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The Promise of Graphene-based Transistors for Democratizing Multiomics Studies

Hsiang-Wei Lu, Alexander A. Kane, Jonathan Parkinson, Yingning Gao, Reza Hajian,
Michael Heltzen, Brett Goldsmith, Kiana Aran

PII: S0956-5663(21)00642-4
DOI: https://doi.org/10.1016/j.bios.2021.113605
Reference: BIOS 113605

To appear in: Biosensors and Bioelectronics

Received Date: 27 May 2021

Revised Date: 22 July 2021
Accepted Date: 29 August 2021

Please cite this article as: Lu, H.-W., Kane, A.A., Parkinson, J., Gao, Y., Hajian, R., Heltzen, M.,
Goldsmith, B., Aran, K., The Promise of Graphene-based Transistors for Democratizing Multiomics
Studies, Biosensors and Bioelectronics, https://doi.org/10.1016/j.bios.2021.113605.

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The Promise of Graphene-based Transistors for Democratizing Multiomics Studies

Hsiang-Wei Lu1,2, Alexander A. Kane2, Jonathan Parkinson2, Yingning Gao2, Reza Hajian1,2,
Michael Heltzen2, Brett Goldsmith2*, Kiana Aran1,2*
1
Keck Graduate Institute, The Claremont Colleges, Claremont, CA, USA
2
Cardea Bio, San Diego, CA, 92121, USA

*To whom correspondence should be addressed: Kiana Aran and Brett R. Goldsmith;
Email: kiana_aran@kgi.edu, brgoldsmith@cardeabio.com

Keywords: graphene field effect transistor, multiomics, digital biosensing, machine learning

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Abstract:

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As biological research has synthesized genomics, proteomics, metabolomics, and
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transcriptomics into systems biology, a new multiomics approach to biological research has
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emerged. Today, multiomics studies are challenging and expensive. An experimental platform

that could unify the multiple omics approaches to measurement could increase access to
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multiomics data by enabling more individual labs to successfully attempt multiomics studies.
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Field effect biosensing based on graphene transistors have gained significant attention as a
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potential unifying technology for such multiomics studies. This review article highlights the

outstanding performance characteristics that makes graphene field effect transistor an attractive

sensing platform for a wide variety of analytes important to system biology. In addition to

many studies demonstrating the biosensing capabilities of graphene field effect transistors, they

are uniquely suited to address the challenges of multiomics studies by providing an integrative

multiplex platform for large scale manufacturing using the well-established processes of

semiconductor industry. Furthermore, the resulting digital data is readily analyzable by machine

learning to derive actionable biological insight to address the challenge of data compatibility

for multiomics studies. A critical stage of systems biology will be democratizing multiomics

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study, and the graphene field effect transistor is uniquely positioned to serve as an accessible

multiomics platform.

1. Introduction

Almost 20 years ago, the Human Genome Project verified something that greatly complicated
the way we understand biology: that proteins are not derived from simple expression of the
genetic codes in nucleic acid sequences. The human genome contains open reading frames that
code for roughly 20,000 canonical proteins. Around 70,000 additional proteins can be created
by using alternative splicing of the DNA code. However, to our best understanding, a single
human cell contains at least 1,000,000 different forms of proteins.(Aebersold et al., 2018)

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The elegance of the Central Dogma of biology that information cannot be transferred from
protein to protein(Pukkila, 2001) has been necessarily augmented by an understanding that

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reality is much more complicated. The definition, and construction of a particular protein is due
to multiple overlapping parameters such as gene expression and post translation modifications,
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which are regulated by interactions with other genetic molecules, methylation, mutation, and
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transcription factors. In addition, the function of the same protein requires multitude of
interactions between other proteins, small molecules, and metabolic markers. Complexity in
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biology comes not only from the complexity of the networks of interactions involved, but also
in the variations in the parts of those networks. This compounded complexity creates limits to
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what can be accomplished via reductionist approaches to biology, as well as limits to the
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usefulness of naive mappings of engineering and computer science concepts into

biology.(Mazzocchi, 2008)

This understanding led to the creation of the field of systems biology, the study of emergent
patterns from the dynamic complexity of biology rather than focus on a particular type of
molecule, such as genomics, proteomics, or metabolomics. An early editorial in Science put it
well: “the pluralism of causes and effects in biological networks is better addressed by
observing, through quantitative measures, multiple components simultaneously, and by
rigorous data integration with mathematical models.”(Sauer et al., 2007) This powerful concept
offers a way forward to understand how observable traits come about in biological systems and
the creation of a new approach to measurement: multiomics.

The current approach to multiomics studies is rooted in a scale up of single omics biological
techniques. In the recent years, collections of large multiomics datasets have enabled population

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studies that captures markers for disease diagnosis, lifestyle, and environmental conditions to
better inform individualized and precise medical treatments.(Emilsson et al., 2018; Mardinoglu
et al., 2018; Price et al., 2017) However, discovery and translating multiomics knowledge
toward effective precision medicine has been slow in comparison to the rate of data
generation.(Ginsburg and Phillips, 2018; Olivier et al., 2019) One major factor is the challenge
to integrate information from independent omics studies. The analysis of multiomics data
requires complex mining of the relations among different genetic molecules using machine
learning methods and multiperspective analysis.(MacArthur et al., 2017)

While tremendous strides in increasing throughput and reducing cost have enabled multiomics
studies through collaborative research, integration of simultaneous measurements from

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multiple biological components is not currently possible. Platforms of the various omics
techniques including genomics(Hindorff et al., 2009; Miles et al., 2020), proteomics(Suhre et

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al., 2017; Zhernakova et al., 2018), transcriptomics(Cloonan et al., 2008; Morin et al., 2008),
metabolomics(Farshidfar et al., 2016; Kim et al., 2009; Wikoff et al., 2009), and
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cytomics(Bernas et al., 2006; Valet, 2005) are used in coordinated studies to produce snapshots
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of data with an intention of creating a holistic system level understanding. These studies can be
quite difficult, normally requiring months or years to accomplish. There are outstanding
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difficulties in experiment planning, data integration, and cost control when performing
multiomics studies. One of the fundamental platform for multiomics studies is mass
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spectrometry (MS) to provide comprehensive analysis of proteomic and metabolomic profile

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of samples.(Beale et al., 2018; Keshishian et al., 2007; Ludwig et al., 2018) While the
throughput of these methods has improved over the years. The detection methods rely on
expensive analytical instruments and experienced personnel to conduct, analyze data and
optimize the assay designs. Even with the heroic efforts of these studies, the dynamic nature
of biology is not addressed by current approaches, and computer models are required to
simulate dynamic responses in between individual time points.(Chaudhary et al., 2018; Conesa
and Beck, 2019; Costello and Martin, 2018; Lin and Lane, 2017; Pinu et al., 2019)

At the same time that multiomics was being developed as an experimental foundation for
systems biology, nanoelectronics was being developed as a foundation to directly link digital
electronics to active biological systems. The digital electrical signal from biological events is
measured by inexpensive electronics and readily analyzed by digital computers common in
research labs. The combination of a unifying multiomics analytical biosensor with machine

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learning algorithms would be a very effective tool for complex biological data collection and
decisive interpretation. Achieving this goal requires advance and maturation of the
bioelectronic platform to enable simple, and reproducible measurements at the hand of
laboratory personnel with minimal knowledge of the underlying technologies. Of the various
designs and materials demonstrated over the 20 years of this effort, graphene transistor-based
sensors are uniquely positioned to provide the capability to detect multiple analytes via an
integrative device with greater accessibility to an academic biology research labs on a wider
scale (Fig 1).

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Fig. 1. Graphene field-effect transistor (gFET) as an integrative platform to provide multi-omics analysis for
cellular and system biology.
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In this review, we will briefly describe the operating principle of field effect biosensors and
contrast the properties of the silicon based and graphene field effect transistors (gFET). The
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second part of this review discusses applications of gFET sensors for the multiomics analysis.
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While most active areas of sensor research focus on a single omics in a study, the versatility of
the technology is illustrated in Table 1 for a wide variety of biological processes in different
sample media. Handling of complex sample materials with consistency is essential to the
success of a multiomics study.(Hasin et al., 2017) To access specific analytes in question,
complex samples obtained from biological specimen will be processed to limit background
signal and interference. Sample preparation procedure before analysis is highly dependent on
the sample matrix such as plasma/serum (Bylda et al., 2014; Luque-Garcia and Neubert, 2007),
urine (Chetwynd et al., 2015; Dhondt et al., 2020), or saliva.(Cuevas-Córdoba and Santiago-
García, 2014; Mias et al., 2021) Plasma/serum is the sample matrix of choice due to the
abundance of analytes across different omics layers.(Pinu et al., 2019) The rich field of sample
processing is still receiving tremendous research attentions with continual developments of new
technologies. The progress are well reviewed by several publications(Bylda et al., 2014; Gama

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and Bottoli, 2017; Kole et al., 2011; Pichon et al., 2014). This review will focus on the multiplex
sensing capability required for multiomics analysis and aspects of scale up manufacturing of
the sensor technology. In addition, in this review we highlight the role of machine learning
models as a crucial tool to fully integrative multiomics analysis.(Shen et al., 2012)

Table 1: Notable gFET biosensing devices for different multiomics analytes.

Multiomics Analyte bioreceptors sample media Refs

DNA/SNP ssDNA PBS/Tris MgCl2 (Hwang et al.,

2016)

Genomic DNA/SNP CRISPR-Cas9 MES (Hajian et al.,

2019a)

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Zika-NS1 Anti-Zika mAb Diluted serum (Afsahi et al.,
2018)

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SARS-CoV-2 Anti-SARS-CoV-2 S Ab complex media (Seo et al.,

Protein/IgE Aptamer
-p PBS
2020)

(Ohno et al.,
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2010)
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Antimicrobial peptide Lipid bilayers PBS (Ang et al.,

2010)

Cellular action potential gFET array PBS (Hess et al.,

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2011; Kireev
et al., 2017)
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Cell sensing gFET array electrolyte (Ang et al.,

2011)
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Glucose Glucose Oxidase PBS (Viswanathan

et al., 2015)

Hormone FAb & PEG Serum (Andoy et al.,

2018)

2. Sensing modes in silicon and graphene field effect transistors

2.1 Sensing Mechanism

Scientists started to develop the in-liquid sensing applications of silicon-based transistors as

early as 1962.(Clark and Lyons, 1962) Adapting the metal-oxide-semiconductor field-effect
transistors (MOSFET) to operation in liquid, the field effect transistors discussed here are
composed of source and drain electrodes connected by semiconducting channels. The
conductance of the channel is modulated by the potential of the liquid gate, which is in itself
measured using a reference electrode (RE) that passes no current. An electrical double layer of
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adsorbed surface charges and ionic screening charges forms at the electrolyte/channel interface,
with a characteristic thickness termed the Debye length.(Israelachvili, 2011)

𝜀𝑘𝑇 1
1) 𝐷 = √ 𝑒 2 𝐼 , 𝐼 = 2 ∑𝑖 𝑐𝑖 𝑧𝑖2

where  is the permittivity of the buffer, k is the Boltzmann constant, T is the temperature, NAv,
e is the electron charge, and I is the ionic strength of the buffer composed of ionic specie i with
density ci and valence zi. This Debye screening length is proportional to the reciprocal of the
square root of the ionic strength, I, and is quite small in physiological solutions, less than 1
nm.(Andoy et al., 2018; Israelachvili, 2011; Ping and Johnson, 2016) The small length gives
the interface an exceptionally high capacitance, allowing for low applied gate voltages and

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therefore stable operation in aqueous environments, required for the monitoring of biological
processes.(Tibaldi et al., 2019) The conductance of the channel is modulated by the potential

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of a liquid gate, which is separated from the channel by a passivation layer put in place to
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prevent chemical oxidation of the silicon by water. Electrolyte-gated field-effect transistors
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such as this have been widely employed in the field of biomedicine.(Casalini et al., 2013) The
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biomolecules influences the biosensor through several mechanisms, all of which contributes
measurable electric signals. In situation where the biomolecule directly interacts with the
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transistor channel, charge transfer can occur between the biomolecule and the channel, thus
affect the electronic properties of the biosensing device. Once the biomolecule is in close
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proximity of the transistor channel, the charged biomolecules can influence the
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transport/scattering of the charge carriers within the channel. Charged biomolecules on the
channel attracts opposite charges toward the channel surface, thus create doping of the channel.
The presence of the charged biomolecules further influences the charge distribution in the
electric double layer which then affects the electronic properties of the transistor channel.

In 1970, the concept of utilizing an ion-sensitive field-effect transistor (ISFET) for detection of
biochemistry (BioFET)(Bergveld, 1970) was introduced. Central to the operation of ISFETs is
the concept of surface buffer capacity, in which an ion-selective membrane covering channel
absorbs the ions of interest.(Bergveld, 2003a) In ISFET based BioFETs, the ion biproducts
(often H+) generated from biomolecular interactions are detected, and the target biomolecules
are not directly detected. To expand the utilization of BioFETs, specialized approaches were
developed where specific biological receptors (or probes) are conjugated on the BioFETs to
directly capture the desired analytes based on bioaffinity.(Forsyth and Devadoss, 2017) The

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binding event is directly transduced, and various BioFET have been developed for the detection
of nucleotides(Estrela et al., 2005; Gonçalves et al., 2008; Sakata et al., 2004), proteins(Caras
and Janata, 1980; R B M Schasfoort et al., 1990; Zayats et al., 2002), and even cellular
activities.(Fromherz et al., 1991; Milgrew et al., 2005) One notable commercially successful
application of this technology is Ion Torrent, which detects H+ production when nucleotides
are incorporated during DNA sequencing.(Rothberg et al., 2011) To expand the utility of
BioFETs, specialized approaches have been developed where specific biological receptors (or
probes) are conjugated on the BioFETs to directly capture the desired analytes.(Freeman et al.,
2007; Marrakchi et al., 2006; Nakako et al., 1986)

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Fig. 2. Illustrations of the analyte/biological receptor interaction effecting the charge distribution on the channel
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surface monitored by (A) BioFET and (B) gFET device. (C) Diagram of an equivalent electrostatic circuit
modeling the maximum potential difference ΔVI that can develop when charges Δσ are bound to the channel
surface at the interface.

In conventional operation, the presence of bound charged biomolecules at the sensing surface
of the transistor will be detected by changes in channel conductance of the FET due to a change
in the effective gate potential. The sensing surface in a BioFET consists of the interface between
a passivating layer, such as silicon oxide, and the solution, as shown in Fig 2A. Using a simple
model, it can be seen that the signal of interest, i.e. the amount of captured biomolecules at the
interface, can be detected as a change in potential ΔVI, which is governed by the capacitance
between the biomolecules and the liquid (CDL), and between the biomolecules and the FET
(CFET) (Kaisti, 2017):
Δ𝜎
2) ∆𝑉𝐼 = 𝐶
𝐷𝐿 +𝐶𝐹𝐸𝑇

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Here, the captured charges from the biomolecules Δσ are compensated by the charges in the
liquid, and the charges in the FET channel, so the FET and the liquid are in parallel from the
perspective of the biomolecules. This is accounted for in the model by the division of the
charges between the two parallel capacitors. Thus, the screening from the liquid represented by
CDL reduces signal, even for molecules adsorbed directly on the interface. To ensure realistic
operating voltages, the passivating gate dielectric must be kept thin while maintaining low
leakage current as a dielectric material. This is a device fabrication challenge. In devices with
very small transistor sizes, CFET is typically much smaller than CDL, and it is often
neglected.(Bousse et al., 1983)

The amount of bound biomolecules is proportional to 𝛥𝜎 and the ratio between the two

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quantities, the charge per biomolecule, is typically known, and depends upon the biochemistry
with dependence on the pH of the aqueous solution and the biomolecules’ isoelectric

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points.(Norde and Lyklema, 1991; R.B.M. Schasfoort et al., 1990) Historically, in commercial
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ISFET applications, the signal of interest is the shift in threshold voltage VT from the addition
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of ΔVI , which is detected directly by measuring the current using an external operational
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amplifier in source and drain follower configuration.(Bergveld, 2003b) The dielectric in

between the channel and the liquid introduces significant background signal due to the
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interposing charge traps between the channel and liquid.(Kutovyi et al., 2020; Liu et al., 2011)

In gFETs, the channel material is a single atomic layer of carbon in the form of graphene, as
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shown in Fig 2B.(Goldsmith et al., 2019; Lerner et al., 2017) Chemical vapor deposition of
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single layer graphene is a popular choice that satisfy both the quality and scalability for sensing
applications. (Kidambi et al., 2018; Kobayashi et al., 2013; Polsen et al., 2015; Yamada et al.,
2012) High quality graphene can also be obtained from exfoliation of graphite with limited
sensing area and scalability due to the serial nature of the operation.(Saltzgaber et al., 2013)
Graphene has ultrahigh native mobility and non-reactivity with ionic buffers. The architecture
of graphene gFET senses the electrical properties at the electrolyte-graphene interface, thus
makes it one of the most potentially sensitive materials for use in bionsensor FETs.(Goldsmith
et al., 2019) The graphene material is extremely sensitive to interactions near its surface due to
its 2-diemntional structure and very high carrier mobility (>2,000 cm2 Vs−1).(Georgakilas et al.,
2016) Its unique structure renders a large biocompatible surface of single atomic thickness
accessible to functionalization. The graphene surface is chemically compatible with biological
electrolytes and so dielectric passivation layers are not required. This enables direct interaction

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with the conduction channel via simple functionalization chemistries.(Nguyen, Emily et al.,
2020)

Rather than using the source-drain follower measurement scheme used for ISFETS, gFET
sensors reported in the literature are usually operated as signal amplifiers, in which the FET
output current response ΔID is measured.(Goldsmith et al., 2019)(Hajian et al., 2019a)(Béraud
et al., 2021)(Andoy et al., 2018)(Hinnemo et al., 2018) In this case the external op-amp will be
configured as a transimpedance amplifier. There are a few reasons for this change in
architecture. The main reason is that graphene is a “zero bandgap” semiconductor. This means
that graphene does not work as a conventional digital transistor, it will not completely turn off
and so the traditional concept of a threshold gate voltage is necessarily replaced with the charge

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neutrality gate voltage, or more conventionally the “Dirac point.” The lack of a bandgap in
graphene can be a benefit when used as a transimpedance amplifier, as effective measurements

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can be made over a much wider range of gate voltages, allowing the biosensor system to adapt
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to the electrical requirements of the biological system rather than subjecting the biological
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system to the electrical requirements of the follower circuit commonly used in silicon ISFETS.
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Using the typical small signal FET model,(Bergveld, 2003a; Goldsmith et al., 2019) combined
with the simple parallel capacitance model from equation 2), the change in drain current from
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a binding event at the surface can be approximated as:

𝑊 𝐶𝐹𝐸𝑇 Δ𝜎
3) ∆𝐼𝐷 = 𝑉𝐷𝑆 𝜇𝑒𝑓𝑓 𝐶
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𝐿 𝐷𝐿 +𝐶𝐹𝐸𝑇

Where W is the channel width, L is the channel length, VDS is the drain-source bias, and µeff is
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the effective mobility incorporating the effects of finite contact resistance. In equations 3, it
can be seen that the overall gain is increased in the current response by a factor equal to the
transconductance gm = (W/L)VDS µeff CFET. Miniaturization of channel to optimize W/L factor
was well established for silicon due to advancement in semiconductor industry. However,
graphene has high intrinsic carrier mobility (µ>2,000 cm2 Vs−1) compared with silicon, leading
to an advantage over Si in amplification mode. This advantage in transconductance is
compounded in biosensing applications because of the increased CFET in graphene compared to
silicon. This is because CFET is a series combination of all the capacitances between the interface
and the channel, which includes the capacitance of the dielectric passivation layer in silicon,
which isn’t present in graphene. In gFETs CFET can be comparable to CDL, leading to a higher
overall gain.(Mackin et al., 2018; Xia et al., 2009) The dielectric also introduces low frequency
noise from the interposing charge traps between the channel and liquid.(Kutovyi et al., 2020;

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Liu et al., 2011) Electronic noise in general can be a difficult problem to overcome. Careful
measurements of the noise in graphene devices relative to equivalent silicon devices shows that
the electronic noise in graphene is lower than that for silicon transistors at the low gate voltages
required for biosensing.(Rumyantsev et al., 2010) This can be surprising to electronics
researchers who are accustomed to thinking of nanomaterials as having intrinsically high 1/f
noise. While carbon nanotubes famously have extremely high 1/f noise, the noise in graphene
devices is much lower.

Beyond the increase in signal gain, gFET’s ability to operate in amplification mode with high
transconductance devices allows for multimodal sensing. Changes in the total gate capacitance
CT and changes in the channel mobility µeff modulate the transconductance and can be detected

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by sweeping the liquid gate voltage to measure the source-to-drain current as a function of the
gate potential, I(Vg), which is the transfer curves of the transistors. This requires a counter

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electrode in the device design in addition to the reference electrode, so that the potential is
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properly measured while sweeping.(Mannik et al., 2006) Changes in capacitance result from
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the displacement of charges in the liquid by the presence of the biomolecules, which can change
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the double layer capacitance of the channel with the liquid gate.(Goldsmith et al., 2019) In this
case, the biomolecules do not need to be necessarily charged to be sensed. Mobility changes
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will be stronger in the opposite limit of strongly charged targets, where charges near the channel
can scatter charge carriers, thereby decreasing the mobility.(Hinnemo et al., 2018)(Andoy et al.,
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2018)
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2.1 Overcoming the challenges of Debye length

The lumped element model from Fig 2A-B is useful conceptually to illustrate differences
between material technologies. The biomolecules in reality will not assemble as uniform layer
of charges right at the interface, but rather will sit some distance away from the FET channel.
Electrolyte buffer forms a double layer at the interface of buffer and the channel surface. The
thickness depends on the ionic strength of the buffer.(Israelachvili, 2011) If the biomolecules
sit much farther away than the Debye length, the ions in solution will nearly completely screen
the charges of the biomolecule, and they will not affect the gate potential of the device. This
challenge is only partially addressed by utilizing smaller bioreceptors such as antibody
fragments(Okamoto et al., 2012) and aptamers(Nakatsuka et al., 2018; Ohno et al., 2010). The
high ionic strength of physiologically relevant fluid can result double layer of < 1nm in
thickness. This effect places a limitation on the application of FET based devices for actual

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biological systems.(R.B.M. Schasfoort et al., 1990) The modern gFET architecture enabled by
the exceptional material properties of graphene address this inherent challenge of conventional
BioFET devices. Modern gFET biosensor designs mitigate Debye screening by employing the
Donnan effect concept from cell biology. Similar to a cell membrane, a semi-permeable layer
of organic molecules is fabricated on top of the semiconductor channel. These layers are
composed of various types of molecules with different functions, including biological receptor
molecules for the analyte of interest, linker molecules if the biological receptor cannot be
directly attached, and blocking molecules such as BSA and PEG to passivate the empty linkers
and channel. (N. Gao et al., 2016; Hajian et al., 2019a; Haustein et al., 2019; Liu et al., 2013;
Palazzo et al., 2015) The semi-permeability requires sufficient surface density that can be

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achieved by appropriate choices in assay parameters for surface functionalization or passivation.

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Characterization by Palazzo et al. shows that Donnan layer can be as far as 20-30D away from

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the channel surface.(Palazzo et al., 2015) Alternatively, Donnan layer can be assembled by
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passivating the channel surface with long chain PEG polymer of 10kDa.(Baldacchini et al.,
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2020) As with the analytes of interest, the organic molecules used in the Donnan layer will
usually be charged when in buffer solution, depending on their isoelectric point and the local
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pH. Larger biomolecules cannot generally penetrate the layer, so the layer is also ion-selective.
A Donnan equilibrium is formed between the immobile charged molecules within the layer and
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the mobile ions in the bulk solution. The potential within the layer, the Donnan potential, is
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sensitive to pH and ionic concentration (Das, 2014; Ohshima, 2009), and this is the potential
directly sensed by the FET. The Donnan potential changes as new biomolecules are bound to
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or release from the layer of biomolecules immobilized on the surface of the gFET.

Adding a Donnan layer to the interface has led to the modern era of biosensor FETs that can
operate even in physiologically relevant, high ionic strength buffers.(Baldacchini et al., 2020;
N. Gao et al., 2016; Gutiérrez-Sanz et al., 2017; Palazzo et al., 2015; Park et al., 2020) The
effect enables sensor to be much more sensitive to bound analyte than under a double layer
alone, because in Donnan equilibrium, the double layer is moved away from the surface of the
FET and instead forms at the Donnan layer interface (Ohshima, 2009). If the biological
receptors are captured within the Donnan layer and so beneath the double layer, the screening
is much reduced because of the decreased concentration of mobile ions.(Haustein et al., 2019)
This sensitivity in high ionic strength buffer was demonstrated by direct detection of thyroid
stimulated hormone in whole serum on gFET sensor.(Andoy et al., 2018) Capacitance mode
sensing also benefits from the presence of the Donnan layer, as CFET must be modified to

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include the series capacitance of the Donnan layer, CDon, which will be smaller than the double
layer capacitance and can therefore limit the total capacitance.. CDon is proportional to the ionic
strength and thereby is more sensitive to changes in ionic concentration than the double layer
capacitance which is proportional to the square root of the ionic strength. The presence of large
biomolecules, even outside the double layer, can cause large shifts in CFET and the
transconductance.(Palazzo et al., 2015) This requires that CDon is the limiting term in CFET, true
for gFETs, but may not be true in silicon BioFETs with passivating layers that themselves limit
CFET.

3. Conjugation of biological receptors to gFET device

In the development of biosensors, selection of an appropriate immobilization technique to

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conjugate the capture biomolecules to the surface of the graphene is critical as it can impact the
sensitivity and specificity of FET sensors.(Andoy et al., 2018; N. Gao et al., 2016; Goldsmith

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et al., 2019) The chemically inert nature of graphene material presents a challenge for direct
covalent functionalization. Linker molecules that have aromatic rings for pi–pi interaction with
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graphene are the most common method to attach functional groups to graphene channel.
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Table 2: Surface functionalization and linker chemistry to incorporate receptors to gFET.

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Linker Name Connection to Connection to Capture Biochemistry required Refs

Graphene Molecule
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PBA Pyrene, non- COOH EDC/NHS to amine (Hajian et

covalent al., 2019a;
Premaratn
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e et al.,
2017; Wei
et al.,
2009; Yüce
and Kurt,
2017)

PBASE Pyrene, non- NHS amine (Gao et al.,

covalent 2018; Seo
et al.,
2020; Yu
et al.,
2019)

Pyrene-NTA Pyrene, non- NTA Cu, Ni, Co ion to His- (Liu et al.,
covalent tag 2010;
Sandoval
et al.,
2020)

Pyrene-maleimide Pyrene, non- maleimide sulfur (Nair et al.,

covalent 2014;
Ravasco et
al., 2019)

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 Click pyrene dibenzocyclooctyne Azide (Sadlowski
et al.,
2018)

Carboxy diazonium Azo, covalent COOH EDC/NHS to amine (Dyke et

al., 2003;
Lerner et
al., 2017;
Yáñez-
Sedeño et
al., 2018)

ssDNA direct adsorption π -π, non-covalent ssDNA DNA hybridization (Cagang et

al., 2016;
Ganguli et
al., 2020;
Hwang et
al., 2020;
Snapp et

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al., 2020)

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Covalent immobilization to a linker is the preferred method of attaching proteins to sensing
surfaces due to the strong and stable linkage.(Banerjee et al., 2005; Kindermann et al., 2003;

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Williams and Blanch, 1994) Amine and carboxyl coupling (between the substrate and protein)
is commonly attained with carbodiimide chemistry that utilizes 1-Ethyl-3-[3-dimethylami-
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nopropyl]-carbodiimide hydrochloride (EDC) combined with succinimidyl esters, such as N-
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hydroxysuccinimide (NHS).(Goddard and Hotchkiss, 2007; Welch et al., 2017) Both of these
chemistries have been widely adapted for gFET biosensors by utilizing PBASE and PBA
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chemistries as described above, where the carboxyl end of linker molecules is utilized for
covalent immobilizations of capture biomolecules to the linker where the linker is non-
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covalently attached to the graphene via its aromatics rings on the other end. Other chemistries
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such as nitrilotriacetic acid (NTA) have been also utilized to attach his-tagged proteins to the
surface of the graphene. (Liu et al., 2010; Sandoval et al., 2020)

The sensing area of gFET is in contact with a liquid sample for sensing of different analytes.
The solution contain significant amounts of electrolytes and other components that cause a
background electrical current. This ionic current is strong enough to affect sensing results from
the specific interaction of an analyte with its receptor. Some components of analyte in the
solution may also interact non-specifically on the graphene channels during the measurement
and cause changes in electrical response of the device. Passivation of the sensing substrate of
graphene channel in gFETs sensors is required to limit these interference. Passivation involves
formation of an outer layer of molecular structures that is as a coating layer with the sensing
substrate. The passivation process should ensure uniform and inactive layer on the areas of
sensor in contact with the liquid. (Gao et al., 2018; Mao et al., 2010) It should also have a

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minimal effect on the electrical signal of the analyte under study. The coverage of graphene
with recognition molecules is often incomplete, in this case passivation must be used to block
non-specific interactions with graphene.(Béraud et al., 2021)

4. gFET sensors for biological analytes

4.1 gFET for genomics

Detection and analysis of DNA is of fundamental interest for the fields of molecular biology,
bioinformatics, genetics and clinical diagnostics.(Bejjani and Shaffer, 2006; Nunez, 2001;
Woolley et al., 1996; Xia, 2013) Current technologies provide the genetic coding in the DNA
sequences by optical interrogation which requires complex workflows and optical instruments.

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This results in limited accessibility of the biological information needed for timely and precise

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medical decisions required for control and treatment at the early outbreak of infectious diseases,
such as the COVID-19 outbreak in early 2020.(Vandenberg et al., 2020) Unlabeled and

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amplification-free electrical detections of DNA by gFETs offer a promising alternative to
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address challenges associated with expensive instrumentation, nucleic acid amplification, and
slow time to results. Graphene offers superior sensitivity to the detection of DNA at low
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concentration and the selectivity to distinguish the sequence information without the need for
nucleic acid amplification methods.(Hajian et al., 2019b) The simplification and sensitivity of
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gFET offers a promising nucleic acid detection platform for DNA and RNA with integrations
with microfluidics.(Gao et al., 2020; Kim et al., 2019)
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Table 3 provides examples of devices for analyzing genomic sequences of DNA and RNA.
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The majority of gFET-based DNA detections are based on immobilization of a varieties of

probe moiety, typically a single stranded DNA (ssDNA) as probe that specifically recognize
complementary target DNA sequences. Hybridization of the immobilized probe with the target
changes the electrical properties of the gFET as described above which can be monitored via
an electronic reader. Sensitivity can be further improved by optimizing the device design by
different probe moiety, increasing the channel to gate surface ratio, and graphene contact
area.(Campos et al., 2019)

Table 3: gFETs for quantification and analysis of genomic sequence

Analyte Probe Moiety Detection Method Detection Range. Refs

Genomic DNA CRISPR-dCas9 Average ISD between 1.7fM – 5.9pM (Hajian et

±100mV al., 2019a)

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 ssDNA(22 - 60nt) PBASE-ssDNA Dirac voltage shift 200nM, (Campos
et al.,
1fM – 100fM (60nt), 2019;
25aM – 10fM Ohno et
al., 2013;
Ping et al.,
2016)

ssDNAa) pi-pi adsorbed Dirac voltage shift N/A (Ganguli et

ssDNA al., 2020;
Hwang et
al., 2020)
a)

ssDNAb) PBASE-Hairpin Dirac voltage shift 100aM – 100pM (Gao et al.,

ssDNA 2018) b)

ssDNA pi-pi adsorbed Dirac voltage shift 100nM (Manohara

ssDNA n et al.,

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2017)

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TP53 ssDNA pi-pi adsorbed Dirac voltage shift 10uM – 1nM (Kim et al.,
ssDNA 2019)

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ssDNA-MBc) PBASE-ssDNA -p gFET impedence 100pM – 10nM (Sun et al.,
2019)
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RNA PBASE-PNA Dirac voltage shift 0.1aM – 1pM (Tian et al.,
2020)
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miRNA pi-pi adsorbed Dirac voltage shift 10fM – 100pM (Gao et al.,
ssDNA 2020)
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ssDNA AuNP decorated Dirac voltage shift/ 0.01nM-500nM (Dong et

channel conductance measurement al., 2010;
Z. Gao et
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al., 2016)

ssDNA AuNP conjugated- Dirac voltage shift 60fM -10pM (Chan et

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ssdNA al., 2016)

a) End point detection by sensing depletion of ssDNA primer for LAMP amplification.

b) ssDNA extended by target triggered self-assembly amplification.

c) ssDNA conjugated to magnetic beads to enable impedence measurement.

PBASE linker chemistry immobilized the probe single-strain DNA (ssDNA) to the graphene
channel. Packed ssDNA of estimated density of 10nm2/ssDNA based on plate capacitor
model(Ohno et al., 2013) can be accomplished by this method. Preferential adsorption of
ssDNA by pi-pi interaction with graphene was also explored as a possible alternative that may
be attractive due to its simplicity. (Hajian et al., 2019a) The strategy offers a sensitive detection
of ssDNA(Hwang et al., 2020) and end point detection for amplification-based assay using
crumpled graphene channel.(Ganguli et al., 2020) In addition to DNA analysis, ability to
analyzing RNA present in the sample provides crucial information regarding gene expression
in response to stimuli. Recently a RNA detection platform was proposed using partially

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adsorbed ssDNA as probe moiety for target RNA.(Gao et al., 2020) Immobilizing peptide
nucleic acid PNA, the neutrally charged probe moiety enhances the binding affinity due the
reduction of repulsion of the negatively charged backbone of DNA and RNA(Genet et al., 2013;
Nielsen et al., 1991). A PNA immobilization on gFET improves the limit of detection for RNA
showing linear range of RNA detection from 1pM to 0.1 aM range(Tian et al., 2020).

Although direct detection of hybridization eliminates the need for labels and optical instruments.
Simple DNA hybridization has limited specificity for sequencing application requiring
specificity to detect single nucleotide polymorphism (SNP). The probe DNA sequence is
limited to short sequence length to increase the discrimination capabilities to single nucleotide
mismatch. Hwang et al. demonstrated improved SNP specific detection by DNA strand

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displacement on gFET of significantly longer strand, up to 47nt length(Hwang et al., 2016).
Enzyme-based methods are still required for further improvement of sensitivity for genomic

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applications.(Nielsen et al., 1991; Olivier, 2005) This requires probe designs, expensive
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reagents, skilled personnel, and specialized infrastructure for sample preparation. By
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immobilizing hairpin DNA probe on graphene channel, Gao et al. accomplishes signal
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amplification using hybridization chain reaction to elongate the short DNA and alleviated the
sensitivity bias against long DNA sequences.(Gao et al., 2018) Introducing noble metallic
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nanoparticles, such as gold (AuNP)(Dong et al., 2010; Z. Gao et al., 2016) on the transistor
channel, or as part of the probe moiety(Chan et al., 2016) act as signal amplification by adding
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electroactive nanoparticles and increase the functionalization capacity of the probe moiety on
the graphene channel. These mechanisms improve the sensitivity or dynamic range of the
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sensor.

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 SIGNAL: +

I-response (%)
Cas
Target Site
SIGNAL: -
0 10 20 30 40
Time (min)

Detection
time

15 min
DNA
gRNA
PBA Linker

Source
Electrode

Genomic DNA
Sample Epoxy
Graphene
Chip layer
Drain
Patient
Electrode

Fig. 3. CRISPR-CHIP is a g FET with and immobilized RNA-guided Cas complex on the graphene channel
between the source and drain electrodes.[111] Copyright (2019) Springer Nature.

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In an effort to eliminate the need for amplification, Hajian et al. utilized a deactivated CRISPR-
Cas9 enzyme to sensitively detect unamplified genomic DNA(Hajian et al., 2019a). The device

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incorporates thousands of RNA-guided Cas nucleases (Fig 3), which interact with a target-p
sequence by scanning the whole genome. The selective association of the target DNA to the
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RNA-guided Cas complex modulates the electrical characteristics of the gFET and results in an
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electrical signal output. While the concentration of DNA in is small, the total charges in the
DNA strand captured by the CRISPR complex is large. The programmability of the Cas by
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association of different guide RNA enables a flexible platform to interrogate various genomic
information from full length genomic DNA. This approach was more recently used to detect
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SNP mutations in unamplified genomic DNA of sickle cell disease and amyotrophic lateral
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sclerosis.(Balderston et al., 2021a) The work opens a new avenues of genomic analysis by
monitoring the nucleotide specific activities in families of Cas complexes.(Bruch et al., 2019;
Dai et al., 2019; Harrington et al., 2018; Kazlauskiene et al., 2016)

4.2 gFET for Proteomics

The detection of protein biomarkers is crucial for the diagnosis and monitoring of
diseases.(Rifai et al., 2006) However, the cost and analysis time of traditional protein detection
techniques are barriers to the universal use of biomarker measurements in health
sciences.(Heath et al., 2009) It is important to understand that any biological assay, including
use of a gFET, is limited by the binding affinity of the capture molecule. An antibody that
cannot bind to a low concentration of antigen will not result in sensitive assay on any platform.
Early efforts to use gFETs for protein detection focused on biologically trivial measurements

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such as detection of BSA.(Ohno et al., 2009) In the 12 years since those first studies, there has
been significant progress, as can be seen in Table 4 below.

Table 4: Detection of biological analytes by varieties of biological receptors on gFET devices.

Analyte Capture Molecule Concentration Electrolyte Refs

lysozyme aptamer 10 nM 0.01X PBS (Ghosh et al., 2018)

thrombin aptamer 30 nM MES (Saltzgaber et al.,

2013)

Heat shock protein Fab 100 pM PBS (Okamoto et al.,

2012)

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Alpha-fetoprotein antibody 0.1 ng/mL 0.01X PBS (Kim et al., 2018)

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Zika NS1 antibody 0.45 nM Dilute serum (Afsahi et al., 2018)

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COV-19 Spike
protein
antibody -p
2.42 copies/mL Transport Media (Seo et al., 2020)
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IgE aptamer 0.29 nM PBS (Ohno et al., 2010)
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DNA topoisomerase dsDNA 300 pM Tris (Zuccaro et al., 2015)

activity
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opioid GPCR 10 pg/mL PBS (Lerner et al., 2014)

dPKC peptide 1 nM PBS (Qvit et al., 2016)

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Small molecules protein 10 µM PBS (Sandoval et al.,

2020)
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Use of aptamers and antibodies immobilized to the graphene surface are common in protein
binding assays on gFETs. One consideration when using antibodies is that the typical height
of an antibody is about 10–15 nm, meaning that the binding sites of antibodies are outside the
typical screening distance in biological buffers. To resolve this issue, Okamoto et al. used a
small size antibody fragment to bring the binding site within the screening distance, as shown
by the Debye length in Fig 4A.

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Fig. 4. (A) Illustration of an antibody and an antigen-binding fragment (Fab) on gFET. (B) Drain current change
versus heat shock protein (HSP) concentration. Copyright (2012) The Japan Society of Applied Physics .

In this study, the change in the conductivity of the gFET during binding of the antibody

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fragment showed a shift indicating binding of a negatively charged molecule to the surface,

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consistent with a negative charge on the fragment in PBS at pH 7.4. (Okamoto et al., 2012) Fig
4B shows the time dependence of source-drain voltage (VSD=1V) and VG =0:05V as the heat

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shock protein at various concentrations were introduced onto the Fab-modified gFETs.
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Studies of binding interactions with gFETs have moved beyond proof of concept in
nanotechnology labs and have been used for biological research. Several notable examples are
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covered here. Using a newly developed dibenzocyclooctyne-pyrene linker, Sadlowski et al.

enabled click chemistry on a gFET device to quantify protein biomarkers labeled by azido-nor-
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leucine from cells expressing a mutant methioyl-tRNA synthetase. (Sadlowski et al., 2018) The
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devices were utilized to rapidly detect the level of Lif-1 and Leptin in heterochronic blood
exchange between young and old mouses in parabiotic systems. This allowed the researchers
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to rapidly and sensitively quantify the relative level of protein biomarkers associated with aging.

Early access to a commercial gFET sensing system enabled Qvit et al. to detect in vitro binding
of candidate peptide therapeutics to delta protein kinase C (dPKC).(Qvit et al., 2016) Measuring
binding of an inhibitor to dPKC is a challenge as this kinase rapidly deteriorates. Use of a gFET
system with the candidate therapeutic bound to the graphene surface enabled fast and accurate
measurement of binding affinity in this challenging biological system.

Sandoval et al. used a gFET system to verify binding affinities of a panel of small molecule
therapeutics in the piperazine drug class to the cell proliferation regulating receptor
mTORC1.(Sandoval et al., 2020) These measurements involve use of high concentrations of
small molecules in buffers supplemented with DMSO. Much of the sensor literature focuses on
evaluating low levels of detection, while practical biological lab work can also require operation
at high concentrations such as those demonstrated here.
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In different application for infectious disease diagnosis, a gFET sensor capable of specific
detection of Zika viral antigens by immobilizing monoclonal antibody on the graphene
channel.(Afsahi et al., 2018) The electronic nature of the sensor enables a portable system that
can perform medical diagnostic in real-time. During the early stage of SARS-CoV-2 pandemic
in 2020, Seo et al. also demonstrated a promising gFET device to detect SARS-CoV-2 virus
utilizing antibody against the SARS-CoV-2 spike protein.(Seo et al., 2020)

4.3 gFET for Extracellular Vesicles and Cells

In addition to detection of circulating protein and nucleic acid biomarkers as discussed in the
above, the ability of gFET to perform label-free detection enables simple and powerful
diagnostics within the field of liquid biopsy For example, exosomes are small extracellular

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vesicles expelled by both healthy and tumorous cells present in blood serum in high abundance
(108-1011 particles/mL).(Grasso et al., 2015) The presence of an exosome with cancer related

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surface biomarkers and its genetic cargo are of significant diagnostic potential.
immobilizing anti-CD63 antibodies on a graphene channel, a back gated gFET demonstrated
By
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specific label-free detection of exosome expressed with CD63 surface markers at
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0.1ug/mL.(Tsang et al., 2019) Hajian et al. utilized an portable liquid gated gFET system to
sensitively detect age specific and cancer exosome.(Hajian et al., 2021) The method potentially
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can be applied toward early-stage cancer diagnosis from sub-milliliter of liquid samples. The
change in electrical properties of the exposed graphene in the presence of exosomes created an
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additional minimum alongside the original Dirac point in the gate voltage transfer curve. Upon
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conjugation with an isotype control, the sensor showed a negligible response in comparison to
an anti-CD63 antibody at high concentration of exosomes, indicating that the functionalized
gFET can specifically detect targeted exosomes.

Similar device design and surface chemistry for exosome detection can be extended toward
monitoring of interactions with cells. An early example of gFET sensing used a device with an
array of graphene sensors to track the flow of cells in a microfluidic channel.(Ang et al., 2011)
In addition to this electrical detection, these sensors were built on top of a glass substrate, which
allowed simultaneous optical and electrical interrogation of the cells in the channel. The
transparent nature of graphene is often emphasized in sensing literature as beneficial to these
kinds of combined optical and electrical measurements. While optical graphene materials and
electronic graphene tools are both now much more accessible in biology labs, combined
systems remain specialty nanotechnology tools. Beyond simple detection of cells, the

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biocompatible nature of graphene enabled gFET sensors for monitoring cardiomyocytes and
neuron activity.(Cohen-Karni et al., 2010; Hébert et al., 2018; Hess et al., 2011; Kireev et al.,
2017) Further development of this technology demonstrated the capability of real-time
recording of electrocorticography on flexible substrates capable of in-vivo measurements in the
near future.(Veliev et al., 2017)

4.4 gFET for Metabolites

Immobilizations of specific enzymes on gFET(Viswanathan et al., 2015) open the new

opportunities in monitoring metabolites and hormones intimately related to patient health.
Nitric Oxide (NO) is involved in a wide range of biological functions in neuro and
cardiovascular systems.(Ignarro et al., 1987; Ohta et al., 1993; Radomski et al., 1990) By

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functionalizing the graphene through pi-pi stacking of hemin chloride, Jiang et al. demonstrated
a gFET device that detects NO in sub-nM concentration against interfering chemicals.(Jiang et

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al., 2013) -p
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An on-chip aptasensor for ochratoxin A (OTA) toxin detection built on a gFET was reported
by Nekrasov et al.(Nekrasov et al., 2019) The results demonstrate fast (within 5 min) response
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to OTA exposure with a linear range of detection between 4 ng/mL and 10 pg/mL, with a
detection limit of 4 pg/mL. Using graphene functionalized by antibody fragments and
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passivated with polyethylene glycol polymers, Andoy et al. demonstrated femtomolar

sensitivity for detection of thyroid stimulating hormone in whole serum.(Andoy et al., 2018)
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The sensitive detection in physiological fluid demonstrates the power of modern gFET devices
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using the Donnan layer to extend sensitivity beyond the Debye screen length.

Sensitive glucose monitoring is always a major challenge in tacking the management and
diagnosis of diabetes. This application has become a common demonstrative test for new
technologies. Huang et al. first reported a gFET functionalized by glucose oxidase or glutamic
dehydrogenase on the graphene channel surface to catalyze glucose or glutamate,
respectively.(Huang et al., 2010) Immobilization of glucose oxidase on a graphene channel
produces H2O2 as a biproduct of the oxidation of glucose. This creates an n-doping reflected
in the negative shift of the Dirac point.(Kwak et al., 2012) Zhang et al. incorporated additional
graphene modified by platinum nanoparticles as gate electrode of the gFET. The enhancement
of the electrocalalytic activity at the gate electrode further improved the sensitivity of glucose
(Viswanathan et al., 2015; Zhang et al., 2015) and dopamine(Zhang et al., 2014) in buffered
solution.

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G-protein-coupled receptors (GPCR) are a family of transmembrane proteins playing a crucial
role in the intracellular signal pathways.(King et al., 2003) GPCRs bind a wide range of target
ligands that are important to the pharmaceutical industry.(Hauser et al., 2017; Overington et al.,
2006) The transmembrane nature of the receptor proteins poses stiff challenges to their
isolation in stable form and integration to sensor devices. A gFET device incorporated a
redesigned µ-opioid receptor with hydrophilic residuals that enables stable manipulation
outside of membrane environment demonstrated sensitive detection of naltrexone down to
pg/mL.(Lerner et al., 2014) Odorant receptors (ORs) are another important class of GPCR that
holds promising applications for food, beverages, environmental monitoring, and disease
diagnostics. Human ORs were chemically attached to gFET to sensitively and selectively detect

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target odorant to fM range.(Park et al., 2012) Multiplex sensors were developed to distinguish

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target odorants in mixtures.(Kwon et al., 2015) Alternatively, ORs were integrated in

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nanovesicles and lipid nanodiscs tethered to gFETs.(Ahn et al., 2016; Murugathas et al., 2020)
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Preserving the transmembrane proteins within their native environments enhances the stability
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and sensitivity to the target ligands.
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4.5 Discussion

The examples above indicate the adaptability of gFETs to a wide range of multiomics analysis
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with minimum need for sensing platform reconfiguration. Therefore, developing highly parallel
multiplexed gFET can provide access to large volume of data, which can ultimately enable us
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to accurately correlate the interplay between biological events in real-time. The efforts toward
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the use of graphene-based biosensors for multi-analyte analysis are at infancy stages.(Ahn et
al., 2016; Kwon et al., 2015; Torrente-Rodríguez et al., 2020) gFET sensor designs are maturing
toward commercialized product for sensing a wide array of analyte of different sizes and types
from metabolites, DNA, proteins, and even exosomes. (Balderston et al., 2021a; Hajian et al.,
2021; Sadlowski et al., 2018; Viswanathan et al., 2015). Sensitive detections on a multiplex
platform will require integration of multiple assay conditions, assay buffer formulations,
surface functionalizations, and instrument gate frequencies. Addressing these challenges will
require sophisticated designs for fluid handling (Donnelly et al., 2018; Khan et al., 2020; Wang
et al., 2019; Xu et al., 2014) while the instrumentation of multiple sensors, each targeting
different analytes, can be addressed through electronic control of different sets of optimized
gate frequencies and functional shapes. The compatibility of gFET manufacturing with the
current semi-conductor industry indicates a great potential and relative ease with which these
sensors can be multiplexed to generate multi-analyte data. Graphene FETs could become an
22
integrative platform for “multiomics” studies, where electronic data acquired using different
“omics” techniques be easily combined for analysis. Having access to this large volume of data
will demand computational power and machine learning algorithms to analyze and extrapolate
meaningful information.

Frequently this data has been analyzed using clustering and other machine learning techniques.
While a comprehensive exploration of data analysis in multiomics is well beyond the scope of
this review, we will briefly consider the potential of machine learning for this field and some
of the unique challenges and opportunities these studies present for biology. The following
section will focus on the importance of computational analysis and machine learning to be
integrated with gFETs to achieve multiomics analysis.

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5. Machine learning techniques for analysis of multiomics data

The promise of machine learning techniques for biology and medicine has been extensively

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explored elsewhere.(Deo, 2015; Rajkomar et al., 2019) Briefly, machine learning algorithms
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are an extension of statistical modeling techniques.(Bzdok et al., 2018) They seek patterns that
provide predictive power and enable modelers to “learn” complex relationships from large
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datasets without the need to specify an expected functional form for the relationship in advance.
These capabilities have revolutionized fields like natural language processing(Collobert and
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Weston, 2008), image analysis(Decencière et al., 2013; Madabhushi and Lee, 2016) and protein
structure prediction(Cheng et al., 2008; Jain et al., 2009), where they have made it possible to
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model complex relationships like the one between the raw pixel values of an image and the
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category of the objects it contains.

Machine learning techniques seem especially appropriate for analysis of multiomics data, where
a complex relationship of unknown functional form is expected between the variables measured
(protein abundance, gene expression etc.) and some output of interest (cancer subtype, disease
progression etc.) Univariate analysis techniques (e.g. association testing in genome-wide
association studies or GWAS) focus on the relevance of single variables in isolation and are
thus insufficient to handle problems where many different variables in combination are required
to predict an outcome.(Deo, 2015; Rajkomar et al., 2019) These types of problems are by
contrast a natural fit for machine learning approaches.

Application of machine learning techniques to multiomics data analysis does however present
several unique challenges. The adage that “garbage in = garbage out” applies here as elsewhere
in statistical modeling, so good data curation and quality control is crucial.(Chen et al., 2019;

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Rajkomar et al., 2019) Multiomics datasets combine data acquired on very different scales with
very different levels of precision. Concatenating the data requires normalization to ensure all
data types are on the same scale and may be counterproductive when the signal to noise in each
data type is already small. Analyzing each data type separately, by contrast, makes it difficult
to integrate the clustering assignments or predictions made using each data type.(Bersanelli et
al., 2016; Wang et al., 2014) In addition to signal quality, data generation on different platforms
inherently introduce complexity to harmonize the data for multiomics data analysis. Electronic
platforms such as a multiplex gFET that generates electronic data from a wide range of
biological interactions simultaneously without labeling can tremendously simplify the
complexity of multiomics data analysis.

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Another challenge lies in the issue of overfitting as it relates to dimensionality. Overfitting
occurs in situations where a model has captured the noise in the data rather than the trend. A

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classic example is fitting a 10th degree polynomial. This highly flexible function is naturally
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prone to capturing the noise in the data, causing its parameters to fluctuate wildly in response
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to small changes in the data used to fit it.
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The problem of overfitting is especially acute when the dimensionality of the data is large
compared to the number of datapoints. The issue with this type of data stems from a well-known
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problem in statistics called the “curse of dimensionality”.(Hastie et al., 2009) The volume of a
space increases exponentially with increasing number of dimensions. The practical implication
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is that small sample, high-dimensional datasets are extremely sparse. These types of datasets
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are frequently encountered in multiomics due to the large number of variables and the expense
of acquiring multiomics data for many patients. Consider a study that acquires data on the
expression levels of 2,000 genes across a hundred patients, for example. Given this data, each
patient could be represented as a datapoint in a dimensional space spanned by 2,000 quantities.

To understand why this creates a challenge, consider three datapoints in a two-dimensional

space, where two of them are randomly assigned to group A and the third to group B. It is easy
to see that no matter the group assignment, we can always find a boundary line that will separate
the two groups. If the number of dimensions is greater than the number of datapoints, we can
find many hyperplanes or linear boundaries that will separate any random two-group
assignment equally well. Most of these boundaries will have captured noise rather than a useful
trend. Hence overfitting is harder to avoid in situations where the number of datapoints is small
and the number of dimensions is large.(Hastie et al., 2009; Teschendorff, 2019) The large

24
number of dimensional space involved in multiomic studies posts a greater challenges to sustain
the scale and expenses for quality data. While tremendous progress was made to establish
quality standards(MAQC Consortium, 2010, 2006) to enable aggregations of data from multiple
studies, the economic cost of these studies remains an issue. Label free detection using a gFET
is attractive in this regard due to its potential for scalable manufacturing and reduction of assay
complexity, leading to greater access to the testing platform. This could allow more biology
researchers to run multiomics studies that properly use a much larger number of samples than
variables being tested.

Frequently, rather than training a model to make predictions, multiomics studies seek rather to
find meaningful subgroups that share some common identifying features. Common techniques

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include clustering and dimension reduction, for which many algorithms exist. Here a special
challenge in multiomics involves ensuring that the clustering is relevant to the problem of

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interest. Imagine that a study hopes to understand the molecular basis for the different outcomes
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observed in patients with a particular disease. Suppose further that the patients in the study can
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be separated easily into two or three subgroups based on some combination of gene expression
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and proteomics data. Statistical correlations must be established from the clusters to the
outcome of interest but more importantly do not reflect variations not directly related. In other
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words, we must ensure that our clustering has clinical significance, and the patient data are
sufficiently annotated.
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Despite these challenges, the promise of machine learning in combination with multiomics is
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clearly substantial. Analysis by machine learning enables integrative understanding of the

patient biology, novel treatment options, and diagnostic biomarkers for chronic diseases (Shen
et al., 2012, 2009; Wang et al., 2014), cardiovascular diseases(Forné et al., 2019; Poss et al.,
2020), and diabetes.(Hathaway et al., 2019; Huang et al., 2015; Lin and Lane, 2017)

Hoadley et al.(Hoadley et al., 2014) acquired five different data types (DNA methylation,
proteomics data, mRNA expression etc.) for 3,527 tumor specimens and clustered each data
type separately. They then clustered the samples using the cluster assignments for each sample
in what they dubbed a “cluster of cluster assignments” approach. This approach avoided some
of the challenges associated with data normalization, since each data type is clustered separately
and only cluster assignments are used in the final clustering. The clustering allows identification
of subtypes that share common genetic mutations and over-expressions of genetic pathways.
Zhang et al.(Zhang et al., 2012) applied a different approach of joint non-negative matrix

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factorization (NMF) algorithm find subsets of the variables in a combination of DNA
methylation, RNA expression and microRNA expression data to identify distinct patient
subgroups and association between different omic activities across a 385 datasets of ovarian
cancer patients.

Machine learning approaches were applied extensively in cardiology to interpret clinical data
such as electrocardiogram, and echocardiography. Machine learning algorithm streamlines the
management of human resources by supports the performance of repetitive tasks. Supervised
learning have shown to accurately identify constrictive physiologies that can lead to coronary
artery diseases using echocardiogram.(Sengupta et al., 2016) In some cases, machine learning
algorithms outperformed traditional prognostic indicators.(Motwani et al., 2016; Raghunath et

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al., 2020) Machine learning analysis of biomarkers from proteomic data have begun to relate
protein profiles to risk of coronary artery diseases.(Bom et al., 2019) Supervised machine

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learning was used to identify panel of plasma proteins for predicting cardiovascular
event.(Williams et al., 2019)
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These are merely a few examples of many applications of machine learning to the analysis of
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multiomics datasets and suggest considerable potential as yet remains untapped. Incorporation
of gFET data with multiomics data from other sources will offer both unique challenges and
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rewards. The natural area of application for gFETs would therefore appear to lie in studies
where a panel of analytes of interest must be monitored. Here, the anticipated reduction in cost,
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elimination in variability caused by complex sample prep and increase in sample throughput
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for gFETs may prove beneficial. For a study of this kind (Fig 5), a multiplexed gFET should be
functionalized with a panel of receptors or antibodies specific to the analytes of interest, with
one receptor or analyte per transistor, so that each analyte can be tracked separately. The
background-subtracted signal could be used for statistical analysis or as input to a machine
learning model. It is important to note that data acquired using different technologies, such as
read counts from RNASeq studies, change in response from baseline for a gFET, or peak areas
from LC-MS metabolomics studies will generally require standardization so that each data type
is on the same scale. The most appropriate normalization for each data type and across multiple
analytes may require careful consideration. The electronic signatures from multiplex gFET can
be easily inspected for anomalies to quality control the measurement dataset. The measurement
dataset is combined with the subject data to generate features. Training of the machine learning
model will be conducted on a subset of features to derive model hyperparameters that produce
sensitive prediction on the training dataset with appropriate cross validations. Multiple models
26
are train to identify an optimal set of features that informs the subject conditions that guides the
grouping of possible outcomes. The resulting models are then validated against a separate test
dataset to gauge the performance. The validated model exhibiting the highest sensitive and
specific prediction of outcome will be deployed to analyze the entire multiomics dataset for the
biological information.

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Fig. 5. The process flow for analyzing multiplex gFET data including (A) preparation of normalized and
standardized electronic data from calibrated multiplex gFET sensors, (B) Training and validation of machine
learning predictive model on a training dataset, and (C) apply prediction on the large scale multiomics dataset.

As discussed, finding actionable insights into complex system biology requires large datasets
that are scarce and expensive. These limitations are the result of scalability and interoperability
of current optical multiomics platform technologies. While each individual omics platform is

27
optimized to generate large quantity of data, interpretations of information in context of system
biology requires domain expertise across different fields that are scarce to address the
heterogeneity and irregularities across datasets.(Miotto et al., 2018) Efforts to develop learning
algorithm to address these needs have made tremendous progress but challenges like data
heterogeneity and relatively small sample size still remain.(Haas et al., 2017) The development
of gFET sensing technologies will accelerate this progress by providing a scalable solution to
integrative measurements across multiple omics domains.

6. Multiplexing, Scaling up and commercialization of gFET

As electronic devices, the potential of gFETs multiplexing capability coupled with machine
learning algorithms is very promising in pushing the boundary of our understanding of biology

f
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in context. The development of gFET design inherited a wealth of experience from well over
30 years of BioFET development. Measurements of over 100 sensors on a chip have been done

r
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at the proof of concept level.(Lerner et al., 2014) The remaining challenge is consistent design
of functionalization interface to address the multitude of analytes in multiomics studies, both in
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the fluidic handling(Khan et al., 2020; Wang et al., 2019; Xu et al., 2014) and surface
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chemistry.(Béraud et al., 2021) Maintaining a consistent device design as a fundamental

platform will be imperative to obtaining reproducible and optimized bioreceptor
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functionalization interfaces.

Above the device design perspective, the promise of gFET as a unifying platform for multiomic
ur

studies cannot be fulfilled if challenges with access to gFET based sensing systems are not
Jo

addressed. Typically, this is discussed as manufacturability or commercialization in the

literature. These terms are stand-ins for the simple question of how a typical biology researcher
acquires access to gFET technology. The previously described opportunities for multiomics
applications have encouraged enthusiasm for broad device development in nanotechnology
research environments. However, proof of concepts alone is not sufficient to impact on the
world unless they are paired with accessibility at commercial scale. This requirement remains
a challenge toward commercial adoption of many nanomaterials as the upscaling of reliable
manufacturing requires organization of nanomaterials across nine orders of magnitude in spatial
dimension. Carbon nanotubes and silicon nanowires have a research history nearly 10 years
older in FET based biosensing.(Syu et al., 2018; Zhang and Lieber, 2016) significant attentions
to address challenges in device assembly and material synthesis are still required to achieve
commercial scalability.

28
Graphene oxide, reduced graphene oxide, (Justino et al., 2017; Peña-Bahamonde et al., 2018)
and graphene nanoflakes (Labroo and Cui, 2014; Muralidharan et al., 2020; Wisitsoraat et al.,
2017) has gained interest as a 2-dimenstional nanomaterial for the manufacturability and
compatibility to a wide range of chemistry. Alternatively, coating or printable ink containing
graphene nanoflakes further improves the scalability of manufacturing. Graphene oxide is a
liquid phase precursor material for obtaining graphene, the electrical quality of the material is
sensitively dependent on the reduction of oxygen functional groups after it is casted on the
substrate. Since no method can complete remove the oxygen functional groups, the material
impurity results defects that degrades the electrical properties.(Pei and Cheng, 2012) While the
simplicity of liquid casting in the fabrication is an attractive feature, the material performance

f
exhibits high sensitivity to batch to batch variations of the liquid precursor and the process

oo
conditions. Further development is required to establish an industry standard for quality control

r
standards necessary to support large scale multiomics studies and the eventual
commercialization.
-p
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The significant factor that enabled the tremendous growth of gFET was the manufacturability
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that allowed greater access to gFETs for researchers and engineers outside of nanotechnology.
na
ur
Jo

Fig. 6. Major advances in commercialization of graphene-based technology toward accessible multiomics

applications from initial discovery, toward electronic grade material, to integration of commercially accessible
devices, and future expansion to multiomics digital biosensing devices.

Graphene sensors have been fabricated at small scale since shortly after the isolation of
graphene by Geim and Novosolov in 2004.(Novoselov et al., 2004) However, manufacturing
of graphene biosensors in a commercial environment only began in earnest ten years later.
Synthesizing electronics quality graphene reproducibly in large quantities had been heavily

29
investigated in the first decade after graphene’s discovery and continues to improve production
scalability while maintaining quality. The most cost effective method of creating electronics
grade graphene has been chemical vapor deposition (CVD).(Kidambi et al., 2018; Kobayashi
et al., 2013; Polsen et al., 2015; Yamada et al., 2012) There are now several commercial
foundries offering services appropriate for nanotechnology researchers(Goldsmith et al., 2019)
and its influences is already being felt in biotechnology (Fig 6). Several commercial tools are
available to perform CVD production with attention of establishing quality control procedures
to drive the commercial adoption of the material.(Deng et al., 2019; Kong et al., 2019) The
outstanding mechanical, electrical properties and compatibility of the production process with
flexible substrates attracted attentions of the electronic industries and several commercial

f
ventures are adopting graphene via roll-to-roll processing for touch screen applications. The

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cost per area for graphene is now below the cost per area for silicon wafers.(Goldsmith et al.,

r
2019) The second hurdle in graphene manufacturing is removing the graphene from its growth
-p
substrate and transferring it to the target substrate, often a silicon wafer. Several non-destructive,
re
low contamination solutions are available, including thermal release tape, pressure sensitive
adhesives, and bubbling transfer.
lP

Although scalable production of gFETs is a crucial step toward increased access and
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commercialization, additional efforts are needed to address challenges to translate the

biosensing research from the laboratory to practical solutions for multiomics applications. To
ur

make these transistors truly usable by non-experts, an entire system of electrical instrumentation
is required to translate the measured raw signals to high level physical parameters. This requires
Jo

layers software development from low level firmware to a user interface, including tools for
experimental design, and organization of experimental data with appropriate annotation of
protocols. Early examples of such systems exist, although not yet capable of multiomics
studies.(Goldsmith et al., 2019)

Extensive and reproducible multiomics studies must be conducted using gFETs to establish
confidence and credible reputations that will facilitate the adoption of this new paradigm shift
in biosensing. Further, development of machine learning models and intelligent analytics is
crucial to facilitate access to biological insight from extensive data generated from the
multiomics studies.

7. Conclusion

30
Multiomics is a challenging idea. Gaining understanding of biology at a system level based on
combining multitudes of simultaneous measurements including pH, mRNA, DNA, and enzyme
activity is going to require far more than “simply” fully realizing gFETs as a multiomics
measurement platform. To understand and overcome the experimental challenges necessary to
standardize and democratize multiomics, we need tools to facilitate more experimentation,
more data, and more creativity.

Many popular biosensing platforms have been commercially available for decades, with over
50 years of developmental histories. In comparison, gFETs have only recently gained attention
as biosensing platform. A tremendous amount of progress has been made that is evident in the
change in the character of gFET sensing literature. Classical gFET literature commonly applied

f
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a large number of samples to a small number of test chips in a repeated fashion. This was
necessary because of the difficulty in constructing a working sensor. Recently, there have been

r
a few studies that follow the statistical conventions of biology research and apply a smaller
-p
number of samples to a larger number of test chips.(Afsahi et al., 2017; Hajian et al., 2019a;
re
Qvit et al., 2016; Sadlowski et al., 2018; Sandoval et al., 2020) The amount of data not only
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support the statistical significance of the final measurements but also provide quality control
measures to guide assay development.(Balderston et al., 2021b) This is a subtle but significant
na

difference. Device designs and analysis are standardizing, while gFET production on a small
scale is maturing. (Goldsmith et al., 2019; Lerner et al., 2017; Ping et al., 2016)
ur

In the coming few years, we expect to see a transition in proof-of-concept studies using gFETs
Jo

from testing a single analyte per sample to testing multiple analytes per sample simultaneously.
The challenges will be the consistent and scalability of functionalizing multitudes of
bioreceptors on the multiplex sensor. The distinguishing characteristic should be that gFETs
can monitor protein, small molecule, and genomic activity together at the same time. This, at
last, would indicate a transition from “promise” to reality for gFETs as a multiomics platform.

Acknowledgements

We acknowledge Dr. Juan Josè Ripoll for helpful advice on multiomics concept
illustrations. Schematics in Fig 1-6 were created with BioRender.com. Funding: This work
was supported by DARPA: HR00112190060, NSF: 2048283, and Cardea sponsored research
to Aran lab.

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Highlights:
 Graphene based field effect transistor (gFET) is a promising platform for democratization
of multiomics analysis.
 The outstanding characteristics of the graphene field effect biosensing are contrasted with
the conventional field effect biosensing.
 A wide range of bioanalysis by gFET, including DNA/RNA, proteins, metabolites,
vesicles, and cellular activities are highlighted.
 gFET simultaneously acquire multiple omics measurements that are readily accessible to
digital analysis by machine learning.
 The scalability of gFET manufacturing by the semiconductor industry further accelerate

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the democratization of multiomics studies.

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Original:

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A scalable multiomics analysis platform is becoming essential with the increase of our
understanding of biology, both in the functional components and the complex interactions
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between them. This review highlights the promise of graphene based electronics to democratize
the access to multiomics analysis. Graphene-based biosensors can bridge the gap between
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modern electronic and biology, which is the future of medicine. The topic of graphene field
effect biosensing is the main focus of the review article as these biosensors have gained much
attention as a promising technology for sensitive analysis of a wide varieties of analyte important
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for understanding of system biology. This review illustrates the versatility by summarize the
innovative works for label-less electronic detection of DNA/RNA, proteins, metabolites,
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vesicles, and cellular activities. With recent progress in understanding of the interfacial physics,
the platform demonstrated capability of performing the analysis from physiologically relevant
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biofluids. Aside from their impressive technical characteristics, the review makes the case for the
technology as an integrative multiomics platform that can address the current challenges of data
analysis by acquiring multiple omics measurements simultaneously that are readily analyzable to
machine learning models. In addition, the attractive potential of these biosensors to leverage the
scalability offered by the semiconductor manufacturing provides a widespread access that can
accelerate the progress of multiomics study for system biology.
Declaration of interests

☐ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☒The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

The authors, Kiana Aran, Brett Goldsmith, and Michael Heltzen are co-founders of Cardea Bio Inc., whose
mission is to commercialize accessible graphene electronic platform for biological sensing, declare
potential conflict of financial interest in the development of multiomics biosensing platform as discussed
in this review manuscript, but have prepared the manuscript with unbiased opinions and breath of
credited references.

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