Microfluidics For Rapid Detection of Live Pathogens

Review
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Microfluidics for Rapid Detection of Live Pathogens

Carla Spatola Rossi, Frederic Coulon, Shaohua Ma, Yu Shrike Zhang, and Zhugen Yang*
year (WHO, 2020). Rapid, sensitive, and

Rapid, sensitive, and selective detection of live pathogens remains a key species-specific detection of pathogens is a
priority for quality control and risk assessment. While conventional methods key component for preventing the spread
often require complicated workflows, costly reagents, lab equipment, and of common diseases. Traditional methods
are time-consuming, rendering them inadequate for field testing and low- for pathogen detection include cell cul-
ture methods and metabolic assays, and
resource settings. Increased attention has been drawn to developing alternative
molecular techniques such as polymerase
low-cost and rapid methods to detect on-site live pathogens in different envi- chain reaction (PCR) and enzyme-linked
ronmental matrices. Among them, microfluidic devices that integrate various immunosorbent assay (ELISA).[1,2] These
laboratory functions in a miniaturized manner have proven to be a promising techniques are well-established and used
tool for the rapid and sensitive detection of pathogens. Herein, the develop- as routine methods in laboratories world-
ment of microfluidic devices specifically designed for the detection of live wide; however, they have significant draw-
backs with respects to their practicality
pathogens is discussed along a concise summary of novel microfluidics sys-
and functionality (Table 1).
tems recently developed, contrasted to conventional methods regarding assay Culture techniques and metabolic
time, the limit of detection, and target organisms. These include a variety of assays are considered the golden standard
micro total analysis systems (µTAS) and microfluidic paper-based analytical for the detection of pathogens and diag-
devices (µPADs) in combination with molecular methods and traditional nosis; nevertheless, these assays can take
days or even weeks to obtain conclusive
live cell detection techniques, such as cell culture, DNA intercalating dyes,
results and are regarded as time-con-
resazurin, and immobilized bioreceptors (e.g., aptamers and capture anti- suming and labour-intensive methods.[3]
bodies). Furthermore, insights on the future perspectives of microfluidics for Furthermore, only a very small percentage
live pathogen detection with a highlight on the rapid and low-cost method of organisms are culturable in labora-
development for field testing are provided. tory conditions whilst the vast majority
enter an unculturable state, leading to
false-positive results that can have grave
implications in relation to public health.[4,5] On the other hand,
1. Introduction
molecular techniques such as PCR and ELISA provide fast test
Infectious diseases are placed in the top ten leading causes of results and species-specific diagnosis but are incapable of dis-
death worldwide, accounting for over four million deaths each tinguishing live from dead pathogens; a major limitation in
many fields such as water quality monitoring, food safety, and
medical diagnostics.
C. Spatola Rossi, F. Coulon, Z. Yang For instance, the detection of live pathogens is crucial for the
School of Water, Energy and Environment monitoring of drinking water quality and ensuring the safety of
Cranfield University a water source. Due to insufficient water networks, millions of
Cranfield MK43 0AL, UK
E-mail: [email protected] people worldwide lack access to clean potable water. It is, there-
S. Ma fore, crucial to have fast and sensitive techniques that can selec-
Tsinghua Shenzhen International Graduate School tively detect viable organisms and identify potential pathogens.
Tsinghua University Only live microorganisms pose a threat to human health, thus
Shenzhen 518055, China selective detection of viable organisms is essential to assess the
Y. S. Zhang safety of a water source and avoid false positive results. To this
Division of Engineering in Medicine
Department of Medicine
end, culturing assays are carried out on a regular basis to iden-
Brigham and Women’s Hospital tify possible microbial contaminants in water.[6,7]
Harvard Medical School In the food industry, both production surfaces and food
Cambridge, MA 02139, USA samples are routinely analyzed in order to assess the produc-
The ORCID identification number(s) for the author(s) of this article tion hygiene and safety of the food products. Approximately
can be found under https://doi.org/10.1002/adfm.202212081. 250 foodborne diseases have been identified so far and it is esti-
© 2023 The Authors. Advanced Functional Materials published by Wiley- mated by the Center for Disease Control (CDC) that each year
VCH GmbH. This is an open access article under the terms of the Crea- 48 million people suffer foodborne illnesses. Microbial testing
tive Commons Attribution License, which permits use, distribution and in every step of food production is therefore a major concern
reproduction in any medium, provided the original work is properly cited.
for the food industry and an essential component in reducing
DOI: 10.1002/adfm.202212081 and eliminating foodborne illnesses. Methods that offer rapid,
Adv. Funct. Mater. 2023, 33, 2212081 2212081 (1 of 17) © 2023 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH
16163028, 2023, 21, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adfm.202212081, Wiley Online Library on [30/06/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
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Table 1. Comparisons of traditional cell culturing techniques, metabolic assays, PCR, and ELISA for pathogen detection.
Live cell detection Specificity Sensitivity Assay cost Assay time Assay procedure
Cell culture Yes Can be genus- or species-specific, −1 High 24 h–5 days, subject to the Complex
High (1 CFU mL , depen-
subject to the selective dant on incubation time) organisms generation time and
culture medium used any further assays to confirm
organism’s genus or species
Metabolic Yes Can be genus or species Moderate (10–106 CFU mL−1, Moderate 1–24 h (typically 4 h of incubation Simple
assays specific, subject to the selective dependant on previous with the reagent of choice)
culture medium used enrichment step)
PCR No Can be genus- or species-specific High (1–10 CFU mL−1) Moderate 2h Simple
ELISA No Can be genus- or species-specific Moderate (10–106 CFU mL−1, Moderate 2h Simple
dependant on previous
enrichment step)
sensitive, and specific detection of microbial pathogens are in highly sensitive and specific technique is of the utmost impor-
high demand, as conventional cell culturing techniques take tance to replace current methodologies.
days to weeks to complete. More importantly, only live micro- As an alternative to conventional techniques, microfluidic
organisms pose a threat to human health, thus selective detec- devices have been developed that incorporate many of the
tion of viable organisms is essential for providing an accurate traditional detection mechanisms such as cell culture, DNA
assessment of food products and hygiene standards. intercalating dyes and metabolic reactions, and that integrate
In the medical field, detection of live pathogens is also essen- the key assay steps into a single operational chip.[12] In recent
tial whilst carrying out regular hygiene tests to comply with years, microfluidics has quickly gained momentum for point-
surface cleanliness standards[8,9] and during drug susceptibility of-care (POC) applications, due to its ability to miniaturize and
testing for diagnostic purposes. With regard to clinical diag- significantly simplify a large number of laboratory processes.
nostics, it is of the utmost importance to utilize a technique Furthermore, the easy and cost-effective production of micro-
that can accurately and selectively detect viable pathogens to fluidic devices, particularly noticeable by paper-based microflu-
provide an accurate diagnosis, allowing medical professionals idics, has proven a key advantange in their application for field
to correctly prescribe and administer the necessary medical testing in resource-limited settings.
treatment. Due to the emergence and rapid spread of antimi- In this review, we will provide a summary of the microflu-
crobialresistant bacteria originating from overuse and misuse idic sensors that have been developed so far for live pathogen
of antibiotics worldwide, accurate diagnosis through drug sus- detection and critically discuss how they compare to conven-
ceptibility testing has become imperative to prevent the spread tional methods with regard to assay time, limit of detection,
of these dangerous organisms.[10] Furthermore, as only viable and target organisms. To the best of our knowledge, this is the
organisms are able to multiply and spread in the environment first review paper on sensors for live pathogen detection, which
and through populations, it is necessary to develop techniques is focused specifically on microfluidic devices.
that can specifically detect live pathogens to control disease out-
breaks and avoid large-scale epidemics.
An overestimation of microbial contamination can lead 2. Conventional Techniques used for Detection of
to unnecessary wastage of food and drinking water, incorrect
Live Pathogens
assessments of production and surface cleanliness, and erro-
neous clinical diagnosis. It is thus essential to employ a tech- 2.1. Culture-Dependant Techniques
nique that can selectively detect live pathogens, as opposed
to the total number of cells, presence of DNA molecules, Despite several drawbacks, such as the inability to detect non-
or protein detection that are not specific indicators of viable culturable bacteria as well as time-consuming and labour-inten-
organisms. sive protocols, culture-based microbiological assays remain the
To accurately distinguish live from dead microorganisms, golden standard for live pathogen detection due to their high
biomarkers such as cell membrane integrity, metabolic activity, sensitivity, specificity, and quantitative capabilities. Several
and RNA synthesis have proven to be effective for cell viability commercially available tests that detect microbial indicators,
testing.[11] Many culture-based and molecular-based techniques such as Escherichia coli (E. coli), are based on cell culturing
are currently available for live cell detection. Nevertheless, there methods. They employ colony-counting techniques, most prob-
is still a need to develop new techniques that can provide auto- able number (MPN), and Presence/Absence assays.[13] These
mation of the workflow, low assay costs, and have the potential techniques rely on the use of specific growth media to selec-
to be utilized for field testing or employed in locations where tively culture and isolate pathogens of interest. The detection
there is no access to laboratory infrastructure. Whether for food of live cells is carried out by growing colonies on solid media,
and water quality analysis, early warning systems for disease measuring the optical density of a liquid culture, or directly
outbreaks, or clinical analysis and drug susceptibility testing, a counting cells using microscopy.
These methods require days or even weeks to produce is based on the entry of the dyes in cells with compromised
results, depending on the ability of the pathogen to form visible membranes, light activation of the intercalating dyes, and sub-
colonies, and while a positive culturing result can be used as sequent irreversible binding of the dyes to the DNA molecules.
conclusive evidence of living organisms, an unsuccessful cul- This technology can be coupled with amplification techniques
turing cannot be used as proof of the absence of live cells in a such as PCR, where the DNA bound to the dyes can no longer
sample.[11] The problem of detecting viable but non-culturable be amplified by DNA polymerase, thus enabling exclusive
bacteria (VNCB) remains one of the most significant chal- detection of DNA contained in cells with intact membranes.
lenges, which may cause false-negative results and dangerous
consequences in clinical diagnostics (e.g., drug susceptibility
testing). Despite being common practice worldwide, very few 2.2.2. Metabolic Activity
of these culturing tests are suitable for low-resource settings
as they require expensive laboratory facilities, and trained per- In metabolically active cells, a number of chemical reactions
sonnel can take days or even weeks to produce results, making occur that can be detected through fluorescent, lumines-
them impractical for specific tasks such as routine water quality cent, or colorimetric means. Adenosine triphosphate (ATP) is
monitoring or for rapidly tracing disease outbreaks.[14] a biomarker that is commonly used for viability testing. ATP
synthesis ceases upon cell death and can therefore be used to
determine the presence of live cells in a sample. Luciferase
2.2. Culture-Independent Techniques enzymes use ATP as a substrate to produce bioluminescence
that can be detected and measured to quantify the number of
Aside from traditional cell culturing techniques, there are live cells in a sample.[21] This method is fast and effective; how-
also numerous other molecular-based methodologies that are ever, exogenous, or non-microbial sources of ATP can lead to
termed culture-independent methods, which are commonly the overestimation of live cells and species-specific detection
used to differentiate live from dead cells. They rely primarily is not possible. With regard to practical considerations, biolu-
on assessing cellular integrity, measuring metabolic activity, minescence detection requires expensive equipment, costly
and detecting RNA.[15] Due to the wide range of cell types and reagents, and trained personnel, and is consequently not well-
sample matrices, there is not a single universal technique that suited for resource-limited settings. Other methods to detect
can be applied to all cases. In terms of practicality, cost-effec- cell respiration include the use of tetrazolium dyes to measure
tiveness, sensitivity, and selectivity, each method has its advan- dehydrogenase activity, and resazurin dye to measure cellular
tages and limitations, and therefore particular attention should oxidoreductase activity. These techniques detect metabolic
be taken when deciding which method to utilize.[16] activity of the cell and thus do not allow the specific identifica-
tion of the live organisms in a complex environmental sample,
they only act as indicators of total live cells.
2.2.1. Membrane Integrity The ability of a cell to multiply is also used as a marker for
live cells. Isotopes such as 13C, 15N, and H218O can be incor-
Membrane integrity is a biomarker commonly used to deter- porated into DNA by metabolically active cells and detected in
mine the viability of cells in a sample. Cells that have lost the a method termed Stable Isotope Probing (SIP).[22] DNA-SIP is
integrity of their membrane and allow passage of otherwise the technique most commonly used, although depending on
non-permeable molecules are considered dead or non-viable. the application and the phylogenetic information one is looking
Fluorescent dyes are widely utilized in various ways to eval- for, DNA, RNA, phospholipid fatty acid, and proteins can be
uate the movement of molecules through membranes and used as targets of SIP labelling.[23] Since only metabolically
assess cell viability.[16] Two types of fluorescent dyes are used active cells that are replicating their DNA are capable of incor-
for cell membrane integrity determination: a) dyes that can porating the labelled isotopes, live microbes can be separated
only permeate compromised cell membranes or b) dyes that from dead and unlabeled microbes. Labelled DNA is separated
can permeate both intact and degraded cell membranes. The from unlabelled DNA by density gradient centrifugation, pro-
discrimination of viable cells in a sample can be accomplished viding an idea of the amount of metabolically active cells in the
using dual-staining kits such as the LIVE/DEAD BacLight Bac- sample.[24] DNA and RNA SIP is often followed by a sequencing
terial Viability kit that uses two fluorophores; one that can per- approach to identify the live cells in the sample. Asides from
meate viable cells and another that can only permeate cells with labelling DNA, proteins can also be labelled by using artificial
compromised cell membranes.[17,18] amino acids in a technique called bioorthogonal noncanonical
The use of fluorescent DNA-intercalating dyes coupled amino acid tagging (BONCAT). These artificial amino acids
with a DNA-amplification method such as PCR or loop-medi- can later be tagged with different reporters, such as fluoro-
ated isothermal amplification (LAMP), has also proven to be an phores and used to detect protein synthesis, a process that can
effective method for detecting live or dead organisms.[19] The only occur in metabolically active cells. BONCAT followed by
method can be applied to detect multiple types of pathogens, rRNA-targeted fluorescence in situ hybridization (FISH),[25]
such as bacteria, viruses, protozoa, and fungi. The technique fluorescence-activated cell sorting (FACS),[26] and 16S rRNA
consists of using DNA-intercalating dyes that can only per- gene sequencing is used to identify live cells in a sample.
meate compromised cell membranes and bind to the DNA. The This method has been used to measure metabolically active
most commonly used intercalating dyes are propidium mono- cells in heterogeneous matrixes such as soils[27] and marine
azide (PMA) and ethidium monoazide (EMA).[20] The technique sediments.[26,28]
In addition to the techniques mentioned above, metabolic detection components that can be integrated into each type of
activity of a cell can also be detected by measuring heat flow. device.
Physical, biological, or chemical processes in the cell are accom- The concept of µTAS was first introduced in 1990, which
panied by heat production or consumption, making heat flow a consisted of a liquid chromatograph on a silicon chip that inte-
good biomarker for viable cells. Isothermal microcalorimetry grated sample pre-treatment, separation, and detection.[39] Cur-
(IMC) using commercial isothermal microcalorimeters can be rently, polydimethylsiloxane (PDSM) is the most widely used
used to measure heat production. substrate for µTAS, however, they can be made using several
different materials such as silicon, glass, quartz, and other
polymers, and the microfluidic channels are typically fabri-
2.2.3. RNA Detection cated by etching and photolithography. The main body of the
device that contains the microfluid channels is connected to
Cellular RNAs are very unstable molecules that are rapidly external units such as pumps, injectors, droplet generators, and
degraded once outside the cell, making them a more appro- microheaters to achieve sampling and detection.[40] µTAS have
priate biomarker for viable cells than DNA.[29] RNA-based gained increased popularity in recent years and many lab-on-
methods have been used in combination with a variety of dif- a-chip (LOC) devices have been developed to scale down one
ferent techniques, such as qPCR, sequencing, transcriptomics or various lab processes in a single chip format by creating 3D
and. In recent years, the isothermal amplification method, structures in PDMS substrates. The involved detection and
nucleic acid sequence-based amplification (NASBA), has readout systems can vary from electrical signals using screen-
become a popular alternative to RT-qPCR for RNA analysis. printed electrodes, to fluorescent and colorimetric visual detec-
It has been used to determine the viability of pathogens such tion. They can be fabricated to contain different materials in a
as E. coli and Vibrio cholerae in water sources.[30–32] The major single device and etched or 3D printed with channels of various
advantage of NASBA compared to RT-PCR is that genomic shapes and sizes that can precisely control the flow of pico-
DNA present in the sample does not lead to false positive litre to microlitre sample and reagent volumes. Due to these
results; as single-stranded RNA is specifically targeted, DNA capabilities, µTAS have been used primarily for biological and
is not involved in the cycle and there is no need add include chemical analysis, as they enable cell transport, confinement,
DNases to reduce DNA contamination, or include an RNA and culture.[41] Their applications include cellomics, medical
extraction step.[33] This is particularly important in low biomass diagnosis, and environmental monitoring and have also been
samples, as DNase treatments often degrade RNA due to con- applied as microreactors for pharmaceutics. Some of the advan-
tamination with RNases.[34] tages of these devices include the use of low sample and rea-
In recent years a technique termed Molecular Viability gent volumes, decreasing assay costs and assay time, simplified
testing (MVT) was developed that uses nutritional stimulation operations, and increased functionality compared to traditional
to induce pre-rRNA synthesis, followed by an RT-qPCR amplifi- assays. Many efforts have been placed to develop microfluidic
cation step to determine the presence of live bacteria in a given chips that can replace current laboratory-based testing tech-
sample.[16] MVT can be conducted by dividing a sample into niques and can be used as POC testing devices to be applied in
two aliquots stimulating one of the aliquots by adding growth low-resource settings.[42]
media and comparing the synthesis of pre-rRNA molecules to µPADS on the other hand, are manufactured using paper
a control aliquot that has not been nutritionally stimulated. If and wax to create microfluidic channels and contain all of the
an increase in pre-rRNA molecules is seen compared to a con- sampling and detection functions within the main body of
trol sample with no nutrition stimulation, then one can infer the device. They provide cost-effective fabrication, with paper
that there are live bacteria in the sample. This technique was being accessible globally and at low cost, quickly becoming an
first used to detect viable bacteria in water by ratio metric detec- attractive choice for developing POC biosensors for pathogen
tion[35] and was later adopted for studies of complex human detection. Paper-based devices are lightweight and portable, can
samples such as serum[36] and milk.[37] be easily stored, transported, and disposed of by incineration,
leaving behind no contaminants and making them ideal for
testing in the field. These devices present good biocompatibility
3. Microfluidic Platforms for Detection of Live and can contain stored reagents within the paper matrix, mini-
mizing sample and reagent pipetting steps during the opera-
Microorganisms
tional procedure and greatly reducing the technical training
Microfluidics refers to the handling of small amounts of fluids requirements.[43]
that are constrained to sub-millimetre structures. Capillary Paper-based microfluidic systems consist of nitrocellulose
forces allow the passive flow of liquid through channels, thus or cellulose supports that are treated with hydrophobic sub-
enabling the transport of substances through a device, and can stances such as wax, PDMS, and ink to contain hydrophilic
be used to process, separate, or mix fluids, depending on the channels to control the flow of liquid within the devices. This
application. It has proven to be a useful tool for the miniaturi- provides a passive flow of liquid without the requirement of
zation of various laboratory processes in a single chip, reducing pumps or valves that more complex microfluidic devices often
sample size, reagent volumes and greatly simplifying workflows employ.[44] The use of capillary force to move liquid from one
and reducing costs.[38] Microfluidic devices can be grouped section of the device to another offers a power-free fluidic trans-
into two main categories: µTAS and µPADs. They differ in port, ideal for POC testing. µPADs have been developed as 3D
their manufacturing method as well as in the sampling and or 2D structures, depending on the requirement to conduct
liquid flow through different reaction zones and achieve an to bind to the active site of the antibodies or aptamers and thus
integrated detection system. To increase the functionality of dead cells will not generate a detectable signal, allowing for
paper-based microfluidic devices while maintaining a reduced selective identification of live cells. In the past decade, several
size, 3D µPADs have been developed. They are manufac- microfluidic devices have been developed to detect live cells by
tured by stacking pieces of paper one on top of the other or assessing membrane integrity through DNA-intercalating dyes
by folding sections of the paper device in a specific order fol- and immobilized bioreceptors such as antibodies or aptamers.
lowing the practice of origami.[45] These devices provide multi
plexing capabilities; a single inlet can lead to multiple detection
areas where each different target is analysed. Splitting pads 3.1.1. DNA-Intercalating Dyes
and connecting pads that contain specific channels embedded
in the paper allow for a vertical and horizontal flow of liquid One of the first microfluidic devices that included the use of an
that can be guided through the different layers of the device intercalating dye, was developed in 2012 to detect live MRSA
and into the detection zone where colorimetric, fluorescent, or cells.[53] A PDMS and glass microfluidic chip was designed that
electrical detection is carried out. Screen-printed electrodes can included EMA pre-treatment, cell lysis, DNA denaturation, and
be embedded in the paper, and fluorescent probes or dyes are on-chip PCR (Figure 1A). The workflow consisted of incubating
often stored in the paper matrix or added to produce a detect- EMA, the sample, and specific probe-conjugated beads on the
able signal. To quantify results, naked eye analysis is being loading chamber of the chip. EMA would penetrate cells with
replaced by smartphone-based technology coupled with an compromised cell walls and covalently bind to dsDNA, ren-
image processing system that can accurately measure the con- dering it incapable of being amplified by PCR. Using a pump,
centration of the target in a sample. the contents of the loading chip were transported to the PCR
Many µPADs and µTAS have been developed to detect path- reaction chamber where on-chip heaters enabled cell lysis and
ogens from a wide range of matrices, including food sources, posterior denaturation of DNA. ssDNA strands were then able
wastewater, drinking water, and human serum and blood to hybridize with the specific probe-conjugated beads that were
amongst others, and have been used for a variety of applications then isolated using a magnet under the chip. PCR reagents
such as food and drinking water quality control, environmental were added to the chip and on-chip amplification was carried
monitoring, and clinical analysis.[46–52] However, the detection out followed by fluorescence measurements. To carry out the
mechanism typically employed by these devices is often based on-chip PCR reaction, a temperature sensor and microheaters
on DNA amplification or protein detection that are not suitable were employed as additional components of the µTAS. With
biomarkers for cell viability, and therefore cannot be used for this technique, they were able to detect 102 CFU mL−1 in only
live pathogen sensing. 2.5 h.
There is an urgent need to develop innovative microfluidic In line with this mechanism of live cell detection, other
devices that can quickly and sensitively distinguish live from devices were later developed that involved on-chip PMA or
dead pathogens. In the following sections, we will discuss EMA treatment coupled with PCR. A PDMS and glass chip
novel microfluidic systems that incorporate traditional live cell was developed capable of detecting viable bacteria from human
detection techniques for the identification of live pathogens. joint samples in < 1 h and with an LOD of 104 CFU mL−1,
Similarly, to conventional methods, these devices rely on whole- showing promising potential for clinical applications.[54] The
cell detection and cell membrane integrity, as well as utilizing chip contained a wash buffer chamber, a PCR reagent chamber,
metabolic biomarkers and cell culturing to assess cell viability. a reaction chamber, and a series of valves and transport units
They show potential for miniaturization and field testing and to enable the flow of liquid from one chamber to the next. The
have proven to be useful to detect a range of clinically relevant system functioned by preloading the chip with vancomycin-
live bacteria, such as E. coli, Salmonella spp., and Campylobacter coated magnetic beads (these were used as a visual probe due
spp., whilst greatly reducing detection times compared to con- to their ability to bind to bacterial cell walls), washing buffer,
ventional techniques, from weeks or days to up < 1 h in some and PCR reagents. The sample that had previously been incu-
cases (Table 2). bated with EMA was then added to the reaction chamber
where, following a brief period of light exposure to activate the
dye, the vancomycin beads captured the bacteria. After 10 min
3.1. Membrane Integrity of incubation period, bacteria-bead complexes were captured by
placing a magnet underneath the chip and PCR inhibitor were
As previously mentioned, current techniques for live bacteria eliminated by pumping the washing buffer into the chamber.
detection heavily rely on assessing the integrity of the cell Finally, PCR was carried out in the reaction chamber where
membrane. This can be performed by employing DNA-inter- only DNA from live cells (DNA not bound to EMA) was ampli-
calating dyes such as PMA and EMA that selectively permeate fied, with the fluorescent signal measured to determine the
cells with compromised cell membranes, typically followed by presence of viable cells.
qPCR to amplify DNA originating from live cells whose mem- Further optimizations of this chip were done to eliminate
branes remain intact. Membrane integrity can also be assessed the PCR step and instead carry out rapid optical detection using
with the use of antibodies and aptamers selected to specifically gold nanoparticles.[55] Bacteria-bead complexes were captured
bind to antigens present in cell membranes, followed by elec- on the chip, followed by cell lysis and denaturing of dsDNA
trochemical detection of the binding event. If the proteins on that was carried out by increasing the temperature to 95 °C. 16S
the membrane have been denatured, they will no longer be able probe-coated gold nanoparticles were then introduced into the
Table 2. Summary of microfluidic devices developed for live pathogen detection, including the detection technique, the target organism, the limit of
detection (LOD) and the assay time.
Technloigies Targets LOD Assay time References

Membrane PDMS and glass substrate chip integrating EMA Methicillin-resistant Staphylo- 102 CFU mL−1 2.5 h [53]
integrity treatment and on-chip PCR coccus aureus (MRSA)
PDMS and glass substrate chip integrating EMA Gram+ and Gram- bacteria 104 CFU mL−1 55 min [54]
treatment and on-chip PCR found in periprosthetic joint
infection
PDMS and glass substrate chip integrating EMA Pseudomonas syringae, Staphylo- 102 CFU mL−1 30 min [55]
treatment and gold nanoparticle probe or on-chip PCR coccus aureus (S. aureus), E. coli,
Enterococcus sp., MRSA
Microfluic droplet platform and fluorescein E. coli and Salmonella N/A N/A [56]
isothiocyanate (FITC)-labeled aptamers
PMA pre-treatment and LAMP amplification on µPAD E. coli 103 CFU mL−1 2h [57]
PMAxx pre-treatment and LAMP amplification Candida. albicans 104 CFU mL−1 <1 h [58]
on a portable chip
Bacterial capture and on-chip PMA Mycobacterium 100 CFU 90 min [59]
treatment tuberculosis (M. tuberculosis)
PMA pre treatment and nucleic acid lateral flow strip E. coli 8.1 × 102 CFU mL−1 or 81 2h [60]
CFU g−1
Impendance sensor on glass substrate with E. coli and Salmonella spp. 10 CFU mL−1 <1 h [61]
immobalized antibodies
Impendance sensor on glass substrate with 2 Salmonella serotypes 300 cells mL−1 <1 h [48]
immobilized antibodies
Impendance sensor on glass substrate with immobilized 3 Salmonella serotypes 7 cells mL−1 <1 h [62]
antibodies
Metabolic lacZ T4 bacteriophage and paper portable culture device E. coli <10 CFU mL−1 5.5 h [63]
activity
ATP detection on µPAD Salmonella 2.6 × 107 CFU mL−1 35 min [64]
Chromogenic reaction and detection on µPAD Campylobacter spp. 10 CFU cm−2 10 h [65]
Picoarray device with rezasurin dye E. coli and Staphylococcus aureus Not provided 3h [66]
Chromogenic reaction and detection on µPAD Coliforms 4
10 CFU ml−1 6h [67]
Microfluidic droplet device and Salmonella 106 CFU mL−1 (1 bacterial <5 h [68]
labeled antibodies. typhimurium (S. typhimurium) cell per
two-to-ten droplets)
Microfluidic droplets containing resazurin on silicon chip . S. typhimurium 50 CFU mL−1 5h [69]
Dip and test array with rezasurin dye Gram- and Gram+ bacteria 10 CFU mL−1 12 h [70]
Chromogenic reaction and detection on µPAD Coliforms 3 CFU mL−1 24 h [71]
Phage-based bioluminescence E. coli 4.1 CFU 100 mL−1 5.5 h [72]
Microfluidic droplets containing resazurin E. coli 2 × 108 CFU mL−1 2h [73]
Chemiluminescence detection in microwell array chip E. coli 560 CFU mL−1 2–4 h [74]
Dipstick and bacteriophage E. coli and S. aureus 250 CFU mL−1 4–8 h [75]
On-chip culture and resazurin dye reaction E. coli 2 CFU 100 mL−1 12 h [76]
Pico-droplet array chip and resazurin dye reaction E. coli 500 CFU mL−1 4h [77]
reaction chamber where they bound to the ssDNA, generating needed, an additional on-chip PCR step was also developed to
a measurable colour change after the addition of hydrochloric produce results in a subsequent 40 min.
acid (Figure 1B). During the denaturing step, EMA-bound DNA Recent advances in the use of intercalating dyes have
remained a double-stranded molecule and could not bind to the been made by coupling PMA with isothermal amplification
gold nanoparticles. Therefore, the visible colour change was methods such as LAMP andrecombinase polymerase amplifica-
due specifically to viable cells in the sample. The whole pro- tion (RPA), thus avoiding the temperature cycling requirements
cess was completed in 30 min and was able to detect as little as of PCR, simplifying workflows and increasing its potential for
102 CFU mL−1. If more information regarding the bacterial low resource setting and field testing applications.[78,79] An ori-
species present or antibiotic characteristics of the bacteria is gami style µPAD was designed that was able to detect only live
Figure 1. A) Schematic illustration of the detection mechanism and a layered view of the microfluidic chip. Reproduced with permission.[53] Copy-
right 2012, AIP Publishing. B) Schematic illustration of the microfluidic chip with its different components and chambers, as well as photographs from
the bottom and upper layers of the device. Reproduced with permission.[55] Copyright 2015, Elsevier. C) Schematic illustration of sample preparation
prior to PMA-LAMP assay on the paper origami device. Reproduced with permission.[57] Copyright 2019, Americal Chemical Society. D) Schematic
illustration of detection procedure using a portable microfluidic chip system. Reproduced with permission.[58] Copyright 2021, Royal Society of Chem-
istry. E) Illustration and photograph of the plastic 3D printed chip with SAR mixers and serpentine incubation channel for on-chip PMA sample
treatment. Reproduced with permission.[80] Copyright 2018, Royal Society of Chemistry.
E. coli and Salmonella cells by carrying out an in-tube sample paper channels and into a compartment where the LAMP
pre-treatment step with PMA dye followed by LAMP amplifica- reagents were added, DNA amplification was carried out at a
tion on the paper device.[57] Following in-tube incubation of the constant temperature of 63 °C and the colorimetric results
sample with PMA and photoactivation of the dye, the sample observed by the naked eye (Figure 1C).
was introduced into the paper device. After successive folding Lin et al. developed a microfluidic chip for multiplexed detec-
steps that enabled the sample to flow through the hydrophilic tion of pathogens, using in-tube pre-treatment of the sample
with PMAxx dye, followed by on-chip LAMP amplification membranes, further work must be done to determine its sensi-
and real-time fluorescence detection on a portable detector tivity to cell viability following other inactivation methods such
(Figure 1D).[58] The total assay time, from sample pre-treatment as UV light exposure, as well as chlorine and antibiotic treat-
to fluorescent detection, was just under 1 h (30 min for PMAxx ment. In terms of practical applications, techniques that use
treatment and 25 min for LAMP reaction), rendering this assay fluorescent dyes have many limitations; The dyes require cold
rapid and suitable for on-site testing. Furthermore, the chip storage, and the incubation periods are also carried out on ice.
consisted of 24 reaction chambers, with species-specific LAMP For intercalating dyes such as PMA and EMA, photoactivation
primers embedded in each reaction well, potentially enabling of the dyes and a centrifugation step are also necessary, adding
extensive multiplexing capabilities. to the complexity of the technique. Furthermore, the duration
Wang et al. developed a PDMS chip that was able to detect and temperature of the incubation and the concentration of the
a total of 100 CFU in 90 min, incorporating on-chip capture dye have to be optimized for each bacterial species and sample
of M. tuberculosis cells using a heparin-binding hemagglutinin matrix tested. This can result in difficulties when multiplexing
antibody and live cell detection via PMA treatment.[59] The chip and generating reproducible results.[84]
contained 12 PCR reaction chambers to further carry out on-
chip PCR and the fluorescence signal was detected with a laser-
induced fluorescent module. In order to automate the process, 3.1.2. Antibodies and Aptamers
micromixers as well as micropumps were used as external
components. Most recently, Wen et al. developed a nucleic acid Bacterial cells present several different surface antigens such
lateral flow strip (NALFS) in combination with PMAxx sample as lipopolysaccharides, carbohydrates and proteins in their
pre-treatment and immunomagnetic separation to detect E. coli membranes that act as biorecognition molecules for antibodies.
in lettuce samples.[60] The NALF assay worked by attaching Monoclonal antibodies synthesised to specifically bind to these
molecules such as fluorescein amidite (FAM) and biotin to regions can therefore be used to detect whole bacterial cells,
species-specific primers, carrying out LAMP amplification and either with optical or electrical sensors. In general, optical sen-
capturing the amplicons with antibodies on the test line. A total sors utilize the ELISA principle, consisting of immobilized
assay time of 2 h was achieved, and the limit of detection was antibodies that bind to the analyte and the addition of a sec-
81 CFU g−1 of contaminated lettuce. ondary antibody, labelled with a fluorophore, or conjugated
PMA/EMA sample treatment is traditionally performed in with an enzyme, that also binds to the analyte and produces
a tube and involves many pipetting steps, UV light exposure an optical signal. Electrochemical biosensors for whole cells
and cold incubation periods. The workflow not only requires include amperometric, potentiometric, and impedimetric types
experienced personnel but also increases the detection time by of detection, with impedimetric being the most popular sensing
≈ 1 h, rendering it impractical for rapid on-site detection. In an technique.
effort to integrate PMA treatment into a microfluidic device, For the detection of whole viable bacterial cells, impedance
Zhu et al. proposed detecting live microbial cells by using a biosensors offer a miniaturized, low-cost, and sensitive detec-
3D printed chip to perform on-chip PMA treatment followed tion mechanism.[85] They consist of glass chips with sets of
by real-time DNA amplification.[80] The chip was made from fluid inlets/outlets and microchannels that contain a focusing
clear plastic 3D printing material and consisted of ten split and region to concentrate bacteria using dielectrophoresis, and a
recombine (SAR) mixers and an incubation channel in ser- cell-sensing region with integrated electrode arrays (IDEAs). In
pentine shape (Figure 1E) The PMA alongside the sample was the IDEA, specific antibodies are immobilized that target anti-
injected into the device in the inlets and later mixed in the SAR gens found on cell surfaces. Live cells contain intact antigens
mixers until they enter the incubation channel. Following incu- that can bind to the antibodies and generate an impedance
bation, the sample was collected and used for real-time PCR. measurement; however, in dead cells these surface antigens are
This technique shows great potential for eliminating in-tube damaged and can no longer be recognized by the antibodies,
pre-treatment with PMA/EMA. If current microfluidic devices producing lower impedance measurements.
that use DNA intercalating can successfully integrate this ser- In recent years, a handful of microfluidic impedance biosen-
pentine structure into their design to perform on-chip PMA/ sors have been developed to detect viable whole bacterial cells.
EMA treatment, the workflow might be further simplified. Recently, Liu et al. developed an impedance biosensor for the
Microfluidic devices have proven useful in coupling the use detection of two different serotypes of Salmonella cells in turkey
of DNA-intercalating dyes into a miniaturized and automated food samples.[48] The biosensor consisted of a glass chip with
workflow to detect viable bacteria, reducing the time for results two detection regions fabricated using microchannels. The
from days or weeks to a few minutes or hours compared to detection regions were made of micro-gaped interdigitated elec-
traditional culturing techniques. Furthermore, DNA interca- trode (IDE) arrays containing immobilized anti-Salmonella anti-
lating dye-based live cell detection in comparison to culturing bodies for impedance measurements to detect the presence/
methods has the major advantage of being able to detect viable absence of cells (Figure 2A). The device contained two sensing
but non-culturable bacteria. Since there is no cell culturing IDE arrays, each with a different antibody for a specific Salmo-
involved in the process, the uncertainty of reporting false nega- nella serotype. The sample was introduced through the antigen
tive results due to VNCB is greatly reduced.[81–83] inlet using a syringe pump, after that it was left for half an hour
However, DNA-intercalating dyes are not devoid of limi- to allow binding of antibodies to Salmonella cells and a subse-
tations; whilst it has shown to be useful for distinguishing quent washing step was performed. The impedance was then
live bacteria from heat-inactivated cells with disrupted cell measured using an impedance analyser and compared to the
Figure 2. A) Photograph and schematic illustration of the microfluidic chip and its components. Reproduced with permission.[48] Copy-
right 2019, PLOS ONE. B) Photograph and schematic illustration of the microfluidic chip and its components. Reproduced with permission.[61]
Copyright 2018, Wiley-VCH. C) Photograph and schematic illustration of the microfluidic chip and its components. Reproduced with permission.[62]
Copyright 2019, Elsevier. D) Schematic illustration of the SELEX process. Reproduced with permission.[86] Copyright 2022, Wiley-VCH.
measurement obtained prior to sample injection. In this way, surface micromachining on a glass substrate.[61] The chip inte-
the difference in the impedance measurement corresponds to grated various electrode arrays used to concentrate the bacterial
the concentration of cells present in the sample. This device cells and direct them to the detection channel where impedance
was used to test live and heat-killed Salmonella cells, showing a measurements were taken on an array of detection electrodes
considerable difference between the impedance measurement (Figure 2B). Specific detection of Salmonella and E. coli cells was
of live and dead cells. achieved by utilizing immobilized antibodies in the detection
Likewise, another microfluidic impedance biosensor was zone, demonstrating its potential to detect different targets with
developed that contained three channels with IDEAs and was good specificity. They were able to differentiate live from dead
able to detect three Salmonella serotypes with a limit of detec- cells by comparing impedance measurements. Dead cells with
tion of 7 cells mL−1 in under 1 h (Figure 2C).[62] In a similar damaged cell surfaces were unable to bind to the immobilized
manner, Abdullah et al. developed a device manufactured by antibodies and failed to produce an impedance measurement
compared to viable cells with intact cell walls. The device was infectious from non-infectious viral particles, widening the
used to detect as little as 10 CFU mL−1 in under 1 h. scope of its application.[88] More work needs to be carried out
In terms of sensitivity and speed, impedimetric biosensors in this field to develop more practical and cost-effective sensing
offer a good alternative for viable cell detection, capable of technologies capable of viability testing. As with any technique
detecting 300 CFU mL−1 or less in under 1 h. This is consider- that is based on assessing cell membrane integrity, it must not
ably faster and more sensitive than techniques based on DNA be overlooked, that false positive results may arise from dead
intercalating dyes. Moreover, microfluidic chips offer great cells that retain an intact cell membrane, overestimating the
multiplexing capabilities compared to other sensing technolo- level of contamination.
gies as a single sample inlet can be later subdivided into mul-
tiple channels depending on the number of targets, offering
a wider sensing capacity, crucial for pathogen detection appli- 3.2. Metabolic Activity and Cell Culture
cations and simultaneous identification of multiple bacterial
species. However, their fabrication is more complicated than There are many commercially available assays that reply on
simpler devices such as paper-based sensors and the use of colorimetric, fluorescent and bioluminescent detection of
antibodies also increases their cost and complexity. Advances metabolic activity or cell division to determine the presence of
in aptamer technology show promise for further reducing the live pathogens in a sample.[91–93] These methods require long
costs and increasing the flexibility of binding sites of imped- assay times and complicated workflows, therefore, microflu-
ance biosensors. idic devices that greatly miniaturize and automize the proto-
In the last few decades the use of aptamers in biosensor cols could potentially be a useful approach to improve live cell
technology has quickly gained popularity as an alternative to detection.
conventional antibodies. Nucleic acid aptamers are short single- ATP is one of the most used biomarkers for cell viability;
stranded DNA or RNA molecules that are selected in vitro to however, little work has been done to incorporate ATP measure-
specifically bind to a target of interest. Compared to antibodies, ments into microfluidic devices. One of the first attempts was
aptamers offer several advantages such as low cost, easy syn- carried out by Lee et al. who developed a biosensor for real-time
thesis, unlimited shelf life, they can be easily modified to detection of ATP from aerosols.[94] The biosensor consisted of
add functionalities and can be selected to bind to almost any an aerosol condensation system, coupled to a silicon microflu-
target. To obtain these highly specific molecules, large libraries idic chip and a bioluminescence sensor. The condensation pro-
of oligonucleotides go through a process termed Sequential Evo- cess allowed the aerosol samples to be quickly concentrated and
lution of Ligands by Exponential Enrichment (SELEX). It that hydrolysed and then inserted into a microfluidic channel where
involves multiples rounds of positive and negative selection of lysis buffer and D-luciferin-luciferase reagents were introduced
the molecules to the target of interest followed by an enrich- through separate inlets. The microfluidic channel served to mix
ment step of the selected aptamers (Figure 2D).[86] Aptamer the reagents with the bacterial cells enabling the reaction of free
targets range from small molecules such as carbohydrates ATP with D-luciferin-luciferase and transport the mixture into
and toxins, to peptides and proteins and even whole cells. The the detection area where an electric circuit was used to measure
ability of aptamers to bind to cell surface structures has proved the bioluminescence intensity (Figure 3A).
particularly useful in detecting live bacteria. Through multiple Jin et al. developed a 3D µPAD to detect the ATP of live
rounds of selection, aptamers can be obtained that specifically bacteria.[64] The paper microfluidic device consisted of two
bind to cell wall structures of bacteria that are not present in zones, a reagent zone, and a testing zone. ATP aptamers were
dead microorganisms whose cell wall has been damaged.[87] immobilized on the reagent zone, followed by the addition of
Based on whole-cell detection and employing electrochemical horseradish peroxidase (HRP)-labeled oligonucleotide, comple-
measurements, several different devices have been developed mentary to the ATP aptamer. Meanwhile, the testing zone was
to detect live pathogens[88–90] showing potential for numerous treated with 3-amino-9-ethylcarbazole (AEC) and polyethylene
applications from environmental monitoring to clinical diag- glycol (PEG). The device functioned by inserting the sample
nosis. Zhang et al. developed a PDMS and glass microfluidic solution into the reagent zone where free ATP from lysed cells
device with immobilized aptamers to selectively bind live E. coli would bind to the ATP aptamer, releasing the HRP-tagged
cells and subsequently detect the fluorescent signal of FITC- DNA. The µPAD was then folded to produce the flow of the
stained cells.[56] A microfluidic channel was carved using soft HRP-tagged DNA into the testing zone. H2O2 was added to the
lithography onto the PDMS slide and contained an input and test zone where AEC was oxidated by HRP/H2O2. This reaction
outlet where the syringe pump was connected to introduce the produced a colour change (from light yellow to red-brown) that
sample. Once the sample was introduced, the FITC-stained was used to determine the concentration of ATP and thus the
bacteria entered the microchannel where they bound to the presence of live bacteria in the sample (Figure 3B). This process
anti-E. Coli aptamers and, after washing steps, the fluorescence involved minimum handling of reagents and rapid detection,
signal was detected using a fluorescent microscope. showing potential for POC applications. However, the limit of
The ability to synthesise aptamers that can selectively bind detection reported was 2.6 × 107 CFU mL−1, very high compared
to surface antigens of live cells offers a great alternative to con- to other microfluidic devices such as all ATP-based assays, it
ventional antibodies. These aptamers can be carefully selected was not a species-specific technique.
by running successive screening rounds using dead cells as Exploiting the use of genus-specific chromatographic reac-
targets to eliminate non-specific binding to dead cells. Further- tions, a 2D µPAD device was developed to selectively detect live
more, the concept could also be transferred to differentiating Campylobacter bacteria by employing a chromogenic substrate
Figure 3. A) Diagram of the experimental setup, including aerosol generation, condensation and detection, and a photograph of the microfluidic device.[94]
B) Schematic illustration of the paper device for ATP detection. Reproduced with permission.[64] Copyright 2015, Elsevier. C) Schematic diagram of the
paper device for colorimetric detection of cells. Reproduced with permission.[65] Copyright 2018, Elsevier. D) Schematic Illustration of droplet microfluidic
chip, droplet collection and cell culture, and counting of fluorescnet droplets. Reproduced with permission.[69] Copyright 2020, Elsevier. E) Diagram of
the workflow for detecting live cells using a picoarray device with resazurin. Reproduced with permission.[66] Copyright 2018, American Chemical Society.
F) Photographs of the dip and test device and loaded with sample and resazurin dye. Reproduced with permission.[70] Copyright 2021, Elsevier.
specific to the campylobacter enzyme α glucosidase.[65] The to detect E. coli, Salmonella spp., and Listeria monocytogenes in
microfluidic paper device consisted of two polyvinyl chlo- agricultural water.[95] Detection of total coliforms, a commonly
ride (PVC) pads pressed together with a filter paper centre used indicator of water contamination, has also been integrated
where the sample was introduced and came into contact with into 2D µPADs that employ β-galactosidase, a coliform-specific
the dried substrate (Figure 3C). Once rehydrated, the substrate enzyme to generate a chromogenic reaction.[67,71,96]
reacted with the enzymes from the live bacteria to produce a Bacteriophages, viruses capable of infecting and replicating
colorimetric reaction that could be detected by the naked eye in host bacteria, are commonly used as a tool for the detection
or using an image processing software to obtain quantita- and identification of bacterial populations. A range of existing
tive results. Unlike commonly used metabolic dyes such as bacteriophages can be engineered with synthetic biology to
resazurin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- contain a reporter gene, rendering them a promising tool for
lium bromide (MTT), this type of enzymatic reaction has the pathogen detection. In recent years increased efforts have
major advantage of being genus- or species-specific depending been made to develop bacteriophage-based assays for clinical
in the combination of enzyme and substrate used and can be diagnostics and drug susceptibility testing. Due to the ability
easily adapted for detection of different pathogens of interest. of phages to only infect live cells, bacteriophage-based assays
The same detection mechanism was employed by Bisha et al. enable the specific identification of viable bacteria. Once the
phage infects its host organism, viable host organisms can a device that could detect viable Salmonella cells directly from
be identified by detecting a fluorescent or luminescent signal food samples, achieving detection limits as low as 50 CFU mL−1
produced by the reporter phage, or by the production of lysis within 5 h.[69] Monodispersed microdroplets were utilized for
plaques produced during cell lysis. For example, Alonzo et al. encapsulating Salmonella cells, enabling single-cell culture.
developed a microfluidic chip able to detect as little as 4.1 CFU Each droplet contained a culture medium and resazurin as a
100 mL−1 in 5.5 h.[72] The chip contained inlet and outlet ports metabolic indicator for live cells. Aerobic respiration in meta-
for sample, phage, and growth media injection, and outlet ports bolicaly active cells produced the reduction of resazurin into the
connected to a waste container. Water samples were first intro- highly fluorescent resorufin, allowing for quick identification of
duced into the chip and filtered via an in-chip polyvinylidene viable cells. The silicon microfluidic chip consisted of a bacte-
difluoride (PVDF) filter that traps the bacteria present in the rial solution inlet, a mineral oil inlet, and an outlet for droplet
sample. Culture media and reporter phages containing the collection. Droplets that exited the collection outlet were then
NanoLuc gene are then added to induce the synthesis of the placed into a 24-well plate for subsequent 5 h culture and anal-
NanoLuc enzyme by viable bacteria. The reporter enzyme is ysis using a fluorescent microscope (Figure 3D).
then transported to a different chamber of the chip containing Similarly, Hsieh et al. developed a device that isolated
a nitrocellulose (NT) membrane that traps the reporter protein, single cells in separate chambers and utilized Rsaezurin dye
and following the addition of substrate and the consequent pro- as an indicator of cell viability.[66] The device consisted of a
duction of luminescence, viable cells can be detected by placing PDMS-based picoarray device (Figure 3E) where the sam-
the chip on a separate detection instrument. ples were loaded alongside the Mueller–Hinton broth and the
Dönmez et al. also utilized bacteriophages for live bacteria resazurin dye. Partitioning oil was injected into the device,
detection but instead of using a reporter phage, they exploited flowing through the channels and separating the sample
the phage’s natural ability to produce lytic plaques.[75] Plaque into picochambers, thus isolating single cells in each pico-
forming units (PFU) are commonly produced in solid agar chamber. Viable cells then reduced the resazurin dye and the
plates and require a laborious procedure and overnight incu- subsequent fluorescent signal was detected using a fluorescent
bation. In this study a microfluidic dipstick was developed for microscope.
fast and simple PFU measurements, achieving a limit of detec- Harmon et al. utilized droplet microfluidics to develop
tion of 250 CFU mL−1 in 4–8 h of incubation depending on the a miniaturized bioreactor to detect viable S. typhimurium
target organism. A major advantage of phage-based assays is cells in under 5 h.[68] Bacterial cells were separated into
their ability to infect bacteria and replicate at a faster rate than droplets that contained growth medium and FITC-labeled
its host, producing large quantities of progeny, reporter pro- anti-S. typhimurium antibody. Following 5 h of incubation,
teins and/or lytic plaques, often rendering bacteriophage-base fluorescence intensity was measured using a fluorescent
detection of viable bacteria, faster, more specific, and more sen- microscope. The sample was separated so as to encapsulate
sitive than traditional culturing techniques. one bacterial cell every two to ten droplets generated. Since
Dyes such as Resazurin (7-Hydroxy-3H-phenoxazin-3-one the fluorescence signal produced by a single cell was not suf-
10-oxide) are commonly used as indicators in cell viability ficient to be detected, they established an optimum incuba-
assays, these assays are based on an enzymatic reaction that tion period of 5 h to allow viable cells to multiply and thus
produces an irreversible colour change or production of fluo- generate a measurable signal. By carrying out a 5 h incuba-
rescence. Resazurin is a phenoxazine dye that is cell-permeable, tion step, they were able to selectively detect viable cells in the
it’s non-toxic to cells, weakly fluorescent, nontoxic, and redox- sample.
sensitive. It has a blue colour that when reduced by meta- Further work on droplet microfluidics was carried out by
bolically active cells turns to resorufin that is pink and highly Akuoko et al. They recently developed a microfluidic setup
fluorescent. Rodoplu et al. developed a microfluidic chip con- capable of encapsulating single bacteria together with a resa-
sisting of a PDMS layer constructed on a glass coverslip with zurin dye to detect live cells within just 2 h of incubation
a culture dish at the bottom (diameter of 20 mm).[76] Water time.[73] The PDSM chip was fabricated using soft lithography
samples containing target bacteria were mixed with antibody- and plasma bonding to create the microfluidic channels, and
bound magnetic particles in Eppendorf tubes and the was mix- was connected to a fluid pump system for droplet generation.
ture introduced into the chip via the sample inlet. The inlet More recently, Needs, Osborn and Edwards developed a “dip
chamber contained a magnet that served to concentrate the and test” device consisting of arrays of microcapillaries that
bacteria in the chip without the need for a vacuum or syringe functions similarly to droplet microfluidics to separate sam-
pump. Culture media with Resazurin dye was then introduced ples into multiple compartments and enumerate live bacteria
into the device and incubated at 37 °C for 12 h. Following the (Figure 3F).[70] The strips of the device are dipped into a 96-well
incubation period, the colorimetric and fluorescent signal was plate containing the sample and resazurin dye. These were
recorded with a portable mobile-phone platform. then taken out and left incubating overnight for end-point col-
Recent advances in droplet microfluidics have enabled the orimetric detection or fluorescent measurements using a fluo-
miniaturization of liquid cell culturing techniques. Contrary rescent microscope.
to traditional cell culturing in agar plates, droplet microfluidic Wu et al. developed a chemiluminescence digital micro-
devices are capable of creating a monodispersed layer of microwell array chip for live E. coli detection.[74] 6-Chloro-4-
droplets, that encapsulate single cells with growth medium and methylumbelliferyl-β-D-glucuronide (6c-MUG) was chosen as
a metabolic indicator such as the resazurin dye or labeled bio- the probe that can be cleaved by the β-D-glucuronidase in E. coli
receptors to quantify cell division. An et al. recently developed to produce a fluorescent signal and achieve fast single bacterial
detection. The chip was fabricated with soft-lithography and 4. Conclusion

PDMS moulding to contain 38 400 microwells of the following
dimension: 30 × 30 × 50 µm. The sample containing E. coli Methods for live pathogen detection are essential in many appli-
and the probe was introduced in the microwell array followed cations ranging from food and water monitoring to disease out-
by thermosetting oil to encapsulate single bacteria in the wells. break detection. Existing laboratory-based techniques for live
If the microwell contained live bacteria, they would multiply pathogen detection, such as cell culturing methods, offer great
during the incubation period and produce β-D-glucuronidase sensitivity and quantitification capabilities, however, are lacking
that would then cleave the probe, resulting in a detectable in species-specific identification, ease-of-use, quick assay times,
fluorescent signal. In a similar manner, Suo et al. used ther- and POC testing applications. Meanwhile, traditional molec-
mosetting oil to encapsulate E. coli cells in individual droplets ular-based techniques such as PCR, count with species-specific
within a PDMS chip and detect viable cells via resazurin dye identification of pathogens and quick assays times, however,
reaction.[77] are unable to distinguish between live and dead cells. To over-
The major advantage of using devices that enable single- come the limitations of traditional technologies, the field of
cell cultures, such as microfluidic droplet chips and micro- microfluidic sensing platforms has widened its scope in the last
fluidic picoarrays, lies in the ability to quantify live bacteria. two decades with the objective of enabling rapid, specific and
These techniques use the Poisson distribution to enumerate on-site live pathogen detection systems.
the bacteria present in the sample. Moreover, due to reduced µPADs and µTAS are miniaturized all-in-one sensing plat-
contaminants, smaller volumes and more efficient mixing, forms that allow sample processing and detection within a
growth rates of bacteria in droplets have proven to be quicker single operational device. To develop a technique that can
than in traditional liquid culture, reducing enrichment times simultaneously possess all the advantages of cell culturing such
and improving overall assay time.[97] As with all metabolic dyes, as live cell detection and high sensitivity, and of molecular-
however there is no certainty with regard to the identity of the based methods such as species-specific detection and quick
bacteria present in the sample as all viable cells are capable of assay times, microfluidics has been combined with cell cul-
reducing resazurin. turing, metabolic and molecular-based assays to develop inno-
Overall, very few microfluidic devices have been developed vative techniques that can overcome the shortcomings of tra-
to detect live pathogens using metabolic activity as a viability ditional methods. However, the challenges associated with the
biomarker. Current attempts exploit ATP production, Resazurin miniaturization of the operational steps whilst still retaining
and MTT reduction, and glucosidase activity. Other metabolic high sensitivity and specificity have resulted difficult to over-
biomarkers such as RNA and protein synthesis or heat produc- come, greatly hindering rapid advances in microfluidic tech-
tion could still be explored for possible viability testing solu- nology for live cell detection.
tions in combination with microfluidics. In many cases, one One of the many obstacles faced during miniaturization is
of the greatest limitations of using metabolic activity as a bio- that microfluidic devices are inherently small and can retain
marker for cell viability is the inability to perform species-spe- reduced volumes of liquid, therefore, the addition of external
cific detection. Further research has yet to be conducted in this pumps and sample enrichment via filtering techniques has to
field to identify possible biomarkers such as proteins and RNA be incorporated for large sample volume analysis. Moreover, for
sequences that confer specific information about an organism’s the use of microfluidic devices in a lab-free setting, innovations
identity. With regard to assay time, culturing and metabolic involving isothermal amplification techniques must also be
assays are inherently restricted by each organism’s duplication incorporated to migrate from PCR-based methods. At present,
time, rendering these assays considerably longer than tech- most devices are still at an early stage of proof of concept where
niques based on membrane integrity. Whilst incubation times more validation is required for their widespread application.
may be longer, the sample enrichment step allows the limit Many µTAS still require the use of laboratory infrastructure and
of detection of these techniques to be lower than membrane expensive equipment such as fluorescent microscopes, there-
integrity-based methods, with some devices having reported the fore, there is still a need to further simplify these devices and
ability to detect < 50 CFU mL−1. their detection mechanism to enable rapid pathogen testing in
In terms of sensitivity and assay time, droplet-based micro- the field.
fluidics shows promising results, having achieved low limits of Microfluidic platforms have the potential to provide a rapid,
detection and greatly reducing the assay time compared to tra- low-cost and easy-to-use species-specific method for sensitive
ditional culturing techniques. Nonetheless, the incubation time live pathogen detection. Advantages of microfluidic devices
compared to traditional resazurin dye assays carried out in a include cost-effective fabrication and low reagent costs, integra-
96-well plate does not differ. Due to the small droplet volumes, tion of multiple functions in an intergrated system, and faster
special consideration has to be taken when optimizing the response times. Despite their advantages, microfluidic systems
volume of culture medium and regulating the pH within the are still lacking in sensitivity and have limited capacity for large
device to promote cell activity. Droplet microfluidics enables sample volumes. For the use of microfluidics in low resource
the use of smaller sample and dye volumes, therefore reducing settings, many limitations such as ease of use, cold storage and
assay costs. Droplet generation simultaneously encapsulates electricity free operations still need to be overcomed to make
both sample and indicator dye which eliminates tedious pipet- them suitable for field testing. When developing new tech-
ting steps, greatly automating the workflow. Furthermore, due niques, it is therefore crucial to take into account their poten-
to the number of microfluidic droplets generated, it has great tial application; for instance, in clinical analysis, having a short
potential for high-hroughput screening assays.[98] assay time might be more important than achieving very low
Table 3. Summary of advantages and limitations of conventional pathogen testing techniques and microfluidic sensors for live pathogen detection.
Technique Aadvantages Limitations

Conventional techniques Cell culture/metabolic High sensitivity Experienced personnel
for pathogen detection assays High sample volume capacity Laboratory infrastructure
Quantitative High cost
Live cell detection Long assay times
Partial specificity
PCR/ELISA High sensitivity Experienced personnel
High specificity Laboratory infrastructure
High sample volume capacity High cost
Quantitative No live cell detection
Quick assay times
Highly specific
Microfluidic sensors for µTAS Minimally trained personnel Laboratory infrastructure
live pathogen detection Reduced sample and reagent volumes Use of external components
Low cost Low sensitivity; many at a proof-of-concept level
Quick assay times Few are quantitative
Complex manufacture
µPADS Minimally trained personnel Low sensitivity; many at a proof-of-concept level
No laboratory infrastructure; suitable for field testing Not quantitative
Simple manufacture
Reduced sample and reagent volumes
Low costQuick assay times
limits of detection, whilst for drinking water monitoring, a high different techniques, such as RNA detection protein synthesis,
sensitivity is crucial for compliying with strict hygene standard. and heat flow monitoring, whose potential to be integrated
Consequently, the trade-offs between assay time, sensitivity, into a microfluidic platform could still be explored. With the
and complexity in relation to their application must be taken recent outbreak of SARS-COV-2, a global effort to improve
into account when comparing these devices’ potential and con- diagnostic techniques for viruses has quickly increased. The
trasting them with traditional methods. use of impedance-based devices that differentiate infectious
We have summarized the advantages and limitations of tra- from non-infectious viral particles based on the integrity of the
ditional pathogen testing techniques and novel microfluidic viral envelope, is an area that requires further investigation and
sensors developed to date for live pathogen detection (Table 3). could potentially be of great use in clinical and epidemiological
In the last few years, there has been a surge in the develop- studies.
ment of microfluidic systems, particularly for E. coli and total
coliform detection for their application in water quality moni-
toring. These systems have exploited the use of microarrays Acknowledgements
and droplet microfluidics in combination with metabolic dyes
to rapidly and specifically detect live cells whilst still retaining The authors acknowledge support from a Royal Academy of Engineering
Frontier Follow-up grant (FF\1920\1\36) and Z.Y. thanks UK NERC
the quantifying capabilities of traditional culturing techniques.
Personal Fellowship grant (NE/R013349/2). The graphical abstract was
Droplet-based or microarray-based techniques have shown the made BioRender.com.
greatest advances in the field and demonstrated the potential to
accurately detect live pathogens in a miniaturized manner.
Recent work in µPADs has rendered them the best option
for point of care applications, as they contain all of the detec-
Conflict of Interest
tion functions within a single device (also namely lab-on-a- The authors declare no conflict of interest.
paper), providing quicker, cheaper, easier operations, enabling
ultra-sensitive and selective multiplexed detection of pathogens
for broad-spectrum applications. Dry reagents that improve the
Keywords
ease of use and increase shelf life are particularly important
to develop devices for POC testing. Paper-based devices have cell viability, microfluidics, pathogen detection, sensors
shown great potential for POC testing as they can integrate all
Received: October 18, 2022
functions within the single device and can be manufactured to
Revised: February 2, 2023
contain dry reagents on the paper substrate, thus improving Published online: March 3, 2023
shelf life and usability.
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Carla Spatola Rossi graduated from the University of Buenos Aires, Argentina in 2020 with
an M.Sc. degree in Biology. She is now a Ph.D. student at Cranfield University, supervised by
Dr. Zhugen Yang and Prof. Frederic Coulon. Carla’s research is focused on developing a paper-
based biosensor for detection of live waterborne pathogens, which aims to develop a point-of-use
paper-based device, sensitive and specific for live pathogen detection, to rapidly monitor drinking
water in low- and middle-income countries.
Frederic Coulon holds a chair in Environmental Chemistry & Microbiology at Cranfield University
(UK) and is recognised for his internationally leading contribution on water, soil and wastewater
treatment, resource recovery and environment, and public communication of environmental sci-
ence and engineering. His work is premised on the understanding that environmental resources
are inextricably intertwined and therefore there is a need of advancing a nexus approach to enable
integrated and sustainable management of water, soil and waste systems.
Shaohua Ma is a tenure-track associate professor of Tsinghua University and currently core-PI

at Tsinghua Shenzhen International Graduate School (SIGS) and Tsinghua-Berkeley Shenzhen
Institute (TBSI). He obtained a bachelor’s degree in engineering from Sun Yat-sen University in
2009 and a Ph.D. degree from Cambridge University (supervisor: Wilhelm Huck) in 2013. After
nearly four years of postdoctoral research at Oxford (supervisor: Hagan Bayley), he joined TBSI
in Sept 2017. His research focuses on organoid engineering, stem cell engineering, microfluidics-
based biofabrication, and computational biomedicine.
Y. Shrike Zhang received a B.Eng. in Biomedical Engineering from Southeast University (2008),
a M.S. in Biomedical Engineering from Washington University in St. Louis (2011), and a Ph.D. in
Biomedical Engineering at Georgia Institute of Technology/Emory University (2013). Dr. Zhang is
currently an Assistant Professor at Harvard Medical School and Associate Bioengineer at Brigham
and Women’s Hospital. Zhang’s research is focused on innovating medical engineering technolo-
gies, including bioprinting, organs-on-chips, microfluidics, and bioanalysis, to recreate functional
tissues and their biomimetic models toward applications in precision medicine.
Zhugen Yang is a Senior Lecturer at Cranfield University in the UK, leading Advanced Sensors
Laboratory for water-environment-health-nexus, after being a Lecturer at the University of
Glasgow. His research focuses on low-cost and rapid sensors and devices for diagnostics,
environmental science, and public health. He received three prestigious Fellowships/awards,
including EU Marie Curie Fellow (2013–2015), UKRI NERC Fellowship (2018–2021), and
Leverhulme Research Leadership Award (2023–2028). He completed his postdoc at the University
of Cambridge and was an EU Marie Curie Fellow at the University of Bath, after receiving his
Ph.D. (2012) at the University of Lyon (Ecole Centrale) in France.

Microfluidics For Rapid Detection of Live Pathogens

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Review

Microfluidics for Rapid Detection of Live Pathogens

year (WHO, 2020). Rapid, sensitive, and

Technloigies Targets LOD Assay time References

detection. The chip was fabricated with soft-lithography and 4. Conclusion

Technique Aadvantages Limitations

Shaohua Ma is a tenure-track associate professor of Tsinghua University and currently core-PI

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