotube transistors for lab-on-a-chip applications,” Lab D. Demarchi and S. Carrara. His main research interests include embedded
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determination of antihypertensive drugs nifedipine and atenolol utilizing for the work centered on the molecular pathways
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Manfredo Quadroni graduated with a degree in
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5774 IEEE SENSORS JOURNAL, VOL. 19, NO. 14, JULY 15, 2019

Marie-Agnès Doucey received the Engineering Danilo Demarchi (M’10–SM’13) received the mas-
degree in biotechnology from the Ecole Supéerieure ter’s and Ph.D. degrees in electronics engineering
de Biotechnologies de Strasbourg (ESBS), France, from the Politecnico di Torino (Polito), Italy, in 1991
and the Ph.D. degree in biochemistry from and 1995, respectively. He is an Associate Professor
the Friedrich-Miescher Institute, Novartis, Basel, with Polito, where he is leading the Micro and Nano
Switzerland. She is currently a Project Leader with Electronic Systems Group, Department of Electron-
the Ludwig Institute for Cancer Research, University ics and Telecommunications. He is also coordinating
of Lausanne. She has more than 20 publications the microelectronics research line at the Center for
in peer-reviewed international journals. Her cur- Space Human Robotics, Istituto Italiano di Tecnolo-
rent work focuses on the tumor microenvironment gia, Polito. He has authored and co-authored two
and the role of monocytes in breast cancer tumor patents and over 150 scientific publications.
development.

Giovanni De Micheli (F’94) is a Professor and the


Director of the Institute of Electrical Engineering,
EPFL, Lausanne, Switzerland. His research interests
include several aspects of design technologies for Sandro Carrara (F’16) received the Diploma
integrated circuits and systems, such as synthesis for degree in electronics, the master’s degree in physics,
emerging technologies, networks on chips, and 3D and the Ph.D. degree in biochemistry and biophysics.
integration. His citation h-index is 93 according to He is a Member of Faculty at EPFL, Lausanne,
Google Scholar. He is a member of the Academia Switzerland, former Professor at the Universities
Europaea and an International Honorary Member of Genoa and Bologna, Italy. Along his carrier,
of the American Academy of Arts and Sciences. he published seven books, one as an author on
He is a member of the Scientific Advisory Board of Bio/CMOS Interfaces (Springer) and, more recently,
IMEC (Leuven, B), CfAED (Dresden, D), and STMicroelectronics. He was a a Handbook of Bioelectronics (Cambridge Univer-
recipient of the 2016 IEEE/CS Harry Goode Award for seminal contributions sity Press). He has more than 250 scientific publica-
to design and design tools of Networks on Chips, the 2016 EDAA Lifetime tions and is the author of 13 patents. His scientific
Achievement Award, the 2012 IEEE/CAS Mac Van Valkenburg Award for interests include electrical phenomena of nano-bio-structured films, and
contributions to theory, practice, and experimentation in design methods and include CMOS design of biochips based on proteins and DNA. He is a
tools, and the 2003 IEEE Emanuel Piore Award for contributions to computer- member of the IEEE Sensors Council, and was a member of the Board
aided synthesis of digital systems. He also received the D. Pederson Award of Governors (BoG) of the IEEE CAS Society. He was a recipient of the
for the best paper of the IEEE T RANSACTIONS ON CAD in 1987 and 2018, IEEE Sensors Council Technical Achievement Award. His work received
and several Best Paper Awards. He has been the Chair of several conferences, several international recognitions as best-cited papers and best conference
including, ICCD (1989), DAC (2000), VLSI SOC (2006), pHealth (2006), papers. He has been the General Chairman of the IEEE BioCAS 2014
DATE (2010), and Memocode (2014). He has been serving IEEE in several Conference. He has been appointed two times as the IEEE Distinguished
capacities, namely: the Division 1 Director (2008–2009), the Co-Founder and Lecturer. He is currently an Editor-in-Chief of the IEEE S ENSORS J OURNAL,
President-Elect of the IEEE Council on EDA (2005–2007), the President the Founder and Editor-in-Chief of the journal BioNanoScience (Springer),
of the IEEE CAS Society (2003), and the Editor-in-Chief of the IEEE and an Associate Editor for the IEEE T RANSACTIONS ON B IOMEDICAL
T RANSACTIONS ON CAD/ICAS (1997–2001). He is a Fellow of ACM. C IRCUITS AND S YSTEMS .

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