Paper Microfluidics For Nucleic Acid Amplification Testing (NAAT) of Infectious Diseases

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Paper microfluidics for nucleic acid amplification

Cite this: DOI: 10.1039/c7lc00013h
testing (NAAT) of infectious diseases
Laura Magro, a Camille Escadafal,b Pierre Garneret,a Batrice Jacquelin,c
Aurlia Kwasiborski,b Jean-Claude Manuguerra,b Fabrice Monti,a
Anavaj Sakuntabhai,d Jessica Vanhomwegen,b Pierre Lafayee and Patrick Tabeling*a
The diagnosis of infectious diseases is entering a new and interesting phase. Technologies based on paper
microfluidics, coupled to developments in isothermal amplification of Nucleic Acids (NAs) raise opportuni-
ties for bringing the methods of molecular biology in the field, in a low setting environment. A lot of work
has been performed in the domain over the last few years and the landscape of contributions is rich and
diverse. Most often, the level of sample preparation differs, along with the sample nature, the amplification
Received 4th January 2017, and detection methods, and the design of the device, among other features. In this review, we attempt to
Accepted 18th May 2017
offer a structured description of the state of the art. The domain is not mature and there exist bottlenecks
DOI: 10.1039/c7lc00013h
that hamper the realization of Nucleic Acid Amplification Tests (NAATs) complying with the constraints of
the field in low and middle income countries. In this domain however, the pace of progress is impressively
rsc.li/loc fast. This review is written for a broad Lab on a Chip audience.
Introduction
a
MMN, Gulliver Laboratory, UMR CNRS 7083, ESPCI Paris, PSL Research
University, Paris, France. E-mail: patrick.tabeling@espci.fr Performing early diagnosis is critical to prevent the propaga-
b
Laboratory for Urgent Response to Biological Threats, Institut Pasteur, Paris, tion of infectious diseases. This is especially true in emergent
France countries, where difficult living conditions and limited health
c
HIV, Inflammation & Persistence Unit, Institut Pasteur, Paris, France
d care cause diseases to propagate more swiftly. The capacity to
Functional Genetics of Infectious Diseases Unit, CNRS URA3012, Institut Pasteur,
Paris, France perform early detection of an infection has been recognized
e
Antibody Engineering Platform, UtechS proteins, Institut Pasteur, Paris, France as critical in the case of Ebola virus disease. In West Africa,
Laura Magro graduated from a Camille Escadafal trained as a

high-level engineering school biochemistry engineer and
ESPCI Paris and defended her worked for 3 years as a research
PhD thesis entitled Paper engineer at the Institut Pasteur
microfluidics: from liquid flow in Senegal on diagnostics for
studies to Ebola virus diagnos- arboviral infections. She joined a
tics in Guinea. Her fields of 2 year fellowship in Public
interest are very broad at the Health Microbiology from the
interface between physics, chem- European CDC based at the
istry and biology. Her PhD work Robert Koch Institute in Ger-
led to 2 publications, 4 patents many and then completed a PhD
and preliminary validation ex- on the development of diagnostic
Laura Magro periments performed on-field in Camille Escadafal methods for arboviral infections.
Guinea, in the Ebola Treatment In 2014, she joined the Labora-
Center of Macenta, along with the Institut Pasteur and the French tory for Urgent Biological Threats at the Institut Pasteur as coor-
Red Cross. In 2015, Laura received a prestigious award from the dinator of the human virology activities of the MediLabSecure pro-
program L'Oral-UNESCO For women in science. ject and coordinated another project on the development of rapid
tests for epidemic-prone viruses by isothermal amplification.
This journal is The Royal Society of Chemistry 2017 Lab Chip

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Tutorial review Lab on a Chip
an outbreak of Ebola Zaire virus disease caused the death of Chain Reaction (PCR) have been performed.3,4 However, be-
more than 11 000 persons in 2014.1 This was mostly due to cause of the lengthy analysis, the non-transportable, expen-
the difficulty and delay in identifying and isolating infected sive equipment, and the necessity to employ trained person-
persons, owing to a lack of rapid, accurate and portable field nel, only a few institutes in Liberia, Guinea and Sierra Leone
diagnostic tests.2 In this context, molecular detection (the most affected countries) could afford using this technol-
methods can play an essential role. In the case of Ebola virus, ogy. Cost, transport and consumable availability have thus
serological techniques are not adapted because they detect represented limiting factors for providing access to molecular
the presence of the virus only once the immunologic re- diagnostics to most of the population.
sponse has taken off, i.e. after five days of infection (see Assessments by the World Health Organization (WHO)
Fig. 1). This is too late to proceed to isolation and hamper show that similar situations occur with other viral, bacterial
the patients from contaminating their environment. By con- or parasitic infections that propagate throughout the world
trast, Nucleic Acid Amplification Tests (NAATs) have on an even larger scale. Tuberculosis (TB) is one of the most
undisputed potential for detecting the virus in the very first deadly infections though curable if quickly diagnosed and
days of the infection, along with an extremely small rate of treated. In 2015, 9.6 million people were infected and 1.5
false positives. In fact, during the Ebola outbreak, sensitive million died due to TB infection. Of these deaths, 95% oc-
NAATs, based on Reverse Transcription (RT) and Polymerase curred in economically developing countries.5 Malaria
Pierre Garneret is a medical stu- Beatrice Jacquelin joined the

dent currently doing a PhD in Institut Pasteur as a research en-
biotechnology following the gineer to work on HIV/AIDS in
French program EdILB by the Michaela Mller-Trutwin's group
Bettencourt Schueller Founda- in 2004. During her under- and
tion in the MMN laboratory post-graduate training that she
(Patrick Tabeling's team) at the performed at The Scripps Re-
ESPCI Paris. His research is ori- search Institute (USA), at the
ented towards the development CNRS and at the Genopole of
of paper microfluidic devices for Evry (France), she developed a
on field applications. One final strong scientific experience in
goal of his research is to set up gene regulation. The focus of her
Pierre Garneret a PAD allowing a multiplexed Beatrice Jacquelin work now is on either factors of
NAAT for Zika, Dengue and susceptibility to HIV-induced in-
Chikungunya simultaneous detection. His medical background as- flammation or factors that allow its control. In 2014, Beatrice
sociated with a physicists' laboratory gives the double skills re- used her background in nucleic acid research to temporarily join
quired for this topic. the Ebola task force (with Pierre Lafaye) and develop a diagnostic
test.
Pierre Lafaye is head of the Anti- Patrick Tabeling is a physicist,

body Engineering Platform at being a specialist of micro-
the Institut Pasteur. His research fluidics. His book (Introduction
topic concerns camel homo- to microfluidics), is cited 700
dimeric antibodies (known as times. He has published 180 ar-
VHH or nanobodies), analyzing ticles in professional journals,
the use of VHH proteins as an filed 10 patents, and has been
alternative to conventional anti- invited to 80 international con-
bodies. He demonstrated that ferences. He is the director of
VHH are naturally able to cross the Institute Pierre Gilles de
the blood brain barrier and to Gennes (IPGG). He received his
bind to an intracellular antigen. doctorate in 1976 and visited
Pierre Lafaye Pierre's ongoing research concerns Patrick Tabeling the University of Chicago (1984
the ability of VHH to neutralize 1985) and UCLA (19992000).
viral infections and their use as diagnostic tools for neurological and He joined the ENS from 1985 to 2000 where he led a team in the
virological diseases. In 2014, Pierre joined the Pasteur EBOLA task Department of Physics, and then joined ESPCI. He is a member of
force and was in charge of the diagnostics of EBOLA virus. the Academia Europaea.
Lab Chip This journal is The Royal Society of Chemistry 2017

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Lab on a Chip Tutorial review
the loop-mediated isothermal amplification (LAMP) that

operates at 6065 C and shows interesting performances in
terms of amplification rates, sensibility and stability.11 But
other schemes, such as Recombinase Protein Amplification

(RPA)12 or Rolling Circle Amplification (RCA)13 offer the ad-
vantage of working at lower temperatures.
Another innovation that occurred in parallel with the devel-
opment of isothermal amplification technologies is paper
microfluidics.14 This technology was invented by G. M.
Whitesides, and its specificity is to work with extremely low cost
paper base, while, in the meantime integrating functionalities
that are as well-performing and as diverse as those in standard
microfluidic devices. The coupling between paper microfluidics
Fig. 1 Diagnostics spectrum. A high concentration of pathogens and isothermal techniques represents in fact a new cross disci-
makes Ebola virus detectable by NAATs from the very beginning of plinary subject. A surge of articles, including hundreds of publi-
infection whereas antibody detection by immunoassays becomes cations, has appeared in the literature over the last few years.
relevant after several days. Reproduced and adapted from the
They convey the promise of realizing a sensitive NA detection
Center for Disease Control and Prevention.
test adapted to the harsh constraints of the field in a point-of-
care (POC) device. At the moment, the work reported in the lit-
disease is due to a parasite transmitted by mosquitoes. Al- erature uses many different approaches, amplification and de-
though, half of the worldwide population is exposed to a risk tection systems, types of samples and preparation levels, and it
of infection, it principally rages in Sub-Saharan Africa. In is sometimes difficult to appreciate the values of the contribu-
2015, 214 million cases and 438 000 deaths were reported tions in a context where many parameters are involved. The
according to the WHO.6 Here again, early diagnostics allows goal of this review is to establish a comprehensive and orga-
adequate treatment and reduces the illness severity and mor- nized description of the state of the art of the topic.
tality. In tropical and sub-tropical areas, dengue virus is This review is organized as follows: it begins with a brief
transmitted to humans through mosquito bites and each tour of paper microfluidics, focusing on the aspects impor-
infected person can pass it to other mosquitoes. Each year, tant for the core of the review, i.e. the combination between
around 500 000 people are affected by a severe form of the NAATs and paper microfluidics. Then the question of sample
disease with a mortality rate of 2.5%.7 In the last decades, preparation is discussed along with the techniques of ampli-
the number of dengue infections has dramatically grown and fication and detection on paper. Perspectives for the future of
explosive outbreaks have occurred. There is no specific treat- the topics are suggested.
ment but early detection and access to proper medical care
lowers fatality rates below 1%. These considerations under- A brief tour of paper microfluidics
line the need for cheap, rapid, accurate and transportable di-
agnostic tools to perform early diagnosis of infectious dis- Paper microfluidics was pioneered by G. M. Whitesides.15 The
eases in developing countries. Note that this remark also idea was to replace standard microfluidic substrates (glass, sili-
concerns the developed world, since massive gatherings of con, plastics or PDMS) by paper. Paper is a cheap material (its
people favor the propagation of infections, a recent example cost is a few hundredths of a cent per sheet) and is available
being the Zika virus that has been introduced in Brazil dur- worldwide, wherever forests and local paper industries exist.
ing the 2014 Soccer world cup.8 Being burnable, it reduces the risk of contamination. Paper is
Up to a recent time, using molecular biology to detect an thus particularly suitable for diagnostic applications in emer-
infection in limited-resource settings was considered as chal- gent nations. One crucial improvement brought to standard
lenging because the only existing techniques were based on paper-based immuno-assays is patterning (Fig. 2). Ref. 16 dem-
PCR. PCR involves thermal cycling, with a regulation of one onstrated that channels along with liquids that are forced to
or two degrees, along with fluorescence reading. These con- flow, can be designed in a way similar to standard micro-
straints impose sophisticated, costly equipment. The situa- fluidics. Fig. 2 shows an illustration of a micro-Paper Analytical
tion has completely changed with the advent of isothermal Device (PAD) for the detection of glucose and Bovine Serum
amplification techniques that amplify DNA strands without Albumin (BSA) on a model sample of urine.14 With this novel
cycling thermally the system. This represents a major instru- patterning capability, paper microfluidics appeared as a new
mental simplification and shortens the time-to-result. Essen- branch of microfluidic technology, holding several promises,
tially, primer design and/or the introduction of new enzymes while using cheaper and more available materials.
replace the role of PCR temperature cycles to denature DNA
strands. There exist different types of isothermal amplifica- Paper-based devices patterning and microfabrication
tions and we refer the reader to these references9,10 for fur- Paper patterning is achieved by different techniques, such as
ther information. The most popular one, at the moment, is photolithography,15,17 cutting,18,19 etching,20 plasma

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treatment21 or printing16,22,23 (Fig. 3a). Thanks to patterning,

the channels are designed, guiding fluids in a way similar to
standard microfluidic devices (Fig. 3b). The patterning tech-
niques differ by their spatial resolution, cost and simplicity.24

Among all these techniques, wax printing is the most popular,
because of its simplicity. Fig. 3b illustrates the technology.
The paper is printed with a commercial wax printer and then
baked on a hot plate for a few minutes. During the baking
process, the wax melts and spreads across the paper sheet.
This allows the hydrophobic walls to be created and form bar-
riers confining the fluids spatially (Fig. 3c). The procedure
takes five minutes and is simple to carry out. The spatial reso-
lution is on the order of one millimeter. Fig. 3d shows a two
dimensional PAD (2D PAD) prepared with this technique.
By stacking and folding, a three dimensional PAD (3D
PAD) can be created. In this case, liquids flow in the hori-
zontal plane, and also vertically, from one layer to the other
Fig. 2 Paper microfluidics introduced by G. M. Whitesides group.
Multiplexed urine analysis on paper: glucose and BSA colorimetric (Fig. 3e). In practice, the junction between paper layers is sta-
quantification through reagents stored in individual spots and bilized with double sided tape or cellulose powder. These 3D
rehydrated by urine flow in paper. Adapted with the permission from devices are simple to realize and represent powerful tools for
ref. 14. Copyright (2009) American Chemical Society. managing fluids at a certain level of complexity. In Fig. 3e,
Fig. 3 Paper based device patterning and microfabrication. a) Paper device fabrication: fabrication can be performed either by cutting the paper
in different ways or by patterning a paper sheet. This can be carried out indirectly with the use of a mask and different exposure techniques or
directly by controlled deposition of the hydrophobic material on the paper. Adapted with the permission from ref. 25. Copyright (2015) WILEYVCH
Verlag GmbH & Co. b) Illustration of a paper-based microfluidic channel. Hydrophilic paper is patterned with two hydrophobic barriers
constraining the flow in the paper channel. The height of the channel is the thickness of the paper and the width is the length between the two
barriers. Adapted with the permission from ref. 14. Copyright (2010) American Chemical Society. c) The quick and easy three-step process of paper
wax patterning. Design (1), wax printing (2) and melting (3) can be achieved in a few minutes. Adapted with the permission from ref. 16. Copyright
(2009) American Chemical Society. d) Paper cross section of the wax printing process for fabricating a 2D PAD. Adapted with the permission from
ref. 14. Copyright (2010) American Chemical Society. e) Stacking of several patterned layers of paper for fabricating a 3D PAD. Adapted with the
permission from ref. 16. Copyright (2009) American Chemical Society.

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we show a four-layer device, which allows multiplexing of the crosslinking, covalent bonding or bioaffinity as displayed in
analysis of a sample with a certain simplicity. Fig. 5b. Covalent bonds enabling cellulose to graft with a bio-
molecule are sketched in Fig. 5c.
In paper, molecules can be immobilized. A review of the

Paper chemistry
strategies currently used is available in the literature.32 Physi-
Paper encompasses a diversity of materials. Although histori- cal immobilization (through electrostatic, hydrophobic, hy-
cally fabricated from cellulose and transformed with coatings drogen bonding...) is the simplest protocol. Its limit is the ab-
and chemical grafting, numerous examples of non-cellulosic sence of control of the biomolecules' orientation, their weak
substrates like membranes and filters now enter under the attachment and their inaccurate localization. Chemical im-
same denomination. In analytical sciences, paper has been mobilization leads to a strong, precise and stable bond but
developed in many formats: pH paper, chromatography paper the procedures are complex.35 These surface treatments can
for component separation, size-defined filters, dipsticks and more easily be achieved with an inkjet printer using inks
Lateral Flow Test (LFT) paper made of nitrocellulose (urine containing DNA, cells, antibodies, hormones or enzymes.36,37
test strips),26 Dried Blood Spot (DBS) cartridges27 and FTA Cellulose based paper is composed of molecules that do
cards28 for the collection and storage of biological samples. not interfere detrimentally with most of the current biomole-
These substrates have in common a hydrophilic porous cules. Because of this property, it is often considered as bio-
microstructure, responsible for two main functions: filtration compatible. Note however that the strict definition of bio-
and transport by capillary pumping. In the early stage of paper compatibility is more demanding. According to Williams38
microfluidics,15 chromatography paper, made of cellulose fi- biocompatibility is the ability of a material to perform with
bers, was the most used material. The medium formed by the an appropriate host response in a specific application. In
fiber tangle is geometrically disordered and complex, and pores this sense, paper is not always biocompatible. For instance,
through which fluids circulate have broad distributions of because of exposure of the hydroxyl groups explained above,
sizes, typically between 5 and 20 m (ref. 29 and 30) (Fig. 4a). paper easily adsorbs proteins through hydrogen bonds. The
The fibers can be treated chemically. FTA cards are good ex- process is enhanced by the porous structure, which yields
amples of cellulose substrates coated with a lysis reagent and high surface to volume ratios. Cellulose pores may thus de-
additional chemicals able to extract and preserve NA from bio- plete reagents from the critical components and in turn in-
logical samples31 (Fig. 4b). Cellulose-made paper can also be hibit chemical reactions that would happen in free environ-
turned into bioactive platforms.32,33 Non cellulosic paper often ments. Examples will be given later for the case of isothermal
offers gains in homogeneity and control. For example, glass fi- amplification.
bers have a monodispersed diameter which, by contrast with
standard paper, do not swell upon contact with liquid (Fig. 4c).
Fiber-free synthetic porous media like Poly-Ether-Sulfone (PES)
Basic paper microfluidics functionalities
membranes have a well-chemically defined surface and a regu-
lar pore size from 0.03 m to 8 m (Fig. 4d). Paper is a rich substrate that naturally integrates several
In cellulose based paper, each fiber consists of a supramo- functionalities:
lecular assembly of several polymer chains of glucose (Fig. 5a). 1. Transport. Ref. 39, 40 and 41 have demonstrated that the
These monomers display several free hydroxyl groups resulting LucasWashburn law42 quantitatively describes the transport of
in a large amount of accessible hydrogen bonds and thereby a aqueous solutions in hydrophilic paper channels. The law stip-
substantial chemical reactivity.34 Oxidation, amination, esterifi- ulates that the flow speed decreases as the inverse of the square
cation, etherification and radical copolymerization of cellulose root of time. This means that paper rapidly imbibes liquids at
are reported in the literature.33 The same authors described short times, i.e. in the first few seconds, slowing down as time
bioactive papers obtained by immobilizing and storing biomol- increases. In practice, according to the LucasWasburn law, the
ecules. This is currently performed by passive adsorption, extent to which the samples or reagents propagate on a paper
Fig. 4 SEM of various types of paper. a) Chromatography paper. Adapted with the permission from ref. 30. b) FTA matrix and trapped DNA31
(image adapted courtesy of GE Healthcare). c) Glass fibers (image adapted courtesy of Sterlitech Corporation). d) PES membrane (image adapted
courtesy of Sterlitech Corporation).

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Fig. 5 Cellulose chemistry. a) Cellulose molecular structure. b) Various strategies for biomolecule immobilization on the cellulose substrate. c)
Covalent bonding by various chemical functions, between a biological target and cellulose fibers. Adapted from ref. 33. Copyright (2014) RSC
publishing.
sheet is limited to 34 centimeters. This limitation represents a time, by locally modifying the porous medium: partially
constraint for the design of paper devices. clogged59 or compressed.60 A valve function can also rely on a
2. Filtration. Pores are between 5 and 50 m in size, and mechanical displacement. This was demonstrated with a fluid-
therefore they arrest objects larger than these dimensions triggered expanding element.61 The actuator is connected on
while the rest of the suspension flows downstream. This fil- demand to a liquid reservoir. When it wicks, the actuator ex-
tering function is useful for the preparation of blood for ana- pands and displaces the main channel. This simple function
lytical purposes43 as discussed later. Surface chemistry obvi- can be used either to connect two channels (on-switch), to dis-
ously plays a role by adsorbing different species, in a specific connect two channels (off-switch) or to change the flow path-
manner. This capability has led paper to serve, for decades, way from one channel to another (diversion switch) as
as chromatographic or extraction units. displayed in Fig. 6c. Another device exploits paper
3. Storage. Storage is a functionality that is crucial for en- deformability.62 The systems include a multilayered structure
abling the portability of the diagnostics, as discussed in which an air spacer can break the fluidic connectivity. By
later.27,44,45 The porous structure of paper and the easy access of pressing the paper on this location, the liquid continuity is re-
the device to external actions, such as micro-pipetting, heating stored and the fluid can flow (Fig. 6d).
and cooling, facilitate the freeze-drying of the reagents. Reagents 6. Multiplexing. A multiplexed assay simultaneously char-
can be uniformly distributed on the paper sheet46 or locally de- acterizes multiple samples in a single run so as to diagnose
posited. The latter possibility led to the achievement of se- several diseases on the same PAD. The importance of this
quences of reactions in ref. 47 and multiple analyses in ref. 48. functionality has recently been underlined.63 Since different
4. Concentrator. Because of their exposure to air, liquids cir- diseases, such as Ebola, dengue or malaria develop similar
culating in paper are subject to evaporation. This property en- symptoms in the first days following the infection, it is cru-
ables paper to concentrate, in a non-specific manner, cial to perform a multiplexed test in order to identify the in-
analytes.4952 In many situations however, concentration is not fection and apply the right treatment. With each test associ-
sought after and it is more appropriate to reduce evaporation.15 ated to two controls (negative and positive), a system
In such cases, devices are enclosed in plastic housings, using including the identification of the three diseases needs nine
adhesive plastic foil53,54 and multilayered paper geometries.55 reactions to be simultaneously performed. Individual paper
5. Valving. The possibility to fold paper has led to the design spots shown in Fig. 7a are equivalent to multiwell plates.46
of actuators. In fact, by folding a paper sheet, one can stop or Several examples of multiplexed structures are shown in
initiate a flow, which is the definition of valving.56 The same is Fig. 7b and c.15,64 Fig. 7d and e show devices using multilay-
true for cutting. Such operations are simple to perform and ered structures55 and folding56 to multiplex with a minimal
they have been used to design complex devices, such as number of pipetting. Other examples are shown in Fig. 7f.65
multiplexed assays. More sophisticated valving systems have
also been reported in the literature: for instance, by inserting a
dissolvable porous bridge between two paper channels57 (see Hybrid technology: paper microfluidics combined with
Fig. 6a). Solid component dissolution has also been exploited microfluidic devices
to induce flow retardation58 (Fig. 6b). Several authors proposed Often, the diagnostics of diseases based on the detection of
to delay liquid flows without changing its composition over NA, from the clinical sample to the readout, involve a number

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Fig. 6 Valve functions in paper devices. a) Stop-flow by a dissolvable bridge. Adapted from ref. 57. Copyright (2013) American Chemical Society.
b) Sequential reagent arrivals thanks to sugar delays. Adapted from ref. 58. Copyright (2013) RSC publishing. c) Expanding actuator for valve func-
tions. Adapted from ref. 61. Copyright (2015) RSC publishing. d) On-button in a multilayered device obtained by air-spacer and paper
deformability. Adapted from ref. 62. Copyright (2010) RSC publishing.
of steps that are difficult to integrate on a single PAD. For this the paper. In a second configuration and for the same objec-
reason, a number of investigators have combined paper micro- tive, a paper disc is introduced anywhere in the microsystems
fluidics with standard lab-on-a-chip technology.6669 A recent through a punched hole closed with a PDMS cork as shown in
work made in our laboratory49 provides examples of such hy- Fig. 8b. In these systems, the liquid injected, either in the paper
brid microsystems, i.e. coupling paper microfluidics and PDMS or in the microchannel elutes the dried samples stored in the
(polydimethylsiloxane) devices. In the first case, a paper strip is paper matrix (Fig. 8c) and therefore conveys the sample in a
inserted as an accessible inlet and liquid flows are driven by a PDMS microfluidic device. Fig. 8d shows a dried fluorescein
negative pressure (i.e. below the ambient pressure) from the sample eluted from paper, driven along a microchannel and
outlet (Fig. 8a). The goal here is to retrieve a sample dried on eventually encapsulated in droplets.
Fig. 7 Paper geometries for multiplexed analysis. a) Hydrophilichydrophobic contrast implemented on paper by photolithography to design
microzone plates. Adapted from ref. 46. Copyright (2009) American Chemical Society. b) Urine analysis in a tree-shaped paper device for simulta-
neous and colorimetric determination of BSA and glucose contents in artificial urine. Adapted from ref. 15. Copyright (2007) Wiley-VCH Verlag
GmbH & Co. c) The many possible geometries for paralleled LFTs in cut paper. Adapted from ref. 64. Copyright (2008) American Chemical Society.
d) Paper and tape multilayered device for rehydration of 1024 detection zones through 4 inlets. Independent trajectories are illustrated with dyes.
Adapted from ref. 55. Copyright (2008) National Academy of Sciences, USA. e) Origami-like device: a patterned paper can be folded and sealed for
liquid flows from one layer to the other. Adapted from ref. 56. Copyright (2011) American Chemical Society. f) Three dimensional hydrophilic pat-
terns in one single paper sheet, obtained by printing and baking wax of various amounts on both sides. Adapted from ref. 65. Copyright (2014)
Springer.

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Sample preparation on paper The large range of viscosities of body fluids
When samples from humans are to be analyzed, they most of- The diversity of biological fluids (blood, urine, sputum...) re-
ten need preparation before being processed with the stan- quires the ability to work with a large range of viscosities:
dard methods of molecular biology.70 This leads to questions from 1 103 to 30 Pa s.8486 In paper, according to the Lu-
in the sample preparation. Sample preparation typically con- casWashburn law,42 where the speed decreases as the inverse
sists of mixing (with reagents), filtration, lysis purification, of the square of the viscosity, reduction can be of one order of
pre-concentration and NA extraction. These steps allow elimi- magnitude. This may limit the practical distance of travel of
nation of the inhibitors of RT and amplification reactions. In the sample to a few millimeters. However, in practice, most
some cases, however, such preparation is not needed or re- authors have found ways to circumvent this difficulty.
mains minimal. For instance, RT-PCR has been demonstrated The case where viscosity does not raise issues is urine,
on crude serum71 and LAMP on untreated preheated blood.72 since its viscosity is 8 mPa s, i.e. only 10 times higher than
It is interesting to note that, in contrast with PCR,73 isother- that of water at 37.5 C.87 Dipstick urine analysis,88 preg-
mal amplification techniques appear more tolerant of the in- nancy lateral flow tests,26 and first PADs15 demonstrated the
hibitors present in the sample.74 Still, in most cases, sample direct handling of urine samples with paper. However, from
preparation is needed and this remains a challenge for paper an infectious disease diagnosis perspective, although being
microfluidic technology.75 In practice, preparation is attractive for its low viscosity and high accessibility, urine
performed externally to the PAD, using automated devices or utility is debated:89 for example, urine composition enables
manual processes. Examples are sputum,76 saliva swabs,77 TB detection only for HIV-infected patients.
stool78 and urine,79 which make use of instruments unfortu- Saliva (8 mPa s (ref. 90)) can also be handled without di-
nately non-transportable in the field. Today, most of the avail- lution. In ref. 91, it is shown that saliva flows in 2 cm long
able commercial POC devices performing sample preparation paper channels within a range of time compatible with diag-
use this type of instrument.75 However, sample preparation is nostic applications (a few minutes). In a recent work how-
in full bloom with a growing number of contributions.8083 ever92 (Fig. 9a), the authors had to include a dilution step
Fig. 8 Hybrid technology devices.49 a) Paper strip inserted at the glassPDMS interface of a microsystem. Liquid flows at the papermicrochannel
junction are obtained thanks to the rehydration of the paper combined with the negative pressure from the outlet of the microsystem. b) A paper
disc can be introduced into a microsystem through any punched hole that can be closed using a PDMS cork. Liquid flows can be handled like
those in any other microchip. c) Elution curves of dried fluorescein samples on paper eluted and measured in microchannels by fluorescence,
according to the flow rate. d) Microscopy visualization of the papermicrochannel junction in a flow-focusing geometry. The water flow from pa-
per is divided into droplets by two oil and surfactant flows.

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allowing flows with longer distance. Dilution is achieved by lance) blood on paper cartridges as displayed in Fig. 10c. Dried
overlapping paper strips, as displayed in Fig. 9b.39 In this samples are stored for days or months afterwards and trans-
case, the dilution ratio could be tuned. Note that over the last ported for analysis in equipped laboratories. It is remarkable
years, there is growing interest in considering salivary tests93 that paper storage does not produce any significant damage on
for HIV, hepatitis C and human papillomavirus, using sero- the biological information. For example, in the case of HIV,
logical methods or NAATs.94 DNA can be stored for 4 years,106 under various conditions.107
Although raw semen is substantially viscous (30 times For some viruses, a diminution of the viral activity, caused by
more than water95), it is still compatible with paper without cell lysis under drying, has been reported.108 This characteristic
dilution, if shorter distances are designed in the paper de- is not general: the hepatitis B virus remains infectious at least
vice.96 Similarly, sputum samples have a quite high viscosity seven days on paper.109 Today, either for early diagnosis or
(2070 mPa s (ref. 97)). Until now, there is no paper device global health monitoring, it is frequent to analyze the presence
operated with sputum samples directly. Today, the existing and concentration of more than 45 analytes contained in the
systems performing NAATs for early diagnostics of tuberculo- blood sample.45 In the NAAT field, several articles report NA ex-
sis98 uses expensive instrumentation: for example, GeneXpert traction and amplification from Dried Blood Spot (DBS) speci-
cost more than US $17 000 and each cartridge US $10. Finally, mens, mainly for HIV diagnostics by RT-PCR110,111 but also for
paper devices for fecal sample analysis for Salmonella detec- detection of other diseases112 such as hemoglobinopathies,
tion99 were demonstrated. Since sample viscosities range Duchenne muscular dystrophy, cystic fibrosis.
from 20 to 2000 Pa s,100 dilution is obviously necessary.
Nucleic acid extraction and purification on paper

Whole blood filtration on paper In standard sample preparation procedures,113,114 dilution
Blood is a vital complex fluid composed of Red Blood Cells and filtration are followed by cell lysis and nucleic acid ex-
(RBCs), white blood cells, platelets and biomolecules diluted traction. NA purification is still required to remove the inhib-
in aqueous solution. Because of its complexity, in most cases itors (for instance RNases) released during cell lysis.
it must be filtered to avoid interferences with inhibitors prior Performing these operations on paper is challenging but, in a
to amplification. Indeed, currently, size-exclusion chromatog- few cases, feasible.
raphy enables the removal of components such as immuno- In a first example,115 PCR amplification in microtubes is
globulin G,101 hemoglobin (in erythrocytes) and lactoferin (in preceded by a sample preparation on paper. Whole blood is
leukocytes)102 that have a major inhibitory effect on PCR first loaded on a filter paper, on top of an absorbent pad as
amplification. displayed in Fig. 11a. Cells that are trapped in the filter are
The porous microstructure of the paper associated with lysed by buffer addition. Released NAs are also captured by
natural capillary pumps, enables spontaneous filtration of surface adsorption whereas other components and inhibitors
RBCs according to the pore size of the material43 (Fig. 10a). are pumped out by the absorbent pad. Finally, the filter
With specific antibodies leading to RBC aggregation, plasma containing trapped DNA is added to the PCR mixture for di-
extraction is made possible even with larger porous media. rect amplification. This sample preparation only requires two
This property is used by several blood typing paper devices as pipetting operations and a very low cost paper assembly that
shown in Fig. 10b:103105 the RBCs' color can either uniformly makes it suitable for on-field applications. The same spotting
spread or locally concentrate in case of aggregation. sample preparation on an FTA membrane was demon-
Note that for decades, blood and paper have been routinely strated on a wide variety of plant pathogens (viruses, bacte-
coupled in maternity hospitals for newborn blood sampling:27 ria, phytoplasma) detected by RT-PCR116 and several proto-
Guthrie cards consist of storing dried finger-prick (or heel cols were compared.117
Fig. 9 Mixing in paper. (a) Modified lateral flow test for salivary diagnostics. Adapted from ref. 92. Copyright (2010) RSC publishing. (b) Mixing by
diffusion between two stacked paper strips according to their length. Adapted from ref. 39. Copyright (2015) RSC publishing.

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Fig. 10 Whole blood on paper. a) Plasma extraction on paper by red blood cell filtration or aggregation. Adapted from ref. 43. Copyright (2012)
RSC publishing. b) Visualization of red blood cell agglutination on antibody coated paper for blood typing. Adapted from ref. 103. Copyright (2012)
American Chemical Society. c) Dried blood spot: fingerstick blood stored on a Guthrie card (adapted from vitas.no).
Another example of cell lysis and NA extraction on paper remaining bacteria, cells, viruses, proteins and lipids close
was demonstrated with Chlamydia trachomatis, using urine to the inlet to be captured, whereas NAs are further trans-
samples.79 To diagnose this sexually transmitted infection by ported in the paper channel. Either directly with an interca-
isothermal amplification (Helicase Dependent Amplification: lating dye or with PCR on small paper parts, it is possible
HDA), NA extraction and isolation is necessary. The idea is to to quantify the amount of NA on different locations. The
mix the sample with a buffer containing components for cell compared results of cellulose and nitrocellulose membranes
lysis and alcohol precipitation of DNA. This mixture is then demonstrated that cellulose better extracts NA (lower limit
added into a pipette tip for which the end is filled with either of detection) but there is less NA chromatographic retention
compressed paper or porous polymer monoliths (Fig. 11b). in nitrocellulose. This sample preparation can be accom-
By pressurizing the liquid, the mixture flows in the porous plished on-field within 5 minutes and can be coupled with
media whereas DNA precipitates remain on top of it. A wash- any other NAATs.
ing step with ethanol enables the elution of DNA from the pa- An origami-based paper device, illustrated in Fig. 11d, uses
per support and its recovery for amplification and detection. the valve function of folding/unfolding several paper layers to
Fabrication of the support material at the end of the pipette perform DNA bacterial extraction from raw viscous samples
tip remains very easy with paper and only requires 10 mi- (pig mucin simulating sputum).118 All reagents (lysis solution,
nutes versus the 20 hours for the polymer. However results of extraction buffer, washing buffers...) are preloaded into the de-
extraction are substantially less reproducible with paper. This vice and dried for storage in order to make it fully ready-to-use
simple device still requires external equipment like a pres- only with nuclease-free water and ethanol. A raw sample can
sure supply to make liquid flows within 510 minutes for be introduced into the central inlet of the device while a cell ly-
each washing step. A natural paper capillary pump could re- sis buffer pad is rehydrated with water. By folding a layer, the
place this function but within longer durations. liquid communication enables the mixing of these compo-
Similarly, Salmonella extraction was demonstrated from nents and the mixture is incubated for 30 minutes. By remov-
poultry packaging liquid, whole blood and fecal samples ing the first layer and folding another one, the lysis stops and
thanks to an initial step of mixing sample with lysis a washing step can start with ethanol addition. These two sim-
buffer.99 This mixture is then loaded on a paper channel as ple operations enable efficient DNA capture on filter paper.
displayed in Fig. 11c. The capillary pump spatially disperses
and conveys the liquid components in the paper channel
according to their size (filter effect of the porous media) Concentration on paper
and to their interaction with the cellulose or nitrocellulose In order to keep the amplification reaction sensitive, it
membrane (chromatography effect, interactions according to may be useful to include concentration steps. So far,
the surface chemistry). Paper properties enable the apart from a few exceptions based on microbeads,119

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Fig. 11 Cell lysis and NA extraction on paper. a) NA extraction and purification from whole blood on a two-layered paper device. Adapted from
ref. 115. Copyright (2009) American Society for Microbiology. b) Filtration of lysed cells and precipitated DNA on a paper support in a pipette tip,
followed by washing steps for elution. Adapted from ref. 79. Copyright (2014) RSC publishing. c) NA isolation through capillary flow on paper.
Adapted from ref. 99. Copyright (2014) RSC publishing. d) Origami paper device for sample lysis and NA extraction from raw viscous samples.
Adapted from ref. 118. Copyright (2012) RSC publishing.
concentration techniques use solvent removal and/or target configuration, the same process can be turned to be more
capture on membranes. These techniques can be specific. For example, in ref. 52, a paper strip is used for
nonspecific and performed on paper thanks to evapora- increasing the initial concentration of the target DNA rela-
tion. In ref. 51, a localized heat source accelerates the sol- tive to the non-specific background (see Fig. 12c). The pa-
vent evaporation process, whereas the capillary pump con- per strip contains an area coated with complementary oli-
stantly renews the material. This is shown in Fig. 12a, in gonucleotides. The target DNA is captured in this area
which a sample dipstick operation and a small heater when the sample flows in the paper, whereas background
concentrate a given analyte at the end of a paper strip. A DNA strains are conveyed downstream. Finally, by cutting
more selective analyte concentration can be obtained with and specifically eluting the capture area, the sample is
specific ionic surface interactions (Fig. 12b).120 In another enriched with target DNA.

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Nucleic acid amplification and nucleic acid and a LAMP reaction is directly performed in it.
detection on paper An FTA card for LAMP amplification within a membrane has
been further used by Zhang et al.129 in a microcapillary sys-
Nucleic acid amplification on paper
tem and by Connelly et al.130 in a sliding device. A bound

Soon after paper microfluidics technology was launched, a glass fiber (PALL grade 8964) has also been reported to allow
question was raised as to whether nucleic acid amplifications a within membrane LAMP and rt-LAMP for various target se-
can be performed directly on paper devices. In this section quences on a dipstick device.131133 The Linnes et al. study
we review the research activity carried out in this area. for LAMP reaction shows that LAMP and RT-LAMP can be
Polymerase chain reaction (PCR). PCR (Polymerase Chain achieved in chromatographic paper, PES and PC (Fig. 13c).121
Reaction) is by far the most popular amplification technique The favorable role of a polyvinyl alcohol (PVA) binder in glass
used in research. To date, it is not clear whether PCR can be fibers, which, depending on the fabrication process, may or
performed in a paper device. Work performed with Fusion 5 not be present, has been pointed out in this study.
membranes playing the role of a paper device, with excess Helicase Dependent Amplification (HDA). HDA (Bio-
liquid (a volume of liquid above the membrane) indicated helix)134,135 is an isothermal amplification reaction working
that PCR can be achieved in the presence of paper.69 None- optimally at 65 C using a strand displacement polymerase
theless, attempts to perform PCR and RT-PCR in cellulose and a helicase helping to unwind and separate the DNA
chromatography paper (CHR), polyethersulfone (PES), poly strands (Fig. 13a). Recently, this reaction has been success-
carbonate membrane (PC), binder free glass fiber (GF) and fully applied to the detection of Mycobacterium tuberculosis
nitrocellulose (NC) were unsuccessful.121 (MTB) on a chromatographic cellulose paper coated with BSA
Recombinase Polymerase Amplification (RPA). RPA to reduce DNA and enzyme adsorption.136 Reagents were
(TwistDx)122,123 is an isothermal amplification reaction opti- dried and stable on paper. HDA has also been studied in
mally working between 3742 C and able to amplify a target more detail by Linnes and colleagues on various matrices.121
sequence using a recombinase, single strand binding proteins The results tend to show that chromatographic cellulose pa-
and a strand displacement polymerase (Fig. 13a). This ampli- per, PES and PC are compatible with HDA. Within mem-
fication reaction has already been adapted for paper-based de- branes, the reactions seem efficient in PES (Fig. 13d).
vices. The first demonstration of compatibility was performed
by Rohrman et al. on a paper and tape device where amplifi-
cation occurs within a cellulose and glass fiber sandwich.124 Techniques of nucleic acid detection on paper
RPA within different bound glass fibers and the paper types Detection in a Lateral Flow Test (LFT) format. Because
used in this work are shown in Fig. 13b. Two other examples LFT is already a useful point-of-care device, it has been
in the literature show RPA and RT-RPA on a cellulose sub- adapted to switch from antibody or antigen recognition to NA
strate (Whatman Chromatography type 1), realized respec- detection in paper devices.138 This combination enables either
tively by Cordray et al. for Plasmodium DNA125 and by Magro the use of DNA amplification for antigenantibody complex
et al. for Ebola virus RNA.126 In ref. 126, the reagents were detection or detection of DNA through a LFT format.26 For in-
freeze-dried on paper for one month before being used. fectious disease diagnostics, the second configuration is more
Loop-mediated isothermal amplification (LAMP). LAMP relevant. A modified LFT, used just after NA amplification, can
(Eiken)127,128 is an isothermal amplification reaction working replace fluorescent detection that relies on external equip-
at 6065 C. It amplifies a target sequence with two or three ment. Dipsticks are very suitable for on-field applications: with
sets of primers and a polymerase with a strand displacement pre-loaded reagents; it can be used by unskilled operators.
activity (Fig. 13a). Due to the configuration of these primers, Visual NA detection on a LFT device has been demon-
the amount of DNA generated is a lot higher than that of strated by modifying RPA primers;12 it can probably be ex-
PCR. First demonstration of LAMP on paper has been tended to other amplification techniques. By grafting forward
conducted by Liu et al. on a plastic/paper hybrid device.68 An and reverse primers with proteins like biotin and carboxy-
FTA modified cellulose membrane is used for isolating the fluorescein (FAM), amplification of the target RNA and
Fig. 12 NA concentration step. a) Evaporative concentration of analytes at the end of a paper strip. Adapted from ref. 51. Copyright (2014)
American Chemical Society. b) Tunable focalization according to the biochemical properties of the porous media surface. Adapted from ref. 120.
Copyright (2013) American Chemical Society. c) A lateral flow strip for target DNA concentration relative to non-specific background DNA.
Adapted from ref. 52. This is an unofficial adaptation of an article that appeared in an ACS publication. ACS has not endorsed the content of this
adaptation or the context of its use.

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Fig. 13 NA amplification on paper. a) Schemes of LAMP, HDA and RPA isothermal amplifications. Adapted from ref. 137. Copyright 2011 Elsevier
b) HIV DNA RPA performed on various types of cellulose and glass fibers. G041 bound glass fiber (at the right side of the gel) and GF/DVA bound
glass fiber seem to be well suited for RPA amplification. Adapted from ref. 124. Copyright (2012) RSC publishing c) RT-LAMP and d) RT-HDA reac-
tions performed on various types of membranes: CHR = chromatography paper, PES = polyethersulfone membrane, PC = polycarbonate mem-
brane, GF = glass microfiber matrix and NC = nitrocellulose membrane. RT-LAMP and RT-HDA are realized respectively for Influenza A and N.
gonorrhoeae RNA strands both with excess liquid and within the membrane. Agarose gel electrophoresis and lateral flow detection (LFD) strips are
shown. Relative LFD test line intensities are displayed on the graphs below each gel. Adapted from ref. 121. Springer Science + Business Media
New York 2016.
complementarities of amplicons produce double-strain DNA flow well conveys reagents in the membrane. Similar proto-
bearing both antigens (Fig. 14a). Without target RNA, no am- cols can be applied on HIV LAMP primers and are compati-
plification occurs and the two primers have no reason to ble with commercial products (Fig. 14b and c).139
match. The associated LFT is built with anti-biotin and anti- Another NA-LFT technique relies on complimentary oligo-
FAM antibodies to form a sandwich format around oligonu- nucleotides.140 In this case, the LFT device is designed with
cleotides. The FAM/anti-FAM complex is associated with gold free gold nanoparticles coated with small DNA strains that
for a visible signal and also as a control line to ensure the match the DNA target (Fig. 14d). The detection zone is also

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coated with small DNA strains that are complementary to the non-specific luminescent reporter associates with any present
other end of the DNA target. The capillary flow conveys the miRNA to form a pink duplex molecule. In the presence of
sample and the nanoparticles towards the location of grafted the target RNA, the capturing molecule (complementary
DNA strains for the formation of the sandwich format. strain of the target) is also involved, matching with lumines-
Detection based on a chemical modification of paper. cent reportermiRNA, changing its color to orange (Fig. 15d).
Capillary pumping, chemical grafting, and DNA hybridization All reagents are only loaded on the polymeric paper (PVDF:
can be integrated in paper-based NA detection devices but in polyvinylidenedifluoride) layer without chemical grafting
a format different from LFT. Schematics of such devices, steps. This facilitates the fabrication but jeopardizes the pos-
presented in Fig. 15 is reminiscent of thin-layer chromatogra- sibility of performing multiplexed analysis.
phy protocols: migration of species on a porous membrane Detection in a microzone plate format. Wax-patterned
that is dipped into a liquid preparation. Chemical treatments technology enables the realization of microzones on paper,
locally modify paper surfaces in order to capture a DNA tar- evoking the 96-multiwell plates currently used in biology. In
get. For example, through alcohol functions, cellulose fiber- this configuration, paper is considered as a substrate, able to
based paper is easily functionalized with oligonucleotides store reagents, and there is essentially no fluid dynamics. In
grafted thanks to amino groups141 (Fig. 15a). With comple- ref. 144, this geometry is used for the detection of Dengue vi-
mentary strains and probe-bearing DNA, the paper enables rus after RT-LAMP amplification is performed in tubes. Here,
rapid detection thanks to the capillary pump and DNA hy- fluorescence is the readout of the amplification process
bridization. The chemical treatment is not necessary global, (Fig. 16a). Recent developments led to the proposal of a col-
it can be local. With several spots grafted with various DNA orimetric version of this protocol.145
sequences, multiplexed detection can be performed on a sin- By assembling several layers of paper microzone plates,
gle device (Fig. 15b and c). Recently, the number of targets fluids and stored reagents are put into contact when the device
has increased from four to eight and the observation could is folded. This provides a method for generating a readout. An
be made with the naked-eye by replacing fluorescent probes example (Fig. 16b) consists of a competitive hybridization be-
with micrometer-sized beads.142 tween a fluorophore-labeled single strain DNA (F1), a smaller
The visual paper-based detection of miRNA has been complementary DNA bearing a quencher (Q1) and a target DNA
reported and is based on duplex and triplex species.143 A matching F1.146 Each component is stored on different layers.
Fig. 14 NA detection on a LFT device. a) Schematics of the RPA principles of product generation and lateral flow strip detection. Adapted from
ref. 12. 2006 Piepenburg et al. b) Picture of commercial Milenia strips for detection of FITC/biotin-labeled amplicons. Adapted from ref. 138.
2014 Singleton et al. c) Picture of commercial Best II cassettes for detection of FITC/biotin-labeled amplicons. Adapted from ref. 138. 2014
Singleton et al. d) Schematics of NALF thanks to nanoparticles coated with template complementary strains. Adapted from ref. 139. Rohrman
et al.

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Fig. 15 NA detection on a thin-layer chromatography plate-like device. a) Illustration of the chemical protocol to graft single strain DNA on a cel-
lulose surface through 1,4-phenylenediisothiocyanate activation. Adapted from ref. 140. Copyright (2012) American Chemical Society. b) Configu-
ration of immobilized oligonucleotide: a negative control (neg), a positive control (S_1) and four target amplicons (T_1, T_2, T_3, T_4). Adapted
from ref. 140. Copyright (2012) American Chemical Society. c) Multiplexed fluorescence detection of DNA hybridization for the various samples.
Adapted from ref. 140. Copyright (2012) American Chemical Society. d) Paper platform for naked eye detection of miRNA by formation of duplex
or triplex species. Adapted from ref. 142. Copyright (2013) American Chemical Society.
Because the target is longer than Q1, the strand displacement tection spot. In the presence of the target, the preferential inter-
kinetics makes the assembly F1-target (fluorescent) more prob- action between the target and aptamers destroys the hydrogel.
able than F1-Q1 (quenched). When folded, the fluorescence sig- The liquid can freely flow towards all the layers of the device,
nal directly provides readout for the target. rehydrating the dye that can be detected by the naked-eye at the
Detection mediated by or exploiting a flow. As said before, end of the paper channel (Fig. 17b). Because aptamers are ver-
the challenge of detection is to reach large sensitivities with satile molecules, multiple biomarkers can be targeted and the
transportable minimal equipment, like a smartphone or the parallelization of the geometry enables the distribution of the
naked eye, in a way similar to paper pregnancy tests. This leads sample towards at least four analysis spots with their own asso-
to the idea developed in ref. 147. In this reference, use is made ciated color. By replacing aptamers with target-matched DNA
of magnetic bead aggregation in the presence of the very long strains, it could be possible to develop similar colorimetric spe-
DNA strands produced by LAMP. The aggregates produce a cific DNA detection.
dark spot while non-agglutinated beads are dispersed by the
flow, thus mimicking in some way a readout technique often
used in LFTs (Fig. 17a). In ref. 148, biomarker detection on pa- Integrated sample-in-answer-out devices
per is demonstrated by using a flow regulator. Without the As seen above, sample preparation, NA amplification and DNA
target, an aptamer-cross-linked hydrogel blocks the liquid flow target detection have been demonstrated on paper. It is unsur-
and the dye stored in the device is not conveyed towards the de- prising that some investigators attempted to integrate all these
Fig. 16 NA detection on microzone wax-patterned devices. a) Fluorescence detection of RT-LAMP products on microzone plate paper. Adapted
from ref. 143. Copyright (2013) RSC publishing. b) Origami paper analytical device: each layer contains a component for competitive hybridization
between a target DNA, a fluorophore-labelled DNA and a quencher bearing DNA. Adapted from ref. 145. Copyright (2013) American Chemical
Society.

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functionalities to build sample-in-answer-out devices. This captured on the paper and removed by the washing opera-
section reviews the main contributions made in this area. tion. Here, paper is used as a capture medium, demonstrat-
Hybrid paper-microsystems. First demonstration of NA ing that amplification is possible in porous media.
amplification on paper has been performed in hybrid devices A more recent amplification ion type of paper is reported
that combine paper and microchips. Amplification of HIV in ref. 152 (see Fig. 19b). It relies on a specific synthetic gene
RNA by RT-LAMP from oral fluids was demonstrated in a dis- network, able to reproduce cell functions like transcription
posable cassette shown in Fig. 18a thanks to washing steps and translation. All reagents are freeze-dried on individual
through an integrated FTA membrane in the micro- paper spots, can be stored and only need rehydration. If the
channel.68 Similarly, in a microcapillary format (Fig. 18b), a target RNA matched synthetic molecular construction, it en-
whole blood droplet is loaded in a trapped FTA mem- ables ribosome activity to translate a repressed gene. This
brane.129 Droplets containing purification reagents, buffers gene translation is responsible for a fluorescence signal or a
and LAMP mixtures, are brought sequentially to the paper to color change after two hours of incubation. Paper appears as
perform all the diagnostics steps. a powerful substrate for reagent storage, amplification and
The recovery of DBS, which relies on passive elution149 in an detection of parallel reactions. The initial publication applied
appropriate buffer, provides another example of a hybrid system to synthetic Ebola virus RNA was then completed by the same
(see Fig. 18c). Examples of active elution and on-line analysis in team with Zika virus detection.153 This work, although based
a microdevice have also appeared in the literature. In ref. 69, a on sophisticated biochemistry, exposing a possible lack of ro-
filter paper containing whole blood is inserted between two lay- bustness in on-field applications, may inspire design of new
ered microchannels. Liquid flows through the paper enable the amplification schemes optimized for NAATs on paper.
rehydration of the sample, removal of inhibitors, extraction of A sliding device was developed to perform sample prepara-
DNA and specific amplification of the biological target. tion, LAMP and NA fluorescence detection.130 It consists of a
In other hybrid microdevices for NAATs, paper plays a sig- paper disc that can be moved to several positions (Fig. 19c). In
nificant role in the visual detection of the amplified prod- the first one, there is an absorbent pad underneath that en-
ucts67 thanks to a LFT or in the reagent storage.150 In the sures capillary suction, forcing the blood sample to flow
same article, the authors argue that paper enables more uni- through the paper disc, removing all inhibitors while the DNA
form reagent distribution and longer conservation of reagent strains are captured. In a second position, buffers complete the
properties than a standard microchip. washing. In the third position, LAMP reagents are added. The
Paper material alone. The first NA amplification on a pa- paper is slid again to a closed location to prevent evaporation
per strip has been reported in ref. 151 (see Fig. 19a). A paper during the 65 C incubation. A final position enables the DNA
strip is coated with DNA-conjugated microgels (D1-MG). The fluorescent intercalating dye to be pipetted and the result be vi-
target DNA is complementary to D1 and to a second detec- sualized. This device, which is autonomous and requires only a
tion DNA (D2). By dipping the paper in a microtube few manual actions, is the most advanced system adapted for
containing the sample and D2, the complex hybridization of on-field diagnostics coupling NAAT and paper technology.
D1target DNAD2 occurs only if the target DNA is present. A The realization of HDA (Helicase Dependant Amplifica-
washing step enables the removal of all the liquids in the tion) from dried reagents stored in paper discs136 shown in
tube, except for the components attached on the microgels. Fig. 19d overcomes the challenge of Mycobacterium tuberculo-
Then, RCA (Rolling Cycle Amplification) reagents and a fluo- sis (MTB) diagnosis in low-income countries. Indeed, the
rescent probe are added in the microtube in order to specifi- large genome GC content (over 65%) makes it non-
cally amplify D2. If the target DNA is not present, D2 is not compatible with most of the isothermal amplifications
Fig. 17 NA detection through a liquid flow. a) Visualization of magnetic bead aggregation on paper in the presence of LAMP products. Adapted
from ref. 146. Copyright (2016) RSC publishing. b) Target-responsive DNA hydrogel that stops the flow in the absence of the target. Adapted from
ref. 147. Copyright (2013) American Chemical Society.

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Fig. 18 Typical hybrid papermicrochip devices. a) FTA membrane in a two layered microchannel for DNA extraction and purification. Adapted
from ref. 68. Copyright (2011) RSC publishing. b) FTA membrane in a microcapillary for DNA extraction and purification. Adapted from ref. 129.
This is an unofficial adaptation of an article that appeared in an ACS publication. ACS has not endorsed the content of this adaptation or the
context of its use. c) Dried sample stored on filter paper is analyzed in a microchip. Adapted from ref. 69. Copyright (2014) RSC publishing.
Fig. 19 NAAT on the paper substrate. a) NAAT on paper strip thanks to DNA-conjugated microgels and RCA. Adapted from ref. 150. Copyright
(2009) RSC publishing. b) Principle of forming an enzyme-free synthetic gene network freeze-dried on paper. Adapted from ref. 151. Copyright
(2014) Cell Press. c) Paper substrate for DNA extraction, LAMP and fluorescence detection through a sliding device. Adapted from ref. 130. Copy-
right (2015) American Chemical Society. d) HDA amplification of Mycobacterium tuberculosis DNA on paper discs. Adapted from ref. 136. Copyright
(2016) RSC publishing.
where no specific denaturation step is operated. The au- reaction authorizes some tolerance to the temperature con-
thors used chromatography paper coated with BSA to pre- ditions (from the optimal 65 C to 50 C) and to the biolog-
vent DNA and enzyme adsorption. Detection relies on fluo- ical inhibitors. Under these conditions, the final experi-
rescence signals or electrophoresis migration. The HDA ments were performed with low-cost heaters like hand

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warmers, on MTB DNA diluted in the viscous environment unskilled operators, the device is not really ready-to-use; it re-
of artificial sputum. quires the user to load all pads with various liquids.
PAD variations. A paper and plastic device can perform The first success in developing a modular paper device that
HIV detection through RPA reaction.124 It uses the origami combines NA isolation from crude biological samples, isother-
principle or valve function: three pads respectively contain mal amplification and Lateral Flow (LF) detection was demon-
the master mix, magnesium acetate and the sample. By fold- strated for Human Papillomavirus DNA detection from cervical
ing the device, the sample pad is enclosed between the two specimens53 (see Fig. 20b). A sample is loaded on a PES mem-
others and the reagents can mix together (Fig. 20a). The au- brane as well as several buffers and washing solutions. An ab-
thors suggest using this paper device in conjunction with sorbent pad, in contact with the membrane, induces the capil-
DBS and LF strips to obtain a full paper diagnostics from lary force responsible for extraction and removal of impurities.
sample-to-answer. However only some RPA reagents are These washing steps enable the precipitation of the DNA that
stored in the device so it still requires several fresh reagent is trapped in the membrane. The absorbent pad can then be
additions. The same research group develops a sliding device discarded. The LAMP mix is added into the membrane to
for RPA reaction, buffer dilution and LF detection.125 Several achieve isothermal amplification. Finally, the cover film is
individual pads are fixed in a plastic housing and contain peeled back to connect the sample port with the LF strip. The
buffers and reagents. Two mobile pads on the layer under- result of amplification can be visually read on the strip. By
neath can selectively be in contact with the fixed pads thanks connecting and disconnecting pieces of paper, all the diagnos-
to the very easy sliding operations and transport the sample tic steps can be performed in a simple device. However, except
and a dilution buffer. If sliding operations are performed by for the LF strip, none of the reagents are stored thus the device
Fig. 20 Multistep operating devices for NAATs on paper. a) RPA of HIV DNA by folding a paper device: sample pad enclosed between a
master mix and magnesium acetate pads. Adapted from ref. 124. Copyright (2012) RSC publishing. b) Modular fluidic paper device for
Human Papillomavirus DNA extraction, amplification (LAMP) and LF detection. Adapted from ref. 53. Copyright (2012) RSC publishing. c)
Two parallel strips for LAMP reaction and LF detection. Adapted from ref. 133. Copyright (2016) RSC publishing. d) Multiplexed detection of
Plasmodium parasites with internal control, from a whole blood droplet in an origami paper device. Adapted from ref. 154. Copyright
(2016) Wiley.

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still relies on several pipetting operations. The idea of why, although publications on POC devices are increasingly ori-
connecting on demand a reaction LAMP pad and a LF strip ented towards emerging countries, only one155 out of 60
for detection was also investigated in a two-parallel strips for- could be evaluated for on-field applications.
mat.133 Here, LAMP occurs in a glass fiber pad, protected by Recently, in our laboratory, we realized a NAAT on a paper
adhesive tape. Removing the disposable tape enables the con- device and brought it to Guinea for detecting Ebola virus in
nection of the LAMP pad to the LF strip (Fig. 20c). clinical samples.126 The device is shown in Fig. 21. The PAD
Sample-to-answer and multiplexed analysis were first made consists of RT-RPA freeze-dried reagents in microzones
in a multilayered device (Fig. 20d) performing Plasmodium designed to be rehydrated and heated at 40 C for the detec-
(parasite responsible for malaria disease) LAMP detection in tion of the presence of sequences specific for Ebola virus in
whole blood.154 Sequential steps of DNA extraction, amplifica- clinical samples (Fig. 21a). This system is combined to a carry-
tion and fluorescence detection on paper required minimal on type of equipment in which all technical elements are an-
handling operations, 45 minutes and low-cost instrumenta- chored in a suitcase, as shown in Fig. 21b. All experiments
tion (heater and handheld flashlight). Three species of Plas- performed included a sample test and two internal controls
modium were detected from a finger-prick volume of human (positive and negative) to ensure no reagent degradation and
blood over 80 patient samples in an operator-blinded study, no external source of contamination (Fig. 21c). Fig. 21d shows
and associated for the first time with an internal control. an extension of this approach to multiplex detection.
Towards on-field validation Summarizing the contributions

Until now, the vast majority of work was carried out in a well- Owing to the diversity of approaches and techniques used in
equipped laboratory. In fact, converting NAATs on paper into a these works, the landscape of the state of the art appears
product usable in the field in a poor setting environment is still multidimensional. This motivated us to raise a synthetic
beyond the state of the art, with the exception of a pioneering overview facilitating a quick and clear understanding of the
work pointing to this direction.155 The task is difficult. The AS- current situation. This is the goal of Table 1 which updates
SURED (Affordable, Sensitive, Specific, User-friendly, Rapid and extends the one proposed by Rodriguez156 by taking into
and Robust, Equipment-free, Delivered) recommendations de- account parameters such as paper substrates, types of ampli-
fined by the World Health Organization (WHO) is constraining. fication reactions and biological targets. Table 1 thus gathers
Extensive work must be done in the laboratory before consider- a set of elements accessible in the series of works published
ing field-evaluation. This includes tests on many samples made in PADs performing NAATs, whether using a hybrid technol-
by untrained operators in blind experiments. Analysis of repro- ogy or not. A few remarks can be made.
ducibility on various samples and variations from the external Amplification step. Over all the published examples and
conditions must also be characterized with statistical tools (Re- all the available techniques of amplification, there is large
ceiver Operating Curves or BlandAltman plots). This explains interest in LAMP reaction that has been reported to be as
Fig. 21 Toward on-field validation of NAAT paper devices for Ebola virus diagnostics.126 a) Freeze-dried RT-RPA reagents on paper. b) Carry-on
equipment for heating step and fluorescence detection. c) Experiments on paper with internal controls to detect viral RNA in clinical samples. d)
Multilayered device to simultaneously perform nine reactions: three sample tests and six associated controls.

Lab Chip
Table 1 Comparison of paper-based devices for NAAT: type of sample considered, reaction of amplification and performances and substrate used
Literature Sample Diagnostics Substrate

Tutorial review
Equipment- # of
Ref. Device Prep. Target Pathogen Fluid free Reaction LOD Storage operations Detection Paper Shape
Paper & Wang PCR DNA B. cereus Extractions PCR 0.1 X 4 Visual Nitrocellulose Strip
Microchip 2006 disposable ng L1 membrane
(ref. 67) cassette
Liu LAMP RNA HIV-1 Saliva RT-lamp 0.1 7 Fluorescence FTA cards Disc
2011 reactor with particle
(ref. 56) isolation per L
membrane
Zhang Integrated X DNA CYP2C19 gene Whole blood LAMP 5000 X 3 Fluorescence FTA cards Disc
2014 microcapillary copies
(ref. 129) per L
Dou Microfluidic DNA N. Meningitidis Cerebrospinal LAMP 3 copies X 3 Fluorescence Chroma- Disc
2014 platform fluid tography
(ref. 150) paper
Gan Microdevice X DNA Amelogenin Whole blood PCR 20 7 Electrophoresis Filter paper Disc
2014 for filter- maker saliva swabs ng L1 FTA cards
(ref. 69) paper DNA
extraction
PAD Ali Bioactive DNA Oligonucleotides Synthetic RCA 10 fM 4 Fluorescence Filter paper Strip
2009 paper strips
(ref. 151)
Pardee Synthetic RNA Ebola, Zika Synthetic X Gene 30 nM X 1 Visual Filter paper Disc
2014/16 gene network
(ref. 151 network
and 152)
Connelly Paper X DNA Escherichia Whole blood LAMP 1 copy 4 Fluorescence FTA cards Disc
2015 machine coli per L
(ref. 130)
Prasad Amplification DNA Mycobacterium Artificial HDA 25 copies X 3 Fluorescent Chroma- Disc
2016 on paper tuberculosis sputum per L electrophoresis tography
(ref. 136) substrate paper + BSA
Rohrman Paper and DNA HIV Whole X RPA 1 copy 6 Visual Glass Disc
2012 plastic blood per L fiber + strip
(ref. 140) device for
RPA HIV
Cordray Paper and DNA Plasmodium Synthetic X RPA 5 copies 8 Visual Glass fiber Disc
2015 plastic per L chroma- + strip
(ref. 125) device tography
or blotted
paper
Magro Experiments RNA Ebola Synthetic/viral RPA Equivalent X 2 Fluorescence Chroma- Disc +
2017 in Guinea & of 34 tography origami
(ref. 126) multiplexed PCR qc paper
device
View Article Online
This journal is The Royal Society of Chemistry 2017

Lab on a Chip
View Article Online
Origami
2 strips
+ strip
Shape
sensitive as the gold standard PCR, while working in an iso-
Disc
thermal way (6065 C). Another amplification technique that
raised attention is RPA that requires conditions close to am-
nitrocellulose
bient temperature (3742 C). Whatever the reaction is, a cru-
filter paper
membrane
membrane
Glass fiber
Substrate
cial point mentioned above is the ability to store dried re-

Paper
Glass
fiber
agents in paper. This leads to several consequences such as
PES
reducing the number of manual operations by heading to-

wards -user-friendly autonomous devices and reducing equip-
Fluorescence
ment dependence, especially cold-chain for reagent preserva-
Storage operations Detection
tion. Reagent storage is demonstrated with various

Visual
Visual
amplification techniques (PCR, LAMP, RPA, HDA) but is

implemented in less than half of the contributions. The limit
of detection announced by the authors either with fresh or
freeze-dried reagents does not seem to be affected by this pa-
# of
rameter, which gives good hope for the systematic use of the
10
freeze-dried configuration.
Type of detection. Despite the large number of solutions
described in the literature to detect NA on paper, PADs
performing NAATs mainly rely on either fluorescence detec-
3000 copies
5 parasites
100 copies
tion based on probes or intercalating dyes (direct adaptation

per L
per L
per L
of microtube assays on paper), or colorimetric LFT applied to

LOD
NA. Both have in common commercial availability. With fluo-

rescence, it seems easier to monitor the amplification reac-
Reaction
tion over time and to run parallel experiments. However, it is

LAMP
LAMP
LAMP
not compatible with the equipment-free requirement. One ex-

ception could be that in ref. 154, where the fluorescence sig-
Equipment-
Diagnostics
nal can be read by the naked-eye thanks to a small and porta-

ble UV-lamp. LFTs give visual readouts through a simple
operation, within a few minutes.
free
Biological target and sample. Most of the amplification

X
techniques on paper are demonstrated on DNA that does not

require a prior step of reverse transcription and is known to
specimen
Synthetic
Cervical
be more stable than RNA. There is a variety of pathogens rep-

Whole
blood
Fluid
resented (HIV, dengue, Ebola virus, Plasmodium...) with ei-

ther clinical samples or model systems (artificial environ-
ment and/or synthetic oligonucleotides). Preparation steps to
papillomavirus
Plasmodium
extract and purify NA from raw body fluids are rarely

Prep. Target Pathogen
performed in the PAD. However, most advanced works dem-

Dengue
Human
onstrate the full NAAT diagnostic line using whole blood to

exhibit results on paper.
The ideal paper device should encompass all these key ele-
DNA
DNA
DNA
ments: a ready-to-use paper functionalized with reagents that

Sample
produce a visible signal and that includes all the preparation

steps to work with raw body fluids. By combining selected
X
contributions from the literature, this is technically possible,

paper fluidics
in particular, with the association of various paper materials.

multiplexed
Rodriguez Integrated
Indeed, with composite paper devices, one may take advan-

Origami
Lamp/lf
Device
based
tage of each component: pumping properties to convey flow,

diag.
capture through a membrane, surface chemistry compatible

with amplification... This is already a reality in the paper
Literature
(ref. 133)
(ref. 153)
(ref. 53)
Table 1 (continued)
microfluidics community as illustrated in the penultimate

Choi
2016
2016
2016
Ref.
column of Table 1.
Xu
However, the integration of sequenced protocols in a

given PAD raises the question of keeping the device sim-
ple with a low number of operations. Manual interventions
proposed in the literature mainly consist of loading

View Article Online
samples, pipetting buffers, folding paper layers or peeling sues that stand beyond the scope of the review, but will cer-
plastic foils, which is feasible in the field. One opportunity tainly play a major role in the future of NAAT on paper.
to improve the feasibility of these operations relies on the
microfluidic functions implemented on the paper such as Authors contributions

valves, dilution and mixing solutions and multiplexed ge-
ometries. This library of technical tools should help in- P. T., J. C. M. and P. L. initiated the collaboration. L. M., P. G.,
crease the level of complexity of the paper devices while B. J. and C. E. prepared the manuscript with the assistance of
keeping them extremely user friendly. P. T. L. M., F. M. and P. T. brought expertise on paper micro-
fluidics and technological developments. J. C. M., A. S., C. E.,
A. K., J. V. and P. L. helped on more biological points of view:
Conclusion isothermal amplifications and sample preparations.
After having embarked the reader on a brief tour in the do- References
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Paper Microfluidics For Nucleic Acid Amplification Testing (NAAT) of Infectious Diseases

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Paper Microfluidics For Nucleic Acid Amplification Testing (NAAT) of Infectious Diseases

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Lab on a Chip

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Paper microfluidics for nucleic acid amplification

Laura Magro graduated from a Camille Escadafal trained as a

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Pierre Garneret is a medical stu- Beatrice Jacquelin joined the

Pierre Lafaye is head of the Anti- Patrick Tabeling is a physicist,

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the loop-mediated isothermal amplification (LAMP) that

other schemes, such as Recombinase Protein Amplification

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treatment21 or printing16,22,23 (Fig. 3a). Thanks to patterning,

niques differ by their spatial resolution, cost and simplicity.24

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In paper, molecules can be immobilized. A review of the

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Lab Chip This journal is The Royal Society of Chemistry 2017

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Sample preparation on paper The large range of viscosities of body fluids

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Nucleic acid extraction and purification on paper

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tem and by Connelly et al.130 in a sliding device. A bound

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Lab Chip This journal is The Royal Society of Chemistry 2017

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Tutorial review Lab on a Chip

Lab Chip This journal is The Royal Society of Chemistry 2017

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Lab Chip This journal is The Royal Society of Chemistry 2017

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Towards on-field validation Summarizing the contributions

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Literature Sample Diagnostics Substrate

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bient temperature (3742 C). Whatever the reaction is, a cru-

cial point mentioned above is the ability to store dried re-

reducing the number of manual operations by heading to-

tion. Reagent storage is demonstrated with various

amplification techniques (PCR, LAMP, RPA, HDA) but is

tion based on probes or intercalating dyes (direct adaptation

of microtube assays on paper), or colorimetric LFT applied to

NA. Both have in common commercial availability. With fluo-