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Smartphone-Based Multiplex 30-minute Nucleic Acid Test of Live
Virus from Nasal Swab Extract
Fu Sun,a Anurup Ganguli,b Judy Nguyen,c Ryan Brisbin,d Krithika Shanmugam,d David L. Hirschberg,c,d
Matthew B. Wheeler,e Rashid Bashir,a,b David M. Nash,f and Brian T. Cunningham*,a,b
Rapid, sensitive and specific detection and reporting of infectious pathogens is important for patient management and
epidemic surveillance. We demonstrated a point-of-care system integrated with a smartphone for detecting live virus from
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x

nasal swab media, using a panel of equine respiratory infectious diseases as a model system for corresponding human
diseases such as COVID-19. Specific nucleic acid sequences of five pathogens were amplified by loop-mediated isothermal
amplification on a microfluidic chip and detected at the end of reactions by the smartphone. Pathogen-spiked horse nasal
swab samples were correctly diagnosed using our system, with a limit of detection comparable to that of the traditional labbased test, polymerase chain reaction, with results achieved in ~30 minutes.

Introduction
Since the SARS-CoV-2 (COVID-19) virus jumped from an animal
reservoir to humans in December 2019, it has rapidly spread
across the world, bringing death, illness, disruption to daily life,
and economic losses to businesses and individuals. A key failure
of the health system across every country has been the ability
to rapidly and accurately diagnose the disease, with
contributing factors that include a limited number of available
test kits, a limited number of certified testing facilities,
combined with the length of time required to obtain a result and
provide information to the patient. The challenges associated
with rapid diagnostic testing contribute to uncertainly
surrounding which individuals should be quarantined, sparse
epidemiological information, and inability to quickly trace
pathogen transmission within/across communities.
The
challenges underlying COVID-19 diagnosis are already well
known from previous encounters with emerging epidemics and
pandemics, such as mosquito-borne diseases (Zika, Dengue,
Chikungunya, Malaria), HIV, and others. Already, the ability to
perform pervasive testing has shown clear benefits to countries
that implement it, such as South Korea, to provide accurate
a. Department

of Electrical and Computer Engineering, University of Illinois at
Urbana-Champaign, Illinois, USA
of Bioengineering, University of Illinois at Urbana-Champaign,
Illinois, USA
c. RAIN Incubator, Tacoma, Washington, USA
d. Department of Interdisciplinary Arts and Sciences & The Center for Urban Waters,
University of Washington Tacoma, Washington, USA
e. Department of Animal Sciences, University of Illinois at Urbana-Champaign,
Illinois, USA
f. Private equine veterinarian, Kentucky, USA
*Corresponding author. E-mail: bcunning@illinois.edu. Tel.: +1 217 333 2301. Fax:
+1 217 244 6375.
Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x
b. Department

information regarding whom to quarantine, which in turn
results in more timely control of disease propagation.
Infectious diseases represent a global challenge for both human
and animal health due to their ability to proliferate rapidly
through direct or indirect contact, insect vectors, and
respiratory inhalation.1, 2 Although the world’s leading causes of
human mortality are shifting toward non-communicable
diseases, infectious diseases still accounted for 8.14 million
deaths in 2017. This represented about 14.6% of total global
deaths, which is comparable to the number of deaths caused by
all cancers.3 Infectious disease transmission within animal
populations raised for food consumption result in substantial
economic loss and is an ongoing threat to food production, as
highlighted by a recent outbreak.4 Additionally, animal
populations kept for companion, racing or entertainment
purposes are comprised of individuals with large sentimental or
economic value, for which rapid point-of-care diagnostic tests
would be particularly valuable. The availability of such tests
would help guide treatment decisions or determine the
necessity of quarantine. The issues facing human and animal
transmission of respiratory diseases are very similar in terms of
collection of samples by nasal swab, laboratory-based assays
using polymerase chain reaction (PCR), and interventions that
suppress disease spread such as quarantine and distancing from
others.
A 2011 outbreak of equine herpesvirus type 1 (EHV1) originated
from an American horse competition and rapidly spread to at
least 242 horse premises in 19 U.S. states, with further spread
to two Canadian provinces.5 Due to shared living facilities,
shared water sources, and physical contact with humans, the
threat of equine infectious diseases is ever-present.6-8 Effective
equine infection surveillance and control should incorporate

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early-stage diagnosis to facilitate early medical intervention,
and provide timely alerts that can contain outbreaks locally.9
Presently, a variety of diagnostic methods are available that can
detect and identify infectious diseases, including direct
microscopic examination, isolation of pathogens in culture,
serological tests of antibody response, and nucleic acid testing
(NAT) such as PCR assays.10, 11 Traditional diagnostic methods
generally require benchtop instruments handled by trained
personnel in a lab within a central facility. While sample
preparation and the assay protocol may only require a few
hours, the time delay imposed by sample delivery, along with
timing uncertainty induced by testing backlogs, holidays, and
other scheduled laboratory closures can cause a significant
delay of results to the veterinarian. Furthermore, because many
infectious diseases present themselves with similar symptoms
and there is also a possibility of co-infection with more than one
pathogen, the need to perform multiple tests to identify
potential pathogens can cause further delays. An improved
capability for pathogen testing would therefore be the ability to
specifically identify multiple pathogens with a single test.
Because available technologies remain expensive (in terms of
capital equipment and reagents), technically challenging, and
labor intensive, there is an urgent need for low-cost portable
platforms that can provide fast, accurate, and multiplex
diagnosis of infectious disease at the point of care.
In the area of human infectious disease testing, well-equipped
laboratories are generally remotely located from low-income,
resource-limited areas.12 To meet the diagnostic needs of lowresource settings, point-of-care (POC) assay technology
platforms have been developed to provide rapid, inexpensive
and portable solutions.13-15 NATs represent an important class
of POC technologies for pathogen sensing that achieve a high
level of specificity through detection of a nucleic acid sequence
that has been carefully selected to identify only one pathogen
species. In addition to high specificity, many NATs are capable
of achieving high sensitivity through the use of enzymatic
amplification of the target nucleic acid sequence. A single
pathogenic DNA sequence can be converted into large numbers
of copies that optionally carry fluorometric or colorimetric tags.
Due to their success in laboratory settings, considerable efforts
have been devoted to performing NATs in POC settings, with
methods based upon PCR among the most prevalent.16-18 The
enzymatic amplification process inherent with PCR requires
repeated heating/cooling cycles, resulting in detection systems
with elaborate thermal control schemes that contribute to
increased cost and complexity. PCR related approaches also
suffer from high false negative rates due to a combination of a
low amount of starting material (one genome copy per viral
particle), instability of the nucleic acid extraction process,
inhibiting substances in the test sample, and quality control
failure of the many reagents.19, 20 Therefore, NATs that utilize
isothermal nucleic acid amplification21, 22 have been
investigated for implementing simple and miniaturized POC
devices. Loop-mediated isothermal amplification (LAMP) is one
such method that amplifies DNA at a constant temperature (60
to 65°C) with only one type of enzyme and four to six primers.23,
24 This method can generate 109 copies of a specific target

sequence in less than an hour. While LAMP primer design is
more complex than PCR primer design, the LAMP process is
generally considered less susceptible to the presence of
materials that inhibit PCR and can operate in unprocessed
samples such as cell lysate. Thus, LAMP-based NATs for
identification of pathogens has been pursued for POC
applications using dedicated readout instruments that detect
fluorescent amplicons in both macrofluidic25, 26 and
microfluidic27, 28 formats.
Recent research has consistently demonstrated that the image
sensors integrated within commercially available smartphones
have sufficient sensitivity for detecting fluorescence in the
contexts of fluorescence microscopy of cells,29 viruses,30 and
bacteria.31 Smartphone cameras are likewise capable of sensing
the fluorescent emission from a wide variety of biological
33
assays,32,
including
LAMP,
within
microfluidic
compartments.27, 28, 34 The advantage of using a smartphone as
the detection instrument for POC analysis is that, it is possible
to take advantage of the integrated optics, image sensor,
computation
power,
user
interface,
and
wireless
communication capabilities of mobile devices, thus minimizing
cost. With assistance from an inexpensive snap-in cradle or clipon instrument, anyone that carries a smartphone would have
the ability to perform testing. Due to the prevalence of cloudbased service systems, one can envision systems that integrate
testing results from large numbers of users over a
geographically distributed area for reporting of new infections
and for epidemiological analysis of disease spread.
In this work, we used a portable smartphone-based instrument
to perform end-point fluorescence detection of LAMP assays on
a microfluidic chip for five bacterial and viral pathogens that
cause equine respiratory infectious diseases and are most
prevalent among horse populations: Streptococcus equi
subspecies equi (S. equi), Streptococcus equi subspecies
zooepidemicus (S. zoo), equine herpesvirus 1 (EHV1), equine
herpesvirus 4 (EHV4) and equine influenza virus (EIV) subtype
H3N8. A qualitative detection threshold was established after
statistical analysis of positive and negative test values. Our
system can detect the specific target nucleic acid sequences in
a multiplex manner without signal crosstalk. Moreover, horse
nasal swab samples spiked with one of the virus targets (EHV1)
were tested by our system to demonstrate its capability in earlystage diagnosis. This work represents, to our knowledge, the
first utilization of smartphone-based LAMP detection of
pathogens for POC application in animal health. Utilizing the
system in the context of equine respiratory diseases represents
a model system for human pathogens such as SARS-CoV-2,
which does not pose biosafety issues, but preserves the main
features of a human COVID-19 testing protocol.

Experimental
LAMP assays
LAMP assays were developed for specific nucleic acid sequences
of the five equine pathogens. Another two LAMP assays
detecting nucleic acid sequences within the genomes of

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Escherichia coli (E. coli) and equine herpesvirus 3 (EHV3) were
used as positive controls for on-chip tests. A set of scrambled
primers that had no target nucleic acid sequence among our
test samples was a negative control. The primers (Table S1, S2)
of these LAMP assays were synthesized by Integrated DNA
Technologies.
The LAMP assays were comprised of the following components
(Table S3): 1.4 mM of deoxynucleoside triphosphates (dNTPs),
1× isothermal amplification buffer (New England Biolabs), 6 mM
of MgSO4 (New England Biolabs), 0.4 M of Betaine (SigmaAldrich), 640 units/mL Bst 2.0 WarmStart DNA Polymerase (New
England Biolabs), 1× EvaGreen dye (Biotium), and a LAMP
primer mix of 0.2 μM of F3 and B3 primers, 1.6 μM of FIP and
BIP primers, and 0.8 μM of LoopF and LoopB primers. To make
a 20 μL final reaction mix, 6.4 μL of template DNA and 1.8 μL of
DEPC-treated water (Invitrogen) was added. The components
were prepared in bulk and stored at −20°C before reactions
were prepared on ice.
Target template sequences were synthesized and cloned in the
pUC57-AMP vector (Genewiz). Cultures of transformed E. coli
were grown overnight and used to extract plasmids. The
concentrations of plasmids were quantified using a Qubit
Fluorometer (Invitrogen) and converted to a copy number using
the plasmid’s length.
Off-chip LAMP assays were carried out in 0.2-mL PCR re-action
tubes, each with 10 μL of reaction mix, in an Eppendorf
Mastercycler Real-Time PCR System. The tubes were incubated
at 65 °C for 60 min in the thermocycler and fluorescence data
was recorded every minute.
PCR assay
The PCR assay for EHV1 was used for DNA quantification in
clinical samples as a gold standard. The PCR standard curve of
EHV1 DNA was established using synthesized EHV1 plasmid at
log concentrations (Fig. S4). The 20 μL PCR reaction mix
consisted of the following components (Table S4): 1× Luna
Universal qPCR Master Mix (New England Biolabs), 5 μL of
template DNA, 4 μL of nuclease-free water, and 0.25 μM of
forward and reverse PCR primers.
Off-chip PCR tests were conducted on the same thermocycler
with the following protocol using its fast ramp speed: 1 min of
initial DNA denaturation at 95 °C, followed by 45 cycles of 95 °C
(15 s of denaturation) and 60 °C (30 s of extension).
Fluorescence data was recorded after each cycle.
Horse nasal swab samples
Six healthy adult horses used in this study were from horse
farms in Champaign, IL, United States. Sterile rayon nasal swabs
(OSOM, SEKISUI-185, Sekisui Diagnostics, Lexington, MA) were
placed 1-2 cm into the nares of each horse to collect nasal
secretions and/or mucus. Each individual swab with sample was
then placed back in its sterile protective cover and transported
to the lab at 4°C. Each swab was then incubated in 1 mL of
phosphate-buffered saline (PBS) solution (Thermo Fisher
Scientific) at 4°C overnight to release nucleic acids. The EHV1
virus stock (USDA 040-EDV) held at -80°C was thawed and
spiked into three of the six sample solutions at different

dilutions (1:10, 1:100, 1:1000) to make three EHV1-positive
samples representing high, moderate and low levels of EHV1
viral load, respectively. We had to make positive samples in this
way because all the horses we had access to were healthy. For
DNA extraction, 200 μL of nasal swab solution was processed by
a high-throughput purification kit (QIAamp DNA Mini Kit,
QIAGEN), with a final elution volume of 100 μL.
The processed swab samples were tested for EHV1 DNA in realtime PCR tests described above (Fig. S5), and the concentration
of EHV1 DNA in each sample (Table S5) was calculated using the
previously established PCR standard curve. The average
concentrations (5.5×104 to 6.3×106 copies/mL) of the positive
swab samples are in the clinical range of EHV1 infections (105 to
107 copies/mL in nasal swab solution from 1 to 6 days, with
lower concentrations afterwards).35
Silicon chip for assays
Silicon microfluidic chips were used to hold LAMP assays as they
are highly stable, have no auto-fluorescence and can be
manufactured efficiently. The chips (25 mm × 15 mm × 0.5 mm)
contained ten parallel flow channels (10 mm × 0.5 mm × 0.2
mm) with a volume of 1 μL each and all sharing the same inlet.
The surface of the chip was thermally oxidized to grow a 200nm layer of SiO2 that served to reduce the potential for bare
silicon to inhibit the amplification process. Full details about
fabrication of the chip can be found in our previous
publication.34
On-chip amplification
For on-chip tests, primers were deposited into the microfluidic
lanes by pipetting and allowed to dry (Fig. 1a). The primers
were reconstituted into solution during the following addition
of LAMP reaction mix. The desired pathogen panel is
“programmed” into the chip by the deposition of specific
primers in each channel. For example, in the multiplex tests for
all five pathogens, Channel 1 was the first positive control with
the primers for the E. coli DNA. In order to assure that Channel
1 provided a positive response, ~40 pg of the E. coli DNA is a
component of the reaction mix. Channel 2 served as a second
positive control by drying down a mixture of the primers and
template DNA (1000 copies) for EHV3. Channel 3 was used as a
negative control deposited with scrambled primers for goldfish
DNA that should have no amplification for any pathogenic DNA
sequence in our panel. The remaining channels were deposited
with primers for pathogenic target sequences or left empty as
needed. The primer solutions injected into the channels result
in a final primer concentration the same as that used for offchip LAMP assays. After deposition, the channels were covered
with a layer of double-sided adhesive (DSA) and sealed by a
piece of cover glass after sample injection (Fig. 1b) to prevent
sample evaporation. The chip was then heated on a hotplate at
65°C to drive the LAMP enzymatic amplification reactions (Fig.
1c).
Fluorescence image capture and analysis
Fluorescence images of amplified chips were captured using the
rear-facing camera of a smartphone (Motorola Nexus 6)

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mounted on a custom-designed cradle.34 The chip was
illuminated with light from an array of eight blue LEDs (485 nm,
#XPEBBL, Cree Inc.) arranged around the perimeter of the chip.
The light from each LED was filtered by a small short pass optical
filter (490 nm, #ZVS0510; Asashi Spectra) placed directly in front
of it. A long pass optical filter (525 nm, #84-744; Edmund Optics)
placed in front of a macro lens (12.5x, #TECHO-LENS-01, TECHO)
allowed only the fluorescence emission of the EvaGreen DNAintercalating dye to reach the camera. During image collection,
the chip was heated to a temperature of ~65 °C by a positive
temperature coefficient (PTC) heater (12 V-80 ˚C, Uxcell) in the
cradle. Each pixel within each image file was described by a
three-dimensional matrix in red, green, blue (RGB) color space.
The RGB intensity component for each pixel was stored as an 8bit unsigned integer ranging from 0 to 255. The fluorescence
emission from the Evagreen dye has a center wavelength of 533
nm, which falls within the spectral range of the green (G)
channel, and thus only the G channel is used for the analysis.
Within each microfluidic channel’s region of interest in the
image field of view, the average value of all the pixels were
calculated. The average intensity obtained from the EHV3
positive control with 1000 copies of DNA was used for intensity
normalization (value = 100) of all the channel intensities within
the same chip.

Results and discussion
Determination of qualitative detection threshold
To identify the most suitable threshold intensity value to
differentiate positive from negative tests, average intensities of

Fig. 1 On-chip detection workflow. a) Deposit controls and target primers into
channels on a cleaned chip and cover it with a transparent double-side adhesive after
reagents are dried; b) Inject LAMP reaction mix from the inlet and seal the chip with
a piece of cover glass; c) Heat the chip at 65°C for LAMP reactions and insert the chip
into the cradle for end-point fluorescence imaging. Typically, results can be retrieved
after 30 minutes.

positive and negative channels after LAMP reactions were
collected for the EHV1 target DNA for concentrations ranging
from 100 to 10000 copies/μL (Fig. 2a). The results of the other
four targets in on-chip experiments were shown in Fig. S2.
Among the ten channels on each chip, two of them were used
for controls, and the other eight channels acted as replicates for
a target. As the target DNA concentration increases, the
distribution of the positive values moves to a higher level and
has a smaller variation (Fig. 2b). The negative channels with
unamplified primers exhibit a low level of background
fluorescence due to the presence of the intercalating dye. There
is a distinct separation between the positive and negative
intensity values (Fig. 2c). Based on the principle of support
vector machine (SVM), the center value (threshold ≈ 74)
between the lowest positive value and highest negative value
was chosen as our positive/negative threshold, from which the
distance to the nearest data points in the two groups is
maximized.
On-chip multiplex detection of pathogen DNA
Fig. 3 and Fig. S3 show smartphone images of the chips after
amplification and corresponding average intensities in each
channel. For multiplex detection of the five targets, Channels 12 were used as positive controls as described previously, with
target sequences integrated within the LAMP reaction mix
(Channel 1) or dried on the chip (Channel 2) before sample
injection, respectively. Both positive control channels displayed
strong fluorescence, indicating successful amplification that can
also be used as references. Channel 3 contained primers used
as a negative control. When comparing Channel 3 and other
unamplified reactions to channels containing no primers at all
(Channel 4 and 10), there were low levels of autofluorescence
present. It was presumed that the background fluorescence was

Fig. 2 On-chip characterization of the equine assays using plasmid DNA. a) Amplified
chips with EHV1 templates at different concentrations (Reagents dried on the chips
from left to right in the image above for Channel 1: positive control, Channel 2:
negative control, Channel 3-10: EHV1 primers); b) The boxplots of average channel
intensities of the above EHV1 samples; c) The boxplots of the overall negative and
positive groups of five test assays, with the qualitative threshold marked by a red
dash line.

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Deleted: The intensities in positive channels reach saturation
with high-concentration templates when the primers are fully
consumed by the amplification process.
Deleted: Despite fluorescence intensity saturation for
positive channels and background fluorescence in negative
channels, t

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due to the binding of the dye with primers. After LAMP
reactions, only the positive controls and the channel with the
primers of the specific target in the sample passed the
qualitative threshold. Because each of the LAMP reaction
occurred independently within its microfluidic lane, the chip can
also be used to detect coinfection with more than one pathogen
(Fig. 3d).

as positive controls, Channel 3 was the negative control,
Channels 4-6 were prepared with the EHV1 primers as three
replicates for the EHV1 assay, and Channels 7-10 were utilized
for the remaining four targets without replicates. Fig. 4 shows
the results for the highest-concentration (400 copies/mL)
negative sample (Fig. 4a-b) and the lowest-concentration
(5.5×104 copies/mL) positive sample (Fig. 4c-d). Our threshold
was able to correctly classify all the positive and negative
samples (the other samples in Fig. S6-9). We also found that
taking end-point pictures of amplified chips at a temperature
higher than 65°C, for example, at 80°C, would decrease the
background fluorescence from negative channels while having
little influence on positive channels, to provide improved overall
contrast. We hypothesize that higher temperature prohibits
formation of primer dimers that can bind to the EvaGreen dye
and produce background fluorescence.

Conclusions

Fig. 3 On-chip multiplex detection of equine respiratory infection pathogen DNA. The
smartphone images of amplified chips and the corresponding channel intensities are
shown for detecting a) S. equi, b) EHV1, c) EIV and d) S. equi accompanied with EIV at
1000 copies/μL. (Reagents dried on the chip for Channel 1: the E. coli positive control
primers, Channel 2: the EHV3 positive control primers and DNA, Channel 3: the
negative control primers, Channel 4 and 10: no primers, Channel 5-9: S. equi, S. zoo,
EHV1, EHV4 and EIV primers, respectively.)

On-chip tests of horse nasal swab samples
The processed horse nasal swab samples were tested on the
chips with a new test layout: Channels 1-2 were again utilized

In summary, this study has demonstrated a smartphone-based
system for rapid and multiplex detection of specific nucleic acids
of five pathogens that cause equine respiratory infectious
diseases. LAMP reactions were performed on a microfluidic chip
with a reaction volume of 1 μL for each assay, and fluorescence
images of the chips were taken by a smartphone in a customized
cradle after isothermal LAMP amplification reaction. The
microfluidic chip can be programmed for different target
pathogens and sensing scenarios by changing the primer sets
deposited in each channel. The integration of multiple
positive/negative experimental controls and experimental
replicates is used to assure that the assay protocol was
performed correctly and can be used to reduce the likelihood of
false positive or false negative results in POC health diagnostic
applications. Our system is able to detect one or more specific
targets simultaneously, which is valuable for coinfection
diagnosis. The sensitivity of the system is adequate for earlystage detection of EHV1 in horse nasal swab samples, down to
5.5×104 copies/mL, which corresponds to about 18 copies per
reaction and is comparable to the limit of detection of a PCR
assay run on a commercial thermocycler. LAMP reactions take
less than 30 minutes for high-concentration samples and the
whole detection process can be finished in an hour with
inexpensive and portable equipment, enabling veterinarians or
physicians to diagnose infections at the point of care and report
outbreaks remotely for efficient epidemic surveillance.
Our efforts are motivated by the urgent need to develop rapid
POC testing for highly contagious human respiratory viruses
such as SARS-CoV-2, that would enable results to be provided
to the patient and physician as early as possible. By using a
smartphone in conjunction with a cradle that enables the
phone’s camera to quickly gather a fluorescent endpoint image
of the LAMP reaction, a positive/negative determination can be
made that incorporates integrated experimental controls and
replicates to assure that the test was performed correctly. By
using the mobile device as a detection instrument, we envision
that data collection can be seamlessly integrated with
telemedicine platforms that facilitate epidemiology reporting

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Commented [SF2]: This answers Reviewer 2’s question: What
was the criteria of considering 65 & 80 degree centigrade
temperature for on-chip detection of the positive horse.

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and sharing test results with a physician. In future work, our
plans include integrating the functions of viral lysis, LAMP buffer
mixing, and LAMP reaction into a single cartridge with the
reagents held within on-cartridge reservoirs. Further, we
envision a detection instrument that clips onto a smartphone,
with mechanical adapters that will align the rear-facing camera
correctly with several popular phone models.

Author contribution
F. Sun, D. L. Hirschberg, R. Bashir and B. T. Cunningham
conceived the idea and designed the study. F.S. designed,
performed the experiments and wrote the manuscript. A.
Ganguli provided the positive control assay. J. Nguyen designed
the negative control primers. R. Brisbin prepared the quantified
plasmids. K. Shanmugam helped J. Nguyen with off-chip
experiments. D. M. Nash suggested the list of target equine
pathogens. M. B. Wheeler provided the horse nasal swab
samples. All offered intellectual inputs.

Conflicts of interest
Intellectual property associated with the technology described
in this manuscript has been licensed by the University of Illinois
to Reliant Immune Diagnostics (Austin, TX), where B. T.
Cunningham serves as a consultant and owns stock options in
the company.

Acknowledgements
The authors are grateful for the funding support provided by
National Science Foundation (NSF) under the grant number
1534126. Any opinions, findings, and conclusions in this work
are those of the authors and do not necessarily reflect the views
of NSF.

Notes and references
1
2
3
4

5

6
7
8
9
Fig. 4 On-chip tests of horse swab samples. The smartphone pictures of the
amplified chip for a negative sample (~400 EHV1 genome copies/mL) and
corresponding average channel intensities were obtained at a) 65 ℃ and b) 80 ℃.
The results for a positive sample (~5.48 × 104 EHV1 genome copies/mL) were also
obtained at c) 65 ℃ and d) 80 ℃. Increasing chip temperature at the imaging time
decreased background fluorescence from unamplified primers and improved
positive/negative contrast. (Reagents dried on the chip for Channel 1: the E. coli
positive control primers, Channel 2: the EHV3 positive control primers and DNA,
Channel 3: the negative control primers, Channel 4~6: EHV1 primers, Channel 7~10:
S. equi, S. zoo, EHV4 and EIV primers, respectively.)

10
11
12

13

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