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This article can be cited before page numbers have been issued, to do this please use: F. Sun, A. Ganguli,
J. Nguyen, R. Brisbin, K. Shanmugam, D. L. Hirschberg, M. B. Wheeler, R. Bashir, D. M. Nash and B. T.
Cunningham, Lab Chip, 2020, DOI: 10.1039/D0LC00304B.
Volume 17
Number 1
7 January 2017
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DOI: 10.1039/D0LC00304B

ARTICLE

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

Lab on a Chip Accepted Manuscript

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
nasal swab media, using a panel of equine respiratory infectious diseases as a model system for corresponding human
Received 00th January 20xx,
diseases such as COVID-19. Specific nucleic acid sequences of five pathogens were amplified by loop-mediated isothermal
Accepted 00th January 20xx
amplification on a microfluidic chip and detected at the end of reactions by the smartphone. Pathogen-spiked horse nasal
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DOI: 10.1039/x0xx00000x swab samples were correctly diagnosed using our system, with a limit of detection comparable to that of the traditional lab-
based test, polymerase chain reaction, with results achieved in ~30 minutes.

provide accurate information regarding whom to quarantine,

Introduction which in turn results in more timely control of disease
propagation.
Since the SARS-CoV-2 (COVID-19) virus jumped from an animal
Infectious diseases represent a global challenge for both
reservoir to humans in December 2019, it has rapidly spread
human and animal health due to their ability to proliferate
across the world, bringing death, illness, disruption to daily life,
rapidly through direct or indirect contact, insect vectors, and
and economic losses to businesses and individuals. A key failure
respiratory inhalation.1, 2 Although the world’s leading causes of
of the health system across every country has been the ability
human mortality are shifting toward non-communicable
to rapidly and accurately diagnose the disease, with
diseases, infectious diseases still accounted for 8.14 million
contributing factors that include a limited number of available
deaths in 2017. This represented about 14.6% of total global
test kits, a limited number of certified testing facilities,
deaths, which is comparable to the number of deaths caused by
combined with the length of time required to obtain a result
all cancers.3 Infectious disease transmission within animal
and provide information to the patient. The challenges
populations raised for food consumption result in substantial
associated with rapid diagnostic testing contribute to
economic loss and is an ongoing threat to food production, as
uncertainly surrounding which individuals should be
highlighted by a recent outbreak.4 Additionally, animal
quarantined, sparse epidemiological information, and inability
populations kept for companion, racing or entertainment
to quickly trace pathogen transmission within/across
purposes are comprised of individuals with large sentimental or
communities. The challenges underlying COVID-19 diagnosis
economic value, for which rapid point-of-care diagnostic tests
are already well known from previous encounters with
would be particularly valuable. The availability of such tests
emerging epidemics and pandemics, such as mosquito-borne
would help guide treatment decisions or determine the
diseases (Zika, Dengue, Chikungunya, Malaria), HIV, and others.
necessity of quarantine. The issues facing human and animal
Already, the ability to perform pervasive testing has shown clear
transmission of respiratory diseases are very similar in terms of
benefits to countries that implement it, such as South Korea, to
collection of samples by nasal swab, laboratory-based assays
using polymerase chain reaction (PCR), and interventions that
a. Department of Electrical and Computer Engineering, University of Illinois at suppress disease spread such as quarantine and distancing from
Urbana-Champaign, Illinois, USA
b. Department of Bioengineering, University of Illinois at Urbana-Champaign, others.
Illinois, USA A 2011 outbreak of equine herpesvirus type 1 (EHV1) originated
c. RAIN Incubator, Tacoma, Washington, USA
d. Department of Interdisciplinary Arts and Sciences & The Center for Urban Waters,
from an American horse competition and rapidly spread to at
University of Washington Tacoma, Washington, USA least 242 horse premises in 19 U.S. states, with further spread
e. Department of Animal Sciences, University of Illinois at Urbana-Champaign,
to two Canadian provinces.5 Due to shared living facilities,
Illinois, USA
f. Private equine veterinarian, Kentucky, USA shared water sources, and physical contact with humans, the
*Corresponding author. E-mail: bcunning@illinois.edu. Tel.: +1 217 333 2301. Fax: threat of equine infectious diseases is ever-present.6-8 Effective
+1 217 244 6375.
Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x
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
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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 low-
resource 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. 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
2 | J. Name., 2012, 00, 1-3
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identification of pathogens has been pursued for Online
View Article POC
DOI: 10.1039/D0LC00304B
applications using dedicated readout instruments that detect
fluorescent amplicons in both macrofluidic 25, 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
assays,32, 33 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
Lab on a Chip Accepted Manuscript
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 clip-
on instrument, anyone that carries a smartphone would have
the ability to perform testing. Due to the prevalence of cloud-
based 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 early-
stage 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. Although our
system was used to detect pathogenic DNAs in this paper for
demonstration, it can be easily adapted for detecting RNA
viruses by using a one-step RT-LAMP protocol which adds
reverse transcriptase to the LAMP reaction mix without
modification to the buffer or reaction conditions.
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
This journal is © The Royal Society of Chemistry 20xx
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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 (Sigma-
Aldrich), 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
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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 2 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
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dilutions (1:10, 1:100, 1:1000) to make three EHV1-positive
View Article Online
samples representing high, moderate and DOI:low
10.1039/D0LC00304B
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 purified solutions from the swab samples were tested by
real-time PCR described above (Fig. S5), and the concentration
of EHV1 DNA in each solution (Table S5) was calculated using
the previously established PCR standard curve. The DNA
concentrations (5.5×104 to 6.3×106 copies/mL) of the purified
solutions from the positive swab samples are in the clinical
Lab on a Chip Accepted Manuscript
range of EHV1 infection, which is reported to be above 105
copies/mL within 6 days post infection and above 104 copies/mL
within 12 days post infection in nasal swab solution.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 200-nm
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
channels 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 off-
chip 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).
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Fluorescence image capture and analysis intensity normalization (value = 100) of all View
theArticle
channel
Online
intensities within the same chip. DOI: 10.1039/D0LC00304B
Fluorescence images of amplified chips were captured using the
rear-facing camera of a smartphone (Motorola Nexus 6)
mounted on a custom-designed cradle.34 The chip was
Results and discussion
illuminated with light from an array of eight blue LEDs (485 nm,
#XPEBBL, Cree Inc.) arranged around the perimeter of the chip. Determination of qualitative detection threshold
The light from each LED was filtered by a small short pass optical
To identify the most suitable threshold intensity value to
filter (490 nm, #ZVS0510; Asashi Spectra) placed directly in front
differentiate positive from negative tests, average intensities of
of it. A long pass optical filter (525 nm, #84-744; Edmund Optics)
positive and negative channels after LAMP reactions were
placed in front of a macro lens (12.5x, #TECHO-LENS-01, TECHO)
collected for the EHV1 target DNA for concentrations ranging
allowed only the fluorescence emission of the EvaGreen DNA-
from 100 to 10000 copies/μL (Fig. 2a). The results of the other
intercalating dye to reach the camera. During image collection,
four targets in on-chip experiments were shown in Fig. S2.

Lab on a Chip Accepted Manuscript

the chip was heated to a temperature of ~65 °C by a positive
Among the ten channels on each chip, two of them were used
temperature coefficient (PTC) heater (12 V-80 ˚C, Uxcell) in the
for controls, and the other eight channels acted as replicates for
cradle. The LED module and the heater were controlled by two
a target. As the target DNA concentration increases, the
separate switches on the cradle body. The LEDs were powered
distribution of the positive values moves to a higher level and
by two AAA batteries (3 V) and the heater was powered by a 9-
has a smaller variation (Fig. 2b). The negative channels with
V battery. A customized Android app was used for imaging with
unamplified primers exhibit a low level of background
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fixed settings (flashlight off, exposure time = 1 s). The app

fluorescence due to the presence of the intercalating dye. There
exported raw images. Each pixel within each image file was
is a distinct separation between the positive and negative
described by a three-dimensional matrix in red, green, blue
intensity values (Fig. 2c). Based on the principle of support
(RGB) color space. The RGB intensity component for each pixel
vector machine (SVM), the center value (threshold ≈ 74)
was stored as an 8-bit unsigned integer ranging from 0 to 255.
between the lowest positive value and highest negative value
The fluorescence emission from the Evagreen dye has a center
was chosen as our positive/negative threshold, from which the
wavelength of 533 nm, which falls within the spectral range of
distance to the nearest data points in the two groups is
the green (G) channel, and thus only the G channel is used for
maximized.
the analysis. Within each microfluidic channel’s region of
interest in the image field of view, the average value of all the On-chip multiplex detection of pathogen DNA
pixels were calculated. The average intensity obtained from the
Fig. 3 and Fig. S3 show smartphone images of the chips after
EHV3 positive control with 1000 copies of DNA was used for
amplification and corresponding average intensities in each
channel. For multiplex detection of the five targets, Channels 1-
2 were used as positive controls as described previously, with

Fig. 2 On-chip characterization of the equine assays using plasmid DNA. a) Amplified
Fig. 1 On-chip detection workflow. a) Deposit controls and target primers into
chips with EHV1 templates at different concentrations (Reagents dried on the chips
channels on a cleaned chip and cover it with a transparent double-side adhesive
from left to right in the image above for Channel 1: positive control, Channel 2:
after reagents are dried; b) Inject LAMP reaction mix from the inlet and seal the
negative control, Channel 3-10: EHV1 primers); b) The boxplots of average channel
chip with a piece of cover glass; c) Heat the chip at 65°C for LAMP reactions and
intensities of the above EHV1 samples; c) The boxplots of the overall negative and
insert the chip into the cradle for end-point fluorescence imaging. Typically, results
positive groups of five test assays, with the qualitative threshold marked by a red
can be retrieved after 30 minutes.
dash line.

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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
due to the binding of the dye with primers. After LAMP
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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. Green bars
represent positive controls, red bars are for negative controls, and blue bars for
tests. Channel 10 was an unused test channel, hence its signal was depicted as a
white bar.)
This journal is © The Royal Society of Chemistry 20xx
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reactions, only the positive controls and the channel withOnline
View Article the
primers of the specific target in theDOI: 10.1039/D0LC00304B
sample passed the
qualitative threshold. Minor to no evaporation of liquid was
observed in channels after amplification and had no effect upon
the qualitative positive/negative determination of a reaction.
As shown in Fig. 3, the shared inlet and the majority of the
linking channels between the inlet and individual channels
remained dark, indicating no cross-contamination between
channels. Because each of the LAMP reaction occurred
independently within its microfluidic channel, the chip can also
be used to detect coinfection with more than one pathogen (Fig.
3d).
Lab on a Chip Accepted Manuscript
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
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 a 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 chips at a higher temperature
after 65°C amplification, for example, at 80°C, would decrease
the background fluorescence from negative channels and have
little influence on positive channels, which provided 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
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 early-
stage 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
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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
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Fig. 4 On-chip tests of horse swab samples. The smartphone pictures were taken at
a) 65 ℃ and b) 80 ℃ of the amplified chip for a negative sample and corresponding
average channel intensities were analysed. The results for a positive sample (~5.48
× 104 EHV1 genome copies/mL) were also imaged at c) 65 ℃ and d) 80 ℃ .
Increasing chip temperature at the imaging time decreased background
fluorescence that may result from primer dimers 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.)
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smartphone in conjunction with a cradle that View enables the
Article Online
DOI: 10.1039/D0LC00304B
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
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,
Lab on a Chip Accepted Manuscript
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
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Lab on a Chip

64x39mm (300 x 300 DPI)

DOI: 10.1039/D0LC00304B
View Article Online

Lab on a Chip Accepted Manuscript

Page 8 of 8
Electronic Supplementary Material (ESI) for Lab on a Chip.
This journal is © The Royal Society of Chemistry 2020

Supporting information for:

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
a
Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Illinois, USA
b
Department 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.

S-1
Table of Contents
Table S1. Sequences of the primers used for detecting five equine pathogens S3
Table S2. Sequences of the primers used in control assays S4
Table S3. Recipe for LAMP reaction mix S4
Table S4. Recipe for PCR reaction mix S5
Fig. S1: Off-chip verification of the LAMP assay specificity S5
Fig. S2: On-chip characterization of four LAMP assays S6
Fig. S3: On-chip multiplex detection of equine pathogen DNA S6
Fig. S4: Off-chip PCR for quantification of EHV1 DNA S7
Fig. S5: Real-time PCR reactions of horse nasal swab samples S7
Table S5: PCR quantification results of horse nasal swab samples S8
Fig. S6: On-chip detection of the positive horse nasal swab sample 1 S9
Fig. S7: On-chip detection of the positive horse nasal swab sample 2 S9
Fig. S8: On-chip detection of the negative horse nasal swab sample 1 S10
Fig. S9: On-chip detection of the negative horse nasal swab sample 2 S10

S-2
 Pathogens Assay Sequences Sequence
Streptococcus equi LAMP F3: AAA ACT AAG TGC CGG TGC
subsp. equi B3: GAG GCG CCT TTT AGA AGA
(S. equi) FIP: TAC GAC TAA CCT CAG AGT TCG CTA TCA GTA TTA GTT GCA ACA AGT G
BIP: CGA CTC CAA GAT TAT CGC GTG ATT GAA CTT TTT GGG CTG ATG A
Loop F: ACA GTT GTC CCT CCC AAC A
Loop B: GCG ATA TAG CCA TAA GTG GAG ATG
Streptococcus equi LAMP F3: AAA GAC CCT CAT GGG AAA T
subsp. zooepidemicus B3: CCT TAG TTG CCG CAT AGG
(S. zoo) FIP: CCT GAC TAA CCA AAT ATA AGC CCT TGA GCT GGA CGA TAA GAC CT
BIP: TGT TGG ACG TAT TTT GGT TGC TCT TCT GAG CCT TCT AAA CCT G
Loop B: GGT GTC ATT ATT AAC ATG GCC TCT
Equine herpesvirus 1 LAMP F3: GGC ATT TAC GTG TGG TCC TT
(EHV1) B3: TCG CGG GCA TTT TTG TAC C
FIP: GTC CAG CAA CGG TGC GTT GTG GCA CGC TCG TTA ACA GT
BIP: CGA GCC TGA AGG GGG AAA ACT GGA GCT GTG TGG AAA GTA GC
Loop F: AGG TTG AGA CGG TAA CGC TG
Loop B: CAC GTG CGT CGT CGC AA
PCR F: GCG CCA GCT GTT TAA CCT TC
R: CGG GCA TTT TTG TAC CAC CG
Equine herpesvirus 4 LAMP F3: CAA GAC GTA ACA ACG GGA GT
(EHV4) B3: CGC AAG TAA CGG CGA TGA
FIP: CGC TCT CCG TTT TCT TCC GAC AAG CCA CCC AGG ATT AGT CAA
BIP: TTA CCC GGA CGG CCT TCC AAC GGG CAT GTC CTC AAC AA
Loop F: GCC TGC TAC TCC GCA TG
Loop B: AGC GTT GTA TAT GAT GCA TCC CCT
Equine influenza virus LAMP F3: ATG CAA TGC TAG GAG ACC
(EIV) B3: AGA AAC TAT CGG CTG ATC C
FIP: TGT CAT ATG GGT AGC AAT TGC TGC AGT ATG AGA ATT GGG ACC T
BIP: GCA TCC TCA GGA ACA TTR GAY TCC GTT TTG AGT GAC AC
Loop F: AAA GCG CTG CTT CTT TCT
Loop B: AGG GAT TCA CAT GGA CAG
Table S1. Sequences of the primers used for detecting five equine pathogens. LAMP primers were designed
for S. equi and S. zoo, targeting the seM and sorD genes respectively.1 The primers for EHV1,2, 3 EHV42 and EIV4
were obtained from literature.

S-3
 Pathogens Assay Sequences Sequence
Escherichia coli LAMP F3: TGA CTA AAA TGT CCC CGG
(E. coli) B3: CGT TCC ATA ATG TTG TAA CCA G
FIP: GAA GCT GGC TAC CGA GAC TCC CAA AAG CAA CAT GAC CGA
BIP: GCG ATC TCT GAA CGG CGA TTC CTG CAA CTG TGA CGA AG
Loop F: GCC GCA TAA TTT AAT GCC TTG TCA
Loop B: ACG CGA AAG ATA CCG CTC T
Equine herpesvirus 3 LAMP F3: AAA ACG GGG ATA ACG AGC
(EHV3) B3: GCG AGA GTA GCT GTT GTG
FIP: CTT CTG CTG TCC GCA CTC AGG ATC TCC GTG GTA CAC C
BIP: GAC CAA CTA CGC GGG TAT CGG ACA ATG AGG CCG ATG AG
Loop F: CTC ATC TTG GGA GTG TGT CTC
Loop B: GGA CCG GAC ACG AGA ATG
Scrambled primers LAMP F3: AGC TTC ATT ATT AGA CCA CTT GC
with no target (SC) B3: GTC TAT TAT TCC GAA TCA CG
FIP: GAG TAT CAC ATG TAA GGA CTT CTA ATA CAA CTT CGT CTC AAT TCC
BIP: GTT ACT GTT GGT ACT ACA ACT AGC TCC TGG TTC TTG TTA TAC AAT A
Loop F: ACT CCG GCG TGA GTA GTA AT
Loop B: CGA TGG GTG TGC AGG ACT CA
Table S2. Sequences of the primers used in control assays. The primers for E. coli5 were from literature. The
EHV3 primers were designed to target the glycoprotein G (gG) gene (GenBank: AF081188.1).

Component Final Concentration 20 µL Reaction

10mM dNTPs 1.4 mM 2.8 μL
10X Isothermal Amplification Buffer 1X 2 μL
5M Betaine 0.4 M 1.6 μL
100mM Magnesium Sulfate Solution 6 mM 1.2 μL
12.5X LAMP Primer Mix 1X 1.6 μL
(2.5 μM of F3 and B3, 20 μM of FIP and BIP,
and 10 μM of Loop F and Loop B primers)
8000 units/mL Bst 2.0 Warmstart DNA Polymerase 640 units/mL 1.6 μL
20X Evagreen Dye 1X 1 μL
DEPC-treated nuclease-free water - 1.8 μL
Template DNA - 6.4 μL
Table S3. Recipe for LAMP reaction mix. For on-chip tests, primer mix was deposited on chips separately, and
it was replaced by nuclease-free water in the reaction mix. Betaine was obtained from Sigma Aldrich. Evagreen
dye was from Biotium. Primers were synthesized by Integrated DNA Technologies (IDT). All the other reagents
were from New England Biolabs.

S-4
 Component Final Concentration 20 µL Reaction
2X Luna Universal qPCR Master Mix 1X 10 μL
Forward primer (10 μM) 0.25 μM 0.5 μL
Reverse primer (10 μM) 0.25 μM 0.5 μL
DEPC-treated nuclease-free water - 4 μL
Template DNA - 5 μL
Table S4. Recipe for PCR reaction mix. Primers were synthesized by Integrated DNA Technologies (IDT). The
qPCR Master Mix is from New England Biolabs.

Fig. S1: Off-chip verification of the LAMP assay specificity. The real-time reaction curves of (a) the E. coli
DNA (a positive control for on-chip tests) against all the other primers to confirm that it won’t interact with other
assays; (b) the SC primers (a set of scrambled primers as a negative control for on-chip tests) against all the other
templates to confirm that it will not amplify under good conditions; (c) the EIV plasmids against the other four
equine assay primers. (d) Summary of the specificity of the LAMP assays.

S-5
Fig. S2: On-chip characterization of four LAMP assays using plasmid DNA templates. The boxplots of the
average channel intensities of positive and negative samples are plotted for a) S. equi, b) S. zoo, c) EHV4, and d)
EIV at 1000 copies/μL. Each boxplot is based on eight experimental duplicates.

Fig. S3: On-chip multiplex detection of equine pathogen DNA. The amplified chips and the corresponding
channel intensities are shown for a) S. zoo and b) EHV4 at 1000 copies/μL.

S-6
Fig. S4: Off-chip PCR for quantification of EHV1 DNA. a) The real-time PCR reaction curves of EHV1 plasmid
at log concentrations; b) The PCR standard curve for quantification of EHV1 genome copies.

Fig. S5: Real-time PCR reactions of six horse swab samples. All nasal swabs were collected from healthy horses
and were negative samples originally. Positive samples (Pos. Swab 1~3) have EHV1 viruses spiked in at decreasing
concentrations. Calculated concentrations of EHV1 genome copies in the samples are shown in Table S5.

S-7
Table S5: PCR quantification results of the horse nasal swab samples. Ct: threshold cycle. The numbers of
EHV1 genome copies were computed using the standard curve in Fig. S4:

# 𝑜𝑓 𝐸𝐻𝑉1 𝑐𝑜𝑝𝑖𝑒𝑠 𝐶𝑡 − 37.385

= 𝑒𝑥𝑝 ( )
𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 −3.562

S-8
Fig. S6: On-chip detection of the positive horse swab sample 1. The smartphone pictures taken at a) 65 ℃ and
b) 80 ℃ of the amplified chip and corresponding average channel intensities.

Fig. S7: On-chip detection of the positive horse swab sample 2. The smartphone pictures taken at a) 65 ℃ and
b) 80 ℃ of the amplified chip and corresponding average channel intensities.

S-9
Fig. S8: On-chip detection of the negative horse swab sample 1. The smartphone pictures taken at a) 65 ℃ and
b) 80 ℃ of the amplified chip and corresponding average channel intensities.

Fig. S9: On-chip detection of the negative horse swab sample 2. The smartphone pictures taken at a) 65 ℃ and
b) 80 ℃ of the amplified chip and corresponding average channel intensities.
S-10
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