Showcasing research from Professor Wen’s laboratory,

Department of Physics, The Hong Kong University of As featured in:

Science and Technology, Hong Kong, China.

Point-of-care testing detection methods for COVID-19

Professor Wen’s group from The Hong Kong University

of Science and Technology used a thin film silicon-based
micro-heater real-time PCR system together with
silicon-glass microfluidic chips, reduced the detection
time of SARS-CoV-2 nucleic acid to less than 40 min.
The portable and rapid SARS-CoV-2 nucleic acid detection
system can reach a temperature rising rate up to 30 °C/s,
and was able to complete the detection with a sensitivity
of up to 500 copies/mL. This article is jointly completed
by Professors Jinbo Wu and Bingpu Zhou from Shanghai See Bingpu Zhou, Jinbo Wu,
Weijia Wen et al.,
University and University of Macau respectively.
Lab Chip, 2021, 21, 1634.

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CRITICAL REVIEW View Journal | View Issue

Point-of-care testing detection methods for

Cite this: Lab Chip, 2021, 21, 1634
COVID-19
Qi Song,†ab Xindi Sun,†c Ziyi Dai,d Yibo Gao,e Xiuqing Gong,c Bingpu Zhou, *d
Jinbo Wu*c and Weijia Wen *a
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

COVID-19 is an acute respiratory disease caused by SARS-CoV-2, which has high transmissibility. People
infected with SARS-CoV-2 can develop symptoms including cough, fever, pneumonia and other complica-
tions, which in severe cases could lead to death. In addition, a proportion of people infected with SARS-
CoV-2 may be asymptomatic. At present, the primary diagnostic method for COVID-19 is reverse
transcription-polymerase chain reaction (RT-PCR), which tests patient samples including nasopharyngeal
swabs, sputum and other lower respiratory tract secretions. Other detection methods, e.g., isothermal
nucleic acid amplification, CRISPR, immunochromatography, enzyme-linked immunosorbent assay (ELISA)
and electrochemical sensors are also in use. As the current testing methods are mostly performed at cen-
tral hospitals and third-party testing centres, the testing systems used mostly employ large, high-through-
put, automated equipment. Given the current situation of the epidemic, point-of-care testing (POCT) is ad-
vantageous in terms of its ease of use, greater approachability on the user's end, more timely detection,
and comparable accuracy and sensitivity, which could reduce the testing load on central hospitals. POCT is
Received 15th November 2020, thus conducive to daily epidemic control and achieving early detection and treatment. This paper summa-
Accepted 21st February 2021
rises the latest research advances in POCT-based SARS-CoV-2 detection methods, compares three cate-
DOI: 10.1039/d0lc01156h
gories of commercially available products, i.e., nucleic acid tests, immunoassays and novel sensors, and
proposes the expectations for the development of POCT-based SARS-CoV-2 detection including greater
rsc.li/loc accessibility, higher sensitivity and lower costs.

1. Introduction outbreak a public health emergency of international concern

(PHEIC). On February 11, 2020, the Coronaviridae Study
At the end of December 2019, several patients with flu-like Group (CSG) of the International Committee on Taxonomy of
symptoms were admitted to hospitals in Wuhan, China. To Viruses named this virus “Severe Acute Respiratory Syndrome
identify the infectious pathogen, patient samples were Coronavirus 2 (SARS-CoV-2)” because of its genetic similarity
subjected to metagenomic RNA sequencing. The results to the severe acute respiratory syndrome coronavirus (SARS-
showed that the pathogen was a novel coronavirus. On Janu- CoV); the 2 viruses belong to the same clade. The disease
ary 30, 2020, the World Health Organization (WHO) caused by SARS-CoV-2 was named coronavirus disease 2019
proclaimed that the pneumonia caused by the newly discov- (COVID-19). Subsequently, the epidemic escalated into a
ered coronavirus was “2019-nCoV acute respiratory disease”. global COVID-19 pandemic. The clinical manifestations of
On January 31, 2020, the WHO declared the novel coronavirus COVID-19 range from mild symptoms, such as cough, fever
and dyspnea, to life-threatening syndromes, such as pneumo-
nia and acute respiratory distress syndrome, and even
a
Department of Physics, The Hong Kong University of Science and Technology, death.1,2 As of the time of writing this article, there were more
Clear Water Bay, Kowloon, Hong Kong, China. E-mail: phwen@ust.hk
b
than 100 million confirmed cases, 2 182 179 deaths. The high
Guangzhou HKUST Fok Ying Tung Research Institute, Guangzhou, Guangdong,
China
infection rate and mortality rate for COVID-19 have caused
c
Materials Genome Institute, Shanghai University, Shanghai, China. substantial losses in terms of life, quality of life and the
E-mail: jinbowu@t.shu.edu.cn global economy.
d
Joint Key Laboratory of the Ministry of Education, Institute of Applied Physics and Chinese researchers found that SARS-CoV-2 originated
Materials Engineering, University of Macau, Macau, China.
from bats, but the intermediate host has not yet been deter-
E-mail: bpzhou@um.edu.mo
e
Shenzhen Shineway Technology Corporation, Shenzhen, Guangdong, China
mined.3 The SARS-CoV-2 genome shares approximately 89%
† These authors contributed equally to this work and should be considered co- sequence homology with bat SARS-like-COVZXC21 and ap-
first authors. proximately 82% sequence homology with human SARS-CoV.4

1634 | Lab Chip, 2021, 21, 1634–1660 This journal is © The Royal Society of Chemistry 2021
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Lab on a Chip Critical review

SARS-CoV-2 enters specific cells through protein–protein in- by infected individuals when they cough/sneeze/talk or
teractions; i.e., the S glycoprotein of SARS-CoV-2 binds to breathe by individuals at close range can also lead to infec-
angiotensin-converting enzyme 2 (ACE2) on the surfaces of tion. Therefore, the early diagnosis of suspected cases is an
some cell types.5 Through processing by TMPRSS2 protease, important task in managing infected individuals and control-
attached virus enters the cells. The S protein consists of 2 ling the spread of pathogens.8 The WHO emphasizes that
functional subunits: S1 and S2. The S1 subunit is binds to early identification and isolation are essential for limiting
host cell receptors, while the S2 subunit fuses the viral mem- human-to-human transmission. Therefore, it is necessary to
brane and cell membrane.6 The structural proteins of SARS- conduct effective large-scale screening using sensitive and
CoV-2, i.e., the envelope (E), membrane (M), helicase (Hel) high-throughput detection methods; this screening approach
and nucleocapsid (N) proteins, are shown in Fig. 1.7 Viral rep- allows the identification, isolation and tracking of individuals
lication requires other auxiliary genes, including open read- without obvious symptoms, facilitates a reduction in second-
ing frame 1a (ORF1a), ORF1b, RNA-dependent RNA polymer- ary infections between close contacts and healthcare workers,
ase (RdRp), and hemagglutinin esterase (HE). The M protein and leads to improvements in local control. In addition, such
is the most abundant protein on viral particles. The E protein screening provides information for regional disease control
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

is the smallest major structural protein of SARS-CoV-2. It is efforts and prevents further spread of infections.
involved in the assembly, release and pathogenesis of the vi- At present, the methods for the detection of COVID-19
rus. The N protein is the main structural protein of SARS- mainly include nucleic acid testing and serological testing.
CoV-2. It is responsible for the transcription and replication These methods are suitable for the in vitro diagnosis of patients
of viral RNA, the packaging of the enveloped genome into vi- with suspected SARS-CoV-2 infection. Nucleic acid testing in-
ral particles, and interactions with the cell cycle of host cells. cludes gene sequencing, reverse transcription-polymerase chain
The N protein is the most abundant phosphorylated protein reaction (RT-PCR) and other methods. In the early stage of the
produced and released during viral infection. It has high im- novel coronavirus outbreak, the genome sequence of SARS-CoV-
munogenicity and can be detected in serum or urine within 2 virus was obtained through gene sequencing, providing an im-
the first two weeks of infection. Release of the N protein by portant basis for subsequent testing.9 RT-PCR is considered the
the virus peaks approximately ten days after infection. gold standard for detecting COVID-19, especially in the early
The main transmission routes of SARS-CoV-2 are contact stage of viral infection. Through targeting the unique RNA se-
transmission and droplet transmission. For example, droplets quence of SARS-CoV-2, the genetic material of the pathogen can
from an infected individual are deposited on the surfaces of be directly detected by RT-PCR.10 However, the currently avail-
objects; if an uninfected individual touches the surface of a able quantitative RT-PCR (RT-qPCR) tests vary considerably in
contaminated object and then touches his/her mucous mem- sensitivity, stability and detection time. Moreover, the test costs
branes of the oral cavity, nasal cavity and eyes, infection may are rather high. These tests are mainly suitable for large and
occur. In addition, direct inhalation of the droplets generated centralized diagnostic laboratories. In addition, the time

Fig. 1 Structural proteins of SARS-CoV-2: envelope (E), membrane (M), spike (S), helicase (Hel) and nucleocapsid (N). Adapted with permission
from ref. 7. Copyright: 2020, the authors.

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Critical review Lab on a Chip

required for sample transportation far exceeds the time spent progress on microfluidic platforms in the context of basic
on testing, further delaying diagnosis.11 Serological testing in- biological research, clinical applications, environmental mon-
volves the use of blood samples to detect viruses. The two main itoring and food safety, focusing on microfluidics-based
antibodies tested in the blood are immunoglobulin G (IgG) and nucleic acid analysis and protein analysis.15 In addition,
immunoglobulin M (IgM).12 Currently, serological testing for Jiang et al. summarized the integration of microfluidics,
COVID-19 mainly involves the direct measurement of antigens nanotechnology, 3D printing and other advanced technolo-
and the indirect detection of antibodies in serum. Enzyme- gies to provide new platforms and tools for biological analy-
linked immunosorbent assay (ELISA) is also a sensitive method sis. A review article published by Qin et al. focused on the
for detecting COVID-19. Imaging also plays an important role in “lab in/on an X” or “LionX” experimental platform and
the diagnosis and treatment of COVID-19, especially in the early discussed nucleic acid testing on an integrated system that
stages of the disease. included sample pretreatment, nucleic acid extraction, ampli-
Due to the rapid development of COVID-19 and the lim- fication and signal detection.16 The review also highlighted
ited laboratory-based molecular detection capacity, a rapid promising diagnostic platforms and technologies that were
point-of-care testing (POCT) method is urgently needed to fa- expected to provide better solutions for the diagnosis of
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

cilitate COVID-19 diagnosis outside of the laboratory setting. COVID-19 and other diseases. On the bases of the aforemen-
POCT methods, such as some rapid tests for an infectious tioned articles, herein, we review existing POCT methods, as
disease diagnosis, provide results within minutes of the test well as POCT methods under development, for detecting
being administered, allowing for rapid decisions about pa- COVID-19; these methods include nucleic acid tests, serologi-
tient care. These devices are low cost and easy to use, provid- cal immunoassay tests, microfluidic platforms and portable
ing sufficient time for the implementation of necessary pre- instruments used for the rapid standard diagnosis of COVID-
ventive or therapeutic measures. So POCT devices can also 19. We also summarize and highlight various fast-detection
extend testing to communities and populations that cannot technologies and portable instruments for the detection of
readily access care. As quoted the POC test guideline from the novel coronavirus.
the United States' Centers for Disease Control and Prevention
(CDC), POC tests are used to diagnose COVID-19 in various 2. POCT based on nucleic acids
settings, such as physician offices, urgent care facilities,
pharmacies, school health clinics, long-term care facilities, To detect SARS-CoV-2 nucleic acid, the first step is to collect
nursing homes, as well as some temporary locations, such as biological fluids from where the virus is located. Fluids are
drive-through sites managed by local organizations.13 In our usually collected using nasopharyngeal swabs and oropharyn-
review article, the POC test, especially for COVID-19 test, geal swabs. The collected fluids are subjected to a series of fil-
mainly refer to a test scenario where a professional could exe- tration and isolation steps to obtain the virus. Microfluidic
cute a fast and frequent test, as the above mentioned. technology allows the miniaturization and integration of
As countries deal with the COVID-19 pandemic in varying nucleic acid purification and large-scale PCR amplification,
ways, one area of agreement is the need to test for the COVID- improving efficiency.14 After RNA extraction, reverse transcrip-
19 virus in as many people as possible. As reported in the UK tase is used to generate the complementary DNA (cDNA) of
media, access to COVID-19 (viral) testing has been limited for the viral RNA. Subsequently, specific regions of the cDNA are
some sections of the population including healthcare profes- amplified, in the presence of DNA probes, by PCR, thereby re-
sionals and careers. There have also been long delays in get- alizing the real-time detection of the amplification process.
ting the results back to the person being tested. A higher fre- Currently, nucleic acid testing for SARS-CoV-2 mainly in-
quency of test, a higher possibility of finding an infectious cludes RT-PCR-, loop-mediated isothermal amplification
people. The most merit of POCT is that it could realize a fast (LAMP)-, and clustered regularly interspaced short palindromic
and high frequency test. We could find out the infectious peo- repeats (CRISPR)/CRISPR-associated proteins (Cas)-based test-
ple immediately rather than delivering the sample to a central ing. This section introduces the latest research results from sci-
test lab and waiting a long time for a test result. entific research teams and commercialized POCT instruments.
POCT doesn't mean a lack of accuracy. Usually, a POCT
machine has the same sensitivity and specificity as the one 2.1 RT-PCR-based tests
used in central lab. Especially, some POCT instruments are PCR is a temperature-controlled biochemical reaction cata-
equipped with automated detection modules by which lyzed by enzymes. PCR instruments are manufactured based
sample-in and answer-out test procedures could be realized. on polymerase and are actually thermocyclers that control 3
This not only decrease the operation complexity, but also in- thermal cycles (namely, denaturation at 95 °C, renaturation at
crease the test sensitivity through an automatic process. 55 °C and extension at 72 °C). Each PCR cycle exponentially
Qin et al. summarized microfluidic technology for the increases the number of DNA fragments, thus producing a
POCT diagnosis of infectious diseases and introduced a fully large number of specific gene fragments in a certain period of
integrated POCT platform that included sample preparation, time. In a conventional desktop thermocycler, automated re-
on-chip nucleic acid analysis, immune analysis, and system actions can be conducted in EP tubes, and temperature differ-
integration.14 Jiang et al. systematically introduced research ences of 20 °C can be attained within 10 s. DNA chips can

1636 | Lab Chip, 2021, 21, 1634–1660 This journal is © The Royal Society of Chemistry 2021
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Lab on a Chip Critical review

attain temperature differences of 20 °C in less than 1 s be- against viral spike protein was incorporated into the patch to
cause of miniaturization, a smaller heat capacity and higher confer viral capture potential with MNs activity. The designed
thermal conductivity, greatly improving efficiency.17 For exam- Mn/AB swab can improve the sampling efficiency and reduce
ple, Wu et al. created a PCR device for rapid DNA amplifica- the “false negative”.23 Sputum, which can be obtained nonin-
tion and detection.18 As shown in Fig. 2, this device consists vasively, serves as a lower respiratory tract specimen. Lin
of an interchangeable PCR chamber, a temperature control et al. diagnosed patients with COVID-19 based on RT-PCR
system, and an optical detection system. The temperature analysis of sputum specimens.24 However, in a series of
control system can achieve high-precision temperature con- COVID-19 cases involving 41 patients, only 28% of the pa-
trol with effective heating/cooling rates. The PCR chip is inde- tients produced sputum suitable for diagnosis.25 RT-PCR re-
pendent of the temperature control system and the optical de- sults from the WHO's Hong Kong laboratory showed that
tection system. There are other microfluidic rapid-heating throat swabs obtained from patients tested positive for
systems based on infrared heating,19 air heating20 and micro- COVID-19.26 A study has shown that saliva has high concor-
wave heating.21 These noncontact systems have higher dance with nasopharyngeal specimens regarding the detec-
heating speeds (65 °C s−1), minimal over- or undershoot (±0.1 tion of coronavirus; the specificity was 90%.27 Moreover, sam-
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

°C) and high temperature accuracy (±0.1 °C).22 pling for saliva tests is easy and can be completed by patients
Currently, RT-PCR is the gold standard for COVID-19 diag- themselves, thus reducing the risk of infection for medical
nosis. Because RT-PCR can potentially detect a single RNA staff. Taking the S gene of SARS-CoV-2 as the target, Kelvin
molecule in one reaction, it is very accurate and sensitive for KW To et al. collected saliva from patients with confirmed
the detection of viral genomes. There are many molecular tar- disease and performed a 1-step RT-qPCR test using an RT-
gets in the sense-strand RNA genome of SARS-CoV-2 that can PCR kit.28 COVID-19 was detected in 11 of the 12 patients.
be used for PCR detection. The standardized scheme custom- RT-PCR primers have been designed to target different re-
ized by the WHO includes sample collection, sample trans- gions of the SARS-CoV-2 genome. Chu et al. developed two
portation, sample testing and test result analysis. Sample col- 1-step RT-PCR methods to detect the ORF1b and N regions of
lection mainly refers to collecting human nasopharyngeal the viral genome.29 To avoid the genetic diversity of
swab samples, oropharyngeal swab samples, sputum samples coronaviruses, primers and probes were designed to react
and saliva samples. Nasopharyngeal swabs and oropharyn- with the novel coronavirus. This method achieved the detec-
geal swabs are used to collect upper respiratory tract speci- tion of SARS-CoV-2. The highly conserved RdRp gene and var-
mens. The collection of these specimens requires medical iable S gene were introduced as the targets in another RT-
staff to be in close contact with patients, which has certain PCR detection study.30 In the RT-PCR-based test developed by
risks. The “false negative” rate of traditional oropharyngeal the German company Tib-Molbiol, the E gene of the SARS-
swab sampling affects the diagnosis results, Chen et al. used CoV-2 genome was used for first-line screening, while the
microneedle (MN) patches to design swabs to improve the RdRp gene was used for confirmatory testing.31 This test was
quality and quantity of virus collection. The antibody (Ab) highly specific for SARS-CoV-2 and had no cross-reactivity
with other coronaviruses. Chan et al. developed three RT-PCR
detection methods that targeted the RdRp/Hel, S and N re-
gions of the SARS-CoV-2 genome and compared their perfor-
mance.32 Among these methods, the COVID-19-RdRp/Hel
method had high sensitivity and specificity.
Shen et al. have developed an integrated microfluidic system
containing a microfluidic chip with a hemagglutinin (HA)/neur-
aminidase (NA) array.33 This system allows automatic virus puri-
fication, virus lysis, one-step RT-PCR and fluorescent signal de-
tection. Magnetic microbeads are used for virus extraction and
isolation, while pneumatically driven microvalves are used for
on-chip liquid control. On this microfluidic platform, up to 12
subtypes of influenza A virus (InfA) can be automatically and si-
multaneously detected in less than 100 min, with an analysis
speed significantly faster than other methods. The structure of
the microfluidic chip is shown in Fig. 3. The chip is 84 mm in
length, 59 mm in width, and 10 mm in depth. It consists of one
thick-film polydimethylsiloxane (PDMS) air control layer, one
Fig. 2 Exchangeable PCR chip and temperature control device (Wu thin-film PDMS liquid channel layer, and a glass substrate (G-
et al.18). (a) Vertical photograph of a PCR chip; (b) back-side view of a
Tech Optoelectronics) to seal the microfluidic chip. The process
heater chip; (c) the upper panel is the optical detection system. The
central panel is the sealed chip. After PCR reagents are injected into
for InfA detection is as follows. First, polysaccharide-modified
the PCR chamber, the PCR chip is placed on the back of the heater magnetic microbeads, a virus-containing sample, RT-PCR re-
chip. The lower panel is the temperature control system. agents, and wash buffer are loaded into the corresponding

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Critical review Lab on a Chip

(IATs), such as LAMP, recombinase polymerase amplification

(RPA), rolling circle amplification (RCA), exponential amplifi-
cation reaction (EXPAR), and strand displacement amplifica-
tion (SDA), allow the amplification of nucleic acids at a con-
stant temperature. IATs are able to overcome the
shortcomings of PCR-based amplification, such as high dena-
turation temperatures and the cumbersome cycling of sam-
ples among different temperatures during amplification. IATs
have been introduced into microfluidics for nucleic acid de-
tection.15 Currently, LAMP and RPA have achieved sensitivi-
ties that no other IAT can achieve when detecting low-copy-
number nucleic acids.
LAMP is a nucleic acid amplification technology that does
not require thermal cycling, and it has the ability to probe
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

multiple targets in a single reaction.36 LAMP synthesizes tar-

get DNA under constant temperature (60 to 65 °C) using spe-
cifically designed primers and DNA polymerase. This DNA
polymerase has strand displacement activity, which improves
the sensitivity and specificity of the results.37 LAMP can rec-
ognize 8 distinct target sites on a target sequence using 6
primers, generating 109 copies of a target sequence.38 The fi-
nal products are stem-loop DNAs with multiple inverted re-
Fig. 3 (a) A picture and (b) a detailed diagram of an integrated peats of the target. The structure of the final products has a
microfluidic chip. Gray, blue, red and green indicate the glass “cauliflower”-like appearance.
substrate, the liquid channel layer, the second air control layer and the Some research reports have shown the advantages of
first air control layer, respectively. The reaction chambers contain LAMP over RT-PCR in terms of sensitivity, accuracy and speci-
primer sets for amplifying specific regions of the HA and NA genes
ficity for SARS-CoV-2 detection. The results reported by Park
such that the RT-PCR-derived signal output can be used for viral
subtyping. Adapted with permission from ref. 33. Copyright: 2019, the et al. have shown that LAMP is specific for the detection of
authors. SARS-CoV-2 and has no cross-reactivity with other respiratory
pathogens such as human coronavirus strains HCoV-OC43
and HCoV-229E.39 Zhang et al. identified SARS-CoV-2 RNA
chambers. The virus is then isolated with microbeads. The mag- from purified RNA or lysed cells from patients employing vi-
netic microbead–virus complexes are collected by applying an sual and colorimetric detection. Using 130 samples, Yang
external magnetic field from the magnet. Subsequently, a ther- et al. were able to directly compare RT-PCR with RT-LAMP.40
moelectric (TE) cooler is placed underneath the chip. Viral RNA It was found that LAMP provided the same clinical diagnosis
is released by thermolysis and transported to the RT-PCR reac- as RT-PCR and displayed similar sensitivity. Moreover, LAMP
tion chamber (40 reaction cycles). Finally, RT-PCR signals are was faster, and the results were easier to read. Other research
detected using an optical detection module. groups reported similar results.41,42 LAMP amplification has
RT-qPCR has certain limitations. For example, sample pu- high sensitivity and specificity when used with samples from
rity must be high, and trained professionals are required. In patients and improves the accuracy of multigene target
addition, RT-PCR testing sometimes fails to detect SARS-CoV- amplification.
2 in the early stages of infection. Some reports have claimed Some research results are based on a visible colorimetric
that false negatives have occurred in subjects for up to 2 response or visual readout. In terms of the visual readout for
weeks.34,35 The possible reason is the difference of viral load, RT-LAMP, Baek et al. employed an assay combining RT-LAMP
and the amount of virus in various patients is different. Dif- and a phenol red colorimetric method, which had a fast de-
ferent course of disease can also lead to differences. It is also tection time of 30 min.43 The RT-LAMP detection primers
possible that the sampling site is not suitable, the specimen were designed to target the nucleocapsid (N) protein gene of
containing virus are not taken or the detection operation is SARS-CoV-2. The resulting RNA limit of detection was 102, i.e.
not standardized. These shortcomings limit the application , close to that for qRT-PCR. A color change in phenol red
of RT-PCR testing under many circumstances. from pink (pH 8.8) to yellow (pH < 8.0) indicates that ampli-
fication had occurred. There was no cross-reactivity with re-
lated coronaviruses, human influenza viruses and other vi-
2.2 LAMP-based tests ruses that cause respiratory diseases. Lu et al. tested a LAMP-
Although RT-PCR is the gold standard for the molecular diag- based detection method that targeted the RdRp gene.44 The
nosis of COVID, it has disadvantages; it is a time-consuming method was able to detect a colorimetric change after the ad-
and laborious method. Isothermal amplification technologies dition of SARS-CoV-2 RNA, indicating a positive result after

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Lab on a Chip Critical review

40 min of amplification. Moreover, the sensitivity of the

method reached 5 copies per microliter. Yin et al. described a
LAMP-based method, iLACO, for the rapid detection of SARS-
Cov-2, producing results consistent with those for RT-PCR.45
A fragment of ORF1ab was selected as the target region, and
the RT-LAMP primers were designed accordingly. The reac-
tion mixture was incubated at 65 °C for 20 min, resulting in Fig. 4 Schematic representation of the single-tube experimental pro-
a color change from pink to light yellow. The fluorescence cedure for Penn-RAMP. COVID-19-Penn-RAMP includes 2 isothermal
signal generated by iLACO was examined under ultraviolet amplification processes. The RPA reaction is performed at 38 °C on
the tube cap, and the LAMP reaction is performed at 63 °C in the tube.
and blue light. A positive signal was visible to the naked eye
The amplification process was monitored using the fluorescent dye
with a colorimetric pH indicator or under blue light with Leuco crystal violet (LCV). Adapted with permission from ref. 49. Copy-
GeneFinder dye. It was found through serial dilution of the right: 2020, the authors.
synthesized ORF1ab primers that iLACO could detect as few
as 10 copies of the ORF1ab gene and that the detection pro-
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

cess could be completed in 20–40 min. Therefore, iLACO is based POC diagnostic equipment and LAMP assay technol-
accurate and easy to implement. C. Yan et al. developed an ogy. This device can be integrated with a smartphone applica-
RT-LAMP assay for the visual detection of SARS-CoV-2.46 The tion to provide a fast, sensitive, and easy-to-use COVID-19 di-
authors correctly identified 58/58 positive and 72/72 negative agnostic tool (Fig. 5). People can collect nasal swab samples
patients; the results were confirmed by parallel RT-PCR test- by themselves; perform LAMP analysis; and observe the visi-
ing. The RT-LAMP analysis of RNA extracts from patient sam- ble colorimetric test results, record the data and share them
ples only required incubation at 63 °C for 60 min. Abbott ID with doctors or professionals via the internet.50 Song et al.
Now™ produced a kit that can detect SARS-CoV-2 within 5 developed a microfluidic platform for the detection of ZIKV.51
min.47,48 This kit has obtained emergency use authorization The process involves saliva sample collection and lysis,
(EUA) certification from the United States Food and Drug Ad- nucleic acid extraction via the isolation membrane of a
ministration (FDA). It uses NEAR for the qualitative detection microfluidic cassette, RT-LAMP amplification and colorimet-
of SARS-CoV-2 RNA and only requires a small portable touch ric DNA detection. Although the operation process can be
screen device, namely, ID Now. It employs an RdRp gene- completed in the same microfluidic chip system, reagents
based molecular detection method, and the test samples can and wash buffers still need to be added manually.
be nasopharyngeal swabs, oropharyngeal swabs or throat Sun et al. intended to use equine respiratory infection dis-
swabs. The kit contains 24 tests as well as negative and posi- eases as the model system for corresponding human diseases
tive controls, and its application is not restricted by location. such as COVID-19 (Fig. 6).52 Namely, the specific nucleic acid se-
Some research groups have combined RPA and LAMP into quences of 5 equine pathogens are LAMP-amplified on a micro-
a two-stage amplification protocol termed RAMP. Song et al. fluidic chip (reaction volume of 1 μL), and the reaction results
used bioinformatic approaches to design primers specific for are detected using a smartphone. The LAMP reaction takes less
the ORF1ab gene in SARS-CoV-2 RNA.49 The study reported a than 30 min, and the entire detection process can be completed
single-stage COVID-19-LAMP in a closed tube and a two-stage
isothermal amplification method (COVID-19-Penn-RAMP) in a
closed tube. COVID-19-Penn-RAMP includes 2 isothermal am-
plification processes (Fig. 4): RPA reaction at 38 °C on the
tube cap and a LAMP reaction at 63 °C in the tube. During the
detection process, a swab is first eluted in water, and the wa-
ter is then heated to above 65 °C. After being added to the
tubes, the samples are subjected to LAMP single-stage ampli-
fication or COVID-19-Penn-RAMP two-stage amplification.
The amplification process is monitored in real-time with the
fluorescent dye Leuco crystal violet (LCV). The performance of
COVID-19-Penn-RAMP is superior to those of COVID-19-PCR
and COVID-19-LAMP. The sensitivity of COVID-19-Penn-RAMP
is 10 times higher than that of LAMP when using purified
samples and 100 times that of LAMP when using minimally Fig. 5 Schematic diagram of the POCT RNA-based COVID-19 diag-
prepared samples. The RAMP method displays high specific- nostic device. In this view, the conceptual workflow of integrating both
ity, similar to LAMP, enhanced sensitivity resulting from two- POCT diagnostics and the LAMP assay with a mobile device is
presented. People at home can collect their own specimen using a na-
stage amplification, and a higher tolerance to inhibitors.
sal swab; after adding specific reagents for the LAMP reaction, the col-
There are also other tools that have application potential orimetric result can be observed. By recording the colorimetric change
for the diagnosis of COVID-19. Yang et al. have described an through a mobile phone, the user can upload the result. Adapted with
RNA-based POC device for COVID-19 that combines paper- permission from ref. 50. Copyright: 2020, the authors.

This journal is © The Royal Society of Chemistry 2021 Lab Chip, 2021, 21, 1634–1660 | 1639
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Critical review Lab on a Chip

within 1 hour using inexpensive and portable equipment. Sili- clipped onto a smartphone. The instrument would have a me-
con microfluidic chips have multiple advantages. These chips chanical adaptor that aligns with the rear-facing camera of sev-
display high stability, have no autofluorescence and can be eral popular mobile phone models.
manufactured efficiently. The structure of a microfluidic chips Wang et al. developed a LAMP-integrated microfluidic chip
is shown in Fig. 6. The chips (25 mm × 15 mm × 0.5 mm) con- system to detect multiple respiratory viruses (Fig. 7).53 The en-
tain 10 parallel flow channels. The channels are 10 mm in tire detection process can be completed within 1 h. This sys-
length, 500 μm in width and 200 μm in depth. The volume of tem is able to specifically recognize influenza A virus subtypes
each channel is 1 μL, and all channels share the same inlet. (H1N1, H3N2, H5N1 and H7N9), influenza B virus and human
The inlet of each chip is an entrance chamber with a diameter adenoviruses with high specificity and high sensitivity. The
of 4 mm, from which the contents are spread to 10 parallel as- microchannels of the chip are approximately 500 μm in width
say channels. There are 2 square markers at the opposite ends and 200 μm in depth. The chip contains 8 independent reac-
of each chip that are used for position alignment during fabrica- tion microchambers (2 × 1 × 1 mm), with a corresponding re-
tion and automated image recognition during measurement. It action volume of approximately 2 μL. The chip system is
has been reported that bare silicon absorbs polymerase, thus closed, which reduces aerosol contamination and reagent
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

inhibiting nucleic acid amplification. Therefore, the surface of evaporation during LAMP. The entire detection process in-
the chip is thermally oxidized to grow a 200 nm layer of SiO2. cludes sample collection, nucleic acid extraction, sample load-
The experimental procedure is as follows: first, the control ing, real-time detection, and signal output. The specific pro-
groups and the targeting primers are deposited into clean chip cess is as follows. First, a throat swab sample is collected
channels. Once the reagents in the microfluidic chip channels from an individual. Nucleic acids are then quickly extracted
dry, the channels are covered with a transparent double-sided from the sample using magnetic nanoparticles. After extrac-
adhesive. Subsequently, LAMP reaction mix is injected from the tion, the nucleic acids are added to the microchambers of the
inlet, and the chip is sealed with cover glass. The chip is heated microfluidic chip. The extracted nucleic acid sample reagents
to 65 °C to drive the LAMP enzymatic amplification reaction are diverted into the 8 microchambers along different chan-
and then inserted into a scaffold for endpoint fluorescence im- nels, which are then covered with film to ensure that LAMP is
aging. Fu Sun and colleagues planned to use this chip for rapid carried out within a sealed chip. The real-time colorimetric
POC detection of SARS-CoV-2. The fluorescent endpoint images detector provides real-time amplification curves based on
of the LAMP reaction are collected using a mobile phone cam- color changes in the chip microchambers. Using this
era. Data collection can be integrated with telemedicine plat-
forms, which would allow sharing the detection results. In addi-
tion, Sun et al. envisaged a detection instrument that could be

Fig. 7 Closed microfluidic chip system for the detection of a variety of

Fig. 6 Ten channel microfluidic chip for multiplexed LAMP detection. respiratory viruses. (a) It consists of 8 independent reaction
(a) The control and the target primer are put into the channel of the microchambers (2 × 1 × 1 mm) with a reaction volume of approximately
chip, which is then covered with double-sided adhesive tape; (b) LAMP 2 μL. The extracted nucleic acid sample reagents are transferred to
reaction mixture is injected at the inlet, and the chip is sealed; (c) the 8 microcavities along different channels and covered with a thin film to
chip is heated to 65 °C for the LAMP reaction, and then the chip is ensure that the LAMP reaction occurs in a sealed chip; (b) real-time de-
inserted into a scaffold for fluorescence imaging. Adapted with permis- tection instrument; (c) real-time amplification curves. Adapted with
sion from ref. 52. Copyright: 2020, the authors. permission from ref. 53. Copyright: 2018, the authors.

1640 | Lab Chip, 2021, 21, 1634–1660 This journal is © The Royal Society of Chemistry 2021
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Lab on a Chip Critical review

microfluidic chip, COVID-19 can be detected rapidly through and accurate fluorescence diagnostic method based on RT-
the use of LAMP primers for SARS-CoV-2. PCR/CRISPR-Cas12a. The CRISPR-FDS methods, includes 3
Compared with traditional technology, LAMP provides steps, the RNA extraction, target amplification, and fluores-
sensitive, accurate and specific results in a short time. It pro- cent signal detection (Fig. 8). Detection by this method re-
vides a potential platform for the POC detection of COVID-19 quires that a sample contains at least 2 copies of the target
in the community. LAMP reduces the time required for trans- RNA sequence; no detectable target signal is produced if the
ferring samples to a centralized laboratory and for standard DNA target amplified by qPCR has less than 5 copies. This as-
RT-PCR testing. In addition, LAMP does not need to be say has a sample-to-answer time of approximately 50 min,
performed in central laboratories by skilled scientific re- and the reagents and equipment are easy to obtain. There-
search and medical personnel, which means that front-line fore, this method has the potential for use in POCT if the re-
medical workers can perform POC testing for COVID-19 upon sults are analyzed with a portable fluorescence reader.58
demand. Moreover, the isothermal platform is low cost and Wang et al. have developed a CRISPR/Cas12a-based assay
does not require expensive reagents or instruments, reducing with a naked-eye readout. This method displays high sensitiv-
the cost of COVID-19 testing. ity and specificity and is capable of detecting as few as 10
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

copies of a viral gene within 45 min. This detection system

provides an ssDNA reporter labeled with a quenched green
2.3 Tests based on the CRISPR/Cas system fluorescence molecule that is cleaved by Cas12a protein in
The CRISPR/Cas system is the immune system of bacteria, the presence of SARS-CoV-2 nucleic acids. As the result, a
fighting against foreign DNA or RNA invasion. Bacteria recog- green fluorescence signal visible to the naked eye is pro-
nize target DNA/RNA and cleave the invading foreign nucleic duced.59 Among the applied SARS-CoV-2 detection systems,
acids through CRISPR RNA (crRNA) and Cas proteins. CRISPR the “All-In-One Dual CRISPR-Cas12a” (AIOD-CRISPR) system
is a powerful gene editing technology that can be used to designed by Ding et al.60 can detect as low as about 5 copies
trim, cut, replace or add to the DNA sequences of organisms. of an RNA target with an incubation time of 40 min. Charles
Therefore, CRISPR/Cas is referred to as “molecular scis- Y. Chiu and colleagues (University of California) have devel-
sors”.54 In recent years, it has been shown that CRISPR and oped a sensitive, rapid, and portable SARS-CoV-2 detection
its related proteins, mainly Cas12a and Cas13, can be used to assay based on CRISPR-Cas12 technology. This assay can be
detect specific nucleic acids in samples. Cas12a and Cas13 run in approximately 30–40 min (ref. 61) and can be used to
bind to RNA or DNA targets, respectively, specified by guide detect SARS-CoV-2 in respiratory swab RNA extracts. It uses
RNA (gRNA) sequences, they can combine with the fluoro- the E gene, N gene and the human RNase P gene as a control
phore quencher DNA probe to produce signal amplification. and consists of an RT-LAMP reaction at 62 °C for 20–30 min
The predicted sequence of COVID-19 was detected by Cas12a and a Cas12 detection reaction at 37 °C for 10 min. The re-
or Cas13, and then the virus was confirmed by cleavage of re- sults are visualized on a lateral flow strip (Fig. 9).
porter molecule.55,56 Recently, multiple detection methods Similarly, CRISPR diagnosis is suitable for RNA molecular di-
combining various isothermal amplification techniques (such agnosis. In 2016, Zhang et al. found that Cas13a would cleave
as LAMP and RPA) and CRISPR have developed into diagnos- any nearby RNA after cleaving the target RNA sequence.62 Such
tic tools for the rapid detection of SARS-CoV-2 RNA. phenomenon is known as “collateral cleavage”. Doudna et al.
Cas12 protein specifically cuts double-stranded DNA. In employed the Cas13a protein for RNA diagnosis.57 However, the
addition, Cas12a has accessory activities. Once crRNA specifi- development of CRISPR-based RNA diagnostic technology has
cally binds to a target sequence, Cas12a not only cleaves the been limited due to low sensitivity. In 2017, Zhang et al. opti-
target sequence but also cuts any single-stranded DNA mized the CRISPR/Cas13a diagnostic platform. The SHERLOCK
(ssDNA) in the system.57 Huang et al. have described a rapid (specific high-sensitivity enzymatic reporter unLOCKing) system

Fig. 8 A CRISPR-based fluorescence diagnosis system for COVID-19 (COVID-19 CRISPR-FDS). (a) Schematic diagram of a CRISPR-FDS assay for
the detection of SARS-CoV-2 RNA; (b) SARS-CoV-2 genome map of COVID-19 CRISPR-FDS target sequences. Adapted with permission from ref.
58. Copyright: 2020, the authors.

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Critical review Lab on a Chip

Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

Fig. 9 CRISPR-based detection. (a) Schematic of the COVID-19 detection process: RNA extraction, reverse transcription, LAMP amplification, and
Cas12-based target gene detection (E, N, and RNase P). These results can be displayed on a horizontal flow strip. (b) Cross flow strip analysis re-
sults: the control line (closest to the sample pad) and the test line (furthest from the sample pad). (c) Example results: a positive result requires the
detection of at least the 2 SARS-CoV-2 gene targets (N gene and E gene). Adapted with permission from ref. 61. Copyright: 2020, the authors.

enhances diagnostic sensitivity by 1 million-fold and is capable Cycler system, integrated and fast real-time fluorescence PCR
of detecting ZIKV or Dengue virus at as low as 1 copy per micro- detection is obtained. Based on that, Cepheid developed a pre-
liter.63 This method is able to consistently detect synthesized processing cartridge that uses the rotation and movement of a
SARS-CoV-2 RNA within a range of 10–100 copies per microliter central plunger to achieve connectivity and liquid movement be-
of input and allows a visual readout of the detection results via tween different chambers on the cartridge and integrated
a lateral flow strip. In addition, the detection process can be nucleic acid extraction and nucleic acid amplification detection
completed within 40 to 57 min after the RNA extraction step.64 on one instrument——GeneXpert.67 In addition, this test can
In February 2020, Zhang et al. employed the CRISPR/Cas13- quickly detect the currently prevalent SARS-CoV-2 within 30
based SHERLOCK technique to detect the novel coronavirus min, with reduced sample preparation duration down to 1
and successfully detected the virus within the range of 10–100 min.68 Femke Wolters et al. compared the Xpert Xpress assay
copies per microliter of input. This method is simple to imple- for SARS-CoV-2 with the traditional in-house RT-PCR method.69
ment. It only requires purified nucleic acid samples and can be In 58 positive samples and 30 negative samples, the Xpert
completed in 1 h. Moreover, this method is able to quickly and Xpress assay results were consistent with those for the tradi-
accurately detect SARS-CoV-2 and exhibits high sensitivity.65 tional in-house RT-PCR method. The limit of detection (LOD)
was 8.26 copies per mL, even lower than described in the prod-
uct manual (250 copies per mL).70 Wei Zhen's group evaluated 3
2.4 Commercial nucleic acids POCT products POCT nucleic acid detection systems using 108 symptomatic pa-
Nucleic acid detection method was the gold standard for the tients' nasopharyngeal swabs and found that the Cepheid Xpert
diagnosis of COVID-19 at the beginning, and it currently has Xpress SARS-CoV-2 Assay had a positive agreement of 98.3%
the most products (Table 1). Some of the products are “all in and a limit of detection of 100 copies per mL, the Abbott ID
one” and can achieve fully automated “sample-in answer-out” NOW COVID-19 Assay had a positive agreement of 87.7% and a
detections, while others mainly focus on the detection pro- limit of detection of 20 000 copies per mL, and the GenMark
cess. The “all in one” products' operations are simpler, while ePlex SARS-CoV-2 Test had a positive agreement of 91.4% and a
the others are more flexible and affordable. limit of detection of 1000 copies per mL.71 In terms of detection
Cepheid used a special reaction tube in the Smart Cycler sys- time, ID NOW was much faster (17 min) when compared with
tem66 to achieve real-time PCR detection in early studies. The re- Cepheid (46 min) and ePlex (approximately 1.5 h).
action tube facilitates optical conduction and rapid heat con- Bosch Healthcare Solutions announced the development
duction, and together with the I-CORE module in the Smart of a fully automated PCR assay that integrated sample

1642 | Lab Chip, 2021, 21, 1634–1660 This journal is © The Royal Society of Chemistry 2021
 Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

Table 1 Commercial nucleic acid POCT products

Positive/ Number of
Detection Detection Detection Negative samples Limit of
No. Device Company technology Sample type duration All-in-one? Regulatory vessel agreement per test detection
Lab on a Chip

1 ID NOW Abbott NEAR NS/OS/NPS 5–13 min Yes FDA EUA Cartridge 93.3%/98.4% 1 125 genome
COVID-1994,95 Diagnostics equivalents
Scarborough, per mL
Inc.
2 VitaPCR A. Menarini Real-time NPS/OS 20 min No CE Tube 100%/100% 1 2730 copies
Platform96 Diagnostics PCR per mL
s.r.l
3 BioFire BioFire Nested NS 45 min Yes FDA EUA, Cartridge 97.1%/99.3% 1–12* Heat-inactivated
Respiratory Diagnostics, multiplex CE virus ATCC
Panel 2.1 LLC PCR VR-1986HK
(RP2.1/2.1-EZ)97,98 500 copies
per mL
Infectious virus

This journal is © The Royal Society of Chemistry 2021

160 copies
per mL
4 Biomeme Biomeme, Inc. Real-time NPS/NS/OS/ 1h No FDA EUA Tube 97.46%/98.51% 9 1800 genome
SARS-CoV-2 PCR NPW-/NA/ equivalent
Real-Time aspirate per mL
RT-PCR Test99
5 Vivalytic VRI Bosch Multiplex NS/OS 39 min Yes CE Cartridge 98%/100% 1 —
Multiplex Test72,100 Healthcare PCR,
Solutions μArray-detection
GmbH
6 Xpert Xpress Cepheid Real-time PCR NPS/NW/ 45 min Yes FDA EUA, Cartridge 100%/100% 1–80* Reference
SARS-CoV-2 Test101,102 aspirate CE material 250
copies per mL
SARS-CoV-2 virus
(USA_WA1/2020)
0.01 PFU mL−1
7 ePlex GenMark eSensor NPS 2h Yes FDA EUA, Cartridge 99.02%/98.41% 1–24* 1000 copies
Respiratory Diagnostics, technology CE per mL, 750
Pathogen Inc. genomic copies
Panel 290,103 per mL
8 iC-COVID19 iCUBATE ARM-PCR NPS/NS/MNS 6h Yes — Cartridge — 1 180 copies
Assay104,105 per mL
9 Automatic Lifereal Real-time OS/ALF/ 1.5 h Yes CE Cartridge 97.62%/100% 1–4* 365 copies
Integrated Biotechnology PCR sputum-/stool per mL
Gene Co., Ltd.
Detection
System106,107
10 Lucira Lucira Health, LAMP NS 30 min Yes FDA EUA Tube 94%/98% 1 900 copies
COVID-19 Inc. per mL
All-In-One
Test kit#108,109
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Lab Chip, 2021, 21, 1634–1660 | 1643

Critical review
 Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

Table 1 (continued)

Positive/ Number of
Detection Detection Detection Negative samples Limit of
No. Device Company technology Sample type duration All-in-one? Regulatory vessel agreement per test detection
11 Microchip Lumex Real-time NS/OS/LRTA/ 50 min No RUO Microfluidic — 7 9000 copies
Critical review

RT-PCR Instruments PCR BLF/NPW/ Chip per mL

COVID-19 NA/aspirate/
Detection sputum
System110
12 ARIES Luminex Real-time NPS 2h Yes FDA EUA Cartridge 100%/100% 1–12* 1500 copies
SARS-CoV-2 Assay111 Corporation PCR per mL
13 Veri-Q MiCo BioMed Real-time NPS/OS/sputum 55 min No CE Microfluidic — 1 —

1644 | Lab Chip, 2021, 21, 1634–1660

PCR 316 Co., Ltd. PCR Chip
COVID-19
Detection Platform112,113
14 LamPORE Oxford Nanopore Nanopore NS/NPS/OS 2h Yes CE Flow cell 99.1%/99.6% 24–480 7000–10 000
assay88,114 Technologies sequencing genome copies
combined per mL of
with LAMP extracted RNA
15 QIAstat-Dx115,116 QIAGEN Real-time NPS 1h Yes FDA EUA, Cartridge 100%/93% 1–4* 500 copies
GmbH PCR TGA, CE per mL
16 iPonatic117,118 Sansure Real-time NPS/OS/ALF/ 15–45 Yes CE, NMPA Tube 98.68%/99.35% 1 Sensitivity
BioTech Inc. PCR Sputum/serum/ min 1 copy, LOD
whole blood/stool less than 2 nM
17 Sherlock Sherlock RT-LAMP NS/NPS/OS/NPW/ 1 h No FDA EUA HybriDetect 97%/100% Observe 6750 copies
CRISPR BioSciences, and CRISPR aspirate/NA/BLF Strips results with per mL VTM
SARS-CoV-2 Kit119,120 Inc. naked eyes,
unlimited
18 PCR Shineway Real-time NS/NPS/OS/ 50 min No CE Microfluidic 99.5%/100% 3 500 copies
Nucleic Acid Technology PCR sputum Chip per mL
Analyzer77,78 Corporation
19 Automatic CPA Ustar CPA OS/sputum 55 min Yes TGA, Tube 98%/95.4% 1–2 —
Nucleic Acid Biotechnologies NMPA
Analyzer121,122 (Hangzhou) Ltd.
20 WizDx F-150 Wizbiosolutions Ultra-fast NPS/OS/ 40 min No CE, MFDS Microfluidic — 10 19.7 copies
Real-time PCR Inc. real-time sputum after RNA Chip per rxn (E gene)
System80,81 PCR extraction 23.4 copies
per rxn
(RdRP gene)

Notes: 1. Sort by first letter of company name. 2. All the carriers (cartridges, microfluidic chips and tubes) are for single use. ‘*’ means one carrier can detect one sample, but the machine
has the ability to test more carriers at one time. 3. ‘#’ means this product is authorized for prescription home use. 4. List of abbreviations: (1) NEAR, nicking endonuclease amplification
reaction; ARM-PCR, amplicon-rescued multiplex PCR; CPA, cross priming amplification; LAMP, loop-mediated isothermal amplification. (2) FDA EUA, U.S. Food & Drug Administration Emer-
gency Use Authorizations; CE, Conformite Europeenne; NMPA, National Medical Products Administration; TGA, Therapeutic Goods Administration; MFDS, Ministry of Food and Drug Safety;
RUO, research use only. (3) NS, nasal swabs; TS, throat swabs; NPS, nasopharyngeal swabs; OS, oropharyngeal swabs; MNS, mid-turbinate nasal swabs; NPW, nasopharyngeal wash; NW, na-
sal wash; ALF, alveolar lavage fluid; BLF, bronchoalveolar lavage; LRTA, lower respiratory tract aspirates; NA, nasal aspirate. https://www.fda.gov/medical-devices/coronavirus-disease-2019-
covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas;; Web: http://app1.nmpa.gov.cn/data_nmpa/face3/dir.html#maodian2;; Web: https://www.finddx.org/covid-19/
pipeline/.
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Lab on a Chip

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Lab on a Chip Critical review

preparation, DNA amplification, detection (ion probe array), reaction chamber was made on silicon wafer by dry etching,
and analysis in a single cartridge.72 A cotton swab (nasopha- and then a glass wafer was bonded to the silicon wafer. A robust
ryngeal or oropharyngeal) is inserted into the cartridge and package and sealing technology were developed to prevent water
instrumentally analyzed. The results are delivered within 2.5 evaporation during a fast PCR thermal cycling. In their previous
h. This test platform, named Vivalytic VRI, allows the selec- researches, SRY gene was successfully detected from saliva sam-
tion of different test samples and analytic methods. A car- ples within 25 min.18 And Escherichia coli and Salmonella were
tridge for COVID-19 detection only is also released and it only detected within 25 minutes, and the detection results were con-
takes 39 min to get a result. However, the diagnostic kit can sistent with the result on Roche LightCycler 480 machine.79
only be used in specific analytical equipment, and many The Shineway system can achieve the SARS-CoV-2 PCR de-
medical institutions do not yet have those devices. tection within 40 min,77 which is only half or less of the time
The FilmArray system invented by BioFire Defense uses a of traditional PCR machines. The dimension of the machine
flexible bag instead of a cartridge to achieve POCT molecular is only 280 mm × 240 mm × 162 mm, and it incorporates a
diagnosis.73,74 Different reagents are preinstalled in different touch-screen control system that eliminates the requirement
parts of the bag. Liquid flow is controlled by squeezing the of a computer. The Shineway system is compatible with third
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

reagent bag with the instrument, and magnetic bead nucleic party reagents. 42 clinical samples of SARS-CoV-2 were tested
acid extraction and nested PCR with melting curve analysis and the specificity was 100%. The detection limit of SARS-
are used to detect viruses. The system can detect more than CoV-2 on this machine was around 500 copies per mL.
20 pathogens at the same time. Zaneeta Dhesi et al. used the Shenzhen Shineway Technology also proposed a complete so-
FilmArray system to detect secondary infections in 94 COVID- lution for POCT SARS-CoV-2 detection, the portable PCR ma-
19 patients in the ICU of 5 UK hospitals.75 They found that chine, a preprocessor and all other accessories were all inte-
the FilmArray system generated more (54% vs. 28%) positive grated into a suitcase, and can be taken to detection site
secondary infection results than traditional bacterial culture easily. This system has been used in inspection vehicles and
did. Using the FilmArray system, Yosuke Hirotsu et al. scre- public places on-site inspection.
ened 191 patients with cold symptoms and found that 32 pa- Wizbio Solutions invented a WizDx F-150 ultrafast real-
tients were infected with at least 1 virus, among which 8 were time PCR system (Fig. 10b),80,81 this system can be used with
infected with only SARS-CoV-2.76 a PCR chip and COVID-19 CrystalMix to detect SARS-CoV-2.
Nature Biotechnology reported an on-site rapid molecular di- The PCR chip is made of a transparent polymer, and the in-
agnostic system (Shineway) for COVID-19 detection (Fig. 10a).77 lets are sealed with paste; 10 samples can be tested together
The system was developed by Wen's group in Hong Kong Uni- on 1 chip. The limit of detection is 19.7 copies per rxn.
versity of Science and Technology and input a mass production WizDx F-150 can control temperature at a rate of 8 °C s−1.
in Shenzhen Shineway Technology Corporation.78 It used a ID NOW COVID-19 system from Abbott Diagnostics, Inc.
novel thin film silicon-based micro-heater for rapid heating and can achieve SARS-CoV-2 detection in less than 13 minutes.48
a silicon-based microfluidic chip for nucleic acid PCR amplifica- The system uses an isothermal nucleic acid amplification
tion.18 The heater and Pt thermal sensor were combined to- technology called “NEAR” (nicking endonuclease amplifica-
gether, which improved the speed of heating and cooling while tion reaction). There is a sample receiver to release nucleic
greatly reduced the complexity of the temperature control sys- acids from the swabs, a test base to do the detection and a
tem. This heater can reach a temperature rising rate up to 30 °C transfer cartridge to remove nucleic acids from sample re-
s−1.79 The microfluidic chip was made of silicon and glass, and ceiver to the test base. Harrington et al. did a comparison of
silicon is a good heat conductor, therefore, the rapid tempera- Abbott ID Now and Abbott m2000 methods for the detection
ture change of the heater can be transmitted to the reaction so- of SARS-CoV-2, and they found that the negative agreement
lution in time and significantly reduce the detection time. The was 99% while positive agreement was 75%.82

Fig. 10 Commercial POCT nucleic acid detection products on SARS-CoV-2. (a) Shineway's portable fast microchip based PCR Analyser (ref. 77);
(b) WizDx F-150 ultra-fast real-time PCR system. Adapted with permission from ref. 81. Copyright: 2020, the websites.

This journal is © The Royal Society of Chemistry 2021 Lab Chip, 2021, 21, 1634–1660 | 1645
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Critical review Lab on a Chip

Sequencing technology is another way to detect SARS-CoV- After the window period, as the antibody concentration in
2 nucleic acids. In the early stage of the COVID-19 outbreak, the patient's body rises, the accuracy of the immune detec-
genome sequencing was used to obtain the genome sequence tion method gradually increases. Antibody screening mainly
information of the virus,83 which provided information for involves IgM and IgG detection. When SARS-CoV-2 invades
the subsequent PCR detection of SARS-CoV-2. Limited by the human body, IgM antibody will appear in about 1 week,
long detection times and high detection costs, sequencing and then IgG antibody will appear in about 1–3 weeks or even
technology has not been applied to SARS-CoV-2 detection on longer;127,128 the concentration will be much higher and du-
a large scale. However, genome sequencing still plays an im- ration much longer than those for IgM.3,129 Immunoassays
portant role in detecting gene mutations,84 researching and can help clinicians determine different stages of viral infec-
analyzing the evolution of SARS-CoV-2,85 researching its path- tion in patients and are effective complementary methods to
ogenic mechanism86 and so on. SARS-CoV-2 nucleic acid detection.130,131
Ming Wang et al. used Oxford Nanopore MinION/GridION Based on different detection techniques, immunoassays
to detect SARS-CoV-2 and other respiratory viruses by nano- mainly can be divided into colloidal gold/immunofluores-
pore sequencing.87 When a nucleic acid passes through the cence chromatography (lateral flow immunoassay, LFA), en-
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

nanopore protein, an electrical signal can be recorded in zyme linked immunosorbent assay (ELISA) and chemilumi-
real-time. In the tests, 22 of 61 suspected COVID-19 samples nescence. Most LFA methods produce test results within 10–
were diagnosed as positive by nanopore sequencing, while 15 min. They have the advantages of simple operation, low
negative or inconclusive results were obtained by RT-qPCR. price, fast detection, etc. ELISA and chemiluminescence are
However, it took 6–10 h for each sequencing test. The results more sensitive and specific than LFA,132,133 but they require
showed that when samples cannot be accurately diagnosed, complex and long protocols as well as large machines.134
sequencing can be a complementary approach to RT-qPCR. However, recently, some studies make POCT via ELISA and
Recently, Oxford Nanopore Technologies developed a chemiluminescence possible, and other methods such as
LamPore assay.88,89 LamPore combines LAMP with nanopore se- SERS and digital immunoassay are also expected to be used
quencing and greatly shortens the detection time. The assay in the detection of SARS-CoV-2.
takes approximately 2 h to test 1–96 samples in 1 single flow cell.
The ePlex SARS-SoV-2 Test system from GenMark Diagnos-
tics also uses a cartridge to achieve nucleic acid extraction 3.1 Lateral flow assay-based tests
and detection.90,91 Different from Cepheid's real-time PCR Benjamin D. Grant et al. used commercially available re-
method, ePlex uses an electrowetting method (EWOD) to ma- agents and developed a half-strip lateral flow assay method
nipulate droplets moving in a planar electrode array. Compet- for POCT SARS-CoV-2 antigen detection (Fig. 11a);135 the as-
itive DNA hybridization and electrochemical detection are say has a limit of detection of 0.65 ng mL−1 recombinant
used in the ePlex system. Katharine Uhteg et al. used clini- antigen. Tian Wen and Chao Huang et al. optimized the pH,
cally negative samples spiked with known concentrations of antigen concentration and several other parameters for the
SARS-CoV-2 RNA as evaluation samples and obtained a limit lateral flow immunoassay strip and obtained specific and sta-
of detection of 600 copies per mL.92 In addition, tests for ble detection results for IgG and IgM antibodies against
both positive and negative samples showed 100% repeatabil- SARS-CoV-2 (Fig. 11b).136,137 The sensitivity and specificity of
ity. A comparative experiment conducted by Wei Zhen et al. IgM detection were 100% and 93.3%, using RT-PCR results
showed that among 104 clinical samples, ePlex showed a as a comparison. Zhenhua Chen et al. used lanthanide-doped
96% positive coincidence rate and a 100% negative coinci-
dence rate, and the detection sensitivity of ePlex was 1000
copies per mL.93

3. POCT based on immunoassay

According to the classification of detection targets, SARS-
CoV-2 immunoassays mainly include antigen detection and
antibody detection.123 Theoretically, SARS-CoV-2 antigen can
be detected once an individual is infected. However, in real-
ity, the sensitivity of antigen detection is lower than that of
RT-PCR method and is better to be used a few days after Fig. 11 Lateral flow assays. (a) A half-strip was constructed using 20
symptom onset.124,125 Different from antigen detection, anti- mm of a nitrocellulose analytical membrane, and 20 mm of wicking
body detection can only be achieved after the body produces pad. Reused with permission from ref. 135. Copyright: 2020, the Amer-
ican Chemical Society. (b) Structure of the LFIA strip and specificity of
an immune response. There is a window period from when
the LFIA strips. Adapted with permission from ref. 136. Copyright:2020,
the virus enters the human body to the time the immune re- the Royal Society of Chemistry. (c) LNP method for detecting anti-
sponse produces antibodies.126 During the immune response, SARS-CoV-2 IgG. Reused with permission from ref. 138. Copy-
detection tools are prone to produce “false negative” results. right:2020, the American Chemical Society.

1646 | Lab Chip, 2021, 21, 1634–1660 This journal is © The Royal Society of Chemistry 2021
 View Article Online

Lab on a Chip Critical review

polystyrene nanoparticles (LNPs) as a fluorescent reporter one. This platform performs chemiluminescence-based ELISA
and detected anti-SARS-CoV-2 IgG in clinical samples. They detection. For malaria, the LOD was 8 ng mL−1. There is a
observed good consistency with RT-PCR results (Fig. 11c).138 capillary pump on the chip, and the capillary force drives the
The use of LFA method to detect COVID-19 has been widely sample through channels and detection regions; no extra
commercialized, and it has low requirements for the detection power supply was required. This research result is expected
environment and instruments, and can realize a on-the-go in- to be used in the detection of COVID-19.
spection and expand the application scenarios. But because the The ELISA method can quantitatively detect SARS-CoV-2,
samples are directly tested without processing, some interfering and the result is more accurate than LFA method. At the
substances may prone to lead to false positive results. To reduce same time, this method has lower requirements for the de-
the false positive rates, optimizing the processing of the strip tection environment than nucleic acid detection method.
materials to enhance the filtering ability of interfering sub- However, due to the complicated operation steps, only parts
stances, and adjusting reagent formulations to enhance the of the operation of ELISA or chemiluminescence were
anti-interference ability are necessary. achieved by microfluidic methods in many researches. Few
have achieved full-process automated POCT ELISA or chemi-
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

luminescence. A large number of commercial chemilumines-

3.2 ELISA-based tests cence products have been introduced to the market. We be-
Siddhartha Tripathi and Amit Agrawal proposed a microfluidic lieve that POCT chemiluminescence devices are also on the
sandwich ELISA system to detect SARS-CoV-2 antibodies. On way to researches and products.
the microfluidic chip, a T-shaped microchannel is used to sepa-
rate plasma from whole blood (Fig. 12a).139–141 The plasma is
then used for a SARS-CoV-2 ELISA. Approximately 10 μL of 3.3 Other methods
plasma can be isolated from 1 mL of whole human blood in ap- In addition to LFA, ELISA, and chemiluminescence, many re-
proximately 3 min. Xudong Fan's group invented a microfluidic searchers have developed other methods to achieve immuno-
ELISA method to quantitatively and sensitively detect SARS- assay of COVID-19.
CoV-2. IgG and viral antigen-S protein in serum were used as Jilie Kong's group developed a point-of-care microfluidic
targets (Fig. 12b).142 The ELISA took 15–20 min to complete. A platform to detect SARS-CoV-2 IgG, IgM and antigen
capillary sensor array with 12 channels was used as an ELISA (Fig. 13a).146 The diagnostic microchips need to be used in a
reactor. An automated system was used to operate the micro- fluorescence detection analyzer, with results available in 15
fluidic chip.143,144 Humanized chimeric SARS-CoV-2 antibodies min. There is a loading chamber, a waste reservoir and a
were used to test the limit of detection, and the LOD was 2 ng fluorescence immunoassay fluid channel on the microchip.
mL−1. In addition, commercial human-cell-expressed SARS- The fluorescence immunoassay fluid channel has a capture
CoV-2 S1 protein can be detected at an LOD of 0.4 ng mL−1. region and a test region. Fluorescent microsphere (FMS)-la-
Sthitodhi Ghosh et al. developed a microchannel capillary beled capture antibodies are printed in the capture region.
flow assay (MCFA) platform.145 The platform comprises an When sample added into the loading chamber, SARS-CoV-2
MCFA chip, a highly-sensitive optical detector and a smartph- biomarkers (IgG/IgM/antigen) specifically bind to the

Fig. 12 (a) The design and experimental photograph of microdevice showing plasma separation. Adapted with permission from ref. 140.
Copyright:2016, Springer Nature Limited. (b) The COVID-19 related immunoassays that were performed with the microfluidic chemiluminescent
ELISA platform. Reused with permission from ref. 142. Copyright: 2020, Elsevier. Reused with permission from ref. 143. Copyright: 2018, the
American Chemical Society.

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Critical review Lab on a Chip

antibody in the capture region. Then, due to capillary effect, DMF platform automatically manipulates reagents to conduct
the “antigen–antibody complexes” and FMS flow to and im- sandwich immunoassays of antigens in samples. SERS is used
mobilize in the test region. After 10 min, the microchip must to detect signals. The DMF-SERS method has an LOD of 74 pg
be centrifuged for 10 s and placed in a portable fluorescence mL−1 and a detection time less than 1 h. After modifying the de-
analyzer to obtain the results. This platform offered a rapid tection kit, the platform, with high sensitivity and automation,
and easy-to-use SARS-CoV-2 detection method. can be used for SARS-CoV-2 detection. Ying Mu's group devel-
Benjamin L. Miller's group reported an arrayed imaging oped a self-compartmentalization bead-based digital immuno-
reflectometry (AIR) platform that can detect 9 antibodies of assay chip.152,153 The reagent in the chip is self-driven. Their
upper respiratory pathogens, including SARS-CoV-2, on 1 group also developed a portable smartphone-based device for
chip (Fig. 13b).147 The microfluidic chip has a silicon dioxide digital PCR detection.154 These two technologies can be used to-
substrate with a thickness specific to an array of antigen gether to obtain ultrasensitive results for SARS-CoV-2 bio-
probes. When antibodies were captured, the light signal can markers on a digital platform.
be reflected and detected by using a CCD camera.148 Each
chip contains a 15-plex array (including a negative control): 1
3.4 Commercial POCT immunoassay products
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target is detected twice, and each antigen is printed in 6

spots. The chip can be placed in 1 well of a 96-well plate. The On the list of FDA's “In Vitro Diagnostics EUAs”, about half
SARS-CoV-2 detection results have a generally good correla- of the serology/antibody tests products use lateral flow
tion with those obtained through ELISA. The AIR assay has method, and antibody detection products is more than anti-
been commercialized as Adarza Ziva, and the commercial in- gen detection products (Table 2).
strument can process 1000 samples in 24 h.149 Weian Zhao's Guangzhou Wondfo's SARS-CoV-2 antibody test uses lateral
group developed a 3D-printable portable imaging platform, flow method to detect IgM and IgG antibody.155,156 The detec-
“TinyArray imager”, to read antigen microarrays for SARS- tion results can be interpreted by naked eyes without any instru-
CoV-2, SARS-1, MERS and 3 other respiratory viruses ment. This is a relatively common method, and it does not rely
(Fig. 13c).150 The imaging platform consists of a camera mod- on detection equipment, so it is particularly suitable for a rapid
ule controlled by Raspberry Pi, 2 LED sources and 2 filters to POCT detection. Jhong-Lin Wu et al. tested four POCT lateral
spectrally select for the emitted fluorescence. The portable flow immunoassays for diagnosis of COVID-19, they found that
imager has a large imaging field of 35 × 26 mm. Positive and the specificity was 100% from the 1st day and after 21 days of
negative COVID-19 sera was used to evaluate the imager. The symptom onset the sensitivity also became 100%.157
TinyArray imager showed good consistency with a commer- Recently, Abbott's antigen detection kit get the FDA EUA ap-
cial imager that is 100× more expensive. These two kinds of proval.158 This BinaxNOW COVID-19 Ag Card works with a nasal
image array platforms are suitable for the rapid detection swab sample, extraction reagent and a credit card-sized reactive
and screening of a variety of antigens or antibodies. card to detect the nucleocapsid protein antigen from SARS-CoV-
SERS method and digital immunoassay chip are two of sen- 2.159 Test results are interpreted visually at 15 minutes. Abbott
sitive and accurate methods in detecting antibodies and anti- also developed an app on mobile phones, the negative detection
gens. But they haven't been used in detecting SARS-CoV-2 yet. result would be shown on the app as a digital health pass.
C. Yang's group combined digital microfluidics (DMF) with sur- Hotgen company invented an up-converting phosphor
face enhanced Raman scattering (SERS) to detect H5N1.151 The immunochromatographic technology (UPT) and used it on

Fig. 13 Other microfluidic immunoassay methods for detecting SARS-CoV-2. (a) Microfluidic platform to detect SARS-CoV-2 IgG, IgM and anti-
gen. Reused with permission from ref. 146. Copyright:2020, the American Chemical Society. (b) Arrayed imaging reflectometry (AIR) platform assay
for antibodies to respiratory viruses. Reused with permission from ref. 147. Copyright: 2020, Elsevier. (c) 3D-printable portable imaging platform
(TinyArray imager) work flow and fluorescence images acquired with the imager and a commercial imager. Adapted with permission from ref. 150.
Copyright:2020, the Royal Society of Chemistry.

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Table 2 Commercial immunoassay POCT products (Since there are too many colloidal gold and immunofluorescence strip products, only some of them are listed in this table)

Detection Detection Results Positive/Negative

Lab on a Chip

No. Device Company target Sample type duration reading Regulatory agreement Detection technology
1 BinaxNOW COVID-19 Abbott Diagnostics Antigen NS 15 min Naked FDA EUA 91.7%/100% Lateral flow
Ag Card#1 159,167 Scarborough, Inc. eye
2 Novel Coronavirus 2019-nCoV Beijing hot view IgM and Serum/plasma 15 min Machine NMPA, CE — Lateral flow,
Antibody Test160 Biotechnology Co., IgG (up-converting phosphor
Ltd. antibody immunochromatographic)
3 WANTAI SARS-CoV-2 Beijing Wantai Biological Total Serum/plasma 15 min Naked FDA EUA, 100%/98.8% Lateral flow
Ab Rapid Test168 Pharmacy Enterprise Co., antibody (dipotassium EDTA, eye CE, TGA (colloidal gold)
Ltd. lithium heparin and
sodium citrate)/VWB
4 BioCheck SARS-CoV-2 IgG and BioCheck, Inc. IgM and Serum 30 min Machine FDA EUA, 99.1%/97.2% Chemiluminescence
IgM Combo test169,170 IgG CE
antibody

This journal is © The Royal Society of Chemistry 2021

5 qSARS-CoV-2 IgG/IgM Cellex Inc. IgM and Serum/plasma 15–20 Naked FDA EUA, 93.8%/96% Lateral flow (colloidal
Rapid Test171,172 IgG (EDTA or citrate)/VWB min eye CE gold)
antibody
6 Ellume COVID-19 Home Ellume Limited Antigen NS 15 min Machine FDA EUA 95%/97% Lateral flow
Test#2 173,174 (fluorophore)
7 SARS-CoV-2 Antibody Test155,175 Guangzhou Wondfo IgM and Whole 15 min Naked NMPA EUA, 45.2%/81.8% Lateral flow
Biotech Co Ltd. IgG blood/serum/plasma eye CE, TGA (colloidal gold)
antibody
8 SARS-CoV-2 IgGIgM Antibody Lumigenex co. LTD IgM and Serum/plasma/whole 10 min Machine — — Lateral flow (time
Rapid Test Kit176 IgG blood resolved fluorescence)
antibody
9 xMAP SARS-CoV-2 Multi-Antigen Luminex Corporation IgG Serum/dipotassium Less Machine FDA EUA, 96.3%/99.3% 96 plate (multiplexed
IgG Assay175,177 antibody EDTA plasma than 3 h Health microsphere-based assay)
Canada
10 LumiraDx SARS-CoV-2 LumiraDx UK Ltd. Antigen NS 12 min Machine FDA EUA, 97.6%/96.6% Microfluidic
Ag Test178 CE immunofluorescence
assay
11 Sofia SARS Antigen FIA163 Quidel Corporation Antigen NPS/NS 15 min Machine FDA EUA, 96.7%/100% Lateral flow
CE immunofluorescent
sandwich assay
12 Accre 6161,179,180 Shenzhen Tisenc Medical IgM and Serum/plasma 22 min Machine — 96.6%/—— Chemiluminescence
Devices Co., Ltd. IgG
antibody
13 Diagnostic Kit for IgM/IgG Zhuhai Livzon Diagnostics IgM and Serum/plasma/VWB 15 min Naked NMPA, CE 90.6%/99.2% Lateral flow
Antibody to Coronavirus Inc. IgG eye (colloidal gold)
(SARS-CoV-2)181 antibody

Notes: 1. Sort by first letter of company name. 2. All the carriers are for single use. 3. ‘#1’ means that this product is authorized for prescription home use; ‘#2’ means that this product is
authorized for non-prescription home use. 4. List of abbreviations: (1) NS, nasal swabs; NPS, nasopharyngeal swabs; VWB, venous whole blood. (2) FDA EUA, U.S. Food & Drug Administra-
tion Emergency Use Authorizations; CE, Conformite Europeenne; NMPA, National Medical Products Administration; NMPA EUA, National Medical Products Administration Emergency Use
Authorizations; TGA, Therapeutic Goods Administration. https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-
diagnostics-euas;; Web: http://app1.nmpa.gov.cn/data_nmpa/face3/dir.html#maodian2;; Web: https://www.finddx.org/covid-19/pipeline/.
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Critical review

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Critical review Lab on a Chip

lateral flow strips.160 The UPT material can absorb long wave target and directly provide sufficient feedback to the end-user
light and emit short wave light. This method has an advantage with optical signals, electrical signals, etc.184,185 With continu-
of low background interference and can achieve no fluorescence ous development in nanoscience and technology, functional
fading. But the tests results must be read by a instrument. The materials and intensified structures have reduced the signal-to-
IgM and IgG antibodies can be tested at the same time in 15 mi- noise ratio and sample-to-answer time for devices. Biosensor-
nutes. And a portable machine is used to read the result in 5 s. based diagnosis is considered an alternative solution for reliev-
At present, Tisenc has developed an IgM and IgG antibody ing the heavy pressure on PCR-based testing, which has been
single person chemiluminescence SARS-CoV-2 detection proved as a promising platform during the pandemic. From
kit.161 Reagent has been pre-stored in a strip and a desktop this perspective, biosensors may be key components in field of
machine was used to automatically drive the fluid to accom- POCT for COVID-19 and lead to wearable devices for facile and
plish the detection. They have tested 90 samples (including rapid transmission from signal collection to detector without
58 cases of nucleic acid positive samples), the IgM clinical co- sophisticated equipment for data analysis.186
incidence rate was more than 90%, IgG clinical coincidence
rate was more than 95%.162 It took 22 min to get the result.
4.1 Plasmonic biosensors
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This is a direction for the development of POCT ELISA and

chemiluminescence detection systems in the future. Based on localized surface plasmon resonance (LSPR) effects
Sofia SARS Antigen FIA from Quidel Corporation is the first from artificial nanostructures, plasmonic biosensors have
device detecting SARS-CoV-2 antigen that get the FDA EUA ap- been considered as important and sensitive tools for
proval.158,163 It uses immunofluorescence-based lateral flow POCT.187,188 Combined with surface functionalization pro-
technology in a sandwich design to detect SARS-CoV-2 antigen cesses, plasmonic nanostructures can therefore enable the
in swabs within 15 min for result release. However, this prod- fast, real-time and label-free detection of analytes even under
uct also need a portable machine to read the results. ultralow concentrations. Developments in material science
Blusense has proposed a BluBox together with ViroTrack have now made it possible to precisely control the morphol-
cartridge to detect COVID-19 IgA + IgM/IgG antibodies.164 ogies of nanomaterials with tunable plasmonic properties
The cartridge only use one drop of blood and can detect and sensitivities.189,190 Alternatively, advanced nano-
COVID-19 in 10 min. In the cartridge, magnetic nanoparticles fabrication techniques, e.g., electron-beam lithography, also
were coated with specific antibodies beforehand. Antigens provide platforms to engineer nanopattern arrays on various
will be trapped and format nanoclusters under a strong mag- substrates as plasmonic sensing devices.191,192 Moitra et al.
netic field. The fluorescence of the nanoclusters will be presented a colorimetric assay based on gold nanoparticles
detected in the machine. Maria Engel Moeller et al. have eval- (AuNPs) that were customized with caps of thiol-modified
uated the BluBox system with ELISA method, in 35 plasma antisense oligonucleotides (ASOs). The functional plasmonic
samples from COVID-19 patients, 29 could be detected by carrier thus enables the specific detection of the N gene (nu-
BluBox.165 And for samples collected 14 days after symptom cleocapsid phosphoprotein) from SARS-CoV-2-infected sam-
appear, the sensitivity of both BluBox system and ELISA was ples through spectral resonance shifts in a fast manner
around 90%, specificity of BluBox system could reach 100%, (within 10 min).193 Interestingly, the authors further incorpo-
while ELISA IgA was 95%. rated RNaseH to cleave the RNA strand from the RNA–DNA
The essence of immunoassay is the specific combination of hybrid, resulting in a naked-eye detectable assay in which ag-
antigens and antibodies. Commercial products have used lat- glomeration among the AuNPs plays an important role
eral flow, chemiluminescence, magnetic nanoparticles capture (Fig. 14a). Via functionalization with complementary DNA re-
and other methods to achieve POCT immunoassay of COVID- ceptors, Qiu et al., took advantage of the combinational
19. Since immunoassay can provide the information of the pa- mechanism from plasmonic photothermal effect and LSPR
tient's disease course, and the accuracy of immunoassays im-
proves, China's National Health Commission has added sero-
logical testing method into the diagnosis standard in
“Diagnosis and Treatment Protocol (version 7)”.166

4. Biosensor-based identification
Apart from the aforementioned methods, there is still an ur-
gent need for rapid and sensitive SARS-CoV-2 identification
techniques as alternative POCT systems.123 Recently, minia-
ture biosensors have exhibited potential as analytical plat-
Fig. 14 (a) Naked-eye detection of SARS-CoV-2 via RNaseH treat-
forms because of their unique characteristics, such as sensi-
ment. Reused with permission from ref. 193. Copyright: 2020, the
tivity, reliable specificity and rapid diagnosis, etc.182,183 A American Chemical Society. (b) Schematic of dual-functional AuNIs as
biosensor, typically composed of a functional receptor, trans- biosensors for SARS-CoV-2 detection. Reused with permission from
ducer, and signal detector/analyzer, can sense the intruded ref. 194. Copyright: 2020, the American Chemical Society.

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Lab on a Chip Critical review

sensing. Apart from the well-known LSPR effect, the Recently, researchers are also investigating alternative
thermoplasmonic heat can further elevate the in situ nucleic electrochemical solutions that can further improve the detec-
acid hybridization temperature, which enhances the detection tion capability, as well as paving the way to reduce the opera-
ability with a lower detection limit of SARS-CoV-2 sequences (as tion procedure or total cost. By introducing advanced 3D
low as 0.22 pM). The two-dimensional gold nanoislands (AuNIs) printing method, Md. Ali et al. created a three-dimensional
can thus serve as a promising solution for the reliable clinical reduced-graphene-oxide electrode and integrated with a
diagnosis of COVID-19 (Fig. 14b).194 Based on previous research, microfluidic device as an electrochemical sensor.200 Such 3D
Murugan et al. proposed a plasmonic fiber-optic absorbance bio- electrodes were functionalized with viral antigens to enable
sensor that can be adapted for a 1-step, wash-free monitoring sensitive detection of antibodies specific to SARS-CoV-2, with
platform for detecting SARS-CoV-2 directly from saliva sam- the detection limit down to 2.8 × 10−15 M. Fabiani et al. ap-
ples.195 As a conceptual idea, the authors envisaged two differ- plied the magnetic beads with the carbon black-based
ent types of biosensors to detect the N protein of SARS-CoV-2 electrode to realize a miniaturized electrochemical sensor for
within 15 min that can well satisfy the urgent demand for rapid SARS-CoV-2 detection (Fig. 15a).201 With assistance from the
and low cost diagnosis under the current situation. Along with magnetic beads, the external magnetic field can provide ad-
Published on 22 February 2021. Downloaded on 5/15/2021 4:57:51 AM.

microfluidic systems, Funari et al. developed an opto- vantages such as promoting the pre-concentration and elimi-
microfluidic chip that uses LSPR to detect SARS-CoV-2 anti- nating the washing step, while preserving the superiorities
bodies.196 When antibodies bind to antigens on the chip, the such as sensitive and reliable detection, anti-interference
peak shift of resonant wavelength from the gold nanospikes from seasonal H1N1 influenza virus, etc. Another two recent
appears for direct monitoring. The results can be obtained in studies also introduce paper as the substrate material for
30 min with an LOD of approximately 0.08 ng mL−1 (∼0.5 electrochemical sensor due to the advantages including low
pM), which allows the quantitative SARS-CoV-2 diagnostics to cost, portability, and disposable property. Via printing
be performed in an easier, cheaper and faster way. electrode patterns on paper, Yakoh et al. produced the
electrochemical device to capture SARS-CoV-2 antibodies for
detection within 30 min (Fig. 15b).202 With the acceptable
4.2 Electrochemical biosensors sensitivity and specificity, the paper-based sensor exhibits as
Electrochemical biosensors are another type of widely-used the potential POCT platform especially for the unique cost-ef-
miniaturized device which attracts much attention thanks to fective, portable and disposable behaviors. Alafeef et al.
the superiorities such as simplicity, low cost, and ease of immobilized the sensing probes to a paper-based electro-
miniaturization. An electrochemical biosensor normally relies chemical platform, thus yielding a device towards the nucleic
on a customized electrode acts as the receptor/transducer for acid testing.203 With precise design of the essential sensing
real-time, specific, and precise target monitoring. The infor- materials (gold nanoparticles), the sensor provides a signifi-
mation from the sensing electrode can be derived to electri- cant improvement in the sensitivity and output signals
cal signals, e.g., potentiometric or amperometric, associated within 5 min. Interestingly, the device can also quantitatively
with the presence of the analyte of interest.197,198 A potential measure the targets in a linear range from 585.4 copies μL−1
solution for the diagnosis of COVID-19 was proposed by to 5.854 × 107 copies per μL with a sensitivity of 231 (copies
Tripathy et al. where a miniaturized electrochemical biosen- per μL)−1, which reveals the potentials to indicate the pro-
sor was fabricated via electrodeposited AuNPs as the trans- gression of SARS-CoV-2 infection after initial infected
ducing element.199 By integrating surface immobilization and confirmation.
smartphone software, the authors claimed that this portable To facilitate the fast result release to end-user and at-
electrochemical analysis system could provide rapid data ac- home diagnosis, the data transmission is another aspect that
quisition, exhibiting the potential as a smart POCT tool to re- should be taken into consideration apart from the electro-
duce the dependence on bulky instrumentation. chemical sensing component. Zhao et al. demonstrated that

Fig. 15 (a) The magnetic beads based assay for COVID-19 detection in untreated saliva. Reused with permission from ref. 201. Copyright: 2021,
Elsevier. (b) Schematic diagram showing the detection principle of paper-based electrochemical biosensor for COVID-19 detection. Reused with
permission from ref. 202. Copyright: 2021, Elsevier.

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Critical review Lab on a Chip

Fig. 16 (a) The COVID-19 ROS diagnosis system consists of needle electrodes coated with functionalized multiwall carbon nanotubes. Reused
with permission from ref. 206. Copyright: 2020, Elsevier. (b) Schematic diagram of operation procedure based on the COVID-19 FET sensor.
Reused with permission from ref. 208. Copyright: 2020, the American Chemical Society.
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the electrochemical sensor can be adapted to a smartphone RapidPlex’ exhibits high sensitivity and low cost detection,
for SARS-CoV-2 RNA detection, which relieves the heavy de- providing the multiplexed information on three key aspects
pendence on large-scale instrument and laboratory pro- of COVID-19 disease (the viral infection, immune response,
cesses.204 Such ‘plug-and-play’ manner can possibly provide and disease severity) with potential of at-home diagnosis.
a portable channel for the customers to assess the test result
conveniently. A recent study further combined the electro-
chemical platform with wireless module to enable the ultra- 4.3 Other methods
rapid identification of COVID-19 based on mass-producible Apart from the aforementioned methodologies based on min-
graphene electrodes.205 The demonstrated ‘SARS-CoV-2 iature biosensors for COVID-19 identification, recent efforts

Table 3 Summary of biosensor-based SARS-CoV-2 detection

Detection Limit of
No. Detection technology Material Detection target Sample type duration Selectivity detection
1 Surface plasmon Gold nanoparticles Nucleic acid Isolated RNA 10 min Against MERS-CoV 180 ng mL−1
resonance and viral RNA
colorimetric assay193
2 Plasmonics and Gold nanoislands Nucleic acid Synthesized — Against SARS-CoV 0.22 pM
photothermal samples
effect194
3 Opto-microfluidic Gold nanospikes Antibodies Diluted human 30 min Against BSA 0.08 ng mL−1
chip196 plasma (bovine serum albumin), (0.5 pM)
IL-6 (interleukin 6),
CRP (C-reactive protein)
4 3D electrochemical Reduced-graphene-oxide Antibodies — Within Against N antibodies, 2.8 × 10 fM
sensor200 nanoflakes seconds IL-6 (interleukin 6)
5 Magnetic beads Magnetic beads and Spike (S) Untreated 30 min Against 2009 H1N1 19 ng mL−1
based biosensor201 carbon black-based protein and saliva influenza and seasonal (S protein)
electrodes nucleocapsid H1N1influenza virus and 8 ng mL−1
(N) protein (N protein)
6 Paper-based Graphene-based Antibodies Human 30 min — 1 ng mL−1
electrochemical materials serum
sensor202
7 Paper-based Gold nanoparticles Nucleic acid COVID-19 <5 min Against MERS-CoV and 6900 copies
electrochemical positive SARS-CoV viral RNA per mL
sensor203 patients
8 Electrochemical Gold@Fe3O4 Nucleic acid Artificial and — Against SARS-CoV, 200 copies
sensor204 nanocomposite clinical RNA MERS-CoV, HCoV-OC43 per mL
samples
9 Electrochemical Multi-wall carbon Reactive oxygen Fresh sputum <30 s — —
sensor206 nanotubes species
10 Nanomaterials-based Gold nanoparticles Disease-specific Exhaled breath In — —
breath sensor207 biomarkers seconds
11 Field-effect Graphene sheet Spike (S) Clinical Real-time — 1 fg mL−1
transistor208 protein samples from electrical
COVID-19 response
patients

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also demonstrate that the detection of other bio-markers or CRISPR method is convenient to implement. LamPORE
advanced sensing mechanisms can be applied as promising launched by Oxford combines the LAMP method with se-
tools. Miripour et al. applied an electrochemical sensor with quencing, which improves the detection efficiency of se-
integrated carbon nanotubes for reactive oxygen species quencing and renders the possibility to use sequencing
(ROS) monitoring, which is based on the fact that an increase methods for the POC detection of SARS-CoV-2. In terms of
in ROS is a side effect of COVID-19.206 Within less than 30 s, immune testing, there are a large number of studies
the electronic device can directly analyze a sputum sample regarding colloidal gold- and fluorescence-based
for real-time ROS, with an accuracy of 94% and a sensitivity immunochromatographic assays and many resulting prod-
of 92% after calibration (Fig. 16a). In view of the volatile or- ucts. Such methods have advantages such as rapid detection
ganic compounds (VOCs) emitted by viral agents, Shan et al. and low cost. There are very few products that use ELISA and
applied gold nanoparticles as the sensor array to detect the chemiluminescence due to the complicated equipment re-
disease-specific biomarkers from exhaled breath.207 Through quired. However, because ELISA and chemiluminescence re-
the surface linking of organic ligands, the device enables the sults are more accurate and reliable than those derived from
reaction with VOCs which finally causes the signal variation chromatographic assays, many researchers are now
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from electric resistance. Even such breath analyzer exhibits conducting POCT studies. In terms of biosensors, new sensor
potential for rapid COVID-19 screening, factors such as envi- detection technologies, such as SPR methods and electro-
ronmental humidity or background diseases can also influ- chemical methods, have fast detection capability and low de-
ence the sensitivity and accuracy that should be considered tection limits, providing alternative solutions for the detec-
before the system to be commercialized. Seo et al. reported a tion of the novel coronavirus. However, due to selectivity
field-effect transistor (FET)-based biosensor for the identifica- limitations, sample preprocessing and preparation costs,
tion of SARS-CoV-2 in clinical samples, where the sensor was there is still substantial room for development. Multiple de-
produced by coating graphene sheets with a specific antibody tection methods complement each other, thereby covering
against SARS-CoV-2 spike protein (Fig. 16b).208 Sensor perfor- multiple stages of COVID-19, such as screening suspected
mance was evaluated using antigen protein, cultured virus, cases, initial infection, disease treatment, and prognosis.
and nasopharyngeal swab specimens from COVID-19 patients However, there is a clear gap between scientific research re-
with a SARS-CoV-2 spike protein detection capability of 1 fg sults and commercial products. Although diverse and novel
mL−1 in phosphate-buffered saline and 100 fg mL−1 in clini- research results are introduced in this review, products in the
cal transport medium. market are mainly concentrated in PCR and LFA detection
Without the requirement of sample pretreatment or label- methods. Lack of stability, high costs and patent restrictions
ing, this method might serve as a suitable platform to realize may be some of the reasons that hinder the commercializa-
the rapid and sensitive diagnosis for COVID-19 or other tion of scientific research achievements. We also hope that
emerging viral diseases (Table 3). more scientific research results can be assisted by enterprises
and transformed into market-oriented competitive products.
5. Conclusion and perspective To date, this epidemic has been developing for more than
one year and without any signs of ending. COVID-19 has high
This article provides a summary analysis of the laboratory infectivity and has now undergone widespread transmission.
methods and commercial products for POCT detection of Moreover, there are patients with asymptomatic infection.
COVID-19. At present, the POCT methods for the detection of High-sensitivity, high-accuracy, rapid, and cost-effective de-
SARS-CoV-2 can be divided into three categories: nucleic acid tection methods are conducive to the early detection of pa-
testing, immune testing, and biosensor testing. In terms of tients in the initial infection stage and patients with asymp-
nucleic acid testing, the products developed by Cepheid, Bio- tomatic infection, rendering it possible to make early
fire and other companies integrate nucleic acid extraction diagnoses and screen close contacts. In addition, the north-
and RT-PCR amplification into a cassette, with automated in- ern hemisphere is entering flu season. The number of pa-
strument operation. These products allow fully automated tients with influenza A, influenza B, and adenovirus infec-
and integrated nucleic acid testing, thus reducing the risk of tions will increase, which will impose challenges regarding
cross-contamination and the requirements for detection con- the diagnosis and treatment of COVID-19. Therefore, rapid
ditions and enhancing the detection efficiency. Due to its fast and high-throughput multiple-screening tests are also very
detection speed and constant temperature, LAMP-based de- important.
tection methods have been used by many researchers in the The existing tests for SARS-CoV-2 are mainly performed by
study of POCT for novel coronavirus detection. These LAMP- large laboratories. Therefore, sample transportation and re-
based methods have achieved fast and convenient nucleic sults reporting take a rather long time. Moreover, the testing
acid detection of the novel coronavirus. And these types of of large numbers of samples easily puts pressure on laborato-
isothermal amplification method are expected to be applied ries and increases the risk of cross-contamination. In view of
to home test. In addition, the CRISPR method uses a chro- the shortcomings and deficiencies of the existing detection
matographic strip to detect target nucleic acids, and the re- methods, the research and development of high-throughput,
sults can be observed with the naked eye. Therefore, the multitarget, fast and automated POCT detection systems

This journal is © The Royal Society of Chemistry 2021 Lab Chip, 2021, 21, 1634–1660 | 1653
 View Article Online

Critical review Lab on a Chip

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