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Article
Integrated graphene oxide purification-lateral flow test strips (iGOP-LFTS) for
direct detection of PCR products with enhanced sensitivity and specificity
Shanglin Li, Yin Gu, Yi Lyu, Yan Jiang, and Peng Liu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02769 • Publication Date (Web): 26 Oct 2017
Downloaded from http://pubs.acs.org on October 27, 2017
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Analytical Chemistry
Integrated graphene oxide purification-lateral flow test strips (iGOPLFTS) for direct detection of PCR products with enhanced sensitivity
and specificity
Shanglin Li,1 Yin Gu,1 Yi Lyu,2 Yan Jiang,2 Peng Liu1,*
1
Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment
of Infectious Diseases, Tsinghua University, Beijing, 100084, China
2
National HIV/HCV Reference Laboratory, National Center for AIDS/STD Control and Prevention, Chinese Center for
Disease Control and Prevention, Beijing, 102206, China
ABSTRACT: An integrated graphene oxide purification-lateral flow test
strip (iGOP-LFTS) was developed for on-strip purifying and visually
detecting polymerase chain reaction (PCR) products with an improved
sensitivity as well as a more stringent specificity. PCR products amplified
with a pair of biotin- and digoxin-labeled primers were directly pipetted
onto GO pads, on which graphene oxide selectively adsorbed residual
primers and primer-dimers with the aid of a running buffer containing
MgCl2 and Tween 20. By stacking up three GO pads to increase the
surface area for adsorption, 83.4% of double-stranded DNA with a length
of 30 bp and 98.6% of 20-nt primers could be removed from a 10-µL DNA
mixture. Since no primers interfered with detection, the increase of the sample loading volume from 5 to 20 µL could improve the
signal-to-noise ratio of the test line 1.6 fold using the iGOP-LFTS while no changes were observed using the conventional LFTS.
The limit of detection of the iGOP-LFTS was determined to be 30 copies of bacteriophage λ-DNA with naked eyes and this limit
could be further decreased to 3 copies by loading 20 µL of the sample, which corresponded to a 1000-fold improvement compared
to that of the LFTS detected by naked eyes. When the ImageJ analysis was employed, a 100-fold decrease of the detection limit can
be obtained. In addition, due to the removal of the primer-dimers, the dim test line observed in the negative control of the LFTS
was eliminated using the iGOP-LFTS. A mock clinical specimen spiked with defective HIV-1 (human immunodeficiency virus)
viruses was successfully analyzed using a two-step reverse transcription-PCR with 30 amplification cycles followed by the iGOPLFTS detection. These significant improvements were achieved without introducing any additional hands-on operations and
instrumentations.
A sensitive yet easy-to-use detection method is indispensable for
realizing nucleic acid testing (NAT) in point-of-care
diagnostics.1,2 While many detection strategies, such as real-time
polymerase chain reaction (PCR),3 electrophoresis,4 microarray,5
mass spectrometry,6 and DNA sequencing,7 have been routinely
employed for nucleic acid analysis, the dependence on bulky
instruments and the complicated operations together with high
costs in reagents and consumables make these methods not
suitable for using outside clinical laboratories.
Lateral flow test strip (LFTS) has been recognized as a promising
detection approach due to its advantages of colorimetric analysis
with naked eyes, simple operations that can be performed by
untrained personnel, and the compact sizes of the paper strips.8-10
One of the most straightforward methods for detecting nucleic
acids with LFTS was achieved by employing biotin and
fluorescein isothiocyanate (FITC) labeled primers for PCR
amplification and then by detecting the double-labeled amplicons
with anti-FITC antibodies on a test strip.11 Since then, this double
labeling scheme as well as the variants have been widely
employed as they were easy to realize and required no additional
hands-on preparations for detection.12,13 However, this method has
two critical problems: first, the residual primers can compete with
the amplicons for the binding sites of the test line on the test strip,
resulting in a low sensitivity. Second, the inevitable primerdimers containing both labels of the primer pair can produce false
positive results.11 Several signal amplification approaches have
been developed to improve the sensitivity of the LFTS, including
fluorescent detection,14,15 electrochemical sensing,16 enzymatic
amplification,17 etc. Meanwhile, antibody-free lateral flow devices
have been successfully invented for the detection of nucleic acids.
These hybridization-based test strips can to some extent eliminate
the interferences of residual primers and primer-dimers due to the
additional sequence recognition by capture oligos.18,19 However,
the abovementioned methods usually either introduce additional
manual operations, such as DNA denaturation and oligo
incubation, or depend on extra instrumentations, undermining the
“dip-to-use” concept of the LFTS.20
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Recently, a variety of nucleic acid amplification methods have
been developed to couple with lateral flow test strips for rapid
genetic analysis with low costs.21-23 For examples, strand
displacement amplification mediated by aptamers was analyzed
using test strips, realizing a sensitivity of 10 CFU (colony forming
unit) of S. enteritidis.24 Loop mediated amplification (LAMP) was
integrated with test strips in a cartridge for the detection of
Mycobacterium tuberculosis genomic DNA.25 Unfortunately, in
contrast to PCR, these amplification methods were still not widely
adopted in clinical diagnosis owing to the lack of the versatility in
amplifying various genetic targets, the proneness to
contamination, and the lengthy process of optimization.
Therefore, a lateral flow test strip that can take the advantages of
PCR while eliminating the interference of primers and primerdimers is highly desired.
Here we describe an integrated graphene oxide purification-lateral
flow test strip (iGOP-LFTS), on which graphene oxide (GO) is
immobilized on three stacked GO pads to purify PCR products
directly, leading to enhanced sensitivity and specificity. It has
been well known that graphene oxide has a much stronger
adsorption to single-stranded DNA (ssDNA) than that to doublestranded DNA (dsDNA).26,27 A variety of biosensors based on the
high affinity of GO to ssDNA have been reported with a high
sensitivity at a low cost.28 More recently, Huang et al.
demonstrated the separation of ssDNA from dsDNA in a liquid
phase using graphene oxide by centrifugation.29 Similarly, we
believe GO immobilized on the LFTS should be able to remove
residual single-stranded primers from PCR products. Moreover, it
has been reported that dsDNA could also bind to GO forming
dsDNA/GO complex in the presence of certain cations, such as
Mg2+, which reduce the electrostatic repulsion between dsDNA
and GO and bind to both dsDNA and carboxylic groups on GO.3032
Stacked chemically converted graphene sheets can also capture
dsDNA under the influence of salts.33 By contrast, some
surfactants, such as Tween 20 and Triton X-100, strongly interfere
with the formation of dsDNA/GO complexes.30,34 We deduced
that the short primer-dimers (usually less than 40 bp) might be
selectively adsorbed by GO from PCR products under a carefully
optimized condition. As a result, this new test strip coupled with
the on-strip GO purification should be able to overcome the
troubles associated with the conventional antibody-based method
without causing any inconvenience to the overall detection
process. We believe the iGOP-LFTS will play a significant role
in point-of-care diagnosis in the future.
EXPERIMENTAL SECTION
Reagents. Streptavidin (S4762), Tween 20, sodium azide, and
tetrachloroauric acid were purchased from Sigma-Aldrich (St.
Louis, MO). Graphene oxide, agarose, and bovine serum albumin
(BSA) were obtained from Aladdin Bio-Chem (Shanghai, China).
Sodium chloride (NaCl), magnesium chloride (MgCl2), and TE
buffer (10 mM Tris, 1 mM EDTA, pH=8.0) were from Sangon
(Shanghai, China). All chemicals were of analytical grade. All of
the primers and the HIV-1 (human immunodeficiency virus)
plasmids were synthesized by Sangon. Anti-digoxin antibody
(ab20814) was purchased from Abcam (Cambridge, MA).
Bacteriophage λ-DNA was obtained from Promega (Madison,
WI). AmpliTaq Gold® 360 Master Mix was from Thermo Fisher
(Waltham, MA). A 20-bp DNA ladder (3420A) and a GeneGreen
nucleic acid dye kit were purchased from TaKaRa (Shiga, Japan)
and Tiangen Biotech (Beijing, China), respectively. Nitrocellulose
membrane (NC membrane, HF13502S25) was obtained from
Merck Millipore (Massachusetts, MA). Glass fiber membrane,
absorbent paper, and plastic backing card were all from Shanghai
Kinbio Tech (Shanghai, China).
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Synthesis and labeling of gold nanoparticles. The 15-nmdiameter gold nanoparticles (AuNPs) were synthesized using the
Frens method which can be found elsewhere.35 Briefly, 100 mL of
0.01% (w/v) tetrachloroauric acid solution was prepared and
heated to boil. Then, 4.5 mL of 1% (w/v) sodium citrate solution
was added with a vigorous mechanical stirring. When the color of
the solution turned into wine red, the heating was stopped and the
solution was continuously stirred for another 10 min. After that,
the solution was cooled to room temperature and stored in a dark
bottle at 4 oC.
For labeling the AuNPs with anti-digoxin antibodies, the pH of
the AuNP solution was first adjusted to 8.5 using 200-mM
K2CO3. Then, the labeling reaction was carried out by adding 10
µL of the anti-digoxin antibody solution at a concentration of 5.78
mg/mL to 5 mL of the AuNPs solution, followed by an incubation
at 37 oC for an hour. After that, a 100-µL sealing solution (10%
BSA and 20 mM Na4B2O7) was added into the reaction and
incubated at room temperature for 0.5 hour to stabilize the gold
nanoparticles. Finally, the excess reagents were removed by
centrifugation for 15 min at 18,000 g, and the AuNPs was
dispersed again with a suspension solution (0.1 M Tris, 10%
sucrose, 5% BSA, 0.25% Tween 20, 0.05% NaN3 pH=8.0).
Preparation of graphene oxide pads and lateral flow test
strips. Sample loading pad: a glass fiber membrane with
dimensions of 50×15×0.3 mm was soaked into a PBS solution
containing 100 mM NaCl and 0.25% Tween 20 for 3 hours and
then dried at 65 oC for 4 hours. After that, the treated glass fiber
membrane was stored in a desiccator until use.
Graphene oxide (GO) pad: first, 1% (w/v) GO stock solution was
diluted by absolute ethyl alcohol into 0.25%. Then, 400 µL of the
0.25% GO solution was pipetted over a glass fiber membrane with
dimensions of 50×15×0.3 mm (corresponding to 0.06 mg on a
3×15×0.3 mm pad) and dried at 50 oC for 3 hours. Finally, a 150µL salt solution containing 20 mM MgCl2, 100 mM NaCl, and 40
mM NaAc (pH=5.0) was added onto the pad and dried at 50 oC
for 0.5 hour.
Conjugation pad: the AuNPs labeled with anti-digoxin antibodies
were first mixed with an equal volume of a conjugation solution
(20 mM Na4B2O7, 2% BSA, 3% sucrose, 600 mM NaCl, 0.2%
Tween 20, and 0.05% NaN3). Then, 160 µL of the mixed solution
was deposited onto a glass fiber conjugation pad with dimensions
of 50×10×0.3 mm, which was dried overnight at 37 oC. Finally,
the prepared conjugation pad was stored in a desiccator at 4 oC.
Nitrocellulose (NC) membrane: a test and a control line were
formed by manually drawing 0.24 µL/strip of 2 mg/mL
streptavidin and 0.24 µL/strip of 2 mg/mL anti-mouse IgG
antibodies onto the NC membrane, respectively. Then, the NC
membrane was dried at 37 oC overnight and stored in a desiccator
until use.
Assembly of lateral flow test strips: to assemble a conventional
lateral flow test strip, a NC membrane was first placed on a plastic
adhesive backing. Then, a conjugation pad was placed next to the
NC membrane with a 2-mm overlap. A sample loading pad was
placed on the backing with a 2-mm overlap with the conjugation
pad. Finally, an absorbent pad was pasted on the other side of the
NC membrane with a 2-mm overlap. After assembly, test strips
were cut to 3 mm wide and stored in a desiccator at 4°C until use.
The assembly of the iGOP-LFTS is similar to that of the
conventional test strip. The only difference is that three GO pads
were stacked up to replace the sample loading pad on the strip.
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Analytical Chemistry
Preparation of PCR samples. For the gel electrophoretic
analysis, a pair of primers (FAM-labeled FFP and NRP listed in
Table S1) was employed to amplify a 106-bp fragment from λDNA. For the LFTS detection, another pair of primers (BFP and
DRP in Table S1), labeled with biotin and digoxin, respectively,
was used for PCR from λ-DNA. A 20-µL PCR mixture was
composed of 0.4 µL of each primer (10 µM), 10 µL of 2×
AmpliTaq Gold® 360 Master Mix, 1 µL of λ-DNA with different
concentrations, and 8.2 µL of DI water. The thermal cycling
protocol included an initial activation of Taq polymerases at 95 oC
for 5 min, followed by 35 cycles of 95 oC for 30 s, 60 oC for 30 s,
and 72 oC for 30 s, and a final extension step at 72 oC for 5 min.
Preparation of standard DNA mixture. The 30-bp doublestranded DNA labeled with FAM was prepared as follows: first,
two 30-nt oligos (FAM-labeled OA and unlabeled OB in Table
S1) were synthesized by Sangon and dissolved in 1× TE buffer to
a concentration of 10 µM. Then, the equal volumes of both oligos
were mixed and heated to 95 oC for 5 min followed by cooling to
room temperature at a rate of 1 oC/min. The solution was stored at
4 oC. To prepare a standard DNA mixture for testing, the 106-bp
amplicons obtained from 1000 copies of λ-DNA with 35 PCR
cycles, the 30-bp dsDNA (5 µM) shown above, and the FAMlabeled primer (10 µM) were mixed together in a volume ratio of
47: 1: 2.
incubated at 37 oC for 15 min, followed by enzyme inactivation at
98 oC for 5 min. In the PCR step, 1 µL of the RT product was
used for amplification using the same protocol as that in the HIV1 plasmid analysis with either 30 or 35 PCR cycles.
Quantitative analysis of gel electrophoreses and lateral flow
test strips. The gel electrophoresis images were taken by the
Azure c150 Biosystem (Dublin, CA) and the intensities of the
DNA bands were measured using the ImageJ software. The
images of the lateral flow test strips were taken by a digital
camera, and then the signal intensities along the central crosssection line of the test strip were measured using the ImageJ. The
peak areas of the test and the control line were calculated and the
peak area ratio between these two lines was subsequently
obtained. The signal-to-noise ratio (S/N) of the test line was also
calculated by dividing the peak height of the test line with the
standard deviation of the baseline signals from the central crosssection line. All the experiments were independently repeated
three times and the results were expressed as means ± standard
deviation.
Optimization of running buffer and immobilized GO. To
optimize the running buffer and the immobilized GO, 10 µL of
the standard DNA mixture was pipetted onto a GO pad,
immediately followed by the loading of 10 µL of the running
buffer. After that, the purified sample was aspirated out from the
side of the GO pad without any incubation. The samples were
then analyzed using agarose gel electrophoresis along with the 20bp DNA ladder.
Preparation of HIV-1 samples. The plasmids containing HIV-1
gag proviral DNA sequences were provided by Sangon and a pair
of primers (SK38 and SK39) was synthesized for amplification.36
A 20-µL PCR mixture was composed of 1 µL of each primer (10
µM), 10 µL of 2× AmpliTaq Gold® 360 Master Mix, 1 µL of the
HIV-1 plasmids with different concentrations, and 7 µL of DI
water. The thermal cycling protocol was the same as that of the λDNA amplification except that the cycle number was increased to
40.
The 8E5 cell line carrying a single defective proviral genome of
HIV-1 was provided by the National HIV/HCV Reference
Laboratory of the Chinese Center for Disease Control and
Prevention. 8E5 cells were cultured in complete RPMI 1640
medium (Gibco, Grand Island, NY) supplemented with 10% (v/v)
fetal bovine serum (PAA Laboratories, Pasching, Austria) and 1%
(v/v) penicillin/streptomycin (Invitrogen, Carlsbad, CA) at 37 °C
with saturated humidity and 5% CO2. When the cells were grown
to about 106 cells/mL, the culture medium containing about 108
copies of defective HIV-1 viruses per µL was collected and
centrifuged at 1000 rpm for 5 min to remove cell debris. The
defective virus has a single-base mutation in the pol gene that
precludes the expression of reverse transcriptase and integrase. A
mock clinical specimen was prepared by spiking healthy human
plasma (obtained with informed consent) with the viruses to a
concentration of ~100 viruses/µL. A two-step reverse
transcription-PCR (RT-PCR) was performed to amplify from the
gag gene with the SK38 and SK39 primer pair. In the first step, a
10-µL reverse transcription mixture containing 1 µL of spiked
human plasma and 9 µL of ReverTra Ace® qPCR RT mix (0.5 µL
of Enzyme Mix, 0.5 µL of Primer Mix, 2 µL of 5× RT buffer, and
6 µL DI water) (FSQ-101, TOYOBO, Osaka, Japan) was
Figure 1. Schematic of the integrated graphene oxide
purification- lateral flow test strip (iGOP-LFTS). (A) Structure of
the iGOP-LFTS. (B) Direct loading of PCR products onto the
stacked GO pad. (C) Purification and detection mechanism of the
iGOP-LFTS.
RESULTS AND DISCUSSION
Purification and detection mechanisms of iGOP-LFTS.
Although diverse amplification methods of nucleic acids have
been developed with many superb properties, such as isothermal
reaction, ultra-high sensitivity, simplified sample preparations,
etc., polymerase chain reaction remains one of the best choices for
clinical diagnosis.37,38 A molecular diagnosis kit based on PCR
can be easily designed and optimized to provide a robust
amplification performance with a high specificity. As a result, a
detection method that requires minimum modifications to the
PCR system is highly desired. In our study, PCR was carried out
using a pair of primers, which were labeled with biotin and
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digoxin, respectively. This simple labeling strategy preserved the
advantages of PCR and enabled the direct use of PCR products for
detection.
As illustrated in Figure 1, the iGOP-LFTS was comprised of four
overlaid pads: three stacked GO pads, a conjugation pad, a
nitrocellulose membrane, and an absorbent pad. The PCR
products were directly loaded onto the GO pads, on which the
primers and the primer-dimers were selectively adsorbed by
graphene oxide. Right after the sample loading, a 100-µL running
buffer (0.1% (w/v) Tween 20, 20 mM MgCl2, 200 mM NaCl, and
80 mM NaAc (pH=5.0)) was pipetted onto the GO pad to drive
the purified amplicons to the conjugation pad, where the gold
nanoparticles with anti-digoxin antibodies bound to the amplicons
via the antibody-antigen interaction. The formed AuNP-DNA
complexes as well as the excess AuNPs were further transferred
to the NC membrane, which contained a test (T line) and a control
line (C line). On the test line, the immobilized streptavidin
captured the biotin-end of the amplicons, leading to the
aggregation of the AuNPs. On the control line, anti-mouse IgG
antibodies captured the excess AuNPs, validating the proper
function of the lateral flow test strip. Since no incubation was
involved, the entire process took only 10-15 min.
Page 4 of 16
Optimization of graphene oxide purification. To optimize the
on-strip purification of PCR products, we envisioned that the
running buffer should play an important role as the large amount
of the buffer provided a liquid phase for the selective DNA
adsorption by graphene oxide. A DNA mixture containing FAMlabeled double- and single-stranded DNA (106-bp amplicons, 30bp synthesized dsDNA, and 20-nt primers shown in Table S1)
was employed as a standard sample for testing. A series of the
running buffers containing different concentrations of MgCl2 and
Tween 20 was evaluated for their purification efficiencies. As
illustrated in Figure S1 and S2, the gel electrophoreses of the
recovered samples from the GO pads followed by the quantitative
analyses determined that the combination of 20 mM MgCl2 and
0.1% (w/v) Tween 20 yielded the best efficiency in terms of the
amplicon-to-primer ratio of recovery. Next, we optimized the
amount of GO pre-dried on the pad and found that, generally
speaking, more graphene oxide in the pads provided higher
purification efficiencies. As shown in Figure 2, 0.12 mg of GO on
a 15×3×0.3 mm pad can remove 97.5% of the 20-nt singlestranded primers from a 10-µL DNA mixture. However, since GO
was simply pre-dried within the pads (Figure S3), such a large
amount of GO could be washed off by the running buffer to
interfere with the downstream detection on the strip. As a result,
the immobilization amount of 0.06 mg was chosen due to its
acceptable efficiency (the amplicon-to-primer ratio = 5.1) and the
negligible interference with the detection.
To further enhance the purification capability of the strip, we
stacked up multiple graphene oxide pads to increase the surface
area for adsorption while maintaining the concentration of GO in
the pads. Figure 3 demonstrated that 83.4% of the 30-bp dsDNA
and 98.6% of the primers could be removed from the 10-µL DNA
mixture using three layers of the GO pads. Although 39.5% of the
106-bp amplicons were lost, the amplicon-to-dsDNA and the
amplicon-to-primer ratios went up to 3.6 and 43.0, respectively.
Therefore, this three-layer design was adopted for purifying PCR
products in the rest of the experiments.
Figure 2. Purification efficiency of different amounts of graphene
oxide immobilized in the pads. (A) Gel electrophoresis image of
recovered DNA mixtures from the GO pads. (B) Relative band
intensities of the 106-bp amplicons, the 30-bp dsDNA, and the
20-nt primers to the positive control (PC). When 0.12 mg of GO
was immobilized on the pad, the best purification can be achieved
with an amplicon-to-primer ratio of 29.0. Unfortunately, such a
large amount of GO on the pad could be washed out to interfere
with the downstream detection. Therefore, 0.06 mg of GO was
immobilized on a pad for the rest of the study.
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Analytical Chemistry
Figure 3. Purification efficiency of one, two, or three stacked
graphene oxide pads. (A) Gel electrophoresis image of recovered
DNA mixtures from stacked GO pads. (B) Relative band
intensities of the 106-bp amplicons, the 30-bp dsDNA, and the
20-nt primers to the positive control. Up to 83.4% of the 30-bp
dsDNA and 98.6% of the 20-nt primers were adsorbed by three
stacked GO pads.
Sample volume effect to sensitivity. Since the residual primers
were eliminated by GO, the increase of the sample loading
volume should lead to the aggregation of more gold nanoparticles
on the test line, resulting in an improve sensitivity of the iGOPLFTS. First, as a comparison, the conventional LFTS without the
GO pad was employed to detect 5, 10, and 20 µL of PCR products
amplified from 1000 copies of λ-DNA with the biotin- and the
digoxin-labeled primer set (Table S1). As shown in Figure 4A, the
dim T lines on all the test strips demonstrated no changes or even
slightly decreases of the intensities with the increase of the sample
volumes, which were confirmed by the quantitative analyses
(Figure 4B). This is because larger volumes of the samples
without purification have more primers, leading to the saturation
of AuNPs and streptavidin. By contrast, the iGOP-LFTS
containing three stacked GO pads can eliminate all residual
primers, resulting in significantly increased signals on the T lines
(Figure 4A). The four-fold volume increase (5 to 20 µL) led to
1.6-fold increase of the signal-to-noise ratios of the test lines and
3-fold increase of the peak area ratios of the test to the control
line, proving the effectiveness of this on-strip purification method
(Figure 4B).
Figure 4. Signal improvement by increasing sample loading
volumes. (A) Photos of the LFTS and the iGOP-LFTS with
different sample loading volumes (5, 10, and 20 µL). (B) Peak
area ratio of test to control line and signal-to-noise ratio of test
line as functions of the sample loading volume. When the loading
volumes were increased from 5 to 20 µL, the peak area ratio and
the S/N were improved 3 and 1.6 fold, respectively, using the
iGOP-LFTS.
Limit of detection. To test the sensitivity of the iGOP-LFTS, we
prepared a series of PCR products amplified from 3000 down to 3
copies of λ-DNA. As illustrated in Figure 5A, the conventional
LFTS without the GO purification can only reach a sensitivity of
3000 copies of template determined by naked eyes. When the
iGOP-LFTS device with three layers of the GO pads was used to
analyze 10-µL PCR products, the limit of detection was decreased
to 30 copies, leading to a 100-fold improvement in the sensitivity.
Interestingly, when 20 µL of the PCR products was loaded onto
the strip, the sensitivity of iGOP-LFTS detected by naked eyes
can reach 3 copies of template. By contrast, the sensitivity of the
conventional LFTS cannot be improved with more samples due to
the competence of residual primers. As a result, a remarkable
1000-fold improvement in sensitivity can be obtained with the
visual detection. In addition, with the aid of an image analysis
software, the sensitivities of both the LFTS and the iGOP-LFTS
could be determined to be 300 and 3 copies, respectively, as
shown in Figure 5B (S/N >3), corresponding to a 100-fold
increase of the limit of detection.
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resolve the problem of the false positives (S/N=1.9) and provide
results without any ambiguity.
Figure 5. Limit of detection of the conventional LFTS and the
iGOP-LFTS. (A) Photos of the strips tested using PCR products
amplified from 3-3000 copies of λ-DNA. The sample loading
volumes (10 or 20 µL) were listed above the photos. * indicates
the limits of detection of the strips determined by naked eyes. (B)
Signal-to-noise ratios of the test lines analyzed with ImageJ.
When 20 µL of PCR products was loaded onto the iGOP-LFTS,
the limit of detection was 3 copies of template, corresponding to a
1000-fold increase compared to the conventional LFTS. *
indicates the limits of detection of the strips determined by image
analysis software (S/N >3).
Enhanced specificity. The false positives caused by primer-dimer
artifacts is one of the most critical drawbacks of the lateral flow
test strip employing the double-labeling scheme for PCR
amplification and detection. To evaluate the capability of the
iGOP-LFTS of resolving the primer-dimer problem, we tested the
visual detection of PCR products amplified from plasmids
containing HIV-1 gag proviral DNA sequences with a slight
primer-dimer issue (primers listed in Table S1).36 Although the
nucleic acid testing of HIV has a shorter window period than that
of the HIV immunoassay,39 the wide adoption of NAT in clinical
diagnosis was still limited due partially to the complicated
operations and the high costs which could be alleviated by the use
of lateral flow test strips. Here we prepared a series of PCR
products amplified from the HIV-1 plasmids from 300 down to 3
copies with 40 cycles. As shown in Figure 6, although the
sensitivity of the LFTS without the GO pads can reach the
detection limit of 3 copies, a dim T line on the strip (S/N=5.5)
appeared in the negative control. This false positive result was
produced by the primer-dimers generated during the
amplification. When the iGOP-LFTS was employed, most of the
primer-dimers were removed by the three stacked GO pads on the
strip and no false positive was observed, validating the
effectiveness of the on-strip purification. The quantitative
analyses of these strips confirmed that the iGOP-LFTS could
Figure 6. Visual detection of HIV-1 plasmids using the
conventional LFTS and the iGOP-LFTS. (A) Photos of the strips
tested using PCR products amplified from 3-300 copies of HIV-1
plasmids. * indicates the limits of detection determined by naked
eyes. A dim test line was observed in the negative control of the
LFTS. (B) Signal-to-noise ratios of the test lines. The S/N in the
negative control of the LFTS was higher than 3, while that in the
iGOP-LFTS is far less than 3. * indicates the limits of detection of
the strips determined by image analyses (S/N >3).
Analysis of mock clinical specimen. To more critically evaluate
the capability of our iGOP-LFTS for HIV testing, we prepared a
mock clinical specimen by spiking healthy human plasma with
defective HIV-1 viruses to a concentration of ~100 viruses/µL.
One-µL specimen was amplified using a two-step RT-PCR
protocol with either 30 or 35 PCR cycles. The products were then
analyzed using the conventional LFTS and the iGOP-LFTS in
parallel. As shown in Figure 7A, the iGOP-LFTS revealed clear
test lines on the strips for both the PCR products amplified with
30 and 35 cycles, while the conventional LFTS can only detect
the sample prepared with 35 PCR cycles. The ImageJ analyses
shown in Figure 7B confirmed that the S/N ratios of all the
positive test lines were higher than 3. This test with a mock
clinical specimen clearly proved the iGOP-LFTS could be used to
detect HIV-1 viruses existing in human plasma.
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the iGOP-LFTS can significantly improve the robustness of the
LFTS-based nucleic acid testing.
CONCLUSION
In summary, we have successfully developed an integrated
graphene oxide purification-lateral flow test strip for the detection
of PCR products directly. Since the residual primers and the
primer-dimer artifacts can be effectively removed by three
stacked GO pads on the strip, the sensitivity of the test strip was
improved up to 1000 fold in visual detection, and the chance of
obtaining false positive results was dramatically reduced. More
importantly, these improvements were achieved without
introducing any additional hands-on operations and instruments.
The “dip-to-use” concept of the lateral flow test strip was well
preserved by this integrated PCR purification and lateral flow
detection method. Our iGOP-LFTS provides a sensitive, easy-touse, and inexpensive detection approach for checking PCR
products in a qualitative or semi-quantitative way. The
combination of the iGOP-LFTS with direct PCR (no nucleic acid
extraction required) will allow the patient’s diagnoses to be
performed in a physician’s office, an ambulance, the home, and
the field by users with minimum trainings.
In the future, it is possible to further enhance the sensitivity and
the specificity of the iGOP-LFTS by permanently immobilizing
GO within the pad and by carefully selecting appropriate
materials of the pads for GO modification. In addition, the
integration of the test strip with nucleic acid extraction and PCR
amplification on a single microfluidic device will resolve the
contamination concern of the current “open-tube” detection by the
strip, realizing a “sample-in-answer-out” analysis.
ASSOCIATED CONTENT
Figure 7. Analysis of spiked clinical specimens containing
defective HIV-1 viruses using the conventional LFTS and the
iGOP-LFTS. (A) Photos of the strips tested using RT-PCR
products amplified from ~100 copes of HIV-1 viruses. * indicates
the limits of detection observed by naked eyes. (B) Signal-tonoise ratios of the test lines. The conventional LFTS can only
detect the PCR products amplified with 35 cycles, while the
iGOP-LFTS allowed the cycle number was reduced to 30. *
indicates the limits of detection analyzed by the ImageJ software.
Supporting Information
Since the lateral flow test strip still depends on the conventional
PCR for sample preparations, PCR parameters, such as primer
design, annealing temperature, and cycle number, can
significantly affect the performance of the strip. For example, the
tests shown in Figure 6 demonstrated that both the conventional
LFTS and the iGOP-LFTS can detect 3 copies of HIV-1 plasmids
amplified in a 20-µL reaction with 40 PCR cycles. However, the
improvement in sensitivity achieved by the cycle number
sacrificed the assay specificity. Primer-dimers or non-specific
amplifications did occur, causing false positives in the LFTS test.
Therefore, a PCR condition that keeps the balance between the
sensitivity and the specificity must be carefully optimized for the
conventional LFTS. In contrast, the iGOP-LFTS has the
capability of eliminating residual primers and primer-dimers. As a
result, a PCR condition that is more towards the sensitivity could
be chosen. For instance, the iGOP-LFTS can detect 3 copies of
HIV-1 plasmids with a 40-cycle amplification while the
interference of the primer-dimers was eliminated. In addition,
Figure 7 illustrated that the iGOP-LFTS can successfully analyze
the mock clinical specimen amplified with only 30 PCR cycles.
Not only did the reduced cycle number lower the chance of
generating non-specific products but also shortened the analytical
time. Overall, the enhanced sensitivity and specificity provided by
Notes
Additional information as noted in text. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Email: [email protected] Phone: +86-10-62798732. Fax:
+86-10-62798732.
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
Financial supports were provided by the National Key Research
and Development Program of China (No. 2016YFC0800703)
from the Ministry of Science and Technology of China.
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