Biosensors and Bioelectronics 171 (2021) 112686

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Biosensors and Bioelectronics

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

Magnetic beads combined with carbon black-based screen-printed

electrodes for COVID-19: A reliable and miniaturized electrochemical
immunosensor for SARS-CoV-2 detection in saliva
Laura Fabiani a, Marco Saroglia b, Giuseppe Galatà c, Riccardo De Santis d, Silvia Fillo d,
Vincenzo Luca d, Giovanni Faggioni d, Nino D’Amore d, Elisa Regalbuto d, Piero Salvatori e,
Genciana Terova b, Danila Moscone a, Florigio Lista d, Fabiana Arduini a, f, *
a
University of Rome “Tor Vergata”, Department of Chemical Science and Technologies, Via della Ricerca Scientifica, 00133, Rome, Italy
b
University of Insubria, Department of Biotechnologies and Life Sciences, Varese, Italy
c
GTS Consulting S.r.l., Via Consolare Pompea 1, 98168, Messina, Italy
d
Scientific Department, Army Medical Center, Rome, Italy
e
UOC, Army Medical Center, Rome, Italy
f
SENSE4MED, Via Renato Rascel 30, 00128, Rome, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: The diffusion of novel SARS-CoV-2 coronavirus over the world generated COVID-19 pandemic event as reported
Immunosensors by World Health Organization on March 2020. The huge issue is the high infectivity and the absence of vaccine
Screen-printed electrodes and customised drugs allowing for hard management of this outbreak, thus a rapid and on site analysis is a need
Carbon black
to contain the spread of COVID-19. Herein, we developed an electrochemical immunoassay for rapid and smart
Magnetic beads
detection of SARS-CoV-2 coronavirus in saliva. The electrochemical assay was conceived for Spike (S) protein or
Differential pulse voltammetry
Nucleocapsid (N) protein detection using magnetic beads as support of immunological chain and secondary
antibody with alkaline phosphatase as immunological label. The enzymatic by-product 1-naphtol was detected
using screen-printed electrodes modified with carbon black nanomaterial. The analytical features of the elec­
trochemical immunoassay were evaluated using the standard solution of S and N protein in buffer solution and
untreated saliva with a detection limit equal to 19 ng/mL and 8 ng/mL in untreated saliva, respectively for S and
N protein. Its effectiveness was assessed using cultured virus in biosafety level 3 and in saliva clinical samples
comparing the data using the nasopharyngeal swab specimens tested with Real-Time PCR. The agreement of the
data, the low detection limit achieved, the rapid analysis (30 min), the miniaturization, and portability of the
instrument combined with the easiness to use and no-invasive sampling, confer to this analytical tool high po­
tentiality for market entry as the first highly sensitive electrochemical immunoassay for SARS-CoV-2 detection in
untreated saliva.

1. Introduction 11 February, WHO named COVID-19 (COrona VIrus Disease 2019) the
disease due to SARS-CoV-2 and one month later WHO reported COVID-
The pandemic event of novel SARS-CoV-2 infection started on 31 19 as a pandemic outbreak, having impact all over the world.
December 2019 when Chinese health authorities reported to World In Europe, the President of European Commission reported as top
Health Organization (WHO) Country Office a pneumonia of unknown priority the safeguarding of the health and well-being of citizens by
cause detected in the city of Wuhan. Later, on 5 January 2020 WHO using any tool available by coordinating all Member States (https://ec.
published the first Disease Outbreak News on the new virus reporting europa.eu/info/live-work-travel-eu/health/coronavirus-response/pu­
the risk assessment and advice as well as the status of patients and the blic-health_en). Several countermeasures are taken to tackle this unex­
public health response on the cluster of pneumonia cases in Wuhan. On pected pandemic event including european strategy to accelerate the

* Corresponding author. University of Rome “Tor Vergata”, Department of Chemical Science and Technologies, Via della Ricerca Scientifica, 00133, Rome, Italy.
E-mail address: [email protected] (F. Arduini).

https://doi.org/10.1016/j.bios.2020.112686
Received 20 July 2020; Received in revised form 28 September 2020; Accepted 2 October 2020
Available online 3 October 2020
0956-5663/© 2020 Elsevier B.V. All rights reserved.
L. Fabiani et al. Biosensors and Bioelectronics 171 (2021) 112686

Fig. 1. The MBs-based assay for SARS-CoV-2 detection in untreated saliva.

Fig. 2. A) ELISA response for two different PAb anti-SARS-CoV-2 1 μg/mL (Sinobiological and ProSci) towards two different Spike proteins coated at 2 ng/mL. B)
Binding curve of colorimetric ELISA for MAb anti-SARS-CoV-2 ranging from 0.12 – 2 μg/mL. Coating of Spike protein: 2 ng/mL. C) Electrochemical response using
the MBs-based assay using CB-based modified electrode (blue line) and bare electrode (black line). The mean value (n = 3) with the corresponding standard deviation
was reported for each measurement. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

development, manufacturing, and deployment of vaccines against manufactured by Chembio Diagnostic Systems, reports that the detec­
COVID-19 and the establishment of the guidelines for high-quality tion of antibodies is carried out using Nucleocapsid protein as target
coronavirus test methodologies which encompasses the analytical (https://chembio.com/dpp-covid-19-igm-igg-system-ous-2/). VITROS
tools for detecting SARS-CoV-2 and those for measuring the immune Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack,
response of the human body to the infection (https://ec.europa.eu/in­ manufactured by Ortho-Clinical Diagnostics, Inc., is an ELISA test using
fo/live-work-travel-eu/health/coronavirus-response/public-health_en). Spike protein as target protein (Ward et al., 2020).
As highlighted by EU, one of the main pillars is related to analysis, Regarding the type of the above-mentioned kits, the EU reported in
indeed the fast and accurate detection represents a huge task for the 2020/C 122 I/01 that “The effectiveness of antibody tests in early COVID-
management of COVID-19, being the key point for identifying the 19 diagnosis is very limited because antibodies become detectable in the pa­
infected people, especially asymptomatic subjects, enabling the correct tient’s blood only several days after infection. This depends on the one hand
countermeasure in timely fashion. on the individual’s immune system and on the other hand on the sensitivity of
To evaluate the immune response of human body to the infection, the technique employed. In addition, antibodies persist for some time after the
different serological tests have been developed and are present in the infection has cleared. They do not give a definite answer on the presence or
market. For instance, qSARS-CoV-2 IgG/IgM Rapid Test is manufactured absence of the SARS-CoV-2 virus and thus they are not suitable to assess if the
by Cellex and it is a lateral flow chromatographic immunoassay to detect tested individual may be contagious for others” (EUROPEAN COMMISSION
IgM and IgG antibodies against the SARS-CoV-2 (https://www.cellex­ COMMUNICATION FROM THE COMMISSION Guidelines on COVID-19
covid.com). However, this kit producer does not report which epitopes in vitro diagnostic tests and their performance (2020/C 122 I/01)).
of virus proteins able to bind IgM and IgG present of the patient’s blood However, the utility of these tests relies on their effectiveness to perform
are used to fabricate the device. COVID-19 IgM/IgG System, large-scale sero-epidemiological population surveys to evaluate and

2
L. Fabiani et al. Biosensors and Bioelectronics 171 (2021) 112686

Fig. 3. A) Study of the signal response for evaluating

PAb concentration. 10 μL of MBs, 200 μL of PAb anti
SARS-CoV-2 0.5, 1, and 2 μg/mL (in PBS) + 200 μL of
PAb-AP anti rabbit IgG 0.5, 1, and 2 μg/mL (in PBS)
+ 300 μL of tested sample, incubation time 30 min
without stirring, 3 washing steps, testing a negative
control and 0.16 μg/mL of S protein. B) Study of the
signal response for evaluating the effect of incubation
time. 10 μL of MBs, 200 μL of PAb anti SARS-CoV-2 1
μg/mL (in PBS) + 200 μL of PAb-AP anti rabbit IgG 1
μg/mL (in PBS) + 300 μL of tested sample, incubation
time (15/30 min) without stirring, 3 washing steps,
testing a negative control, 0.16 and 2.5 μg/mL of S
protein. C) Study of the signal response as function of
mixing during the incubation step. 10 μL of MBs, 200
μL of PAb anti SARS-CoV-2 1 μg/mL (in PBS) + 200
μL of PAb-AP anti rabbit IgG 1 μg/mL (in PBS) + 300
μL of tested sample, incubation time 30 min with and
without stirring, 3 washing steps, testing a negative
control and 0.16 and 2.5 μg/mL of S protein. D) Study
of the signal response for evaluating the effect of
number of washing steps. 10 μL of MBs, 200 μL of PAb
anti SARS-CoV-2 1 μg/mL (in PBS) + 200 μL of PAb-
AP anti rabbit IgG 1 μg/mL (in PBS) + 300 μL of
tested sample, incubation time 30 min without stir­
ring, 1/2/3 washing steps, testing a negative control
and 0.16 μg/mL of S protein. The mean value (n = 3)
with the corresponding standard deviation was re­
ported for each measurement.

guide the management when the pandemic is under control, neverthe­ including transmembrane Spike (S), Envelope (E), and Membrane (M)
less they cannot exclude that the patient having developed an immu­ proteins. S protein is cleaved by a host cell furin-like protease into two
nitary defence is still contagious. separate polypeptides S1 and S2 and it is characterized by an affinity
Regarding the detection of the virus, the quantification of SARS-CoV- with the human angiotensin-converting enzyme 2 (hACE2) used as a
2 at low level is the most challenging issue in the rapid diagnosis of receptor to infect human cells (Verdecchia et al., 2020; Xia et al., 2020).
COVID-19, because the detection of SARS-CoV-2 at the start of infection E protein is found in small quantities embedded within the host-
is the main and successful approach for an effective COVID-19 man­ derived viral envelope membrane while M protein is the structural
agement to limit the spread of this viral contagious. protein with two different conformations to promote membrane curva­
For detecting SARS-CoV-2, the approach recommended by the WHO ture as well as bind to the nucleocapsid (J Alsaadi et al., 2019; Bhowmik
and the European Centre for Disease Prevention and Control (ECDC) is et al., 2020; Coutard et al., 2020).
related to the quantification of virus genetic material by reverse tran­ SARS-CoV-2 is also characterised by highly immunogenic phospho­
scription polymerase chain reaction in nasal or throat secretions (i.e. protein namely the nucleocapsid (N) protein which binds the viral
swabs). The currently available kits based on real-time PCR technology genome in beads on a string type conformation. N protein of coronavirus
offers the highest sensitivity to detect the presence of SARS-CoV-2 virus. is often used as a marker in diagnostic assays (Fehr and Perlman, 2015).
However, in case of samples with low viral load, the repetition of the test Other authors also indicate N antigen of SARS-CoV-2 for reliable diag­
may be required for delivering a definitive diagnosis (Cui and Zhou, nosis of severe acute respiratory syndrome in patients (Di et al., 2005).
2020). Actually, it is considered as positive for SARS-CoV-2 RNA the More recently, Diao et al. reported N protein-based assay for accurate
specimen with a limit of 38/40 cycle threshold (CT) (corresponding to and rapid diagnosis of COVID-19, measuring N protein in urine as well
5610 virus copies/mL) (Wyllie et al., 2020; Wang et al., 2020). The main as nasofaringeal swab within 10 min with fluorescence immunochro­
drawbacks of this method include the long analysis time i.e. at least 3 h, matographic assay. After comparing their N protein detection with data
laboratory set-up, and skilled personnel. obtained on the same samples with the nucleic acids test, authors
In addition to PCR for SARS-CoV-2 RNA detection, ELISA for anti­ concluded that their N antigen detection method may guarantee early
body analysis have been developed, but both these methods are char­ diagnosis in hospitals, moreover it may be used in large-scale screening
acterised by some limitations (Lassaunière et al., 2020; Liu et al., 2020). in community (Diao et al., 2020).
As widely reported in literature, immunosensors are characterised by The N and S2 proteins share a highly conserved structure with SARS-
sensitive, cost-effective, and accurate detection (Khristunova et al., CoV, while S1 subunit is less conserved and more highly specific to
2019, 2020), and the detection of antigens such as viral proteins, which SARS-CoV-2, thus these proteins can be used as antigens for rapid assay
is the basis to develop smart and reliable biosensing systems, can be an development (Ward et al., 2020). Seo et al. developed a field-effect
added value for the management of SARS-CoV-2 outbreak (Moral­ transistor–based device for detecting SARS-CoV-2 by using as target
es-Narváez and Dincer, 2020; Mahapatra and Chandra, 2020). analyte SARS-CoV-2 S protein and the antibody for the S protein as
However, all the developed methods are tests based on serum anal­ biocomponent immobilised on the graphene sheets coating on
ysis, while our method uses saliva as specimen, which is a non-invasive field-effect transistor (Seo et al., 2020). This biosensor was tested in
sample and which accuracy has been demonstrated, through PCR nasopharyngeal swab specimens from COVID-19 patients and cultured
methods, to be comparable with nasopharyngeal sampling method virus, observing a detection limit equal to 100 fg/mL and 1.6 × 101
(Alizagar et al., 2020; Azzi et al., 2020). PFU/mL, respectively. In the market, for antigen detection a kit namely
In detail, SARS-CoV-2 is a coronavirus with size ranges 50–200 nm COVID-19 antigen Respi-Strip is available, which consists in a lateral
diameter and consists of positive-sense single-stranded ribonucleic acid flow assay for qualitative detection of virus in a nasopharyngeal or nasal
(RNA), characterized by an envelope with superficial glycoproteins sample with a LOD value of 5 × 103 PFU/mL (https://www.intermed.

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L. Fabiani et al. Biosensors and Bioelectronics 171 (2021) 112686

Fig. 4. A) Electrochemical calibration curve (inset the

voltammograms) using the optimized parameters for S
protein detection in buffer (black line) and in un­
treated saliva (red line) (n = 3). B) Electrochemical
calibration curve (inset the voltammograms) for N
protein detection in buffer (black line) and untreated
saliva (red line) (n = 3). C) Voltammograms obtained
without (black line) and with (blue line) cultured
SARS-CoV-2 virus at concentration 6.5 PFU/mL using
MBs-based immunoassay for S protein. D) Voltam­
mograms obtained without (black line) and with (blue
line) cultured SARS-CoV-2 virus at concentration 6.5 x
104 PFU/mL using MBs-based immunoassay for N
protein. E) Exclusivity test on seasonal influenza virus
A (H1N1) (102.9 TCID50 mL− 1) and Influenza 2009
pH1N1 virus (104.15 TCID50 mL− 1) in comparison to
SARS-CoV-2 (6.5 PFU/mL) using assay for S protein
(n = 3). F) Exclusivity test on seasonal influenza virus
A (H1N1) (102.9 TCID50 mL− 1) and 2009 influenza
virus pH1N1 (104.15 TCID50 mL− 1) in comparison to
SARS-CoV-2 (6.5 x 104 PFU/mL) using assay for N
protein (n = 3). (For interpretation of the references
to color in this figure legend, the reader is referred to
the Web version of this article.)

be/en/professional-products/laboratory-diagnostics/serology-amp-vir­ the electrochemical biosensors based on the use of screen-printed elec­

ology/rapid-test/coris-covid-19-ag-respi-strip.html). trochemical cell, i.e. screen-printed electrodes (SPEs), have attracted a
These biosensing tools are conceived to be easily used in respect of huge attention for the miniaturization of both the electrochemical
PCR, however they require nasopharyngeal swab specimen, thus the sensor as well as the reader (Caratelli et al., 2020), the sensitivity, the
sampling needs to be carried out by skilled personnel. capability to work in complex matrices, and the easiness to use (Arduini
Taking into account that the seasonal influenza virus is present in et al., 2016). In addition, SPEs are easily modified with nanomaterials
salivary liquid, To et al. (2020) investigated and reported that also namely gold nanoparticles, graphene, and carbon black during the
SARS-CoV-2 is present in the saliva of 91,7% of patients, demonstrating mass-production to improve the electroanalytical performances and
that saliva is a promising non-invasive specimen for diagnosis, moni­ increasing the sensitivity of biosensors (Antuña-Jiménez et al., 2020;
toring, and infection control in patients with COVID-19. Indeed, simple Arduini et al., 2020; Cinti and Arduini, 2017).
sputum samples are processed with in-house 1-step real-time reverse The magnetic beads-based electrochemical assay is an antibody-
transcription–quantitative polymerase chain reaction (RT-qPCR) assay based assay in which magnetic beads (MBs) are used as support for
targeting the S gene of SARS-CoV-2 (Chan et al., 2020). In addition, the immunological chain with the following advantages i) the possibility
another study reported that between nasopharyngeal and saliva sam­ to load high amount of capture antibody thanks to the high surface-to-
ples, saliva yielded greater sensitivity with the huge advantage of volume ratio, allowing for an augmented sensitivity; ii) the use of
self-sample collection of saliva with less variability in respect to naso­ magnetic field to move MBs in a selected volume eliminating with a
paryngeal samples (Wyllie et al., 2020). Among the different biosensors, simple washing step any matrix during the measurement; iii) the pre-

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L. Fabiani et al. Biosensors and Bioelectronics 171 (2021) 112686

Table 1 mg/mL), which recognized N protein, were purchased from Biorbyt

Clinical samples analyzed using MBs-based immunoassay and RT-PCR. * freezed (UK); high affinity Anti-Rabbit IgG Antibody Alkaline Phosphatase-
samples. labelled (PAb-AP, 1 mg/mL) that binds rabbit antibodies and anti-
Sample Spike protein N protein MBs- RT- CT number mouse IgG-AP (MAb-AP, 1 mg/mL) that binds mouse antibodies were
MBs-assay assay PCR from Vector Laboratories (USA); the bovine serum albumin (BSA), used
Patient#1 + + + 38 (for both as blocking agent for covered MBs or plates to reduce the aspecific ad­
ORF and N) sorptions, the electrochemical AP substrates 1-Naphthyl phosphate
Patient#2 + – + 38 for ORF, disodium salt, the optical AP substrate 4-Nitrophenyl phosphate diso­
negative for N
dium salt (PNPP), diethanolamine, tween 20, sodium azide and all other
Patient#3 – – – NO CT
Patient#4 – – – NO CT reagents were obtained from Sigma (USA). Maxisorp™ surface, 96well
Patient#5* + + + 37 for ORF and polystyrene microtitre plates, were purchased from Nunc (Roskilde,
33 for N Denmark). Dynabeads® Pan Mouse IgG pre-coated with anti-Mouse IgG
Patient#6* + + + 35 for ORF and able to bind monoclonal antibody (supplied as a suspension containing 4
33 for N
Patient#7* + + + 31 for ORF and
x 108 beads/mL in phosphate buffered saline, pH 7.4) was from Life
28 for N Technologies (USA). 25 μL of MBs are able to bind ~1 μg of antibody. A
Patient#8* + No tested for low + 31 for ORF and rotary shaker and a magnetic rack/particle concentrator were from
volume of sample 27 for N Dynal Biotech (USA). Phosphate saline buffer (PBS) = 0.0015 M
Patient#9* NO CT
+ + –
KH2PO4, 0.0081 M Na2HPO4, 0.137 M NaCl, 0,0027 M KCl, pH 7.4;
Patient#10* – – – NO CT
Patient#11* – – – NO CT Washing Buffer = PBS+0.05 % Tween 20; Storage buffer = PBS + 0.02%
Patient#12* – – – NO CT NaN3, and DEA buffer = DEA–MgCl2–KCl buffer = 0.97 M DEA+1 mM
Patient#13* – No tested for low – NO CT MgCl2+0.1 M KCl pH 9.6 were used as buffer solution.
volume of sample Saliva and Nasopharyngeal swab samples were collected with the
Patient#14* No tested for low NO CT
express consent, free and with informed agreement, to such collection
– –
volume of sample
Patient#15* – No tested for low – NO CT and use, of the person from whom this material was taken, according to
volume of sample the current legislation.
Patient#16 + – – NO CT
Patient#17 31 for ORF and
+ + +
2.2. Screen-printed electrode production and modification
30 for N
Patient#18 – – – NO CT
Patient#19 – – – NO CT SPEs were produced onto a transparent and flexible polyester sup­
Patient#20 – – – NO CT port by employing 245 DEK (Weymouth, UK) serigraphic printer. A
Patient#21 – – – NO CT three-electrode cell was realised by using a graphite-based ink (Elec­
Patient#22 NO CT
trodag 423 SS) for the working electrode (surface area equal to 0.07
– – –
Patient#23 – – – NO CT
Patient#24 – – – NO CT cm2) and counter-electrodes, and a silver-based ink (Electrodag 6033
SS) for the pseudo-reference electrode. The SPEs were modified with 6
μL of CB N220 dispersion (1 mg/mL in N,N-dimethylformamide:distilled
concentration of MBs on the working electrode surface using a minia­ H2O 1:1 v/v), by a three-step consecutive drop-casting procedure of 2 μL
turized customized magnetic tool for increasing the enzymatic by- on the working electrode. The optimization of modification procedure
product in close contact to the working electrode surface and thus the and morphological characterisation of CB-modified SPEs by the scan­
sensitive of electrochemical measurement (Pedrero et al., 2012). ning electron microscope were reported in our previous article (Arduini
In this study, in order to have a miniaturized electroanalytical de­ et al., 2012).
vice, we developed the first sensitive electrochemical biosensor for
SARS-CoV-2 detection in saliva using a MBs-based electrochemical assay 2.3. ELISA procedure
and carbon black-based SPE as sensor combined with PALM SENS
portable potentiostat as reader. Both SARS-CoV-2 proteins namely S This test was performed to verify the capability of the antibodies to
protein and N protein were used as target analyte developing a sandwich bind target proteins. 100 μL of both S proteins (from Sinobiological and
assay with immobilised antibodies for S or N proteins on MBs. The from ProSci) 2 ng/mL prepared in PBS buffer were added in each well of
binding was evaluated by using secondary antibody labelled with a microtitre-plate and left overnight at 4 ◦ C. Then, the coated plate was
alkaline phosphatase enzyme. The developed MBs-based assay was blocked with 100 μL of 3% (w/v) BSA in PBS and incubated for 1 h at
tested in buffer solution, saliva samples, and cultured SARS-CoV-2 virus, 37 ◦ C. Binding curves for each anti-SARS-CoV antibody were con­
as well as in saliva clinical samples, comparing the data with the ones structed by adding into the wells 100 μL of different antibody dilutions
obtained by RT-PCR using nasopharyngeal swab specimen. prepared in PBS and ranging from 2 μg/mL to 0.5 μg/mL (we have tested
all the antibody purchased), for 1 h of incubation at 37 ◦ C. The labelling
2. Materials and methods step involves the addition of 100 μL of anti-rabbit IgG-AP 2 μg/mL and
an incubation for 1 h at 37 ◦ C (when we test MAb antibody, the labelling
2.1. Reagents, solutions and samples step involves anti-mouse IgG-AP 2 μg/mL). Between each step, a three-
cycle washing procedure, using 150 μL of PBS +0.05% Tween 20, was
The target SARS-CoV Spike (subunit S1) protein (0.5 mg/mL), the performed. Finally, 100 μL of PNPP solution (2 mg/mL in DEA buffer, pH
Monoloclonal antibody anti SARS-CoV (produced in mouse, 1 mg/mL) 9.6) were used and the absorbance was read (at 415 nm) after 30 min of
and the Polyclonal antibody SARS-CoV Spike (produced in rabbit, 1 mg/ incubation. To evaluate the efficiency of MBs to tether antibodies, a
mL) which recognizes the S protein, were purchased from Sinobiological competitive ELISA was performed: 100 μL of 10 μg/mL of MAb anti-
(Germany); the target SARS Coronavirus 2019 Spike Recombinant SARS-CoV-2 (Sinobiological) prepared in PBS buffer were added in
protein (1 mg/mL) and the Polyclonal Antibody SARS-CoV-2 to Spike S1 each well of microtitre-plate and left overnight at 4 ◦ C. Then, the coated
(produced in rabbit, 0.1 mg) were from ProSci (USA); the target SARS- plate was blocked with 100 μL of 3% (w/v) BSA in PBS and incubated for
CoV-2 Nucleocapside protein was from Acrobiosystem (USA, 0.6 mg/ 1 h at 37 ◦ C. Competition curves for anti-SARS-CoV antibody were
mL); Monoclonal antibody to SARS-CoV-2 (produced in mouse, 3.4 mg/ constructed by adding into the wells 100 μL containing different anti­
mL) and Polyclonal antibody to SARS-CoV-2 (produced in rabbit, 7.8 body concentrations prepared in PBS ranging from 0.005 to 20 μg/mL +

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L. Fabiani et al. Biosensors and Bioelectronics 171 (2021) 112686

Table 2
Overview of biosensors for SARS-CoV-2 detection.
BIOSENSOR METHOD ANALYTE SAMPLE ANALYTICAL REF.
FEATURES

CARMEN-Cas13a Microwell-array system for color-coded Amplified nucleic acid Plasma, nasal or Attomolar sensitivity Ackerman et al., 2020
droplets of CRISPR detection reagents throat swabs
(fluorescent signal)
CRISPR-Chip Label-free electrical detection Unamplified nucleic acid Buccal swab – https://cardeabio.
com/crispr-chip/
CRISPR-powered assays RT-PCR CRISPR-Cas12a fluorescent reporter Amplified nucleic acid Nasal swab LOD: 2 copy /μL Huang et al., 2020
assay
Dual-Functional Biosensor combining the plasmonic Unamplified nucleic acid – LOD: 0.22 pM Qiu et al., 2020
Plasmonic photothermal (PPT) effect and localized
Photothermal surface plasmon resonance (LSPR) sensing
Biosensors transduction
CONVAT Optical biosensor Unamplified nucleic acid Nasal or saliva – https://optics.
swabs org/news/11/4/13
FET biosensor Label-free, real-time electrical detection Spike protein Nasopharyngeal LOD: 100 fg /mL Seo, G. et al., 2020
with graphene-based FET functionalized swabs
with antibody
Portable, Ultra-Rapid and Bioeectric recognition assay based on Spike protein LOD: 1 fg /mL Mavrikou et al., 2020
Ultra-Sensitive Cell- mammalian Vero cells, which were
Based Biosensor engineered by electroinserting the human
chimeric spike S1 antibody
Toroidal plasmonic Miniaturized plasmonic immunosensor Spike protein LOD: 4.2 fmol Ahmadivand et al.,
metasensors based on toroidal electrodynamics concept Miniaturized plasmonic 2020
immunosensor based on
toroidal electrodynamics
concept
Fluorescence Fluorescence immunochromatographic Nucleocapsid protein Nasopharyngeal Sensitivity: 68%, Diao, B. et al., 2020
immunochromato- assay in blind analysis swab, Specificity: 100%,
graphic assay Urine Accuracy: 72%
Electrochemical Magnetic bead-based immunosensor Spike and Nucleocapsid Untreated saliva 19 ng/mL and 8 ng/mL This work
immunosensor combined with carbon black-modified protein in untreated saliva,
screen-printed electrode respectively for S and N
protein

anti-mouse IgG-AP 0.5 μg/mL in an incubation for 1 h at 37 ◦ C. Between 1. shake and transfer 10 μL of the coated and blocked beads sus­
each step, a three-cycle washing procedure, using 150 μL of PBS +0.05% pension (stored at 4 ◦ C) into 2 mL tube (in number required for
Tween 20, was performed. Finally, 100 μL of PNPP solution (2 mg/mL in the samples to analyze);
DEA buffer, pH 9.6) were used and the absorbance was read (at 415 nm) 2. wash 2 times with 1 mL PBS, every time shaking and discarding
after 15 min of incubation. the supernatant by placing for 1 min a magnet outside the tube in
a lateral position;
2.4. Electrochemical immunoassay 3. add consecutively 200 μL of PAb anti SARS-CoV-2 1 μg/mL in PBS
(produced in rabbit) + 200 μL of PAb-AP anti rabbit IgG 1 μg/mL
The electrochemical MBs-based assay involves three sequential in PBS + 300 μL of sample;
procedures: I) a preliminary blocking-coating procedure of the Dyna­ 4. incubate for 30 min at RT;
beads® Pan Mouse IgG (to store them at 4 ◦ C until use for several 5. wash 2 times with 1 mL PBS +0.05% Tween 20 and one time with
months); II) an immunoassay procedure (in which the classical PBS, every time shaking and discarding the supernatant by using
sequential incubations for the immuno-recognition events are merged in the magnet as above,
a single incubation of 30 min); III) an electrochemical detection using III) Electrochemical measurements: At the end of the immunoassay
SPEs. procedure, the beads (with their immunological chain) were
resuspended in 100 μL of DEA buffer and 20 μL of these suspen­
I) Blocking-coating procedure: 250 μL of MBs 4 x 108 beads/mL was sion (three replicates for each sample) were drop cast on the
pipetted into 2 mL tube and washed twice with 1 mL of PBS pH 7.4 working electrode and magnetically concentrated onto the sur­
and the MBs were blocked incubating them in 1 mL of PBS pH 7.4 + face through the magnet positioned just under the working
3% BSA for 30 min at room temperature (RT) with slow tilt rotation electrode. The immune complex was then revealed by adding 70
(using Dynal sample mixer). Then the supernatant was discarded and μl of 1- naphthyl phosphate (5 mg/mL, prepared in
500 μL of PBS, containing 10 μg MAb anti SARS-CoV-2, were added DEA–MgCl2–KCl buffer pH 9.6) into each well. Waiting 2 min for
to the beads and incubated for 30 min at RT with slow tilt rotation. the enzymatic reaction, the electroactive enzymatic product (1-
The blocked and coated MBs were washed twice with 1 mL of PBS. naphthol) is measured by applying differential pulse voltamme­
Finally, they were resuspended in 250 μL of PBS +0.02% NaN3, and try (DPV) with the following parameters, based on our previous
stored for up to several months at 4 ◦ C. work (Fabiani et al., 2019): Ebegin = − 0.2 V; Eend = 0.4 V; Estep
= 0.016 V; Epulse = 0.05 V; tpulse = 0.06 s; scan rate = 0.016
Between each washing step, the Eppendorf tube containing the V/s.
magnetic beads was placed in the Dynal MPC and after 30 s the super­
natant was discarded. The electrochemical measurements were performed by a portable
potentiostat, PalmSens3 (Palm Instrument, The Netherlands), connected
II) Immunoassay procedure: to a personal computer.

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L. Fabiani et al. Biosensors and Bioelectronics 171 (2021) 112686

2.5. SARS-CoV-2 virus propagation The concentration of infectious viruses was determined by the 50%
tissue culture infectious dose (TCID50) by 10-fold serial titration in
SARS-CoV-2 was passaged once in Vero cells to generate a virus master MDCK cells.
stock used to generate a virus working stock. The virus was propagated in
Vero cells cultured in minimum essential medium (MEM) containing 2% 3. Results and discussions
(w/v) fetal bovine serum (Euroclone S.p.A.). After infection, virus stock
was collected by centrifuging the culture supernatants of infected Vero 3.1. Electrochemical MBs-based set-up
cells at 600 g for 5 min. The clarified supernatant was supplemented to 20%
with fetal bovine serum (w/v), frozen and kept at − 80 ◦ C until use. The The high sensitivity of electrochemical detection combined with the
concentration of infectious virus was determined by plaque-forming titre miniaturization is the key of this work to reach the overriding goal for a
assay. Virus propagation, virus isolation or neutralization assays of cost-effective and reliable high sensitivity detection of SARS-CoV-2
SARS-CoV-2 needs to be conducted in a bio-safety Level-3 facility using a no-invasive and attractive biological fluid namely saliva.
according to WHO laboratory biosafety guidance (https://www.who.int/ To accomplish this task, we have exploited MBs as support for
publications/i/item/laboratory-biosafety-guidance-related-to-coronaviru immunological chain with their outstanding features as well as SPE
s-disease-(covid-19https://www.who.int/publications/i/item/laboratory modified with the nanomaterial carbon black (CB) to reach improved
-biosafety-guidance-related-to-coronavirus-disease-(covid-19)). sensitivity as demonstrated in our previous papers for several electro­
active compounds (Arduini et al., 2010, 2011, 2012, 2020; Talarico
2.6. SARS-CoV-2 quantification by real-time PCR et al., 2015) (Fig. 1).
For making a simple and easy analysis, saliva is used by simply
Nasopharyngeal swabs collected and tested from Italian Scientific adding the sputum in the tube previously loaded with the reagents
dept. of Army Medical Center for SARS-CoV-2. Viral RNA was extracted needed for the measurement, without requiring no-extra task to the end-
from 300 μL of swab’s medium using a Maxwell RSC Viral Total Nucleic users.
Acid Purification Kit on Maxwell RSC Instrument (Promega, USA). Total After the selected minutes for immunological chain construction,
nucleic acid was eluted in a final volume of 50 μL of nuclease-free water. washing steps are required followed by the addition of MBs on the
Then, one step Real-Time PCR was performed using Novel Coranavirus working electrode surface and the enzymatic substrate i.e. 1- naphthyl
(2019-nCoV) Nucleic Acid Diagnostic Kit (Sansure Biotech Inc., China) phosphate.
on LC480 instrument (Roche Diagnostics, Germany). This test utilizes To develop a highly performant analytical tool in terms of sensitivity
2019-CoV ORF1ab and N protein genes as target regions and an internal and selectivity, we start to optimize the assay using specific antibody for
control, which monitors the presence of PCR inhibitors. Reaction S1 subunit of S protein, taking into account that several S proteins are
mixture contained 13 μL of 2019-nCoV-PCR Mix, 2 μL 2019-nCoV-PCR present on each viral particles.
and 5 μL of extract viral RNA to reach to final volume of 20 μL. The
cycling program was as follows: Reverse transcription at 50 ◦ C for 30 3.2. Electrochemical MBs-assay optimization for spike protein detection
min; cDNA denaturation at 95 ◦ C for 1 min and 45 cycles of PCR at 95 ◦ C
for 15 s and 60 ◦ C for 30 s (signal acquisition). FAM, ROX and Cy5 The use of the high sensitive antibodies is one of the main task to
channels were selected for ORF1ab, N and the internal control detection, develop high sensitive and selective analytical device, thus for the se­
respectively. lection of antibodies, spectrophotometric ELISA was carried out to assess
the reactivity of MAb and two different PAb (Sinobiological, Germany
2.7. Electrochemical measurement of SARS- CoV-2 in biosafety level 3 and ProSci, USA) towards two different S proteins namely SARS Coro­
environment navirus 2019 Spike Recombinant protein (1000–1200 aa) and Recom­
binant Spike protein SARS-CoV Spike protein, S1 subunit. For all optical
To date it is impossible to relate the protein concentration to the total measurements, the secondary antibody labelled with alkaline phosphate
viral load, so, to evaluate the sensitivity of our assay on native virus, we enzyme was added subsequently using 4-nitrophenyl phosphate as
tested SARS-CoV-2 directly in electrochemical MBs-based assay in enzymatic substrate and monitoring the binding through the formation
Biosafety Level 3 laboratory. Starting from a SARS-CoV-2 aliquot of 6.5 x of enzymatic by-product 4-nitrophenol at 415 nm. In Fig. 2A the
105 PFU/mL, we prepared five 1:10 v/v serial dilutions in PBS and response of ELISA was reported testing the two PAb and two S proteins
incubated for 30 min 300 μL of each dilution (and a negative control) and demonstrating the better affinity between PAb belonging from
with 10 μL of MBs (precoated with MAb anti SARS-CoV and washed), Sinobiological, Germany and Recombinant Spike protein SARS-CoV, S1
200 μL of PAb anti-SARS-CoV 1 μg/mL in PBS and 200 μL of PAb-AP 1 subunit from Sinobiological, Germany, thus these reagents were selected
μg/mL in PBS. The test was performed on N protein and S protein in for further studies. Subsequently, MAb was tested towards this S protein
parallel using antibodies that recognize protein N and S, respectively. in ELISA and the binding curve was reported in Fig. 2B, demonstrating
After the washing step, we proceeded with the electrochemical mea­ high affinity.
surement. All the apparatus used were covered in plastic to avoid any For the electrochemical measurement, the secondary antibody
contamination. labelled with alkaline phosphate enzyme was used with 1-naphthyl
phosphate as enzymatic substrate, monitoring the formation of enzy­
2.8. 2009 H1N1 influenza pandemic and seasonal H1N1 influenza virus matic by-product 1-naphtol at around 0.1 V by using differential pulse
propagation for selectivity test voltammetry. The use of CB-based SPE has allowed to increase the
sensitivity as reported in Fig. 2C. Indeed, the enzymatic by-product was
2009 H1N1 influenza pandemic (2009 pH1N1) and seasonal H1N1 detected with increased sensitivity of ca. 4 folds at lower potential 0.1 V
influenza virus master stocks were passaged in Madin-Darby canine using CB-modified electrode (blue line) in respect to 0.2 V using un­
kidney (MDCK) to generate virus working stocks. The viruses were modified electrode (black line). To evaluate how many MAbs were
propagated in MDCK cultured in minimum essential medium (MEM) tethered on beads, we have measured the solution after the incubation of
containing 2% (w/v) fetal bovine serum (Euroclone S.p.A.), 2 μg/mL 125 μL of MBs with 5 μg of MAb by means of spectrophotometric ELISA,
trypsin-TPCK (Merck, Germany). After infection, virus stocks were with the aim to estimate the amount of MAb unbounded. In detail, the
collected by centrifuging the culture supernatants of infected MDCK amount of MAb tethered on beads was calculated by interpolating the
cells at 600 g for 5 min. The clarified supernatants were supplemented to data (red point) within the competition curve reported in Fig. S1,
20% with fetal bovine serum (w/v), frozen and kept at − 80 ◦ C until use. funding that 125 μL of MBs capture 2.6 μg of antibody, thus each MB

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L. Fabiani et al. Biosensors and Bioelectronics 171 (2021) 112686

tethers ~2 × 105 antibodies. This number is also consistent with the N protein in standard solutions, different concentrations of N protein
spherical surface of one bead (~63.6 μm2) and the dimension of one Ab diluted in 0.015 M phosphate buffer +0.137 M NaCl +0.0027 M KCl pH
(~10 nm) (Reth, 2013). = 7.4 ranging from 0.01 to 0.6 μg/mL were tested (Fig. 4B), using the
The construction of immunological chain requires the optimization previously optimized parameters, observing a sigmoidal behavior
of the concentration of PAb i.e. the antibody free in solution, the time of described by non-linear four-parameter logistic calibration plot Eq. (1)
reaction between antigen and antibodies, the mode of reaction (i.e. tilt observing a detection limit of 4 ng/mL. The matrix effect due to the
rotation or static condition), and number of washing steps. analysis in saliva without any pre-treatment was evaluated by con­
PAb antibody is free in solution, thus it affects the response of the struction the calibration curve in untreated saliva, observing well-
assay being the element that completes the sandwich immunological defined sigmoidal behavior with lower intensity of peak current due
format. We tested amounts equal to 0.5, 1, and 2 μg/mL observing the the matrix effect with detection limit equal to 8 ng/mL.
highest signal using 1 μg/mL, thus this value was selected for further
studies (Fig. 3A). This experiment showed that 0.5 μg/mL of antibody 3.5. Measurement of SARS-CoV-2 virus
was not a sufficient concentration to bind all the proteins present, while
2 μg/mL of antibody probably creates a steric hindrance that did not The structural studies on SARS-CoV-2 reported in literature until
allow the correct binding (in the figure the positive signal is lower than now are not able to furnish unequivocal information related to the
the one obtained with 1 μg/mL of antibody). In Fig. 3B the effect of number of S proteins present on each virus (Ke et al., 2020; Kiss et al.,
incubation time was evaluated demonstrating that 15 min were not 2020), thus it is difficult to calculate the number of viruses detected
sufficient for a good sensitivity, thus 30 min were selected. Because the using the calibration curve for S protein. To evaluate the response of our
assay involves a single incubation step, a minimum time of 30 min is assay for native virus, we tested the native virus ranging from 6.5 x 105
required to correctly create the antibody-antigen-antibody bonds. to 6.5 PFU/mL in Biosafety Level 3 environment. In Fig. 4 C/D, the
Within the aim to develop a device to be easily customised for the end- voltammograms show the signal related to virus concentration in respect
user, the effect of stirring was evaluated. As depicted in Fig. 3C, the to the signal of negative control using the electrochemical MBs-assay for
lower concentration i.e. 0.16 μg/mL demonstrated high signal in stirring S protein and N protein, respectively.
condition, with a signal significantly different from the blank signal. As depicted in Fig. 4C, when the test is carried out using antibodies
However, in the case of the higher concentration 2.5 μg/mL, the same directed against S protein, the sensitivity of the assay is excellent (can
sensitivity was observed. This happens because the probability of detect 6.5 PFU/mL), in respect to the assay N protein (Fig. 4D). Instead,
binding at high concentrations is greater than the one at low concen­ in case of N protein-based assay the sensitivity is lower (can be detect 6.5
trations, being the assay carried out in static condition. Taking into x 103 PFU/mL), because the amount of N protein is significantly lower
account that the device has been designed for an easy miniaturization, than the amount of S protein in SARS-CoV-2 virus.
the measurement without stirring was selected for the rest of work. To
reduce the tasks required by end-users for the analysis, the effect of 3.6. Exclusivity study
number of washing steps needed to avoid the aspecific absorption on the
MBs was evaluated, choosing two washing steps for the construction of To assess the selectivity of the proposed MBs-based assay, an ex­
the calibration curve (Fig. 3D). Under the optimized condition, the effect clusivity test was performed analyzing seasonal influenza virus A
of pH on the assay was evaluated testing DEA buffer in a pH range (H1N1) (102.9 TCID50 mL− 1) and 2009 influenza virus pH1N1 (104.15
comprised between 8.5 and 10, observing the highest sensitivity at pH = TCID50 mL− 1), comparing the results with the SARS-CoV-2 previously
9.6, thus this value was selected (Fig. S2). analyzed (section 3.5). These tests were carried out using the assay
directed against the S protein (Fig. 4E) and the assay directed against the
N protein (Fig. 4F). In both cases, the results show a 100% of exclusivity.
3.3. Analytical features of electrochemical MBs-based assay for spike
protein detection in standard solution and saliva
3.7. Measurement of SARS-CoV-2 in clinical samples
To assess the analytical features of the developed assay for measuring
To assess the effectiveness of the MBs-based assay using clinical
S protein in standard solutions, different concentrations of S protein
samples, saliva, and nasopharyngeal swabs were tested using MBs-assay
diluted in 0.015 M phosphate buffer +0.137 M NaCl +0.0027 M KCl pH
and Real-Time PCR, respectively. In detail, we tested saliva samples
= 7.4 ranging from 0.04 to 10 μg/mL were tested, observing a sigmodal
using fresh samples and freezed samples, observing a reduction of signal
behavior (Fig. 4A) described by non-linear four-parameter logistic
in case of freezed sample for both positive and negative patients, thus we
calibration plot as follows:
decided to set 1.8 μA as threshold in case of unfreezed samples, while in
a− d case of freezed sample the threshold selected was 1 μA. We suggested the
f (x) = ( )b + d (1)
1 + xc use of fresh saliva sampled after drinking a glass of water, with the aim
of easy sampling without any treatment to match the requirement of the
point of care system. As depicted in Table 1, we have observed an
where a and d are the asymptotic maximum and minimum values, c is
agreement in 22/24 samples, which is a very satisfactory result, taking
the value of x at the inflection point and b is the slope, obtaining a
into account the low viral load detected as well as the analytical features
detection limit equal to 14 ng/mL, calculated as blank signal + 3 stan­
of biosensors reported in literature (Table 2).
dard deviation (SD). The matrix effect due to the analysis in saliva
without any pre-treatment was evaluated by constructing the calibration
4. Conclusions
curve in untreated saliva (Fig. 4A), observing the same sigmodal
behavior with lower intensity of peak current due the matrix effect. The
The COVID-19 pandemic event declared by World Health Organi­
calibration curve in saliva was described by non-linear four-parameter
zation on March 2020 has required great effort by the scientific com­
logistic calibration plot with detection limit equal to 19 ng/mL.
munity, including the research activity for the development of rapid
assay for SARS-CoV-2. Herein, we developed a smart immunosensor for
3.4. Analytical features of electrochemical MBs-based assay for N SARS-CoV-2 detection in saliva by combining the use of MBs as support
detection in standard solution and saliva for immunological chain and carbon black-based SPEs for sensitive and
reliable detection. This sensor configuration demonstrated the capa­
To assess the analytical features of the developed assay for measuring bility to detect S and N proteins in untreated saliva with a detection limit

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L. Fabiani et al. Biosensors and Bioelectronics 171 (2021) 112686

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CRediT authorship contribution statement COMMUNICATION from the COMMISSION Guidelines on COVID-19 in Vitro Diagnostic
Tests and Their Performance (2020/C 122 I/01).
Fabiani, L., Delibato, E., Volpe, G., Piermarini, S., De Medici, D., Palleschi, G., 2019.
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Experimental investigation, Methodology, Data curation, Formal anal­ Huang, Z., Tian, D., Liu, Y., Lin, Z., Lyon, C., Lai, W., Fusco, D., Drouin, A., Yin, X., Hu, T.,
ysis, Writing - review & editing. Silvia Fillo: Experimental investiga­ Ning, B., 2020. Biosens. Bioelectron. 164, 112316.
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tion, Methodology, Data curation, Formal analysis, Writing - review &
Khristunova, E., Barek, J., Kratochvil, B., Korotkova, E., Dorozhko, E., Vyskocil, V., 2020.
editing. Vincenzo Luca: Experimental investigation, Methodology, Bioelectrochemistry 135, 121136.
Formal analysis. Giovanni Faggioni: Data curation, Formal analysis, Khristunova, Y., Korotkova, E., Kratochvil, B., Barek, J., Dorozhko, E., Vyskocil, V.,
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Declaration of competing interest Qiu, G., Gai, Z., Tao, Y., Schmitt, J., Kullak-Ublick, G.A., Wang, J., 2020. ACS Nano 14,
5268–5277.
The authors declare that they have no known competing financial Reth, M., 2013. Nat. Immunol. 14, 765–767.
Seo, G., Lee, G., Kim, M.J., Baek, S.H., Choi, M., Ku, K.B., Lee, C.S., Jun, S., Park, D.,
interests or personal relationships that could have appeared to influence
Kim, H.G., Kim, S.J., Lee, J.O., Kim, B.T., Park, E.C., Kim, S., 2020. ACS Nano 14,
the work reported in this paper. 5135–5142.
Talarico, D., Arduini, F., Constantino, A., Del Carlo, M., Compagnone, D., Moscone, D.,
Appendix A. Supplementary data Palleschi, G., 2015. Electrochem. Commun. 60, 78–82.
To, K.K., Tsang, O.T., Chik-Yan Yip, C., Chan, K.H., Wu, T.C., Chan, J., Leung, W.S.,
Chik, T.S., Choi, C.Y., Kandamby, D.H., Lung, D.C., Tam, A.R., Poon, R.W., Fung, A.
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org/10.1016/j.bios.2020.112686. ciaa149.
Verdecchia, P., Cavallini, C., Spanevello, A., Angeli, F., 2020. Eur. J. Intern. Med. 76,
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