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Thread-based isotachophoresis for DNA extraction

Cite this: Lab Chip, 2021, 21, 2565
and purification from biological samples†
Liang Chen,ab Joan M. Cabot *abc and Brett Paull ab

A rapid, low-cost, and disposable microfluidic thread-based isotachophoresis method was developed for
the purification and preconcentration of nucleic acids from biological samples, prior to their extraction and
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Received 5th March 2021,
Accepted 30th April 2021
DOI: 10.1039/d1lc00179e
rsc.li/loc
successful analysis using quantitative polymerase chain reaction (qPCR). This approach extracts and
concentrates protein-free DNA from the terminating electrolyte buffer, via a continuous sampling
approach, resulting in significant focussing of the extracted DNA upon a 6 cm length nylon thread. The
platform was optimised using the preconcentration of a fluorescent dye, showing a 600-fold concentration
capacity within <5 min. The system was then applied to the one-step extraction of lambda DNA – an E. coli
bacteriophage – spiked into whole blood, exhibiting the exclusion of PCR inhibitors. The extraction
efficiency from the thread material following concentration was consistent, between 94.4–113.9%. The
determination of lambda DNA in whole blood was achieved within a linear range of 1.0–1 × 105 fg μL−1 (20–
2 × 106 copies per μL). This technique demonstrates great potential for the development of thread-based
affordable analytical and diagnostic devices based upon DNA and RNA isolation.

1. Introduction in PCR-mediated amplification.9 Therefore, selective and

Nucleic acids (NAs) have long been recognised as ideal
biomolecular markers in clinical and analytical applications for
diagnostics and tracking the spread of infections.1–3 A
significant potential example application of this is liquid
biopsy in cancer diagnosis. Liquid biopsy offers a non-invasive
procedure for sample collection, without the resection of tissue
from the biopsy for testing, and is complimentary to the
common tissue biopsy techniques. Current DNA detection
methods mainly rely on polymerase chain reaction based
assays (qPCR),4 blotting,5 next generation sequencing,6
microarray approaches7 and ‘clusters of regularly interspaced
short palindromic repeats’ based methods (CRISPR).8 However,
each of these advanced techniques are susceptible to
contamination. The typical complexity of biological matrices
(e.g., proteins, lipids and carbohydrates, drugs) in samples,
such as blood, urine and saliva, can directly affect the
fluorescent signal response, reducing method accuracy,
precision and overall sensitivity. For example, the haemoglobin
and humic acids within cell lysates can reduce qPCR efficiency
a
Australian Centre for Research on Separation Science (ACROSS), School of
Natural Sciences, University of Tasmania, Private Bag 75, Hobart 7001, Australia
b
ARC Centre of Excellence for Electromaterials Sciences (ACES), School of Natural
Sciences, University of Tasmania, Hobart, Tasmania 7001, Australia
c
Diagnostic Devices Unit, Leitat Technology Center, Innovació 2, Terrassa,
Barcelona 08225, Spain. E-mail: jmcabot@leitat.org
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1lc00179e
specific extraction methods to isolate sufficiently abundant
NAs from limited volumes of original sample are crucial for
improved downstream detection and analysis.
Conventional NAs extraction and purification methods are
mainly achieved by various liquid-phase extraction (LPE)10–12
and solid-phase extraction methods (SPE).13–16 However, most
LPE and SPE methods are typically labour-intensive, requiring
well-trained operators to complete a sequence of tedious steps
(e.g., mixing, centrifugation and separation), which prohibits
automation. Most also require large sample volumes from
hundreds of microliters to several millilitres and involve the
use of toxic reagents (phenol, chloroform, EtBr, etc.).17 In
recent years, microfluidic devices18–22 have been developed to
reduce sample consumption and enable limited automation.
For example, Thompson et al. incorporated the EA1 enzyme
into a hybrid laminated polyester and PMMA layered device
for DNA extraction in 3 minutes from a swab sample.23
Campos et al. developed a UV/O3 activated polymer-based
microfluidic solid-phase extraction device for the selective
extraction of cell-free DNA from plasma with recoveries
>90%.24 Despite these recent advances in miniaturization
and automation of NA extraction, significant challenges
remain, such as the need for highly specific materials for
target isolation, the need for biocompatible surface
chemistries, and the requirement for complex micro-pumping
and valving systems. With this said, the development of low-
cost, rapid, automated, and user-friendly methods for NA
isolation continues to be an opportunity of great significance.

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Paper
Gel electrophoresis and capillary electrophoresis are
considered gold standard techniques for the separation,
isolation, and processing of DNA, as both deliver advantages
of high efficiency, high throughput, reproducibility and
automation. As a mode of electrophoresis, isotachophoresis
(ITP) has gained increasing popularity for the selective
separation and concentration of NAs over recent years.17,25–28
This method applies an electric field to control the
movement of target molecules within a discontinuous
electrolyte system, established by terminating electrolyte (TE)
ions, whose moves slower than the sample ions, and leading
electrolyte (LE) ions which moves faster than the sample
ions. For example, Wu et al. reported a disposable integrated
microfluidic device for the separation and pre-concentration
of DNA fragments with ITP hyphenated gel electrophoresis,
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followed by electroelution on-chip for extraction and
purification.29 In addition, a low-cost paper-based
microfluidic device was developed by Rosenfeld et al. for
DNA focussing using electroosmotically balanced ITP, which
delivered an 20 000-fold increase in concentration within a
processing time of ∼6 min.30 Indeed, ITP has been shown to
generally deliver high selectivity, sensitivity and robustness,
providing potential enrichment factors up to one million-
fold.31
Although some paper- and microchip-based ITP methods
have demonstrated feasibility and potential for extraction of
several DNA strands and biomarker analysis,30,32–35 the
search for ever simpler or indeed robust alternative
approaches remains a hot topic. Thus herein, we present a
novel cost-effective, disposable microfluidic thread-based ITP
focussing method for the extraction and preconcentration of
DNA from within biological samples. Threads do not require
hydrophobic wax barriers like in paper-based ITP
methods,30,36,37 and they have no problems with air bubbles,
channel obstruction or clogging, as often the case in
microfluidic chips with closed channels. The approach
described herein improves subsequent qPCR detection
methods without the requirement for complex sample
treatments. This microscale thread-based device (μTAD) for
ITP was based upon commercially available nylon thread
held within a simple 3D printed platform. A fluorescent dye
was used as a model to investigate ITP focussing efficacy on-
thread. The effectiveness of our proposed thread-based
approach for DNA extraction was evaluated by the
determination of lambda DNA in whole blood, with the
subsequent qPCR results demonstrating effective purification
via the ITP process.
2. Experimental section
2.1 Reagents and materials
Tris-(hydroxyl methyl) amino-methane (TRIS), 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
hydrochloric acid, and fluorescein sodium salt, each of
analytical reagent grade, were obtained from Sigma-Aldrich
(New South Wales, Australia). Lambda DNA and SYBR™
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Green PCR Master Mix were purchased from Thermo
Scientific™ (Victoria, Australia). The forward (5′-CGGCGTCAA
AAAGAACTTCC-3′) and reserve (5′- GCATCCTGAATGCAGCCA
TA-3′) primer sequence reagents were purchased from
Integrated DNA Technologies (Coralville, IA USA). Solutions
were prepared in water from a Milli-Q Water Plus system
from Millipore (Bedford, MA, USA), with a resistivity of 18.2
MΩ cm.
100% nylon (diameter (Ø) 500 μm, woolly nylon stretches
overlocking thread, QA Thread, China) was used for thread-
based microfluidics, due to its superior electrophoretic
performance (electroosmotic flow of 6.09 × 10−8 m2 V−1 s−1),38
and physical uniformity. Diameters of the wetted threads
were measured in 10 different samples using an objective-
type inverted microscope (Nikon Eclipse TE2000). Threads
were washed in Milli-Q water and sonicated for 10 minutes 3
times to remove impurities on the surface. Subsequently,
threads were plasma treated by a vacuum plasma reactor
(K1050X plasma asher (Quorum Emitech, UK)) to increase
the wicking property,39,40 facilitating the application of the
sample.
2.2 Microfluidic thread-based analytical devices (μTAD)
fabrication
A PMMA base of 12 cm × 8 cm × 0.5 cm (width × depth ×
height) was prepared using a commercial laser cutting system
(Full Spectrum, Las Vegas, NV, USA) (or 3D printed when
stated), to enable the arrangement of the thread-based
microfluidics system. The base was accurately designed using
Draftsight CAD software (SolidWorks Corp., Dassault
Systemes, France) to fit a microscope slide support and a
Dino-Lite handheld digital microscope (AM4113T-GFBW,
Clarkson, WA, Australia). Two square holes were also cut
uniformly in the left and right side of the platform, providing
positions for the installation of buffer reservoirs.
Buffer reservoirs (Fig. 1a) were designed with SolidWorks
CAD software (SolidWorks Corp., Dassault Systemes, France)
and printed using an Eden 260VS 3D printer (Stratasys, MN,
USA) with VeroClear build material, and SUP707 water
soluble support. The support material was removed by
agitation in 2% NaOH for 2 hours, followed by water for 4–6
hours. Finally, buffer reservoirs were rinsed and subsequently
soaked for 1 day in Milli-Q water. The buffer reservoirs were
removable and could be ultrasonically washed with 2%
NaOH, followed by water rinse, to reuse them. The design of
each buffer reservoir was according to our previous work.38
Briefly, each buffer reservoir has a ring to tie in the thread,
two symmetrical horizontal rollers to guide the thread
downwards, allowing immersion in the small volume of
buffer, two perpendicular cylinders to support the electrodes,
a basin for the storage of buffer with a maximum volume of
500 μL, a convex squared column (5 mm depth 3 mm
squared) at the bottom to hold into the platform.
The platform assembly involved just two steps (Fig. 1b).
Firstly, inserting buffer reservoirs into the PMMA base
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Fig. 1 Design, photograph, and schematic of thread-based ITP experiment. a) CAD design of buffer reservoir. b) Photograph of the assembled 3D
printed fluidic platform for thread-based ITP. c) Schematic of the distribution of leading, terminating, target solute ions and the placement of the
electrodes.
according to the desired thread length. Secondly, tightening
of the thread across to each buffer reservoir in the following
order. Namely, the thread enters from the front part in a
straight line, passes directly underneath the horizontal
rollers, and then is tied off using the loop at the back. In all
experiments, threads were kept wet with buffer solutions
(leading electrolyte) during the separation process.
2.3 Experimental setup
Fluorescence images were obtained using a USB microscope
AM4113T-GFBW (Dino-Lite Premier, Clarkson, WA, Australia)
equipped with a blue light-emitting diode for excitation and a
510 nm emission filter. The microscope was controlled using
DinoCapture 2.0 software. For the quantification of
fluorescent targets, fluorescence intensities of images and
videos were processed with ImageJ software, analysing the
region of interest (ROI) and then monitoring the mean
fluorescence intensity value of the ROI versus time.
Background fluorescence was deducted by subtracting the
blank. All the electromigration images were obtained using an
inverted fluorescence microscope (Ti-U, Nikon, Tokyo, Japan)
integrated with a Nikon high-definition colour charge-coupled
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device (CCD) camera head (Digital Sight DS-Fi1c, Nikon,
Japan) and operated with NIS-Elements BR 3.10 software
(Melville, NY, USA). The electropherogram was acquired by a
photomultiplier tube (PMT) (Hamamatsu Photonics KK,
Hamamatsu, Japan) connected to the microscope. Data
acquisition was made using an Agilent interface (35900E)
connected to a laptop and operated by Agilent ChemStation
software (Agilent Technologies, Waldbronn, Germany). The
microscope objective was fixed at 30X and the focused thread
images were obtained by adjusting the distance. The
numerical aperture was 0.5. The exposure time was controlled
by the luma value and was set at 74.
An in-house built four channel high voltage power supply
(CSL Electronic Workshop, University of Tasmania, Hobart,
Australia) was used for the introduction of voltage for all the
thread-based ITP experiments. Experiments were carried out
in anodic mode, in which the cathode was placed within the
inlet buffer reservoir and the anode was in the outlet
reservoir. The system was controlled using a 12-Bit, 10 KS/s
multifunction DAQ system (USB-6008 OEM, National
Instruments, Austin, TX, USA). A Rotor-Gene Q 5plex Platform
(Qiagen, Germantown, USA) was used as the qPCR cycler with
green channel (excitation 470 ± 10 and 510 ± 5 nm).
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2.4 Thread-based isotachophoresis operation
A schematic of the thread-based ITP process is shown as
Fig. 1c. Two buffer solutions were prepared for the ITP
experiments, specifically LE and TE. 5 mM TRIS/2.5 mM
HEPES was used as the TE solution and 20 mM TRIS/10
mM HCl as the LE solution. Chloride was selected as main
anion for LE (high mobility ion) and HEPES for TE (low
mobility ion). Therefore, all negatively charged molecules
with mobilities higher than TE ions and lower than LE ions
will be focused between the LE and TE boundary, including
the model molecules used here (lambda-DNA and
fluorescein). It should be noted that other DNA strands with
apparent mobilities between TE and LE would also be
focused within this zone. After assembling the thread-based
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ITP setup and inserting the platinum electrodes, the thread
was soaked with LE and 500 μL of TE-analyte mixture was
placed in the inlet (left chamber), and 500 μL LE in the
outlet (right chamber) (Fig. 1). Before applying any voltage,
this system was kept for 1 minute to achieve equilibration,
reducing any capillary action along the thread. Finally,
constant voltage or current was applied to initiate the ITP
process.
For the investigation of focussing and stacking,
fluorescein (sodium salt) was used as the model analyte due
to its high quantum yield. A continuous injection approach
was applied in each experiment, adding the analyte into the
TE solution at a concentration of 1 pg μL−1 within the left
reservoir. Within comparison to a finite sample injection
approach, where samples are introduced by manual pipetting
onto a wetted fibre, the continuous injection mode provides
greater reproducibility.30 A constant current of + 300 μA was
applied for all experiments, unless stated otherwise. For the
determination of recovery experiment with lambda-DNA
focussing, a range of 0.001–10 pg μL−1 (20–2 × 105 copies per
μL approximately) of lambda-DNA was mixed with TE
solution. The DNA present within the TE solution was
gradually depleted from the reservoir and focussed into a
narrow band upon the thread between reservoirs throughout
the ITP process. To monitor ITP band, DNA was previously
labelled with SYTO 9 (0.5 μM) – a dsDNA-specific fluorescent
dye. To collect the purified DNA, the thread was cut with
scissors between x = 4.5 and 5.5 cm, after focussing for 5
min, after which time the focussed band position is known
to be centred at x = 5.00 ± 0.24 cm (n = 6). Standard deviation
was calculated from repeated focussing experimental curves,
as shown in section 3.2. To extract the solution from the
thread, the cut section was introduced into a small empty
Eppendorf tube impaled by a needle (0.9 mm diameter,
extraction tube) and the latter into a larger empty Eppendorf
(receiving tub). Subsequently, both Eppendorf tubes were
centrifugated at 2000 rpm for 2 min. The solution held
within the thread was then separated from the nylon and
collected in the larger Eppendorf for posterior qPCR analysis.
The precise arrangement of the two Eppendorfs is detailed in
section 3.2.
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2.5 Whole blood lysate
The lysate of whole blood samples were prepared as per
previous works with slight modification.32 Briefly, cell lysate
was carried out using only 25 μL of fresh human blood from
a volunteer and diluted up to 500 μL with lysis buffer. For
preparation of the lysate sample, this solution was then held
at 65 degrees for 10 min within a water bath. The lysis buffer
was composed of the TE solution, with 1% of Triton x-100
(non-ionic surfactant), 1% of proteinase K, and 40 mM of
dithiothreitol as a reducing agent. The final blood lysate
solution was kept on ice until required.
2.6 Quantitative polymerase chain reaction (qPCR)
Off-thread qPCR was applied for the detection and
verification of extracted DNA, concentrated using thread-
based ITP. Composition of the PCR solution consisted of 10
μL μM SYBR Green PCR master mix, obtained from Thermo
Fisher Scientific, 1 μL of 10 μM lambda F, 1 μL of 10 μM
lambda R, and 8 μL of sample (with lambda DNA going from
0.001–100 pg μL−1 (20–2 × 106 copies per μL) in LE). The total
volume used for the qPCR processing was 20 μL. DNA
primers were from Integrated DNA technologies (IDT,
Coralville, Iowa, USA). DNase free water and materials were
used in all experiments. qPCR was performed on a Rotor-
gene Q system, with the following thermal profile: initial
heating at 95 °C for 3 minutes, followed by 40 cycles,
including 10 s denaturation at 95 °C,15 s annealing at 60 °C,
and 20 s extension at 72 °C. The fluorescence was measured
after denaturation and extension in each cycle and the PCR
was stopped after 40 cycles.
3. Results and discussion
3.1 ITP focussing on microfluidic thread-based analytical
devices
ITP is an electrophoretic method applied to electrokinetically
isolate and preconcentrate ions at the TE/LE boundary. The
technique is a powerful approach to isolate target analytes
from within complex sample matrices, separated by
differential size and charge. ITP is highly dependent upon
analyte mobility and the ionic conductance of the LE and TE
buffers, as well as the applied voltage. For a given distance,
target solute ions accumulation rate (stacking) is higher and
solute focussing zone migrates faster, when higher currents
are generated. However, the solute ions accumulation rate
plateaus at certain high currents, whereupon dispersion
effects begin to dominate.41 In addition, high currents induce
Joule heating, which can dramatically affect the separation
process, causing such problems as water evaporation, band
broadening, and even burnout of the thread itself.42
Consequently, herein we designed a platform with a thread
length of 6 cm and maximum thread diameter of 500 μm.
After tightening of the thread across the platform, the set-up
allows for the generation of a low current of 300 μA, at which
the adverse effects produced by Joule heating were reduced
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to a negligible level. Obviously, lower electric currents can
also be applied, but at the expense of higher focussing times.
Khurana et al. reported that reducing the TE conductivity
(buffer concentration) increases the local field strength in TE
zone and thus results in a higher influx of target solute ions
into the TE/LE interface.41 However, the buffering capacity is
also reduced and thus there is a simultaneous reduction in
repeatability and robustness. In addition, too low a TE
concentration could potentially cause the thread to overheat
and even burn at the TE zone. Herein, we chose HEPES
(mobility: 23.50 cm2 V−1 s−1) as trailing ion and a mixture of
5 mM TRIS/2.5 mM HEPES as the TE solution, to maintain a
balance of high solute focussing rates and sufficient buffer
capacity. In ITP, the solute focussing zone migration speed is
determined by mobility of leading ions and by the local
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concentration. However, it does not show a significant
increase in the influx of solute ions into the focussing zone
because the difference in mobilities between solute ions and
TE decreases due to the influence of high ionic strength,
resulting in a lower focussing rate. Therefore, we chose Cl−
(mobility: 79.10 cm2 V−1 s−1) as the leading ion and a mixture
of 20 mM TRIS/10 mM HCl was used as the LE, to obtain a
high ionic solute focussing rate and appropriate migration
velocity. DNA is negatively charge due to phosphate with a
mobility faster than HEPES and slower than Cl−, thus DNA
ions will be stacked at the boundary between the TE and the
LE. As this work was performed in anodic mode, positive and
neutral molecules do not migrate onto the thread and
therefore remain in the TE reservoir.
On-thread ITP was demonstrated herein using a

electric field in the leading zone (LE). Bočker et al. suggested
that, under given conditions, LE concentration above a
certain critical value is required to facilitate the
isotachophoretic process, as at low LE concentrations
hydroxyl groups could penetrate from TE zone into LE zone
and negatively affect the isotachophoretic migration.43 The
stacking increases slightly with the increasing LE
fluorescent model compound (negatively charged
fluorescein). Fig. 2a shows a thread isotachopherogram of
fluorescein (1 pg μL−1), obtained by focussing the fluorescent
microscope at a point on the thread 5 cm from the TE
reservoir. A focussed narrow solute band was observed, with
the focussed fluorescein band migrating past the thread
detection point (5 cm) after 300 s. After passage of its ITP

Fig. 2 a) Isotachopherogram on thread of 1 pg μL−1 fluorescent dye (fluorescein). Inverted fluorescent microscope was placed at 5 cm from the
TE reservoir and signal was measured by a PMT connected to the objective and process by Agilent software. The narrow band can be seen at 300
s from the start of the electrophoretic process. b) Concentration factor (Cf) of the solute band as a function of the time, c) signal intensity
(maximum) and bandwidth of solute band versus ITP running time and d) raw intensity images corresponding to focussing time. 1 pg μL−1 of
fluorescein was continuously injected from a 500 μL reservoir. The intensity of the plots b) and c) were calculate using ImageJ and the pictures
were taken from the handheld microscope. A constant voltage of + 900 V was applied for these experiments. Scale bars are 1 mm and data from
B to D were treated with image processing (ImageJ).

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zone, there was an increase in background signal due to the

continuous injection of fluorescein from the TE reservoir.
Fig. 2b shows the concentration factor (Cf) of fluorescent dye
at the ITP interface as a function of time during ITP
focussing along the thread. The data was collected every 30 s.
The concentration factor increased with time, reaching over
600-fold in 200 s. The Cf was not measured after 200 s due to
the saturation of the detector signal. Cf was calculated by
Cpeak/C0, being Cpeak the concentration at the maximum
intensity of the band and C0 the initial concentration of
analyte at the TE. Cpeak was calculated from a standard Fig. 3 Plot of lambda-DNA band position versus time. Solid lines
calibration curve obtained by measuring the intensity of 5 represent the results (five repeats), with data were collected at
different threads wetted with a known concentration of intervals of 2.5 mm. Dotted line is a linear function fitted to the
fluorescein within the range of 50 to 1000 pg uL−1. The slope, average of experimental data, showing that the sample plug position is
approx. Proportional to the experimental time.
intercept and r 2 were 0.3155, 4.851, and 0.9894, respectively.
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The intensity from the picture's ROI was converted to

concentration of fluorescein. These results demonstrate the
successful preconcentration and isolation of fluorescein from
the TE buffer reservoir. The experiment confirmed that
anionic targets could be concentrated from the TE buffer
reservoir upon a 6 cm long thread using ITP. Both fluorescent
signal intensity and bandwidth of the solute band were also
monitored throughout the ITP process (Fig. 2c and d). As
above, signal increased with the ITP running time.
Nevertheless, no significant increase in the signal intensity
was observed post-200 s due to the saturation of the detector
signal, as commented before. The width of the fluorescein
band first increased to a maximum and then decreased as
the plug migrated towards the LE reservoir. Here we see the
solute anions migrate to the TE/LE boundary at a uniform
velocity, and so the solute bandwidth increases with the
amount of target solutes extracted from the TE reservoir.
After the ions reach to the boundary, the migration rate slows
due to the difference in local electric field, wherein solute
ions are focussed in a narrow band between the TE and LE.
3.2 DNA purification and concentration on thread
The extraction of lambda DNA – an E. coli bacteriophage – was
used to demonstrate the applicability of the μTAD for the
purification and concentration of DNA. Initially, the TE buffer
was spiked with a concentration of 1 pg μL−1 of lambda-DNA.
Upon initiation of ITP, the on-thread position of the DNA band
was monitored with time and the migration velocity obtained.
The data was collected from 0 cm to 6 cm following 5 replicate
runs and it is shown within Fig. 3. The migration and position
of the DNA band corresponded linearly with time, with an
average velocity of 10.4 ± 0.5 mm min−1 along the thread. This
figure shows that the velocity of the band along the thread is
relatively constant with time and it is in accordance with
Khurana's theory that target solute ions migrate in joint zones
at the same speed, determined by the mobility of leading ion
and the applied electric field.41 This observation is significant,
as it allows for accurate prediction of the position of the solute
band without the need to continuously monitor or indeed label
with fluorescent tags.
Herein the actual migration of fluorescently labelled DNA
along the nylon thread can be seen within with ESI† as Movie
M1. As shown in Fig. 4a, following ITP focussing, the final
focussed DNA bandwidth was less than 500 μm. This isolated
DNA band could then be cut out from the thread and
analysed using qPCR, according to the procedure detailed in
section 2.4. Fig. 4b illustrates the Eppendorf arrangement for
DNA extraction and collection from the cut section of nylon
thread.
3.3 DNA extraction from nylon thread
qPCR is a powerful analytical method for the quantification
of gene expression, however, the existence of inhibitory
compounds in complex biological (and environmental)
samples can be detrimental to the success of PCR
amplification. PCR inhibitors can impact amplification
through reducing the DNA polymerase activity or binding
with the nucleic acids.44 Some substances can also inhibit
the effectiveness of a PCR assay by quenching the
fluorescence of double-stranded (ds) DNA binding dyes.45
Fig. 4 a) Photographs of lambda-DNA after ITP focussing on nylon
thread. b) Illustration of the set-up for centrifugal DNA extraction from
nylon thread. A 0.2 μL Eppendorf (small) with the wetted thread was
placed inside another one of 0.5 mL (large). The 200 μL Eppendorf was
previously impaled by a needle. For a better illustration, the water
contained in the thread was mixed with purple food dye. After
centrifugation, the purple solution was separated from the thread.

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Herein it was first necessary to confirm that the use of
nylon itself, as the ITP substrate, does not affect the
subsequent amplification of DNA, and that DNA can be
quantitatively extracted from the nylon surface. To confirm
this, 1 cm lengths of nylon thread were soaked for 1 h in
standard solutions (n = 5) spiked with lambda-DNA, at
concentrations between 0.001–10 pg μL−1. Subsequently, the
solution retained within the thread was separated by
centrifugation, as detailed within the Experimental section.
These solutions were then analysed using qPCR and
compared with fresh lambda-DNA standard solutions. The
results in the form of intensity versus number of cycle plots
are shown in Fig. 5a. The recoveries were calculated fixing
the Ct (threshold cycle) value at 0.6. The extraction efficiency,
(recovery) shown in Fig. 5b from the thread material
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remained consistently between 94.4–113.9% across the
complete range of concentrations explored, giving an average
of 102.1 ± 7.7%. Therefore, it can be concluded that any
Fig. 5 qPCR curves from the amplification of lambda-DNA. a). qPCR
analysis for lambda-DNA at different concentration from 0.001–10 pg
μL−1 (20–2 × 105 copies per μL). The black solid lines represent qPCR of
extracted DNA from thread using centrifugation and dotted lines are
the direct amplification of standard solutions of DNA. The grey solid
line is the amplification of master mix with 1 pg μL−1 (2 × 104 copies
per μL) of lambda DNA together with a thread of 0.5 cm long. The grey
dashed line is the blank without DNA and contained the leading
electrolyte, the master mix, and a thread of 0.5 cm long. b) The
recovery after extraction of DNA from thread material. The recovery
remained consistently between 94.4–113.9% across the 4 orders of
magnitude, giving a consistent average of 102.1 ± 7.7%.
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specific adsorption on nylon was negligible during the DNA
extraction.
Direct PCR without removing the thread clearly inhibited
the amplification as it can be seen in the grey solid line of
Fig. 5a. The nylon thread itself may quench the fluorescence
of the DNA amplification causing a significant decrease in
the intensity. Therefore, it is necessary to separate the sample
liquid from the nylon thread before performing qPCR
analysis.
3.4 Purification and determination of λDNA in whole blood
As a proof of concept, microfluidic thread-based ITP
focussing was used for the determination of 500 fg μL−1 (104
copies per μL) spiked lambda-DNA in whole blood. Fig. 6
shows the qPCR results of on-thread ITP purified DNA and
qPCR of unprocessed blood lysate. The on-thread ITP
extracted DNA was successfully amplified, just before the 30
cycles with a recovery of 97.4%. Whereas the unprocessed
lysate showed inhibited or low amplification. The
amplification was negligible with the blank (leading
electrolyte with master mix and no DNA) after 40 cycles.
Blood dilution can sometimes weaken the effect of
inhibitory compounds. However, it has the drawback of
having to reduce the concentration of the target DNA as well.
In this work, even a 100-fold dilution of the lysate was not
enough. Fig. 6 shows that direct amplification of diluted
blood lysate provided a signal improvement, however, the
intensity of DNA remained below threshold even after 40
cycles of amplification. This demonstrated the need of a
Fig. 6 qPCR analysis for lambda-DNA. Blue lines represent the
amplification of ITP DNA extract in blood lysate (light blue) and ITP
DNA extract from 100 times diluted blood lysate (dark blue). Dotted
lines (black) correspond to the standard qPCR calibration at 6 different
concentrations going from 0.001 to 100 pg μL−1 (20–2 × 106 copies per
μL). Direct qPCR amplification of DNA in human blood lysate (red) and
lysate diluted 100 times (yellow). In green are blank DNA with leading
electrolyte (free of lambda DNA). Human blood sample was spike with
500 fg μL−1 (104 copies per μL) of targeted DNA. Diluted sample was
diluted 100-fold with trailing electrolyte.
Lab Chip, 2021, 21, 2565–2573 | 2571

Paper
clean-up method to separate interfering substances in sample
matrices. The on-thread ITP was able to exclude the neutral,
positive charged or those with negative charge with lower
electrophoretic mobility than terminating ions from the
blood sample. The DNA blanks from the LE buffer (6 green
solid lines) did not reach the threshold.
Furthermore, the nylon thread not only acted as a
separation channel, but also as a size-exclusion filter for large
particles within blood. Compared with chip or capillary based
ITP focussing, in which the ITP zones must be eluted and
reach their respective reservoirs for post collection, this μTAD
has the considerable advantage of facile subsequent DNA
collection, not only by cutting the desired section, but also to
the direct access to the concentrated DNA material. In
addition, this μTAD was performed under ambient conditions
Published on 07 May 2021. Downloaded on 2/2/2023 9:02:40 PM.
and the solutes were separated on the surface of thread,
which enables the combination of this method with ambient
mass spectrometry (e.g., desorption electrospray ionization)
for fast high throughput detection.46 This experiment can also
be extrapolated to DNA based biomarkers determination in
liquid biopsy for cancer diagnosis, prognosis prediction and
drug resistance monitoring. For example, the study of DNA
methylation in sputum and blood for the early detection of
lung cancer showed the potential to reduce mortality rates.47
Usually, larger sampling volume are required for these
diagnostics. This could be addressed by using larger buffer
reservoirs, reduced dilution effect on the mixture between TE
and sample, or even placing the outlet reservoirs in higher
positions to introduce a counter flow to prolong the DNA
extraction and stacking time. Apart from these benefits, due
to the low-cost and ease of fabrication of such thread-based
ITP devices, high throughput analysis could be achieved using
multiplexed platform, in which the multiple buffer reservoirs
share the same electrode, as stated in our previous work.38
4. Conclusion
A low-cost and versatile microfluidic platform was developed
using commercially available threads for the purification and
preconcentration of low abundant nucleic acids in biological
samples. A fluorescein sodium salt was used as the model for
the study of focussing behaviour of ITP on thread. A
focussing ratio of 600 was achieved within 5 minutes under
optimised experimental conditions. The advantage of threads
for the concentrated solute collection was also showed.
Separated solutes were collected by simply cutting and
centrifuging of the threads for further analysis. As a proof-of-
concept study, this thread-based ITP was applied for the
extraction of lambda DNA from whole blood, with
applicability to the determination of biomarkers in clinical
diagnostics for tracking the spread of infections or liquid
biopsy. This technique showed good performance in the
selective extraction and preconcentrate of the target
biomolecules from whole blood, demonstrating the ability for
the development of affordable analytical and diagnostic
devices. As no complicated and expensive equipment was
2572 | Lab Chip, 2021, 21, 2565–2573
View Article Online
Lab on a Chip
required, this approach also has great potential in the
development of multiplexed platform, allowing the
simultaneous processing of different samples in parallel.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work is supported by Australian Research Council (ARC)
Centre of Excellence for Electromaterials Science (ACES)
(Project CE140100012) and ARC Discovery Project
DP170102572. JMC acknowledges the European Union's
Horizon 2020 research and innovation programme under
Marie Skłodowska-Curie 801342 (TecniospringINDUSTRY)
and the Government of Catalonia's Agency for Business
Competitiveness (ACCIÓ). The authors thank the service from
Central Science Laboratory (CSL) for providing qPCR
equipment and 3D printed facilities specially Sharee
McCammon and Adam Smolenski.
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