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A simple cassette as point-of-care diagnostic

device for naked-eye colorimetric bacteria
Cite this: Analyst, 2014, 139, 482
detection†
Mohammadali Safavieh,a Minhaz Uddin Ahmed,ab Esen Sokullu,a Andy Ng,a
Liliana Braescuac and Mohammed Zourob*d

Received 1st October 2013
Accepted 19th October 2013
DOI: 10.1039/c3an01859h
www.rsc.org/analyst
Effective pathogen detection is necessary for treatment of infectious diseases. Point of care (POC) devices
have tremendously improved the global human heath. However, design criteria for sample processing POC
devices for pathogen detection in limited infrastructure are challenging and can make a significant
contribution to global health by providing rapid and sensitive detection of bacteria in food, water, and
patient samples. In this paper, we demonstrate a novel portable POC diagnostic device that is simple to
assemble for genetic detection of bacterial pathogens by isothermal DNA amplification. The device is
fabricated with very low production cost, using simple methods and easy-to-access materials on a
flexible ribbon polyethylene substrate. We showed that the device is capable of detection of 30 CFU
mL
1 of E. coli and 200 CFU mL
1 of S. aureus in less than 1 hour. Through numerical simulations, we
estimated that the device can be extended to high-throughput detection simultaneously performing a
minimum of 36 analyses. This robust and sensitive detection device can be assembled and operated by
non-specialist personnel, particularly for multiple bacterial pathogen detections in low-resource settings.

immunoassays in the LFT format. Although these LFTs have

Introduction
Infectious diseases account for one third of patient deaths
around the world.1 Most of these diseases are easily treatable by
current medications if they have been diagnosed properly at the
right time. Modern medical diagnostics are the cornerstone of
global health and have revolutionized medicine by providing
rapid, accurate and easy-to-use bedside tests.1,2 It is estimated
that medical diagnostics represent only 3–5% of the total health
care cost, yet their contribution to correct health care decisions
reaches beyond 70%.3 Their signicant impact has led to
improvement of health care, particularly in areas with poor and
low resources such as villages in developing countries. Despite
the tremendous achievements in development of diagnostic
devices, most bacterial pathogen detections still rely heavily on
culturing and immunoassays laminar ow tests (LFTs). A
signicant number of recent POC devices adopt the
a
Institut national de la recherche scientique, Énergie, Matériaux et
Télécommunications (INRS-EMT), Université du Québec, 1650 Boul. Lionel Boulet,
Varennes, Québec J3X 1S2, Canada
b
Faculty of Science, University of Brunei Darussalam, Jalan Tungku Link, Gadong BE
1410, Brunei Darussalam
c
Department of Computer Science, West University of Timisoara, Blvd. V. Parvan 4,
Timisoara-300223, Romania
d
Center of Biomedical Engineering, Vincent Building, Craneld University, Bedfordshire
MK43 0AL, UK. E-mail: m.zourob@craneld.ac.uk; Tel: +44 (0)1234 758318
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3an01859h
provided rapid and accurate results, they are still facing major
shortcomings in bacterial pathogen detection such as the lack of
sensitivity and specicity.4 On the other hand, the advancement
of polymerase chain reaction (PCR) techniques has provided both
high sensitivity and specicity, which revolutionized genetic and
molecular diagnostics. Several POC devices have been developed
using PCR and RT-PCR-based pathogen identication.5–9 Never-
theless, major barriers have prevented widespread application of
these devices. Namely, POC applications of PCR-based detections
need to overcome the requirement of costly thermal cyclers,
engineering difficulties on heat transfers and sample contami-
nation. In order to circumvent the problems associated with
traditional PCR, isothermal amplications such as LAMP,10
RPA,11 HDA,12 and RCA13 have been introduced for POC tech-
nologies. These new nucleic acid amplication techniques do not
require complex instrumentation while providing comparable or
even higher sensitivity and specicity to traditional PCR.
However, some of these techniques either are not sensitive or due
to low specicity require elaborate techniques to increase speci-
city. In contrast, LAMP is a robust, highly sensitive and specic
technique that amplies target DNA in less than 1 h.14
Various technologies have been developed in the past decade
in order to constrain and manipulate minute volumes of liquid
within a microuidic chip.15 Microuidics has recently received
a lot of attention in POC technology since it provides the ability
of sample purication and its integration with the detection
system. Enclosing the entire analysis within a microuidic

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system can also reduce the potential of contamination and

human error as well as minimize the consumption of patient
samples.16–18 However, despite having extraordinary benets in
POC technology it has several drawbacks. Microuidic systems
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generally require costly and time-consuming fabrication

processes inside dust-free cleanroom facilities. Several efforts
have been made by Vullev and his group to fabricate micro-
uidics without using cleanroom facilities.19–21 Although most
of the elastomers used are inherently inexpensive, the processes
which make them compatible for device function signicantly
increase fabrication cost.22 These difficulties make the fabrica-
tion technology difficult to implement in developing countries Fig. 1 Overview of cassette device and its components. (A) Schematic
of the cassette. The cassette consists of two aluminium reels to
and low-resource areas.
provide and collect samples, a heat controller and a heater to provide
Currently, clinical diagnostic laboratories and hospitals are precise temperature for amplification and a flexible substrate where
overloaded with a large number of samples and tests to be the chambers are fabricated. (B) Image of the cassette device. (C)
performed. Modern technologies such as optical detection,23 Image of the flexible substrate.
electrochemical sensing,24 and mass spectroscopy identica-
tion25 have led to increased throughput, sensitivity and reli-
ability in diagnostic technology. However these technologies Cassette assembling and operation
require additional costly and complex instruments such as The cassette comprises of two collector and provider reels with a
electrodes or optical chips. Most importantly, these techniques diameter of 150 mm and a thickness of 11 mm and a exible
require specic readers for signal processing and interpreta- substrate. The collector provider is connected to an electric
tion. Various biochip platforms have been introduced recently heater and a controller unit to provide precise temperature
based on genomic amplication for bacteria detection from (66  C). A 5 mL bacteria real sample with a 20 mL LAMP master
samples to results, using optical detection,23 electrochemical mix was thoroughly mixed initially and applied to each reservoir
detection,26,27 and integration with microarrays followed by of the exible substrate. Then, the chambers were covered by
image analysis.28 However, they have high fabrication cost, are tape and the ribbon was rolled into the collector reel for
integrated with optical/electrochemical readers and are not subsequent DNA amplication. Aer the amplication process
high throughput. Consequently they are not suitable for is over, the ribbon is extended to visualize the assay’s result.
multiplex analysis. In contrast, colorimetric detection does not Fig. 1 shows the schematic of the cassette’s operation.
require any reader. Furthermore, interpretation of data is
straightforward. For example, a number of colorimetric dyes Flexible substrate fabrication
such as hydroxy naphthol blue (HNB),29 SYBR Green,30 calcein,31
A exible substrate was fabricated as shown in Fig. 2 and
and propidium iodide32 have been used to indicate positive
provided reservoirs with a uniform volume of 35 mL each. A
signals generated by genetic-based detection. A POC device that
polyethylene strip was used as a substrate with a width of
can simultaneously analyze multiple samples with high sensi-
tivity and specicity with easy-to-interpret results will meet the
current clinical diagnostic demand.
Here, we develop a novel POC device using genetic-based
bacterial detection and colorimetric read-out. The device
employs LAMP for specic bacterial pathogen detection. Using
HNB and calcein dyes, positive LAMP detection is indicated by an
unambiguous color change. The entire analysis is performed and
enclosed upon a exible plastic substrate in a reel-to-reel cassette
format that is readily available. We rst demonstrate the
assembly of the cassette and fabrication of the chambers inside
the exible substrate. Colorimetric LAMP assays enclosed in the
cassette are then performed for E. coli as a Gram-negative model
and S. aureus as a Gram-positive bacteria real sample. Finally,
using numerical simulation, we have studied heat transfer and Fig. 2 Fabrication of a flexible substrate. (A) The flexible substrate
temperature analysis around the heater reel to estimate the consists of three layers of polyethylene ribbons and double-sided
possible number of sample analyses simultaneously. tapes. (B) The plastic ribbons are attached on top of each other using
double-sided tape. (C) The ribbon was punched to provide reservoirs
with uniform dimensions. (D) The residues at the bottom layer were
Materials and methods removed to increase the flexibility of the ribbon. (E) A white paper was
attached at the bottom of the ribbon in order to enhance visualization
E. coli and S. aureus cultures were freshly prepared by overnight of the assay’s result. (F) After applying the samples to each reservoir,
culturing (12 hours) in 2% LB broth media and stored at 4  C. samples were covered using tape.

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10 mm and a thickness of 3 mm. Three layers of polystyrene
strips were cut and attached on top of each other using double
sided Kapton® tape. One-sided tape was used as the bottom
layer and white-colored paper was used to enhance the obser-
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detection is an easy-to-use, low-cost and qualitative technique
to conrm the presence of DNA amplicons. In this work, we
used HNB, which develops a purple color in the presence of
Mg2+. In the amplication process, a signicant amount of

vation of assay results. insoluble magnesium pyrophosphate is produced which causes

major reduction of Mg2+ in the solution. This reduction
LAMP reaction changed the color of HNB from purple to blue.29 We have added
25 mL of LAMP solution including 5 mL of bacteria sample with
A LAMP protocol was used to amplify the Tuf gene of E. coli and
concentrations ranging from 3  108 CFU mL
1 to 30 CFU
amplicons are detected using hydronaphtol blue (HNB) dye.
mL
1. Aer 1 h of DNA amplication, the ribbon was expanded
The protocol was optimized in order to be compatible with
to visualize the result. We could detect E. coli at a concentration
primers used in our assays. 20 mL of master mix composed of
as low as 30 CFU mL
1. The advantage of this process is that it
3.2 mL of 0.6 mM betaine, 2.5 mL of 10 Thermopol buffer, 1 mL
does not require any reader/accessories and all the data can be
of Bst Polymerase (1600 units), 8000 U mL
1, 1.25 mL of 5 mM
interpreted by the naked eye. Fig. 3 shows the result of the
MgSO4, 0.6 mM concentration 0.6 mL of 0.6 mM dNTP, 0.15 mL of
colorimetric assay. In order to perform more precise analysis,
120 mM HNB, 0.25 mL of 0.2 mM outer primers (F3 and B3), 2.0 mL
samples were extracted from the reservoirs and UV light
of 1.6 mM inner primers (FIP and BIP) and 1.0 mL of 1.0 mM loop
absorbance was conducted. We have scanned UV light in
primers (LF and LB).
various wavelengths ranging from 230 nm to 700 nm. As shown
An optimized LAMP protocol was used to amplify the Mcat
in Fig. 3B, there was a major difference in absorbance at 655 nm
gene of S. aureus. All materials and concentrations are identical
between the negative control and positive samples. This
to the E. coli Tuf gene amplication except that we used 0.75 mL
of 3 mM of MgSO4, 0.4 mL of 0.4 mM dNTP, 3.125 mL solution of
200 mM calcein and 4 mM MnCl2. For post processing sample
analysis, a NanoDrop 2000 spectrometer (Thermo Scientic,
Delaware, USA) and a NanoDrop 3300 (Thermo Scientic) were
used. Table S1 from ESI† shows the primer sequences of the
LAMP reaction for Tuf gene amplication in E. coli and Mcat
gene for S. aureus, respectively.

Finite element method modeling

A 3D time dependant heat transfer numerical model was
implemented with a nite element method (FEM) using COM-
SOL Multiphysics 3.5 (MA, USA). The time-dependent temper-
ature proles at the surface of different layers were computed
using the general heat transfer module for a 60 min period.
Boundary conditions were set as follows: for reel perimeter T ¼
66  C; far from the cassette TInf ¼ 25  C. Internal boundary
conditions were set based on continuity of heat transfer
between different layers and environments. The heat transfer
simulation was run for various ribbon rounds around the
heater.

Results
Colorimetric detection of E. coli
Detection of bacteria in real samples based on genomic
amplication has been challenging due to the multi-step
process required: cell lysis, DNA extraction, purication and
amplication.33 In our previous study,34–36 we have shown that
we could detect E. coli bacteria in various media without puri- Fig. 3 E. coli detection assay on a flexible substrate. (A) Colorimetric
cation by lysis and amplication of the target DNA in one assay of E. coli using HNB dye. Various analyses were conducted for
microuidic reaction chamber. Subsequent detection of DNA different E. coli concentrations ranging from 30 to 3  108 CFU mL
1.
amplicons was detected electrochemically. Despite providing a The limit of detection (LOD) was found to be 30 CFU mL
1. The
positive samples are blue and negative tests are purple. The specificity
rapid, highly sensitive chip for bacteria detection, it has limi-
of the assay was tested using S. aureus DNA as the negative control. (B)
tations for high throughput analysis of many samples in the UV absorbance scanning of different samples from 620–700 nm. (C)
low-income budget countries due to multistep operation as well UV peak absorbance was measured at 650 nm to show the difference
as high fabrication cost. On the other hand, colorimetric between negative and positive samples.

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Paper Analyst

difference is due to the color change of HNB from purple to blue

as well as the increased turbidity in the samples containing
DNA amplicons (Fig. 3C). Since the chambers are of adequate
size, direct observation by the human eye is straightforward
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without any accessories, allowing unambiguous distinction

between negative and positive results. The assembly and, in
particular, the operation of the device is also very easy.
Personnel without specialized skills would appreciate the
simplicity of the device’s construction and its ease of operation.

Colorimetric detection of S. aureus

Detection of the Gram-positive bacteria S. aureus requires a
more rigid lysis technique since Gram-positive bacteria are
more resistant to lysis in comparison to Gram-negative bacteria.
Lysis techniques for Gram-positive bacteria include chemical,
thermal, electrical and mechanical.37,38 Chemical lysis is a low-
cost method but a post-processing step is required to remove
the inhibitor agents from the lysis solution, which complicates
the subsequent amplication process within the same
chamber. Electrical and mechanical techniques require expen-
sive apparatus as well as costly fabrication processes and
therefore are not suitable for a low-cost system. On the other
hand, thermal shock lysis is simple, rapid, effective and easy to
use. Thermal shock lysis does not require any extra instru-
ments. It can employ the same heater used for the amplication
process already integrated to the device. To exploit this process,
we loaded 5 mL of S. aureus with different concentrations in each
chamber and covered the ribbon with tape. The chamber was
then rolled into the collector reel and was heated for 5 min at
90  C. Subsequently the samples were rolled back, uncovered
and 20 mL of LAMP solution was applied to the chamber and
mixed properly by pipetting and covered by tape again. The
samples were rolled into the collector and heated at 65  C for
DNA amplication by LAMP. Calcein dye was used for qualita-
tive detection, which changed from yellow to green through
uorescence emission in the presence of amplicon. Fig. 4 shows
the colorimetric assay results for various S. aureus concentra-
tions ranging from 2  108 to 200 CFU mL
1. Fig. 4 S. aureus detection assay. (A) Colorimetric detection of
With this technique, we could detect 200 CFU mL
1 of S. aureus using calcein. The LOD is 200 CFU mL
1. The specificity of
the assay was tested with E. coli DNA as the negative control. (B)
S. aureus in real samples without any pretreatment in less than
Fluorescence emission scan from 500–590 nm. (C) Peak fluorescence
1 h. Further analysis of the samples was conducted by uores- emissions are measured for different S. aureus concentrations and
cence measurement due to uorescence emission of the calcein negative controls.
in positive samples.
We scanned uorescence emission at various wavelengths
between 500 and 580 nm. As shown in Fig. 4B, there is a marked
Understanding the heat transfer in the provider reel is key to
difference in uorescent emission at 510 nm. This difference
obtain more details on the device’s performance for further
can be observed in Fig. 3C when comparing the negative
optimization. We performed numerical simulations to under-
controls and various samples with different concentrations of
stand the heat transfer and temperature proles around the
S. aureus bacteria. The specicity of the assay was also examined
aluminum reel and the exible substrate.
using E. coli and it was conrmed that the LAMP primers used in
From this information, we can determine the number of
this study are highly specic to the target Mcat gene of S. aureus.
rolls of the ribbon that can be rolled around the aluminum reel.
Aluminum reel (thermal conductivity K ¼ 205 W m
1 k
1,
Heat transfer effect on cassette reel density r ¼ 2700 kg m
3, heat capacity Cp ¼ 897 J kg
1 K
1) acts
The LAMP method operates in the temperature range between as the source of the heat. Polyethylene ribbons (K ¼ 0.42 W m
1
60  C and 66  C. When samples in the chambers reach this k
1, r ¼ 900 kg m
3, Cp ¼ 2307.2 J kg
1 k
1) and the air (K ¼
temperature range the amplication process is initiated. 0.0257 W m
1 k
1, r ¼ 1.205 kg m
3, Cp ¼ 1005 J kg
1 k
1) were

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Analyst
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Fig. 5 Numerical heat-transfer simulation around the cassette reel
and ribbon. (A) 3D time-dependent heat transfer simulation was per-
formed for different rounds of ribbons around the heater reel. The
heater can provide heat to maintain a minimum of 60  C for at least
50 min, required for amplification and colorimetric detection. (B)
Temperature profile around the heater and ribbon.
set as the environment materials. 3D time-dependant heat
transfer FEM modeling was carried out for various ribbon cycles
with a 3 mm thickness for each round, to understand whether
the heater can provide enough heat to reach 60–66  C for a
minimum of 50 min. Fig. 5 shows the result of heat transfer
simulation in the cassette. Temperature proles at the surface
of each layer have been calculated with respect to time for
60 min. For each prole, a transient time is required to reach
the steady state temperature at the surface. For the rst layer,
the transition time is very short (2 min). By adding the number
of layers, the transition time increases and eventually reaches to
the level that cannot provide 50 min consistent time in the
amplication temperature range. This trend is shown in Fig. 5A.
We have found that the heating element can provide enough
heat for 3 rounds of ribbons, equivalent to at least 36 sample
lyses and amplications.
Conclusions
In this paper, we have demonstrated a novel, easy-to-fabricate,
easy-to-assemble diagnostic device capable of bacteria and
pathogens detection with high sensitivity and specicity. This
device can detect Gram-negative bacteria as well as a number of
Gram-positive bacteria with the same characteristic as S. aureus.
This device does not require any expensive reader to process and
interpret results. Since the device requires only one temperature
486 | Analyst, 2014, 139, 482–487
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Paper
controller, manufacturing cost is extremely low. This device can
be integrated with an electricity free heating cartridge39,40 and is
particularly attractive for bacterial pathogen detection in low-
resource areas. In addition, this approach can also be imple-
mented in a high-throughput manner, with high potential to
meet the increasingly heavy demand for disease diagnostics in
large health care centers. However, several problems need to be
considered in order to be applied as POC devices. Usually a real
sample does not have a pure sample of bacteria but instead, a
mixture of various species/strains. If the assay predetermines
the target species/strain based on the primer design, then DNA
from other species/strains present in the sample, are not
susceptible to amplication by the specic primers used.
Therefore, the pathogens will be undetected which could be a
dangerous approach for the clinician. A potential solution to
this would be using multiple primers to target as many possible
species/strains that could be present in the sample. However,
then the cost of assay will go higher and this should be
considered for POC applications in resource-depleted areas.
Acknowledgements
The authors wish to acknowledge National Science and Engi-
neering Council of Canada (NSERC) for nancial support. M.S.
wishes to thank the ISS NSERC-CREATE fellowship and Fonds
de Recherché du Québec – Nature et Technologies (FQRNT)
doctoral scholarship. L.B. thanks West University of Timisoara
for providing access to the soware facilities.
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