Ultra Sensitive Detection of Influenza A
Virus Based on Cdse/Zns Quantum Dots
Feng Wu1,2#, Mao Mao1#, Qian Liu2, Lei Shi3, Yu Cen2,
Zhifeng Qin3 and Lan Ma2*
1Key Laboratory for Special Functional Materials, Henan University, Kaifeng 475004, P. R. China
2Division of Life Science and Health, Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055, P. R. China
3Shenzhen Entry-Exit Inspection and Quarantine Bureau of the People’s Republic of China (SZCIQ), Shenzhen, 518045, P.R.
#Authors contributed equally to this article
Lan Ma, Division of Life Science and Health, Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055, P.
R. China, E-mail:
Received: 24 November, 2016; Accepted: 16 December, 2016; Published: 28 December, 2016
An ultrasensitive lateral flow immunoassay system (LFIAS)
was established for the detection of influenza A virus. In this LFIAS,
hydrophilic dihydrazide-modified CdSe/ZnSquantom dots (QDs)
were conjugated with specific antibodies and used as fluorescent
labels, and a pair of matched anti-nucleoprotein of influenza A virus
antibodies were used to form a sandwich immunoassay. The QDs
were in conjugation with the fragment crystallizable region (Fc
region) of specific influenza A virus antibodies through aldehydehydrazide
covalent chemistry, conferring high sensitivity. The
antibodies used for detection are specific for the most conserved and
popular nucleoprotein of influenza A virus and ensure the accuracy
and specificity. The QDs-LFIAS can analyze the nasal-pharyngeal
swab samples through simple steps and get results within 15 min.
Detection of nasal-pharyngeal swab samples makes it more rapid
and convenient, and it is highly efficient for identification of influenza
infection and improves influenza patients’ management. The limit
of detection of this QDs-LFIAS for recombinant nucleoprotein of
influenza A virus was 0.01 ng/ mL, which was 1000-fold higher
than the sensitivity of colloidal gold method. The detection of actual
patient samples indicated that the QDs-LFIAS had a high compliance
with real-time PCR.
Keywords: Lateral flow immunoassay; Influenza A virus; CdSe/
ZnS; Quantum dots
Influenza viruses circulate each year and cause mild to severe
illness and even death in humans. Type A influenza can outbreaks
in seasonal and regional, and the viruses are susceptible to
mutation. On the basis of the antigenic nature of the surface
glycoproteins, hemagglutinin (HA) and neuraminidase (NA),
type A influenza viruses are subdivided into several subtypes .
At present, the seasonal influenza A virus subtypes caused by
human infection is influenza A (H1N1) and A (H3N2) . Avian
influenza viruses such as A (H5N1) A (H5N6), A (H7N9) and A
(H9N2) can sometimes spread to domestic poultry and cause severe outbreaks disease, and they can cause serious infections
To date, numerous analytical methods have being used to
detect influenza A virus. According to the type of detection target,
these methods could be categorized into, for example, virus
isolation and identification, nucleic acid-based detection, antigen
detection, and antibody detection [3-8]. Most of these methods
need demanding conditions and professional operations, and
some of the detection processes is time-consuming. For these
reasons, some new biosensors based on nano materials such as
gold nanoparticles, magnetic nano beads and quantum dots (QDs)
have been coming to the forefront [9-12].Developing a sensitive,
specific and fast biosensor for diagnosing the influenza virus at
the early stages of infection is a challenge. Lateral flow immune
assay method has been widely used in detection of infectious
diseases with the advantages of rapid, easy to operate, and low
cost . However, traditional method such as colloidal gold
lateral flow tests has limitations when high sensitivity is needed
.QDs have been developed to replace colloidal gold owing
to their excellent optical properties such as high fluorescence
efficiency, wide range of excitation wavelength, narrow and
symmetric emission spectra, and QDs based lateral flow tests
have been proved higher sensitivity and reproducibility for
detection of pathogen, characteristic protein and even nucleic
In this paper, we present an ultrasensitive lateral flow
immunoassay (LFIA) for detecting influenza A virus. This immune
sensor is based on site-specific covalent binding with the Fc end
of influenza A virus antibodies to CdSe/ ZnS QDs. The carboxylfunctionalized
CdSe/ ZnS QDs conjugated with antibodies via
an amide bond often result in random links of the antibody
structure in the QD conjugates and block certain antigenbinding
sites. In order to obtain site-specific linking, we modified
carboxyl-functionalized QDs with adipicdihydrazide (ADH) and
oxidized the carbohydrate groups on the antibody’s Fc regionto obtain reactive aldehyde groups, the oxidized antibodies can
then conjugated with dihydrazide-modified QDs . The Fc
region of the antibody was linked to the QDs surface with the
fragment antigen-binding region (Fab region) facing outward
as shown in Figure 1A. The antibodies used for detection by the
developed sensor are specific for the most conserved and popular
nucleoprotein of influenza A virus
Materials and Methods
Sodium periodates, Ethylene glycol, Adipic acid dihydrazide
(ADH), D-(+)-Trehalose dehydrates, 2-(N-morpholino) ethane
sulfonic acid (MES), and D-(+)-glucose were purchased
from Sigma-Aldrich (Shanghai, China). 1-Ethyl-3-[3-
dimethylaminopropyl] carbodiimide hydrochloride (EDC) was
purchased from Thermo Fisher Scientific, Inc. (Waltham, MA.
U.S.A.). Sodium phosphate dibasic, sodium phosphate monobasic
monohydrate, sodium acetate, bovine serum albumin fraction V
(BSA), and Tween-20were purchased from Shanghai Sangon Ltd.
(Shanghai, China). Goat anti-mouse IgG antibody was purchased
from Arista Biologicals, Inc.(Allentown, PA. U.S.A.).Antinucleoprotein
of influenza A virus antibodies (mAb IgG) and
recombinant nucleoprotein of influenza A virus were obtained
from Life Science Division, Tsinghua University. Influenza A
subtype viruses HI test antigens were purchased from Harbin
Weike Biotechnology Development Company (Habin, China).
Influenza B subtype viruses, Measles virus, Mumps virus,
Rubella virus, Varicella zoster virus, Staphylococcus aureus and
Pseudomonas aeruginosa test antigens were purchased from
China food and Drug Inspection Institute. All chemical reagents used were of analytical grade without any further purification.
Conjugation of influenza A virus antibodies to QDs via
oxidized Fc-carbohydrate groups
Hydrophilic carboxyl-functionalized CdSe/ Zn SQDs were
prepared according to the previous work of our group [20-22].
The carboxyl-functionalized QDs were dihydrazide-modified
by ADH. Briefly, 5 mg of the QDs were mixed with 5 mM EDC
and 5 mM ADH in 0.01 M phosphate-buffered saline (PBS) and
then incubated for 4 h at room temperature. After washing
and centrifuged three times at 20,000 g for 0.5 h, the QDs were
dispersed in 0.01 M PBS buffer. Influenza A virus antibodies were
oxidized by sodium periodate [19, 23]. Briefly,0.1 M sodium
periodate dissolved in 0.1 M sodium acetate buffer (pH=5.5), and
100 μL of the solution was added to 1 ml of the antibody in 0.01
M PBS buffer(1.5 mg/mL), then incubated in the dark for 0.5h
at 0°C. The reaction was terminated by adding 100 μL ethylene
glycol. Oxidized antibodies were purified by dialysis overnight
against 0.01 M PBS buffer (pH=7.2). To form QDs-Ab conjugates,
0.3 mg of oxidized influenza A virus antibodies were mixed
with5mg dihydrazide-modified QDs, and then incubated for 2
h at room temperature. For blocking residual active coupling
sites, 1% glucose solution was added and incubated for 0.5 h
at room temperature. The conjugated QDs-Ab were purified by
centrifugation at 20,000 g for 3 times, and stored in 0.01 M PBS
buffer at 4°C before use.
Preparation of QDs-LFIA strips
In order to prepare the QDs-LFIA strips, the antibodies
were dispensed onto the nitrocellulose membrane while the
conjugated QDs-Ab was dispensed onto the sample pad using
the XYZ Dispensing System (Bio Dot Inc, Irvine, CA). Briefly,
Figure 1: Schematic rep%esentation of the QDs-LFIAS (A) Conjugation of antibodies to QDs via oxidized Fc-carbohydrate groups (B) Analytical
representation of the QDs-LFIAS
anti-influenza A virus coating antibody was dispensed onto the
nitrocellulose membrane at 2 mg/ mL as the test line, and the
goat anti-mouse IgG was dispensed at 0.5 mg/ mL as the control
line. The dispensed nitrocellulose membranes were dried at 37°C
in a vacuum oven for 4 h. The sample pad was pretreated by PBS
buffer containing D-(+)-trehalose dehydrate (1%, w/ v), BSA (1%,
w/v) and Tween-20 (0.1%, w/v), and dried at 37°C for 3 h. The
conjugated QDs-Ab was dispensed at a ratio of 10 μL/ cm onto
the pretreated sample pad and dried at 37°C for 3 h. The QDs-
LFIA strip was assembled in its standard configuration as shown
in Figure 1B. The completed QDs-LFIA was cut into individual 3.5
mm strips and stored in sealed and dry condition.
Sixty micro liter of analyte was added onto the sample port of
QDs-LFIA strip, the captured fluorescence intensity of test line and
control line were scanned by a fluorescence test strip scanning
device after a 15 min reaction. The captured fluorescence QDs
on the test line and control line produced a bright fluorescent
band in response to 365 nm ultraviolet excitation, and the
fluorescence intensity at 620 nm were detected by the device
. As shown in Figure 1B, once influenza A virus was in the
sample, the QDs-Abbound the influenza A virus specifically and
were later captured by the second anti-influenza A virus antibody
at the test line, and QDs-Ab without influenza A virus bound
were captured by the goat anti-mouse IgG at the control line, the
captured QDs-Ab aggregated into a fluorescent band under 365
nm ultra violet excitation. The fluorescence intensity of test line
was closely correlated with the concentration of captured QDs-
Ab and virus complex..The cutoff value was obtained by detecting
50 negative samples, the average of fluorescence intensity was
calculated, and two times of the average value was defined as the
cutoff value. The calculated cut off value is 102 a.u. Fluorescence
intensity of 102 a.u. or above was defined as positive, while
less than 102 a.u. was negative. The limit of detection (LOD)
was estimated by analyzing samples at various concentrations.
Briefly, the recombinant nucleoprotein of influenza A virus was
ten-fold diluted by 20 mM PBS (1000, 100, 10, 1, 0.1, 0.01 and
0.001 ng/ mL) and test separately, 20 mM PBS was test as a
negative control. The fluorescence intensity of test line was
detected while the LOD was calculated. The LOD was defined as
the lowest concentration of recombinant nucleoprotein whose
fluorescence intensity was the minimum above the cutoff value.
We also test the reproducibility of QDs-LFIA strip by analyzing
20 replicates of recombinant nucleoprotein sample sat different
concentrations (1, 10 and 100 ng/ mL). The specificity and
cross-reactivity was analyzed by detecting influenza A virus
subtypes(H1N1, H3N2, H5N1 re-4/6, H7N9, H9N2 re-2,)HI test
antigens, influenza B virus subtypes(1704 strain, Victoria strain,
Yamagata strain),Measles virus, Mumps virus, Rubella virus,
Varicella zoster virus, Staphylococcus aureus and Pseudomonas
Practical field sample tests
Sixty samples (human throat swabs collected into sterile
Hanks’ balanced salt solution viral transport media)—which were collected and preserved by Shenzhen International
Travel Health Care Center, Shenzhen Entry-Exit Inspection
and Quarantine Bureau—were assayed using QDs-LFIA
strips. Commercial influenza A antigen rapid diagnostic test
kit (colloidal gold) was conducted in parallel, and real-time
PCR assay was used as a reference method to evaluate the
accuracy of QDs- LFIAS. A pair of specific primers (Forward
Primer 5’-GACCRATCCTGTCACCTCTGAC-3’, Reverse Primer
5’-AGGGCATTYTGGACAAAKCGTCTA-3’) and a probe (FAMTGCAGTCCTCGCTCACTGGGCACG-
BHQ1) for detecting influenza
A virus was used in real-time PCR assay.
Results and discussion
Conjugation of antibodies to QDs via oxidized Fccarbohydrate
The reaction mechanism of hydrophilic carboxylfunctionalized
CdSe/ ZnS QDs has been previously described in
the literature [24, 25]. The dynamic light scattering analysis in
Figure 2 shows that the average hydrodynamic size of carboxylfunctionalized
CdSe/ Zn SQDs was 42.86 nm with Stdev 18.72
nm, and this size increased to 109.5 nm with Stdev 52.16 nm after
dihydrazide modified and conjugation with antibodies, which
indicated that antibodies were successful conjugated.
Limit of detection of QDs-LFIA strip
The LOD was estimated by analyzing recombinant
nucleoprotein of influenza A virus samples at various
concentrations, the fluorescence intensity of test line was scanned
by fluorescence test strip scanner after 15 min of addition of the
samples. The diluted recombinant nucleoprotein of influenza A
virus samples was also tested by commercial influenza A antigen
rapid diagnostic test kit (colloidal gold). As demonstrated by
Figure3A, with the recombinant nucleoprotein concentration
increasing, the fluorescent band of test line at 0.1 ng/ mL was
still visible. The commercial influenza A antigen rapid diagnostic
test kit (colloidal gold) could only detect the recombinant
nucleoprotein at 10 ng/ mL concentration through Figure 3B. As
show in Figure 3C, employing the fluorescence test strip scanning
device, the recombinant nucleoprotein could be detected at 0.01
ng/ mL by QDs-LFIA strips, which was 1000-fold higher than the
sensitivity of colloidal gold method.
Figure 2:(A) Images of tested QDs-LFIAS in response to excitation with
365 nm ultraviolet light. (B) Images of tested commercial influenza A
antigen rapid diagnostic test strip (colloidal gold). (C). Fluorescence intensity
scans at different concentrations of recombinant nucleoprotein
of influenza A virus measured by the fluorescence strip scanning device.
Shows the fluorescence from test line of QDs-LFIA strips at 0.1ng/mL
was still visible. B shows the test line of commercial influenza A antigen
rapid diagnostic test kit (colloidal gold) at 10 ng/mL was visible. C
shows the test line of QDs-LFIA strips at 0.01 ng/mL can be detected by
fluorescence test strip scanning device.
Reproducibility of QDs-LFIA strip
The reproducibility of this QDs-LFIA strip was analysed
by detecting 20 replicates of the recombinant nucleoprotein at
various concentrations (1, 10 and 100 ng/mL). The fluorescence
intensity of test line was detected while the relative standard
deviations (RSD) were later calculated. Table 1 shows the RSD
results and the RSD values were below 8%, demonstrating the
QDs-LFIA strip had good reproducibility.
Specificity and cross-reactivity of QDs-LFIA strip
The specificity and cross-reactivity of the QDs-LFIA strip was
analyzed by detecting influenza A virus subtypes (H1N1, H3N2,
H5N1 re-4/6, H7N9, H9N2 re-2,) HI test antigens, influenza B
virus subtypes(1704 strain, Victoria strain, Yamagata strain),
Measles virus, Mumps virus, Rubella virus, Varicella zoster virus,
Staphylococcus aureus and Pseudomonas aeruginosa. Figure 4A
shows that the QDs-LFIA strip could detect all the subtypes of
influenza A virus used but none of other type antigens. We also
tested the influenza A virus subtypes with concentration ranging
from 1/512 to 16HAU. As demonstrated in Figure4B, the QDs-
LFIA could detect influenza A virus subtype H1N1 at 1/256 HAU,
influenza A virus subtype H5N1 at 1/32 HAU, influenza A virus
subtype H3N2, H7N9 and H9N2 at 1/128 HAU. It demonstrated
that the QD-LFIAS could detect influenza A subtype viruses with
high sensitivity and specificity.
Practical field sample tests
Sixty samples (human throat swabs collected into sterile
Hanks’ balanced salt solution viral transport media) were
detected using QDs-LFIAS. Table 2 shows that all positive
samples with low real-time PCR threshold cycle (Ct ≤ 30) were
detected by QDs- LFIAS with a high accuracy. For the positive
samples with high real-time PCR threshold cycle (Ct ˃ 30), three
were detected as negative by QDs-LFIAS, but all were detected
as negative by commercial influenza A antigen rapid diagnostic
test kit (colloidal gold). The results indicated that the QDs-LFIAS
had an accuracy of 95%, while that of the commercial influenza
A antigen rapid diagnostic test kit (colloidal gold) was 56.7%
compared with real-time PCR.
Figure 3: DLS data of QDs and conjugated QDs-Ab. (A) Carboxyl-functionalized
QDs. (B) Antibody conjugated QDs. Average hydrodynamic
size of CdSe/ZnS QDs was 42.86 nm and this size increased to 109.5 nm
after conjugation with antibodies
Table 1: Reproducibility Tests of QDs-LFIAS. 20 replicates of the
recombinant nucleoprotein at various concentrations (1, 10 and 100
ng/mL) were test by QDs-LFIAS. The relative standard deviations (RSD)
were then calculated accordingly.
Fluorescence intensity average (a.u.)
Figure 4:(A) Specificity tests of QDs-LFIAS. (B) Fluorescence intensity
scans at different concentrations of influenzaA virus subtypes. Shows
QDs-LFIAS could detect all the subtypes of influenza A virus used but
none of other type antigens. B shows QDs-LFIAS could detect the subtypes
of influenza A virus with high sensitivity.
Table 2: Practical field sample Tests using QDs-LFIAS. Sixty human
throat swab samples were detected using QDs- LFIAS, commercial
influenza A antigen rapid diagnostic test kit and real-time PCR. Compared
with real-time PCR, the QDs-LFIAS had an accuracy of 95%, while that of
the commercial infl uenzaA antigen rapid diagnostic test kit (colloidal
gold) was 56.7%.
Number of samples
Commercial rapid diagnostic test kit result(P/N)
Real-time PCR result(P/N)
Real-time PCR positive result
Ct > 30
Real-time PCR positive result
Ct ≤ 30
At the early stage of influenza A virus infection, the symptoms
of patients were similar to those of common cold and it was
difficult and insufficient to use these common symptoms for the
diagnosis of type A influenza virus infection . Besides, the
influenza A virus titer was very low during the early infection.
So, high sensitivity is the key for diagnosing the influenza virus
at the early stages of infection. Real-time PCR method has ultrahigh
accuracy but time consuming, as point-of-care testing method, the QDs-LFIAS was proved to detect human throat swab
samples more sensitive and accurate than colloidal gold method.
Detection of nasal-pharyngeal swab samples makes it more
rapid and convenient, and it is highly efficient for monitoring
and prevention of influenza outbreak in the hospital emergency,
port quarantine, schools and also in-home health care. QDs-
LFIAS is a low-cost technique with small amount of immunoassay
reagents consumed, and results can be objectively determined
by a handheld device. According to our results above, the QDs-
LFIAS only requires one step and provides results in 15 min. This
simple and less time consuming method could be used in on-site
tests, clinical diagnosis and early treatments of influenza virus
The purpose of this study was to develop an ultrasensitive,
rapid and low cost lateral flow immune sensor for influenza A
virus preliminary screening. We developed a QDs-LFIAS method,
which rapidly analyzed the sample through one steps. The
results were objectively analyzed by an inexpensive, portable
device within 15min. The LOD of this QDs-LFIAS for recombinant
nucleoprotein of influenza A virus was 0.01ng/ mL, which was
1000-fold higher than the sensitivity of colloidal gold method.
The QDs-LFIAS could detect influenza A virus subtype H1N1 at
a concentration of 1/ 256 HAU, influenza A virus subtype H5N1
at 1/ 32 HAU, influenza A virus subtype H3N2, H7N9 and H9N2
at 1/128 HAU. It demonstrated that the QD-LFIAS could detect
influenza A virus subtypes with high sensitivity and specificity. This
was more sensitive than that of traditional point-of-care testing
methods. The specificity and reproducibility were shown to be
good. Real patient samples demonstrated that the QDs-LFIAS had
high accuracy, and detection of nasal-pharyngeal swab samples
makes it more rapid and efficient for identification of influenza
infection and improves influenza patients’ management
This work was supported by the following sources:
the National High Technology Research and Development
Program of China (863 Program, NO. 2013AA032204), Science
and Technology Planning Project of Guangdong Province
(2012B031500003), Shenzhen strategic emerging industry
development special funds (JSGG20140716144254155)
Conflict of interest
The authors declare that there is no conflict of interest
regarding the publication of this paper.
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