Journal of Chromatography A 1704 (2023) 464117

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Review article

Microfluidic paper and thread-based separations: Chromatography and

electrophoresis
Bahram Hemmateenejad∗ , Elmira Rafatmah, Zahra Shojaeifard
Chemistry Department, Shiraz University, Shiraz, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Paper and thread are widely used as the substrates for fabricating low-cost, disposable, and portable mi-
Received 6 April 2023 crofluidic analytical devices used in clinical, environmental, and food safety monitoring. Concerning sepa-
Revised 25 May 2023
ration methods including chromatography and electrophoresis, these substrates provide unique platforms
Accepted 30 May 2023
for developing portable devices. This review focuses on summarizing recent research on the miniatur-
Available online 2 June 2023
ization of the separation techniques using paper and thread. Preconcentration, purification, desalination,
Keywords: and separation of various analytes are achievable using electrophoresis and chromatography methods in-
Paper tegrated with modified or unmodified paper/thread wicking channels. A variety of 2D and 3D designs of
Thread paper/thread platforms for zone electrophoresis, capillary electrophoresis, and modified/unmodified chro-
Chromatography matography are discussed with emphasis on their limitation and improvements. The current progress in
Electrophoresis the signal amplification strategies such as isoelectric focusing, isotachophoresis, ion concentration polar-
Microfluidic
ization, isoelectric focusing, and stacking methods in paper-based devices are reviewed. Different strate-
gies for chromatographic separations based on paper/thread will be explained. The separation of target
species from complex samples and their determination by integration with other analytical methods like
spectroscopy and electrochemistry are well-listed. Furthermore, the innovations for plasma and cell sep-
aration from blood as an important human biofluid are presented, and the related paper/thread modifi-
cation methods are explored.
© 2023 Elsevier B.V. All rights reserved.

1. Introduction substrates by different driving forces such as wicking action, grav-

There is a large demand for portable diagnostic equipment for
point-of-care testing and for measurements at remote areas with
limited health infrastructure and resource restrictions. Microfluidic
devices, which are worked based on the manipulation and con-
trol of fluids physically confined to submillimeter dimensions, can
be considered an ideal platform to fabricate portable diagnostic
devices. Conventional microfluidic devices are fabricated by ma-
nipulating microchannels inside the substrates like glass or dif-
ferent plastics and the solutions flow through channels using ex-
ternal pumps. Considering global health, the use of environmen-
tally friendly substrates has attracted much attention. On the other
hand, to be compatible with point-of-care testing, devices without
the need to power sources (such as electric pumps) are preferred.
Paper and thread are two biocompatible, accessible, portable and
low-cost substrates, which have widely been used for fabricating
microfluidic devices. In microfluidic paper or thread-based analyt-
ical devices (μPADs and μTADs), the fluid can transport on these
∗
Corresponding author.
E-mail address: hemmatb@shirazu.ac.ir (B. Hemmateenejad).
itational force, and electrophoretic force [1,2].
Microfluidic analytical devices can be applied as sensors or
biosensors with a different applications in point-of-care diagnos-
tic [3], health monitoring [4], environmental [5,6], and food safety
[7,8]. Accordingly, different analytical sensing strategies based on
the paper substrate have been invented [9–11]. Analytical chemists
used the wicking feature of the paper to develop lateral flow as-
says, most specially immunoassays [12,13]. Using different paper
patterning methods, one can create multi-channels microfluidics
for multi-assays too. Multi-channel paper microfluidics has also
been used to fabricate electronic tongues on the paper substrate,
where an array of cross-reactive chemo-sensor reagents are de-
posited at the end of the created hydrophilic channels on the pa-
per. Such devices produce multivariate data, which are analyzed by
the chemometrics method to extract unique signature patterns for
the individual analytes or systems of complex mixtures of the an-
alytes [5,14,15].
Paper-based titration can be considered as an innovative ap-
plication of paper microfluidics. In these techniques, all titration
steps are installed on a single piece of patterned paper. Hydrophilic
channels are created on the paper substrate in a flower (star)-like
shape. Standard solutions and indicators are pre-deposited at the

https://doi.org/10.1016/j.chroma.2023.464117
0021-9673/© 2023 Elsevier B.V. All rights reserved.
B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard Journal of Chromatography A 1704 (2023) 464117

end of the channels. Titration is started by adding some microliters the previous articles, which were relevant to the topic of this re-
amount of sample solution to the center of the device, which fol- view, were also considered. Since thread-based microfluidics share
low through the channels, and reach to the standard solutions and very similar experimental basis and analytical features with paper-
the indicators [16,17]. based methods, we will review thread-based separation methods
Similar to paper-based electronic tongues, paper-based elec- too. The review will begin with electrophoresis and then will be
tronic noses have also found special interest in sensitive and se- continued with chromatography. In recent years, special attention
lective detection of the volatile compounds emitted from different has been noted toward the separation of blood cells and blood
sources (foods, environment, or biological specimens) [18–21]. For plasma based on paper and thread microfluidics. Therefore, at the
example, different paper-based optical nose devices have been fab- end of the article, recent advances in this important topic will be
ricated to detect and identify volatile organic compounds emitted reviewed too.
from human urine, blood, or exalted breath aiming to detect dif-
ferent kinds of cancer, non-invasively [22,23].
2. Electrophoresis
Besides to development of sensing and diagnostic devices based
on a paper substrate, the paper has a long history in separation
Electrophoresis is the migration and separation of charged ions
and chromatography. Cellulose-based supports became one of the
under the influence of an electric field. Separation, enrichment, de-
popular stationary phases in separation techniques since the dis-
salination, and purification of target analytes are achievable by this
covery of paper chromatography in the 1940s [24]. Paper is ex-
method. There are different electrophoresis techniques according
tremely cheap, biocompatible, easy to employ, store, and transport.
to their supporting media, instrumentation, or separation mech-
In addition, its thinness and flexibility make it possible to be used
anism. Electrophoresis can be classified into gel electrophoresis,
in different designs. Moreover, chemical modification of the paper
zone electrophoresis, free-flow electrophoresis, and capillary elec-
surface, due to the chemical reactivity of the hydroxyl groups on
trophoresis. Each group can be divided into subcategory techniques
the paper surface, makes it an ideal candidate for preparing mod-
as depicted in Fig. 1.
ified stationary phases. The wicking ability of paper/thread sub-
Supporting media in gel electrophoresis can be agarose or poly-
strates eliminates the need for pumps to drive fluid flow. The pos-
acrylamide gel while solid supporting material such as paper or
sibility of the flow through capillary action in these substrates can
cellulose acetate is used for zone electrophoresis. Free-flow elec-
simply reduce the device cost and also complexity.
trophoresis is conducted in liquid media while capillary elec-
Passive fluid transport in paper-based devices is controlled by
trophoresis is performed in a capillary tube [29]. Like other ana-
the fabrication of hydrophobic barriers and hydrophilic channels.
lytical techniques, paper/thread-based electrophoresis microfluidics
A wide range of paper patterning had been reported that can be
have been investigated by various research teams. Paper/thread-
classified into physical and chemical methods. Hydrophobic barri-
based electrophoresis has the advantage over chromatography-
ers can be patterned by physical methods such as wax pattern-
wicking devices due to its resolving power [30]. The initial reports
ing, plotting, inkjet etching, flexographic printing, stamping, cut-
on the paper-based electrophoresis platform go back to 2014. The
ting and shaping, screen printing, spraying, and drawing. As an al-
developed origami paper-based electrophoretic device was oper-
ternative to physical methods, photolithography, plasma treatment,
ated at low voltages due to its thin configuration. A driving voltage
chemical vapor-phase deposition, and wet etching can be used for
of 10 V was enough for the separation of bovine serum proteins
chemical paper hydrophobization [25–27]. Considering the advan-
and fluorescent molecules through the Whatman paper layers [31].
tages of paper chromatography and μPAD technology, their combi-
In the same year, the separation and measurement of serine, aspar-
nation was introduced as one of the significant methods for sepa-
tic acid, and lysine amino acids by coupling the wireless electro-
rating various analytes. The separated species can be detected and
generated chemiluminescence on the copper/gold bipolar electrode
determined by various analytical techniques such as electrochem-
with paper-based electrophoresis was also reported [32].
istry, colorimetry, and mass spectroscopy.
The development of electrophoresis devices continued from
μTADs have significant advantages such as high mechanical
then on. Various materials including cellulose, cellulose acetate,
strength, flexibility, and high performance without needing any hy-
glass/cotton fibers, mineral, and nitrocellulose paper substrates
drophobic barrier and modification capability in wet conditions.
have been used in the fabrication of electrophoresis channels.
Accordingly, attentions have been directed toward using thread as
Moreover, nylon, polyester, and cotton threads were the other op-
an alternative substrate to papers. Threads can be divided into
tions for the design and fabrication of microfluidic capillary elec-
two groups based on their preparation: Natural (cotton, wool, silk,
trophoresis sensing devices [33–37]. A numerical model for the
linen, etc.) or artificial (nylon, polyester, polyurethane, polyethy-
electromigration in paper-based electrophoresis and electrophoret-
lene, polypropylene, acrylic, etc.). The production of natural threads
ically driven dispersive transport mechanism was also introduced
is more complicated, time-consuming, and leads to irregular sur-
and validated [38].
face morphologies than manufactured threads with regular and
smoother surfaces [28]. Both natural threads and artificial threads
are applicable in designing μTADs. However, in selecting the type 2.1. Zone electrophoresis
of thread, some parameters such as their surface chemistry, struc-
ture, and morphology should be considered. Similar to paper, the The most popular geometry used in paper-based zone elec-
surface chemistry features of threads can be improved by modi- trophoresis is the crossed-channel design. The sample injection
fications. Accordingly, μTADs have become one of the interesting is performed in the horizontal line of “T” geometry and separa-
substrates and stationary phases for separation due to their mi- tion will be completed in the vertical line. Control of fluid by
crofluidic properties, and modifiability, requiring small samples, electrokinetic flow for multistep electrophoretic separation (sample
reagents, or mobile phase. However, researches in microfluidic loading/injection, and electrophoretic separation) on the crossed-
thread-based chromatography has not grown as paper-based de- channel paper-based device was reported for the first time in
vices did. 2016. The effect of buffer properties such as pH and concentra-
In this article, we will review the separation methods, most tion, channel width, and separation time on the electrophore-
importantly chromatographic and electrophoresis, based on paper sis separation efficiency was studied and discussed [39]. Low ad-
and thread. Our search was based on Scopus database and we fo- sorption filter membrane materials including WhatmanTM cellulose
cused on papers that published in 2015 or later. However, some of acetate, DuraporeTM polyvinylidene fluoride, and MFMilliporeTM

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B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard Journal of Chromatography A 1704 (2023) 464117

Fig. 1. Electrophoresis classification based on the supporting media.

Fig. 2. (A) Schematic representation of the laminated paper-based setup for zonal electrophoresis (adapted with permission from [40]), (B) The structure of paper-based
electrophoresis with electrodes for capacitively coupled contactless conductivity measurements (reprinted with permission from [41]), origami structure of electrophoresis
device for separation of pI markers (reprinted with permission from [42]).

mixed cellulose ester membranes were all evaluated for zonal elec-
trophoretic separations. Filter membrane substrates were cut by a
CO2 direct write laser to reduce the total thermal mass of the de-
vice. A laminate structure (Fig. 2(A)) with cross-T geometries and
an applied voltage of 400 V was used for amino acid separations.
The lamination of the device improved the heat dissipation while
the more porous substrate contributes to conductive heat dissipa-
tion. The possibility of fluorescence detection in these substrates
was studied using fluorescein and Nile blue. According to this re-
search, the WhatmanTM cellulose acetate membrane showed a bet-
ter response for fluorescence detection and electrophoretic separa-
tion compared to cellulose ester (MF-Millipore), and polyvinylidene
fluoride (Durapore PVDF) substrates [40].
A capacitively coupled contactless conductivity detection
method coupled with cross-shape geometry channels made by
Whatman chromatographic paper was used for separation of
bovine serum albumin and creatinine. As shown in Fig. 2(B),
the paper substrate was cut by laser and then laminated.
Pencil-drawn electrodes were used for injection and separa-
tion steps. The position of electrodes, running electrolyte, and

3
B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard
Fig. 3. (A) The commercial HemeChip reader, and (B) Gazelle-Multispectral. Reprinted with permission from [45,46].
type of paper were optimized for attaining better separation
resolution [41].
Later on, an origami paper-based electrophoresis separation
with a low applied voltage of 5 V was developed with Whatman
filter paper for pI markers in 2019. The pH distribution of the de-
vice was visualized by pH test paper. It was shown that a long
run time will shift the pH value in the channel. The sample was
dropped in the center of the origami structure (Fig. 2(C)) and the
separation was followed by fluorescent imaging [42].
The material used for the hydrophobic channel barriers can af-
fect the flow, zeta potential, and therefore the peak broadening.
This was concluded from the electrophoretic laminated parafilm
paper used for zone electrophoresis with colorimetric and fluores-
cence detection. Chlorophenol red and indigo carmine with a sep-
aration potential of −600 V, fluorescein isothiocyanate (FITC), and
FITC-labeled l-glutamic acid (FITC-Glu) with a separation potential
of −450 V were successfully detected. To control the effect of Joule
heating on the Whatman filter paper, pre-wetting time and applied
voltage were optimized [43].
Recently, paper electrophoresis has been available in the form
of commercial devices. Paper-based microchip electrophoresis was
developed and used for the separation and determination of gly-
cosylated hemoglobin components as an indicator of the glucose
level in the blood. The sensor was based on affinity electrophore-
sis and used a plastic-backed cellulose acetate paper strip for sep-
aration. An external power source of 70 V was applied and a cus-
tomized data analysis algorithm was written for the quantifica-
tion of glycosylated hemoglobin from the captured UV images. The
designed sensor had been commercialized as the HemeChip mi-
crochip electrophoresis platform with the product name Gazelle
[44].
Gurkan et al. reported the methodologies for characterizing the
HemeChip system (Fig. 3(A)) of pH and temperature shifts during
the electrophoretic separations. Temperature and pH shift or gra-
dients in the separation channel can affect the accuracy and re-
producibility of the electrophoresis platforms. Water electrolysis at
electrode surfaces and joule heating effects can change the pH,
temperature, and separation stability. Temperature and pH-tracking
systems with the aim of digital and infrared imaging systems were
4
Journal of Chromatography A 1704 (2023) 464117
used for obtaining calibration curves and investigation of tempera-
ture and pH distributions. The effects of applied potentials, electro-
chemical reactions, and conduction/diffusion on the non-ideal pH
and temperature shifts was discussed. It was shown that the pre-
treatment of the paper can mitigate the pH and temperature shift
[45].
The Gazelle-Multispectral platform (Fig. 3(B)) is the next gen-
eration of commercial Gazelle electrophoresis. Multispectral imag-
ing had been combined with Gazelle paper-based hemoglobin
electrophoresis for newborn hemoglobin variant screening. Whole
blood specimens were analyzed for separation and determina-
tion of hemoglobin variants. Normal hemoglobin A (Hb A), sickle
hemoglobin S (Hb S), Hemoglobin C, and fetal hemoglobin (Hb
F) were separated based on their charge-to-mass ratio at pH 8.4.
The image capturing system and data analysis algorithm integrated
with the device can automatically quantify the percentages of
hemoglobin variants [46].
Since Wei’s report on thread-based electrophoresis in 2012
[47,48], researches have continued in this area. Thread-based zone
electrophoresis using various threads was studied for separation
and fluorescence determination of riboflavin in urine samples. Ny-
lon bundle (NYL), silk (SE), cotton (CO), wool (WO), acrylic (AC),
50% acrylic/50% nylon (AC/NYL), polyester (PES), and waxed dental
tape (WT) were checked for the effect of polarity, chemical compo-
sition, porosity and their swelling properties on thread-based zone
electrophoresis. A nylon bundle showed the best condition for the
electrophoresis process [35]. Also, electrophoresis features suitable
for classroom demonstrations were developed by Cai et al. for the
separation of food dyes (carmine and sunset yellow). In this report,
paper and cotton threads were used for sample loading whereas
platinum electrodes were applied for separation steps [49,50].
2.2. Capillary electrophoresis
Pretreatment and preconcentration of samples in paper/thread-
based devices can effectively enhance the sensitivity of the mea-
suring device. Capillary electrophoresis and related techniques
such as isoelectric focusing, field amplified sample stacking, ion
concentration polarization, and isotachophoresis can be conducted
B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard
Fig. 4. (A) Structure and fabrication of cross-designed paper-based capillary electrophoresis with polyacrylamide gel sieving matrix (adapted with permission from [53]). (B)
representation of paper-based semi-dry electrophoresis (reprinted with permission from [54]).
simultaneously by wicking microfluidics for preconcentration, sep-
aration, and determination. That’s why these methods are cate-
gorized as online preconcentration techniques [51]. As an exam-
ple of paper-based capillary electrophoresis, the separation of viral
long dsRNA from long and short dsRNA mixtures was reported by
Jeong et al. in 2021. Long viral double-stranded RNA can be used
as a virus marker in infectious disease diagnostics. Au anode and
cathode, a polylactic acid housing, and a paper separation channel
made from Whatman cellulose chromatography paper were com-
ponents of the electrophoresis device. Due to the inverse relation-
ship between the electrophoretic mobility and the molecular size,
long RNAs moved slower in the channel. The electric field and the
channel width were optimized for obtaining narrow peaks and bet-
ter separation resolution. Weak electrophoretic mobility and strong
electroosmotic flow in too low or too high electric fields can gener-
ate wide and ambiguous bands. The authors confirmed the width-
dependent electrophoretic behavior of RNA. Large amounts of RNA
could’nt pass through the narrow channels and diffusion of short
ssRNA could result in peak broadening in too wide channels [52].
In the following subsections, different branches of the capillary
electrophoresis (as depicted in Fig. 1) will be explained, separately.
2.2.1. Gel electrophoresis
Cross-designed electrophoresis made from mineral paper and
polyacrylamide gel sieving matrix was used for polymerase chain
reaction amplicons of Escherichia coli O157:H7 and Staphylococ-
cus aureus (Fig. 4(A)). With the separation voltage of 1500 V, the
fluorescence signal of the analytes was followed [53]. In another
research, Hennig et al. introduced an open paper-based semi-dry
electrophoresis approach, which was impressively faster than a
conventional slab-gel protein electrophoresis. An agarose gel-based
sieving matrix was printed into a cellulose acetate membrane for
electrophoresis (application of a constant voltage of 100 V through
the carbon pins for 15 min). Post-electrophoretic immunoprob-
ing was also performed by transferring the separated proteins
onto a protein-binding poly(vinylidene difluoride) membrane. The
schematic representation of the semi-dry electrophoresis device is
shown in Fig. 4(B) [54].
2.2.2. Ion concentration polarization (ICP)
Different mobilities of ions in ion-selective membranes un-
der an electric field provide the appropriate condition for ion
concentration polarization. Ion depletion and enrichment zones
are formed due to selective charge transport. The first reports
on paper-based ICP date back to 2015, when Hepatitis B virus
DNA targets were detected by nitrocellulose paper-based ICP
(Fig. 5(A)). Three modes of the device including transport with-
out ICP, switching-polarity ICP, and direct ICP were tested. Since
5
Journal of Chromatography A 1704 (2023) 464117
the competition between electrophoretic migration and electroos-
motic flow defines the net movement of anions, the behavior of
electrophoretic/electroosmotic flow and the separation process was
discussed in each mode [55].
Nafion is the most used interface material in ICP. In 2016, the
separated Nafion layer instead of pipetting/infiltrating was used in
paper-based ICP in order to improve the stability of the lateral
fluid flow [56]. In the continuation of developments in this field,
the applied voltage for ICP processes was reduced by the three-
dimensional (3D) design of the paper-based device. A 100-fold
enhancement factor was achieved for fluorescein isothiocyanate
with an external driving voltage of 40 V [57]. The 3D design of
paper-based ICP (Fig. 5(B)) was reported for nondestructive isola-
tion of microvesicle/exosome biomarkers, simultaneous enrichment
and spatial separation of microspheres [58–60].
The application of ion exchange membranes for ICP in paper-
based devices was first introduced by Gao et al. in 2018 [61]. Also,
Wu et al. showed that a simultaneous electric field and pH gra-
dient can result in the enrichment and separation of ionic and
amphoteric species in the paper substrate. In order to produce
pH variation through the channel, a polymeric cation exchange
membrane was placed on the glass fiber paper channel instead
of Nafion [62]. Concurrency of the electric field and pH gradient
in the channels was also introduced in a cross-type ICP interface
for stable clean-up and preconcentration of salty samples such as
blood and urine. Detection was achieved by cutting the final stack-
ing bands and rinsing them for mass spectrometry analyses. The
reported setup can be applied for the purification of salty samples
even with NaCl concentrations up to 400 mM [63].
2.2.3. Isotachophoresis
Leading and trailing of electrolytes with their respective ions
have high and low magnitudes of mobility in the presence of an
electric field, respectively. According to this mobility and electro-
migration variation, an electric field gradient will be formed in the
interface between the electrolytes. The selective focusing of sam-
ple ions can be achieved through the presence of the electric field
gradient in the channel [64]. Isolation and determination of target
species such as bacteria, proteins, and blood nucleic acid had been
investigated using microfluidic isotachophoresis [65,66].
Similar to the other capillary electrophoresis techniques,
origami paper-based device produced the availability of low-
voltage isotachophoresis enrichment with improved collection and
extraction efficiency. DNA focusing with a paper-based isota-
chophoresis device was presented by Crooks et al. in 2015 with a
3D design [67]. Another DNA determination was discussed by an-
ionic stationary nitrocellulose paper-based isotachophoresis. Elec-
troosmotic flow and high zeta potential of nitrocellulose repre-
B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard
Fig. 5. Schematic representation of paper-based ICP device (A), paper-based isotachophoresis device (B), paper-based fast isoelectric focusing (C), 3D origami ICP precon-
centrator (D), thread-based isotachophoresis (E), paper isoelectric focusing with inkjet-printed silver electrodes (F), thread-based capillary electrophoresis (G), thread-based
capillary electrophoresis platform with capacitively coupled contactless conductivity detection (H), and thread-based electrophoretic device with electrochemical detection
(I). (Reprinted with permission from [47,55,58,69–71,81–83]).
sented an enhancement effect on the isotachophoresis focusing.
Different shapes of electrodes for operating at lower voltages were
investigated. Simultaneous analysis of 12 different analytes by mul-
tiplexing the platform have been discussed [68]. The electroki-
netic focusing of both positive and negative-charged plasma pro-
teins with a cationic isotachophoresis approach was studied, re-
cently. The reported investigations showed that the shorter the pa-
per channel length, the more productive concentrating effect will
be. The isoelectric point change of the complex formed by im-
munobinding of target protein with antibody was approved. This
produced the condition to modify the electromobility of analytes
in order to focus the mixture of charged analytes simultaneously.
The representation of the device setup is provided in Fig. 5(C) [69].
2.2.4. Isoelectric focusing
In the presence of an electric field and a pH gradient, ampho-
teric molecules such as proteins can be separated according to dif-
ference in their isoelectric point. The pH gradients are generated
using carrier ampholytes (different zwitterions with various iso-
electric points) although ampholytes-free platforms are also possi-
ble. The paper-based isoelectric focusing devices were firstly fab-
ricated with inkjet-printed silver electrodes and Whatman chro-
matography paper by Jokinen et al. aiming to enrich and sepa-
rate cytochrome c and myoglobin. Minimization of the evaporation
rate during the period of separation time was reported using the
absorbent pads in the cathode and anode poles (Fig. 5(D)). Joule
heating was also controlled by stepwise increasing the focusing
voltage [70].
Ampholyte-free isoelectric focusing on a paper-based analyt-
ical device was introduced by Yang et al., who used a tunable
pH gradient, induced by a DC voltage. The effect of paper sub-
strates and electroosmotic flow was visually investigated and opti-
mized. Desalination of protein high-salt samples was essential for
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Journal of Chromatography A 1704 (2023) 464117
this experiment. Also, this developed setup (Fig. 5(E)) was cou-
pled with matrix-assisted laser desorption ionization time-of-flight
mass spectrometry for offline protein identifications [71,72]. Sep-
aration and enrichment of proteins in multi-paper channels us-
ing an isoelectric focusing mechanism was reported by Yu et al.
A paper sheet of 0.5 mm thickness was used for eliminating the
evaporation and drying of the sample solution during the analy-
sis. Polyvinylpyrrolidone was added to the solution to suppress the
electroosmotic flow, sample adsorption, and band broadening. To
maintain a balance between migration time and the produced elec-
tric heat, a low pre-focusing voltage and a high focusing voltage
were applied in an stepwise manner [73].
Low-voltage separation and enrichment of proteins in both low-
salt and high-salt samples were achieved very recently using a 3D
paper-based isoelectric focusing. The 3D separation channel and
reservoirs were made from cellulose acetate membranes (with a
low protein adsorption feature) and chromatography paper, respec-
tively. A stable pH gradient and electromigration was achieved by a
3D configuration of the device. Clinical urine microalbumin and C-
reactive protein were investigated using this platform. Colorimetric
and coupled lateral flow strip was used for quantifications of the
analyte [36].
2.2.5. Field amplified sample stacking
Among electrophoretic stacking techniques based on concentra-
tion adjustment, field-amplified sample stacking is the most re-
ported method for sample enrichment in paper-based devices. By
the presence of a conductivity gradient in the background elec-
trolyte with high conductivity, the sample will be concentrated
into a short zone. The first report on running of field amplified
sample stacking on the paper substrate was done by Ma et al.,
who utilized a battery-driven power supply, platinum wire elec-
trodes, glass fiber paper, and a smartphone fluorescence imaging
B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard
system for the construction of the setup. DNA preconcentration
with 10 0 0-fold signal improvement was achieved [74]. The mech-
anism of field-amplified sample stacking on paper was further in-
vestigated by this research group. They showed that the acid-base
titration reaction of the ions in the background electrolyte and wa-
ter electrolysis has the main role in the establishment of the elec-
tric field gradient [75]. There are other reports from this group
about field amplified sample stacking on paper substrate. The fluo-
rescent whitening agent in a napkin and toilet paper was detected
with a 160-fold enhanced factor [76]. The possibility of the elec-
trokinetic stacking of electrically neutral analytes was reported by
the same authors. By the formation of the micelle band on the pa-
per fluidic channel, analyte species are swept by the micelle. Rho-
damine B as the visual probe was detected with 30-fold enhance-
ment in sensitivity [76].
Field-amplified sample stacking was applied for preconcentra-
tion before electrophoresis measurement on the Whatman chro-
matography paper chip. A 266-fold enhancement in the detection
limit for metal ions determination was achieved utilizing a hy-
bridized field-amplified sample stacking with a moving reaction
boundary [77]. Stacking and separation of food dyes and macro-
molecule proteins on the glass fiber paper were done and op-
timized by controlling the level of electroosmotic flow and the
electric field gradient. The modifiers of polyvinylpyrrolidone and
hydroxyethylcellulose were used to suppress the electroosmotic
flow. The electric field gradient was controlled by aiming the back-
ground electrolyte concentration [78]. Simultaneous electrokinetic
stacking and separation of both anionic and cationic species were
reported by Liu et al. By using a background electrolyte contain-
ing a weak acid and a weak base salt, two field gradients were
developed in the glass fiber paper channel. The importance of the
type of background electrolyte, the effect of electroosmotic flow,
and stacking efficiency were studied and discussed [79].
Electrokinetic pre-concentration by electrokinetic stacking on
a paper-based microfluidic device using glass fiber paper and a
multi-channel star-shape design was introduced a year ago. Eight
T-shaped copper-clad gold electrodes were used as the edge elec-
trodes. A voltage of 190 V was applied through the central elec-
trode and these edge electrodes. Stacking bands of the anionic ana-
lytes can be formed in each channel. It was shown that by decreas-
ing the liquid capacity of the cathode reservoirs, stacking can be
completed in a shorter time. Pigments from beverages and amino
acids from serum were tested by this device applying the colori-
metric or mass spectrometry (MS/MS) detection methods [37].
Thread-based capillary electrophoresis for separation and quan-
tification of chloric, bromic, and iodic ions was developed by many
researchers (Fig. 5(F)). They applied polyester threads for thread-
based microfluidic electrophoresis. The effects of thread diame-
ter and surface treatment were studied. Wettability and electro-
osmotic flow mobility of the threads were modified by oxygen
plasma. It was shown that plasma treatment of polyester threads
can improve the Joule heating effect [47]. Also, commercial eye
drops and energy drinks of Red Bull ingredients were analyzed
by a polyester thread microfluidic electrophoretic system with
mass spectroscopy detection. Electrospray ionization was applied
for ionizing the separated species at the end of the channel. Sput-
tered copper and gold layers were used as electrodes on the PMMA
substrate [80].
In further studies, capacitively coupled contactless conductivity
detection was used for thread-based capillary electrophoresis by
Quero et al. in 2019 (Fig. 5(G)). Separation and detection of cations
(sodium, potassium, and lithium ions) were studied. An injection
voltage of +700 V and separation voltage of 1 kV was performed
using platinum electrodes while enameled copper wires were af-
fixed for conductivity detection. The authors demonstrated elec-
troosmotic flow inversion in textile threads for the first time [82].
7
Journal of Chromatography A 1704 (2023) 464117
The effects of buffer conductivity, pH, and separation voltage on
the electroosmotic flow was studied using thermal imaging. White
100% polyester threads were used for separation and electrochem-
ical investigation of the pyocyanin as a P. aeruginosa marker and
methylene blue as a probe of pathogen detection (Fig. 5(H)). Pen-
cil graphite electrodes were used in the end-channel arrangement
of the thread-based platform. It was shown that higher electrolyte
concentrations increased the electroosmotic flow variability [81].
There are few reports on using threads as the channel for
isotachophoresis. Thread-based isoelectric focusing was coupled
with on-thread desorption electrospray ionization mass spectrom-
etry for the separation and enrichment of proteins and alkaloids
(Fig. 5(I)). The effect of thread moisture, trapping thread knot ma-
terial, and diameter on the device performance have been stud-
ied [83,84]. Ambient ionization mass spectrometry in combination
with isotachophoresis using nano-functionalized threads channel
was reported in 2022. The nylon thread modified by graphene ox-
ide was used for trapping and determination of the urine alkaloids
with high sensitivity [85].
3. Chromatography
In chromatography, the separation is achieved by differential in-
teraction of the analytes with the stationary and mobile phases.
Because of the wicking character of paper or thread, irrespec-
tive of electrophoresis and the other liquid chromatographic meth-
ods, the paper/thread-based chromatography does not require ex-
ternal power or pump. The mobile phase, which flows automati-
cally through paper/thread pores, carries the analytes in the sam-
ple pre-deposited on the substrate. The important advantage of
microfluidic paper-based chromatography with respect to conven-
tional paper chromatography is using a very low amount of mo-
bile phase. Also, the possibility of miniaturization and automation
should be considered too. As mentioned earlier, one of the great
advantages of paper chromatography is the simplicity of provid-
ing a chemically-modified paper substrate. In the next subsections,
the developed chromatographic separation methods on the modi-
fied and unmodified paper/thread substrates will be reviewed, sep-
arately.
3.1. Unmodified paper/thread-based chromatography
The first microfluidic paper-based chromatography has been re-
ported by Carvalhal et al. [86] in 2010. They coupled patterned
Whatman cellulose paper (grade 1) as a chromatographic column
into the interface of a thin-layer gold electrochemical microcell
for the separation and detection of ascorbic acids and uric acids.
Fig. 6(A) shows the integrated device containing the chromato-
graphic and electrochemical parts. After spotting 2 μL of the sam-
ple’s solution onto the paper, it soaked in the mobile phase (ac-
etate buffer) to follow through the paper by capillary action. Ac-
cording to the affinity of analytes toward the eluent and paper, the
separation proceeds throughout the paper until reaching the elec-
trodes and is detected by the amperometric method. In a similar
study, this group used the Whatman P81 paper in a combination
with gold electrodes for the separation and detection of paraceta-
mol and 4-aminophenol [87]. Murphy et al. [88] applied a sim-
ilar integrated separation and electrochemical device considering
the effect of physical properties of different filter paper substrates
for the separation and detection of dopamine and ascorbic acid.
Various cellulose filter papers from different manufacturers such
as Whatman (Whatman 4, 5, and P81) and VWR (VWR 413) were
used to construct the μPCs. The role of surface carboxyl groups
as weak ion-exchangers in the analyte separation that is changed
with the paper grade and type of manufacture was explained.
B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard
Fig. 6. Schematic representation of integrated the paper-based separation and electrochemical device (A), TLC and PAD connector separation design coupled by colorimetric
method (B), folding μPAD to generate a hydrophilic channel for separation of food additive (C), microfluidic MCD chip and UV microanalysis device (D). Reproduced with
permission from ref. [82,89–91].
Similar to previous works, separation of a free dye (pure fluo-
rescein isothiocyanate) from a protein-dye mixture (pure fluores-
cein isothiocyanate-BSA) was achieved based on their size in the
common channel dimension. Simple geometry designs and cap-
illary action have been used to control the flow of the mobile
phase through channels [89]. Also, the chromatographic paper in-
tegrated electrochemical detection method was used to separate
9 -tetrahydrocannabinol from cannabidiol of cannabis oil without
any interference. The electrochemical detection parts were contain-
ing cobalt-phthalocyanine modified screen-printed graphene elec-
trodes embedded on the chromatographic paper as separation part.
Microliter volume of mobile phase (20% methanol in hexane) was
applied and separation was completed after 20 min. In contrast to
previous research, the separated parts were cut and eluted with
phosphate buffer solution (pH 7) to move them from the chro-
matography paper into the electrodes as the detection part [90].
Picking a separate spot for post-analysis might decrease the mi-
crofluidic paper-based chromatography resolution which makes it
undesirable in comparison to similar methods.
An interesting approach in microfluidic paper-based separation
methods is their integration with mass spectroscopy for sensitive
detections. Lin group [91] designed a 2D paper-based microflu-
idic cassette to separate, enriched, and also detect the separated
analytes using electrospray ionization mass spectroscopy. The de-
signed cassette was produced with a 3D printer containing a mi-
crofluidic paper holder, a sample inlet trench, and point contact for
developing a buffer inlet. To increase the ion intensity up to 100-
fold in MS, the developing solution guided to the slot channels on
the side wing to elute with a second eluent for 2nd-dimensional
paper chromatography. The concentrated sample plugs were di-
rected to the paper electrospray ionization. Paper electrospray ion-
8
Journal of Chromatography A 1704 (2023) 464117
ization tip containing electrodes were contacted on the filter paper
to induce an electric field for electrospray ionization. Finally, the
ionized separated analytes were screened by MS. This method was
used for the separation and determination of pesticides in fresh
green onion [91], and caffeine and vitamin B6 and its metabolites
in the urine samples [92].
The paper chromatography devices can be coupled by colori-
metric method for easy, chip, and fast detection of the separated
analytes. For example, Dawan et al. [93] used a combination of
TLC and paper chromatography to separate and colorimetrically de-
termine capsaicinoids in chili extract. The TLC sheet was embed-
ded in a hexane-ethyl acetate (50:50, v/v) chromatographic cham-
ber to separate the analytes. In order to detect the separated cap-
saicinoids, the TLC part was contacted to a pre-deposited Folin-
Ciocalteu reagent onto a circular Whatman filter paper via a PVC
sheet connector. The TLC paper was immersed in ethanol to elute
the capsaicinoids from the TLC plate to the reagents in circular pa-
per yielding the blue color due to the interaction of phenol with
the Folin-Ciocalteu reagent (Fig. 6(B)).
In order to get high resolution in multi-dimensional microflu-
idic paper-based chromatography, and avoid picking the sepa-
rated spots for post-colorimetric determination, Gharaghani et al.
[94] designed a three-dimensional microfluidic device. The de-
signed origami device contained 23 layers of folded paper. A hy-
drophilic microchannel was embedded in the middle of the papers
as a column for the analyte and mobile phase injection and inter-
action. The unfolded μPADs and the color intensity of the two azo
dyes at a certain paper layer, demonstrated the separation of ana-
lytes (Fig. 6C). The device was used for separation and determina-
tion of two food additive azo dyes (Tartrazine and Indigo carmine)
in commercial food products. They decreased the volume of the
B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard
Fig. 7. Schematic representation of modified paper-based substrate with silane functional groups (A), phenyl isocyanate (B) and Ehrlich’s reagent (C). (Reprinted with per-
mission from ref [96–98]).
mobile phase to ∼12 μL and separation time to less than a minute
by their proposed design.
In further studies, the microfluidic paper-based chromatography
were coupled with a UV microanalysis device to separate and de-
termine two common artificial sweeteners, saccharin sodium and
acesulfame potassium in 16 commercial food samples. The Advan-
tec chromatography paper as the stationary phase and the mixture
of water, ethyl acetate, acetic acid, and ethanol as the mobile phase
were used (12 μL). The images of the separated SAC and Ace-K
were captured by a CMOS camera while illuminated by a UV light
source (254 nm) (Fig. 6(D)). To evaluate the efficiency of the sep-
aration method, theoretical plate number (Nplate) and resolution
(Rs) were investigated [95].
3.2. Modified microfluidic paper and thread-based analytical devices
In order to simulate the liquid stationary phase, paper-based
substrates can be modified by polar and non-polar functional
groups. Hashemi Hedeshi et al. [96] used different silane func-
tional groups modified paper-based substrates for extracting the
antidepressant drugs, organophosphates, and triazine toxins with
different polarity. A sandwiched-format microfluidic platform was
designed to hold unmodified/modified paper sheets for han-
dling aquatic samples. Among four different silane-based reagents
namely hexadecyltrimethoxysilane (HDTMS), phenyltrimethoxysi-
lane (PTES), (3-aminopropyl) triethoxysilane (APTES), and 3–(2,3–
epoxypropoxy) propyltrimethoxysilane (EPPTMOS), PTES showed
the best performance. As shown in Fig. 7(A), the sample solutions
were transferred into the square-wave-shaped channel of microflu-
idic by a syringe pump. The extracts of the microfluidic device
were then transferred to gas chromatography-mass spectrometry
for quantitation of the analytes.
Non-covalent modifications with hydrophilic and hydropho-
bic materials cannot support long-term stability for normal and
reverse-phase stationary phases. In addition, most of the eluents
have affinities with them. In this regard, Ochiai and his workers
[97], investigated the effect of hydrophobic covalent modifiers for
the separation of various dyes to simulate a reverse HPLC sepa-
ration in paper chromatography. They investigated the efficiency
of separation in filter papers that were modified with phenyl iso-
cyanate (PI), acetic anhydride, and benzoyl chloride under con-
trolled conditions. Among the investigated modifiers, PI showed
the best effect on separation due to its higher hydrophobicity
which caused the larger difference in the polarity of the eluents.
Also, the behavior of a thread stationary phase that is modified
with PI for the separation of brilliant Blue and tartrazine has been
studied. As expected, the PI-modified threat demonstrated greater
separation of the dyes compared to the unmodified one (Fig. 7(B)).
A dip and read modified paper-based analytical device for col-
orimetric separation and quantitative analysis of indole in shrimp
was designed using cellulose paper modified with Ehrlich’s reagent
consisting of 4-(dimethylamino) benzaldehyde and pdimethy-
9
Journal of Chromatography A 1704 (2023) 464117
laminobenzaldehyde to produce a violet product with indole. As
shown in Fig. 7(C), by immersing the modified paper in the ethyl
acetate extracted solution of shrimp, the solvent flow through the
reaction zone, producing the violet color product of indole while
the interfering orange-colored astaxanthin was separated and ac-
cumulated in the corners of the paper [98].
In 2018, Agustini et al. [99] proposed an integrated microflu-
idic thread-based electroanalytical system for the separation of
dopamine and ascorbic acid in tear samples through an ion ex-
change mechanism. The treated cotton thread was used as a sub-
stitute for the chromatography column. The treatment was based
on the dehydration of citric acid solution under heating to incor-
porate two free carboxyl groups through an esterification reaction
with the hydroxyl groups of the cellulose surface. To detect the
separated analytes, two electrodes were embedded in a specific
portion of the modified thread by gold deposition. In this method,
the mobile phase flowed by the capillary action and gravitational
forces. Also, the authors separated ascorbic acid and epinephrine
by a modified thread and determined them by gold electrodes.
4. Blood separation using μPADs and μTADs
Human biofluid samples, such as urine [100], blood, tear
[101] and saliva [102] have the potential to offer accurate and inva-
sive approach to identify the health condition. Among the bioflu-
ids, blood is the most studied human biofluid used as the health
indicator [103]. For example, FDA has approved the blood tests that
are used in clinical diagnosis of diabetes, HIV, anemia, cancer coro-
nary heart disease, and etc. [104–106]. Most of the biochemical
tests are usually carried out in serum or plasma that lead to the re-
quiring of a step for separating interference of the blood cells, most
importantly the red blood cells (RBCs), from the plasma. The con-
ventional RBCs separating methods (centrifugation and filtration)
are time-consuming, requiring large blood volume (mL), expensive
equipment, and skilled workers. As there is a great demand to sim-
plify clinical tests for home measurements. Usually, The first step
for designing lab on chip devices for the whole blood tests is sepa-
rating the plasma form other interference. In this regard, designing
miniaturized separating and diagnostic devices aiming to reduce
the required sample volume, simplify the preparation processing,
and shorten the measuring time, are of high significance in clinical
chemistry investigations.
Microfluidics devices are one of the interesting alternative can-
didates toward the conventional method that can provide an ap-
propriate platform for RBCs separation and plasma extraction due
to their inexpensive, less blood volume requirement, and fast re-
sponse time. Microfluidic blood cell separation is divided into two
active and passive separation methods. Active separation methods
applied external forces such as magnetic force, acoustic force, or
dielectrophoretic force to control the blood fluid in a specific di-
rection. While passive microfluidic separation methods are based
on the physical properties (shape, size, or rigidity) of cells [107].
B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard Journal of Chromatography A 1704 (2023) 464117

Table 1
Microfluidic paper and thread-based techniques used for passive blood cell separation from plasma.

Separation Blood
Paper type Modifier Time (s) Volume (μL) Application Detection method ref

μPAD Whatman No. 1 and – 00–200 8–20 – – [108]

MF1, LF1 and VF2
separation membranes
Whatman No. 1 and VF1, – Alkaline phosphatase, Colorimetric [121]
VF2, MF1, Fusion 5, GX, aspartate
GR, GF, separation aminotransferase, and
membranes total serum protein
Whatman No. 1 anti-A, anti-B and 300 7 Glucose assay Colorimetric [112]
anti-D
Whatman No. 1 and – 240 200 Glucose assay Electrochemistry [122]
VF1, VF2 separation
membranes
H channel printed on PBS solution 200 50 Glucose assay Colorimetric [109]
Whatman No. 4
Polysulfone asymmetric 600 70 ferritin, retinol-binding Colorimetric [110]
membrane GR VIVID, FR-1 protein and C-reactive
filter pad, Fusion 5 protein
separation membranes
Whatman No. 4 NaCl 180 Glucose assay Colorimetric [123]
Whatman No. 1, 2, 5 Zinc oxide 600 3 – – [124]
nanorods.
Synthetic paper anti-A, -B, and -AB 316 90 – – [125]
VF2 NaCl 220 300 – – [126]
filter paper
Whatman No. 2 anti-A, anti-D and 120–180 27.5 ABO and Rh blood Colorimetric [113]
anti-B typing -
μTAD silk and cotton Antibody 0.5 – ABO and Rh blood Colorimetric [117]
typing
Polyester thread Antibody 60 2 ABO and Rh blood Colorimetric [118]
typing
cotton EDTA 120 3 albumin assay Colorimetric [119]
cotton NaCl 150 2 therapeutic antibody bioluminescence [120]
cetuximab

Since passive microfluidic separation methods are simpler, cheaper,
have the capability of continues operation, and consider almost all
aspects of a clinical assay, they are more noticeable in further stud-
ies.
We categorize the passive separation techniques into two modi-
fied and unmodified papers and treads used for plasma separation.
Table 1 demonstrates several modified and unmodified microfluidic
paper and thread-based chromatography devices capable of sepa-
rating plasma from whole blood samples. Most unmodified papers
are easy and available candidates that used blood separation mem-
branes for RBC separation.
In 2012, Songjaroen et al. [108] designed a microfluidic device
to separate plasma from the whole blood in a single step and then
quantify plasma proteins (Fig. 8(A)). They used the combination of
a patterned Whatman No.1 paper with different plasma separation
membranes (PSM), three of which including MF1, LF1, and VF2 are
polyvinyl alcohol-bound glass fiber filters, and the fourth mem-
brane, VF1, is a binder-free glass fiber filter. Among them, the LF1
membrane showed better performance in blood separation. Hu-
man serum protein of separated plasma was detected using the
bromocresol green colorimetric assay. In 2015, a two-dimensional
paper-based network was introduced as an H-channel to separate
plasma from whole blood on a simple paper substrate without
needing any modification (Fig. 8(B)) [109]. In further research, a
polysulfone filtration membrane was combined with a differen-
tiation pad with a slightly larger pore size than red blood cells
to slow down its movement toward the serum and prevent the
formation of the sediment layer on the filtration membrane. The
proposed arrangement improved the separation yield by as much
as 80%. Also, another nitrocellulose membrane was considered in
the arrangement as a calibration pad to store the constant per-
meated serum capacity and control output volume, omitting the
need for pipetting (Fig. 8(C)) [110]. In order to avoid agglomeration
of RBCs on embedded PSM in the microfluidic paper, Park et al.
[111] coated the PSM with parylene C to prepare it for 3D printing.
The coated PSM was superimposed on the filter paper (Fig. 8(D)).
A reservoir and a detection zone were printed on the bottom and
top of the filter paper as RBC and plasma holder, respectively.
In the second group of passive microfluidic blood separation
devices, different modification methods were used to replaced
PSM (Table 1). In 2012, Yang and his coworkers [112] proposed
a μPAD modified by spotting agglutinating antibodies (anti-A, B)
for plasma separation. It should be mentioned that the interac-
tion of antibody with its difference antigen leads to the agglu-
tinate. By agglomeration of the RBCs of the whole blood in the
center of the μPAD, the separated plasma is transported laterally
into the periphery of the device. The separated plasma was used
for glucose concentration measurement with colorimetric assay
(Fig. 9(A)).
In another research, antibodies modified three-dimensional
origami paper microfluidic device was used for RGB separating and
ABO and Rh blood type detection [113]. A Whatman paper contain-
ing four hydrophilic circle zones was folded to 13 layers (Fig. 9(B)).
The two first layers were considered as sample and distribution
zones. In layers 3 to 8, three of the circles were treated with an-
tibodies A, B, and D while the fourth circle was the control. The
basis of the proposed design was RBC agglutination through the
interaction of antibodies and related blood antigens. Clearly, the
plasma movement in agglomerated groups was slower than non-
agglutinated RBCs, which caused going through fewer layers. Since
the movements through the layers of each channel were specific
for each blood type, counting the number of layers in which the
blood moved in each channel led to the identification of the blood
type.

10
B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard Journal of Chromatography A 1704 (2023) 464117

Fig. 8. Schematic representation of unmodified microfluidic paper-based chromatography for separation of RBCs from plasma. Paper-based substrate with printed Whatman
paper design (A), two-dimensional H- channels (B) membrane filtration membrane (C), coated the paper separation membrane with parylene C (D). Reproduced with
permission from ref. [108–111].

Fig. 9. Schematic representation of modified microfluidic paper-based chromatography for separation of RBCs from plasma, modified paper-based substrate with antibodies
(A and B), mixture of EDTA and chitosan (C), NaCl modified the VF2 pad (D). (Reproduced with permission from ref. [112–115]).

11
B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard
Fig. 10. Scheme of the fabrication process of the μTAD for plasma separation from whole blood while modifying with related antibodies (A), different method for addition
of EDTA (B), and NaCl. Reproduced with permission from ref. [116,119,120].
Another paper modifier of two-dimensional microfluidic paper-
based chromatography was chitosan which could separate only a
small amount of sample (1 μL) from its plasma. After confirming
the aggregation effect of chitosan, RBC isolation on the mixture
of chitosan and EDTA functionalization in open channels, single
wax barriers, and double wax barriers were evaluated. As shown in
Fig. 9(C), 4 μL of the PBS diluted blood (1 μL of blood and 3 μL of
buffer) was separated better on EDTA-and-chitosan-functionalized
channels than unmodified channels [114].
Burgos-Flórez et al. [115] used two filter papers modified with
NaCl as whole blood collection and plasma retention zones. They
modified the VF2 polyvinyl alcohol-bound glass fiber filter paper
with NaCl to induce RBC aggregation and speed up plasma wicking.
The presence of NaCl generated a hypertonic medium that caused
osmotic pressure on RBCs, losing their water and aggregating. The
VF2 filter paper is conducted to the MF1 polyvinyl alcohol-bound
glass fiber filter paper applied in accumulation and biomarker de-
tection. The microfluidic channel size in the overlapping interface
of two filter papers was reduced such that could induce the re-
moval of the particles sizing larger than 2–3 mm (Fig. 9(D)).
Using a thread-based blood grouping device for separating the
agglutinated RBCs from the plasma was initiated by Shen and
coworkers in 2011 [116]. The basis of their proposed method was
the formation of large and discrete agglutinated RBCs in the inter-
fiber spaces of the antibodies-modified thread. This agglomeration
leads to preventing RBCs’ capillary wicking force while serum con-
tinues wicking forward. Polyester threads were treated with blood
grouping antibodies: Anti-A and anti-B and anti-D. The colorimet-
ric detection of separated plasma was according to the commer-
cial colors of Anti-A (transparent cyan) and anti-B (yellow). Since
anti-D is a clear solution, water-soluble inks (cyan and magenta)
were added for visual detection. The gap between agglomerated
RBC and mixture of dyes with the plasma demonstrated the type
of blood group (Fig 10(A)). In the continuation of the previous
research, this group investigated the effect of different threads
(silk and cotton) with different morphologies on the separation
properties of RBCs for blood typing detection. The silk fiber con-
tains a smooth surface with regular inter-fiber gaps. In contrast
to silk, the cotton threads are made of hollows with a few intra-
12
Journal of Chromatography A 1704 (2023) 464117
fiber gaps on their surface [117]. In parallel research, a polyester
thread-based blood typing method was developed by modifying it
with antibodies and two fluorescent dyes, rhodamine B and flu-
orescein isothiocyanate, to investigate the behavior and location
of RBCs and plasma by a fluorescent confocal microscope, respec-
tively. The results confirmed the first hypothesis regarding the
separation of the whole blood into three RBCs, plasma, and dye
zones [118].
Ulum et al. investigated the effect of Ethylenediaminetetraacetic
acid (EDTA) anticoagulant solution as a modifier of cotton thread
in wicking the whole blood sample and its plasma separation. The
cotton threads were placed in glass slides and 3 μL of whole blood
without and with several ratios of blood-10% EDTA were dropped
on them (Fig. 10(B)). The threads, which were modified with EDTA
and dried in the refrigerator before dropping the blood, demon-
strated a longer wicking length and better blood plasma separa-
tion. The performance and applicability of the separated plasma
were confirmed by comparing it with the conventional method
and albumin assay, respectively [119]. Shimazu et al. [120] pre-
treated the thread with NaCl to separate the plasma of whole
blood for therapeutic antibody cetuximab detection based on bi-
oluminescence resonance energy transfer. The ability of two types
of threads (cotton and vinylon) toward whole blood transportation
was examined. Due to the porosity and wettability of the viny-
lon thread, it showed better blood penetration and longer wick-
ing distance. Also, the effect of three common thread modifiers for
plasma separation namely NaCl, CaCl2 and EDTA was investigated.
In all cases, increasing in concentration of the modifiers caused the
longer plasma distance. However, NaCl was selected due to its high
stability, and plasma separation over a short thread distance. The
designed μTADs platform was used for colorimetric therapeutic an-
tibody cetuximab detection by 2 μL of the whole blood (Fig. 10(C)).
5. Conclusion
The review demonstrates the great potential of paper/thread-
based devices for fabricating miniaturized and portable separation
devices. An interesting aspect of the separation methods based
on paper/thread is easy coupling of which with various detec-
B. Hemmateenejad, E. Rafatmah and Z. Shojaeifard
tion methods, more importantly, electrochemical and colorimetric
methods. By choosing proper designs in electrophoresis, the re-
quired applied potential could be lowered down to the tenth of
volts. Running chromatographic separations in microfluidic forms
could decrease solvent consumption and running time dramati-
cally. Separation of blood plasma from blood cells can be consid-
ered as a very important application of paper/thread-based mi-
crofluidic analytical devices, which offers efficient separations in
some minutes without needing external power source/expensive
instrument. This guarantees the application of such devices in re-
mote areas with low accessibility to clinical facilities.
In spite of great and fast achievements in the paper/thread-
based microfluidic separation methods, there are many open
doors for further improvements. The development of integrated
separation-detection methods for diagnosis is largely demanding.
Since the 3D origami methods represented promising results in
both electrophoresis and chromatographic separations, it is re-
quired to develop methods for precise alignment of the hydrophilic
areas to make separation channels. Furthermore, the separations in
the microfluidic channels of paper depend largely on the geometry
of the patterns used and the type of the utilized method for paper
patterning. In this regard, the edge of the paper, which is in direct
contact with the sample solution/mobile phase, can affect signifi-
cantly the separation process. Therefore, more research is required
to unravel the effect of the paper patterning method on separa-
tion processes. Finally, extension of the current theoretical basis of
the separation process on multilayer paper devices and thread sub-
strates is required.
CRediT authorship contribution statement
Bahram Hemmateenejad: Conceptualization, Supervision,
Writing - review & editing. Zahra Shojaeifard: Methodology,
Investigation, Writing – original draf. Elmira Rafatmah: Method-
ology, Investigation, Writing – original draft.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Data availability
No data was used for the research described in the article.
Acknowledgment
Financial support of this work by Shiraz University Research
Council is highly appreciated.
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