of 35
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SARS-CoV-2 RapidPlex: A Graphene-based Multiplexed Telemedicine Platform for
Rapid and Low-Cost COVID-19 Diagnosis and Monitoring
Rebeca M. Torrente-Rodríguez, Heather Lukas, Jiaobing Tu, Jihong Min, Yiran Yang,
Changhao Xu, Harry B. Rossiter, Wei Gao
PII:
S2590-2385(20)30553-1
DOI:
https://doi.org/10.1016/j.matt.2020.09.027
Reference:
MATT 460
To appear in:
Matter
Received Date:
1 September 2020
Revised Date:
20 September 2020
Accepted Date:
29 September 2020
Please cite this article as: Torrente-Rodríguez RM, Lukas H, Tu J, Min J, Yang Y, Xu C, Rossiter
HB, Gao W, SARS-CoV-2 RapidPlex: A Graphene-based Multiplexed Telemedicine Platform for
Rapid and Low-Cost COVID-19 Diagnosis and Monitoring,
Matter
(2020), doi:
https://doi.org/10.1016/
j.matt.2020.09.027
.
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SARS-CoV-2 RapidPlex: A Graphene-based Multiplexed
Telemedicine Platform for
Rapid and Low-Cost COVID-19 Diagnosis and Monitorin
g
Rebeca M. Torrente-Rodríguez,
1,3
Heather Lukas,
1,3
Jiaobing Tu,
1
Jihong Min,
1
Yiran Yang,
1
Changhao Xu,
1
Harry B. Rossiter,
2
Wei Gao
1,4,*
1
Andrew and Peggy Cherng Department of Medical Engin
eering, California Institute of
Technology, Pasadena, California, 91125, USA
2
Rehabilitation Clinical Trials Center, Division of
Respiratory and Critical Care Physiology
and Medicine, The Lundquist Institute for Biomedica
l Innovation at Harbor-UCLA Medical
Center, Torrance, California, 90502, USA
3
These authors contributed equally to this work
4
Lead contact
*Email: weigao@caltech.edu
SUMMARY
The COVID-19 pandemic is an ongoing global challeng
e for public health systems.
Ultrasensitive and early identification of infectio
n is critical to prevent widespread COVID-
19 infection by presymptomatic and asymptomatic ind
ividuals, especially in the community
and in-home settings. We demonstrate a multiplexed,
portable, wireless electrochemical
platform for ultra-rapid detection of COVID-19: the
SARS-CoV-2 RapidPlex. It detects viral
antigen nucleocapsid protein, IgM and IgG antibodie
s, as well as the inflammatory biomarker
C-reactive protein, based on our mass-producible la
ser-engraved graphene electrodes. We
demonstrate ultrasensitive, highly selective, and r
apid electrochemical detection in the
physiologically relevant ranges. We successfully ev
aluated the applicability of our SARS-
CoV-2 RapidPlex platform with COVID-19 positive and
negative blood and saliva samples.
Based on this pilot study, our multiplexed immunose
nsor platform may allow for high
frequency at-home testing for COVID-19 telemedicine
diagnosis and monitoring.
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INTRODUCTION
On March 11, 2020, the World Health Organization ch
aracterized the COVID-19 outbreak as
a pandemic. Six months later, the global health cri
sis had continued with over 30 million
confirmed cases of novel coronavirus globally – ove
r 22% of these were in the United
States.
1
It is estimated that 14-20% of patients will devel
op severe illness requiring
hospitalization.
2
Initial efforts to mitigate the spread through sta
te-mandated “stay-at-home”
orders appeared effective, however, reopening of th
e US economy resulted in renewed
exponential spread of novel coronavirus, as predict
ed.
3
It is estimated that the US GDP will
suffer losses upwards of $45.3 billion during a flu
-like pandemic without available vaccines.
4
Safe reopening of the economy, schools and universi
ties requires multiple approaches to
mitigate the risks associated with COVID-19, includ
ing simple, affordable and effective test-
and-trace measures.
Containing the spread is difficult due to the chall
enges in identifying infectious individuals.
Most COVID-19 community spread may occur in the abs
ence of symptoms. Peak viremia
may be at the end of the incubation period, allowin
g for a transmission-sufficient viral load 1-
2 days prior to symptom onset.
3
Additionally, due to the unknown duration and prev
alence of
asymptomatic cases, the true reproduction number ma
y be under-estimated.
5,6
Reported
incidence of asymptomatic patients ranges from 17.9
% to 30.8%.
7,8
Increased access to COVID-19 testing has allowed in
creased monitoring of the community
spread, but several diagnostic challenges remain. C
urrently, the standard testing method is
viral nucleic acid real-time polymerase chain react
ion (RT-PCR), which is a slow
process
9
and requires expensive equipment and trained techn
icians for nasopharyngeal swab
sample collection and analysis.
9
In addition, the sheer volume of testing required
is
overwhelming the ability for healthcare systems to
report RT-PCR results to patients, causing,
in some states, delays of ~7-10 days to inform posi
tive
10
findings and enact isolation and
monitoring protocols. Despite the recent advances o
n point-of-care (POC) rapid RT-PCR
test,
11–15
nucleic acid tests are also known to produce false
negatives, which may limit
containment strategies and access to treatment.
16
An additional consideration for RT-PCR is
that it only identifies active carriers of the viru
s. Identifying convalescent persons based on
COVID-19 antibody presentation is equally important
as it may provide health officials with
crucial information regarding the potential impact
of reopening measures.
17
Serologic assays
detect circulating antibodies specific to SARS-CoV-
2 antigens, including the nucleocapsid
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protein and the outer spike protein.
9,18
However, it is not possible to differentiate betwe
en
asymptomatic carriers and immune persons using anti
body detection. Therefore, to
effectively mitigate the risks of COVID-19 communit
y spread, systems are required that
determine simultaneously both the viral and serolog
ic status of an individual. Moreover,
recent studies show correlation between circulating
inflammatory biomarker concentration
and COVID-19 severity.
19
Increased C-reactive protein (CRP) concentration i
s found in
patients diagnosed with COVID-19 pneumonia and is a
ssociated with increasing
severity, suggesting a role in diagnosis and progno
sis of COVID-19 patients.
20,21
There is a clear and urgent need for a highly sensi
tive, rapid, inexpensive, telemedicine
COVID-19 test that can identify a patient’s past an
d present infection status.
22
There has been
progress towards POC COVID-19 testing, but all comm
ercially available test kits provide
only qualitative results. Quantitative analysis of
COVID-19 biomarkers using a telemedicine
device may provide predictive information of diseas
e severity and provide seroconversion
information regarding disease time course. Electroc
hemical biosensors, in this regard, are
advantageous due to their rapid detection efficacy
and ease of use for POC applications.
23–27
Simple, safe and effective COVID-19 sample collecti
on has proved challenging given current
assay requirements. Saliva-compatible POC assays wo
uld be advantageous since saliva
contains rich information and can be easily and non
-invasively collected by patients
themselves for telemedicine testing.
28
Here, we present a novel multiplexed, portable, wir
eless electrochemical platform for ultra-
rapid detection of COVID-19: SARS-CoV-2 RapidPlex (
Figure 1
). This platform
quantitatively detects biomarkers specific to COVID
-19 in both blood and saliva including
SARS-CoV-2 nucleocapsid protein (NP), specific immu
noglobulins (Igs) against SARS-
CoV-2 spike protein (S1) (S1-IgM and S1-IgG), and C
RP, within physiologically relevant
ranges. The platform uses capture antigens and anti
bodies immobilized on mass-producible,
low-cost laser-engraved graphene (LEG)
29,30
electrodes. This multiplexed platform tracks the
infection progression by diagnosing the stage of th
e disease, allowing for the clear
identification of individuals who are infectious, v
ulnerable, and/or immune (
Table 1
). The
main features of SARS-CoV-2 RapidPlex are high sens
itivity, low cost, ultra-fast detection,
wireless remote and multiplexed sensing that provid
es information on three key aspects of
COVID-19 disease: viral infection (NP),
31
immune response (IgG and IgM),
9
and disease
severity (CRP).
19–21
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RESULTS AND DISCUSSION
Design of the SARS-CoV-2 RapidPlex Platform
As illustrated in
Figure 1A
, SARS-CoV-2 RapidPlex is composed of four graphene
working
electrodes (WEs), a Ag/AgCl reference electrode (RE
), and a graphene counter electrode
(CE), all of them patterned on a polyimide (PI) sub
strate via CO
2
laser engraving, a fast,
high-throughput, and cost-effective production meth
od (
Figure 1B
and
1C
). Our group has
recently demonstrated the use of mesoporous graphen
e structure fabricated by laser
engraving for high performance and low-cost biosens
ing.
29,30
The materials cost for the
unmodified RapidPlex platform is within $0.05; addi
tional chemical and reagent costs for the
multiplexed sensor preparation are at the level of
dollars depending on the order sizes.
Detection of selected target proteins (NP and CRP)
and specific immunoglobulins (S1-IgG
and S1-IgM) is achieved through sandwich- and indir
ect-based immunosensing strategies
onto the LEG electrodes, respectively. The superior
properties of graphene, in terms of high
charge mobility and surface area together with the
high sensitivity and selectivity of sensing
strategies involving both capture and detector rece
ptors, make our device (
Figure 1D
) a
highly convenient tool for the rapid, accurate, and
stage-specific COVID-19 infection
detection in blood as well as in non-invasive biofl
uid samples, such as saliva.
Electrochemical Characterization of SARS-CoV-2 Rapi
dPlex Platform
Functionalization and modification steps carried ou
t on the LEG surfaces for the covalent
attachment of each of the specific receptors requir
ed for the development of our SARS-CoV-
2 RapidPlex platform is schematized in
Figure 2A
. 1-pyrenebutyric acid (PBA) is used as the
linker to anchor the required receptors to the grap
hene layer. Although attachment of
functional groups directly on the sp
2
carbon atom surface is one of the common ways to
functionalize graphene, these methods are associate
d with the requirement of defects or edges
in the sensor material, which could alter its speci
fic physical properties.
32,33
In contrast,
introduction of functional groups on the sensing la
yer by means of pyrene derivatives is
preferred here as it does not disrupt the conjugati
on of the graphene sheets and improves its
stability.
34,35
PBA consisting of a pyrene group that contains
π
-electrons and a carboxylic
group is used to functionalize graphene layers via
π
-stacking and hydrophobic interactions.
The pyrene units of PBA strongly interact with grap
hene layers in the way that original
structure and properties of the graphene are well m
aintained. The functional moieties
contained in each PBA molecule allow the preparatio
n of the affinity-based biosensing
platform through the covalent coupling between the
carboxylic groups on PBA units and the
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–NH
2
groups of the respective capture receptors (specif
ic antibodies or capture proteins).
Blocking of unreacted sites with bovine serum album
in (BSA) impedes the non-specific
adsorption of other molecules involved in each assa
y configuration or circulating in the
sample of interest.
Differential pulse voltammetry (DPV) and open circu
it potential-electrochemical impedance
spectroscopy (OCP-EIS) techniques are employed to e
lectrochemically characterize and
prove the stepwise self-assembled processes in both
assay configurations for the detection of
selected target molecules. DPV readings reflect low
er peak current intensity after each
modification step related to S1-Ig assay due to the
hindered diffusion of the redox label to the
electrode surface derived from both the carboxyl gr
oups and the attached proteins and
biological macromolecules (
Figure 2B
). At the same time, resistance in the Nyquist plot
s
from OCP-EIS is increased after each functionalizat
ion step (
Figure 2C
). The successful
anchorage of PBA was also verified with scanning el
ectron microscopy (SEM) (
Figure S1
).
Electrochemical characterization of the sandwich as
say-based sensor modification using CRP
protein as a model molecule and the aforementioned
techniques are presented in
Figure S2
.
To preserve the native structure and properties of
the bound biomolecules, PBA was chosen
as a heterobifunctional linker, effectively prevent
ing the direct interaction between large
biomolecules and the graphene surface.
33
In order to verify this selection, CRP and SARS-
CoV-2 specific IgG assay configurations were constr
ucted on graphene electrodes
functionalized with PBA and another common linker,
1H-pyrrole-1-propionic acid (PPA).
30
Greater signal-to-blank (S/B) ratios were observed
for both assays where PBA was used as a
linker support (
Figure 2D
), mainly due to a significant decrease in the sign
als obtained in the
absence of the corresponding target molecule when P
BA was used instead of PPA. Together
with an optimal blocking strategy, PBA can be used
for the immobilization of specific
biomolecular probes (e.g. antibodies, proteins, etc
.) while avoiding non-specific adsorptions
in the context of immunoassays.
36
The orientation of modified antigenic proteins on s
olid surfaces is strongly associated with
their activity and reactivity. Specific anti-His an
tibodies can be used to orient the
immobilization of antigenic receptors containing hi
stidine residues, but this implies an
additional step compared with their direct attachme
nt on the sensing layer, as it is
schematized in
Figure S3A
. Our results show no significant differences in as
say performance
for IgG detection on PBA-graphene electrodes covale
ntly functionalized with the specific
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coating protein (direct immobilization) and with an
ti-His antibodies for the previous capture
of the polyhistidine-tag specific coating protein (
oriented immobilization) (
Figure S3B
),
proving that random protein orientation does not in
terfere with the epitope accessibility for
effective recognition by specific target antibodies
. This is in agreement with other reports
confirming that His-tagged fusion antigens can be d
irectly immobilized on different surfaces
with protein orientations completely compatible wit
h antibody recognition.
37–40
In order to
simplify and reduce the cost and time of the assay,
direct immobilization of S1 protein was
carried out for specific Ig detection.
Considering that rapid target binding is essential
to the successful implementation of our
proposed platform as a POC system, we investigated
how target (or sample) incubation time
affects the response of each biosensor comprising o
ur SARS-CoV-2 RapidPlex platform.
Figure 2E
summarizes the amperometric signals obtained for e
ach of the four sensing units
at different incubation times (1, 5 and 10 minutes)
in the absence (blank, B) and in the
presence (S) of 500 pg mL
-1
, 250 ng mL
-1
, and 50 ng mL
-1
of NP, SARS-CoV-2 specific IgG
and IgM isotypes, and CRP, respectively. It is impo
rtant to note that although a 10-minute
incubation time was selected for most of the studie
s here in order to ensure the highest
sensitivity for the determination of ultra-low leve
ls of each target molecule, a significant
difference between the absence and the presence of
each of the corresponding targets is
obtained with just 1-minute incubation time. This p
rovides one of the major advantages of
our SARS-CoV-2 RapidPlex system as a rapid POC devi
ce for SARS-CoV-2 infection
monitoring with the required sensitivity for both p
rotein and Ig determination. ELISA,
17,41–44
nucleic acid amplification,
45–49
mass spectrometry,
50
or even combinations
51
have been
reported very recently for determination of the pro
posed SARS-CoV-2 specific target
molecules, among others. However, most of these met
hods show crucial pitfalls, mainly in
terms of sample preparation, complexity, and expens
ive and bulky equipment requirements,
that make them still highly difficult to be impleme
nted as POC systems. Our device provides
an attractive alternative to standard assays for pr
otein determination, such as ELISA, because
of its multiplexing capabilities, remote functional
ity and short sample-to-answer time.
Evaluation of Analytical Performance of the SARS-Co
V-2 RapidPlex
The performance of each biosensor contained in the
SARS-CoV-2 RapidPlex was
characterized in phosphate-buffered saline (PBS) so
lutions supplemented with 1.0% of
bovine serum albumin (BSA) by measuring the amperom
etric readout in the presence of
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increased concentrations of NP, S1-IgG, S1-IgM, and
CRP (
Figure 3
). The selected
strategies for NP viral antigen and CRP proteins ar
e based on double sandwich and sandwich
configurations, respectively, as illustrated in
Figure 3A
. The sandwich-based immunoassays
for antigen detection are, in general, highly sensi
tive due to the involvement of two different
antibodies as capture and detector entities. Accord
ing to the low levels that must be reached
for NP and CRP in diluted serum and saliva (pg mL
-1
to ng mL
-1
), we think these strategies
are the most suitable to be implemented on our plat
form. Variation of cathodic currents with
the concentration for NP and CRP in buffered soluti
ons are presented in
Figure 3B
and
3C
,
respectively. S1-IgG and S1-IgM were detected based
on indirect immunoassays (
Figure 3D
),
which are considered highly suitable for detection
of circulating macromolecules in antisera
and other biofluids.
Figure 3E
and
3F
show the calibration curves for S1 specific Ig
determination (S1-IgG and S1-IgM, respectively) in
buffered solutions. Reproducibility was
demonstrated through the relative standard deviatio
n (RSD) values obtained with different
biosensors prepared in the same manner on different
days. RSD values of 6.3%, 8.4%, 6.0%
and 7.6% for 20 ng mL
-1
CRP, 250 ng mL
-1
S1-IgG, 250 ng mL
-1
S1-IgM and 500 pg mL
-1
NP antigen (n=5) demonstrate good reproducibility i
n both device preparation and signal
transduction.
In addition, the sensors showed stable responses ov
er a 5-day storage period at
4
°
C (
Figure S5
). We did not observe significant slope variations
between data obtained in
properly diluted human serum and in buffered soluti
ons for the determination of each target
analyte (for instance, the slope sensitivity value
(16.28 nA mL ng
-1
) obtained for CRP as
model analyte in PBS buffered solutions is nearly t
he same as that in diluted serum samples
from a healthy volunteer (16.64 nA mL ng
-1
)); therefore, accurate quantification of the
proposed target analytes can be carried out by cond
ucting a simple interpolation of the
cathodic readings obtained for each sample tested i
n the corresponding calibration curve
constructed in buffered solution.
Since diagnostic sensitivity and specificity of ser
oprevalence studies can be improved by
using a mixture of antigenic proteins instead of a
single protein,
52,53
we modified graphene
with a mixture of SARS-CoV-2 related antigens, NP a
nd S1, to capture specific
immunoglobulin isotypes against both antigens in th
e same WE. A calibration curve for (NP
+ S1)-IgG detection is shown in
Figure S4
. Thus, this methodology can be tailored for
detecting isotype-specific IgG (or IgM) or a combin
ation of both Ig isotypes in the same
sensing surface to better capture total Ig concentr
ation and thus increase assay sensitivity
across the patient population.
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Investigation of the Selectivity and Multiplexed Pe
rformance of the SARS-CoV-2
RapidPlex
Human biofluids contain a complex and variable mixt
ure of circulating molecules that could
interfere with molecular sensing. In addition, negl
igible crosstalk between different working
surfaces is an essential requirement to perform mul
tiplexed detection readings accurately and
meaningfully. Therefore, selectivity and crosstalk
of the SARS-CoV-2 RapidPlex platform
were evaluated. Amperometric readings obtained for
each developed biosensor against non-
target molecules are presented in
Figure 4A
. We evaluated the specific binding for SARS-
CoV-2 biomarkers in comparison to biomarkers of sim
ilar coronaviruses, including SARS-
CoV and MERS-CoV. We observed no significant cross-
reaction for NP, S1-IgG, S1-IgM
and CRP assays in the presence of each tested inter
ferent, including SARS-CoV-2 S1, SARS-
CoV S1, and CRP (for NP assay), SARS-CoV-2 NP-IgG,
SARS-CoV IgG, MERS-CoV IgG,
S1-IgG, and negative controls containing mixtures o
f IgG and IgM against both MERS-CoV
and SARS-CoV (for S1-IgG and S1-IgM assays), and BN
P, NP, SARS-CoV NP and SARS-
CoV S1 (for CRP assay), respectively. However, SARS
-CoV NP viral antigen interferent
provided a cathodic current corresponding to ~80% o
f the raw current obtained for the
detection of the specific NP antigen. Spike, envelo
pe, and membrane SARS-CoV-2 proteins
share 76-95% sequence identity with those of SARS-C
oV. This percentage homology is
reduced to 30-40% for MERS-CoV. Similarly, since SA
RS-CoV-2 NP is 90% identical to
SARS-CoV NP,
17,54–56
the interference observed from SARS-CoV NP antigen
was expected.
However, the lack of selectivity in this particular
case is not a major concern due to the
absence of new SARS-CoV cases detected recently; th
erefore, it can be inferred that this
interference will not produce a barrier for selecti
ve SARS-CoV-2 NP determination in real
samples. We further evaluated the amperometric-deri
ved concentrations with absorbance-
derived concentrations collected via ELISA. As it i
s presented in
Figure 4B
, the results from
our functionalized electrochemical biosensor were l
inearly correlated (r = 0.955) with the
results using the same reagents in a traditional EL
ISA protocol.
Once the performance and selectivity of each constr
ucted biosensor was individually and
exhaustively evaluated, we demonstrate the multiple
xing capabilities of our four-working-
electrode (4WEs) graphene array device designed wit
h a Ag/AgCl RE and a graphene CE.
The block diagram showing the functional units that
comprise the integrated electronic
system is illustrated in
Figure 4C
and
4D
. Amperometric readings from the four channels are
concurrently taken and data is wirelessly transmitt
ed to a user device over Bluetooth Low
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Energy. The electronic system, including the printe
d circuit board (PCB) and a lithium-ion
polymer battery, is 20 × 35 × 7.3 mm in dimension.
The compact device can perform
amperometric measurements continuously for over 5 h
ours in a single charge.
With the objective of demonstrating the utility of
our SARS-CoV-2 RapidPlex array for
multiplexed and simultaneous quantification of sele
cted target molecules, we evaluated the
potential cross-reaction resulting from the diffusi
on of signal substances between adjacent
immunosurfaces. For this, each of the four convenie
ntly functionalized working surfaces
were incubated with buffered solutions containing s
ignificantly high concentration of each of
the selected targets, followed by the corresponding
detector receptors in each case. The
absence of cross-talk between the adjacent working
electrodes is verified from the
experimental readings in buffered solutions contain
ing 1.0 ng mL
-1
NP antigen (I), 250 ng
mL
-1
S1 specific IgG (II) and -IgM (III), and 50 ng mL
-1
CRP (IV) (
Figure 4E
). As
envisaged, significantly higher signal was obtained
when each target was specifically
captured and further labeled by its tracer antibody
in the corresponding functionalized
immunosurface. These results, in conjunction with t
hose from
Figure 4A
demonstrate the
feasibility of the developed SARS-CoV-2 RapidPlex p
latform for fast, selective and reliable
determination of NP, S1-IgG and S1-IgM isotypes, an
d CRP in one single experiment. It
should be noted that since IgG and IgM have similar
binding mechanisms to viral antigens
and individual quantification of Igs require no mix
ing of the specific detector labels,
individual droplets were used on IgG and IgM sensin
g electrodes during modification and
labelling.
Detection of SARS-CoV-2 Related Selected Targets in
Human Biospecimens
To prove the utility of our device in a more comple
x and real scenario, we evaluated the
multiplexed capabilities of SARS-CoV-2 RapidPlex in
representative serum samples from
COVID-19 RT-PCR negative and positive subjects. Sen
sor data from the serum samples of a
RT-PCR negative subject (
Figure 5A
) and a RT-PCR positive patient (
Figure 5B
) show
minimal cross-talk in a real and complex sample mat
rix, indicating the efficient functionality
of SARS-CoV-2 RapidPlex to simultaneously different
iate the overexpressed presence of
SARS-CoV-2 related target reporters in COVID-19 pos
itive specimens. Moreover, the
SARS-CoV-2 RapidPlex device is able to provide sign
ificant positive readings for all targets
after incubating the COVID-19 positive serum sample
for just 1 minute (
Figure 5C
and
Figure S5
): The maintained high signal in positive patient s
amples demonstrates the great
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potential in future translation of the SARS-CoV-2 R
apidPlex device as an ultra-fast POC
remote diagnostic tool.
To further investigate NP, S1-IgG, S1-IgM, and CRP
response to SARS-CoV-2 infection
using our LEG-based biosensors, each target molecul
e was measured in serum and saliva
samples from RT-PCR confirmed COVID-19 positive and
negative subjects. Obtained results
were plotted as the ratio between the amperometric
readings for each sample tested (S) and
the respective blank (B) in each case to compare ta
rget detection in different concentration
ranges. Using the graphene sensors, a total of 17 C
OVID-19 RT-PCR tested serum samples
(10 positive, 7 negative) were assayed, and a total
of 8 COVID-19 RT-PCR tested saliva
samples (5 positive, 3 negative) were analyzed (
Table S1
).
Results from
Figure 5D
and
5E
corroborate that, as expected, compared to RT-PCR n
egative
subjects, RT-PCR positive COVID-19 patients show si
gnificantly elevated levels of the
selected targets in both serum and saliva samples,
with median S/B ratios of 10.53, 11.62,
10.67 and 12.39 in serum, and 2.81, 3.24, 1.62, and
1.76 in saliva, for NP, S1-IgG, S1-IgM,
and CRP, respectively. We observed a concentration
of NP in the range of 0.1-0.8
μ
g mL
-1
and 0.5–2.0 ng mL
-1
in COVID-19 patient serum and saliva, respectively
; S1-IgG in the
range of 20–40
μ
g mL
-1
and 0.2–0.5
μ
g mL
-1
in COVID-19 patient serum and saliva,
respectively; S1-IgM in the range of 20–50
μ
g mL
-1
and 0.6–5.0
μ
g mL
-1
in COVID-19
patient serum and saliva, respectively; and CRP in
the range of 10–20
μ
g mL
-1
and 0.1–0.5
μ
g mL
-1
in COVID-19 patient serum and saliva, respectively
. The factor that all the positive
samples show much higher signals compared to negati
ve samples proves the real utility for
the accurate evaluation of the COVID-19 biomarkers
in biofluids using our LEG-based
biosensors. In particular, the observed significant
presence of COVID-19 biomarkers in
saliva demonstrates the great utility of this biofl
uid as a valuable source for non-invasively
diagnosing and monitoring SARS-CoV-2 infection.
With the aim to confirm the relationship between th
e levels of inflammatory biomarkers
involved in the cytokine storm directly associated
with disease progression, severity and
outcome in COVID-19,
57–62
we evaluated the variation of serum CRP levels in
RT-PCR
negative subjects (n=7) and RT-PCR positive COVID-1
9 patients who were classified
clinically according to disease severity as asympto
matic (n=2), mild (n=5), moderate (n=2).
As shown in
Figure 5F
, we observed a positive association between CRP co
ncentration and
COVID-19 symptom severity grade, consistent with th
e recent literature reports.
20,61
Future
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