Anal Bioanal Chem DOI 10.1007/s00216-014-7756-1
Lab-on-paper-based devices using chemiluminescence and electrogenerated chemiluminescence detection Lei Ge & Jinghua Yu & Shenguang Ge & Mei Yan
Received: 16 January 2014 / Revised: 3 March 2014 / Accepted: 7 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract As an analytical support, paper, being low cost, highly abundant, of high porosity, disposable or biodegradable, and easy to use, store, transport, and print, has excellent chemical compatibility with many applications. Since the first microfluidic paper-based analytical device (μ-PAD or lab-onpaper) was proposed, the paper-based assay has never attracted as much attention as it does now. There has recently been rapidly increasing interest in using sensitive luminescence methods, for example chemiluminescence (CL) and electrogenerated chemiluminescence (ECL), as the detection strategy for lab-on-paper devices. Because of their intrinsic characteristics, CL and ECL provide outstanding performance while retaining the simplicity, low cost, multifunctionality, versatility, flexibility, and disposability of μ-PADs. The objective of this review is to cover the development of labon-paper-based devices using CL and ECL detection, including fabrication of paper devices, construction of sensing interfaces, signal amplification strategies, external instruments used, and applications. We believe that lab-on-paper devices with CL and ECL detection methods will meet the diverse requirements of point-of-care diagnosis.
Keywords Chemiluminescence . Electrochemiluminescence . Lab-on-paper
Published in the topical collection Analytical Bioluminescence and Chemiluminescence with guest editors Elisa Michelini and Mara Mirasoli. L. Ge : J. Yu (*) : S. Ge : M. Yan Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China e-mail: [email protected]
Introduction Compared with standard laboratory testing, point-of-care (POC) diagnostics are rapid, simple, and inexpensive, and thus have great accessibility to resource-limited settings . POC diagnostics are therefore essential to initiate and scale up on-site medical care for the prevention and control of infectious disease [2–5]. Because of their evident advantages, microfluidic analytical systems [6–11] have been widely used in developing POC diagnostics to address global health problems. Microfluidic technology, also referred to as “lab-on-achip” or “micro-total-analysis system” (μTAS), includes systems capable of integrating, into a miniaturized device, all the analytical stages usually performed in a laboratory: sample pretreatment, sample and/or reagent transport, mixing, reaction, separation, and detection. Microfluidic technology seeks to improve analytical performance by reducing analysis time, reducing consumption of sample and reagents, reducing the risk of contamination, consuming less power, and increasing reliability, functionality, and sensitivity through automation, integrating multiplexing analysis, and, especially, portability, thus enabling point-of-care applications. For example, microfluidic devices, coupled with different functional units (e.g. pumps, valves, and reactors) and integrated into a miniaturized analytical system , can manipulate small volumes of fluid . Therefore, microfluidic devices substantially reduce consumption of samples and reagents, complexity of operation procedures, and length of assay time, without compromising specificity and sensitivity [11, 12]. However, most microfluidic devices, which are made of glass, silicon, and polymers including poly(dimethylsiloxane) and poly(methylmethacrylate) , require both complex fabrication processes and use of external instruments. Paper-based analytical techniques date back to the early 20th century; the development of paper chromatography led to the Nobel Prize in chemistry in 1952. This was the first time
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that a paper strip was turned into a commercial product, and has been followed by high-impact lateral-flow devices including the pregnancy test strip , which uses colloidal gold for the readout. In recent years, paper has attracted increasing interest as an option for replacing conventional rigid materials, for example glass and silicon, in microfluidic devices [14–27]. The motivation is very clear, and the growing interest in paper-based microfluidics is driven by several factors: 1. Paper, as an industrial product, is extremely cheap, ubiquitously available, and derived from renewable resources. Paper-based products can be produced in small or large quantities using several different methods; 2. Paper is combustible [15, 28], so paper-related devices can be economically and safely disposed of by an incinerator. Compared with disposal of plastic micro-plates contaminated with biological substances in laboratory tests, incineration of used paper substrates enables more sanitary disposal, eliminating hazardous biological substances; 3. Paper is thin, lightweight and flexible, making paperbased devices easy to transport and distribute. 4. Paper is biocompatible and biodegradable ; 5. Paper provides a large surface area for storing reagents [17, 25]. The paper substrates have a porous structure, facilitating immobilization of sensing materials and diffusion of analytes; 6. Assays performed on paper require only small sample volumes, which is important for samples with limited availability, for example tears, saliva, urine from newborn infants, and blood from finger pricks ; 7. The porous structure of filter paper simplifies a microfluidic system because it provides spontaneous capillary-flow energy to transport solutions . Thus, such accessories as a pump are not needed; and 8. Paper can be patterned by traditional photolithography processes, or by printing methods for mass production. Microfluidic paper-based analytical devices (μ-PADs or lab-on-paper) were pioneered by Whitesides et al. at Harvard University in 2007 , and combine the simplicity and low cost of paper-strip tests with some of the capabilities of conventional lab-on-chip devices. These devices stand out as a new class of POC diagnostic device that are inexpensive, easy to use, lightweight for easy transportation, and compatible with biological samples. The advantages of paper-based devices enable new applications, and make them suitable for use in locations with resource-limited settings and in emergency situations, military field assignments, home healthcare, and developing countries . To date, most μ-PADs have used a colorimetric readout method [17–26] based on visually comparing the color intensity of the reaction spots, either by naked eye or using camera phones and portable scanners. With the
use of a fluidic timer, more accurate μ-PAD assays were achieved by colorimetric methods [31–34]. However, for μPADs, a detection strategy with high sensitivity and selectivity is still needed for when determining the level of an analyte in a real, complex biological sample is important for simple, rapid, low-cost POC testing, public health, and environmental monitoring. There is an emerging trend of establishing new analytical methods, including electrochemical methods [35–39], fluorescent methods [40–44], chemiluminescent methods [45–49], electrochemiluminescent methods [50–55], photoelectrochemical methods [56–58], and surfaceenhanced Raman scattering [59–62], for use with μ-PADs, which not only retain the simplicity, low cost, portability, and disposability of paper-based analytical devices, but also provide new opportunities and directions in developing precise and sensitive diagnostic devices. Chemiluminescence (CL) is a detection system based on the production of electromagnetic radiation (ultraviolet (UV), visible (Vis) or infrared (IR)) observed when a photochemical reaction yields an electrically excited intermediate or product, which either luminesces (direct CL) or donates its energy to another molecule that then luminesces (indirect or sensitized CL). CL has proved to be a superior analytical tool for detection and quantification of a wide variety of biological materials, including cells , microorganisms , proteins , DNA , RNA , and other analytes , because of its low detection limit, wide linear range, and high compatibility with micromachining technology . For CL-based detection, in contrast with other optical techniques including absorption, fluorescence, and phosphorescence spectroscopy, no excitation source and no optical filters are required . The usefulness of CL also results from the absence of unwanted background luminescence compared with that of absorption and fluorescence methods. This reduces the background signal, the probability of the light source being unstable, and, especially, the effects of stray light associated with fluorescence, and thus leads to improved limits of detection. For these reasons, as shown in Table 1, CL-based detection has become a quite useful tool for use with μ-PADs (denoted as μPCLDs) [45–49]. Unlike colorimetric readout, the light readout for μ-PCLDs requires a photomultiplier detector, which is more complex than the camera phones [71–75] and portable scanners [76–81] used by accurate colorimetric assays using μ-PADs; however, the high sensitivity and low background of light readout still make CL detection an attractive alternative for μ-PADs. The photochemical reactions for CL detection involve the oxidation of an organic dye by a strong oxidizing agent in the presence of a catalyst (chemical or biological). The dyes used in μ-PADs are luminol [45, 47–49, 82–85], carbon dots , and rhodanine [86, 87], which are not luminescent in the ground state (before an oxidation reaction). The oxidation of dyes with hydrogen peroxide (oxidizing agent) in the presence of a catalyst converts the ground state
Lab-on-paper-based devices using chemi-/electrochemi-luminescence Table 1 A summary of μ-PCLDs Fabrication method
M4NRASPa; H2O2 M4NRASP; H2O2
Uric acid Glucose Uric acid Wax printing Horseradish peroxidase Carcinoembryonic antigen Luminol; H2O2 p-iodophenol Wax printing Ag nanoparticlesAlpha-fetoprotein luminol; H2O2 Carcinoma antigen 153 Carcinoma antigen 199 Carcinoembryonic antigen Wax Horseradish peroxidase Alpha-fetoprotein screen-printing Luminol; H2O2 Carcinoma antigen 125 p-iodophenol Carcinoembryonic antigen Wax printing TiO2 nanoparticles; Prostate-specific antigen Luminol; H2O2 Cutting Horseradish peroxidase Cotinine Luminol; H2O2 2,4-Db Cutting Tobacco peroxidase; Luminol; H2O2 Wax KMnO4; carbon dots; Single-stranded DNA screen-printing nanoporous gold Dichlorvos Cutting Luminol H2O2 a
3-p-nitrylphenyl-5-(4 -methyl-2 -sulfonophenylazo)rhodanine
Detection limit Linear range
Urate oxidase Glucose oxidase Urate oxidase Antibody
1.9 mmol L−1 0.14 mmol L−1 0.52 mmol L−1 6.5 pg mL−1
2.6 to 49.0 mmol L−1 0.42 to 50 mmol L−1 1.4 to 47 mmol L−1 0.01 to 30.0 ng mL−1
2.5 to 110 ng mL−1 1.0 to 100 U mL−1 0.5 to 150 U mL−1 0.1 to 130 ng mL−1 0.1 to 35.0 ng mL−1 0.5 to 80 U mL−1 0.1 to 70 ng mL−1 0.001 to 20 ng mL−1
1.0 ng mL−1 0.4 U mL−1 0.06 U mL−1 0.02 ng mL−1 0.06 ng mL−1 0.33 U mL 0.05 ng mL−1 0.8 pg mL−1
5.0 ng mL−1
0.01 to 1.0 μg mL−1
Molecularly 1.0 pmol L−1 5.0 pmol L−1 to 10.0 μmol L−1  imprinted polymer Complementary 0.856 amol L−1 1.0 amol L−1 to 10.0 fmol L−1  single-stranded DNA Paper chromatography 3.6 ng mL−1 0.01 to 1.0 μg mL−1 
of dyes into an activated state (chemically induced electronic excited states). A strong emission can be observed as a result of the decay of the excited states back to the ground state. Electrogenerated chemiluminescence (also called electrochemiluminescence, and abbreviated ECL) is a form of CL involving the production of a sensitive light signal by species, generated at an electrode, that can undergo highly energetic electron-transfer reactions . However, luminescence in CL is initiated and controlled by the mixing of necessary reagents; in contrast, luminescence in ECL is initiated and controlled by changing an electrode potential with the aid of an electrochemical workstation. Although the electrochemical workstation is complicated and needs a skilled operator, the time and position of the sensitive light-emitting reaction can be controlled exactly by this method, in contrast with conventional chemiluminescent methods. By controlling the time, light emission can be delayed until such events as immune or enzyme-catalyzed reactions have taken place. Control over position can be used to confine light emission to a region that is precisely located with respect to the detector, improving sensitivity by increasing the signal-to-noise ratio. Furthermore, ECL can be more selective than CL, because the generation of excited states in ECL can be selectively controlled by varying the electrode potential. Since the ECL assay
was first constructed for μ-PADs (denoted as μ-PECLDs) by Shen , several novel μ-PECLDs have been used and their performance investigated (Table 2). For example, application of a voltage to a paper-based electrode in the presence of an ECL luminophore, e.g. Ru(bpy) 3 2+ (where bpy = 2,2 bipyridine) [50, 53, 55, 89–94], quantum dots [54, 89, 90, 92, 95, 96], [4,4 -(2,5-dimethoxy-1,4-phenylene)bis(ethyne2,1-diyl) dibenzoic acid] [97–99], (bis-2,2 -bipyridyl) ruthenium polyvinylpyridine , or luminol [52, 90, 100], results in light emission and enables detection of the ECL emitter, ECL co-reactant, or ECL catalyst at very low concentrations. At present, almost all ECL analytical systems used with μPECLDs are based on co-reactant ECL technology. Therefore, understanding the ECL mechanisms of the relevant systems is important. Co-reactant ECL is frequently generated by onedirectional potential scanning at an electrode, in a solution containing emitter in the presence of a deliberately added reagent (co-reactant). Depending on the polarity of the applied potential, both the emitter and the co-reactant species can be first oxidized [51, 98] or reduced [54, 92, 96] at the electrode to form radicals, and intermediates formed from the coreactant then decompose to produce a powerful reducing or oxidizing species that reacts with the oxidized or reduced emitter, producing the excited states that emit light.
CdTe; H2O2; nanoporous gold Luminol; H2O2; nanoporous gold Ru(bpy)32+; H2O2; nanoporous gold CdTe; K2S2O8; nanoporous silver P-acidb; tri-n-propylamine nanoporous silver RuPVPc; guanine
Electrochemical workstation Electrochemical workstation Electrochemical workstation
P-acidb; tri-n-propylamine Au nanoparticlesRu(bpy)32+; tri-n-propylamine Si nanospherecarbon dots Luminol-H2O2
Screen-printed carbon electrode on paper
Screen-printed carbon electrode on paper Gold-paper electrode Paper electrode
Silver-paper electrode GrapheneAu nanoparticlepaper electrode Screen-printed carbon electrode on paper Carbon tape-indium tin oxide glass Screen-printed carbon electrode on paper Screen-printed carbon electrode on paper Carbon nanotubespaper electrode
Screen-printed carbon electrode on paper
Commercial screen-printed electrode
4,4 -(2,5-dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl)dibenzoic acid
Alkenyl ketene dimer
Ru(bpy)32+; tri-n-propylamine Carbon dots; K2S2O8;
Au nanoparticlesluminuol; H2O2
Electrochemical workstation Nickel-metal hydride rechargeable battery
Electrochemical workstation Lithium battery
Table 2 A summary of μ-PECLDs
Adenosine triphosphate Hg2+
Carcinoembryonic antigen Carcinoma antigen 153 Alpha-fetoprotein Carcinoma antigen 199 Carcinoembryonic antigen
Carcinoma antigen 125
Human chorionic gonadotropin
Prostate-specific antigen Carcinoembryonic antigen
Carcinoma antigen 125
2-(dibutylamino)ethanol Nicotinamide adeninedinucleotide Alpha-fetoprotein Carcinoma antigen 125 Carcinoma antigen 199 Carcinoembryonic antigen Alpha-fetoprotein
– Glucose oxidase
0.005 to 4000 mU
10 nmol L−1 to 10 μmol L−1 0.1 mmol L−1 to 3 mmol L−1
1.7 nmol L−1 0.1 mmol L−1
to 1.0 μmol L
30.0 pmol L−1 to 1.0 μmol L−1
0.5 nmol L 10 pmol L−1
0.2 nmol L
0.5 pmol L−1 to 7.0 nmol L−1
0.1 pmol L−1
0.005 to 50 ng mL−1
4.0 pg mL−1 5.0 mU mL−1 0.02 ng mL 6.0 mU mL−1 0.001 ng mL−1
0.01 to 100 U mL−1
7.4 mU mL−1
0.1 mmol L−1 to 100 mmol L−1
0.003 to 20 ng mL 0.001 to 10 ng mL−1
150 nmol L−1
to 50 ng mL−1
to 50 U mL
0.5 pg mL−1 to 20 ng mL−1
1.0 pg mL
5.0 mU mL
0.15 to 12.5 μmol L−1
1.0 pg mL 0.8 pg mL−1
0.12 pg mL−1
0.8 pg mL
1.2 mU mL
0.5 to 110 ng mL−1 1.0 to 100 U mL−1 0.5 to 100 U mL−1 1.0 to 100 ng mL−1 3.0 pg mL−1 to 50 ng mL−1
0.15 ng mL−1 0.6 U mL−1 0.17 U mL−1 0.5 ng mL−1 1.0 pg mL−1 −1
0.2 mmol L−1 to 10 mmol L−1
72 μmol L−1
3 μmol L−1 to 5 mmol L−1
0.9 μmol L−1
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Lab-on-paper-based devices using chemi-/electrochemi-luminescence
Fabrication of μ-PCLDs and μ-PECLDs Paper-based microfluidic devices were fabricated primarily by patterning and manipulation of the hydrophilic property of paper [20, 101, 102]. Patterning paper into regions of hydrophilic channels demarcated by hydrophobic barriers (or air) creates microfluidic devices that enable distribution of a sample into multiple, spatially segregated regions, enabling multiple simultaneous assays (or replicates of an assay) on a single device. In virtually all cases, hydrophilic flow channels in μPADs are defined by the presence of hydrophobic barriers based on photoresist [30, 35–37, 52, 71, 103–111], wax [31, 33, 43, 51, 73, 76, 78, 112–128], polystyrene [129, 130], alkyl ketene dimer [53, 131–134], polydimethylsiloxane [135–137], poly(o-nitrobenzyl methacrylate) , fluorochemicals [139–141], methylsilsesquioxane , dipropylene glycol methyl ether acetate or acrylic polymer , poly(hydroxybutyrate) , toner [54, 145–147], resins , acrylate , octadecyltrichlorosilane , or air [47, 86, 87, 151–166]. Barrier fabrication methods include photolithography [103, 105], a variety of printing methods that form hydrophobic barriers [112, 129, 135, 149], cutting [47, 140, 157, 161, 162], chemical vapor deposition  or plotting , and the chemical , plasma [53, 131–134], O3 , or laser etching [163, 168] of completely hydrophobized paper to regenerate hydrophilic channels. Each fabrication method has advantages and limitations. Photolithography Wang and coworkers developed microfluidic ECL sensing techniques based on photolithography (Fig. 1(i)) [52, 55]. Photolithography shares some fundamental principles with photography, in that the pattern in the etching resist is created by exposing it to light in the form of a projected image, using an optical mask. It can create extremely small patterns (down to a few tens of nanometers in size), and the lowest possible feature sizes are determined by the diffraction barrier of the light used. Photolithography can produce hydrophilic features in paper with high resolution between hydrophilic and hydrophobic areas (~200 μm minimum barrier-line width) . As shown in Fig. 1(i), Wang and coworkers [52, 55] cut the filter paper to a suitable size, and the paper is then impregnated with photoresist and selectively polymerized by exposure to UV light through a high-resolution printed photomask. The unexposed portion of the paper is then washed away with commercial washing liquor to obtain the hydrophilic area. Finally, the device is washed with deionized water and dried under nitrogen flow. This procedure is comparable to a high-precision version of the method used
Fig. 1 (i) Pictures of the fabrication process of an origami μ-PECLD based on photolithography: (a) Bare filter paper; (b) polymerized photoresist patterns on paper; (c) size cutting. Adapted from Ref.  with permission from The Royal Society of Chemistry. (ii) A 96-paper microzone plate for CL ELISA was fabricated by wax printing using a wax printer: (a) bare A4 paper; (b) wax printer; (c) wax-printed paper; (d) wax-penetrated paper after baking; (e) Image showing the water-control ability of the paper microzone plate. Adapted from Ref.  with permission from The Royal Society of Chemistry. (iii) A 6×3 paper multimicrozone plate for CL ELISA was fabricated through wax screenprinting: (1) Screen (200 mesh of nylon on an aluminium frame) and bare paper; (2) the screen is closely placed onto the paper surface; (3), (4) solid wax is used as squeegee and rubbed through the screen stencil to print (5) wax patterns on paper surface; (6) the wax-screen-printed paper is placed in an oven set at 130 °C for 150 s. Adapted from Ref.  with permission from Elsevier. (iv) Heat transfer of wax: (a) Patterning of two pieces of folded wax paper using a puncher and a sharp blade; (b) two filter papers are patterned by heat pressing between these folded-waxpaper templates at 350 °C for 60 s. Adapted with permission from . Copyright (2013) American Chemical Society
to make printed circuit boards. It is used because it affords exact control over the shape and size of the objects created, and because it can create patterns over an entire surface.
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Wax Another patterning method deposits wax on paper by drawing , dropping , solid printer [112, 114], screen-printing [83, 116], stamp printing , or heat transfer . The method based on the wax printer seems to be most popular for the fabrication of μ-PCLDs [45, 46, 82–87] and μ-PECLDs [52, 89, 90, 93–95, 98, 99], because of its simplicity, speed, and compatibility with aqueous solutions. As shown in Fig. 1(ii), the wax-printed paper is heated to re-melt the wax, which penetrates the paper to generate complete hydrophobic barriers. A set of microfluidic devices can be fabricated in less than 5 min. Although wax printing has a lower resolution than photolithography (~550 μm minimum channel width ), it has three main advantages over photolithography: 1. The material preparation and manufacturing cost of the wax printing method are lower than those of photolithography, which include organic solvents, expensive photoresist, and photolithography equipment, for example the UV exposure system; 2. Extra treatment, for example plasma oxidation , is not necessary to increase the hydrophilicity of the patterned paper, because paper does not retain any of the undissolved photoresist in the hydrophilic region; and 3. The simplicity, low cost, and speed of the wax-printing method make it very attractive for mass production of μPADs. Moreover, researchers without access to photolithographic facilities can use this technique for rapid development of new prototypes of μ-PADs. One limitation of the wax-printing method is the need for tools that are rare in the laboratories of developing countries, for example wax printers. Therefore, Yu et al. presented a paper-based CL ELISA  and DNA sensor  using wax screen-printing (Fig. 1(iii)) as a low-cost, simple, and rapid method of fabricating paper devices, requiring minimum external instrumentation (their work used only a silk screen, obtained from a local printing shop), for use in developing countries. Rusling et al.  further reduced the external instrumentation required to construct a paper-based ECL device through heat transfer of wax from a patterned wax paper onto the target filter paper (Fig. 1(iv)). Cutting Some μ-PCLDs [47–49, 82, 87] were created by forming physical boundaries (rather than hydrophobic boundaries), using a knife plotter or craft-cutting plotter to precisely cut chromatographic paper into suitable shapes to conduct microfluidic channels. In these devices, the physical boundaries formed by cutting restrict the flow of a fluid in a specific area. μ-PCLDs based on physical boundaries usually suffer
from a common problem: the fact that paper has a relatively low mechanical strength, especially when wet. Dealing with this weakness is an important concern, because paper microfluidic devices are, by definition, required to function when wet. To address this problem, Wang et al.  attached the μ-PCLDs to plastic surfaces, using double-sided tape to support the device (Fig. 2(i)). The μ-PCLDs could also be encased between two layers of adhesive tape [48, 49, 86, 87] to increase the mechanical strength (Fig. 2(ii and iii)). Liu [47, 49] reported a simple fabrication method for producing μPCLDs by craft cutting and lamination, in a way similar to that used in making an identification (ID) card (Fig. 2(iv)). The method uses a digital craft cutter to generate physical boundaries of paper according to the design of the device, followed by a roll laminator to produce laminated μ-PCLDs. These laminated μ-PCLDs have increased mechanical strength and stability, especially when organic solvents are required during an assay. Other patterning methods Shen et al.  used alkenyl ketene dimer, which has two hydrocarbon chains of C16–C20, with a C=C double bond in each hydrocarbon chain, as the cellulose hydrophobization agent to create the hydrophobic barrier in μ-PECLDs (Fig. 3(i)). The cost of the liquid alkenyl ketene dimer for each paper-fluidic device is approximately 0.000002 Euro. The electronically generated paper-fluidic patterns were printed onto A4 filter paper with an alkenyl ketene dimer– heptane solution (2 %v/v), using a reconstructed commercial digital-inkjet printer (Canon Pixma ip4500). The printed filterpaper sheets were then heated in an oven at 100 °C for 8 min to cure the alkenyl ketene dimer onto the cellulose fibers. The advantage of the alkenyl-ketene-dimer barrier is that it enables precise patterns to be created on paper surfaces without affecting their flexibility or surface topography. Shi et al.  revealed that toner could be used to pattern paper for μPECLD fabrication (Fig. 3(ii)). Toner is a complex powder composite, usually used in laser printers to form an image on polyester film or wax paper. In the laser printers, the toner starts off as a powder, becomes a fluid, and ends up as a solid structure bonded to the paper surface . In the next step, the toner-printed paper is heated to re-melt the toner, which consequently diffuses (vertically and horizontally) by capillarity through the porous paper, generating hydrophobic barriers across the paper. Toner-based devices can be fabricated directly using common laser printers.
Preparation of the electrode system of μ-PECLDs μ-PECLD is one category of μ-PCLD with electrodes. ECL is the process whereby species generated on or in electrodes undergo high-energy electron-transfer reactions to form
Lab-on-paper-based devices using chemi-/electrochemi-luminescence
Fig. 2 (i) Molecularly imprinted polymer-modified paper disks were attached to plastic surfaces by double-sided tape for high-throughput CL detection. Adapted from Ref.  with permission from Elsevier. (ii) The paper pad, cut from the paper chromatographic strip, was encased between two layers of adhesive tape for CL readout after chromatographic separation. Adapted from Ref.  with permission from Elsevier. (iii) μ-PCLD was fabricated, through the successive assembly of bare paper,
enzyme-modified paper channels, and CL-reagent-modified paper pads between two layers of adhesive tape, for enzyme-based assay. Adapted from Ref.  with permission from The Royal Society of Chemistry. (iv) The sheared paper strip was laminated between aligned cover and bottom polyester film using a laminator. The cover film had an opening similar to the pattern of the paper strip. Adapted with permission from Ref. . Copyright (2013) American Chemical Society
excited states that emit light . A typical electrode-based ECL sensing unit for μ-PECLDs is composed of three electrodes: a working electrode (WE), a counter electrode (CE), and a reference electrode (RE) (Fig. 4(i–iv)) [53–55, 89–91, 93, 98, 99]. A two-electrode ECL system (working electrode (WE) and counter electrode (CE), Fig. 4(v–vii)) has been used with μ-PECLDs, with a commercial battery [92, 93, 97], a primary battery , or a cell phone  used as a simple external energy supply instead of expensive electrochemical workstations [172, 173]. To realize the combination of paper device with electrode, Delaney et al. [50, 53] directly laminated their paper device onto a commercial Zensor screen-
printed three-electrode plate. Similarly, Shi et al.  attached the unpatterned area of the paper device onto a quantum-dotmodified indium-tin-oxide glass, which was used as the working electrode. An Ag or AgCl wire and a Pt wire were fixed on the unpatterned area with a clasp and used as the reference electrode and the counter electrode, respectively. To further minimize the size of the μ-PECLDs and reduce the cost, the electrodes should be directly screen-printed on paper [35, 36, 104, 118]. For example, Yu’s group [89, 90, 92–94, 98] and other groups [51, 52, 55] directly screenprinted electrodes onto μ-PECLDs (Fig. 4(i–iv)). Usually, the counter electrode and reference electrode were screen-
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printed onto the same paper layer, whereas the working electrode was screen-printed onto another paper layer (Fig. 4(i–iv)). The advantages of this configuration include: 1. The separated three-paper-electrode system is very beneficial for the high integration (for multiplex assay) and various distributional pattern of working electrodes on paper, removing the need to consider the position and pattern of the reference and counter electrode; 2. Bulk operations on or in working electrodes can be realized without considering the effect on, and possible contamination of, the reference and counter electrode.
Fig. 3 (i) The μ-PECLDs are produced in bulk on A4 paper by a conventional inkjet printer, using alkenyl ketene dimer as hydrophobization agent. Individual μ-PECLDs are then cut out and the hydrophilic portion filled with a 10 mmol L−1 Ru(bpy)32+ solution. Adapted with permission from Ref. . Copyright (2011) American Chemical Society. (ii) The front and back sides of the μ-PECLD, printed with toner (black part) after treatment at 150 °C for 300 min. The yellow part is the diffused area of the marker solution. Adapted with permission from Ref. . Copyright (2012) American Chemical Society
Furthermore, in these multiplex μ-PECLDs [51, 90, 92, 94, 97], different working electrodes can share the same reference and counter electrodes. The electrodes used in these μ-PECLDs are prepared from conducting inks (carbon ink and Ag or AgCl ink) by screen-printing. Because of the small size of the paper device, the silver wires and contact pads used in conventional screenprinted electrodes [35, 174] are unnecessary, and can be directly replaced by carbon ink and Ag or AgCl ink, respectively. This will be very important for the further development of this device for low-cost applications.
Fig. 4 Electrodes screen-printed on μ-PECLDs. (i–iv) Examples of paper-based three-electrode ECL system. The RE and CE were screenprinted on the same paper pad, whereas the WE(s) was (or were) screenprinted on another paper pad, connecting with RE and CE after stacking and folding of the paper pads. (i) Adapted from Ref.  with permission from Elsevier. (ii) Adapted from Ref.  with permission from The Royal Society of Chemistry. (iii) Adapted with permission from Ref. .
Copyright (2012) American Chemical Society. (iv) Adapted from Ref.  with permission from Elsevier. (v–vii) Examples of paper-based twoelectrode system for battery-induced ECL detection. (v) Adapted from Ref.  with permission from The Royal Society of Chemistry. (vi) Adapted from Ref.  with permission from The Royal Society of Chemistry. (vii) Adapted from Ref.  with permission from The Royal Society of Chemistry
Lab-on-paper-based devices using chemi-/electrochemi-luminescence
Sensing interfaces of μ-PCLDs and μ-PECLDs Almost all selective assays using lab-on-paper devices rely on highly specific recognition events for detection of their target analytes. The essential function of the sensing interface of labon-paper devices is to enable immobilization of the probe– target complex in such a way that the binding event induces a usable signal for read-out. The minimum elements of any assay using lab-on-paper include a molecular recognition probe and signal transducer that can be coupled to an appropriate read-out strategy. The sensing interfaces of μ-PCLDs and μ-PECLDs are created by using chemical modification or physical absorption to immobilize different functional molecule probes, for example luminescence reagents [48–55], enzymes [86, 87], antibodies [47, 90–92], nucleic acids [46, 89, 98], or a molecularly imprinted polymer , in the defined hydrophilic paper area [52, 53, 82, 89, 90, 92, 98] or on or in the working electrode [55, 91, 93, 94]. After this, the sensing interfaces should usually be blocked with, for example, bovine serum albumin or 6-Mercapto-l-hexanol, to cover the possible remaining active sites and prevent nonspecific adsorption. Enzyme assays using μ-PCLDs Use of enzymes as the biological recognition element was very popular in the development of enzyme-based μ-PADs, because of their high binding specificity and catalytic activity towards their corresponding substrate. A μ-PAD with enzyme assay is an analytical device that combines an enzyme with a transducer to produce a signal proportional to the target analyte concentration. This signal can result from a change in the absorption, the release of product, or other properties, brought about by the reaction catalyzed by the enzyme. For a practical enzyme-based μ-PAD, the most important aspect was significant improvement in selectivity, at least under well-controlled conditions. Glucose oxidase [30, 87, 175, 176], cholesteroloxidase , urate oxidase , horseradish peroxidase , tyrosinase , lactate oxidase [35, 178], and cholesterol oxidase  are among the enzymes that have been used in μ-PADs. Yu et al. [86, 87] developed the first enzyme-based μPCLDs for the simultaneous detection of glucose and uric acid, with convincing performance. In their work, the enzymes (glucose oxidase or urate oxidase) and CL reagent are immobilized in bulk into the porous structure of paper, through a simple physical-absorption technique (immersion), constructing bioactive paper channels and CL-detection areas (Fig. 2(iii)), respectively. When the sample solution (glucose and uric acid solution) migrates through the bioactive paper channels, enzyme–substrate reactions between the target compounds and the immobilized enzymes occur. The hydrogen peroxide generated continues to migrate along the bioactive
paper channels, finally reaching the CL detection areas to induce the CL reaction with the preloaded CL reagent (3-pnitrylphenyl-5-(4 -methyl-2 -sulfonophenylazo) rhodanine), resulting in CL emission. Immunoassays using μ-PECLDs and μ-PCLDs Antigen–antibody-binding-based immunoassay, as a rapid, sensitive, and cost-effective analytical technique, is widely used in lab-on-paper devices [80, 143, 162, 164, 179], because of its high sensitivity, high selectivity, rapid detection, and ability to analyze difficult matrices without extensive pretreatment. Enzyme-linked immunosorbent assays, as the most extensively used types of immunoassay, were first developed for μ-PADs by Cheng et al. . They also revealed that fast immunoassay could be realized using paper, because the highsurface-to-volume-ratio, porous paper fibers accelerate immunoreaction. Thus, microfluidic paper-based immunodevices have been widely used to develop assays with a variety of detection strategies, including colorimetric [117, 143, 164] and fluorescence [180, 181]. In recent years, CL immunoassay and ECL immunoassay have become very popular for use with μ-PADs for clinical and environmental analysis, because of their high sensitivity and wide dynamic range. Multiplex-sandwich CL immunoassays were first introduced into μ-PADs by Yu’s group [45, 83–85], to achieve simultaneous detection of different protein macromolecules. The CL–immunoassay interface was constructed by immobilizing the capture antibodies onto the cellulose fibers of paper, through chitosan coating and glutaraldehyde crosslinking (Fig. 5(i)). The retention and adhesion of chitosan onto the cellulose fibers of paper occurred because of the different charges of the chitosan and cellulose. These sandwich immunoassays contain two kinds of antibody: the capture antibody and signal antibody. The capture antibody, immobilized in paper, can specifically recognize and bond with the antigen, for example alpha-fetoprotein, carcinoma antigens, and carcinoembryonic antigen. Then the signal antibody, conjugated with horseradish peroxidase, can also bond with the captured antigen, forming the antibody–antigen–antibody sandwich immunocomplex. After removal of the unbound signal antibodies, the horseradish-peroxidase labels can catalyze the CL reaction and enhance CL emission, thus revealing the existence of antigens. Similarly, Liu et al.  developed a single-channel competitive immunoassay using μ-PAD for detecting small-molecule analytes. Only the capture antibody, immobilized in one end of the paper channel through chitosan coating and glutaraldehyde cross-linking (Fig. 5(ii)), was used to competitively capture the analyte and HRP-labeled analyte. Although the stability and reproducibility of the above microfluidic paper-based CL immunodevices were relatively satisfactory, the fabrication procedures contained three
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Fig. 5 Examples of the modified paper device for CL immunoassay. (i) Capture antibodies were immobilized in the hydrophilic paper zone through chitosan coating and glutaraldehyde cross-linking, followed by BSA blocking. Then the capture antibody–antigen–signal antibody immunocomplex was formed in the paper zone for sandwich CL immunoassay. Adapted from Ref.  with permission from Elsevier. (ii) (a) The folded detection zone of the laminated μ-PCLD was modified by chitosan, then cross-linked with glutaraldehyde, followed by conjugation with anticotinine. Nonspecific binding sites were then blocked by BSA;
(b) A mixture containing a cotinine sample and a fixed amount of cotinine-HRP was applied to the folded detection zone for competitive CL immunoassay. Reprinted with permission from Ref. . Copyright (2013) American Chemical Society. (iii) Periodate oxidation converts the 1,2-dihydroxyl (glycol) groups in the hydrophilic paper zone to paired aldehyde groups; these react with the amino group of capture antibodies, thus immobilizing the capture antibodies. Reproduced from Ref.  with permission from The Royal Society of Chemistry
complex steps: chitosan coating, glutaraldehyde cross-linking, and Schiff-base reducing by NaBH3CN. To simplify the fabrication procedures for the sensing interface, Wang et al.  developed a simple and stable modification strategy for constructing sandwich CL immunoassays for μ-PADs. Based on periodate oxidation, they directly converted the 1,2dihydroxyl (glycol) groups on the cellulose fibers to paired aldehyde groups, without the need for chitosan coating and glutaraldehyde cross-linking (Fig. 5(iii)). For sandwich ECL immunoassays using μ-PADs, antibodies should be immobilized on the electrode surface. Yan et al.  and Ge et al.  developed a single-channel  and a multiplex  ECL sandwich immunoassay, respectively, by immobilizing capture antibodies onto the surface of the screen-printed carbon working electrode through chitosan coating and glutaraldehyde cross-linking (Fig. 6(i)). Several nanomaterials were used to modify the surface of the screenprinted carbon working electrode to enhance the conductivity and surface area of the electrode interface. Wang et al. prepared a gold-nanoparticle (AuNP)-modified screen-printed carbon working electrode (SPCWE)  and a nanoporous-gold (NPG)-modified SPCWE  on μ-PAD
to immobilize the capture antibodies. AuNP or NPG were first modified with L-cysteine, and the capture antibodies were then immobilized on AuNP or NPG with the aid of carbodiimide and N-hydroxysuccinimide (Fig. 6(ii)). In addition, composite nanomaterials, for example AuNP–graphene  and gold–silver–graphene , were used to modify the surface of the SPCWE for covalent immobilization of capture antibodies (Fig. 6(iii)). Furthermore, to accelerate the sandwich immunoreaction in μ-PECLDs, the sandwich immunocomplexes were assembled into the porous paper zones on the back of the SPCWEs [90, 92, 96, 97]. To ensure sufficient efficiency and sensitivity of the electrochemical reactions in the insulating paper zone, materials including AuNP , AgNP [90, 96], carbon nanotubes , and/or graphene nanosheets  are used to modify the paper zone. For example, Wang et al.  and Li et al.  reported, respectively, the use of multi-walled carbon nanotubes (MWCNTs) and graphene oxide to modify cellulose fibers in the paper zone. In Wang’s work, MWCNTs in the form of small bundles or single tubes were assembled on paper fibers through physical adsorption. Chitosan was then used to coat the MWCNT-modified paper fibers, both to avoid
Lab-on-paper-based devices using chemi-/electrochemi-luminescence
Fig. 6 Examples of modifications on the surface of SPCWE for ECL sandwich immunoassays. (i) The surface of SPCWE was first coated with chitosan, then cross-linked with glutaraldehyde, followed by immobilization with capture antibodies. Nonspecific binding sites were then blocked by BSA. The capture antibody–antigen–signal antibody immunocomplex was finally established on the electrode surface. Reprinted from Ref.  with permission from Elsevier. (ii) L-cysteinecapped AuNP were modified on the electrode surface, to enhance both the surface area and conductivity of SPCWE, and to cross-link capture antibodies. Reprinted from Ref.  with permission from Elsevier. (iii) The preparation procedure of gold–silver–graphene nanocomposite (a) for modification of the electrode surface (c), and of quantum dot– carbon microsphere-labeled signal antibody (b) for signal amplification (c). Reproduced from Ref.  with permission from The Royal Society of Chemistry
leakage of MWCNTs, and to provide active amino-groups to immobilize capture antibodies through glutaraldehyde crosslinking. In Li’s work, paper fibers were first modified with a graphene oxide–chitosan composite (Fig. 7(i)), which was then used as substrate to immobilize antibodies through AuNP cross-linking. Furthermore, Li et al. [90, 96] developed a porous silver-paper electrode, through the growth of an AgNP layer from AgNP seeds on the surface of cellulose fibers in the paper zone, to immobilize capture antibodies in the paper zone through chemical absorption between AgNP and the−NH2 groups of capture antibodies (Fig. 7(ii)). Because of the high
Fig. 7 Examples of modifications in the paper zone of SPCWE for ECL sandwich immunoassays. (i) (A) Bare μ-PECLD: (a) wax-printed hydrophobic area, (b) unprinted hydrophilic paper zone; (B) μ-PECLD after screen-printing of the electrode and modification of the hydrophilic paper zone with graphene oxide–chitosan–AuNP nanocomposite; (C) The two hydrophilic paper zones were immobilized with different capture antibodies, and blocked with BSA; (D) after capturing, washing, and incubating with corresponding signal antibody. Reprinted from Ref.  with permission from Elsevier. (ii) The growth of an AgNP layer from AgNP seeds on the surface of cellulose fibers in the paper zone, to support the capture antibody–antigen–signal antibody immunocomplex. Reproduced from Ref.  with permission from The Royal Society of Chemistry
surface-area-to-weight ratio (9.5 m2 g−1)  and porous structure of paper and the high conductivity of Ag, the active surface area and the sensitivity of this μ-PECLD were remarkably improved. Nucleic acid-based assays in μ-PECLDs and μ-PCLDs Compared with antigen and/or antibody-based immunoassay, nucleic acid assay is more suitable for early detection of genetic and infectious diseases by use of μ-PADs [41, 43, 46, 75, 124, 182 185], because the base-pairing interactions between complementary sequences are more specific and robust than the interaction between antigen and antibody. Typically, a nucleic acid assay using μ-PADs requires two types of oligonucleotide probe: detector probe and capture
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probe. Detector and capture probes are both complementary with their target nucleic-acid sequence, and the detector probe is combined with a tag to make the reaction visible or measurable. The tag can be used for colorimetric assays [75, 182–187], electrochemical measurements , or fluorescent reporting [43, 124, 189–192] using μ-PADs. Because the ability to sense and to detect ultralow concentrations of specific DNA sequences is important in clinical diagnostics, gene therapy, food safety, environment, and biodefense applications, CL nucleic-acid assay for μ-PADs promises great potential for highly sensitive and reliable diagnosis. For example, Wang et al.  reported CL DNA assays in a paper microzone plate, in which multiple detection zones for different DNA samples can be implemented in the same paper Plate. N,N -Disuccinimidyl carbonate, a carbonyl group containing two NHS esters, can activate the hydroxylic groups of the cellulose fibers in the paper detection zone, forming an amine-reactive succinimidyl carbonate derivative to immobilize the amine-containing DNA capture probes through stable carbamate linkages under mild conditions (Fig. 8i). Functional nucleic acids, especially aptamers, are nucleic acids whose functions include nucleic-acid hybridization . Aptamers are chosen as molecular probes to functionalize μ-PADs because of their salient properties, including high affinity and specificity, ease of chemical modification, good stability, and low immunogenicity. Aptamerbased μ-PADs can be used to specifically detect a wide range of analytes, from ions to proteins [34, 56, 57, 89, 98, 175]. More importantly, aptamers offer remarkable flexibility and convenience in the design of their structures, which has led to novel sensing strategies for μ-PADs that have high sensitivity and selectivity. Furthermore, their small size and versatility enable efficient immobilization at high density, which is of vital importance in multiplexing miniaturized systems. Zhang et al.  developed a signal-on aptamer-based μPECLD to achieve simultaneous detection of two different metal ions in a single paper electrode through covalent immobilization of the corresponding aptamer, tagged with a terminal ECL label, into the paper zone of the SPCWE, based on periodate oxidation (Fig. 8(iii)). The long, flexible, modified aptamer chain prevented electrical contact of the ECL label with the electrode. Upon binding to their target metal ions, the immobilized aptamers fold their flexible, single-stranded chains into well-defined three-dimensional structures; this behavior enabled the electrical communication of the ECL label with the electrode, and produced a positive signal. To obtain high specificity and introduce amplification factors to improve sensitivity, sandwich configurations are often used in aptamer-based μ-PECLD. Yan et al.  reported a selective and sensitive sandwich ECL aptasensor for μ-PADs, requiring only a single aptamer sequence. They created this sandwich ECL aptasensor by splitting the aptamer into two fragments, one of which is attached to a gold-paper electrode
via alkane thiol self-assembled monolayer chemistry, and the second of which is modified with the ECL labels (Fig. 8(ii)). The gold-paper electrode was fabricated through the growth of an AuNP layer from AuNP seeds on the surface of cellulose fibers in the paper zone of the SPCWE. In the presence of the target analyte, the two fragments were hybridized together to form a rigid three-dimensional structure with the aid of the target. Target-induced association of the two fragments thus increases the concentration of ECL labels in the paper electrode, which can be readily monitored via ECL emission. Rusling’s group  used DNA as an indicator to detect potential genotoxic pollutant activity in environmental samples through the forming of a thin film on paper containing the CL reagent, enzyme, and DNA, using layer-by-layer film fabrication. Genotoxicity here refers to damage to the immobilized DNA by reactive chemicals and their metabolites. This disrupts the double helix, and guanines (the only major reactants in the ECL process) in damaged DNA are more accessible to the ECL reagents. The catalytic oxidation of these guanines is thus faster for damaged DNA than intact DNA, and consequently the ECL light output is increased. Molecularly imprinted polymers for μ-PCLD Molecular imprinting is a well-known and widely used tool for producing synthetic bio-mimetic receptors for target molecules . The target molecule, or a derivative thereof, acts as a template around which interacting and cross-linking monomers are arranged and polymerized to form a cast-like shell. After removal of the template, binding cavities appear which are complementary in shape, size, and position of chemical functionalities with respect to the template, and the resulting material can specifically recognize and bind its target. In comparison with natural bio-recognition materials, for example antibodies or DNA, molecularly imprinted polymers (MIPs) offer advantages including superior physical and chemical stability, specific recognition, ease of mass preparation, and ready availability, and thus have found applications in a wide range of fields, including separation, sensors, and catalysis. Wang et al.  developed a molecularly imprinted polymer-grafted paper-based multi-disk micro-disk plate (PMIP-MMP) for CL detection of pesticide. The MIP-grafted paper disks were prepared by a simple in-situ polymerization of an MIP layer on the surface of cellulose fibers in paper (Fig. 8(iv)). Before the in-situ polymerization of MIPs, bare paper disks were first activated using a silane-coupling technique to introduce C=C groups onto the surface of cellulose fibers, making use of the abundant –OH groups per anhydroglucose unit of cellulose. Like the zones in a conventional paper microzone plate , each MIP-grafted paper disk on the P-MIP-MMP can be used to run an independent assay, and the design of the plate facilitates parallel processing of large numbers of samples. An indirect competitive assay
Lab-on-paper-based devices using chemi-/electrochemi-luminescence Fig. 8 (i) N,N -disuccinimidyl carbonate was used to activate the hydroxyl group on cellulose fibers in the paper zone to an amine-reactive succinimidyl carbonate derivative, for immobilization of NH2− DNA capture probes in the paper zone. Reproduced from Ref.  with permission from The Royal Society of Chemistry. (ii) Growth of an AuNP layer from AuNP seeds on the surface of cellulose fibers in the paper zone, for the immobilization of HS− aptamers through chemical absorption between AuNP and HS− of aptamers. Reproduced from Ref.  with permission from The Royal Society of Chemistry. (iii) Immobilization of aptamers in the bare paper zone of the paper electrode, through covalent bonding between the NH2− of aptamers and the in-situgenerated aldehyde groups on the cellulose fibers. Reprinted from Ref.  with permission from Elsevier. (iv) After silanization of the surfaces of cellulose fibers, insitu polymerization of the MIP layer on the surface of cellulose fibers was performed. Then, binding sites on the MIP surface were created through eluting the template molecule, followed by blocking and labeling. Adapted from Ref.  with permission from Elsevier
format, based on competition between the free analyte in the sample solution and the immobilized analyte–tobacco peroxidase conjugate in the grafted MIP layer in the paper disk, was used to quantify the analyte in the sample.
Signal amplification in μ-PCLDs and μ-PECLDs The increasing demand for early-stage disease screening requires ultrasensitive detection of biologically relevant species at an extremely low level of expression, which has led to intense research efforts into signal-amplification strategies for enhancing detection sensitivity. Some successful signalamplification strategies have been developed for μ-PADs, including enrichment of analytes , enlargement of colorimetric nanoparticles [77, 158], use of novel probes [40, 197], construction of new device architecture , and incorporation of nanomaterials to increase the upload of tags [37, 39, 57, 58, 198, 199]. The last approach is particularly popular for
use with μ-PCLDs and μ-PECLDs [45, 46, 89, 95, 98–100] for introducing multiple signal tags per binding event. For example, carbon microspheres [90, 95], silica nanospheres , AuNP [89, 100], AgNP , mesoporous Pt-Ag alloy nanoparticles [98, 99], nanoporous gold [46, 90], nanoporous silver [96, 97], and multiwalled carbon nanotubes  have all been used with μ-PCLDs or μ-PECLDs as the carrier for a large load of signal tags, including quantum dots [46, 89, 92, 95, 96], Ru(bpy)32+ [89, 92, 93], phenyleneethynylene derivative [97–99], luminol [85, 100], and/or catalysts [45, 90].
Analytical instruments used with μ-PCLDs and μ-PECLDs For CL detection by μ-PCLDs, only a detector, usually a photomultiplier [47–49, 85, 87], should be used for detecting light emission from μ-PCLDs. However, an extra electrochemical workstation [51, 54, 55, 89, 91, 98] must be used
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with μ-PECLDs to induce the electrochemical reaction generating ECL emission. To develop μ-PECLDs for low-cost and disposable applications, batteries, including primary batteries  (Fig. 9(i)), lithium batteries [92, 97] (Fig. 9(ii)), nickel–metal hydride rechargeable batteries  (Fig. 9(iii)), and mobile phone batteries  (Fig. 9(v)), have been used to initiate the ECL detections (constant potential), enabling the usual electrochemical workstation to be abandoned. To precisely control the potential value and enable sensitive and exact ECL detection, Yu and coworkers [92, 97] developed a voltage-tunable power device, able to preset and/or control output voltage from the battery with a resolution of 0.01 V (Fig. 9(ii)). For μ-PECLDs to be truly “simple,” however, the signal should be capable of being detected quantitatively without recourse to a dedicated scientific instrument. Although photomultipliers are widely available in miniaturized form,
they are somewhat expensive. The use of commonplace, lowcost, small, and portable instruments for luminescence detection with μ-PADs can be regarded as a new trend in this field. Cell-phone cameras [71, 185, 200] and scanners [80, 106, 136, 201] are relatively inexpensive, off-the-shelf solutions, able to provide quantitative luminescence detection with imaging processing. Delaney et al. [50, 53] developed a portable and low-cost cell phone apparatus to measure and/or image the ECL emission from μ-PECLD with the aid of a cell-phone camera (Fig. 9(iv)). Ambient light is completely eliminated by fixing the paper device flush against the camera lens with the aid of a simple holder (Fig. 9(v)), offering a more sensitive means of detection. Quantitative analysis was achieved using the sum of the red pixel intensities in the RGB image, obtained from the mobile phone camera.
Applications of μ-PCLDs and μ-PECLDs Applications of μ-PADs have been frequently related to bioassays. Rapid diagnostic tests for people in underdeveloped regions worldwide are regarded as a promising application for μ-PADs [17–26]. Most diagnostic applications of μ-PCLDs and μ-PECLDs have focused on DNA , small-molecule detection [47–54, 86, 87], or metal ions . Cancer is the leading cause of death in the developed world, and ranks second in developing countries. Approximately 12.7 million kinds of cancer have been diagnosed, and 7.6 million people worldwide die of cancer every year . Delayed diagnosis contributes to poor quality of life and low survival . There is therefore much potential to make use of μ-PCLDs and μ-PECLDs for analyzing protein biomarkers [45, 83, 84, 90–97, 99, 100] in cancer-related applications: simplicity, affordability, sensitivity, specificity, rapidity, and robustness are all essential requirements for cancer diagnosis. Fig. 9 Examples of the analytical instruments used with μ-PCLDs and μ-PECLDs. (i) A stable, simple environmentally friendly, and noblemetal-free primary battery (C|FeCl3|NaCl|AlCl3|Al) was constructed on paper to induce the ECL reaction in μ-PECLD. The open-circuit voltage of the battery can be adjusted within a wide range by changing the amount of FeCl3 used. Adapted from Ref.  with permission from The Royal Society of Chemistry. (ii) A voltage-tunable power device, consisting of a 3 V lithium battery, three light-emitting diodes, and a keyboard, was used to induce the ECL reaction in μ-PECLD. The output voltage coordinates with the preset output voltage, with a resolution of 0.01 V. Adapted from Ref.  with permission from The Royal Society of Chemistry. (iii) A 1.2 V rechargeable nickel-metal hydride battery was used to directly induce the ECL reaction in μ-PECLD, without a controller. Adapted from Ref.  with permission from The Royal Society of Chemistry. (iv) A cell phone equipped with a camera for imaging the ECL emission. Adapted with permission from Ref. . Copyright (2011) American Chemical Society. (v) A cell phone can perform the basic functions of a potentiostat, controlling an applied potential to generate ECL emission, and can image the resultant ECL signal using the camera in video mode. Reprinted from Ref.  with permission from Elsevier
Conclusions and future perspective CL and ECL methods have been widely used to develop labon-paper analytical devices because of their high sensitivity, wide linear range, simple instrumentation, and high compatibility with micromachining technology. Current technology in the field of lab-on-paper devices using CL and ECL detection was summarized in this review. Through the combination of CL and ECL methods, paper-based assays are becoming more sensitive, more accurate, and multi-functional. There are several types of analytical strategy available for use with μPCLDs or μ-PECLDs, including enzyme-based assay, immunoassay, DNA-based assay, and MIP-based assay. The choice of analytical strategy for μ-PCLDs or μ-PECLDs depends on the application.
Lab-on-paper-based devices using chemi-/electrochemi-luminescence
However, the potential of μ-PCLDs and μ-PECLDs will not be maximized until the multiple operation steps of the sensitive and specific assay mode—for example, the sandwich assay—of μ-PCLDs and μ-PECLDs are thoroughly simplified for end users. For example, both Lutz’s group  and Fu’s group  used an automatic sandwich reaction on μPADs to achieve sensitive colorimetric readout just through addition of solutions. Furthermore, few of the μ-PCLDs and μ-PECLDs developed have reached the market, which has been mainly attributed to the dependence on laboratory instruments, including the photomultiplier and electrochemical workstation. Although the electrochemical workstation has been replaced by batteries [52, 92, 93], researchers should make efforts to provide alternative tools, for example mobile phones, for unskilled people to use to directly or indirectly read and interpret the luminescence results from μ-PCLDs and μ-PECLDs in real time. Attention also needs to be given to other aspects, including both signal amplification and the integration of a variety of processes (including sample collection and target separation) into a single μ-PCLD and μ-PECLD technique containing multiple functional elements. Establishing a high-throughput electrode array for μ-PECLD, and ensuring high specificity and sensitivity for detecting extremely small volumes without significantly perturbing the sample, are still challenges. A wide range of new luminophores, nanomaterials, and catalytic compounds are expected to continuously advance CL and ECL detection by lab-on-paper devices, enabling highly sensitive detection. Finally, the applicability of μ-PCLDs and μPECLDs is not restricted to diagnostic assays. It is believed that these devices could also be investigated for use in homeland security , pharmaceutical assay , environmental monitoring [47, 51, 89], food safety [48, 49, 82], and other point-of-care applications . Acknowledgments This work was financially supported by Natural Science Research Foundation of China (21175058, 21277058); Natural Science Foundation of Shandong Province, China (ZR2012BZ002).
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