Electrochemical assay to detect influenza viruses and measure drug susceptibility

Article abstract of DOI:10.1002/anie.201412164

An electrochemical assay has been designed to rapidly diagnose influenza viruses. Exposure of a glucose-bearing substrate to influenza viruses or its enzyme, neuraminidase (NA), releases glucose, which was detected amperometrically. Two methods were used

Full text of DOI:10.1002/anie.201412164

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201412164
German Edition: DOI: 10.1002/ange.201412164
Biosensors
Electrochemical Assay to Detect Influenza Viruses and Measure Drug
Susceptibility**
Xiaohu Zhang, Abasaheb N. Dhawane, Joyce Sweeney, Yun He, Mugdha Vasireddi, and
Suri S. Iyer*
Dedicated to Professor Malcolm Chisholm on the occasion of his 69th birthday.
Abstract: An electrochemical assay has been designed to
rapidly diagnose influenza viruses. Exposure of a glucose-
bearing substrate to influenza viruses or its enzyme, neurami-
nidase (NA), releases glucose, which was detected ampero-
metrically. Two methods were used to detect released glucose.
First, we used a standard glucose blood meter to detect two
viral NAs and three influenza strains. We also demonstrated
drug susceptibility of two antivirals, Zanamivir and Oseltami-
vir, using the assay. Finally, we used disposable test strips to
detect nineteen H1N1 and H3N2 influenza strains using this
assay in one hour. The limit and range of detection of this first
generation assay is 102 and 102–108 plaque forming units (pfu),
respectively. Current user-friendly glucose meters can be
repurposed to detect influenza viruses.
kit require a laboratory setting.[6] Antibody-based tests can be
variable as it is highly dependent on antibody purification,
bioconjugation, and the quality controls established by the
manufacturer. Indeed, the CDC does not recommend the use
of these tests unless it is supported by more accurate
techniques.[7] Colorimetric tests such as the ZstatFlu test
provide a visual readout, but as is the case with several optical
tests, the readout is prone to human error and is not
sensitive.[8] All these tests are expensive and none of them
can measure drug susceptibility rapidly in a point-of-care
setting. The lack of good rapid diagnostic tests leads to
asymptomatic treatment and overuse of drugs, which
increases drug resistance. Indeed, reports of resistance to
Oseltamivir, the preferred antiviral for influenza, have been
reported.[9]
I
nfluenza virus is a highly contagious virus. The US Centers
Influenza has two major surface glycoproteins, hemaglut-
tinin (HA) and neuraminidase (NA). Hemagluttinnin is
implicated in viral entry and neuraminidase is the enzyme
that cleaves N-acetyl neuraminic acid (sialic acid) from the
surface of host cells to release viral progeny.[10] There are
approximately 50–100 copies of NA on the viral surface as
determined by immunogold labeling and cryoelectron tomog-
raphy.[11] Since NA is present as a tetramer, there are
approximately 200–400 individual units capable of cleaving
sialic acids, which makes it a suitable target for biosensing
applications.
for Disease Control (CDC) estimate that seasonal influenza is
responsible for over 200000 hospitalizations and 30000
deaths in the US.[1] Pandemics, although infrequent, can
cause significant devastation. In 2009, the H1N1 “swine flu”
outbreak infected people in more than 200 countries within
weeks of the initial outbreak.[2] Measuring drug susceptibility
is equally important since antivirals are most efficacious when
administered before onset of infection[3] and the virus has
a high mutation rate, approximately 1.5 ” 10À5 mutations per
nucleotide per infectious cycle.[4]
Diagnostics for influenza viruses include nucleic acid
amplification tests (NAAT) and antibody and fluorescence
tests. NAATs such as the Xpert Flu tests are highly selective
and sensitive but require sophisticated instruments; the
correct primers and can be cost prohibitive for use in primary
care facilities, resource poor areas, and homes.[5] Chemilumi-
nescence-based tests such as the Amplex Red Neuraminidase
Our strategy was to develop and expose substrates that
would release glucose upon action of NA (Figure 1a). The
released glucose can be measured amperometrically. A
similar approach has been used to detect other enzymes, for
example b-galactosidase[12] and a-amylase.[13] Herein, we
demonstrate detection of viral NA and nineteen unique
strains of influenza and demonstrate drug susceptibility of the
two antivirals, Zanamivir and Oseltamivir (Figure 1b). These
results were validated using rRT–PCR (real-time reverse
transcription–polymerase chain reaction) and plaque assays.
We choose to measure glucose concentration after 1 hour of
incubation using disposable test strips, however, a continuous
measurement system can also be designed.
[*] X. Zhang, Dr. A. N. Dhawane, J. Sweeney, Dr. Y. He, Dr. M. Vasireddi,
Prof. S. S. Iyer
788 Petit Science Center, Department of Chemistry
Center for Diagnostics and Therapeutics
Georgia State University, Atlanta, GA-30302 (USA)
E-mail: siyer@gsu.edu
We synthesized a sialic acid derivative (SG1) where sialic
acid is attached to glucose at the 6-position (Scheme 1).
Briefly, benzyl 2,3,4-tri-O-benzyl-a/b-d-glucopyranoside
(1a,b) was synthesized using a modified procedure and
reacted with the known N-acetyl-5-N,4-O-carbonyl-protected
thiosialoside donor (2)[14] to yield 3a,b. Exclusive a sialoside
was obtained, which was confirmed by NMR spectrosco-
[**] We are grateful to NIH-NIAID (R01-AI089450) for funding. We thank
BEI Resources, NIAID, Manassas, VA for the viral strains and Dr.
Didier Merlin for kind use of his instruments.
Supporting information for this article (including the synthesis of
SG1, characterization data of intermediates and the final com-
pound, and details of the assays) is available on the WWW under
http://dx.doi.org/10.1002/anie.201412164.
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electrode and used this electrochemical cell to develop
a standard curve[16] (see Figure S1 in the Supporting Informa-
tion).
SG1 (0.5 mm) was dissolved in PBS buffer and tested for
the presence of glucose. In the absence of enzymes, there is no
glucose released (Figure 2a; sample N, negative control, no
NA or virus added). Membrane-free influenza viral NA from
two different strains (N1 from H5N1A/Anhui/1/2005 and N2
from A/Babol/36/2005) was incubated for 2 h at room
temperature with SG1. The sample was analyzed for the
presence of glucose directly without further sample prepara-
tion. Glucose was released as determined by the current (i)
measured amperometrically, which indicated that complete
cleavage of SG1 had occurred (Figure 2a, samples A, B). The
positive control (Figure 2a, sample P) was glucose as the only
analyte in PBS buffer. Next, we tested three influenza
strains, H1N1A/Brisbane/59/2007, H3N2A/Aichi/2/1968,
and H3N2A/HongKong/8/68, which were quantified by
plaque assays and rRT-PCR (Figure S2). Introduction of
UV-inactivated virus (100 mL) to SG1 and incubation for 2 h
Figure 1. Detection of influenza virus using SG1. a) Workflow and
scheme for electrochemical detection of influenza virus. b) Measure-
ment of drug susceptibility. c) Influenza NA cleaves SG1 to release
glucose.
Figure 2. a) Detection of influenza virus or viral NA. SG1 (0.5 mm)
was incubated with membrane-free soluble N1 NA (sample A, strain
H5N1A/Anhui/1/2005) or N2 NA (sample B, strain H3N2A/Babol/36/
2005) or three different UV-inactivated influenza strains, H3N2A/
Aichi/2/1968 (sample C), H1N1A/Brisbane/59/2007 (sample D), or
H3N2A/HongKong/8/68 (sample E) for two hours. The negative con-
trol where no virus or NA was added (sample N) did not show any
noticeable current and the positive control (sample P) was d-glucose
at 0.5 mm. b) Drug susceptibility studies. 10 ng of Zanamivir or
Oseltamivir (Carbosynth, USA, San Diego, CA) were premixed with the
strains (as detailed in (a)) for 30 min at RT before addition of SG1.
c) Studies with bacterial NA (BNA). BNA cleaves SG1 to release
glucose, however, Zanamivir does not inhibit BNA and glucose is
released when BNA was premixed with Zanamivir and incubated with
SG1. d) Studies with human samples. NS denotes nasal swab only,
and shows that no glucose is present. NSV denotes a nasal swab
spiked with 105 pfu of H1N1A/Brisbane/59/2007 and added to SG1.
The positive signal indicates there are no matrix effects. In (a–d), the
y axis shows current (i) in amperes measured after 100 s using an
amperometric i–t curve at a working potential of 0.00 V. All experi-
ments were performed in triplicate independently on different days.
Scheme 1. Synthesis of SG1. Reagents and conditions: a) TfOH, NIS,
CH2Cl2, À608C, 2 h, 75%; b) i) NaOMe, MeOH, RT, 30 min; ii)
Pd(OH)2/C/H2, EtOH, RT, 12 h; iii) 0.05n NaOH in H2O, RT, 4 h, 93%
yield over three steps. TfOH=trifluoromethanesulfonic acid. NIS=
N-iodosuccinimide.
py.[14a,15] Next, a three-step procedure was performed. First,
ZemplØn deacetylation conditions were used to remove the
acetates and regioselectively open the oxazolidinone ring to
obtain the N-acetamido group.[21] This was followed by
hydrogenation to remove the benzyl groups and the resulting
product was saponified to produce SG1 in excellent yield. To
measure glucose, we developed a three-electrode electro-
chemical cell comprising a reference, working and counter
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Table 1: Electrochemical detection of 19 influenza strains and validation
with rRT–PCR and cell-culture plaque assays.
resulted in the release of glucose (Figure 2a, samples C, D,
and E). The cleavage was confirmed by mass spectral analysis.
The mass spectra of the control where there is no virus or NA
revealed a peak at m/z 494.1497 (M+Na, positive ion)
corresponding to uncleaved SG1. A new peak emerges at
m/z 181.0711 (M+1, positive ion) corresponding to glucose
when virus or NA is added.
To test for drug susceptibility, we premixed FDA-
approved NA inhibitors Zanamivir or Oseltamivir with the
virus or NA for 30 min before introducing SG1. If the strains
are not resistant to the antivirals, they are expected to block
the action of NA and a signal for glucose should not be
detected. As seen in Figure 2b, the three strains and NA are
completely inhibited by the antivirals. Thus, we determined
drug susceptibility of these strains within a sample results in
a test time of less than 2 hours. This is highly significant
because drug susceptibility for influenza viruses using geno-
typic and/or phenotypic methods requires sophisticated
instruments, trained personnel, and several hours to
complete.[17]
To differentiate between bacteria/human NA that are also
expected to cleave SG1, we exploited known differences in
the binding pocket of NAs. Bacteria/human NA have
a smaller binding pocket and cannot accommodate larger
groups at the 4-position of sialic acid, as confirmed by X-ray
structures and functional assays with Zanamivir and Oselta-
mivir.[10] We, and others, have exploited this feature to
develop inhibitors[10,18] and substrates[19] that are highly
specific for influenza NA. As expected, bacterial NA
(BNA) from Clostridium perfringens and viral NA cleave
SG1 (Figure 2c, sample BNA). However, when Zanamivir
was premixed with both NAs, only BNA cleaves SG1 and not
the viral NA (VNA) because the antiviral is specific for VNA
(Figure 2c). Therefore, we can distinguish between BNA and
viral NA using the antivirals. An alternate approach is to
introduce larger groups at the 4-position of sialic acid to make
the substrate highly specific for viral NA instead of using
Zanamivir; the syntheses of these next-generation substrates
are ongoing.
Next, we were interested in determining if nasal or throat
swabs, the standard source of clinical samples for influenza
viruses, have a background level of glucose. We found glucose
is absent in the nose or throat by testing samples obtained
from healthy human volunteers. Glucose is released when the
samples are spiked with known concentrations of the virus
(Figure 2d). Thus, this assay could be used to test influenza in
nasal and/or throat swabs.
Influenza strains
Plaque
Ct[b] i [10À8 A][c]
Assay[a]
No virus + SG1
No glucose
b-Galactosidase
a-Mannosidase
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
3.6Æ2.2
3.4Æ1.4
4.2Æ1.9
4.7Æ1.6
Glucose (1 mm)
n/a
n/a 124.9Æ2.3
A/Wilson-Smith/1933 (H1N1)
A/PuertoRico/8/1934 (H1N1)
A/HongKong/8/1968 (H3N2)
A/Aichi/2/1968 (H3N2)
A/Beijing/262/1995 (H1N1)
A/Nanchang/933/1995 (H3N2)
A/Sydney/5/1997 (H3N2)
A/NewCaledonia/20/1999 (H1N1)
A/SolomonIslands/3/2006 (H1N1)
A/Uruguay/716/2007 (H3N2)
A/Brisbane/59/2007 (H1N1)
A/Brisbane/10/2007 (H3N2)
A/California/07/2009 (H1N1)
A/New York/18/2009 (H1N1)
A/San Diego/1/2009 (H1N1)
A/Wisconsin/629-D02452/2009
(H1N1)
4.2”106
1.4”104
1.5”105
1.0”105
3.5”105
7.0”105
8.0”103
4.4”107
1.1”109
1.2”107
2.5”107
2.2”106
3.6”106
3.5”105
1.2”105
6.0”104
18
15
22
15
99.5Æ4.4
13.4Æ2.0
64.4Æ5.2
31.3Æ5.3
21 118.1Æ11.6
18
15.1Æ2.8
29 115.7Æ4.3
12
11
17.7Æ2.6
79.7Æ5.0
20 132.4Æ6.3
12 106.2Æ5.1
21
13
17
78.6Æ4.9
13.4Æ1.5
74.4Æ5.6
19 108.5Æ12.0
13
98.9Æ5.7
A/Wisconsin/15/2009 (H3N2)
A/Brownsville/31H/2009 (H1N1)
A/Victoria/361/2011 (H3N2)
5.0”103
4.0”103
7.0”106
26 107.8Æ5.9
22
26
91.7Æ9.7
94.5Æ2.3
[a] Reported in pfu mLÀ1. [b] rRT–PCR was performed using 100 mL of
virus to measure Ct, that is, the cycle number at which fluorescence is
above the threshold (background). Ct is inversely proportional to the
number of amplicons. [c] Measured by electrochemical assay. 100 mL of
virus was mixed with 100 mL of PBS buffer with SG1 for 1 hour at 378C.
Glucose concentration was determined using 20 mL of this solution.
2’-(4-methylumbelliferyl)-a-d-N-acetylneuraminic
acid
(MUNANA; Figure S4). As expected, the rate of cleavage
of A/PuertoRico/8/1934 (H1N1), A/California/07/2009
(H1N1), and A/New Caledonia/20/1999 (H1N1) strains are
slower than the A/Beijing/262/1995 (H1N1) strain. We note
that, despite variations in printed electrodes from different
manufacturer or different batches from the same manufac-
turer, the assay detects all strains. We also determined the
analytical sensitivity using one of the strains using this
rudimentary setup (Figure S3). The limit of detection and
range is 102 and 102–108 pfu, respectively. As multiple studies
have reported that patients (n > 50) suffering from influenza
typically harbor 103–108 pfumLÀ1 in the nose/throat,[20] this
assay could be useful for rapid detection in a primary-care
setting.
To summarize, we have developed an electrochemical
assay that releases glucose upon introduction of influenza
viruses. We successfully detected 19 influenza strains. The
assay can be used to measure drug susceptibility rapidly,
a significant advantage over current genotypic and pheno-
typic methods that take time, resources, and a laboratory
environment.[17] The assay can be integrated into current
glucose meters by repurposing the instruments to test nasal or
throat swabs for influenza. As glucose meters with disposable
test strips are user friendly, ubiquitous, and inexpensive, this
To improve assay performance, we used disposable
printed electrodes (CH instruments, Austin, Texas) for the
next set of experiments (Figure S3). This experimental setup
requires only 20 mL of solution, similar to commercial
disposable test strips used in blood glucose meters. We
obtained 19 H1N1 and H3N2 influenza strains that spanned
eighty years, from 1933 to 2011, including strains from the
most recent 2009 pandemic. All strains were detected in one
hour, which demonstrates broad specificity. These results
were validated using rRT–PCR and plaque assays (Table 1).
Some strains cleaved SG1 slowly; we corroborated the results
by measuring the slow release using a fluorescent substrate,
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