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Adv Biochem Eng Biotechnol
https://doi.org/10.1007/10_2020_128
©Springer Nature Switzerland AG 2020
Microfluidics for Environmental
Applications
Ting Wang, Cecilia Yu, and Xing Xie
Contents
1 Introduction
2 Applications of Microfluidics in Environmental Science and Engineering
2.1 Microfluidics Used for Contaminant Analysis
2.2 Microfluidics Used for Microorganism Detection
2.3 Microfluidics Used as Research Platforms
3 Perspectives on Microfluidics’Applications in Environmental Science
and Engineering
References
Abstract Microfluidic and lab-on-a-chip systems have become increasingly impor-
tant tools across many research fields in recent years. As a result of their small size
and precise flow control, as well as their ability to enable in situ process visualiza-
tion, microfluidic systems are increasingly finding applications in environmental
science and engineering. Broadly speaking, their main present applications within
these fields include use as sensors for water contaminant analysis (e.g., heavy metals
and organic pollutants), as tools for microorganism detection (e.g., virus and bacte-
ria), and as platforms for the investigation of environment-related problems (e.g.,
bacteria electron transfer and biofilm formation). This chapter aims to review the
applications of microfluidics in environmental science and engineering –with a
particular focus on the foregoing topics. The advantages and limitations of
microfluidics when compared to traditional methods are also surveyed, and several
perspectives on the future of research and development into microfluidics for
environmental applications are offered.
T. Wang, C. Yu, and X. Xie (*)
School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA,
USA
e-mail: xing.xie@ce.gatech.edu
Graphical Abstract
Keywords Bacterial electron transfer, Biofilm formation, Contaminant analysis,
Environmental science and engineering, Lab-on-a-chip, Microfluidics,
Microorganism detection
1 Introduction
Microfluidics, or “lab-on-a-chip”, is the science and technology of systems that are
made using integrated circuits and/or miniaturized fluidic channels designed to
realize different functions via electrical signals and/or flow manipulation
[1]. When feature size and flow volume are shrunk down to microscale, surface
area dramatically increases –which significantly improves the efficiency of molec-
ular diffusion and heat transfer [1,2]. As a result of their properties, microfluidics are
increasingly finding applications in disparate areas of multidisciplinary research,
including chemical [3,4], biological [5,6], medical (e.g., drug delivery) [2], and
engineering (e.g., material synthesis) [7]fields.
Environmental science and engineering is a discipline for understanding
environment-related processes and dealing with environment-related issues –such
as understanding the conditions of environmental contamination and/or finding ways
to affect environment remediation and protection. Some of the most widely studied
environmental topics currently include pollution monitoring and analysis, research
into the effects of pollutants on ecologies and human health, technologies of
pollution treatment and removal, and microorganism-related challenges (such as
the spread of antibiotic resistance).
Microfluidic and lab-on-a-chip devices are gaining increasing attention in this
field due to their usefulness as tools for (by way of example) pollutant sensing,
microorganism detection, and general environment-related process investigation
(Fig. 1). Microfluidic devices offer several remarkable advantages over more con-
ventional methods: for instance, they can more readily be used as portable detectors
or analyzers due to their small size, thereby enabling on-site pollution detection and
monitoring. Lab-on-a-chip devices have also provided research platforms for in situ
and real-time observation of microorganisms and for visualization of other
T. Wang et al.
environmental processes. In this chapter, we will review some of the primary
applications of microfluidic and lab-on-a-chip devices in environmental science
and engineering. Finally, advantages, limitations, and perspectives on future devel-
opment in this area will be discussed.
2 Applications of Microfluidics in Environmental Science
and Engineering
2.1 Microfluidics Used for Contaminant Analysis
Conventional methods for conducting water pollutant analysis use advanced and
complex instruments, such as inductively coupled plasma mass spectrometry
(ICP-MS) for metal ions detection; high-performance liquid chromatography
(HPLC) and gas chromatography-mass spectrometry (GC-MS) for organic com-
pounds detection; and –ultraviolet-visible spectroscopy (UV-Vis) for nitrate or
nitrite detection. Compared to these traditional analytical techniques, microfluidic
systems and lab-on-a-chip sensors possess several significant advantages, namely,
greater portability for on-site monitoring, smaller required sample volume, shorter
reaction time, and better process control. Optical and electrochemical methods are
the two main approaches that are typically used for pollutant detection in
Fig. 1 Environmental applications of microfluidic and lab-on-a-chip devices (some images are
from the Internet)
Microfluidics for Environmental Applications
microfluidic devices and sensors [8]. Common optical methods include fluorescent,
colorimetric, surface plasmon resonance, and surface-enhanced Raman scattering
(SERS) [8]. The detection techniques used in electrochemical sensors consist of
amperometry, voltammetry, conductometry, and potentiometry [9]. With the sensing
device miniaturization and sample volume decrease, electrochemical methods offer
an inherent advantage over optical approaches. Since electrochemical methods rely
on the concentration instead of absolute amount of the analyte, the sensitivity is
independent of the sample volume, and more accurate determinations could be
achieved due to the higher surface-area-to-volume ratios of the small probes [10].
2.1.1 Heavy Metal Ion Analysis
Water contamination by heavy metals is a severe environmental problem with
significant implications for public health. Indeed, many lab-on-a-chip-based sensors
have been developed specifically to facilitate the detection of a variety of metal ions,
including Hg (II) ions [11], Pd (II) ions [12], Cd (II) ions [13,14], Cu (II) ions
[15,16], and other metal ions [17,18].
Wang et al. have developed a microfluidic device for quantitative analysis of trace
Hg (II) ions (Hg
2+
) based on surface-enhanced Raman scattering (SERS) [19]. A
sample containing Hg
2+
was mixed with gold nanoparticles while flowing through a
wandering channel (Fig. 2a, b). The gold nanoparticles had rhodamine B dye
molecules attached on the surface. Due to the strong affinity between Hg
2+
and
gold nanoparticles, the rhodamine B attached on the gold particles could be replaced
by Hg
2+
(Fig. 2c), causing a change in the SERS signal of rhodamine B in a function
of the concentration of Hg
2+
. The SERS changing was characterized by a Raman
microscope system. The concentration analysis range of Hg
2+
was estimated to be
between 0.1 and 0.5 μg/L.
Another microfluidic device has been developed for continuous and on-site
monitoring of Pb (II) ions (Pb
2+
)[20]. The device is composed of cyclic olefin
copolymer microfluidic channels, with silver working and counter-electrodes. The
Pb
2+
measurement was achieved by square-wave anodic stripping voltammetry
(SWASV) technique. Specifically, water sample containing Pb
2+
was first injected
into the channel through an inlet. Under a certain voltage, Pb (II) ions were deposited
Fig. 2 Schematics of the microfluidic system for Hg ions detection. (a) Schematic of the
microfluidic device for Hg
2+
detection. (b) Photograph of the channel during operation.
(c) Schematic of Hg
2+
sensing mechanism based on the replacement of RB dye molecules through
the reduction of Hg
2+
on the surface of Au nanoparticles (reprinted by permission from Springer
Nature Customer Service Centre GmbH: Springer, [19]. Copyright (2009))
T. Wang et al.
onto the Ag electrode via electrodeposition, and then plated metal was oxidized off
from the Ag electrode using a square-wave anodic potential sweep. The whole
electrochemical reaction is presented by Pb
2+
+2e
⟷Pb. The current generated
during the stripping process was measured to identify and quantify Pb (II) ions. The
detection limit was 0.55 ppb, and the correlation coefficient is 0.998 within the
concentration range of 1–1,000 ppb. Furthermore, the detection performance
remained stable after 43 consecutive measurements, demonstrating the sensor’s
reusability and great potential for real-world applications.
Microfluidic systems for water arsenic detection using both colorimetric methods
and electrochemical methods have also been developed [10]. In addition, biological
detection methods have been pioneered as well. A strain of genetically modified
Escherichia coli (E. coli) was used as reporter bacteria for arsenic detection in a
microfluidic device [21]. Polydimethylsiloxane (PDMS) was used to fabricate the
microchannels. The bacteria were encapsulated in agarose beads and packed into
small cages in the microchannels. When water sample containing arsenic flowed
through the cages, the bacteria exposed to arsenic could produce green fluorescent
proteins. The fluorescence was imaged with a microscope and processed for inten-
sity analysis. The rate of fluorescence signal increase was linearly proportional to the
arsenic concentration within the range of 0–50 μg/L. More microfluidic systems for
arsenic detection were reviewed in [10].
In addition to standard silicon-based sensors, paper-based microfluidics have also
been developed for metal analysis (reviewed in [22]). Paper-based microfluidics are
paper substrates patterned as channels and barriers to realize different functions.
Compared to traditional PMDS and glass or silicon-based microfluidics, the paper-
based microfluidic devices are more cost-efficient [22]. By combining eight
pyridylazo compounds, a paper-based microfluidic device could discriminate eight
different heavy-metal ions (Hg
2+
,Cd
2+
,Pb
2+
,Ag
+
,Ni
2+
,Cu
2+
,Zn
2+
, and Co
2+
)at
concentrations as low as 50 μM[23].
2.1.2 Organic Compound Analysis
Potentially toxic organic compounds –such as phenolic compounds and pesticides –
are widely used across many industries. Unfortunately, some of these organic
compounds may also cause water contamination, due to wastewater discharge or
leaching from soil. Microfluidic sensors for organic matter have been developed
based on different detection mechanisms, including amperometry [24], enzyme-
based techniques [25–27], and electrophoresis [28,29]. A lab-on-a-chip device
with layer-by-layer printing of quantum dot (QD)/enzyme microarrays was fabri-
cated for organophosphorus pesticide (OP) detection [30]. Layer-by-layer
microarrays of QDs/poly (dimethyldiallyl ammonium chloride) (PDDA) and acetyl-
cholinesterase enzyme (AChE) were fabricated on a glass slide using inkjet (Fig. 3).
Water samples and acetylthiocholine (ATCh) were added to the chip for OPs
detection. AChE catalyzes the hydrolysis of ATCh, generating thiocholine (TCh),
which can dissociate the electron-hole pair of QDs and quench the fluorescence.
Microfluidics for Environmental Applications
When OPs are present, the activity of AChE was inhibited; thus the fluorescence of
QDs will not be quenched. A detection limit of 1 μg/L of Ops was achieved with this
device, which was much lower than levels specified by standard tests and other
colorimetric detection methods.
2.1.3 Nitrate and Ammonia Analysis
Nitrate and nitrite are ubiquitous water contaminants in both surface and groundwa-
ter, and they each can impose harmful effects on human health. A miniaturized
microfluidic sensor has been developed to facilitate nitrate determination using a
double-potential-step chronocoulometry (DPSC) method [31]. Two potential steps,
E
1
and E
2
, were applied sequentially to obtain oxygen reduction charge Q
1
and both
nitrate and oxygen reduction charge Q
2
. The nitrate reduction charge was calculated
by subtracting Q1 from Q2, which is directly related to nitrate concentration in the
sample. A silver sensing electrode, silver oxide reference electrode, and platinum
counter electrode were then deposited on a silicon substrate. A polyimide passiv-
ation layer was also deposited to prevent short circuit and improve reliability. The
microchannels were fabricated via deep reactive ion etching, which enabled the
flow-through analysis. The lower and upper detection limit for nitrate were 4–75 μM
and 500–2,000 μM, and the linearity (R
2
) was >0.99. Other microfluidic-based
sensors for nitrate [32,33] and ammonia [34,35] analysis were also reported.
Lab-on-a-chip systems have also found applications in a wide variety of disparate
environments, including marine pollution analysis [36–39], air pollutant detection
[40,41], and bioaerosol monitoring [42–44].
Fig. 3 The schematic of the fabrication process of the OPs detection chip and the image of QDs
after OPs are added. (Reprinted from [30]. Copyright (2016), with permission from Elsevier)
T. Wang et al.
2.2 Microfluidics Used for Microorganism Detection
Pathogen contamination of drinking water remains a serious public health concern
worldwide, especially in less developed areas. Waterborne pathogens can include
bacteria, viruses, and some protozoa. Some of these biological agents are highly
infectious and resistant to water treatment processes and accordingly pose a severe
risk to human health. Different detection approaches have been developed to
facilitate pathogen detection on-chip, such as nanomechanical cantilever sensing
[45,46]; surface-enhanced Raman spectroscopy [47]; impedance-based sensing
[48]; amplification-based sensing, including PCR [49–56] and loop-mediated iso-
thermal amplification [52]; and quartz crystal microbalance-based sensing [57]. Both
optical signals [58–61] and electrical signals [62,63] are used in microorganism
sensing techniques. The applications of microfluidics in waterborne pathogen detec-
tion are reviewed in [64]. Microfluidics for pathogen detection are also being
developed as point-of-care devices, for diagnostic purpose [65]. Although the
samples analyzed in diagnostic devices (e.g., saliva and blood) are different from
environmental samples, the detection techniques and approaches are still valuable as
references.
2.2.1 Virus Detection
An ultrasensitive virus detection sensor based on the Young interferometer has been
reported [66]. The sensor is a silicon chip consisting of four light channels. Si
3
N
4
and SiO
2
layers were deposited on a silicon substrate via chemical vapor deposition.
The SiO
2
layers were etched to form windows for antibody functionalization and
virus detection. The Si
3
N
4
layer beneath served as a pathway for light (Fig. 4a).
Monochromatic light from a laser source was coupled to an optical channel and
guided into the four parallel channels (Fig. 4b). Antibodies for different viruses’
detection were coated onto the channels. The light interfered on a screen after exiting
from the four waveguide channels, generating an interference pattern. Virus binding
to the antibody would be probed by the evanescent field of the guided modes, thus
causing a phase change which could be measured as a change in the interference
pattern. The pattern reflects the amount of the viruses bonded on the antibodies.
Figure 4c shows the specific detection of herpes simplex virus type 1 (HSV-1)
realized by the specific reaction between HSV-1 and the antibodies. The sensor
specifically and sensitively detected HSV-1 with the concentration as low as 850 par-
ticles/mL and the detection sensitivity of the sensor was estimated to approach one
single HSV-1 particle.
Microfluidics for Environmental Applications
Fig. 4 (a) Schematic of the sensor for virus detection. 1, 2, and 3 are the measuring channels, and 4 is the reference channel (reprinted with permission from
[66]). Copyright (2007) American Chemical Society]. (b) Cross section of the chip along the direction of the channels (Adapted form [67]). (c) Specific and
selection detection of HSV-1. The figures indicate phase changes as a function of time. The phase change does not increase when human serum albumin (HSA)
is added but increases only after HSV-1 is added, which is due to the specific interactions between HSV-1 and α-HSV-1gG (reprinted with permission from
[66]. Copyright (2007) American Chemical Society)
T. Wang et al.
2.2.2 Bacteria Detection
Bacteria are another kind of major waterborne pathogen that poses risk to human
health. Mannoor et al. have reported a microfluidic system for real-time on-chip
bacteria detection using impedance spectroscopy [68]. A gold electrode array was
deposited onto a silicon substrate via standard microfabrication methods. The flow
channel for real-time monitoring was fabricated using PDMS and bonded to the
substrate. The electrode surface was functionalized with magainin I, which is a kind
of antimicrobial peptide (AMPs) used for bacteria binding. When the bacteria
contained in water samples were recognized by the AMPs and bonded to the
electrode surface, the impedance of the electrode array changed, which was analyzed
by a spectrum analyzer. Since the binding activity was directly proportional to the
variation of impedance, the bacteria concentration could be analyzed. The detection
limit of E. coli was about 1 bacterium/μL. The system showed sufficient selectivity
toward pathogenic and Gram-negative bacteria, and also maintained broad detection
capability for other bacteria. Furthermore, the flow system enabled real-time bacteria
monitoring for a continuous water sample.
2.2.3 Protozoa Detection
In addition to viruses and bacteria, protozoa –especially some parasites –can pose a
significant risk to human health. Cryptosporidium is one of the parasites of greatest
concern on this front, due to its low infection dose and resistance to common water
treatment approaches [69]. Several techniques have been integrated to miniaturized
fluidic chips for Cryptosporidium detection, including optical methods such as target
trapping combined with immunofluorescence or microscopy detection; mass-based
methods such as quartz crystal microbalance sensing and cantilever sensing; and
electrical techniques such as bioimpedance and dielectrophoresis methods. The
detection of cryptosporidium in microfluidic devices is reviewed in [69].
2.3 Microfluidics Used as Research Platforms
Understanding environmental-related natural processes is another important compo-
nent of environmental science and engineering. These widely studied processes are
encompassed within, but certainly not restricted to, the fields of environmental
microbiology, ecotoxicology, and contaminant transportation. Microfluidic and
lab-on-a-chip devices are increasingly being used in environmental research labora-
tories, since they provide ideal research platforms for in situ and real-time observa-
tion. The combination of lab-on-a-chip devices and observation techniques (such as
microscopy) enables in situ visualization,characterization, and simulation of a wide
Microfluidics for Environmental Applications
range of environment-related processes, thus becoming a valuable investigation
approach in environmental studies.
2.3.1 Mechanisms of Bacteria Electron Transfer
Microbial fuel cells, which use microorganisms colonizing electrodes to catalyze
electrochemical reactions and convert chemical energy into electrical power, are
being intensively studied in the environmental technology field since they possess
the potential capability of converting organic or inorganic waste into power via an
environmentally friendly microbiological process [70,71]. Understanding the mech-
anisms of electron transfer from bacteria to electrode is accordingly imperative for
the further development of potential microbial fuel cells.
Three possible electron transfer pathways have been proposed: via direct contact,
via conduct pili, and via diffusion of soluble redox-active molecules serving as
“electron shuttles”[72]. Jiang et al. have reported a lab-on-a-chip device with
microelectrodes as a platform to investigate the electron transfer between
Shewanella oneidensis and electrodes [73]. Finger-shape electrodes were defined
by photolithography and deposited onto a cover glass using metal evaporation and
lift-off methods. A passive Si
3
N
4
layer was deposited by chemical vapor deposition
and patterned to have nanoscale openings on one electrode and a big opening on the
other electrode (Fig. 5a, b). The nanoholes were small enough to prevent direct
contact between bacteria and the electrode but allowed the indirect contact through
pili or diffusion of extracellular redox-active molecules. A SU-8 (a commonly used
epoxy-based negative photoresist) chamber was fabricated to improve reliability and
environmental control. In situ cell image/tracking with a microscope and current
recording revealed that the currents could be detected even without direct contact
between bacteria and electrodes, suggesting that electron transfer was realized by pili
or a mediator’s diffusion. In addition, the removal of the diffusible mediators caused
a rapid drop of the current, which further supported that electron transfer occurs
predominantly by diffusion of mediators.
Fig. 5 Schematic of the design of the electrodes for Shewanella electron transfer study. (a) The
silicon nitride insulating layer (blue) with nanoholes or large window openings is deposited over
electrodes (yellow) to prevent or enable direct contact with bacteria (orange). (b) SEM images of the
bacteria cells on the electrodes with nanoholes (left) and large window openings (right). (Reprinted
with permission from [73], Proceedings of the National Academy of Sciences)
T. Wang et al.
For other kinds of bacteria, however, the electron transfer mechanism may need
to be altered. A similar lab-on-a-chip device was fabricated to probe the charge
transport from Geobacter sulfurreducens to electrode [74]. Gold electrodes were
deposited via metal evaporation and lift-off. Thick SU-8 was then fabricated to form
wells around the electrodes to allow direct contact between bacteria and electrodes
(Fig. 6a, b). Simultaneous recording of cell position and currents indicated that the
contact of a cell to the electrode directly caused a stepwise increasing of current
(Fig. 6c). The current of a single Geobacter was 92 fA, and the current density was
estimated to be ~10
6
Am
3
. In addition, when the diffusible redox mediators were
removed, the current was not affected. These measurements together indicated that,
different from Shewanella, the electron transfer between Geobacter and electrode
was mainly due to direct contact. Ding et al. reported a nanoelectronics lab-on-a-chip
system to investigate the electrical conductivity of both Shewanella and Geobacter
and indicated that electrochemical electron transfer at the cell/electrode interface was
the origin of the conductive current for both microbes [75].
As researchers have started to gain a deeper understanding of bacterial electron
transfer, the role of bacterial self-assembled nanostructures for extracellular electron
transfer has also garnered increased attention. For example, to elucidate the effects of
microenvironment on the intercellular microbial nanostructures (nanowires)
Fig. 6 (a) Schematic of experimental design for Geobacter sulfurreducens electron transfer. (b)
SEM image of a well containing two finger electrodes. (c) In situ microscopy images of Geobacter
cells around and on the measured electrode and the current changes at the same time. The cell that
contacts the electrode at the same time with the current increases is marked in red. (Reprinted by
permission from Springer Nature Customer Service Centre GmbH: Springer, [74]. Copyright
(2013))
Microfluidics for Environmental Applications
formation, a one-dimensional core/shell bacterial cable has been developed –which
allows rational control of the microenvironments [76]. The fabrication method of
this cable was different from common microfluidics fabrication processes. The cable
was generated through a flow-focusing device with coaxially aligned glass capil-
laries and multiple inlets for different solutions. Bacteria solution flow was focused
into a narrow stream, and alginate was injected to the device to form the scaffolding
for bacteria encapsulation. A Ca
2+
containing sheath flow was exploited to cross-link
alginate to become a solid hydrogel (Fig. 7a). The results revealed that the formation
of intercellular structures is closely related to the fiber diameters. More densely and
closely packed bacteria produced more self-assembling microbial nanowires, which
directly increased the extracellular electron transfer efficiency (Fig. 7b). Further-
more, lack of electron acceptors can enhance the production of the nanowires
(Fig. 7b).
2.3.2 Biofilm Formation
Biofilm formation is a natural process that occurs during bacteria growth. On one
hand, biofilms play important roles in some environmental engineering processes,
including in wastewater biological treatment and microbial fuel cells. However,
biofilm can also cause environmental and public health problems –including by
contaminating or clogging drinking water pipelines or fouling water treatment
systems. As a result, the process of biofilm formation is gaining more attention in
environmental science and engineering. Drescher et al. have developed
a microfluidic device to investigate biofilm formation in fluidic channels [77]. A
meandering microfluidic channel was fabricated with PDMS and sealed with a cover
glass. Pseudomonas aeruginosa bacterial solution flowed through the microfluidic
channel and the biofilm formation process in the channel was observed with a
microscope. This work demonstrated that the 3D biofilm streamers that bridged
the space between obstacles and corners caused major clogging of the channel,
instead of the biofilm attached on the inner surface. The 3D biofilm streamer was first
Fig. 7 (a) Schematics of the flow-focusing device for core/shell bacterial fiber generation. The
bacteria-containing core stream (brown) is focused before entering the alginate shell stream
(yellow), and then a CaCl
2
sheath flow is introduced to cross-link the alginate to form the cord.
(b) SEM images of high (left) and low (middle) bacteria density networks as well as high-density
networks cultured in electron acceptor rich conditions (right). (Reprinted with permission from
[76]. Copyright (2018) American Chemical Society)
T. Wang et al.
formed by the extracellular matrix shed from the attached bacteria and then worked
as a network to catch the flowing bacteria and biomass, leading to a rapid clogging.
With this microfluidic chip that enabled in situ observation of the biofilm formation,
this work demonstrated a biofilm formation process which is independent of and
much faster than bacteria growth. The results also suggested that the biofilm
streamers may contribute more to the clogging of flow through systems such as
water pipelines.
During biofilm formation, the bacteria within microbial communities can sense
chemical signals from other cells and regulate their own gene expression as a
response. This process is referred to as quorum sensing, and it is an important factor
in regulating biofilm formation that is a current subject of intense study in the
environmental microbial field. Flickinger et al. have reported a lab-on-a-chip plat-
form to study quorum sensing between microbial communities [78]. The lab-on-a-
chip device contained an array of spatially confined chambers fabricated with poly
(ethylene glycol) diacrylate (PEGDA) on a silanized cover glass using a PDMS mold
(Fig. 8a). Pseudomonas aeruginosa (P. aeruginosa) was used as a model bacterial
strain and inoculated in the center chambers for biofilm growth (Fig. 8b). The
molecule regulators secreted from the biofilm for quorum sensing, homoserine
lactones (HSLs), can diffuse inside (filled with 15% PEGDA) and between the
PEGDA chamber to form spatial and temporal gradients, thus enabling analysis of
the relationship between the diffusion of HSLs and formation of nascent new biofilm
(Fig. 8c). The results showed that HSL was detected by the bacteria cells within a
distance of 8 mm. The new biofilm growth within 3 mm away from the existing
biofilm, where the HSL concentration was higher than 1 μM, was enhanced due to
the detection of HSL, while further biofilms were not affected. In addition to regular
on-chip chambers, 3D cavities with various geometries were fabricated using 3D
printing strategy to study the mechanisms of community regulation [79].
Fig. 8 (a) An image of the chamber for quorum sensing study with hydrogel chamber wall (stained
with red dye) on a glass coverslip (upper). An image of the PDMS stamp used to make the chamber
(lower). (b) The center chamber was inoculated with P. aeruginosa.(c) Schematic of the experi-
ment. HSL diffuse through the hydrogel chamber wall, which is detected by biofilm in each
chamber. (Reprinted with permission from [78]. Copyright (2011) American Chemical Society)
Microfluidics for Environmental Applications
2.3.3 Antibiotic Resistance Gene Transfer
It is now known that horizontal gene transfer is an important pathway by which
antibiotic resistance spreads from one organism to another. Microfluidic devices are
promising platforms for facilitating gene transfer study, since they enable the in situ
and real-time monitoring of the process dynamics. A microfluidic device was
reported to investigate the plasmid-mediated horizontal gene transfer within the
same species and between different species [80]. The microfluidic chip consisted
of a cover glass with a layer of agarose and a PDMS cover on top (Fig. 9a). A drop of
mixed bacteria solution containing the gene donor strain (Pseudomonas putida
harboring an antibiotic resistance plasmid) and recipient strain (E. coli or bacteria
extracted form activated sludge) was sandwiched between the agarose and cover
glass. The PDMS cover had a channel in it for broth delivery and waste removal for
bacteria growth (Fig. 9a). The gene donor bacteria carried plasmid RP4, which was
labeled with GFP, but also tagged with red fluorescent genes that repress the
expression of GFP. So, the donor bacteria emitted red fluorescence. When the
plasmids were transferred to acceptors, the acceptors would emit green fluorescent
from GFP carried with the plasmids. The gene horizontal transfer process on the chip
was monitored with a fluorescence microscope. The results showed that the hori-
zontal gene transfer was highly dependent on the structure and composition of the
biofilm. The plasmids were first successfully transferred from donor species Pseu-
domonas putida to acceptor E. coli. Within the pure E. coli colony, the transfer from
the first transconjugants to other cells was very efficient, leading to a cascading gene
spread within the single-strain biofilms (Fig. 9b). In comparison, for the activated
sludge biofilm consisting of different species, vertical gene transfer appeared to be
the dominant route instead of horizontal transfer (Fig. 9c). It is also found that many
species that showed horizontal gene transfer were associated with human pathogens.
Fig. 9 (a) Schematic of the device to study antibiotic resistant genes transfer (upper) and the device
setup (lower). (b) Gene spread in pure E. coli culture. (c) Gene spread in activated sludge
community. For both (b) and (c), the donor cells P. putida KT2440 are red; normal E. coli or
active sludge cells are colorless, while transconjugants emit green fluorescence. (Reprinted with
permission from [80], https://pubs.acs.org/doi/abs/10.1021/acs.est.8b03281. Copyright (2018)
American Chemical Society. Further permissions related to the material excerpted should be
directed to the ACS)
T. Wang et al.
Other microfluidic systems for gene transfer and antimicrobial resistance related
studies were also reported, including using microfluidic devices to study gene
transfer on the single-cell level [81], dissect horizontal and vertical gene transfer
[82], test antimicrobial susceptibility [83], and investigate the modulation of antibi-
otics on horizontal gene transfer [84]. More studies are reviewed in [85].
2.3.4 Electroporation
Electroporation is the phenomenon whereby pores form on a cell membrane when
the cell is exposed to an external electric field. It is commonly used to control cell
membrane permeability when molecular intracellular transfer is desired. In addition,
electroporation is also a widely used method for cell inactivation and lysis.
Researchers in environmental fields are also increasingly exploring the possibility
of using electroporation as a bacteria inactivation approach for drinking water
disinfection [86–89] and hazardous wastewater decontamination [90]. Understand-
ing the electroporation process has therefore become another important environmen-
tal study topic. Since the actual formation of these pores is difficult to observe,
Sengel et at. have developed a lab-on-a-chip device to image the dynamics of
individual electropores [91]. The experimental setups are shown in Fig. 10a.A
cover glass was coated with agarose and placed in a recess. Lipid solution was
added and associated with the agarose to form a lipid monolayer. An aqueous droplet
with lipid monolayer was brought onto the cover glass. Two monolayers at the
contact area formed a lipid bilayer, which is similar to cell membrane. Two elec-
trodes were placed at the two sides of the lipid bilayer to monitor the current, and the
pore formation was labeled by a fluorescent dye and recorded with a fluorescence
microscope (Fig. 10b). With this platform, researchers found several interesting
phenomena of electroporation. When the potential difference across the bilayer
reached 100 mV, the membrane permeability started to change. With higher
Fig. 10 Schematic of the experimental setup. (a) A lipid bilayer is formed on the interface of the
droplet and the substrate. When a pore is formed due to electroporation, Ca
2+
ions flow into the
drop, which could be detected by the Ca
2+
sensitive dye fluo-8 and visualized by a microscopy. (b)
A microscopy image of pores formed on lipid bilayer. (Adapted and reprinted with permission form
[91], Proceedings of the National Academy of Sciences)
Microfluidics for Environmental Applications
transmembrane potential, larger pores formed, but a large number of small pores still
existed. In addition, the pores fluctuated (opened and closed) in a variety of modes,
and higher potential did not lead to more stable pores. Two adjacent pores did not
tend to combine, while anti-combination was found since the potential across the
lipid bilayer would be released when a nearby pore gets larger. A lab-on-a-chip
device to rapidly determine the electroporation threshold for bacteria inactivation
was also reported [92].
2.3.5 On-Chip Toxicity Test
Ecotoxicology focuses on identifying the toxicity level and impact of environmental
pollutants on creatures and human health. Compared to traditional toxicity testing
approaches, the emerging on-chip toxicity tests enabled by microfluidics are signif-
icantly more compact, convenient, and labor-efficient –and as a result they are
quickly gaining substantial attention in this field.
Fine particles are major pollutants in the air, which makes them crucial indicators
of general air quality. A microfluidic device aiming at recognizing the toxicity of fine
particle matter (PM
2.5
) on human lung epithelial cells was reported [93]. A porous
membrane was bonded to the PDMS chamber, and the human lung epithelial cell
line (BEAS-2B) was cultured on the membrane. Medium flowed under the mem-
brane to replenish nutrient for cell growth (Fig. 11). The cell viability remained
above 98% after 21 days of culturing, which demonstrated that this lab-on-a-chip is
capable of retaining the viability of cells for the toxicity test. The air liquid interface
mimicked the pulmonary natural microenvironment, which enabled the in vitro
cytotoxicity test. Particles were added to the cells using an aerosol nebulizer, and
the cytotoxicity was analyzed by several different approaches after exposure. The
results showed that some metabolic pathways of the cells contributing to inflamma-
tion reactions were activated after the exposure. The cell apoptosis rate was also
increased from 3.8% to 66.7% after 24 h of exposure. This configuration is also
applicable for cytotoxicity test of other pollutants.
Engineered metal nanoparticles are being used for a variety of applications in
many different fields. However, the potential hazards of these nanoparticles to
human health and environment remain topics of hot debate and active research. To
investigate the effects of silver nanoparticles on microorganisms’behavior, a
Fig. 11 Schematic of the designed microfluidic chip for ambient particle toxicity test. (Reprinted
from [93]. Copyright (2019) with permission from Elsevier)
T. Wang et al.
microfluidic device was reported to study the swimming response of algae to silver
nanoparticles [94]. PDMS containing microchannels was fabricated via soft lithog-
raphy and bonded to a glass slide, forming the microfluidic device. The microfluidic
device used in this study was previously designed for a bacterial chemotaxis test,
which contained a concentration-gradient generator and a chemotaxis observation
channel [95]. Solution with and without silver nanoparticles were added to the two
inlets, respectively. After flowing through the mixing part, nanoparticle concentra-
tion gradient was created and maintained in the observation channel. Algae was then
added to the observation channel and exposed to the nanoparticle gradient. The algae
swimming response was observed and recorded with a microscope. The results
showed that algae moved away from the area containing 10
8
silver particles/mL,
but no significant aversive swimming was found to gold nanoparticles at the same
concentration. The toxicity of the released Ag ions may be the main reason leading
to the avoidance behavior.
Microfluidics also have applications in aquatic toxicity tests on bacteria [96,97],
nematodes [98], crustacea [99,100], and fish embryo [101]. More applications and
future perspective were discussed in the review paper [102].
3 Perspectives on Microfluidics’Applications
in Environmental Science and Engineering
Microfluidic and lab-on-a-chip devices are increasingly being used as tools in the
fields of environmental science and engineering. Miniaturized microfluidic or
lab-on-a-chip devices evidence remarkable sensing abilities, because sensing elec-
trodes can be miniaturized without losing sensitivity and the configurations are
compatible with thin layer operations [9]. Flow manipulation and compound sepa-
ration can also be enabled by incorporating additional electrodes in existing chan-
nels, without adding additional parts. When used as detection equipment, lab-on-a-
chip systems offer several comparative advantages over traditional mechanisms –
including shorter analysis time, smaller sample volume, and online and real-time
monitoring. All of these benefits are attributable to their small size, precise flow
control, and low cost compared to traditional instruments. Some sensors have
already been commercialized, such as test strips based on electrochemistry for
arsenic detection [103] and sensors based on stripping square-wave voltammetry
for metal analysis [104]. A DNA electrochemical biosensor has been combined with
sample processing platforms for online pathogens monitoring in natural water
[105]. IBM is also working on the development of sensors for environmental
pollution detection, such as methane leakage [106]. Since real-world samples are
often complex and signal characterization systems are still required for the systems,
real-world implementation of these detection devices remains limited in some
instances. In addition to further improving the performance of sensors, future studies
will undoubtedly focus on developing more integrated systems that combine
Microfluidics for Environmental Applications
sampling, pre-treatment, and signal interpretation on a single chip –which will
increase the viability of on-site, real-time applications across a wide variety of real-
world settings. Furthermore, exploring cost-efficient materials (e.g., paper-based
devices), simpler fabrication processes and easier operation approaches will also
undoubtedly continue to bring the cost associated with these devices down, which
will also further facilitate the feasibility of on-site and point-of-use applications.
But compared to their use as sensors and analyzers, microfluidic devices offer
even more remarkable advantages as research platforms for environment-related
process investigation. Their miniaturized size and the flexible configurations for
realizing various functions provide them with unique capabilities for visualizing and
unveiling the secrets of numerous environmental-related processes, which are not
comparable by other approaches. Therefore, we believe the future growth of lab-on-
a-chip devices as research platforms will be focused on exploring novel and clever
designs to realize more functions based on different investigation purposes. Nano
structures, such as nanoholes, nanoparticles, nanowires, and coating layers with
nanoscale thickness, are providing lab-on-a-chip devices with more features and
functions. Nanofabrication techniques, including electron beam lithography and
atomic layer deposition, are becoming widely used for chip fabrication. In addition,
more and more lab-on-chip devices for research purpose are not restricted to
standard chamber or channel on-chip configurations. Various 3D geometries are
enabled by thriving 3D fabrication techniques, such as 3D printing, two photon
polymerization, and micron/submicron stereolithography. To improve the capabili-
ties of lab-on-a-chip devices, the performance of their basic functions, such as flow
control, cell manipulation, cell culturing, and target tracking, is also worth improv-
ing. Finding the environmental problems and processes that could be investigated
using lab-on-a-chip platforms is also important. In addition to visualizing small-scale
process, such as bacterial-related phenomena mentioned in Sect. 2.3, lab-on-a-chip
systems are also ideal for mimicking and simulating large-scale ecological pro-
cesses, such as fate and transport of nanoparticles in soil and groundwater
[107,108]. The findings of the on-chip simulations could provide valuable experi-
mental data for modeling and further on-site studies.
Acknowledgments The authors would like to acknowledge the US National Science Foundation
[grant numbers CBET 1845354]. The authors would like to thank Dr. Janina Bahnemann for her
contribution to this chapter.
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