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Ultrasensitive and Highly Specific Lateral Flow
Assays for Point-of-Care Diagnosis
Yilin Liu, Li Zhan, Zhenpeng Qin, James Sackrison, and John C. Bischof*
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ABSTRACT: Lateral flow assays (LFAs) are paper-based point-of-care (POC)
diagnostic tools that are widely used because of their low cost, ease of use, and
rapid format. Unfortunately, traditional commercial LFAs have significantly
poorer sensitivities (μM) and specificities than standard laboratory tests
(enzyme-linked immunosorbent assay, ELISA: pM−fM; polymerase chain
reaction, PCR: aM), thus limiting their impact in disease control. In this
Perspective, we review the evolving efforts to increase the sensitivity and
specificity of LFAs. Recent work to improve the sensitivity through assay
improvement includes optimization of the assay kinetics and signal
amplification by either reader systems or additional reagents. Together,
these efforts have produced LFAs with ELISA-level sensitivities (pM−fM). In addition, sample preamplification can be applied
to both nucleic acids (direct amplification) and other analytes (indirect amplification) prior to LFA testing, which can lead to
PCR-level (aM) sensitivity. However, these amplification strategies also increase the detection time and assay complexity,
which inhibits the large-scale POC use of LFAs. Perspectives to achieve future rapid (<30 min), ultrasensitive (PCR-level),
and “sample-to-answer”POC diagnostics are also provided. In the case of LFA specificity, recent research efforts have focused
on high-affinity molecules and assay optimization to reduce nonspecific binding. Furthermore, novel highly specific molecules,
such as CRISPR/Cas systems, can be integrated into diagnosis with LFAs to produce not only ultrasensitive but also highly
specific POC diagnostics. In summary, with continuing improvements, LFAs may soon offer performance at the POC that is
competitive with laboratory techniques while retaining a rapid format.
Although infectious diseases have always posed global
threats, there is no clearer current example of the need
for inexpensive and high-performing (i.e., low rates of
false negatives (FNs) and positives (FPs)) point-of-care
(POC) diagnostics than with the global pandemic of SARS-
CoV-2 (i.e., COVID-19). As a single serological antibody or
antigen test can only indicate past or recent exposure to SARS-
CoV-2, multiple and broad testing throughout the population
will be needed to identify “hot spots”and to control the
disease effectively.
1−5
In an ideal situation, at-risk individuals
would be tested regularly (i.e., weekly or daily) to enable timely
isolation and to minimize virus transmission among the
community.
1−5
Unfortunately, this need cannot be met using
the current primary diagnostic tools, such as reverse tran-
scription polymerase chain reaction (RT-PCR). Although RT-
PCR has excellent sensitivity and specificity, it cannot be used
as a POC test because this method requires trained staffin
laboratories equipped with specialized thermal cycling equip-
ment and strict environmental conditions to prevent
contamination.
6−8
In addition, the long turnaround time
(hours to days) and high cost of RT-PCR (100−200 USD per
COVID-19 swab test) compared to other rapid (<15 min)
diagnostic tools, such as lateral flow assays (LFAs; <$50 per
COVID-19 swab test),
9,10
restrict its deployment in POC
settings. These costs can be expected to drop further because
other commercialized LFAs, such as human chorionic
gonadotropin (pregnancy) LFAs, are <$1 per test.
Because LFAs are arguably the cheapest, fastest, and easiest
to use paper-based POC tests,
11−15
they exhibit promise as a
tool for achieving global pandemic control by enabling the
rapid screening of infections. In addition to the detection of
SARS-CoV-2, LFAs have also been widely applied in
biomedicine, food contaminant and toxic chemical detection,
and environmental monitoring.
12
In biomedical diagnosis, an
important advantage of this technology is that it enables the
decentralization of laboratory testing to POC sites. Some
important examples of LFAs are those used for the rapid
diagnosis of influenza,
16,17
Streptococcus,
18,19
and many other
viral and bacterial infections.
20−22
The Centers for Disease
Perspective
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Control and Prevention estimates that 9−45 million influenza
infections occur in the United States each year, leading to
140,000−810,000 hospitalizations and 12,000−61,000 deaths
per year.
23
Rapid influenza LFA testing provides results within
minutes, thus enabling timely clinical decisions and medical
treatment. This rapid diagnosis, in turn, provides enormous
healthcare benefits, including slowing disease transmission,
reducing the number of hospitalizations, decreasing down-
stream treatment costs, and minimizing antibiotic
use.
16,18,19,24−26
However, traditionally built commercial LFAs have several
limitations, including poorer sensitivity (more FNs) and lower
specificity (more FPs) than laboratory tests (see the sensitivity
comparison in Figure 1A). These limitations also hinder the
diagnosis and transmission control of diseases such as SARS-
CoV-2,
22
influenza,
17,27
Streptococcus,
18,19
and HIV
28,29
by
LFAs, thus maintaining the requirement for diagnostic
confirmation by more complex laboratory tests such as PCR
and enzyme-linked immunosorbent assay (ELISA). For
example, even though commercial SARS-CoV-2 LFAs are
available, the clinical assay accuracy was much lower (positive
predictive value: 11%−50%) than the claimed sensitivity
(87%−97.5%) and specificity (100%), which limited the
impact that LFAs have on pandemic control and manage-
ment.
22
Similar issues have also been reported elsewhere.
30,31
To address these limitations, extensive efforts have been
invested into improving the sensitivity and specificity of LFAs
to achieve more accurate and higher-performing POC tests.
Two important strategies for improving the sensitivity include
assay improvement
12,13,32
and sample enrichment. As
summarized in Figure 1A, signal amplification can improve
the detection sensitivity by several orders of magnitude, which
enables LFAs to achieve ELISA-level sensitivities. Further,
sample preamplification with an LFA readout provides access
to PCR-level sensitivity. Unfortunately, many of these
techniques for improving LFA sensitivity require longer assay
times (Figure 1A).
32,33
Therefore, balancing the sensitivity and
assay time poses a major challenge in the development of
future POC diagnostics (PCR-level sensitivity within 30 min),
as indicated in Figure 1A. The specificity of LFAs is also
important and is mainly improved through assay optimization
and the identification and use of high-affinity and highly
specific reagents. Figure 2 provides an overview of a sandwich
LFA structure and the sample enrichment, assay optimization,
and signal amplification methods for enhancing the sensitivity
and specificity of LFAs.
In the following sections, we discuss strategies for improving
the sensitivity (Improving Sensitivity) and specificity (Improv-
ing Specificity) of LFAs in detail. We also provide perspectives
on the further improvements needed to extend the existing
technologies into future POC diagnostics in each subsection.
Figure 1. Comparison of the analytical sensitivity and diagnostic speed of signal-amplified lateral flow assays (LFAs) with those of other
diagnostic tools. (A) Comparison of signal-amplified LFAs with emerging isothermal nucleic acid amplification diagnostics, digital enzyme-
linked immunosorbent assay (dELISA), and commercial diagnostic tools. (B) Estimated limit of detection ranges and detection times for
different signal-amplified LFAs. A detailed literature summary of the data provided in (B) is shown in Table S2. The conversion of units
between g/mL and molarity (M) is achieved with the molar weight of the target analytes. Chemically enhanced LFAs include LFAs that use
novel labels and enhancing LFA reagents. Photothermal LFA methods include thermal contrast,
43,44
photoacoustic imaging,
45,46
photothermal laser speckle imaging,
47
and thermal photonic lock-in imaging.
48
SERS: surface-enhanced Raman scattering; PCR: polymerase
chain reaction; POC: point of care.
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B
Note that the bulk of the work reviewed here is based on
prototyped laboratory LFAs from published data instead of
commercial LFAs. We also note that this Perspective focuses
mainly on protein- and nucleic-acid-based sandwich LFAs (see
the structure in Figure 2A). However, the principles and
techniques discussed here are not limited to biomolecule
detection and can be extended to other targets, such as
extracellular vesicles,
34−36
bacteria,
37,38
viruses,
39
fungi,
40
foodborne pathogens and toxins,
41
and toxic pollutants.
42
IMPROVING SENSITIVITY BY ASSAY IMPROVEMENT
The sensitivity of LFAs can be substantially improved via
either assay improvement or sample enrichment. Assay
improvement includes the optimization of the fundamental
assay kinetics (Figure 2C(1)) and signal amplification methods
by either chemical enhancement and reader use (Figure 2D).
The perspectives on their future improvements was also
discussed.
Optimization of the Assay Kinetics. Exploring the assay
kinetics (i.e., the transport and reaction kinetics) is a
fundamental step in LFA development and is essential for
enhancing the LFA sensitivity (see Figure 2C(1)). These
kinetics ultimately impact the specific binding (SB) and
nonspecific binding (NSB) events, which, in turn, determine
the sensitivity and specificity of the assay.
49−51
Therefore, the
goals of optimizing the assay kinetics are to (a) maximize SB
and (b) minimize NSB. Quantitative assay optimization can be
achieved by maximizing the signal-to-noise ratio (SB/NSB).
51
In this subsection, we first introduce the fundamentals of assay
Figure 2. Overview of strategies to improve the sensitivity and specificity of lateral flow assays (LFAs). (A) Schematic of a sandwich LFA. (B)
Sample enrichment: The analyte in a sample can be preconcentrated and/or amplified to enhance the detection limit. (C) Assay
optimization: Assay performance can be improved by altering the assay kinetics and reagents. (D) Signal amplification: Signals from LFAs
can be directly enhanced by either chemical enhancement methods or the use of readers. Related nomenclature is summarized in Table S1.
MNP: magnetic nanoparticle; GNP: gold nanoparticle; CNT: carbon nanotube. Figure C(1) was reprinted with permission from ref 49.
Copyright 2017 American Chemical Society.
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C
kinetics and then discuss methods to increase SB and to
minimize NSB. Related work is tabulated in Table S3.
The kinetics of transport and reaction are characterized by
the Péclet number (Pe) and the Damköhler number (Da),
respectively, which are defined as
==
P
eUR
D
convection rate
diffusion rate (1)
==
′
Da Ck R
D
reaction rate
diffusion rate
Ron
(2)
where Uis the velocity of the moving fluids through the LFA
membrane, Ris the characteristic size of the pores inside the
membrane, Dis the diffusivity of the molecules and
conjugation labels transported in the fluids, and CRis the
concentration of capture molecules in the test region.
49
The
term kon
′is the effective forward immunoreaction rate constant
for the conjugated label in the test region and is assumed to be
kon
′=nkon, where kon is the forward reaction rate constant of
the single antibody−antigen immunoreaction and nis the
effective number of antigens per label particle.
49,52
According to the Pe and Da estimations in Table 1, LFAs are
limited by the reaction rate,
49,52
and, thus, improving the
reaction efficiency is the most critical step toward maximizing
SB and boosting the sensitivity of LFAs. As shown in Table 1,
the transport of molecules and labels is limited by the diffusion
rate (≪convection rate, Pe =10−102), and the surface
reaction is limited by the reaction rate (≪diffusion rate, Da =
10−4−10−1). This improvement in reaction efficiency can be
achieved by increasing the reaction rate, which is proportional
to the reaction rate constant (reaction kinetics) and reactant
concentration and/or by increasing the reaction time.
Boosting the Reaction Rate by Increasing the Reaction
Kinetics. There are several reports on increasing the reaction
kinetics associated with forming the conjugation/antigen/
capture antibody ternary (sandwich ternary). Liang et al. found
that the sandwich ternary forms more slowly when an antigen
first binds with a conjugated label and then a capture antibody
in a premixing flow than when it first binds with a capture
antibody and then a conjugated label in a sequential flow.
60
As
a result, the limit of detection (LoD) for malarial protein from
a sequential flow was reported to be 4- to 10-fold lower than
that obtained from a premixing flow.
60
Note that longer assay
times can be a drawback of sequential flow. For nucleic acid
hybridization, the hybridization kinetics are strongly correlated
to the ionic strength at a salt concentration below 0.2 M.
61
Thus, He et al. added a saline barrier in the membrane before
the test region to accelerate the hybridization reaction,
although this addition also slowed the flow velocity and
increased the assay time.
62
As a result, they achieved a 10-fold
increase in detection sensitivity without changing the LFA
format and procedures.
62
The above-mentioned work is
summarized in Table S3.
Boosting the Reaction Rate by Increasing the Reactant
Concentration. For most immunoreactions between antigens
and antibodies, the reaction rate constant is relatively
unchangeable. Thus, concentrating the reactants can be an
effective alternative way to increase the reaction rate and,
subsequently, to increase the number of captured labels in the
test region. First, analytes in a sample can be preconcentrated
before the sample is introduced to an LFA test (see Figure
2B(1)). For example, Sharma et al. used magnetic separation
together to preconcentrate analytes in a sample prior to lateral
flow and achieved a 10-fold increase in sensitivity.
63
Mashayekhi et al. reported that analytes predetected by labels
can also be preconcentrated in a micelle-poor layer by adding
Triton X-114, a nonionic surfactant, into the sample to form a
two-phase micellar system.
64
This method was reported to
provide an ca. 10-fold decrease in the LoD.
64
Second, analytes can also be concentrated during the flow
period of an LFA. For instance, isotachophoresis (concentrat-
ing flowing ionic analytes with an applied electrical field) helps
to preconcentrate the antigen−conjugation complex and to
enhance the transport kinetics in LFAs. As a result, the surface
reaction rate and equilibrium binding of labels are dramatically
increased in the test region.
56
Experimental results show that
this technique improves the LoD by up to 400-fold.
56
Third, increasing the number of effective binding sites for
the conjugated labels can augment the reaction rate and thus
lower the LoD. This increase in binding sites can be realized
through either label design or the specific orientation of
detection molecules. For example, the size of the gold
nanoparticle (GNP) labels can be increased,
49
or the particle
surface can be functionalized with multiple layers
65
to enable
the loading of more detection molecules, resulting in increased
numbers of binding sites. Of note, the particle size and coating
are limited by the need to maintain the particles’stability and
diffusivity in flow through the porous membrane; otherwise,
staining or background noise due to settling out and/or NSB
may occur.
49
Enforcing a specific orientation of the detection
molecules on labels’surface through improved conjugation
methods was reported to generate more effective binding sites
than were achieved with randomly oriented conjugation
through traditional physical adsorption.
66−68
Aspecific
orientation can be achieved through either covalent binding
mediated by a chemical layer (e.g., PEGylation) or bioaffinity
binding mediated by a biomolecular layer (e.g., protein A and
G, biotin−streptavidin coupling, DNA-directed immobiliza-
tion).
66−68
Furthermore, the coverage of the detection
molecules should be optimized to minimize any steric
hindrance created by a dense layer of detection molecules
and to maximize the affinity for the analyte.
69
Fourth, the number of effective binding sites in the test
region can be increased. For instance, regular capture
molecules at the test line can be substituted with three-
dimensional (3D) “proteinticle”probes in which multiple
peptides are self-assembled and oriented to give a substantial
Table 1. Estimated Values of Pe and Da in LFAs
parameters estimated range of
values ref
characteristic pore size
of membrane L(m) (3−20) ×10−653,54
average fluid flow
velocity U(ms−1) (0.5−3) ×10−449,55
diffusivity (antigen,
particles) D(m2s−1)10
−12−10−10 49,55
association rate
constant kon
′(M−1s−1)
a
103−10549,52,
56−59
concentration of
capture antibodies CR(mol m−2)10
−10−10−849
Péclet number
=
P
e
UL
D
10−102(≫1
diffusion limit) 49,52
Damköhler number =
′
Da Ck L
D
Ron 10−4−10−1(≪1
reaction limit) 49,52
a
1M
−1s−1=10
−3mol m−3s−1.
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enhancement in reactivity, leading to a 4- to 8-fold improve-
ment in sensitivity.
70
Modifying the membrane with cellulose
nanofibers to enable the loading of more capture molecules can
also boost the detection sensitivity by 20-fold.
71
Cellulose
nanofibers were also reported to bring the capture molecules
closer to the surface, thus increasing the colorimetric intensity
of the captured labels by 36.5%.
72
Increasing the Reaction Time. Increasing the reaction time
can also augment the number of captured labels in the test
region. For example, putting cotton threads into the membrane
can slow the flow rate and improve the detection sensitivity by
4-fold.
73
Adding a stacking pad between the membrane and the
conjugation pad can also lengthen the reaction time, leading to
1.1- to 2-fold decreases in the LoD.
74
In addition to maximizing SB by the aforementioned
methods, minimizing NSB is also important to improve the
sensitivity of LFAs because NSB can interfere with the
detection of very low concentration analytes and pose limits to
sensitivity improvements. Thus, reducing NSB in combination
with performing other assay optimization methods can
improve the LFA sensitivity. For example, the PEGylation of
40 nm gold nanospheres (GNSs) led to a large decrease in
NSB, which otherwise arises from the aggregation of unstable
citrate-stabilized GNSs.
75
As a result, the detection sensitivity
for bisphenol A increased by 12.5-fold.
75
Likewise, introducing
a silica coating on GNPs significantly lowered the background
noise by imparting high particle stability.
76
This coating also
increased the surface area, which enabled the loading of more
antibodies.
76
Subsequently, the LoD for alpha-fetoprotein by
silica-coated GNPs was lowered by 30-fold.
76
In another
example, coating GNPs with polydopamine increased the
particles’tolerance to pH and ionic strength, thereby reducing
possible NSBs.
77
This coating also augmented the antibody
loading efficiency. As a result, the sensitivity of the label-
optimized LFAs was 10-fold higher than that achieved with
traditional, bare GNP labels.
77
A summary of related work is
provided in Table S3.
In summary, optimizing the assay kinetics is a fundamental
step toward improving LFAs that can achieve up to
hundred(s)-fold increase in sensitivity (see Table S3 and
Figure S1). Even greater sensitivity improvements can then be
achieved by amplifying the signals from test regions (see
Tables S2 and S3 and Figure S1), as discussed in the following
two subsections.
Chemical Enhancement. In addition to optimization of
the assay kinetics, sensitivity can also be extensively improved
by amplifying the signal from the test region. Increasing the
colorimetric contrast of the positive test region through
chemical enhancement is a direct and straightforward way to
amplify the signal while maintaining the convenience of visual
detection. This enhanced contrast can be realized through label
design and the use of enhancing LFA solutions (see Figure 2
D(1)). Related work is summarized in Table S3.
Label Design. Label design usually replaces the traditional
small (ca.20−40 nm) GNSs employed in LFAs with other
labels that have stronger colorimetric contrast while maintain-
ing the traditional LFA format. Stronger contrast can be
achieved by modifying the structure and size of the GNPs or
by replacing the GNPs with particle clusters or particles made
of another metal, metal oxide, or organic material, as shown in
Figure 2 D(1). For example, a label of GNP-decorated silica
nanorods (i.e., microsized silica nanorods coated with colloidal
gold), achieved a ca. 50-fold lower LoD in the detection of
rabbit IgG than traditional GNSs.
78
Similarly, polystyrene
microbeads have been used as a support for nm-scale GNSs to
enhance the colorimetric contrast of the test region; this design
improved the detection sensitivity for the influenza virus H3
subtype by 64-fold over that achieved with 10 nm GNP-based
LFAs and 16-fold over that achieved with 30 nm GNP-based
LFAs.
79
In another study, the use of gold nanopopcorn
achieved a 5-fold lower LoD for procalcitonin relative to that
obtained from LFAs that used 20 nm GNPs.
80
Strikingly, the
use of carbon nanotubes (CNTs) as a label in the detection of
rabbit IgG decreased the detection limit by 3 orders of
magnitude relative to the use of the traditional GNP-based
LFAs. This improvement arises from the higher aspect ratio of
CNTs, which enables the loading of more detection antibodies
and thereby improves the immunoreaction rate.
81
The label
design approach preserves the LFA benefits of a rapid
response, simple use, and low cost without changing any
assay formats or steps.
Use of Enhancing Lateral Flow Assay Reagents.
Enhancing LFA reagents can be applied to induce catalytic
or other chemical reactions in the test region after a normal
assay to amplify the colorimetric contrast. Catalytic amplifica-
tion is usually achieved by using an enzyme or nanozyme to
catalyze oxidation−reduction reactions in the test region. The
most widely used enzyme is horseradish peroxidase (HRP),
which catalyzes the oxidation of an organic substrate in the
presence of hydrogen peroxide. In LFAs, HRP is linked to
detection molecules that are conjugated with labels to be
captured in the test region. After a sample is run on the LFA, a
wash step is performed to remove excess labels from the
membrane. Finally, solutions of the HRP substrate and H2O2
are flowed through the LFA to achieve enzymatic amplification
and produce a strong color enhancement or chemilumines-
cence in the test region.
82
Parolo et al. reported an increase in
sensitivity of up to 1 order of magnitude over conventional
GNP-based LFAs in the detection of human IgG by applying
enzymatic amplification.
83
Recently, nanozymes (i.e., nanomaterial-based artificial
enzymes) have been discovered and rapidly developed as
direct surrogates of natural enzymes (i.e., protein-based) for
catalysis. Compared with natural enzymes, nanozymes have
advantages of higher catalytic stability, easier modification
process, and lower manufacturing cost, among others (see ref
84 for a comprehensive review of the design of nanozymes,
their applications in diagnosis and therapeutics, and the
outlook of this technology).
84
Some nanozymes have already
been used as labels in LFAs. For example, a Pt nanocatalyst
(Au@Pt core@shell structure) achieved an LoD for p24 spiked
in sera as low as ca. 0.8 pg/mL (ca. 33 fM), which is even
better than the sensitivity of commercial ELISA (>1 pg/mL,
>42 fM).
85
Similarly, other LFAs have used Au@Pt core@shell
nanostructures, such as Pt-decorated GNPs and Pt−Au
nanoflowers, for catalytic amplification and achieved LoDs
that are approximately 100-fold lower than those of conven-
tional GNP-based LFAs.
86,87
Other chemical enhancement techniques include silver
enhancement, double gold conjugation, and induced gold
aggregation. In the silver enhancement method, Ag is
nucleated on captured GNPs in the test region by flowing
Ag-reducing reagents through an LFA after a normal assay.
The resulting Ag layer on the GNP label surface amplifies the
color intensity of the test region. With this method, a
sensitivity gain of approximately 10-fold relative to that
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without Ag enhancement has been achieved.
80,88
Similarly, for
double gold conjugation, secondary GNPs are introduced to
bind with the primary GNPs that are already captured within
the test region, which results in an enhanced color intensity.
This binding can be achieved by making use of the high
biotin−streptavidin binding affinity
89
or by employing the
reaction between primary and secondary antibodies, which is
similar to the basis of indirect ELISA.
90
The double gold
conjugation method has been reported to increase the
detection sensitivity for hepatitis B surface antigen by
approximately 30-fold.
89
The induced gold aggregation
method is similar to the double gold conjugation approach,
but more GNPs can be coated on the captured GNPs with this
method, thus better amplifying the color intensity. For
example, the direct use of aggregated MNPs as labels was
found to improve the detection sensitivity by 40-fold over that
achieved with monodisperse MNPs, although large aggregates
have a slower transport velocity and thus require a longer assay
time.
91
Alternatively, using a liposome-encapsulating reagent to
aggregate additional GNPs onto the captured GNPs in the test
region achieved a 1000-fold improvement in sensitivity.
92
More impressively, adopting DNA−GNPs to induce the 3D
growth of GNP aggregates led to a decrease in the LoD by 4
orders of magnitude relative to that without signal
amplification.
93
These enhancements are also noted in Table
S3. Importantly, although these approaches increase the
sensitivity, they come with a reduction in the ability to
quantify the signal (i.e., the originally captured labels) due to
the inconsistency associated with coupling multiple reaction
steps.
Reader Use. In addition to the chemical enhancement
method, LFA signals can also be amplified with the use of
readers. With readers, captured nanoparticle (NP) labels in the
test region are excited by an external physical stimulus, such as
laser light, an electric potential, or a magnetic field (see Figure
2D(2)) to produce an amplified signal. The amplified signal is
then detected by sensitive optical/electrical/magnetic sensors
that can discern tiny signal differences over the background. By
applying intense external fields and sensitive sensors, reader
systems can enhance the detection sensitivity by several orders
of magnitude over the traditional visual readout and rival the
sensitivity of laboratory-based ELISA (see comparison in
Figure 1A). Additionally, quantification of a signal (i.e., the
amount of label) can be achieved with reader systems because
the signal intensity is generally proportional to the number of
NPs captured in the test region, which also correlates with the
amount of target analytes. Multiple types of readers are
available depending on the excitation method, including
fluorescence,
42,94
surface-enhanced Raman scattering
(SERS),
95,96
photothermal (i.e., thermal contrast,
43,44
photo-
acoustic imaging,
45,46
photothermal laser speckle imaging,
47
and thermal photonic lock-in imaging
48
), electrochemistry,
97
and magnetic amplification.
98,99
Due to the significant benefits associated with reader use,
these systems have attracted substantial attention and are being
applied in next-generation LFA technologies, as described in
multiple reviews.
11−15,32,100,101
Nguyen et al. reviewed the
majority of these reader systems and discussed their working
mechanism, setup, development, and detection sensitivity.
13
Kim et al. summarized various NPs and their roles in different
reader-assisted LFAs and compared their detection sensitivity
to those of commercial tools such as traditional commercial
LFAs, ELISA, and PCR.
100
Ye et al. extensively reviewed signal
amplification methods based on the laser excitation of
plasmonic NPs.
15
In addition to the working mechanism and
the advancements of the reader systems, the authors discussed
how to bridge the gap between laboratory readers and mature
commercial needs.
15
In addition to standard electronic readers,
smartphone-based readers are also emerging as a promising
POC technology, and additional details are discussed in other
reviews.
12,102−104
Future of Assay Improvement. In this section, we
provide a comprehensive comparison of the LFA techniques
discussed above and offer perspectives on the likelihood of
those techniques becoming mature products or meeting future
POC diagnostic needs. The sensitivities and detection times of
the techniques are systematically compared with those of the
existing commercial tests (commercial LFAs, ELISA, and
PCR) in Figure 1A. A detailed comparison of the various signal
amplification methods (labels, reagents, and readers) is
presented in Figure 1B with the corresponding literature
summarized in Table S2. Their advantages, disadvantages, and
potential for application in future ultrasensitive (sub-fM),
rapid, easy-to-use, low-cost, and multiplexed POC diagnostics
are discussed.
The sensitivity comparison shows that a substantial
improvement was achieved with these advanced LFAs, but
there remains a need for integrated assay optimization to
reduce performance variations within the same technique.
Figure 1A shows that the sensitivity of novel LFAs is 1−9
orders of magnitude better than that of traditional commercial
LFAs and that it overlaps with the sensitivity of ELISA. The
sensitivity of some of the techniques, such as fluorescence
LFA
105
and electrochemical paper-based diagnostics,
106
can
even reach the fM level (the lower limit of the ELISA
sensitivity). However, Figure 1B also indicates that there is a
large variation in the LoD (3−6 orders of magnitude) within
the same technique. This variation results from insufficient
optimization of the assay. During an assay, NSB of the labels
inevitably occurs along with SB in the test region. When signal
amplification is performed, both the signal (from SB) and the
noise (from NSB) are amplified; thus, the number of FNs is
reduced, but the number of FPs may also increase. In other
words, the sensitivity improves at the cost of the specificity. In
particular, for low target concentrations, the amount of label
captured through NSB can be comparable to or even larger
than that captured by SB. In that case, further increasing the
intensity of the external excitation field cannot discriminate the
signal from the noise, and instead, it creates FPs. This issue has
been reported for LFAs that use thermal contrast amplifica-
tion
51
as well as silver enhancement.
107
As a result, the efficacy
of signal amplification can be limited by the NSB of labels.
To address this limitation, an integrated assay optimization
method that is compatible with the signal amplification
technique is needed (see flowchart in Figure 3). This
optimization method is different from the traditional LFA
development protocol,
108,109
because the latter does not
include signal amplification in the iterative assay optimization
process. In contrast, in this integrated assay optimization
approach, an assay optimization step is performed after signal
amplification specifically to reduce NSB. After such optimiza-
tion, a more intense applied field or enhancing LFA reagents
(e.g., silver enhancement) can be used to enhance the detection
sensitivity further until the specificity suffers (i.e., the number
of FPs increases). This iterative assay optimization and signal
amplification approach can be continued until the sensitivity
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and specificity are sufficiently high. To achieve quantitative
assay optimization, Zhan et al. showed how the signal-to-noise
ratio (i.e., SB/NSB ratio) can be maximized.
51
This ratio is
related to the signal versus noise in the test region when the
assay is run in the presence and absence of the target analytes.
Alternatively, the control lines can be precoated with target
molecules,
51
thus giving SB-dependent signals. The SB/NSB
ratio can be quantified by an imaging tool or by readers. To
achieve an even higher sensitivity, multiple sensitivity-
enhancing methods can be combined in the same LFA,
although integrated assay optimization is still needed.
In addition to sensitivity, speed, ease of use, and cost are
important metrics in the evaluation of techniques for
application in POC diagnostics. A summary of the advantages,
disadvantages, and necessary future improvements of LFAs is
provided in Table 2 (with specific innovations in Tables S2
and S3), and a detailed comparison of their detection times is
shown in Figure 1B. As noted in Table 2,chemical
enhancement maintains the advantage of rapid visual
detection. More specifically, label design usually does not
impact the assay time, although the diffusion issues of large
labels and particle-dependent NSB should be considered. In
contrast, the use of enhancing LFA reagents can significantly
add to the assay time, assay complexity, and imprecision
because additional steps are needed to deliver the various
reagents. The assay time can then range from near 20 min
85
to
approximately 1 h.
110,111
To address this increase in assay time,
some researchers have sought to automate the multiple
solution-delivery steps by designing novel LFA structures.
For example, a two-dimensional paper network with internally
set timings was designed to automate a multistep assay,
111
but
its assay time was still long (ca. 1 h).
111
Another upgraded
device took advantage of a polymer that swelled upon the
addition of water to automate the sequential delivery of the
immunoreaction and amplification reagents. This approach
achieved assay completion within 20 min.
112
Further improve-
ments can be made by simultaneously miniaturizing the device,
automating all assay steps, and reducing the assay time by
exploring the reaction kinetics.
Similarly, external signal amplification with readers can also
add to the detection time and cost of an assay, although this
method has the additional advantage of providing reproducible
quantitative readouts. The increase in detection time
introduced by most readers is usually <20 min except for the
electrochemical measurement. The total time consumed by the
electrochemical method is usually <30 min,
106,113,114
but some
assays with multiple steps can take over 1 h.
115
Many efforts
have been made to reduce the scanning time of these readers.
For example, the fastest SERS reader can scan a test region in
only 5 s,
96
and a thermal contrast reader with a scanning time
of <1 min is being developed.
116
Some of the efforts to reduce
scanning time are extensively discussed in a separate review.
15
The reliability and reproducibility of the reader performance in
various POC conditions also remain unknown. Therefore,
further work on miniaturization, cost reduction, and validation
through clinical trials is needed to make these readers better
suited for POC applications.
In addition, there is a strong drive to develop multiplexed
LFAs that are capable of simultaneously detecting multiple
analytes due to the associated advantages of a reduced cost,
Figure 3. Integrated assay optimization with signal amplification
methods. The traditional lateral flow assay (LFA) development
steps are described in other work.
108,109
FPs: false positives; FNs:
false negatives.
Table 2. Advantages, Disadvantages, and Necessary Future Improvements of Lateral Flow Assays
signal amplification advantages disadvantages future improvements
chemical enhancement sensitivity increase NSB
51
miniaturization
fast visual readout diffusion issues with large labels
49
automation
adds time and complexity
110,111
simplification
may not be quantified
76
integrated assay optimization (SB/NSB)
multiple analyte detection
combination approaches
novel signal processing methods
readers sensitivity increase NSB
51
clinical validation
quantification
13,15,100
adds steps and time
13,15,100
miniaturization
reproducibility
13,15,100
adds cost
13,15,100
cost reduction
complex structure (electrochemical)
114,121
integrated assay optimization (SB/NSB)
multiple analyte detection
combination approaches
novel signal-processing methods
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lower sample volume, and the ability to discriminate rapidly
between diseases with common symptoms. However, several
challenges with multiplexed LFAs remain, including cross-
reactivity between affinity molecules for different target
analytes, physical limitations of multiplexed test regions in an
LFA strip, and difficulty in clinical validation, which requires
patients with multiple co-infections.
117
More in-depth
discussion on the development, challenges, and opportunities
of multiplexed LFAs can be found in other work and
reviews.
12,103,117−119
Note that vertical flow assays (VFAs),
which operate under similar fundamental principles as LFAs
but have a different flow direction, can increase the
multiplexing capability and reduce cross-reaction effects.
97,120
A detailed comparison of LFAs and VFAs is available in other
reviews.
103,104
Finally, novel signal-processing methods are emerging to
enable sub-fM detection sensitivity. The signals from LFAs are
usually read in an analog format, which intrinsically limits the
upper limit of sensitivity improvements, that is, the readers
require millions of labels to be accumulated to generate a
detectable signal intensity. As a result, it is difficult for these
techniques to achieve sub-fM sensitivities similar to that of
ELISA, as shown in Figure 1A. In contrast, digital ELISA
(dELISA) enables the detection of sub-fM to aM concen-
trations (i.e., PCR-level sensitivity or single-molecule detection
in 1 μL of solution) owing to its digitized signal acquisition
method.
122,123
dELISA can count the signal from each enzyme
label after immunoreaction.
122,123
The state-of-the-art com-
mercial dELISA system is the signal-molecule array (Simoa)
and the corresponding Simoa HD-1 Analyzer.
122,124
The
Simoa enables partitioning of hundreds to thousands of
paramagnetic beads into femtoliter wells. Each well can hold at
most one bead, thus concentrating the immunoreaction
product from a single target molecule in a femtoliter
droplet.
122,124
By counting the signals from each well, dELISA
can achieve a ca. 1000-fold improvement in sensitivity over
conventional ELISA.
122,124
However, the equipment costs ca.
100,000 USD, and a testing operation takes ca.1hto
complete, although multiple samples can be loaded togeth-
er.
124,125
To lower the cost and detection time, many efforts
have focused on upgrading the Simoa platform; these efforts
include droplet dELISA,
126
droplet-free dELISA,
127
mobile
dELISA,
125
and dropcast single-molecule arrays.
128
Of note,
these platforms are not yet ready for POC use because of
either the complexity of their fabrication and use or the long
turnaround times required for signal processing. Nevertheless,
it is valuable to consider combining the signal acquisition
method of dELISA with existing LFAs to develop a cheaper
and simpler (sample-to-answer) digital detection platform for
ultrasensitive (sub-fM or aM) POC diagnosis. This goal may
be achieved by taking advantage of porous membranes that
allow automated fluid flow, reagent storage, mixing, and
volumetric reactions and transparent plastic containers that
enable imaging-assisted digital signal acquisition.
Miller et al. proposed another signal processing method for
spin-enhanced LFAs to enable sub-fM detection of labels.
129
The authors modulated the fluorescence intensity of nano-
diamond labels by a microwave field (spin manipulation),
hence the fluorescence from labels could be separated from
background autofluorescence in the frequency domain with
lock-in analysis. As a result, this method achieved a 105-fold
increase in sensitivity for the avidin−biotin (direct binding)
Figure 4. Indirect sample preamplification for the polymerase chain reaction level detection of proteins and bacteria. (A) Use of
nanoparticle-based biobarcodes and magnetic microparticles for the ultrasensitive detection of proteins.
135
(B) Ultrasensitive and DNA
extraction-free detection of salmonella based on aptamer-mediated strand displacement and amplification.
137
(A) Reprinted with permission
from ref 135. Copyright 2003 AAAS. (B) Reprinted with permission from ref 137. Copyright 2014 Elsevier.
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assay than traditional GNP-based LFAs.
129
Note that in
sandwich LFAs, NSB of nanodiamond labels in the test region
can pose a limit to the sensitivity improvement. Thus,
integrated assay optimization (Figure 3) will still be needed.
Overall, after assay optimization, device upgrade, and
validation, this method holds promise to be adapted for future
ultrasensitive POC use.
IMPROVING SENSITIVITY BY SAMPLE
PREAMPLIFICATION
In addition to improving LFAs, preamplifying the analyte
(Figure 2 B(2)) is also an effective way to boost sensitivity.
Both nucleic acids (direct amplification) and other analytes
(indirect amplification) can be amplified prior to LFA testing,
leading to PCR-level (aM) sensitivity.
Amplifying Nucleic Acid Analytes. The gold standard
and most widely used sample preamplification tool is PCR,
which can achieve aM detection sensitivities for target DNA/
RNA (see Figure 1A).
100
However, PCR cannot be used in
POC settings due to the need for specialized equipment,
trained operators, and a strictly controlled working environ-
ment.
6
To address this limitation, isothermal amplification
methods, which can amplify nucleic acids at a constant
temperature without the need for thermal cycling, have been
proposed as a substitute for PCR with the goal of
decentralizing ultrasensitive DNA/RNA diagnosis from the
laboratory to POC settings. Multiple isothermal amplification
methods are under development, and some are capable of
completing the preamplification step within 30 min, as featured
in many recent reviews,
130−132
and are being commercial-
ized.
132−134
The readout format of these methods can be either
real-time or end point. The real-time readout is usually based
on fluorescence and needs an excitation light source and a
photodetector, which is not ideal for POC tests due to the
added cost and energy consumption of this equipment. In
contrast, LFAs can utilize a simple end point readout. In
summary, as shown in Figure 1A, analyte amplification with an
LFA can yield PCR-level results at the cost of a somewhat
greater complexity, potentially reduced dynamic range, and
longer time duration (>30 min) compared to typical LFAs.
Amplifying Analyte-Specific Aptamers. Unlike DNA
and RNA, other targets such as proteins cannot be directly
amplified and therefore are difficult, if not impossible, to
achieve PCR-level detection by LFAs. However, novel
methods for amplifying target-related DNA or target-specific
aptamers have been proposed, as shown in Figure 4.Figure 4A
presents the biobarcode amplification method proposed by the
Mirkin group.
135
In this assay, target proteins are dually
recognized by antibody-conjugated GNPs and magnetic
microparticles. The GNPs are also pre-encoded with
biobarcode DNA in addition to detection antibodies. After
magnetic separation, the biobarcode DNA fragments are
released from the NPs, after which they are amplified and
detected. The detection of the biobarcode DNA indicates the
presence of the target proteins. This method lowered the
detection limit for the protein of interest to as low as 30 aM
(within PCR-level sensitivity). A similar method was also
applied to the detection of target DNA with an LoD of 0.5 aM,
which is also a PCR-level sensitivity.
136
Figure 4B shows
another example of indirect amplification in which aptamers
are developed to specifically recognize a target and become
amplified after magnetic separation.
137
In this method, target
bacteria are first recognized by two aptamers. One of the
aptamers is linked to magnetic beads to enable magnetic
separation, while the other is amplified and detected afterward.
This method achieved a detection limit for Salmonella
enteritidis of as low as 10 colony forming units without DNA
extraction.
137
However, these assays require multiple steps and
take hours to complete. Furthermore, the extensive manual
manipulation required in this technique may compromise the
reproducibility. Therefore, further automation and reduction of
the assay time are needed to make this technique suitable for
future large-scale POC use.
The Future of Sample Preamplification. Although the
amplification of targets or target-specific aptamers provides
access to PCR-level detection sensitivities, challenges remain in
further advancing these technologies to achieve future
ultrasensitive and highly specific POC testing, which requires
that they be simple (sample-to-answer), rapid (<30 min), and
inexpensive.
First, further investigation is needed to develop robust
“sample-to-answer”devicesthatcanintegrateallsteps,
including DNA/RNA extraction, amplification, and end point
readout. The inclusion of multiple steps increases the assay
complexity, time, and labor and contributes to a lack of
robustness and inaccuracy. To address these drawbacks,
microfluidic platforms for automating these assay steps are in
development.
134,138
However, these platforms usually require
electrical pumps or centrifuges.
134,138
To eliminate the need
for electrical equipment, microfluidic platforms such as 3D-
structured microfluidic paper,
139,140
capillary tubes,
141
and
vacuum-powered microfluidic chips
142
have been developed.
Although these are intriguing developments, these assays
require careful further development and validation to ensure
that they are robust.
Second, the added sample preamplification step increases
the total detection time. As shown in Figure 1A, most systems
that combine isothermal amplification with fast readout still
take 1/2−4 h to complete. The reaction itself has been studied
in depth to improve the kinetics of the amplification step. For
example, among the various isothermal methods, recombinase
polymerase amplification (RPA),
143
loop-mediated isothermal
amplification (LAMP),
144,145
and exponential amplification
reaction (EXPAR)
146,147
can complete the amplification
reaction within 1 h. Further, some real-time RPA detection
methods can be completed within 10−20 min.
148−150
More
examples are summarized in a recent review article.
143
Note
that the sensitivity may be compromised by cutting down the
preamplification time and, thus, combining sample preampli-
fication with an existing assay improvement method for the
end point LFA readout may be a promising avenue.
Third, nonspecific sample preamplification can occur, which
makes the assay less specific. This issue has been reported with
isothermal methods
134,151−153
such as LAMP,
154
RPA,
151,152
rolling circle amplification (RCA),
134
helicase-dependent
amplification (HDA),
134
and EXPAR.
153
To minimize non-
specific sample preamplification, it is critical to optimize the
primer design and assay conditions.
133,151
Another promising
way to maintain specificity is with the newly discovered
clustered regularly interspaced short palindromic repeats
(CRISPR) and CRISPR-associated (Cas) systems.
155,156
The
CRISPR/Cas systems are highly specific to target sequences,
thus enabling highly specific detection.
157
However, challenges
with incorporating the CRISPR/Cas reaction into future
“sample-to-answer”POC devices remain.
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Fourth, the dynamic detection range of analytes may be
reduced by sample preamplification due to saturation in the
exponential amplification step.
158
This reduction can be
problematic because the quantification of analytes in a wide
concentration range is important to clinical treatment. To
address this limitation, the amount of analyte and primers in
the amplification step needs to be optimized.
Finally, other intrinsic issues related to sample preamplifi-
cation also need to be addressed. These issues include the
tolerance to impurities and inhibitors, the presence of
amplification bias, and the occurrence of cross reactions in
multiplexed amplification. More details about and insights into
these issues can be found in other reviews.
133,159
Above all,
sample treatment (purification), device innovation (reagent
mixing to enhance the reaction), and reagent optimization and
screening (for multiplexed sample preamplification and
detection) will need to be further investigated to make assays
robust for POC use.
To summarize the methods for improving sensitivity,
advanced assay improvement techniques (assay kinetics
optimization, chemical enhancement, and reader use) enable
LFAs to achieve ELISA-level (pM−fM level) sensitivities,
although further effort is needed to bring them to successful
commercialization for POC use. The combination of sample
preamplification with an LFA readout also holds promise for
future rapid (<30 min) and ultrasensitive (PCR-level or aM-
level) POC diagnostics after the challenges mentioned above
are addressed.
IMPROVING SPECIFICITY
Specificity is equally important as sensitivity in the develop-
ment of a robust POC diagnostic test. Improving the specificity
relies on not only minimizing the NSB but also screening for
the most specificaffinity molecules.
Assay Optimization. Assay optimization to reduce the
NSB that results in an FP readout is critical to improving the
specificity of LFAs. The NSB of labels in the test region usually
arises from the following sources:
•Presence of NSB substances in a sample that are capable
of binding to the affinity molecules in LFAs
160
•Nonspecific interactions between the conjugated labels
and the capture antibodies and/or membrane
51,75,161
•Physical trapping of the conjugated labels (especially
aggregates of unstable labels) in the porous membra-
ne
49,51,75
To address these issues, the following approaches are
outlined in the sections below.
Reducing Substances Capable of Nonspecific Binding.
Multiple methods are available for treating samples to reduce
substances capable of NSB, as summarized in Table 3. For
example, when detecting analytes from whole blood, blood
cells and other large particles are usually separated from the
blood before the LFA is run by adding a filtration pad before
the conjugation pad
162
or by centrifuging the sample. Also,
preheating urine was found to reduce the activity of thermally
labile biomolecules, thus decreasing the number of FPs
obtained when detecting cryptococcal antigen by LFAs.
163
For DNA/RNA LFAs with a sample preamplification step,
FPs can be produced by the primers/probes and side products
from the preamplification step, such as primer dimers.
160,164,165
Such FPs can be reduced by optimizing the primer and probe
sequences in the preamplification step
151,152,166,167
and the
capture and detection sequences in the LFA.
168
Other special
treatments can also be used to prevent FPs. For example, Li et
al. used a graphene oxide pad to filter residual primers and
primer dimers to increase the specificity.
160
Similarly, running
LFAs at 37 °C was reported to reduce the nonspecific
adsorption of nucleic acid-conjugated labels to the test region
(see Table 3).
168
Other Assay Optimization Methods. Other typical assay
optimization methods for minimizing NSB include:
•Surface modification/blocking of labels
•Optimizing the label size and concentration
169
•Screening the running buffer
•Membrane blocking
The work related to other assay optimization methods is
summarized in Table 3 and discussed in detail below. Of note,
these assay modification methods can also impact SB; thus, the
signal-to-noise ratio (i.e., SB/NSB ratio)
51
should also be
evaluated and maximized.
First, the surface modification/blocking of labels prevents
label aggregation-induced NSB by blocking NSB sites and
increasing the hydrophilicity and stability of the labels. Typical
blockers and stabilizers (see Table S1 for definition) include
proteins (e.g., bovine serum albumin (BSA)) and sug-
ars.
170,173,174
The surface functionalization of labels, such as
through PEGylation
28
and hydroxylation,
174
can also increase
the particle stability and prevent nonspecific electrostatic
interactions. Second, the label size and concentration
simultaneously affect both SB and NSB.
49,169
Smaller labels
showed lower signal intensity and sensitivity compared to
larger ones, whereas labels that were too large had diffusion
and NSB problems.
49,169
Likewise, insufficient label concen-
tration led to low signal intensity, whereas too high of a
concentration resulted in NSB and background noise.
168
Thus,
label optimization is needed to maximize the SB/NSB ratio for
the best assay performance.
51
Third, the composition of the
running buffer (see Table S1 and Figure 2C(2)) has proven to
be important for the overall LFA performance.
51,171
For
instance, surfactants are added to the running buffer to
Table 3. Innovations for Reducing NSB to Improve
Specificity
a
approaches consequences ref
Reducing Substances Capable of NSB
blood cell pad filter out blood cells 162
heat fresh urine inactive biomolecules 163
graphene oxide pad filter out primers and primer-dimers 160
primer and probe
sequence reduce side products in sample
preamplification 151,152,
166,167
detection/capture
sequence reduce NSB to nontarget sequences 168
elevate LFA
temperature reduce NSB of conjugated GNPs at
test regions 168
Other Assay Optimization Methods
NP surface coating stabilize NPs to avoid aggregation 75
optimal label size and
concentration reduce NSB of labels at test regions 169
blocking conjugates reduce NSB of labels to nontarget
molecules and membrane 170
blocking membrane reduce NSB of proteins to membrane 51,171,172
assay running buffer
screening maximize SB and minimize NSB;
preserve NP stability 51,171,173
a
LFA: lateral flow assay; NP: nanoparticle, SB: specific binding.
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increase the hydrophilicity of biomolecules and membranes
and to reduce nonspecific hydrophobic interactions; however,
an excessively high concentration of surfactant adversely affects
SB.
173
The pH, ionic strength, blockers, and stabilizers in the
running buffer need to be tuned to preserve the stability of the
labels and to reduce interference from heterophilic antibodies
in patient samples while maintaining a high SB efficiency.
Again, the SB/NSB ratio can serve as a quantitative metric for
screening the running buffers.
51
Finally, membrane treatment
prior to performing the assay is optional to reduce NSB by
blocking possible NSB sites in the membrane. For example, De
Puig et al. found that treating the membrane with human
serum reduced FPs significantly.
172
Highly SpecificAffinity Molecules. Optimal affinity
molecules provide maximal specificity (i.e., maximal SB while
minimizing NSB). The selection and screening of appropriate
affinity molecules (i.e., antibody or aptamer) for its analyte (i.e.,
protein, DNA, or RNA) are critical to assay perform-
ance.
175−177
For standard single protein detection, antibody
pair screening is usually performed by reagent suppliers. In
multiplexed assays, the degree of NSB is proportional to the
number of target analytes and antibodies.
12,178
Hence, more
extensive characterization and careful selection of antibodies
are needed. For example, Bosch et al. screened monoclonal
antibodies in order to develop a multiplex LFA that could
distinguish antigens from four serotypes of the Dengue and
Zika viruses.
179
More detailed discussions are available in
reviews on multiplexing POC diagnostics.
12,180
Although protein/antibody-based LFAs have a rich history,
LFAs based on different analyte/affinity molecules show
promise for improving LFA performance. New affinity
molecules, such as nanobodies, short peptides, and aptamers,
can improve the LFA specificity and sensitivity due to their
excellent reaction kinetics and selectivity for target analytes. A
comparison of the available affinity molecules is provided in
Table 4. A nanobody (ca. 15 kDa) is an antibody fragment (see
Figure 2C(2)) that can be generated by recombinant methods.
Due to their small size and concave shape, nanobodies can
recognize and capture cryptic epitopes from antigens that are
difficult to access with whole antibodies (usually 150
kDa).
12,181,182
Furthermore, due to the presence of fewer
charged groups, nanobodies have fewer problems with cross
reactions than whole antibodies.
183
Short peptides (5−20
amino acid residues) share a similar size and advantage as
nanobodies (ca. 15 kDa). Aptamers are single-stranded
oligonucleotides (usually 20−60 nucleotides, approximately
13−39 kDa) and are commonly selected by the systematic
evolution of ligands by exponential enrichment process.
184
Compared with whole antibodies, nanobodies, and short
peptides, aptamers are more thermally and chemically stable,
have less batch-to-batch variability, better high-affinity kinetics,
and lower dissociation constants, and they are easier to orient
upon immobilization, thus providing more consistent and
accurate assays.
185−187
Furthermore, antibodies, nanobodies,
and short peptides can detect only immunogenic molecules
(i.e., proteins and haptens), whereas aptamers can detect any
type of target regardless of whether it has immunogenic
properties. Also, the better stability of aptamers compared to
other affinity molecules enables aptamers to retain high affinity
and specificity in different conditions.
185−187
Aptamers are,
therefore, promising as substitutes for antibodies.
185−187
However, antibodies have the advantage of being able to
detect larger targets than other affinity molecules due to their
larger molecular size (ca. 150 kDa for antibodies versus usually
<40 kDa for the other affinity molecules). In addition, the
production method, cost, and commercialization can also affect
the use of affinity molecules in applications. For example,
aptamers are used less frequently than antibodies because
aptamer selection and production are still being commercial-
ized, whereas antibody generation has a much longer history
Table 4. Comparison of Affinity Molecules
type antibody nanobody short
peptide aptamer
target anaytes
186
immunogenic: proteins, haptens any target, including ions, non-immunogenic or toxic targets, and
cells
target size
186
larger than others smaller than antibodies
affinity
12
high high high higher
dissociation constant,
12
(M)
a
10−7−10−910−6−10−11 10−6−10−810−9−10−12
batch difference
186
varied less varied uniform
stability
186,187
sensitive to heat, pH more pH and heat stable
than antibodies tolerant to heat, pH, salt, and chelating agents
187
oriented immobilization
15
difficult, requires multiple
steps easier than antibodies easy
a
Note: Biotin−avidin interaction has a dissociation constant of about 1.3 ×10−15 M.
189
Table 5. Different Cas Effectors in CRISPR/Cas Systems and Example Diagnostic Platforms
a
types Cas9 (dCas9) Cas12 Cas13a
target dsDNA ssDNA, dsDNA ssRNA
cleavage target only target and other sequences (i.e., collateral cleavage)
diagnosing
platforms RHC,
201
NASBA,
202
PC reporter,
203
CAS-EXPAR,
204
CASLFA
195
DETCTR,
196,205
HOLMES
200,206
SHERLOCK,
158,199,207
HUDSON +
SHERLOCK
197
a
dCas9: catalytically dead Cas9 (inactive endonuclease); dsDNA: double-stranded DNA; ssDNA: single-stranded DNA; ssRNA: single-stranded
RNA; RHC: RCA-CRISPR-split-HRP; NASBA: nucleic acid sequence-based amplification; PC reporter: Paired dCas9 PC reporter; CAS-EXPAR:
CRISPR/Cas9 triggered isothermal exponential amplification reaction; CASLFA: CRISPR/Cas9-mediated lateral flow nucleic acid assay;
DETCTR: DNA endonuclease-targeted CRISPR trans reporter; HOLMES: 1 h low-cost multipurpose highly efficient system; SHERLOCK:
specific high-sensitivity enzymatic reporter unlocking; HUDSON: heating unextracted diagnostic samples to obliterate nucleases.
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K
(ca. 120 years for antibodies versus ca. 20 years for aptamers)
and is already commercialized.
188
Comprehensive comparisons
and reviews of the applications of affinity molecules have been
provided in other work.
12,185−187
Promising Highly Specific CRISPR/Cas Systems.
CRISPR/Cas recognition is among the most promising,
novel, and effective solutions to nonspecific nucleic acid
diagnostic problems. CRISPR/Cas systems have a high
binding specificity for target sequences and can even identify
single nucleotide polymorphisms.
157
Multiple Cas effectors
(Cas9, Cas12, Cas13) have been discovered since the first
discovery of Cas9 in 2013
190,191
and have led to the 2020
Nobel Prize in Chemistry.
192
Table 5 briefly summarizes the
characteristics of different Cas effectors and their reported
diagnostic platforms. The working mechanisms and diagnostic
applications of these CRISPR/Cas systems have been
comprehensively reviewed in other work.
157,193
Briefly, Cas
effectors use CRISPR RNA (crRNA) as a guide to recognize a
target DNA/RNA and then perform target-dependent
cleavage. The Cas12 and Cas13 effectors collaterally cleave
the other DNA or RNA reporters in the system into small
pieces, whereas Cas9 cleaves only the target dsDNA.
Substantial effort has been devoted to implementing
CRISPR/Cas reactions in diagnostic platforms to achieve
highly specific detection. Here, we limit our discussion to the
LFA-based diagnostic platforms that hold promise for
adaptation into future POC tests. For Cas9 effectors, the
release of cleaved DNA is extremely slow and can take up to
several hours.
194,195
To take advantage of this slow release,
Wang et al. proposed a CRISPR/Cas9-mediated lateral flow
nucleic acid assay (CASLFA) format (shown in Figure 5A)
where the Cas9 effector was directly incorporated in the
LFA.
195
In this CASLFA format, DNA-conjugated GNPs
recognize a universal loop sequence from the crRNA (i.e.,
CRISPR RNA) in a CRISPR/Cas9-DNA ternary complex,
while the biotinylated DNA amplicon from that complex binds
to the streptavidin-coated membrane to form a sandwich
assay.
195
With amplified DNAs, the CASLFA format can reach
a detection limit near the level of PCR (hundreds of copies of
genomes per μL sample) with high specificity (100% for 110
clinical samples compared with the gold standard PCR results)
within 1 h.
195
With regard to Cas12 and Cas13 effectors, the
CRISPR/Cas reaction can simply be added after sample
preamplification (by either PCR or isothermal methods) and
before the LFA readout.
158,196,197
An example of a Cas12a-
mediated diagnostic platform is shown in Figure 5B. In the
Cas12a reaction, the presence of target sequences triggers the
autocleavage of genome probes that are terminated by biotin
and a fluorophore (FAM).
198
The LFAs are designed to detect
the presence or absence of the autocleavage products. If not
Figure 5. Lateral flow assay (LFA) readout formats in different Cas effector-mediated diagnostic platforms. (A) CRISPR/Cas9-mediated
lateral flow nucleic acid assay
195
(sgRNA: single-guide RNA). (B) Cas12a-mediated DNA preamplification which cleaves biotin and FAM-
labeled probes with an LFA readout.
198
In the absence of target DNAs, the LFA’s control line will deplete the available gold nanoparticle
(Au-NP) labels, leaving an empty test line.
198
(A) Reprinted from ref 195. Copyright 2020 American Chemical Society. (B) Adapted from ref
198, whose images are licensed under Creative Commons Attribution License 4.0 (CC BY).
ACS Nano www.acsnano.org Perspective
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cleaved, the control line with streptavidin depletes the biotin-
linked sequence, leaving the test line blank.
198
If cleaved, the
control line still binds with the biotin-linked sequence, while
the cleaved FAM forms a sandwich assay on the test line.
198
As
a result, the assay showed 100% sensitivity and specificity for
20 clinical samples that were previously characterized by PCR.
This diagnostic principle has also been applied in other Cas12
and Cas13-mediated bioassays, such as DETECTR,
196
SHERLOCK,
158
and HUDSON.
197
Note that these CRISPR/Cas-mediated diagnostic platforms
can identify both DNA and RNA targets and are not limited by
the target types listed in Table 5 when coupled with sample
preamplification. This flexibility arises because the preampli-
fication step can convert between nucleic acid types (tran-
scription for DNA to RNA and reverse transcription (RT) for
RNA to DNA). For example, by using RT-recombinase
polymerase amplification (RT-RPA), the Cas13-mediated
SHERLOCK test can detect both RNAs and DNAs.
199
Also,
by designing different CRISPR/Cas systems paired with
various end labels, multiplex diagnosis can be achieved.
158
However, the assay time required for CRISPR/Cas
recognition needs to be optimized, and the kinetics of target
DNA/RNA recognition by Cas effectors and of nucleic acid
cleavage require further study. For example, the reaction of
Cas12b with target DNAs and the cleavage of probes takes
approximately 30 min, which can add to the total assay time.
200
Thus, optimization to incorporate the CRISPR/Cas recog-
nition step while maintaining a rapid test will be an important
topic of further research. Also, as mentioned earlier, a “sample-
to-answer”instrument that miniaturizes and automates all the
reaction steps, including DNA/RNA extraction, amplification,
CRISPR/Cas reaction, and readout, needs to be developed to
achieve a future POC testing platform.
CONCLUSIONS AND PROSPECTS
Lateral flow assays continue to be among the most common
POC tests globally due to their quick readout, low cost, and
ease of use, with notable drawbacks being low sensitivity, low
specificity, and lack of quantitation. Recent work on assay
improvement and sample enrichment by preamplification is
now addressing these remaining drawbacks and making LFAs
competitive with more expensive and time-consuming
laboratory tests (e.g., ELISA and PCR). For instance, assay
kinetics optimization and signal amplification (by chemical
enhancement and reader use) enable LFAs to achieve ELISA-
level sensitivities (pM−fM). Further improvements through
approaches such as isothermal preamplification (direct or
indirect) prior to the LFA readout are now poised to achieve
even greater sensitivity. Furthermore, the specificity, which is
of paramount importance to the LFA performance, can be
enhanced through assay optimization and the identification
and use of highly specificaffinity molecules. With these
improvements in sensitivity and specificity, clear proof of
concept exists that ultrasensitive (aM) highly specific LFAs for
POC diagnostics can be achieved. Nevertheless, the integration
of sample treatment, preamplification, highly specificaffinity
molecule reactions (e.g., CRISPR/Cas), and rapid LFA readout
at an affordable cost are important challenges to be met.
Ultimately, with the growing knowledge and tools reviewed
here, we believe that these challenges will be met such that
LFAs will soon achieve laboratory testing performance while
maintaining their advantages for rapid and large-scale POC
use.
ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsnano.0c10035.
More tables and figures that summarize the nomencla-
ture and descriptions, limits of detection of various
signal-amplified LFAs, innovative work on assay kinetic
optimization and chemical enhancement, and the
comparison of sensitivity range increases imparted by
various methods (PDF)
AUTHOR INFORMATION
Corresponding Author
John C. Bischof −Department of Mechanical Engineering,
University of Minnesota, Minneapolis, Minnesota 55455,
United States; Department of Biomedical Engineering and
Director, Institute of Engineering in Medicine, University of
Minnesota, Minneapolis, Minnesota 55455, United States;
orcid.org/0000-0001-6726-7111; Email: bischof@
umn.edu
Authors
Yilin Liu −Department of Mechanical Engineering, University
of Minnesota, Minneapolis, Minnesota 55455, United States;
orcid.org/0000-0002-8521-8943
Li Zhan −Department of Mechanical Engineering, University
of Minnesota, Minneapolis, Minnesota 55455, United States
Zhenpeng Qin −Department of Mechanical Engineering,
Department of Bioengineering, and Center for Advanced Pain
Studies, University of Texas at Dallas, Richardson, Texas
75080, United States; Department of Surgery, University of
Texas Southwestern Medical Center, Dallas, Texas 75390,
United States; orcid.org/0000-0003-3406-3045
James Sackrison −3984 Hunters Hill Way, Minnetonka,
Minnesota 55345, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsnano.0c10035
Notes
Theauthorsdeclarethefollowingcompetingfinancial
interest(s): John Bischof is a founder and James Sackrison
an employee of Vigilant Diagnostics. This company is focused
on commercialization of thermal contrast amplification readers
and lateral flow assays.
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation
(CBET- 2029474) and the Medtronic-Bakken Endowed Chair
to J.C.B.
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