Review Article
Point-of-Care Diagnoses and Assays Based on Lateral Flow Test

Miroslav Pohanka

Faculty of Military Health Sciences, University of Defense, Trebesska 1575, Hradec Kralove CZ-50001, Czech Republic

Correspondence should be addressed to Miroslav Pohanka; miroslav.pohanka@gmail.com

Received 26 November 2020; Revised 5 January 2021; Accepted 11 January 2021; Published 20 January 2021

Academic Editor: Adil Denizli

Copyright © 2021 Miroslav Pohanka. )is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Analytical devices for point-of-care diagnoses are highly desired and would improve quality of life when first diagnoses are made
early and pathologies are recognized soon. Lateral flow tests (LFTs) are such tools that can be easily performed without specific
equipment, skills, or experiences. )is review is focused on the use of LFT in point-of-care diagnoses. )e principle of the assay is
explained, and new materials like nanoparticles for labeling, new recognition molecules for interaction with an analyte, and new
additional instrumentation like signal scaling by a smartphone camera are described and discussed. Advantages of the LFTdevices
as well as their limitations are described and discussed here considering actual papers that are properly cited.

1. Introduction

)ere are standard laboratory methods (chromatographic,
mass, immunochemical, genetical, etc.) suitable for the
analysis of various compounds and substances. Although the
aforementioned laboratory methods exert superior features,
they are quite expensive for both purchasing and cost per
one assay. Moreover, education related to the type of analysis
or experiences at least are required for staff controlling the
instruments.

In the bioanalytical approaches, there is a little different
situation to the standard analytical methods in the central
laboratories. While the dominant part of analyses is expected
to be made in clinical laboratories, it is also necessary to
perform some analyses outside. )e term point-of-care
testing has emerged in current medicine. It can be explained
as a simple test suitable to be finished including data
evaluation in the home conditions by a patient or by a
caregiver with no education in the bioanalyses or similar
disciplines. Disposable urine test strips for multiple bio-
chemical parameters and glucose biosensors for a fast gly-
cemia assay can be mentioned as the standard commercial
devices. Research on diagnostical biosensors is ongoing, and
a number of new biosensor devices suitable for point-of-care
testing have been investigated [1–5]. Other types of point-of-

care tests like the colorimetric one based on a digital camera
are developed [6–9].

)e current review is focused on lateral flow immu-
nochromatographic assays also known as lateral flow tests
(LFTs) and their use in point-of-care. )e LFT has started
quite a long ago, and many analytical and diagnostical
methods have been developed on the platform. In recent
time, further improvement was achieved due to the use of
advanced materials like colored nanoparticles. )e recent
progress in the field of LFTis surveyed here, and the progress
is analyzed and discussed. )e actual literature on LFT is
summarized here.

2. LFT Development as a Standard Platform

Various paper tests for measuring a wide scale of parameters
like pH or thin-layer chromatography assay have been ex-
tensively researched since the beginning of modern chem-
istry. Chromatography as a general method is connected
with the work of Russian scientist Mikhail Tsvet in the early
1900s, and thin-layer chromatography was first reported by
Russian scientists Izmailov and Shreiber in 1938 [10].
Further research on immunoassays including the latex fix-
ation test provided a simple analytical tool significantly

Hindawi
International Journal of Analytical Chemistry
Volume 2021, Article ID 6685619, 9 pages
https://doi.org/10.1155/2021/6685619

mailto:miroslav.pohanka@gmail.com
https://orcid.org/0000-0001-8804-8356
https://creativecommons.org/licenses/by/4.0/
https://creativecommons.org/licenses/by/4.0/
https://doi.org/10.1155/2021/6685619


improving and simplifying the previous methods [11, 12].
)e first LFT devices were developed as an outcome of
knowledge from previous methods and a series of patents
applied in the 1980s. LFTs for proving pregnancy by the
assay of human chorionic gonadotropin in urine were the
first commercial tests working on the lateral flow principle
and using specific antibodies against the hormone [13].

)e original types of LFT were based on the recognition
capacity of antibodies that served as a recognition part of the
assay. )e general principle is shown in Figure 1 and can be
described as follows: the assay is performed on a sheet-
shaped matrix from paper, cellulose, etc., that contains freely
adsorbed antibodies labeled by color or fluorescent mark
and specific against the analyte on the first end. )e matrix
also contains two zones with immobilized antibodies on the
second end: the first zone contains antibodies specific to the
analyte, and the second zone immobilized antibodies specific
against free-labeled antibodies. A liquid sample is applied on
the first end and the analyte presented in the sample
interacting with the labeled antibodies. )e complex analyte-
labeled antibody and the unreacted antibodies are carried by
the lateral flow on and in the hydrophilic matrix. )e
complex analyte-labeled antibody is captured on the first
zone forming a colored spot visible by a naked eye. )e
unreacted labeled antibodies interact with the second zone
and form a visible spot as well. )e coloration caused by an
analyte is frequently called the test line, while the spot by
unreacted antibodies is frequently called the control line.

As seen from the principle of the LFT, it is a simple
method suitable for simple assay relying on a naked eye, no
specific instrumentation is necessary, and even liquid sample
can be measured directly without further treatment. It is
typically suitable for field applications [14]. )ough LFTcan
be called by a synonym lateral flow immunochromato-
graphic assay and antibodies are relevant and are also the
most traditional recognition part in them, other recognition
molecules fully replacing the antibodies can be also em-
bedded. Aptamers can be exampled [15].

Gravidity tests for semiquantitative determination of
human chorionic gonadotropin in urine are well known and
are mass-produced types of LFT [16]. However, other types
of LFT are currently available in the market, and many of
them serve for the purpose of point-of-care diagnosis. )e
LFT kits for the detection of lipoarabinomannan in urine as
a marker of Mycobacterium tuberculosis and diagnosis of
tuberculosis disease [17, 18], detection of antibodies re-
sponsible for allergic reactions like the antibodies causing
allergic bronchopulmonary aspergillosis [19, 20], detection
of allergenic substances like peanut and hazelnut in food
products [21], diagnosis of coronavirus disease 2019
(COVID-19) including antigens and specific antibodies
[22–25], detection of antibodies against Brucella sp. to di-
agnose brucellosis [26], peste des petits ruminants virus
disease diagnosis by antigen detection in fecal and nasal
swab samples [27], measuring of C reactive protein in the
blood, blood plasma, and serum [28], and assay of biological
warfare agents and toxins like Bacillus anthracis, Escherichia
coli O157:H7, staphylococcal enterotoxin B, ricin, botuli-
num toxin, Francisella tularensis, and Yersinia pestis [29–32]

can be mentioned as examples of commercially available
devices. )e appearance of a commercially available LFT is
depicted in Figure 2.

)e standard methods have a lack in the inability to
measure the exact concentration of the analyte, and the
assays can be performed as a semiquantitative test only. )e
spots formed on the LFTmatrix are typically narrow which is
optimal for coloration scaling by a naked eye but not an ideal
solution for colorimetry. Digital cameras including the
cameras integrated into smartphones are considered as the
future tool for coloration scaling in various analytical
protocols [8, 33–36]. )e assays can be also improved by spot
design or by manufacturing more positive spots with an
unequal affinity toward analyte placing in the LFT strip, so
the concentration of analyte would be better estimated by a
naked eye. )e next evolution of LFT should be made with
regard to the measuring platforms making the formerly
qualitative or semiquantitative tests to be the quantitative
ones.

2.1. Current Trends in LFT Construction in Point-of-Care
Diagnosis. )ough the original LFT devices from the 1980s
are the functional ones, further improvement is desired to
improve their analytical specifications and reduce costs.
Comparing to the original devices from the 1980s, the
currently researched and developed LFT contains typically
alterations in selected recognition molecule and substance
responsible for the visualization of the interaction with the
analyte. Evolution of materials for matrix manufacturing,
overall design resolving problems with manipulation by an
unskilled worker, and improving LFT package to make it
have long-term stability can be mentioned as the other areas
of improvement. )e evolution of LFTs is not of course
related to devices for diagnosis only because the platform
gained overall popularity in analytical chemistry and various
applications are known for this moment.

)e immunoassay-based point-of-care diagnostic tool
was, for instance, described for COVID-19. )e researchers
investigated seroprevalence for COVID-19 using standard
enzyme-linked immunosorbent assay (ELISA) and com-
pared it with a standard LFT based on antibodies labeled by
colloidal gold [37]. )e LFT and ELISA mutually correlated
and the authors concluded their work by a recommendation
that LFTis suitable for point-of-care in the healthcare setting
and COVID-19 monitoring. In another study on COVID-
19, an LFT device for immunoglobulins (Ig) M and G in
blood was constructed using selenium nanoparticles labeled
SARS-CoV-2 nucleoproteins causing interaction with IgM
and IgG antibodies [38]. )e assay exerted a limit of de-
tection of 20 ng/ml for IgM and 5 ng/ml for IgG in a 10-
minute lasting assay. Other types of nanoparticles can be also
used for an LFT immunoassay. For instance, LFT based on
carbon nanoparticles conjugated with p48 protein was de-
veloped for the diagnosis of mycoplasma caused by Myco-
plasma bovis [39]. )e assay exerted 100% specificity and no
cross-reactivity with antibodies to other bovine pathogens. A
full correlation with ELISA was also reached. An LFT test
using monoclonal antibodies labeled by gold nanoparticles

2 International Journal of Analytical Chemistry



was developed by Liu and coworkers for assay of dinitolmide
in chicken tissue [40]. )e researchers reported a limit of
detection of 2.5 μg/kg for chicken tissue containing dini-
tolmide, and the assay was fully comparable to liquid
chromatography and ELISA. Gold nanoparticles (gold
spheres with size 30 and 100 nm or gold-silica shells with size
150 nm) and antibody-based detection were also used in the
development of an LFT for the human immunodeficiency
virus [41]. )e gold nanoparticles were covered by a
monoclonal antibody against protein p24 of the human
immunodeficiency virus, and the whole assay was made in a
standard manner. Signal was recorded by thermal contrast
reading using an IR camera and laser. )e assay was highly
sensitive as a limit of detection of 8 pg/ml of p24 was
achieved.

)e relevance of LFT can be largely perceived in the
recent events when a fast test for the diagnosis of COVID-19
was demanded. )e standard diagnosis of COVID-19 was
based on the polymerase chain reaction (presence of
pathogen) and ELISA (prove of antibodies), but the tests
have to be performed in specialized laboratories, and they
require a quite long time to be finished. LFTs were suc-
cessfully introduced as an alternative to the polymerase
chain reaction and ELISA, and they were proved to be
suitable for routine diagnosis based on the detection of
COVID-19 antigen. )ough they are not a replacement of
polymerase chain reaction and ELISA, they were proved to
be a suitable tool to be developed in a short period and used
wherever necessary.

Concurrently, the recognition capability of antibodies
suitable for antigens measuring respective antigens when
antibodies are assayed is frequently replaced by the use of
aptamers by many assays. Extensive progress in aptamer
preparation has been reached in recent years, and the
aptamers typically comprise ribonucleic acid (RNA),
deoxyribonucleic acid (DNA), peptides, or proteins [42–48].
)eir potency for LFT development was recognized as well
[49]. Aptamers found their way to the construction of LFT,
and they have become a relevant recognition molecule in
LFT devices’ development. In a work by Tripathi and co-
workers, the LFT test was constructed for the point-of-care
cancer diagnosis by the measurement of marker CA125 level
[50]. )e researchers used aptamer linked with gold
nanoparticles serving as peroxidase mimetic and optimized
the assay for the detection of CA125 in human serum. )e
assay had a limit of detection of 3.71 U/ml and correlated
with ELISA. A DNA aptamer for residual penicillin antibiotic
ampicillin was prepared by Lin and coworkers [51]. )e
researchers used hexachlorofluorescein for oligonucleotide
labeling and were able to detect ampicillin in a range of 10 to
200 ng/l and a limit of quantification of 2.71 ng/l when water
sample was analyzed. DNA aptamer was also used for the
detection of dopamine in urine [52]. In this study, dopamine
duplex DNA aptamers were conjugated to 40 nm gold
nanoparticles, and LFT was performed in a standard manner
resulting in a limit of detection of 50 ng/ml. Protein

(a) (b)

Capillary flow

(c)

Figure 1: General principle of an LFT immunoassay. (a) Sample with an analyte (circle) is added to the pad where a labeled antibody (Y
shaped) already exists. (b) Analyte and a labeled antibody are carried by a lateral flow (arrow) and they can mutually interact. (c) Complex of
analyte-labeled antibody and the labeled antibody are captured on test spot (analyte) or control spot (unreacted antibody) forming colored
lines.

BA

RC

CB

YP

se

Figure 2: Appearance of commercial LFT devices. In the upper
part, there is LFT for human chorionic gonadotropin; in the down
part, there is a photograph of LFT for the contemporary deter-
mination of five biological warfare agents Bacillus anthracis, ricin,
Clostridium botulinum/botulinum toxin, Yersinia pestis, and
staphylococcal enterotoxin B by Pro Strips (Advnt Biotechnologies,
Phoenix, AZ, USA).

International Journal of Analytical Chemistry 3



osteopontin representing a new marker for cancer was de-
tected by a biotinylated aptamer and then streptavidin
modified gold nanoparticles served for the visualization
purpose [53]. )e assay had a limit of detection of 0.1 ng/ml
with dynamic detection of osteopontin in a range from 10 to
500 ng/ml for a measuring time of 5 minutes. Aptamer-based
LFT was chosen by Ali and coworkers for the early diagnosis
of type-2 diabetes by assay of protein vaspin using fluorescent
upconverting nanoparticles [54]. Vaspin was recognized in a
range of 0.1–55 ng/ml with a limit of detection of 39 pg/ml.
LFT does not have to be targeted on defined molecules only.
In a work by Yu and coworkers, LFT was developed for
tumor-derived exosomes for rapid diagnosis of lung cell
cancer [55]. Aptamer specific to CD63 on exosome surface
and gold nanoparticles giving good visualization and ob-
servability of the assay by a naked eye were used for the LFT
construction. )e assay was proved on exosomes isolated
from human lung carcinoma cells in a dilution of 6.4 × 109

particles/ml. Quantum dots are another material that can
serve for the purpose of reaction visualization and macro-
molecules labeling [56–59]. An LFT method based on CdTe
quantum dots was, for instance, constructed by Lu and
coworkers in order to detect Shiga toxin type II [60]. )e
authors also worked with gold nanoparticles as a material for
labeling antibodies. While labeling by quantum dots pro-
vided a limit of detection of 25 ng/ml, the LFT based on CdTe
quantum dots had a five times lower limit of detection: 5 ng/
ml. )e LFT tests can be improved by connecting with other
techniques improving samples and sensitivity increased. For
instance, detection of Escherichia coli O157:H7 was per-
formed in two steps: the first step was based on bacterium
captured by immunomagnetic nanoparticles and monoclo-
nal antibody conjugate with beta-lactamase and gold
nanoparticles; the second step contained penicillin solution
application and hydrolysis by the beta-lactamase [61]. )e
final test containing LFT consisted of an ultrasensitive
penicillin immunochromatographic test strip. )e assay had
a limit of detection of 137 CFU/ml. Further investigation is
also focused on other types of recognition parts and labels
like molecularly imprinted polymers [62] and the use of
liposome enhanced signal amplification [63]. Highly fluo-
rescent europium chelate loaded silica nanoparticles can be
mentioned as another type of label [64]. Research on matrix
composition is also demanded, and it can improve analytical
properties. Nitrocellulose or nitrocellulose coated with
nanocolloids appears to be promising [40, 65]. An overview
of the aforementioned LFTs is given in Table 1.

Considering the current trends, it is clear that the new
directions in LFT research are focused on two major areas.
Firstly, new recognition elements are researched. Secondly,
new types of nanoparticles are used for LFT construction. All
the new materials can improve the final analytical parameters
of a final LFT, but the suitability of the particular materials will
depend on the type of assay and other conditions. )ere
probably will never be an ideal recognition element or a label
for any assay scenarios. Molecularly imprinted polymers and
aptamers can be prospective recognition elements, but anti-
bodies will probably remain an irreplaceable part of many
commercial LFTs. Standard chemical labels like fluorescein will

also remain a part of standard LFTs though nanoparticles like
the gold one or quantum dots will probably represent a better
alternative gaining higher popularity in the future praxis.

Considering the current options in analytical chemistry,
LFT remains probably the major tool for point-of-care
specific diagnosis of various pathologies besides the devices
like Clark glucose biosensor and simple urine colorimetric
test strips. Compared to the other tests, LFT has quite high
versatility for the diagnosis of good presumptions to be used
under point-of-care conditions; on the other hand, LFT has a
limitation on the molecular weight of analyte because the
assay is on an affinity principle. Analytes with low molecular
weight are not suitable for a standard immunoassay, and a
competitive format is the only possibility of how to use an
immunoassay for the analysis of a small compound.
However, the new types of recognition elements like
aptamers bring improvement and even LFT for small
molecules like dinitolmide, ampicillin, and dopamine can be
seen in the examples of new research on LFT.

2.2. Instrumentation of LFT for Point-of-Care Diagnosis.
LFTmethods are typically intended to be either qualitative or
semiquantitative, and the coloration is determined by a
naked eye. If the assay is performed as a semiquantitative,
the found range of value is highly inaccurate. )e overall
simplicity of the method and no necessity to use an analytical
device, electricity, or elaborative sample manipulation are
the major advantages of LFT. On the other hand, there are
disadvantages as well. )e scaling of coloration by a naked
eye is highly subjective and also depends on ambient light
conditions. )e subjective perception of color may be a
problem when the point-of-care diagnosis is performed by
elderly or disabled people. Development of coloration
readers suitable for standard LFT is a way of how to improve
the assay. )e reader devices are especially desired in point-
of-care testing [66]. )e improved LFT assays are quanti-
tative or at least semiquantitative with acceptable accuracy of
concentration range determination.

In the current time, broad attention is given to digital
photography because of the good availability of cameras and
their integration into smartphones. Standard cameras in-
tegrated into smartphones are able to provide at least 8-bit
digital photography in a format like jpg and have infor-
mation about color depth for the 8-bit photography equal to
256 variables for each channel. Better cameras giving figures
in 12, 14, 16, and more bits and providing raw data from the
digital sensor are also widely available in the market. Digital
photography is highly suitable for colorimetry by color
depth analysis and colorimetric tests performed on a thin-
layer-like paper-based assays and detector strips like the pH
and others can be recorded this way, and the digital pho-
tography-assisted assay is well suitable for point-of-care
testing [7, 36, 67–69]. Digital photography has also its
limitations making the assay inaccurate under some con-
ditions. In the first point, the light source has to be men-
tioned. )e light conditions are crucial when a sensor is
photographed; integrated light-emitting diodes can have
problems with the light temperature setting. )ere can be

4 International Journal of Analytical Chemistry



also problems with the setting of white balance and color
temperature in the camera. Problems with lens quality can
also play a role when a cheap camera is used for point-of-
care testing. Nevertheless, the use of digital cameras in
personal diagnosis is considered as the next direction of
research and application into praxis [70–75].

Instrumental analysis of spots formed in LFT was in-
troduced in several applications. In the aforementioned
study by Zhan and coworkers, detection of p24 protein of the
human immunodeficiency virus was made using thermal
contrast reading [41]. )e spot on LFT was formed by a
complex of gold nanoparticle conjugate with p24 from a
sample and capturing zone on the LFT pad. )e spot was
focused by laser with wavelength 532 or 800 nm with power
adjusted in the range from 10 to 500 mW. )e alighted spot
was recorded, and temperature measured by an IR camera
and digital data for further processing and signal scaling
were recorded. Digital camera containing complementary
metal-oxide-semiconductor (CMOS) chip served for the
spot color recording in the study by Jahanpeyma and co-
workers [76]. )e researchers tested their LFTdevice for the
hybridization of DNA, and visualization was made by the
application of a biotinylated detector probe in the presence
of peroxidase-streptavidin conjugate. Just the peroxidase
was responsible for the chemiluminescence reaction
recorded by a camera. )e assay was tested for proving the
16S rRNA gene from Escherichia coli, and the lowest reached
limit of detection was equal to 1.5 pmol/l. )e use of

peroxidase-catalyzed reaction in an LFT was also described
in a paper by Mirasoli and coworkers [77]. )ey adopted
their method for the detection of fumonisin in food samples,
and the mycotoxin was detected in a range of 2.5–500 μg/l
with a limit of detection of 2.5 μg/l for an assay lasting for 25
minutes. )e detection signal was evaluated by a charge-
couple device (CCD) camera. )e authors stated that the
peroxidase reaction generating chemiluminescence products
is more suitable for quantitative LFT assay than an assay
where colloidal gold is used instead of peroxidase. )e digital
scaling of coloration can be even made by simpler devices
than cameras. Digital scanner was selected as an analytical
tool in the work by Posthuma-Trumpie and coworkers [78].
)e authors successfully performed a standard LFT test for
progesterone assay using antibodies and carbon colloid as a
label and the LFTstrips scanned and analyzed digitally. Spots
on an LFT test can be evaluated, and coloration was de-
termined by a smartphone camera which makes the assays
more available to most people. A smartphone camera assay
based on an LFT was investigated for the detection of
mercury [79]. )e assay comprised of the use of streptavidin-
biotinylated DNA probes modified with gold nanoparticles
and adsorbing mercury was proved with a limit of detection
of 2.53 nmol/l. In another smartphone application, uricemia
(uric acid content in the blood) was measured by a com-
bination of an LFT where coloration was initiated Prussian
blue nanoparticles as artificial nanozymes and standard
smartphone for spots characterization [80]. )e assay

Table 1: Overview of LFT in point-of-care diagnosis.

Assay/diagnosis of a
pathology

Type of recognition part
Type of label attached to the

recognition part
Analytical specifications Reference

COVID-19 Antibody Colloidal gold
Full correlation with ELISA for clinical

samples testing
[37]

COVID-19-specific
antibodies recognition

SARS-CoV-2
nucleoproteins

Selenium nanoparticles
20 ng/ml for IgM and 5 ng/ml for IgG

in 10 minutes
[38]

Mycoplasma p48 protein Carbon nanoparticles
100% specificity, no cross-reactivity,

full correlation with ELISA
[39]

Dinitolmide in tissue Monoclonal antibody Gold nanoparticles
Limit of detection of 2.5 μg/kg for

chicken tissue containing dinitolmide
[40]

Protein p24 of human
immunodeficiency virus

Monoclonal antibody Gold nanoparticles Limit of detection of 8 pg/ml [41]

Diagnosis of cancer by
CA125 assay

Aptamer Gold nanoparticles Limit of detection of 3.71 U/ml [50]

Ampicillin in water DNA aptamer Hexachlorofluorescein Limit of quantification of 2.71 ng/l [51]
Dopamine in urine DNA aptamer Gold nanoparticles Limit of detection of 50 ng/ml [52]

Cancer marker osteopontin Biotinylated aptamer
Conjugate streptavidin-gold

nanoparticles

Limit of detection 0.1 ng/ml, with a
dynamic range of 10 to 500 ng/ml,

time per assay 5 minutes
[53]

Assay of vaspin as an early
marker of type-2 diabetes

Aptamer
Fluorescent upconverting

nanoparticles
Limit of detection for vaspin of 39 pg/

ml
[54]

Exosomes for rapid diagnosis
of lung cell cancer

Aptamer specific to
CD63 on exosomes

surface
Gold nanoparticles Detection of 6.4 ×109 exosomes/ml [55]

Shiga toxin type II Antibodies
Gold nanoparticles and CdSe

quantum dots

25 ng/ml (labeling by gold
nanoparticles), 5 ng/ml (labeling by

quantum dots)
[60]

International Journal of Analytical Chemistry 5



exerted a linear range of 1.5–8.5 mg/dl for uric acid. An
overview of the types of instrumentation applicable for an
LFT assay is shown in Table 2.

3. Conclusions

LFT devices and kits can be found in the current market as
standard devices, and new improved types are researched.
)ree major directions of improvement can be observed
when the current research is compared with the traditional
LFT devices on immunoassay principle: (1) new labels and
materials including nanoparticles providing contrast col-
oration, (2) new recognition molecules selectively inter-
acting with analytes, and (3) types of instrumentation
making the LFT-based assays quantitative from the origi-
nally qualitative one. All the facts make the LFTa significant
tool in point-of-care diagnostic where it can be performed
for multiple diagnoses or analyses of harmful substances or
microorganisms. Overall simplicity and growing sensitivity
allow making LFT a tool for a wide number of markers.
)ough the traditional analytes in an LFT assay were
molecules with higher molecular weight, it is expected that
the LFT will become a universal tool even for analytes with
lower molecular weight and make the assay more universal.
)e relevance of LFTwas also evident during the COVID-19
crisis when the tests COVID antibodies and antigens were
urgently developed and marketed in a quite short time.

Data Availability

No data were used to support this study.

Conflicts of Interest

)e authors declare that they have no conflicts of interest.

Acknowledgments

)is work was supported by a long-term organization de-
velopment plan “Medical Aspects of Weapons of Mass
Destruction” (Faculty of Military Health Sciences, Univer-
sity of Defense, Czech Republic) and Technological Agency
od Czech Republic (TACR) (TH03030336).

References

[1] P. Babaie, A. Saadati, and M. Hasanzadeh, “Recent progress
and challenges on the bioassay of pathogenic bacteria,”
Journal of Biomedical Materials Research Part B: Applied
Biomaterials, vol. 14, Article ID 34723, 2020.

[2] S. Menon, M. R. Mathew, S. Sam, K. Keerthi, and
K. G. Kumar, “Recent advances and challenges in electro-
chemical biosensors for emerging and re-emerging infectious
diseases,” Journal of Electroanalytical Chemistry, vol. 878,
Article ID 114596, 2020.

[3] Q. H. Nguyen and M. I. Kim, “Nanomaterial-mediated paper-
based biosensors for colorimetric pathogen detection,” TrAC
Trends in Analytical Chemistry, vol. 132, Article ID 116038,
2020.

[4] M. Pohanka, “)e piezoelectric biosensors: principles and
applications, a review,” International Journal of Electro-
chemical Science, vol. 12, pp. 496–506, 2017.

[5] B. Senf, W.-H. Yeo, and J.-H. Kim, “Recent advances in
portable biosensors for biomarker detection in body fluids,”
Biosensors, vol. 10, no. 9, p. 127, 2020.

[6] S.-u. Hassan, A. Tariq, Z. Noreen et al., “Capillary-driven flow
microfluidics combined with smartphone detection: an
emerging tool for point-of-care diagnostics,” Diagnostics,
vol. 10, no. 8, p. 509, 2020.

[7] D. S. Y. Ong and M. Poljak, “Smartphones as mobile mi-
crobiological laboratories,” Clinical Microbiology and Infec-
tion, vol. 26, no. 4, pp. 421–424, 2020.

Table 2: LFT signal recording by an analytical instrument.

Type of assay Type of instrumentation
Physical principle of the instrumental

assay
Reference

LFT for p24 protein from human
immunodeficiency virus, gold nanoparticles
conjugated with a monoclonal antibody against p24
were used

Laser alighting specific spots,
IR camera visually recording

temperature
)ermal contrast reading [41]

Detection of 16S rRNA gene from Escherichia coli
by hybridization and use of biotinylated probe and
peroxidase conjugated with streptavidin,
peroxidase was responsible for the
chemiluminescent reaction

Digital camera with CMOS
chip

Digital camera recorded
chemiluminescence provided by

peroxidase
[76]

Detection of fumonisin, labeling of antibodies by
peroxidase allows performing chemiluminescent
chemical reaction that is instrumentally measured

Digital camera with CCD chip
Digital camera recorded

chemiluminescence provided by
peroxidase

[77]

LFT immunoassay of progesterone based on
labeling by carbon colloid

Digital scanner
Strips were scanned and digital data

analyzed
[78]

)e LFTassay comprised of the use of streptavidin-
biotinylated DNA probes modified with gold
nanoparticles

Smartphone camera
Detected spots were photographed by a
smartphone camera, and coloration was

measured
[79]

LFT where coloration was initiated Prussian blue
nanoparticles as artificial nanozymes

Smartphone camera
Detected spots were photographed by a
smartphone camera, and coloration was

measured
[80]

6 International Journal of Analytical Chemistry



[8] M. Pohanka, “Small camera as a handheld colorimetric tool in
the analytical chemistry,” Chemical Papers, vol. 71, no. 9,
pp. 1553–1561, 2017c.

[9] H. A. Watson, R. M. Tribe, and A. H. Shennan, “)e role of
medical smartphone apps in clinical decision-support: a lit-
erature review,” Artificial Intelligence in Medicine, vol. 100,
Article ID 101707, 2019.

[10] N. A. Izmailov and M. S. Schraiber, “Spot chromatographic
adsorption analysis and its application in pharmacy,” Com-
munication I Farmatsiya, vol. 3, pp. 1–7, 1936.

[11] F. Klein and M. B. Janssens, “Standardisation of serological
tests for rheumatoid factor measurement,” Annals of the
Rheumatic Diseases, vol. 46, no. 9, pp. 674–680, 1987.

[12] J. M. Singer, S. C. Edberg, R. L. Markowitz, J. D. Glickman,
L. Miller, and R. Marchitelli, “Performance of latex-fixation
kits used for serologic diagnosis of rheumatoid factor in
rheumatoid arthritis sera,” American Journal of Clinical Pa-
thology, vol. 72, no. 4, pp. 597–603, 1979.

[13] A. E. Urusov, A. V. Zherdev, and B. B. Dzantiev, “Towards
lateral flow quantitative assays: detection approaches,” Bio-
sensors, vol. 9, no. 3, p. 89, 2019.

[14] B. O׳Farrell, “Lateral flow technology for field-based appli-
cations-basics and advanced developments,” Topics in Com-
panion Animal Medicine, vol. 30, no. 4, pp. 139–147, 2015.

[15] A. Chen and S. Yang, “Replacing antibodies with aptamers in
lateral flow immunoassay,” Biosensors and Bioelectronics,
vol. 71, pp. 230–242, 2015.

[16] Y. Zhang, X. Liu, L. Wang et al., “Improvement in detection
limit for lateral flow assay of biomacromolecules by test-zone
pre-enrichment,” Scientific Reports, vol. 10, no. 1, p. 9604,
2020.

[17] S. D. Lawn, “Point-of-care detection of lipoarabinomannan
(LAM) in urine for diagnosis of HIV-associated tuberculosis:
a state of the art review,” BMC Infectious Diseases, vol. 12,
no. 1, pp. 1471–2334, 2012.

[18] M. Shah, C. Hanrahan, Z. Y. Wang et al., “Lateral flow urine
lipoarabinomannan assay for detecting active tuberculosis in
HIV-positive adults,” Cochrane Database of Systematic Re-
views, vol. 10, p. CD011420, 2016.

[19] E. S. Hunter, I. D. Page, M. D. Richardson, and
D. W. Denning, “Evaluation of the LDBio Aspergillus ICT
lateral flow assay for serodiagnosis of allergic broncho-
pulmonary aspergillosis,” PLoS One, vol. 15, 2020.

[20] P. Schubert-Ullrich, J. Rudolf, P. Ansari et al., “Commer-
cialized rapid immunoanalytical tests for determination of
allergenic food proteins: an overview,” Analytical and Bio-
analytical Chemistry, vol. 395, no. 1, pp. 69–81, 2009.

[21] M. Röder, S. Vieths, and T. Holzhauser, “Commercial lateral
flow devices for rapid detection of peanut (Arachis hypogaea)
and hazelnut (Corylus avellana) cross-contamination in the
industrial production of cookies,” Analytical and Bio-
analytical Chemistry, vol. 395, no. 1, pp. 103–109, 2009.

[22] F. Cui and H. S. Zhou, “Diagnostic methods and potential
portable biosensors for coronavirus disease 2019,” Biosensors
and Bioelectronics, vol. 165, Article ID 112349, 2020.

[23] H. A. Hussein, R. Y. A. Hassan, M. Chino, and F. Febbraio,
“Point-of-Care diagnostics of COVID-19: from current work
to future perspectives,” Sensors, vol. 20, no. 15, p. 4289, 2020.

[24] Z. Lyu, M. Harada Sassa, T. Fujitani, and K. H. Harada,
“Serological tests for SARS-CoV-2 coronavirus by commer-
cially available point-of-care and laboratory diagnostics in
pre-COVID-19 samples in Japan,” Diseases, vol. 8, no. 4, p. 36,
2020.

[25] L. Weidner, S. Gänsdorfer, S. Unterweger et al., “Quantifi-
cation of SARS-CoV-2 antibodies with eight commercially
available immunoassays,” Journal of Clinical Virology,
vol. 129, Article ID 104540, 2020.

[26] D. M. Pfukenyi, E. Meletis, B. Modise et al., “Evaluation of the
sensitivity and specificity of the lateral flow assay, Rose Bengal
test and the complement fixation test for the diagnosis of
brucellosis in cattle using Bayesian latent class analysis,”
Preventive Veterinary Medicine, vol. 181, Article ID 105075,
2020.

[27] S. Halecker, S. Joseph, R. Mohammed et al., “Comparative
evaluation of different antigen detection methods for the
detection of peste des petits ruminants virus,” Transboundary
and Emerging Diseases, vol. 5, Article ID 13660, 2020.

[28] J. C. Wei, Y. M. Gao, G. Q. Li et al., “A lateral flow immu-
nochromatographic assay (LFIA) strip reader based on
scheduler and 8051 IP core,” Automatika, vol. 57, no. 4,
pp. 1079–1088, 2016.

[29] F. Gessler, S. Pagel-Wieder, M.-A. Avondet, and H. Böhnel,
“Evaluation of lateral flow assays for the detection of botu-
linum neurotoxin type A and their application in laboratory
diagnosis of botulism,” Diagnostic Microbiology and Infectious
Disease, vol. 57, no. 3, pp. 243–249, 2007.

[30] H.-L. Hsu, C.-C. Chuang, C.-C. Liang et al., “Rapid and
sensitive detection of Yersinia pestis by lateral-flow assay in
simulated clinical samples,” BMC Infectious Diseases, vol. 18,
no. 1, p. 402, 2018.

[31] M. Pohanka, “Current trends in the biosensors for biological
warfare agents assay,” Materials, vol. 12, no. 14, p. 2303, 2019.

[32] I. Ziegler, P. Vollmar, M. Knüpfer, P. Braun, and K. Stoecker,
“Reevaluating limits of detection of 12 lateral flow immu-
noassays for the detection of Yersinia pestis, Francisella
tularensis , and Bacillus anthracis spores using viable risk
group-3 strains,” Journal of Applied Microbiology, 2020.

[33] E. Frantz, H. Li, and A. J. Steckl, “Quantitative hematocrit
measurement of whole blood in a point-of-care lateral flow
device using a smartphone flow tracking app,” Biosensors and
Bioelectronics, vol. 163, Article ID 112300, 2020.

[34] Y. Jung, Y. Heo, J. J. Lee, A. Deering, and E. Bae, “Smart-
phone-based lateral flow imaging system for detection of
food-borne bacteria E.coli O157:H7,” Journal of Microbio-
logical Methods, vol. 168, Article ID 105800, 2020.

[35] M. Pohanka, “Photography by cameras integrated in smart-
phones as a tool for analytical chemistry represented by an
butyrylcholinesterase activity assay,” Sensors, vol. 15, no. 6,
pp. 13752–13762, 2015.

[36] M. Pohanka, “Colorimetric hand-held sensors and biosensors
with a small digital camera as signal recorder, a review,”
Reviews in Analytical Chemistry, vol. 39, no. 1, p. 20, 2020.

[37] S. Pickering, G. Betancor, R. P. Galão et al., “Comparative
assessment of multiple COVID-19 serological technologies
supports continued evaluation of point-of-care lateral flow
assays in hospital and community healthcare settings,” PLOS
Pathogens, vol. 16, no. 9, Article ID e1008817, 2020.

[38] Z. Wang, Z. Zheng, H. Hu et al., “A point-of-care selenium
nanoparticle-based test for the combined detection of anti-
SARS-CoV-2 IgM and IgG in human serum and blood,” Lab
on a Chip, vol. 20, no. 22, p. 4255, 2020.

[39] F. Shi, Y. Zhao, Y. Sun, and C. Chen, “Development and
application of a colloidal carbon test strip for the detection of
antibodies against Mycoplasma bovis,” World Journal of
Microbiology and Biotechnology, vol. 36, no. 10, p. 157, 2020.

[40] C. Liu, S. Fang, Y. Tian et al., “Rapid detection of Escherichia
coli O157:H7 in milk, bread, and jelly by lac dye coloration-

International Journal of Analytical Chemistry 7



based bidirectional lateral flow immunoassay strip,” Journal of
Food Safety, 2020.

[41] L. Zhan, T. Granade, Y. Liu et al., “Development and opti-
mization of thermal contrast amplification lateral flow im-
munoassays for ultrasensitive HIV p24 protein detection,”
Microsystems & Nanoengineering, vol. 6, no. 1, p. 54, 2020.

[42] F. Farshchi and M. Hasanzadeh, “Nanomaterial based apta-
sensing of prostate specific antigen (PSA): recent progress and
challenges in efficient diagnosis of prostate cancer using
biomedicine,” Biomedicine & Pharmacotherapy, vol. 132,
Article ID 110878, 2020.

[43] Y. Guo, F. Yang, Y. Yao et al., “Novel Au-tetrahedral aptamer
nanostructure for the electrochemiluminescence detection of
acetamiprid,” Journal of Hazardous Materials, vol. 401, Article
ID 123794, 2021.

[44] N. Cheng, Y. Liu, O. Mukama et al., “A signal-enhanced and
sensitive lateral flow aptasensor for the rapid detection of
PDGF-BB,” RSC Advances, vol. 10, no. 32, pp. 18601–18607,
2020.

[45] T. N. Navien, R. )evendran, H. Y. Hamdani, T.-H. Tang, and
M. Citartan, “In silico molecular docking in DNA aptamer
development,” Biochimie, vol. 180, pp. 54–67, 2021.

[46] Y. Ning, J. Hu, and F. Lu, “Aptamers used for biosensors and
targeted therapy,” Biomedicine & Pharmacotherapy, vol. 132,
Article ID 110902, 2020.

[47] F. Xue, Y. Chen, Y. Wen et al., “Isolation of extracellular
vesicles with multivalent aptamers,” ;e Analyst, vol. 146,
no. 1, p. 253, 2020.

[48] T. Yang, X. Yang, X. Guo et al., “A novel fluorometric
aptasensor based on carbon nanocomposite for sensitive
detection of Escherichia coli O157:H7 in milk,” Journal of
Dairy Science, vol. 103, no. 9, pp. 7879–7889, 2020.

[49] R. Reid, B. Chatterjee, S. J. Das, S. Ghosh, and T. K. Sharma,
“Application of aptamers as molecular recognition elements
in lateral flow assays,” Analytical Biochemistry, vol. 593,
Article ID 113574, 2020.

[50] P. Tripathi, A. Kumar, M. Sachan, S. Gupta, and S. Nara,
“Aptamer-gold nanozyme based competitive lateral flow assay
for rapid detection of CA125 in human serum,” Biosensors
and Bioelectronics, vol. 165, Article ID 112368, 2020.

[51] H. Lin, F. Fang, J. Zang et al., “A fluorescent sensor-assisted
paper-based competitive lateral flow immunoassay for the
rapid and sensitive detection of ampicillin in hospital
wastewater,” Micromachines, vol. 11, no. 4, p. 431, 2020.

[52] S. Dalirirad and A. J. Steckl, “Lateral flow assay using aptamer-
based sensing for on-site detection of dopamine in urine,”
Analytical Biochemistry, vol. 596, Article ID 113637, 2020.

[53] O. Mukama, W. Wu, J. Wu et al., “A highly sensitive and
specific lateral flow aptasensor for the detection of human
osteopontin,” Talanta, vol. 210, Article ID 120624, 2020.

[54] M. Ali, M. Sajid, M. A. U. Khalid et al., “A fluorescent lateral
flow biosensor for the quantitative detection of Vaspin using
upconverting nanoparticles,” Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy, vol. 226, Article ID
117610, 2020.

[55] Q. Yu, Q. Zhao, S. Wang et al., “Development of a lateral flow
aptamer assay strip for facile identification of theranostic
exosomes isolated from human lung carcinoma cells,” Ana-
lytical Biochemistry, vol. 594, Article ID 113591, 2020.

[56] J. Cao, B. Zhu, K. Zheng et al., “Recent progress in NIR-II
contrast agent for biological imaging,” Frontiers in Bioengi-
neering and Biotechnology, vol. 7, p. 487, 2020.

[57] M. Ganesan and P. Nagaraaj, “Quantum dots as nanosensors
for detection of toxics: a literature review,” Analytical
Methods, vol. 12, no. 35, pp. 4254–4275, 2020.

[58] M. Pan, X. Xie, K. Liu, J. Yang, L. Hong, and S. Wang,
“Fluorescent carbon quantum dots-synthesis, functionaliza-
tion and sensing application in food analysis,” Nanomaterials,
vol. 10, no. 5, p. 930, 2020.

[59] M. Pohanka, “Quantum dots in the therapy: current trends
and perspectives,” Mini Rev Med Chem, vol. 20, p. 20, 2017.

[60] T. Lu, K.-D. Zhu, C. Huang et al., “Rapid detection of Shiga
toxin type II using lateral flow immunochromatography test
strips of colorimetry and fluorimetry,” ;e Analyst, vol. 145,
no. 1, pp. 76–82, 2020.

[61] W. Chen, S. Shan, J. Peng et al., “Sensitive and hook effect-free
lateral flow assay integrated with cascade signal transduction
system,” Sensors and Actuators B: Chemical, vol. 321, Article
ID 128465, 2020.

[62] Y. He, S. Hong, M. Wang et al., “Development of fluorescent
lateral flow test strips based on an electrospun molecularly
imprinted membrane for detection of triazophos residues in
tap water,” New Journal of Chemistry, vol. 44, no. 15,
pp. 6026–6036, 2020.

[63] S. Kim and H. D. Sikes, “Liposome-enhanced polymerization-
based signal amplification for highly sensitive naked-eye
biodetection in paper-based sensors,” ACS Applied Materials
& Interfaces, vol. 11, no. 31, pp. 28469–28477, 2019.

[64] X. Xia, Y. Xu, X. Zhao, and Q. Li, “Lateral flow immunoassay
using europium chelate-loaded silica nanoparticles as labels,”
Clinical Chemistry, vol. 55, no. 1, pp. 179–182, 2009.

[65] P. Manta, R. Nagraik, A. Sharma et al., “Optical density
optimization of malaria Pan rapid diagnostic test strips for
improved test zone band intensity,” Diagnostics, vol. 10,
no. 11, p. 880, 2020.

[66] R. Xiao, L. Lu, Z. Rong et al., “Portable and multiplexed lateral
flow immunoassay reader based on SERS for highly sensitive
point-of-care testing,” Biosensors and Bioelectronics, vol. 168,
Article ID 112524, 2020.

[67] G. M. Fernandes, W. R. Silva, D. N. Barreto et al., “Novel
approaches for colorimetric measurements in analytical
chemistry - a review,” Analytica Chimica Acta, vol. 1135,
pp. 187–203, 2020.

[68] H. Chen, Z. Li, L. Zhang, P. Sawaya, J. Shi, and P. Wang,
“Quantitation of femtomolar-level protein biomarkers using a
simple microbubbling digital assay and bright-field smart-
phone imaging,” Angewandte Chemie International Edition,
vol. 58, no. 39, pp. 13922–13928, 2019.

[69] M. Pohanka, J. Zakova, and I. Sedlacek, “Digital camera-based
lipase biosensor for the determination of paraoxon,” Sensors
and Actuators B: Chemical, vol. 273, pp. 610–615, 2018.

[70] L. Harendarcikova and J. Petr, “Smartphones & microfluidics:
marriage for the future,” Electrophoresis, vol. 39, pp. 1319–
1328, 2018.

[71] I. Nishidate, M. Minakawa, D. McDuff et al., “Simple and
affordable imaging of multiple physiological parameters with
RGB camera-based diffuse reflectance spectroscopy,” Bio-
medical Optics Express, vol. 11, no. 2, pp. 1073–1091, 2020.

[72] D. Quesada-González and A. Merkoçi, “Mobile phone-based
biosensing: an emerging “diagnostic and communication”
technology,” Biosensors and Bioelectronics, vol. 92, pp. 549–
562, 2017.

[73] T. Sergeyeva, D. Yarynka, L. Dubey et al., “Sensor based on
molecularly imprinted polymer membranes and smartphone
for detection of Fusarium contamination in cereals,” Sensors,
vol. 20, no. 15, p. 4304, 2020.

8 International Journal of Analytical Chemistry



[74] K. Tomimuro, K. Tenda, Y. Ni, Y. Hiruta, M. Merkx, and
D. Citterio, “)read-based bioluminescent sensor for
detecting multiple antibodies in a single drop of whole blood,”
ACS Sensors, vol. 5, no. 6, pp. 1786–1794, 2020.

[75] A. K. Yetisen, M. S. Akram, and C. R. Lowe, “Paper-based
microfluidic point-of-care diagnostic devices,” Lab on a Chip,
vol. 13, no. 12, pp. 2210–2251, 2013.

[76] F. Jahanpeyma, M. Forouzandeh, M. J. Rasaee, and N. Shoaie,
“An enzymatic paper-based biosensor for ultrasensitive de-
tection of DNA,” Frontiers in Bioscience (Scholar Edition),
vol. 11, pp. 122–135, 2019.

[77] M. Mirasoli, A. Buragina, L. S. Dolci et al., “Chem-
iluminescence-based biosensor for fumonisins quantitative
detection in maize samples,” Biosensors and Bioelectronics,
vol. 32, no. 1, pp. 283–287, 2012.

[78] G. A. Posthuma-Trumpie, J. Korf, and A. van Amerongen,
“Development of a competitive lateral flow immunoassay for
progesterone: influence of coating conjugates and buffer
components,” Analytical and Bioanalytical Chemistry,
vol. 392, no. 6, pp. 1215–1223, 2008.

[79] Z. Guo, Y. Kang, S. Liang, and J. Zhang, “Detection of Hg(II)
in adsorption experiment by a lateral flow biosensor based on
streptavidin-biotinylated DNA probes modified gold nano-
particles and smartphone reader,” Environmental Pollution,
vol. 266, Article ID 115389, 2020.

[80] N.-S. Li, Y.-T. Chen, Y.-P. Hsu et al., “Mobile healthcare
system based on the combination of a lateral flow pad and
smartphone for rapid detection of uric acid in whole blood,”
Biosensors and Bioelectronics, vol. 164, Article ID 112309,
2020.

International Journal of Analytical Chemistry 9