Fabrication techniques for microfluidic paper-based analytical devices and their applications for biological testing: A review

Author’s Accepted Manuscript Fabrication techniques for microfluidic paper-based analytical devices and their applications for biological testing: A review Yanyan Xia, Jin Si, Zhiyang Li www.elsevier.com/locate/bios

PII: DOI: Reference:

S0956-5663(15)30496-6 http://dx.doi.org/10.1016/j.bios.2015.10.032 BIOS8068

To appear in: Biosensors and Bioelectronic Received date: 15 July 2015 Revised date: 27 September 2015 Accepted date: 10 October 2015 Cite this article as: Yanyan Xia, Jin Si and Zhiyang Li, Fabrication techniques for microfluidic paper-based analytical devices and their applications for biological testing: A review, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.10.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Fabrication techniques for microfluidic paper-based analytical devices and their applications for biological testing: a review Yanyan Xiaa, Jin Sib,*, Zhiyang Lia,*

a

Department of Laboratory Medicine, the Second Affiliated Hospital, Nanjing

Medical University, Nanjing 210011, China

b

Department of Laboratory Medicine, Drum Tower Clinical College, Nanjing Medical

University, Nanjing 210008, China

*Corresponding author: Jin Si, Tel: 86-25-58509923; E-mail: [email protected]

Zhiyang Li, Tel: 86-25-58509924; E-mail: [email protected]

ABSTRACT Paper is increasingly recognized as a user-friendly and ubiquitous substrate for construction of microfluidic devices. Microfluidic paper-based analytical devices (μPADs) provide an alternative technology for development of affordable, portable, disposable and low-cost diagnostic tools for improving point of care testing (POCT) and disease screening in the developing world, especially in those countries with noor low-infrastructure and limited trained medical and health professionals. We in this

review present fabrication techniques for microfluidic devices and their respective applications for biological detection as reported to date. These include: (i) fabrication techniques: examples of devices fabricated by using two-dimensional (2D) and threedimensional (3D) methods; (ii) detection application: biochemical, immunological and molecular detection by incorporating efficient detection methods such as, colorimetric

detection,

electrochemical

detection,

fluorescence

detection,

chemiluminescence (CL) detection, electrochemiluninescence (ECL) detection, photoelectrochemi (PEC) detection and so on. In addition, main advantages, disadvantages and future trends for the devices are also discussed in this review. Key words: Point of care testing; Microfluidics; Paper; Biochemical; Immunological; Molecular

1. Introduction ................................................................................................................ 4

2. Fabrication techniques ............................................................................................... 5

2.1. Fabrication of 2D paper-based microfluidics .................................................. 6

2.1.1. Wax printing ................................................................................................. 6

2.1.2. Inkjet printing ............................................................................................... 8

2.1.3. Photolithography ........................................................................................ 10

2.1.4. Flexographic printing ................................................................................. 12

2.1.5. Plasma treatment......................................................................................... 13

2.1.6. Laser treatment ........................................................................................... 14

2.1.7. Wet etching ................................................................................................. 15

2.1.8. Screen-printing ........................................................................................... 15

2.1.9. Wax screen-printing ................................................................................... 16

2.2. Fabrication of 3D paper-based microfluidic .................................................. 17

3. Application platforms .............................................................................................. 20

3.1. Biochemical detection ................................................................................... 20

3.2. Immunological detection ............................................................................... 31

3.3. Molecular detection ....................................................................................... 39

3.4. Other detection methods ................................................................................ 45

4. Conclusions .............................................................................................................. 46

Acknowledgments........................................................................................................ 48

References .................................................................................................................... 48

1. Introduction

Microfluidic paper-based analytical devices (μPADs) were introduced in 2007 (Martinez et al., 2007). They have hydrophilic/hydrophobic micro-channel networks and associated analytical devices which can enable fluid handling and quantitative analysis for their potential applications in medicine, healthcare and environmental monitoring (Hu et al., 2014). The μPADs also have the ability to perform laboratory operations on micro-scale, using miniaturized equipment, hence having their significant stimulated concern as a multiplexable point of care testing (POCT) platform (Bier and Schumacher, 2013). When compared with the conventional microfluidic analytical devices which are fabricated by silicon, glass and superpolymer as their substrates, the μPADs, fabricated by paper, are affordable, userfriendly, ubiquitous and do not require external instruments and complex fabrication processes. They hence are providing a common platform for prototyping new POCT (Chen et al., 2015; Barbosa et al., 2015; Fan et al., 2015; Martins et al., 2015; Tan et al., 2015), particularly using in limited resource environments (Phillips and Lewis, 2014; Gubala et al., 2012; Warsinke, 2009; Peeling et al., 2006). The μPADs can enable fluid handling and quantitative analysis when applied in medicine, healthcare and environmental monitoring (Hu et al., 2014). Coupled with different fabrication methods and functional diagnostic equipments, to fabricate miniaturized portable

medical tools, the μPADs have had many new developments recently (Zhang et al., 2015b; Li et al., 2015). There has been more than 100 articles involving the μPADs that have been published during 2014 to 2015. We therefore focus on the twodimensional (2D) and three-dimensional (3D) fabrication methods, and their respective application for biochemical, immunological and molecular detection, incorporating efficient detection methods, such as colorimetry, electrochemistry, fluorescence,

chemiluminescence

(CL),

electrochemiluninescence

(ECL),

photoelectrochemistry (PEC) etc. In addition, the main advantages, disadvantages and future trends for the devices are also discussed.

2. Fabrication techniques

Microfluidic paper-based analytical devices can be fabricated by using 2D (Balu et al., 2009; Fu et al., 2010; Kauffman et al., 2010; Lutz et al., 2011) or 3D (Han et al., 2013; Kalisha and Tsutsui, 2014; Lewis et al., 2012; Li et al., 2014c; Liu and Crooks, 2011; Martinez et al., 2010a; Martinez et al., 2008a; Mosadegh et al., 2014; Phillips and Thom, 2013; Schilling et al., 2013) methods, to transport fluids in both horizontal and vertical dimensions depending on complexity of the diagnostic application.

2.1. Fabrication of 2D paper-based microfluidic devices

This treatment changes specific areas (or lines) of cellulose paper from being hydrophilic to hydrophobic. The two parallel hydrophobic lines act as channels, because hydrophilic sample solution cannot penetrate the hydrophobic line or barrier and consequently the liquid flows in the channels owing to capillary action. Overall, there have been nine reported techniques in the literature for fabrication of paper-based microfluidic devices (Elsharkawy et al., 2014; Rosenfeld and Bercovici, 2014; Sones et al., 2014; Viola et al., 2013): These include: (1) wax printing, (2) inkjet printing, (3) photolithography, (4) flexographic printing, (5) plasma treatment, (6) laser treatment, (7) wet etching, (8) screen-printing, and (9) wax screen-printing.

2.1.1. Wax printing

The wax-patterning method (Cai et al., 2013; Li et al., 2014d; Zhong et al., 2012) has following merits; a simple fabrication process (printing and baking), its rapid (510 min), inexpensive (both wax and paper are cheap and easy to obtain), and environmentally friendly (no use of organic solvents throughout the fabrication process). The paper and wax can also be easily disposed of by burning (Fig. 1) (Lu et al., 2010). The wax printing involves the fewest number of steps and is best suited for

fabricating large numbers (> 100) of paper-based analytical devices in a single batch. The wax-based micropatterning technology is therefore very useful for prototyping paper-based microfluidic devices to implement low cost bioassays in remote settings (Carrilho et al., 2009a). Three different ways for wax patterning have been introduced, these include: (i) painting with a wax pen, (ii) printing with a normal inkjet printer, followed by tracing by painting with a wax pen (iii) direct printing by a wax printer (Lu et al., 2009). The printing process is easy to operate and can be finished within 5-10 min without use of a clean room, ultraviolet (UV) lamp, organic solvent, etc.

Fig. 1. Schematic illustration of fabrication processes for paper-based microfluidics in a nitrocellulose (NC) membrane by wax printing (Lu et al., 2010).

2.1.2. Inkjet printing

This is a new fabrication method for the paper-based microfluidic devices which incorporates the paper sizing chemistry with digital inkjet printing technique (Li et al., 2010a). The Inkjet printing can deliver biomolecules and indicator reagents with precision into the microfluidic patterns, to form biological/chemical sensing zones within the patterns and also complete sensing devices. The potential for combining paper sizing chemistry and inkjet printing to produce paper-based sensors has proved to be of low cost and commercial volume. Fig. 2 illustrates the schematic presentation of fabrication process for the inkjet printed microfluidic multianalyte chemical sensing paper. The paper based-fluidic devices, which are fabricated by inkjet printing and spraying of conductive hydrophobic electrodes/valves, in conjunction with conductive hydrophilic electrodes, are able to stop the fluid front from phosphate buffered saline (Koo et al., 2013). The hydrophobic valves are then actuated by an applied potential which alters the fluorinated monolayer on the electrode. When the applied potential between the electrodes is increased, the amount of time needed for the front fluid to pass the valve also decreases, because the monolayer is altered faster. The hydrophobic barriers, comprised of wax in the paper-based microfluidic systems, are restricted to constrain aqueous surfactants solutions which are frequently used in

biological assays, while the inkjet which is printed onto the pure cellulose paper by curing silicone resins, using inexpensive thermal inkjet printers, are able to resist penetration by surfactant solutions (Rajendra et al., 2014). The omniphobic "fluoroalkylated paper" can be used as substrate for inkjet printing of aqueous inks that can be used to print high resolution and conductive patterns that remain conductive after folding and exposure to common solvents (Lessing et al., 2014). This inkjet printing fabrication method can be scaled up and adapted for use as high speed, high volume and low cost commercial printing technology (Li et al., 2010b). For the inkjet-printed surface-enhanced Raman spectroscopy substrate for analyte detection, measurements show that the technique is quantitative and repeatable across multiple swabs and dipsticks (Carrilho et al., 2009a; Yu and White, 2013).

Fig. 2. Schematic representation of fabrication process for the inkjet printed microfluidic multianalyte chemical sensing paper, featuring microfluidic channels connecting a central sample inlet area with three different sensing areas and a reference area (Abe et al., 2008).

2.1.3. Photolithography

Fabrication of the paper-based plates by patterning sheets of paper into hydrophilic zones surrounded by hydrophobic polymeric barriers can be done using photolithography. The photolithography can use an inexpensive formulation photo resistant that allows rapid (15 min) prototyping of paper-based plates (Fig. 3) (Carrilho et al., 2009b). One publication evaluated a novel and facile fabrication method for the μPADs using flash foam stamp lithography and compared it to common fabrication methods, such as wax printing and inkjet printing. This publication demonstrated that the lithography method is convenient, quick, and cheap (He et al., 2014). Other publications described new ways to fabricate paper-based microfluidic devices using the following: (i) Hydroxypropyl cellulose; which is rendered photo cross-linkable by grafting it with methylacrylic anhydride whose linkages also render the cross-linked construct hydrolytically degradable. The Hydroxypropyl cellulose is then cross-linked via the photolithography-based fabrication process, making it hydrolytically degradable and biocompatible, thus enabling cell migration and proliferation, and therefore constituting an ideal candidate for long-term cell culture and implantable tissue scaffold applications (Qi et al., 2014). (ii) Fast Lithographic Activation of Sheets; this is also a rapid method for laboratory prototyping of microfluidic devices on paper. It is based on

photolithography but requires a UV lamp and hotplate only. The patterning can even be performed in sunlight when the UV lamp and hotplate are unavailable and no clean room or special facilities are required (Martinez et al., 2008b). Photolithographically defined channels exhibit high background while wax printed channels show very low background (Dungchai et al., 2011). The photolithography requires organic solvents, expensive photoresists and photolithography equipment.

Fig. 3. Paper plates for multizone assays produced using photolithography (Carrilho et al., 2009b). (A) Image of a 96-zone plate after application using a range of different dyes solution volumes in alternating zones, this image demonstrates the fluidic isolation of the zones. (B) Image of a 384-zone plate after application using 1-10 μL of the same solutions as in part A, the two zones in the fifth and sixth rows show small breaches in the hydrophobic walls. (C) Alternative design of a 96-zone paper plate incorporating distribution or connection channels between zones. (D) Image

showing the 96-zone plate with volumes of liquid up to 55 μL that were completely restrained from spreading over the paper by hydrophobic barrier. (E) Similar experiment demonstrating that the smaller zones in the 384-zone plates hold at least 10 μL of liquid. (F) Time-lapse images of the details shown in part C, illustrating the mixing of two solutions and reaction, the color development is apparent within minutes after application of the two solutions.

2.1.4. Flexographic printing

This is a simple method based on flexographic printing of polystyrene to form liquid guiding boundaries and layers on paper substrates, allowing formation of hydrophobic barrier structures that partially or completely penetrate through the substrate (Olkkonen et al., 2010). This unique property enables one to form very thin fluidic channels on paper that lead to reduced sample volumes required in point of care diagnostic devices. The described method is compatible with roll-to-roll flexography units found in many printing houses, making it an ideal method for largescale production of paper-based fluidic structures. Fig. 4 illustrates the schematic illustration of the flexography unit.

Fig. 4. (a) Schematic illustration of the flexography unit used in the study. (b) Relief patterns in the printing plate defining the hydrophobic regions to be formed into paper (Olkkonen et al., 2010).

2.1.5. Plasma treatment

Fabrication of the μPADs via plasma treatment is done as follows: paper is firstly hydrophobized via octadecyltrichlorosilane (OTS) silanization and then the OTS silanized paper is regionally selected and plasma-treated via a mask with channel network. The plasma-exposed area of the paper is turned to hydrophilic channel network due to degradation of hydrophobic OTS molecules coupled to the paper's cellulose fibres before (Yan et al., 2014). When the effect from the plasma-treatment time on hydrophilicity of paper was investigated, the water contact angle was dramatically decreased from 133.9 degrees +/- 1.3 degrees to 0 degrees, with prolongation of the plasma treatment time from 0 s to 30 s. Moreover, the depth of

wettable channel could also increase to nearly the thickness of the paper after treatment for 30 s. However, a well known problem for plasma treatment is that the substrate under the mask is often over stretched, causing the treated pattern to be bigger than the mask. The treatment intensity and time should then be controlled to find reproducible channel width. The paper devices made using the plasma treatment have an advantage over the barrier design in that, simple functional elements, such as switches, filters, and separators, can be easily built into the microfluidic system (Li et al., 2008).

2.1.6. Laser treatment

A laser-based fabrication procedure that uses polymerisation of a photopolymer has successfully guided the flow of fluids and allowed containment of fluids in wells. The minimum width for the hydrophobic barriers that prevents fluid leakage was found to be 120 μm in a conducted study, and the minimum width for the fluidic channels that can be formed was 80 μm, which is the smallest reported so far for the paper-based microfluidic (Sones et al., 2014). Besides, the CO2 laser method involves only one operation for cutting a piece of paper by laser according to a predesigned pattern (Nie et al., 2013), This method is versatile and allows for controlled throughcutting and ablative etching of NC substrates (Spicar-Mihalic et al., 2013). In

addition, the laser system is able to selectively modify the surface structure and property of several papers (Chitnis et al., 2011) and can cut a variety of components that are useful in the fabrication of paper-based devices, including cellulose wicking pads, glass fiber source pads and Mylar-based substrates for the device housing.

2.1.7. Wet etching

The fabrication process for the μPADs by selective wet etching of hydrophobic filter paper consists of two steps (Cai et al., 2014). First, the hydrophilic filter paper is hydrophobically patterned using trimethoxyoctadecylsilane solution as the patterning agent. Next, a paper mask penetrated with NaOH solution (containing 30% glycerol) is aligned onto the hydrophobic filter paper, allowing the etching of the silanized filter paper by the etching reagent. The masked region then becomes highly hydrophilic, whereas the unmasked region remains highly hydrophobic. The hydrophilic channels, reservoirs, and detection zones are thus generated and delimited by the hydrophobic barriers.

2.1.8. Screen-printing

Carbon electrodes screen-printed directly on cellulose paper can be employed to perform bipolar electrochemistry (Renault et al., 2013). In addition, an array of 18 screen-printed bipolar electrodes can be simultaneously controlled using a single pair

of driving electrodes. The electrochemical state of the bipolar electrodes is read-out using electro-generated CL and this demonstrates the feasibility of the coupling bipolar electrochemistry for the μPADs to perform highly multiplexed and low-cost measurements.

2.1.9. Wax screen-printing

Wax screen-printing is fabricated by two simple steps as follows: (1) printing patterns of solid wax on the surface of paper using a simple screen-printing method and common household supplies, and (2) melting the wax into paper to form complete hydrophobic barriers using a hot plate. This is schematically shown in Fig. 5. The final widths of the hydrophobic barrier and hydrophilic channel are in the range of 1200-1800 mm and 550-1000 mm, respectively, at optimal melting temperature and time. The conventional wax printing needs a wax printer (at $2500 US) but printing screens are cheap (at $5 US or 200 Thai Baht per 100 cm2) and can easily be obtained from around the world. Besides, the wax is low-cost and can be purchased anywhere in the world, and is also environmentally friendly. In addition, the wax screen-printing method is accomplished without the use of clean room, UV lamp, organic solvents, or complexed instrumentation. Finally, the major advantage of this method over previous methods is that it requires only a common hot plate (or similar surface) and common

printing screen that can be produced anywhere in the world, making it ideal for fabrication of the μPADs in developing countries (Dungchai et al., 2011).

Fig. 5. Schematic diagram of fabrication step for wax screen-printing method (Dungchai et al., 2011)

2.2. Fabrication of 3D paper-based microfluidic devices

3D paper-based microfluidic devices are fabricated by stacking alternating layers of paper and water-impermeable double-sided adhesive tape, both patterned in ways that channel the flow of fluid within and between layers of paper (Martinez et al., 2008a). The hydrophobic polymer patterned into the paper demarcates the channels through which the fluids move laterally (Martinez et al., 2008b; Martinez et al., 2007) and the layers of water-impermeable double sided tape separates the channels in the neighboring layers of paper. The holes cut into the tape and allow fluids to flow vertically. The layer in the 3D μPADs can be made using different papers and the

multiple functionalities provided by the different types of paper can therefore be combined into a single device (Martinez et al., 2010b). The cost of material is approximately $0.03 or $0.003 per square centimeter per layer of paper. Tape and dyes’ aqueous solutions (20 μL aliquots) can weaken the length of these channels in ≈5 min (Martinez et al., 2008a). The used dynamic mask photo curing which was generated by a desktop stereolithography 3D printer to fabricate the μPADs showed a leap forward in terms of time saved in one study. Since all the hydrophobic barriers were cured at a time, the fabrication process could be completed in only 2 min, no matter how complex the patterns were (He et al., 2015). Comparisons of fabrication techniques for the microfluidic paper-based analytical devices are listed in Table 1. Table 1 Comparisons of fabrication techniques for microfluidic paper-based analytical devices Fabrication

Patterning

Hydrophobic

Volumes of

techniques

agents

barrier

sample (μL)

Stored time

3 months under normal conditions

100 μm width Wax (Lu et al., Wax printing

2 (Lu et al., (Lu et al.,

2014 )

2014 ) 2014 )

at room temperature (Lu et al., 2014 )

Permanent

Drawbacks

Simple and rapid (5-10 Requires expensive wax min) fabrication process, printers and an extra environmentally friendly heating step (Lu et al., (Cai et al., 2013; Li et al., 2010) 2014d) Can be scaled up,

marker ink

Inkjet printing

Advantages

At least 6 months

inexpensive thermal inkjet

(Xu et al.,

550 μm width

10 femtomoles

when stored at

printers ($60) (Rajendra et

Not suitable for mass

2015);

(Yamada et al.,

(Yu and White,

room temperature

al., 2014); print high

fabrication (Yu and White,

Hexadecenyl

2015)

2010)

(Yamada et al.,

resolution and conductive

2010)

2015)

patterns (Lessing et al.,

succinic anhydride (Yu

2014)

and White, 2010) Requires organic solvents,

Photoresist

0.5 mm width

(Carrilho et al.,

(Carrilho et al.,

2009b)

2009b)

Photolithograp hy

Rapid (15 min), high

expensive photoresists

5 (Carrilho et

resolution of microfluidic

(SU-8 is approximately

al., 2009b)

channels (Carrilho et al.,

$800/L; SC photoresist is

2009b)

about $100/L) (Dungchai et al., 2011)

Thin fluidic channels, At least 400

leading to reduced sample

Requires two prints of

Polystyrene Flexographic

μm width

0.2 (Olkkonen

volumes, allows direct roll-

polystyrene solution;

(Olkkonen et

et al., 2010)

to-roll production in

requires different printing

existing printing houses

plates (Li et al., 2012 )

(Olkkonen et printing al., 2010) al., 2010)

(Olkkonen et al., 2010) Alkyl ketene

The substrate under a mask is <1.5 mm in width

Plasma Treatment dimer (Li et al.,

Cheap patterning agent (Li et 2 (Li et al., 2008)

often overetching (Li et al.,

(Li et al., 2008)

al., 2008)

2008)

2008) Do not allow lateral flow of 120 μm width

Versatile, easy controlled

(Sones et al.,

(Spicar-Mihalic et al., 2013);

Any paper with a

fluids, requires extra coating

hydrophobic Laser treatment

for liquid flow, it is not well 2014); 150 μm

3 (Sones et al.,

selectively modify the surface

width (Spicar-

2014)

structure and property of

surface coating

suited for a scale up to very

(Chitnis et al.,

high throughput mass Mihalic et al.,

several papers (Chitnis et al.,

2013)

2011)

2011)

production of devices (Li et al., 2012)

Trimethoxyoctade

No expensive facilities and The printing apparatus must be 0.8 (Cai et al.,

Wet etching

cylsilane (Cai et

materials are used (Zhang et

customized (Zhang et al.,

al., 2015a)

2015a)

2014) al., 2014)

Low resolution of Varnish paint Screenprinting

microfluidic channels, 500 μm width

Produces devices with

(Sun et al.,

simple process (Li et al.,

2015)

2012)

solution, roof

requires different printing

sealant (Sun et

screens for creating

al., 2015)

different patterns (Li et al., 2012) Printing screens are cheap ($5 US or 200 Thai Baht Diameter of 6

○

Wax mm /1300 μm

Wax screen-

(Dungchai et al., 2011)

per 100 cm2),

at least 5 weeks

environmentally friendly, it

(Wang et al., 2012)

requires only a common

1 (Dungchai et

(Dungchai et printing

Patterned mesh is

At 4 C (sealed) for

necessary, making it

al., 2011)

inadequate for prototyping

al., 2011)

(Dungchai et al., 2011) hot plate (Dungchai et al., 2011)

3. Application platforms

The main application of the μPADs is to provide low-cost, easy-to use, and portable analytical platforms for assays, either multi-analyte or semi-quantitative (even quantitative), in order to provide people living in the developing world with affordable disease diagnosis which is environmentally friendly (Li et al., 2012; Zhang et al., 2015a). According to their reaction mechanisms, these tests can be categorized into biochemical, immunological, and molecular detections.

3.1. Biochemical detection

Paper-based biochemical detection has been applied to test many analytes. When sample solutions from sampling zones reach detection zones in the patterned paper, a chemical reaction, like acid-alkali reaction, or precipitation reaction, or redox, or enzymatic reaction occurs between the target compound and immobilized reagents and a signal is developed. The signal can then be detected by colorimetric (Wang et al., 2010; Li et al., 2014a), electrochemical (Dungchai et al., 2009), fluorescent (Gong et al., 2014), chemiluminiscence (CL) (Ge et al., 2014), Electrochemiluminiscence (ECL) (Wu et al., 2015b; Yan et al., 2013), or photoelectrochemical (PEC) methods (Ge et al., 2013). The comparisons between the proposed detection methods for biochemical testing using the microfluidic paper-based analytical devices are listed in

Table 2, which includes the fabrication techniques, cost of the devices, time required to complete the detection, analytes, detection limit, linear range and required equipments.

Table 2 Comparisons between proposed detection methods for biochemical detection using microfluidic paper-based analytical devices Detection Detection

Fabrication

methods

technique

Others(Advantages/Dra Detection

Costs

Time

Analyte

Linear range

Equipments

wbacks)

limit (min) Be visible to the naked eye (Lopez-Ruiz et al., H2O2

Wax printing

$3 (Wang

0.65 Mm

0.65~300

Equipment-

2014); interference from

(Zhang et al.,

mM (Zhang

Free (Wang et

competing metal ions

2014b)

et al., 2014b)

al., 2014a)

(Zhang et al., 2013)

(Zhang et Colorimetric

(Wang et al.,

et al., al.,

2014a)

2014a) 2014b)

Insensitive to ambient Cd and Pb Wax printing

$1 (Scida

4.6 (Scida

Electrochemic

0.25 ng

0.25~7.5 ng

Potentiostat

illumination conditions

(Rattanarat

(Rattanarat

(Rattanarat et

and impurities in the

et al., 2014)

et al., 2014)

al., 2014)

samples (Zhao et al.,

(Rattanara (Rattanarat et

et al.,

et al.,

al

t et al., al., 2014)

2014)

2014) 2014)

2013; Nie et al., 2010) β-D$0.15

30 (Thom

galactosid

0.7 nM

0.7~12 nM

(Thom et

et al.,

ase

(Thom et al.,

(Thom et al.,

al., 2014)

2014)

(Thom et

2014)

2014)

Wax printing Fluorescent (Thom et al., 2014)

Camera-

Interference from

equipped

competing metal ions

cellular phone

(Zhang et al., 2013)

(Thom et al., al., 2014) 2014) Computerized Uric acid

Chemilumines

Cutting

2.6~49.0 1.9Mm (Yu

(Yu et al., cence

method (Yu et

et al., 2011a) 2011a)

al., 2011a)

ultraweak

Independent of ambient

luminescence

light (Delaney et al.,

analyzer (Yu

2011);

mM (Yu et al., 2011a)

et al., 2011a)

Petroff-

Combination of the

Hausser cell

advantages from the

counter (Wu et

luminescence and

al., 2015a);

electrochemical

photomultiplie

techniques (Wu et al.,

r tube (Li et

2015a)

Human 1.0~450×107

breastade Wax printing Electrochemilu

250 cells/Ml nocarcino

(Wu et al., ninescence

cells/mL (Wu et al.,

macells 2015a)

(Wu et al., 2015a)

(Wu et al.,

2015a)

2015a) al., 2013)

Scanning

Have the advantages

electron

from the optical

microscopy,

methods and

Electrochemic

electrochemical sensors

al Workstation

(Sun et al., 2014b)

Pentachlo Wax-screenrophenol Photoelectroch

printing (Sun

0.01~100 4 pg/Ml (Sun

(Sun et emical

et al., 2014a)

ng/mL (Sun et al., 2014a)

al.,

et al., 2014a)

2014a) (Sun et al., 2014a)

The colorimetric reaction is the most commonly used assay method in the μPADs, due to its easy operation and straightforward signal readout. It is based on colored compounds generated by unknown to those generated by known analyte concentrations. The intensity of the color can then be recorded by scanners or cameras which transmit the digitized readout off-site for quantitative analysis (Hossain et al., 2009; Khan et al., 2010). The chemical reaction is the common cause for generating color change. Zhou et al. developed paper-based colorimetric biosensing platform, utilizing cross-linked siloxane 3-aminopropyltriethoxysilane (APTMS) as probe for the detection of a broad range of targets, including H2O2 and glucose. When the APTMS was cross-linked with glutaraldehyde (GA), the resulting complex (APTMS–

GA) displayed brick-red color, and the visual color change was observed when the complex reacted with H2O2. By integrating the APTMS–GA complex with filter paper, the modified paper enabled quantitative detection of H2O2 and with immobilization of glucose oxidase (GOx) onto the modified paper, glucose could be detected through the detection of enzymatically generated H2O2 (Zhou et al., 2014). Lopez-Ruiz fabricated a smartphone-based simultaneous pH and nitrite colorimetric determination μPADs. The device consisted of one main central area (sampling area) and seven sensing areas with independent channels as shown in Fig. 6B. The pH sensing areas used two different pH indicators, phenol red and chlorophenol red. The nitrite sensitive areas are based on the Griess reaction and also one blank for reference during the colorimetric detection. When the analytes are dropped into the central sampling area of the microfluidic device, the solution flows toward the seven sensing areas, because of the capillarity of the filter paper and barriers created by the stamped indelible ink. When the microfluidic device is dry, the uniform and stable color can be easily detected by the Android application installed on the smartphone. It is therefore possible to perform multi-detection of the areas in one single experiment using one single microfluidic device without the need for external processing elements (LopezRuiz et al., 2014). This multi-detection idea was also used to detect urine (Martinez et al., 2008c), uric acid, glucose, and lactate (Dungchai et al., 2010).

Fig. 6. Smartphone-based simultaneous pH and nitrite colorimetric determination device (Lopez-Ruiz et al., 2014). (A) Microfluidic device with reagents placed in each sensing area. (B) Microfluidic device with reagents placed in each sensing area. Nanoparticle colorimetric sensing approaches have also been used in the paperbased colorimetric analytical devices, because their extinction coefficients are higher than common dyes (Ngo et al., 2012). Nath et al. (2014) designed a compound of Au nanoparticles (AuNPs) coupled with thioctic acid (TA) and thioguanine (TG) molecules which could show visible bluish-black colour precipitate due to the formation of nanoparticle aggregates through transverse diffusive mixing of Au-TATG with As3+ ions on the paper substrate. Ratnarathorn et al. (2012) investigated the silver nanoparticles (AgNPs) colorimetric sensing of Cu2+ by the μPADs. The AgNPs colorimetric sensing for the detection of Cu2+ was characterized by UV–visible spectroscopy. The –SH groups on homocysteine and dithiothreitol were used to

modify the AgNPs surface, whereas the –COOH and –NH2 functional groups had strong affinity to Cu2+ relative to other ions in solution. The plasmon resonance absorption peak intensity at 404 nm decreased and a new red-shifted band at 502 nm occurred in the presence of Cu2+. Paper devices coated with the modified AgNPs solution change from yellow to orange and green-brown color after the addition of Cu2+ due to nanoparticle aggregation (Ratnarathorn et al., 2012). Furthermore, another study reported that curcumin nanoparticles nanonization and low solubility in solutions, to chelate the metal ions and form the complexes (Pourreza and Golmohammadi, 2015). Many paper-based electrochemical microfluidic devices in conjunction with electro-analytical sensors demonstrated much lower limit of detection than colorimetric assays and electrochemical detection was insensitive to ambient illumination conditions and impurities in the samples, making it particularly suitable for use in field and/or dirty environments (Zhao et al., 2013; Maxwell et al., 2013; Silva et al., 2014). Dungchai and Tan fabricated the electrodes by screen printing technology on the paper-based microfluidic devices for determination of glucose and metal ion (Dungchai et al., 2009; Tan et al., 2010). Yang et al. also developed a miniaturized paper-based microfluidic electrochemical enzymatic biosensing platform by measuring the H2O2 for detecting glucose. Villarrubia et al. introduced the

reagentless and non-reagentless nicotinamide adenine dinucleotide-dependent glucose dehydrogenase bioanodes, integrating multi walled nanotubes-bucky papers (Villarrubia et al., 2014). Fluorescence assay is another kind of optical method which possesses inherently much higher sensitivity than the colorimetric methods. Thom et al. (2014) described a point-of-care assay strategy in which fluorescence in the visible region was used as readout while a camera-equipped cellular phone was used to capture the fluorescence response and quantify the assay. The small molecule reagent was transformed from weakly fluorescent to highly fluorescent when exposed to a specific enzyme biomarker. The resulting visible fluorescence was digitized by photographing the assay region using a camera-equipped cellular phone. Upconversion fluorescence is a type of anti-Stokes luminescence emitted from upconversion phosphors that can decrease serious background fluorescence from additives and scattering light in the paper substrate which considerably improves the robustness and sensitivity of the fluorescence assays. The upconversion phosphors tagged with specific probes are spotted to the test zones on the μPADs, followed by the introduction of assay targets. The upconversion fluorescence measurements are directly conducted on the test zones after completion of the probe-to-target reactions without any post-treatments. The combination of the μPADs with upconversion fluorescence assay is therefore likely to

afford a promising tool for the POCT with expected simplicity, accuracy, and sensitivity, hence promoting the application of the μPADs in clinical diagnosis (He and Liu, 2013). The CL method can be combined with the μPADs to establish novel CL μPADs biosensor made of the final biosensor that is inexpensive and highly sensitive (Yu et al., 2011a). Yu et al. (2011b) also designed a novel microfluidic paper-based CL analytical device with a simultaneous and quantitative response for glucose and uric acid determination by differing the distances that the two samples traveled. The new device-holder was designed at the bottom of the cassette to fix the position of the μPADs. The cassette could then be shut with a black metallic cover that has an injection hole for sample injection. When the μPADs was put into the holder, the sample injection area was aligned to the photomultiplier of the analyzer. For detection, the sample solution migrated towards the CL detection area, to obtain the CL signal, which was recorded using a computer. This CL detection method was also used to detect L-cysteine (Liu et al., 2014) and serine, aspartic acid, and lysine (Ge et al., 2014). Microfluidic paper-based ECL has significantly grown in importance in recent years, due to combination of advantages from the luminescence and electrochemical techniques, such as high sensitivity and wide dynamic concentration response range.

It is also potentially- and spatially controlled. Wu et al. developed a microfluidic paper-based electrochemiluminescence origami cyto-device in which aptamers modified 3D macroporous Au-paper electrodes were employed as working electrodes and efficient platforms for the specific capture of cancer cells. Owing to the effective disproportion of hydrogen peroxide and specific recognition of mannose on the cell surface, concanavalin-A conjugated porous AuPd alloy nanoparticles were introduced into this device as catalytically promoted nanolabels for the peroxydisulfate ECL system. Under the optimal conditions, the proposed device exhibited excellent analytical performance, with good stability, reproducibility, and accuracy towards the cyto-sensing of four types of cancer cells, indicating its potential for applications to facilitate effective and multiple early cancer diagnosis and clinical treatment (Wu et al., 2015a). The detection of toxic heavy metals has been always a subject of considerable research due to environmental contamination and many current techniques, such as fluorescence and colorimetry, have some limitations, including poor sensitivity and selectivity, interference from competing metal ions, and restriction to single metal ion detection. For these reasons, Zhang et al. proposed a 3D microfluidic paper-based analytical device for simultaneous ECL nanoprobes detection for lead ion and mercury ion based on oligonucleotide, providing a platform for low cost POCT analysis and extending the application of ECL field (Zhang et al.,

2013). A mobile camera phone can also be used to detect the luminescence from the sensors when the paper substrate is aligned and fixed onto the face of the screen-printed electrodes,

by laminating with transparent plastic. A drop of samples is introduced

through a small aperture in the plastic at the base of the channel when the detection zone is fully wetted and the sensors are placed close to the lens of the camera phone when a potential of 1.25 V is applied, the resulting emission is then captured and analyzed. Finally, the ubiquity, power and connectivity of the mobile phones create opportunities to enhance health-care outcomes in the developing world via concepts such as telemedicine and e-health (Fig. 8) (Delaney et al. 2011).

Fig. 7. Fabrication and operation of a paper-based microfluidic ECL sensor (Delaney et al. 2011). (a) The paper microfluidics are produced in bulk using a conventional inkjet printer. (b) Individual paper fluidic elements are cut to size and hydrophilic portion filled with a 10 mM tris (2, 20 bipyridyl) ruthenium (II) solution before drying. (c) Paper substrate is then aligned and fixed onto the face of the screen-printed electrodes. (d) A potential of 1.25 V is applied and resulting emission is captured and analyzed.

By coupling the photoirradiation with electrochemical detection, such PEC sensors draw the advantages from the optical methods and electrochemical sensors, thus showing great promise for analytical applications (Sun et al., 2014b). This technique therefore shows promising analytical applications and has attracted considerable research interest (Wang et al., 2015b). The PEC method was able to be introduced into the μPADs and further amplification of the generated photocurrents, an all-solid-state paper supercapacitor, was constructed and integrated into the μPADs, to collect and store the generated photocurrents. The stored electrical energy could be released instantaneously through the digital multimeter to obtain an amplified and digital multimeter detectable current, as well as a higher sensitivity than the direct photocurrent measurements, allowing the expensive and sophisticated electrochemical workstation or lock-in amplifier to be abandoned (Fig. 9) (Ge et al., 2013).

Fig. 8. Schematic illustration of the photocurrent generation mechanism in the modified paper sample zone of the paper working electrode under (A) An external physical light source. (B) An internal CL light source. (C) Storage of the generated photocurrent in the paper supercapacitor for 60s. (D) Instantaneous release of the stored electrical energy through the digital multimeter once the switch was closed (Ge et al., 2013).

3.2. Immunological detection

Immunological detection is a method that uses immunoassay technique for multiple applications, such as human chorionic gonadotropin (Apilux et al., 2013; Schonhorn et al., 2014), Escherichia coli O157:H7 (Reinholt et al., 2014), Rabbit IgG (Gerbers et al., 2014), goat anti-rabbit IgG (Bai et al., 2013), and red blood cells agglutination (Yang et al., 2012). In clinical detection, it is mainly used to detect humoral antibodies or antigenic substances through an antigen-antibody reaction. The papers are modified chemically in this technique and combine with functional groups to covalently bond with small molecules and proteins. The comparisons of proposed detection methods for immunological detection in the microfluidic paper-based analytical devices are listed in Table 3. Table 3

Comparisons of proposed detection methods for immunological detection in microfluidic paper-based analytical devices Detection

Fabrication

methods

technique

Colorimetric

Wax printing

Time

Analyte

Detection limit

Linear range

Equipments Ref.

Carcinoembry 0.03 ng/mL

0.1~20.0 ng/mL

Scanner

onic antigen

Liu et al., 2015

Cancerantigen Electrochemical Electrochemic

Screen

125 and

0.02 and 0.04

2s al

printing

Li et al., 2014c 0.08~0.10 mU/mL

carcinomaanti

workstation,

mU/Ml spectrometer

gen 199 Photolithograp

Circulating

Nikon inverted

hy

tumor cells

microscopy

Fluorescent

Wu et al., 2014

Modulus Chemilumines

26.7 fmol~267 wax printing

30 min

Anti-HCV

267 amol

cence

Ge et al., 2012a; Microplate

amol

Mu et al., 2014 Multimode Reader

r-fetoprotein, carcinoma

electrochemilu Wax printing ninescence

antigen 125,

0.15 ng/mL,

0.5~100 ng/mL,

carcinoma

0.6 U/mL,

1.0~100 U/mL,

Scanning electron

antigen 199

0.17U/mL, and

0.5~100 U/mL, and

microscopy

and

0.5 ng/mL

1.0~100 ng/mL

30 min Ge et al., 2012b

carcinoembryo nic antigen Paper Photoelectroch

Carcinoembry Wax printing

emical

0.1 pg/mL~5

supercapacitor,

μg/mL

terminal digital

0.065 pg/mL onic antigen

Wang et al., 2015a

multimeter detector

Mu et al. (2014) used irreplaceable merits of multiplex microfluidic paper-based immunoassay for the serologic detection of IgG antibody against hepatitis C virus (anti-HCV) (Fig. 10). The paper was manually patterned by craft punch patterning under ambient temperature, and the craft punch patterning was inexpensive (less than

$2). The “petals” could be used as multiple detection zones, and the radial shape would prevent it from cross contamination by using air barriers between the detection zones. The liquids could be pipeted either directly on each detection zones, shown by a series of red dye. The former consumes fewer samples while the latter is less laborintensive. This method demonstrates remarkable merits, for example, the material cost for one detection zone in a multiplex paper-based device is estimated to be onetwenty-fifth of one well in the 96-well enzyme-linked immunosorbent assay (ELISA), and the required serum is as low as 6 nL per detection zone, which is approximately 2000 times lower 10 and 20 μL serum in ELISA.

Fig. 9. Indirect-ELISA on patterned NC for quantitative detection of mouse IgG (Mu et al., 2014). (A) Schematics of indirect ELISA procedures. (B) Comparison of CL signals from 1 μg/mL mouse IgG, protran BA85 is preferred for immobilizing proteins over 20 kDa and thus shows higher signal than others. (C and D) CL image and plot from different pipetting volumes of mouse IgG that show a linear relationship (R2 = 0.988). (E and F) CL image and plot from mouse IgG from 668

fmol (200 μg/mL) to 2.67 amol (0.8 ng/mL) that fits well with the hill equation (R2 = 0.999). (G and H) CL image and plot from mouse IgG from 26.7 fmol (8 μg/mL) to 13.3 amol (4 ng/mL). The fm and am indicate fmol and amol, respectively. Paper-based electrochemical cyto-device with scalable and economical fabrication method was designed and fabricated in one study to demonstrate the sensitive monitoring of cancer cells using the μPADs (Su et al., 2014). In this work, the aptamers modified 3D macroporous Au-paper electrode was fabricated and employed as the working electrode for specific and efficient cancer cells’ capture. The sequential in-electrode 3D cell culture showed enhanced capture capacity for cancer cells and good biocompatibility for preserving the activity of the captured living cells. Compared

with

the

conventional

detection

methods,

the

colorimetric

(semiquantitative “yes” or “no” answer is insufficient for early and accurate cancer diagnosis) and fluorescent methods (expensive, time-cost, and requiring advanced instrumentation), the electrochemical detection is quantitative and simple for the field use. The paper-based microfluidic electrochemical immune device integrated with the nanobioprobes onto graphene film for ultrasensitive multiplexed detection of four kinds of cancer biomarkers. The paper was impregnated with SU-8 3010 photoresist and the region soaked by the photoresist was impermeable to liquid, whereas the photoresist-eluted region remained hydrophilic. Horseradish peroxidase and antibody

co-immobilized silica nanoparticles and graphene were used to achieve dual signal amplification (Fig. 11). The lowest detectable concentrations for the four cancer biomarkers were 0.001, 0.005, 0.001, and 0.005 ng/mL, respectively (Wu et al., 2013).

Fig. 10. Paper-based microfluidic electrochemical immunodevice integrated with nanobioprobes onto graphene film for ultrasensitive multiplexed detection of cancer biomarkers (Wu et al., 2013). (A) Preparation of nanobioprobes through the coimmobilization of horseradish peroxidase and antibody onto monodispersed SiO2 nanoparticles. (B) Schematic representation of fabrication and assay procedure used to prepare the microfluidic paper-based electrochemical immunodevice and alphafetoprotein are provided as an example. Reagent storage has been a long-standing challenge for diagnostics, especially those reagents designed for low resource settings and point of care applications. In

general, the stability of a reagent relies on careful temperature control, often by refrigeration, which is costly and often unavailable in these remote settings. Poor reagent integrity can negatively affect the reproducibility and reliability of an assay. Given the recent interest in paper-based devices designed for quantitative analysis in the point of care settings, a better understanding of reagent stability on filter paper is critical for proper device use and its longevity. Wu et al. (2014) presented an independent method to examine the stability of reconstituted antibodies that were stored on filter paper using flow cytometry. They validated the method by measuring the activity as measured by the mean fluorescence intensity of antibodies stored with known stabilizers. Furthermore, they demonstrated the potential for the method to screen the influence of other paper treatments and storage processes on antibody stability, which may be applicable to the storage of reagents on paper in general. Microfluidic

paper-based

CL-ELISA

device

which

combines

typical

immunoassay format with the CL for high-throughput and reusable point-of-care testing has been developed (Liu et al., 2013a; Wang et al., 2012). Chitosan was used to modify the μPADs, to covalently immobilize the antibodies on the μPADs. The sandwich CL-ELISA on the μPADs can therefore be easily realized for further development of this technique in sensitive, specific and low-cost application as shown in Fig. 12. The CL immunoassays that bring advantages of simplicity and low cost for

the devices and also sensitivity and selectivity for the detection method have been developed (Liu et al., 2013a).

Fig. 11. Device preparation and sandwich CL-ELISA on μPADs (Liu et al., 2013a). Although the ECL combines the advantages of CL and electrochemistry, and also shows wide dynamic concentration response range and high sensitivity, the indispensable expensive electrochemical workstations have limited their applications in the remote regions of developing countries. Li et al. (2013) integrated the battery and voltage-controller as a voltage-tunable power device to trigger the ECL on the immunodevice instead of the traditional electrochemical workstations and multiplexed immunoassays. These were also used to simplify the operations, provide good sensitivity in early diagnosis of diseases, and shorten analytical time. In addition, Ge et al. (2012) demonstrated the 3D paper-based ECL device based on wax-patterned technology and screen-printed paper-electrodes. This work could not only make contribution to further expand detection mode on the μPADs but also could be easily

integrated and combined with the recently emerging class of paper electronics, to further develop simple and portable POCT device without device-holder in their future work. As a photovoltaic conversion method, the PEC principle has been utilized extensively in the diagnostic systems (Wang et al., 2013a; Wang et al., 2013b). The development of titanium dioxide (TiO2) based composites in the visible-light activated the PEC biosensing that could largely increase the solar light utilization efficiency and reduce the destructive effect from the UV light. Besides, the cadmium sulfide (CdS) quantum dots are photoelectrochemically active in the visible range and have been successfully used in the PEC immunosensors. Hence, Wang et al. (2015b) fabricated a sensitive PEC immunosensor based on the CdS/TiO2 hybrid modified electrodes, to detect the carcinoembryonic antigen which greatly improved the photocurrent intensity of the CdS quantum dots. The effective matching of energy levels between the conduction bands of the CdS and TiO2 allowed fast electron injection from the excited CdS to TiO2 upon irradiation, which inhibited the recombination process of electron-hole pairs and prompted the PEC performance. N(aminobutyl)-N-(ethylisoluminol) and glucose oxidase linked to AuNPs for signal amplification could greatly enhance the sensitivity.

3.3. Molecular detection

Microfluidic paper-based molecular detection assays rely on the sequencespecific detection of nucleic acid hybridization. The capture probe targets the sequence with a tag or change in the concentration of the capture probe makes the reaction visible or measurable. A salt-induced colorimetric sensing strategy employing unmodified 13 nm AuNPs and a paper assay platform for tuberculosis diagnosis was reported in one study (Tsai et al., 2013). The solid wax was printed on the chromatography paper to form the hydrophobic barrier and then the unknown extracted human DNA sequences were hybridized with detection oligonucleotide sequences, followed by addition of the AuNPs colloid and triggering of the colorimetric sensing with a sodium chloride solution. If the extracted DNA sequences consisted of target sequences, the detection oligonucleotide sequences would hybridize with them and only a few ssDNA sequences would be absorbed on the AuNP surface to avoid aggregation after the addition of salt. In the absence of the target sequences, the color of the mixture remains red after hybridization and does not change. The status of the aggregation of AuNPs depends on the surface charge and steric effect on the AuNPs surfaces that can be manipulated by introducing salt into the solution or binding biomolecules via electrostatic adsorption. Wang et al. (2014b) introduced a mediator-less and compartment less glucose/air

enzymatic biofuel cell into the μPADs with AuNPs and platinum nanoparticles (PtNPs)-modified paper electrodes as the anodic and cathodic substrates, respectively, to implement the self-powered DNA detection. They used the wax as the paper hydrophobization and insulation agent to construct a hydrophobic barrier on the chromatography paper. The electrons generated at the anode flew through an external circuit into the PtNPs-modified cathode that catalyzed the reduction of oxygen with the participation of protons. In addition, the generated current could be collected and stored by the paper supercapacitor. The supercapacitor was then automatically shortened under the control of a switch to output an instantaneously amplified current which could be sensitively detected by the terminal digital multi-meter detector (Fig. 13). Besides, Lu et al. (2012) also introduced an electrochemical DNA sensor into a folding paper-based on the AuNPs/grapheme modified screen-printed on the working paper electrode before. The fabrication process for the device consisted of waxprinting, baking the wax-patterned sheet, screen-printing electrodes, followed by cutting. The whole fabrication process took less than 10 min. In addtion, 5 μL of graphene nanosheets was initially deposited on the electrode surface, after which the AuNPs were modified on the graphene via the interaction between the amino groups and Au.

Fig. 12. Schematic diagram of the fabrication of the (A) Paper anodic electrode. (B) Paper cathodic electrode. (C) Circuit diagram of the entire system (Wang et al., 2014b). Fabrication of the μPADs by wax printing for capturing and detection of the DNA hybrids on the paper via the anchoring of antibodies with fusions of carbohydrate binding modules and ZZ-domains are also the suitable platforms for the development of molecular diagnostic assays. Patterns of hydrophobic barriers are designed as black lines on the white background using drawing software and printed with the wax printer and sample liquid to travel the length of the microchannel for approximately 5 min (Fig. 14). The fluorescence intensity of the test zones are measured after scanning the μPADs and the fluorescent signals can be visible for the DNA concentration as low as 1 pmol in a 15 μL sample volume (Rosa et al., 2014).

Fig. 13. Schematic representation of μPADs (Rosa et al., 2014). (A) Schematic illustration of general structure of wax-printed μPADs and methodology used to detect biotin-labeled DNA strands. (B) Black lines on a white background subsequently. (C) Melted on a hot plate, the scanning electron microscopy images show that the tangle of fibers characteristic of cellulose is kept after the film of (D) printed wax is (E) melted. Wang et al. (2014c) fabricated an ultrasensitive CL detection method with reusable POCT and quantitative response for DNA on μPADs in another study. Sodium periodate was used to form covalent bonds between the μPADs and captured DNA. The developed paper-based CL immunodevice, combined with a typical luminol-H2O2-horse radish peroxidase CL system, showed excellent analytical performance for the detection of DNA. Mani et al. (2013) reported a microfluidic paper based electrochemical devices made by heat transfer of wax paper templates for detection of specific pollutant

compounds. The ECL is generated by electrochemically oxidizing ruthenium polyvinylpyridine in the presence of DNA as the co-reactant (Fig. 15). It’s applied when measuring the presence of genotoxic equivalents in environmental samples. Its analytical end point is for detecting the DNA damage from the metabolites produced in the device using the ECL output measured with a charge-coupled device camera (Mani. et al. 2013). Li et al. (2014b) developed a simple and sensitive ECL DNA sensor based on graphene-modified porous Au-paper working electrode in another study. In addition, Wang et al. (2013c) detected DNA by using the graphene-modified porous Au-paper electrode as working electrode, to obtain enhanced PEC responses. The quantification mechanism for this strategy was based on the charging of the supercapacitor which was constructed on the paper-based analytical platform through a simple “drawing and soaking” method by the generated photocurrent. The supercapacitor was automatically shortened under the control of a novel built-in fluidic delay-switch to output an instantaneously amplified current after a fixed period which could be sensitively detected by the digital multi-meter. At optimal conditions, this paper-based analytical platform could detect DNA at femtomolar concentrations. This approach also showed excellent specificity towards the single nucleotide mismatches. The comparisons of proposed methods for the molecular detection using microfluidic paper-based analytical devices are listed in Table 4.

Fig. 14. Schematic representation of μPADs with the working electrode printed on filter paper underneath the paper channel and spots underneath holes in this channel that are coated with DNA-enzyme-ruthenium polyvinylpyridine and DNA films (Mani et al., 2013). Table 4 Comparison of proposed detection methods for molecular detection in microfluidic paper-based analytical devices Detection

Fabrication

Ref. Time

methods

Analyte

Detection limit

Linear range

Equipment

technique Tuberculosis

Colorimetric

Wax printing

1h

mycobacterium

2.6nM

smartphone Tsai et al., 2013

target sequences

10.0 fm~100 nm; Electrochemic

6.3 fm; 2 × Wax printing

Target-ssDNA

al

−16

10

Laser Fluorescent

mmol/L

8× 10−16~5 × 10−10 mmol/L

Electrochemical Wang et al., 2014b; workstation Lu et al., 2012

Neisseria

~3 DNA

Fluorescence

meningitidis

copies (or 7.4

microscope

45 min treatment

Dou et al., 2014

fg)

Jin et al., 2015

Chemilumines ATP

1 μM

Benzo[a]-pyrene;

∼150 nM; 8.5

Wax printing

0.5~10 μM

Optical scanner

cence 0.15~12.5 μM; 4.0 Electrochemilu

Several

ninescence

Charge-coupled x 10-17~5.0 x 10-11

Heat pressing min

target DNA

x 10

-18

device camera

M M

Mani et al., 2013; Li et al., 2014b

Internal CLlight source, paper Photoelectroch Wax printing

Target-ssDNA

15 fM

50.0 fM~100 nM

supercapacitor,

Wang et al., 2013c

emical terminal digital multi-meter

3.4. Other detection methods

Other detection methods have been researched and presented in addition to the above-discussed methods. These methods include: calorimetric method for glucose determination (Davaji and Lee, 2014); spectrophotometric method for phosphate and food dyes determination (Gáspár and Bácsi, 2009; Jayawardane et al., 2014); mass spectrometry for acetylcholine hydrolysis determination (Zhang et al., 2014a), rhodamine 6G and L-phenylalanine determination (Liu et al., 2013b); potentiometric methods metal ions determination (Cui et al., 2014; Lisak et al., 2015), pH (Lei and Yang, 2013; Lisak et al., 2015), polyvinylamine and potassium polyvinylsulfate determination (Leung et al., 2010). All these methods provide new platforms for diagnosis of diseases.

4. Conclusions

As presented in this review, the μPADs have been employed for development of POCT, due to their potential for disposable, integrated and user-friendly diagnostic platforms as discussed. Production of the μPADs is advantageous because of the following reasons: (i) the devices have low-cost (Gan et al., 2014; Jarujamrus et al., 2012), they are lightweight, portable (Lan et al., 2013), time-dependent (Sana et al., 2014), energy efficient (with no pump or external equipment needed for running the assays), robust (Kasten et al., 2008), and can also be used as diagnostic tools in lowresource environments worldwide (Funes-Huacca et al., 2012; Kim et al., 2014). (ii) the devices do not require clean room facilities (Fan et al., 2013), (iii) the devices only require microliter volumes of fluid which can be obtained from a finger stick (Pollock et al., 2013), (iv) multiple assays can be developed simultaneously (Lopez-Ruiz et al., 2014), (v) and they can be integrated with telemedicine for health-care services in resource-limited settings (Wang et al., 2011). However, the μPADs do have limitations, for example; (i) evaporation and retention of the sample can cause sample delivery inefficiencies in the transport process (Li et al., 2012), (ii) more complex and multiple steps involved in the analysis, such as premixing of samples before the final reaction, can make them not suitable for home based healthcare (Martinez. et al. 2010b), (iii) existing techniques for whole-

genome/transcriptome amplification prior to sequencing suffer from bias and nonspecific products (Lisowski et al., 2013).

Research on the μPADs is currently still at an early stage andsignificant research efforts are therefore needed to make the μPADs practical and genuinely useful, and to particularly also expand their capabilities for POCT. For the fabrication of the μPADs, further studies on structure of the paper and printing technology for decreasing evaporation and retention of the sample will significantly be beneficial to the future development of the μPADs. Moreover, the paper surface energy, capillary wicking and vertical flow may need further investigation to gain more precise control of the samples flow (Chen et al., 2012; Chumo et al., 2013; Glavan et al., 2013; Jin et al., 2015). The paper-based power sources and switch may also be the most relevant parameters which may attract further investigation (Chen et al., 2014; Esquivel et al., 2014; Fraiwan and Choi, 2014; Li et al., 2015; Reid et al., 2013; Thom et al., 2013; Thom et al., 2012; Zhang and Zha, 2012). For the μPADs’ detection methodology, the enhanced sensitivity, selectivity, specificity and easy operation is the next direction of our research efforts. More studies on the bioactive paper, incorporating new and biofunctional nanomaterials into the paper, will invite new ideas for enhancing the sensitivity and selectivity of the diagnostic devices. Molecular detection is another detection method for improving the

sensitivity and specificity of the μPADs, although Rodriguez et al. (2015) only reported the paper-based chromatographic lateral molecular detection experiments which still provide a reference for future development of molecular detection based on the μPADs. We believe that the μPADs, integrated with nucleic acid extraction, isothermal amplification and detection results while overcoming the aforementioned disadvantages will be the focus for future research and applications.

Acknowledgments

We acknowledge the financial support from the National Natural Science Foundation of China (81472831, 61201033), the Medical Key Talent Foundation of Jiangsu Province (RC2011081), the Medical Key Science and Technology Development Projects of Nanjing (ZKX11176), the Talents Planning of Six Summit Fields of Jiangsu Province (2013-WSN-054, 2013-WSN-056), and the Science and Technology Development Fund of Nanjing Medical University (2014NJMU138).

References

Abe, K., Suzuki, K., Citterio, D., 2008. Anal. Chem. 80, 6928–6934. Apilux, A., Ukita, Y., Chikae, M., Chailapakul, O., Takamura, Y., 2013. Lab Chip 13(1), 126-135.

Bai, P., Luo, Y., Li, Y., Yu, X-D., Chen, H-Y., 2013. Chinese J. Anal. Chem. 41(1), 20-24. Balu, B., Berry, A.D., Hess, D.W., Breedveld, V., 2009. Lab Chip 9(21), 3066-3075. Barbosa, A.I., Gehlot, P., Sidapra, K., Edwards, A.D., Reis, N.M., 2015. Biosens. Bioelectron. 70, 5-14. Bier, FF., Schumacher, S., 2013. Adv. Biochem. Eng. Biot. 133, 1-14. Cai, L., Xu, C., Lin, S., Luo, J., Wu, M., Yang, F., 2014. Biomicrofluidics 8(5). Cai, L.F., Wu, Y.Y., Xu, C.X., Chen, Z.F., 2013. J. Chem. Educ. 90(2), 232-234. Campas, M., Carpentier, R., Rouillon, R., 2008. Biotechnol. Adv. 26(4), 370-378. Carrilho, E., Martinez, A.W., Whitesides, G.M., 2009a. Anal. Chem. 81(16), 70917095. Carrilho, E., Phillips, S.T., Vella, S.J., Martinez, A.W., Whitesides, G.M., 2009b. Anal. Chem. 81, 5990–5998. Chen, H., Cogswell, J., Anagnostopoulos, C., Faghri, M., 2012. Lab Chip 12(16), 2909-2913. Chen, S.S., Hu, C.W., Yu, I.F., Liao, Y.C., Yang, J.T., 2014. Lab Chip 14(12), 21242130. Chen, Y., Zilberman, Y., Mostafalu, P., Sonkusale, S.R., 2015. Biosens. Bioelectron. 67, 477-484.

Chitnis, G., Ding, Z.W., Chang, C.L., Savran, C.A., Ziaie, B., 2011. Lab Chip 11(6), 1161-1165. Chumo, B., Muluneh, M., Issadore, D., 2013. Biomicrofluidics 7(6), 7. Craighead, H., 2006. Nature 442(7101), 387-393. Cui, J.W., Lisak, G., Strzalkowska, S., Bobacka, J., 2014. Analyst 139(9), 2133-2136. Davaji, B., Lee, C.H., 2014. Biosens. Bioelectron. 59, 120-126. Delaney, J.L., Hogan, C.F., Tian, J., Shen, W., 2011. Anal. Chem. 83(4), 1300-1306. DeMello, A.J., 2006. Nature 442(7101), 394-402. Dou, M., Dominguez, D.C., Li, X., Sanchez, J., Scott, G., 2014. Anal. Chem. 86(15), 7978-7986. Dungchai, W., Chailapakul, O., Henry, C.S., 2010. Anal. Chim. Acta 674(2), 227233. Dungchai, W., Chailapakul, O., Henry, C.S., 2011. Analyst 136(1), 77-82. Dungchai, W., Chailapakul, O., Henry, C.S., 2009. Anal. Chem. 81, 5821–5826. El-Ali, J., Sorger, P.K., Jensen, K.F., 2006. Nature 442, 403-411 Elsharkawy, M., Schutzius, T.M., Megaridis, C.M., 2014. Lab Chip 14(6), 1168-1175. Esquivel, J.P., Del Campo, F.J., Gomes de la Fuente, J.L., Rojas, S., Sabate, N., 2014. Energ. Environ. Sci. 7(5), 1744-1749.

Fan, X.Y., Jia, C.P., Yang, J., Li, G., Mao, H.J., Jin, Q.H., Zhao, J.L., 2015. Biosens. Bioelectron. 71, 380-386. Fan, Y.Q., Li, H.W., Yi, Y., Foulds, I.G., 2013. J. Micro-Nanolith. Mem. 12(4), 6. Fraiwan, A., Choi, S., 2014. PCCP 16(47), 26288-26293. Freeman, P., Girsh, J., Willlner, L., 2013. ACS Appl. Mater. Inter. 5(8), 2815-2834. Fu, E., Lutz, B., Kauffman, P., Yager, P., 2010. Lab Chip 10(7), 918-920. Funes-Huacca, M., Wu, A., Szepesvari, E., Rajendran, P., Kwan-Wong, N., Razgulin, A., Shen, Y., Kagira, J., Campbell, R., Derda, R., 2012. Lab Chip 12(21), 42694278. Gáspár, A., Bácsi, I., 2009. Microchem. J. 92(1), 83-86. Gan, W.P., Zhuang, B., Zhang, P.F., Han, J.P., Li, C.X., Liu, P., 2014. Lab Chip 14(19), 3719-3728. Ge, L., Wang, P., Ge, S., Li, N., Yu, J., Yan, M., Huang, J., 2013. Anal. Chem. 85, 3961-3970. Ge, L., Wang, S.W., Ge, S.G., Yu, J.H., Yan, M., Li, N.Q., Huang, J.D., 2014. Chem. Commun. 50(43), 5699-5702. Ge, L., Wang, S.M., Song, X.R., Ge, S.G., Yu, J.H., 2012a. Lab chip 12(17), 31503158.

Gerbers, R., Foellscher, W., Chen, H., Anagnostopoulos, C., Faghri, M., 2014. Lab Chip 14(20), 4042-4049. Glavan, A.C., Martinez, R.V., Maxwell, E.J., Subramaniam, A.B., Nunes, R.M.D., Soh, S., Whitesides, G.M., 2013. Lab Chip 13(15), 2922-2930. Gong, M.M., Zhang, P., MacDonald, B.D., Sinton, D., 2014. Anal. Chem. 86, 80908097. Gubala, V., Harris, L.F., Ricco, A.J., Tan, M.X., Williams, D.E., 2012. Anal. Chem. 84, 487–515. Han, Y.L., Wang, W.Q., Hu, J., Huang, G.Y., Wang, S.Q., Lee, W.G., Lu, T.J., Xu, F., 2013. Lab Chip 13(24), 4745-4749. He, M., Liu, Z., 2013. Anal. Chem. 85(24), 11691-11694. He, Y., Wu, W.B., Fu, J.Z., 2015. Rsc Adv. 5(4), 2694-2701. He, Y., Wu, Y., Xiao, X., Fu, J.Z., Xue, G.H., 2014. Rsc Adv. 4(109), 63860-63865. Hossain, S.M.Z., Luckham, R.E., McFadden, M.J., Brennan, J.D., 2009. Anal. Chem. 81, 9055–9064. Hu, J., Wang, SQ., Wang, L., Li, F., Pingguan-Murphy, B., Lu, TJ., Xu, F., 2014. Biosens. Bioelectron. 54, 585-597. Jarujamrus, P., Tian, J.F., Li, X., Siripinyanond, A., Shiowatana, J., Shen, W., 2012. Analyst 137(9), 2205-2210.

Jayawardane, B.M., Wongwilai, W., Grudpan, K., Kolev, S.D., Heaven, M.W., Nash, D.M., McKelvie, I.D., 2014. J. Environ. Qual. 43(3), 1081-1085. Jin, S.Q., Guo, S.M., Zuo, P., Ye, B.C., 2015. Biosens. Bioelectron. 63, 379-383. Kalisha, B., Tsutsui, H., 2014. Lab Chip 14(22), 4354-4361. Kasten, A.M., Sulkin, J.D., Leisher, P.O., McElfresh, D.K., Vacar, D., Choquette, K.D., 2008. Ieee J. Sel. Top. Quant. 14(4), 1123-1131. Kauffman, P., Fu, E., Lutz, B., Yager, P., 2010. Lab Chip 10(19), 2614-2617. Khan, M.S., Li, X., Shen, W., Garnier, G., 2010. Colloid. Surface. B. 75(1), 239-246. Kim, Y.T., Jung, J.H., Choi, Y.K., Seo, T.S., 2014. Biosens. Bioelectron. 61, 485-490. Koo, C.K., He, F., Nugen, S.R., 2013. Analyst 138(17), 4998-5004. Lan, W.J., Maxwell, E.J., Parolo, C., Bwambok, D.K., Subramaniam, A.B., Whitesides, G.M., 2013. Lab Chip 13(20), 4103-4108. Lei, K.F., Yang, S.I., 2013. J. Nanosci. Nanotechnol. 13(10), 6917-6923. Lessing, J., Glavan, A.C., Walker, S.B., Keplinger, C., Lewis, J.A., Whitesides, G.M., 2014. Adv. Mater. 26(27), 4677-+. Leung, V., Shehata, A.-A.M., Filipe, C.D.M., Pelton, R., 2010. A. 364(1-3), 16-18. Lewis, G.G., DiTucci, M.J., Baker, M.S., Phillips, S.T., 2012. Lab Chip 12(15), 26302633. Li, H., Han, D., Pauletti, G.M., Steckl, A.J., 2014a. Lab Chip 14(20), 4035-4041.

Li, M., Wang, Y.H., Zhang, Y., Yu, J.H., Ge, S.G., Yan, M., 2014b. Biosens. Bioelectron. 59, 307-313. Lisak, G., Cui, J., Bobacka, J., 2015. Sensor. Actuat. B-Chem. 207, 933-939. Lisowski, P., Zarzycki, P.K., 2013. Chromatographia 76, 1201-1214. Li, S., Wang, Y., Ge, S., Yu, J., Yan, M., 2015. Biosens. Bioelectron. 71, 18-24. Liu, H., Crooks, R.M., 2011. J. Am. Chem. Soc. 133(44), 17564-17566. Liu, W., Cassano, C.L., Xu, X., Fan, Z.H., 2013a. Anal. Chem. 85(21), 10270-10276. Liu, W., Guo, Y., Zhao, M., Li, H., Zhang, Z., 2015. Anal. Chem. 87(15), 7951-7957. Li, W., Li, M., Ge, S., Yan, M., Huang, J., Yu, J., 2013. Anal. Chim. Acta 767, 66-74. Li, W.P., Li, L., Ge, S.G., Song, X.R., Ge, L., Yan, M., Yu, J.H., 2014c. Biosens. Bioelectron. 56, 167-173. Liu, W., Luo, J., Guo, Y., Kou, J., Li, B., Zhang, Z., 2014. Talanta 120, 336-341. Liu, W., Mao, S.F., Wu, J., Lin, J.M., 2013b. Analyst 138(7), 2163-2170. Li, X., Ballerini, D.R., Shen, W., 2012. Biomicrofluidics 6(1), 11301-1130113. Li, X., Tian, J.F., Shen, W., 2010ba. Cellulose 17(3), 649-659. Li, X., Tian, J., Garnier, G., Shen, W., 2010ab. Surface. B. 76(2), 564-570. Li, X., Tian, J., Nguyen, T., Shen, W., 2008. Anal. Chem. 80, 9131–9134. Li, Z.A., Hou, L.Y., Zhang, W.Y., Zhu, L., 2014d. Anal. Methods 6(3), 878-885.

Lopez-Ruiz, N., Curto, V.F., Erenas, M.M., Benito-Lopez, F., Diamond, D., Palma, A.J., Capitan-Vallvey, L.F., 2014. Anal. Chem. 86(19), 9554-9562. Lu, J., Ge, S., Ge, L., Yan, M., Yu, J., 2012. Electrochim. Acta 80, 334– 341. Lu, Y., Shi, W., Jiang, L., Qin, J., Lin, B., 2009. Electrophoresis 30(9), 1497-1500. Lu, Y., Shi, W., Qin, J., Lin, B., 2010. Anal. Chem. 82, 329–335. Lutz, B.R., Trinh, P., Ball, C., Fu, E., Yager, P., 2011. Lab Chip 11(24), 4274-4278. Mani, V., Kadimisetty, K., Malla, S., Joshi, A.A., Rusling, J.F., 2013. Environ. Sci. Technol. 47, 1937-1944. Martinez, A.W., Phillips, S.T., Nie, Z.H., Cheng, C.M., Carrilho, E., Wiley, B.J., Whitesides, G.M., 2010a. Lab Chip 10(19), 2499-2504.

Martinez, A.W., Phillips, S.T., Whitesides, G.M., 2010b. Anal. Chem. 82, 3-10.

Martinez, A.W., Phillips, S.T., Whitesides, G.M., 2008a. P. Natl. Acad. Sci. U. S. A. 105(50), 19606-19611. Martinez, A.W., Phillips, S.T., Wiley, B.J., Gupta, M., Whitesides, G.M., 2008b. Lab Chip 8(12), 2146-2150. Martinez, A.W., Phillips., D.S.T., Butte., D.M.J., Whitesides., P.G.M., 2007. Angew. Chem. Int. Ed. Engl. 46(8), 1318–1320. Martinez, A.W., Phillips, S.T., Carrilho, E., Thomas, S.W., Sindi, H., Whitesides, G.M., 2008c. Anal. Chem. 80, 3699–3707.

Martins, D., Levicky, R., Song, Y.A., 2015. Biosens. Bioelectron. 72, 87-94. Maxwell, E.J., Mazzeo, A.D., Whitesides, G.M., 2013. MRS Bull. 38(4), 309-314. Mosadegh, B., Dabiri, B.E., Lockett, M.R., Derda, R., Campbell, P., Parker, K.K., Whitesides, G.M., 2014. Adv. Healthc. Mater. 3(7), 1036-1043. Mu, X., Zhang, L., Chang, S., Cui, W., Zheng, Z., 2014. Anal. Chem. 86(11), 53385344. Nath, P., Arun, RK., Chanda, N., 2014. RSC Adv. 4(103), 59558-59561. Nie, J.F., Liang, Y.Z., Zhang, Y., Le, S.W., Li, D.N., Zhang, S.B., 2013. Analyst 138(2), 671-676. Nie, Z., Deiss, F., Liu, X., Akbulut, O., Whitesides, G. M., 2010. Lab Chip 10(22), 3163. Ngo, Y.H., Li, D., Simon, G.P., Garnier, G., 2012. Langmuir 28, 8782-8790. Olkkonen, J., Lehtinen, K., Erho, T., 2010. Anal. Chem. 82, 10246–10250. Peeling, R.W., Holmes, K.K., Mabey, D., Ronald, A., 2006. Sex. Transm. Infect. 82. Phillips, S.T., Lewis, G.G., 2014. Expert Rev. Mol. Diagn. 14(2), 123-125. Phillips, S.T., Thom, N.K., 2013. Methods in molecular biology (Clifton, N.J.) 949, 185-196. Pike, J., Godbert, S., Johnson, S., 2013. informa healthcare 7, 435-441. Pollock, N.R., Colby, D., Rolland, J.P., 2013. Clin. Gastroenterol. H. 11(5), 478-482.

Pourreza, N., Golmohammadi, H., 2015. Talanta 131, 136-141. Qi, A.S., Hoo, S.P., Friend, J., Yeo, L., Yue, Z.L., Chan, P.P.Y., 2014. Adv. Healthc. Mater. 3(4), 543-554. Rajendra, V., Sicard, C., Brennan, J.D., Brook, M.A., 2014. Analyst 139(24), 63616365. Ratnarathorn, N., Chailapakul, O., Henry, C.S., Dungchai, W., 2012. Talanta 99, 552557. Rattanarat, P., Dungchai, W., Cate, D., Volckens, J., Chailapakul, O., Henry, C.S., 2014. Anal. Chem. 86(7), 3555-3562. Reid, R.C., Giroud, F., Minteer, S.D., Gale, B.K., 2013. J. Electrochem. Soc. 160(9), H612-H619. Reinholt, S.J., Sonnenfeldt, A., Naik, A., Frey, M.W., Baeumner, A.J., 2014. Anal. Bioanal. Chem. 406(14), 3297-3304. Renault, C., Scida, K., Knust, K.N., Fosdick, S.E., Crooks, R.M., 2013. J. Electrochem. Sci. Te. 4(4), 146-152. Rodriguez, N.M., Linnes, J.C., Fan, A., Ellenson, C.K., Pollock, N.R., Klapperich, C.M., 2015. Anal. Chem. 87(15), 7872-7879. osa, A.M.M., Louro, A. ., Martins, .A.M., In cio, .o., Azevedo, A.M., Prazeres, D.M.F., 2014. Anal. Chem. 86, 4340-4347.

Rosenfeld, T., Bercovici, M., 2014. Lab Chip 14(23), 4465-4474. Sana, J.A., Aleah, H., Vincent, L., Clemence, S., Kevin, P., Robert, P., John D, B., Carlos D.M, F., 2014. Lab Chip 14(1), 229-236. Schilling, K.M., Jauregui, D., Martinez, A.W., 2013. Lab Chip 13(4), 628-631. Schonhorn, J.E., Fernandes, S.C., Rajaratnam, A., Deraney, R.N., Rolland, J.P., Mace, C.R., 2014. Lab Chip 14(24), 4653-4658. Scida, K., Cunningham, J.C., Renault, C., Richards, I., Crooks, R.M., 2014. Anal. Chem. 86(13), 6501-6507 Silva, E., Santhiago, M., Barragan, J.T.C., Kubota, L.T., 2014. Anal. Methods 6(15), 6133-6136. Sones, C.L., Katis, I.N., He, P.J.W., Mills, B., Namiq, M.F., Shardlow, P., Ibsen, M., Eason, R.W., 2014. Lab Chip 14(23), 4567-4574. Spicar-Mihalic, P., Toley, B., Houghtaling, J., Liang, T., Yager, P., Fu, E., 2013. J. Micromech. Microeng. 23(6), 6. Su, M., Ge, L., Ge, S., Li, N., Yu, J., Yan, M., Huang, J., 2014. Anal. Chim. Acta 847, 1-9. Sun, G., Wang, P., Ge, S., Ge, L., Yu, J., Yan, M., 2014a. Biosens. Bioelectron. 56, 97-103.

Sun, G.Q., Wang, P.P., Ge, S.G., Ge, L., Yu, J.H., Yan, M., 2014b. Biosens. Bioelectron. 56, 97-103. Sun, J.Y., Cheng, C.M., Liao, Y.C., 2015. Anal. Sci. 31(3), 145-151 Tan, S.N., Ge, L., Wang, W., 2010. Anal. Chem. 82, 8844–8847. Tan, Y.F., Tang, T.T., Xu, H.S., Zhu, C.Q., Cunningham, B.T., 2015. Biosens. Bioelectron. 73, 32-40. Thom, N.K., Lewis, G.G., DiTucci, M.J., Phillips, S.T., 2013. Rsc Adv. 3(19), 68886895. Thom, N.K., Lewis, G.G., Yeung, K., Phillips, S.T., 2014. RSC Adv. 4(3), 13341340. Thom, N.K., Yeung, K., Pillion, M.B., Phillips, S.T., 2012. Lab Chip 12(10), 17681770. Tsai, T.T., Shen, S.W., Cheng, C.M., Chen, C.F., 2013. Sci. Technol. Adv. Mat. 14, 044404. Villarrubia, C.W.N., Artyushkova, K., Garcia, S.O., Atanassov, P., 2014. J. Electrochem. Soc. 161(13), H3020-H3028. Viola, I., Ghofraniha, N., Zacheo, A., Arima, V., Conti, C., Gigli, G., 2013. J. Mater. Chem. C 1(48), 8128-8133.

Wang, H., Li, Y.J., Wei, J.F., Xu, J.R., Wang, Y.H., Zheng, G.X., 2014a. Anal. Bioanal. Chem. 406(12), 2799-2807. Wang, P. P., Ge, L., Ge, S.G., Yu, J.H., Yan, M., Huang, J.D., 2013a. Chem. Commun. 49(32), 3294-3296. Wang, P. P., Sun, G.Q., Ge, L., Ge, S.G., Song, X.R., Yan, M., Yu, J.H., 2013b. Chem. Commun. 49(88), 10400-10402. Wang, S., Ge, L., Song, X., Yu, J., Ge, S., Huang, J., Zeng, F., 2012. Biosens. Bioelectron. 31(1), 212-218. Wang, W., Wu, W.Y., Wang, W., Zhu, J.J., 2010. J. Chromatogr. A 1217(24), 38963899. Wang, Y., Ge, L., Ma, C., Kong, Q., Yan, M., Ge, S., Yu, J., 2014b. Chem-Eur. J. 20(39), 12453-12462. Wang, Y.H., Ge, L., Wang, P.P., Yan, M., Ge, S.G., Li, N.Q., Yu, J.H., Huang, J.D., 2013c. Lab Chip 13(19), 3945-3955. Wang, Y., Liu, H., Wang, P., Yu, J., Ge, S., Yan, M., 2015a. Sensor. Actuat. B-Chem. 208, 546–553. Wang, S.Q., Zhao., X.H., Khimji, I., Akbas, R., Qiu, W.L., Edwards, D., Cramer, D.W., Ye, B., Demirci, U., 2011. Lab Chip 11, 3411-3418.

Wang, Y.H., Liu, H.Y., Wang, P.P., Yu, J.H., Ge, S.G., Yan, M., 2015b. Sensor. Actuat. B-chem. 208, 546–553. Wang, Y.H., Wang, S.M., Ge, S.G., Wang, S.W., Yan, M., Zang, D.J., Yu, J.H., 2014c. Monatsh. Chem. 145, 129–135. Warsinke, A., 2009. Anal. Bioanal. Chem. 393(5), 1393-1405. Whitesides, G.M., 2006. Nature 442(7101), 368-373. Wu, G., Srivastava, J., Zaman, M.H., 2014a. Anal. Biochem. 449, 147-154. Wu, L., Ma, C., Ge, L., Kong, Q., Yan, M., Ge, S., Yu, J., 2015a. Biosens. Bioelectron. 63, 450-457. Wu, L.D., Ma, C., Ge, L., Kong, Q.K., Yan, M., Ge, S.G., Yu, J.H., 2015b. Biosens. Bioelectron. 63, 450-457. Wu, Y., Xue, P., Hui, K.M., Kang, Y., 2014b. ChemElectroChem 1(4), 722-727. Wu, Y., Xue, P., Kang, Y., Hui, K.M., 2013. Anal. Chem. 85, 8661-8668. Xu, C., Cai, L., Zhong, M., Zheng, S., 2015. RSC Adv. 5(7), 4770-4773. Yager, P., Edwards, T., Fu, E., Helton, K., Nelson, K., Tam, M.R., Weigl, B.H., 2006. Nature 442(7101), 412-418. Yamada, K., Henares, T.G., Suzuki, K., Citterio, D., 2015. Angew. Chem. Int. Edit. 54(18), 5294-5310

Yan, C.F., Yu, S.Y., Jiang, Y., He, Q.H., Chen, H.W., 2014. Acta Chim. Sinica 72(10), 1099-1104. Yan, J.X., Yan, M., Ge, L., Yu, J.H., Ge, S.G., Huang, J.D., 2013. Chem. Commun. 49(14), 1383-1385. Yang, X.X., Forouzan, O., Brown, T.P., Shevkoplyas, S.S., 2012. Lab Chip 12(2), 274-280. Yetisen, A.K., Akram, M.S., Lowe, C.R., 2013. Lab chip 13(12), 2210-2251. Yu, J., Wang, S., Ge, L., Ge, S., 2011a. Biosens. Bioelectron. 26(7), 3284-3289. Yu, J.H., Ge, L., Huang, J.D., Wang, S.M., Ge, S.G., 2011b. Lab Chip 11(7), 12861291. Yu, W.W., White, I.M., 2010. Anal. Chem. 82, 9626–9630. Yu, W.W., White, I.M., 2013. Analyst 138(4), 1020-1025. Zhang, A.L., Zha, Y., 2012. Ferroelectrics 437, 149-159. Zhang, L., Wang, W., Ju, X-J., Xie, R., Liu, Zh., Chu, L-Y., 2015a. RSC Adv. 5, 5638-5646. Zhang, M., Ge, L., Ge, S., Yan, M., Yu, J., Huang, J., Liu, S., 2013. Biosens. Bioelectron. 41, 544-550. Zhang, Y.D., Li, H.F., Ma, Y., Lin, J.M., 2014a. Analyst 139(5), 1023-1029. Zhang, Y.L., Zuo, P., Ye, B.C., 2015b. Biosens. Bioelectron. 68, 14-19.

Zhang, Y., Zhou, C., Nie, J., Le, S., Qin, Q., Liu, F., Li, Y., Li, J., 2014b. Anal. Chem. 86(4), 2005-2012. Zhao, C., Thuo, M.M., Liu, X., 2013. Sci. Technol. Adv. Mat. 14(5), 054402. Zhong, Z.W., Wang, Z.P., Huang, G.X.D., 2012. Microsyst. Technol. 18(5), 649-659. Zhou, M., Yang, M.H., Zhou, F.M., 2014. Biosens. Bioelectron. 55, 39-43.

Highlights We presented the fabrication techniques of paper-based microfluidic devices, including two-dimensional and three-dimensional methods. We summarized the application in biochemical, immunological and molecular detection. The main advantages, disadvantages and future trends for the devices were discussed.