Opinion
Paper – a potential platform in pharmaceutical development Yi-Hsun Chen1,2*, Zong-Keng Kuo1,3*, and Chao-Min Cheng1 1
Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 300, Taiwan Pharmacokinetic Technology Department, Center of Excellence for Drug Development, Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu 300, Taiwan 3 Pharmacodynamics Technology Department, Center of Excellence for Drug Development, Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu 300, Taiwan 2
Paper is predominantly composed of cellulose fibers that have an inherent ability to wick fluids by capillary action; it provides an interesting diagnostic platform that is inexpensive, easily obtained, and eco-friendly. Paper has been used in various types of biologically relevant applications including paper-based molecular assays, paper-based ELISA (P-ELISA), paper-based nucleic acid assays, and paper-based cell assays. Based on recent successes with the use of paper as a platform, we contend that paper is not only very suitable for diagnostics but could provide a more advantageous platform than current plastics-based platforms for drug discovery, and would be useful for accomplishing in vitro precompound screening steps while offering a possible solution to several economic obstacles inherent in the pharmaceutical industry. Paper – a potential platform to adjust the resource structure of pharmaceutical development Pharmaceutical companies develop well-established workflows to keep pace with the timelines for unmet medical needs. They usually devote all their resources to developing new drugs for prolonging life, improving quality of life, avoiding the adverse effects of drug treatment, and increasing convenience for people who require medications [1]. Launching a new drug to market is widely known to cost more than a billion dollars from concept to execution. Companies have refined the drug-development process to take a drug from benchtop to the market, while minimizing any waste in funding or resources as a result of trial and error. The drug-development process can usually be divided into six stages: target identification and validation, hit selection, lead identification [2], lead optimization [3], drug nomination, and clinical trials. The early stages of research and development focus on the molecular targets of disease, pre-compound synthesis, and screening, which are costly and very important in the pursuit of a successful product [4–6]. In early drug-screening stages such as hit selection, Corresponding author: Cheng, C.-M. ([email protected]). Keywords: bioassay; diagnostics; paper-based microfluidic device; point-of-care diagnostics; clinically based diagnostics in hospital; translational medicine. * These authors contributed equally. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.11.004
4
Trends in Biotechnology, January 2015, Vol. 33, No. 1
pharmaceutical companies have developed and have long employed a variety of molecular or cell based assays to test amounts of pre-compounds. They have also attempted to minimize cost and economize manpower with the use of automatic, high-throughput systems. However, the automatic high-throughput system is a costly instrument that wastes considerable resources and materials, including but not limited to the use of expensive reagents [e.g., monoclonal antibodies (mAbs), enzymes, and substrates]. These disadvantages make biological assays very expensive and create a high threshold for launching pharmaceutical industry research. Paper has been successfully used for biological assays in recent years [7–19], and many review articles have summarized how well these versatile paper-based platforms perform in biological assays, and several advantages over conventional platforms have been demonstrated [20,21]. For instance, paper-based platforms can replace current in vitro assays including cell assays, and thus can overcome the high cost of microtiter plates. Furthermore, such assays can be implemented not only for hit selection but also for lead selection, lead optimization, candidate nomination, and even clinical trials. Based on these promising results, we will try, in this manuscript, to expand upon the benefits of paper-based assays and provide a perspective on using paper-based assays as an alternative to existing tools in the pharmaceutical industry (Figure 1). While we highlight assay advantages, it is also important to note that paper-based platforms are economically advantageous for use in developing and underdeveloped countries. Benefits of paper-based diagnostics in current translational-medicine applications Paper is affordable, abundant, disposable, and compatible with large-scale manufacturing processes for the production of microfluidic devices. Paper, essentially a thin sheet of cellulose fibers, possesses advantages that are compatible with many bioassays [20,22]. Paper is thin, lightweight, amenable to long-term storage, and of low cost [22]; paper is also easily disposed of by incineration, which is more ecofriendly than plastics. Paper is usually white, and is therefore suitable for colorimetric assays. Paper can be easily modified chemically and can be conjugated with many biomolecules, including peptides and nucleotides, and
Opinion
Trends in Biotechnology January 2015, Vol. 33, No. 1
Plate type
Plascs
Paper
Plaorm cost
High
Low
Eco-friendly
No
Yes
Storage space
Large
Small
Time of tesng
Long
Short
Reagent volume
High
Low
3D in cell culture
No
Yes
Integraon with HTS
Yes
Yes
Potenal of paper-based assay to accelerate drug discovery
New drug approved
Clinical trials Candidate nominaon paper vs plascs
10
5
Lead opmizaon paper vs plascs
8
4 Lead idenficaon paper vs plascs
Target discovery/ validaon
Small molecule/mAb drug
1
0
Ye ars
10
?
100
Paper-based in vitro assays
2 1 10 0 00
3D cell culture in paper
4
2
Com poun d nu mbe r
Hit selecon paper vs plascs
Ye ars
Paper-based cell assays
6
3
Paper-based assays for animal study
Real meframe of drug discovery TRENDS in Biotechnology
Figure 1. Adjusting the resource structure of drug development with paper-based platforms. Paper has distinct advantages over plastic (top left). P-ELISA, paper-based cell assays, and paper-based animal studies (bottom left) have the potential to accelerate the rate of drug discovery and thus decrease the amount of time required to achieve drug approval (right). Abbreviations: HTS, high-throughput screening; mAB, monoclonal antibody; P-ELISA, paper-based ELISA.
can be customized to meet special needs [21]. Patterned barriers (e.g., 96-well and 384-well formats for highthroughput use) can be easily created on paper using wax printing (Box 1) [23], microchannels can easily be fabricated on paper, and some multiplex and labor-consuming steps can be simplified by leveraging the microfluidic advantages of paper (Box 1) [24]. Based on these characteristics and advantages, paper can be used in many bioassays, including molecular assays, P-ELISA, cell culture studies, and more [8–17,19,21,25–27]. Diagnostic assays are the most achievable application for paper-based platforms, and these have already been used to examine clinical samples such as blood, saliva, tears, aqueous humor, seminal fluid, and more [10,11,17,26,28– 32]. Highly versatile, paper-based micro-fluidic devices (mPADs) have several advantages because they require small sample and reagent volumes, and can be rapidly and conveniently integrated into portable instruments that use diverse analytical detection approaches including colorimetry, fluorescence and chemiluminescence, electrochemical methods, or transmittance [33]. We will highlight current studies that use paper bioassay platforms for ELISA (Table 1) and cell based assays, and discuss their benefits as superior replacements for current plastic-based bioassay platforms in the pharmaceutical industry. Paper-based ELISA assays Ligand-binding assays (LBA) have developed for the detection or quantification of molecules based on immunological
affinity. ELISA, the most widely used LBA, employs signal amplification via a specific antibody combined with highturnover catalytic enzymes and an enzymatic substrate that produces a detectable signal. ELISA is an efficient method for the routine assessment of large numbers of samples, for example in the quantification of drugs [34] and hormones [35], and provides a fundamental tool for measuring drugs and biomarkers in in vitro and in vivo samples during compound screening, animal studies, and clinical trials. ELISA assays are routinely used for drug screening of over 100 000 compounds. Although ELISA can provide highthroughput performance with rapid runs, the assays rely on high sample volume and use multiple unique reagents. PELISA provides several distinct advantages over conventional ELISA, such as sample conservation, economical use of reagents, and time and labor savings. P-ELISA was first used to determine immunoglobulin G (IgG) and HIV antigen titers via colorimetric assay [16]. PELISA can reduce reagent requirements to 1/25 of the volume needed for current microtiter plate processes, and can reduce the reaction time to 1/5 of that required for conventional ELISA [16]. P-ELISA is also a powerful tool for quantifying drugs and biomarkers in living samples. For example, P-ELISA using antibody against vascular endothelial growth factor (VEGF) has been used to measure VEGF concentrations in aqueous humor. In humans, only 200 mL of aqueous humor can be collected from the anterior chamber before threat of anterior chamber collapse. For P-ELISA, only 2 mL of aqueous humor is 5
Opinion
Trends in Biotechnology January 2015, Vol. 33, No. 1
Box 1. Paper-based devices Paper and other related membranes composed of chemically modified cellulose or other materials (for example, nitrocellulose) have been used in diverse biochemical assays. Three major platforms have been used: dipsticks, lateral flow assays (LFAs), and microfluidic paper-based analytical devices (mPADs). The simplest platform is the dipstick format, in which analytes are added to paper coated with chemicals, the chemical reaction proceeds, and a color change indicates analyte concentration. This process is illustrated, for example, by pH test strips (Figure IA). LFA is a simple method for detecting analytes and uses capillary flow of samples onto antibodycoated paper or other materials. In this process, specific analytes are captured by an antibody and a test line is revealed, as with pregnancy
test strips that detect hCG (human chorionic gonadotropin; Figure IB). First introduced by the Whiteside group, mPADs employ waxpatterned channels that can be designed easily using graphics software, printed using a wax-printer, and wicked into the paper by applying heat (Figure IC). They take advantage of capillary flow in wax-patterned channels to create a variety of interesting paper-based microfluidic devices that have been successfully applied in the detection of many different analytes (Figure ID). In an example of accommodation to existing technological formats, wax-patterned paper has been used to create a 96-well format that has been successfully used for paper-based ELISA, cell culture assays, and more.
(B)
(A) 4.5
5.0
5.5
5.75
6.0
6.25
(C)
Cross-secon of paper
6.5
Posive Very acidic 6.75
7.0
7.25
Acidic 7.5
8.0
8.5
Test zone
HCG anbody
9.0
Negave Control anbody Opmal
Print paerns on paper Wax Heat paper
Too alkaline
Wax barrier
(D)
Mulplex chip
Heat paper
Perform assays
Channel
Design and print
Wax
Test zone
Mul-well plate TRENDS in Biotechnology
Figure I. Paper-based diagnostic devices. (A) Dipsticks: diagnostic pH test dipsticks. (http://www.phionbalance.com). (B) LFAs: HCG pregnancy test strip. (C) The use of a wax-pattern to create mPADs. Modified from [24]. (D) Different mPADs.
needed for each test zone of a paper-based 96-well plate. Furthermore, considerably less detection probe, for example anti-VEGF mAb, is needed. In ophthalmological research using P-ELISA it was found that mean aqueous VEGF levels differed between patients with proliferative diabetic retinopathy (n = 14), age-related macular degeneration (n = 17), and retinal vein occlusion (n = 10). VEGF increases to 740.1 pg/mL, 383 pg/mL, and 219.4 pg/mL, respectively; these values were higher than in samples from control patients (n = 13). Senile cataract (VEGF levels as low as 14.4 pg/mL) was used as a further control [26]. This platform may elicit a paradigm shift regarding current diagnostics and treatment. Medical practitioners are provided with a useful and convenient tool to monitor ocular diseases, and retinal ischemic patient compliance is increased. Several paper-based platforms have been developed for disease diagnostics that should improve drug discovery of several diseases including, but not limited to, cancer [7,36], infectious viral diseases [12,37], dry eye disease 6
[29], post-traumatic stress disorder [38], and bullous pemphigoid (BP) [30]. Great advances have been made with mAb-based protein therapeutics such as infliximab and adalimumab for rheumatoid arthritis, and bevacizumab, cetuximab, panitumumab, and trastuzumab for cancer. Such mAb therapies can be highly effective and are becoming increasingly important in the treatment of inflammatory syndromes and cancer. The sticking point in the evaluation of mAb pharmacodynamics and pharmacokinetics is ELISA. Specifically, determinations of drug exposure (area under the curve, AUC), half-life (T1/2), Cmax, and anti-drug antibody production are crucial for improving efficacy and safety following mAb treatment, and all are ELISA-focused. Replacing traditional approaches with P-ELISA here could reap great benefits in terms of cost and speed. Cell based assays in paper The potential of every candidate drug must be evaluated in cell based assays before entering in vivo studies. Many aspects of cell based assays could be improved. In particular,
Biomarker
ALT (alanine transaminase) AST (aspartate transaminase) ALP (alkaline phosphatase) Cholesterol Nitrite
Fields in paper-based platform Metabolic assay
Ketone
Glucose Lactate Uric acid Albumin
Neuropeptide Y (NPY) VEGF (vascular endothelial growth factor) Lactoferrin Serotype-2 dengue HCV (hepatitis C virus) a
P-ELISA
Sample
Application
mPAD dynamic range
LoD a
Cut-off value/reference in clinics
Refs
Liver injury
Serum
Point-of-care diagnostics
0–400 U/L
53 U/L
>40 U/L
[17]
Liver injury Liver injury
Serum Serum
44–200 U/L
84 U/L; 44 U/L 44 U/L
>40 U/L 30–120 U/L
[11,17] [11]
Biliary obstruction/ hypophosphatemia Metabolic disease End-stage renal disease Diabetic ketoacidosis/ end-stage renal disease Renal disease/ metabolic disease Metabolic disease Metabolic disease Renal disease/ metabolic disease Hepatocellular carcinoma Ovarian cancer
Serum
15–1000 U/L
15 U/L
30–120 U/L
[11]
Plasma Salivary (artificial) Urine (artificial)
0.8–6.5 mM 5–2000 mM
0.67 mM 5 mM
[18] [32]
5–16 mM
0.5 mM
>5.2 mM 1–40 mM (normal), 40–160 mM (patients) 1.9 mM for ‘low’, between 2.9 and 3.9 mM for ‘moderate’, and 7.8 mM for ‘high’
0–20 mM/ 3–50 mM 0–50 mM 0–32 mM 0.38–60 mM; 0.38–7.5 mM 0.1–35 ng/mL
2.5 mM/2.8 mM
>0.8 mM
[22,32]
0.36 0.03 mM 1.38 0.13 mM 0.38 mM
>3.5 mM Abnormal range, >0.4 mM; 4 mM)
[28] [28] [11,22]
0.06 ng/mL
>25 ng/mL
[7]
Serum
0.5–80 U/mL
0.33 U/mL
>35 U/mL
[7]
Colorectal cancer
Serum
0.1–70.0 ng/mL
0.05 ng/mL
>5 ng/mL
[7]
Prostate cancer
Serum
0.5 50 mg/L
360.2 ng/L
>4 mg/L
[36]
Bullous pemphigoid
Serum or blister fluid Saliva
1–50 mg/mL
Aqueous humor
10
Tear
0.5–3 mg/mL
0.3 mg/mL
Serum Serum
100 pg/mL–14 mg/mL 26.7 fmol–267 amol
100 pg/mL 6.7 amol (1 pg)
Post-traumatic stress disorder (PTSD) Retinal ischemia
Disorders of the corneal epithelium Infectious disease Infectious disease
Urine (artificial) Serum Serum Urine (artificial) Serum
Clinic-based diagnostics
[30]
10 pM–100 nM 14
to 10
[32]
6
g/mL
AMD, age-related macular degeneration; LoD, limit of detection; PDR, proliferative diabetic retinopathy; RVO, retinal vein occlusion.
1 pM 10
14
g/mL
50 100 pM (normal), 400 1400 pM (stress)
[38]
14.4 8.5 pg/mL (normal), PDR (740.1 267.7 pg/mL), AMD (383 155.5 pg/mL), and RVO (219.4 92.1 pg/mL) a
[26]
[29] 4 ng/mL (conventional ELISA)
[12,27] [37]
7
Trends in Biotechnology January 2015, Vol. 33, No. 1
AFP (a-fetoprotein) CA125 (cancer antigen 125) CEA (carcinoembryonic antigen) PSA (prostate-specific antigen) NC16A
Disease/condition
Opinion
Table 1. Applications of clinically related biomarkers in translational medicine through esystems
Opinion it would be desirable to shorten the translational gaps between in vitro and in vivo studies, and to further increase the efficiency and lower the cost of cell based assays. Currently, 96-well plates have been the most useful tool for highthroughput cell based assays, but cells can only grow as a 2D monolayer on these plates, a far cry from physiological conditions, which have 3D structure and nutrient and biosignal gradients. In recent years, paper has been successfully demonstrated as a novel 3D platform for cell culture. By stacking layers of paper and impregnating the layers with cells, 3D structures can be created that allow the examination of cellular responses to oxygen and nutrient gradients in a high-throughput manner, and subsequent destacking has provided further valuable information [13,14,25]. Furthermore, using Teflon-patterned paper has enabled parallel flow-through synthesis of peptides on paper, and significantly ameliorated issues regarding low yield for chemical reactions compared to SPOT synthesis. Automated spotting of different amino acids on paper for peptide synthesis has enabled the production of paper-based peptide arrays which allow cell responses to different peptides to be observed efficiently in a highthroughput manner [39]. In addition to eukaryotic cell culture, prokaryotic cell culture has been performed using a paper-based platform. In such a device, bacterial growth rates, antibiotic responses, and phage amplification were comparable to those observed on agar plates or in shaking cultures [40,41]. In summary, paper not only shows comparable results in cell culture to conventional microplates but also provides a 3D structure for cell culture, demonstrating that paper can act as a viable alternative to plastic-based platforms. Concluding remarks and future perspectives In the period covered by this manuscript there has been increased use of paper to develop low-cost devices dedicated to diagnostic and clinical assays. Moreover, using paperbased platforms in many bioassays has been shown to provide comparable results to traditional platforms, including molecular assays, ELISA, nucleic acid-based assays, and cell based assays. Based on previous successful studies using paper, we feel that paper could provide an advantageous platform for accomplishing in vitro pre-compound screening steps, and could offer a solution to many economic obstacles inherent in the pharmaceutical industry. The high cost of drug development in the pharmaceutical industry impedes progress in generating novel drugs, especially in underdeveloped countries. Even in some developing countries, such as Taiwan, where there are excellent insurance and medical services, pharmaceutical companies are small and cannot always invest heavily in drug-discovery projects. Pharmaceutical companies have attempted to minimize cost and economize manpower with the use of automated high-throughput systems. However, such systems are still unattainable for small companies, especially given the material requirements. Moreover, it is still necessary to enhance accuracy and precision in high-throughput screening. One strategy to overcome such performance problems might be to leverage the hydrophobic and hydrophilic properties of patterned, wax-printed paper to guide a loading sample to the test zone in a high-throughput system. 8
Trends in Biotechnology January 2015, Vol. 33, No. 1
We believe that using paper as an assay platform to substitute for existing assay platforms would open up new possibilities in the pharmaceutical and biotechnological communities. Further, this approach offers great potential for drug development in the pharmaceutical industry, especially in less economically developed nations or in small pharmaceutical offices connected to healthcare centers. This could ultimately impact upon drug development and delivery for rare disorders in local communities, and improve healthcare for those currently suffering from diseases without drugs. Acknowledgments We would like to thank the National Science Council of Taiwan for financially supporting this research under Contract NSC 101-2628-E-007011-MY3 (to C.-M.C.).
References 1 Aronson, J.K. et al. (2012) Defining rewardable innovation in drug therapy. Nat. Rev. Drug Discov. 11, 253–254 2 Bleicher, K.H. et al. (2003) Hit and lead generation: beyond highthroughput screening. Nat. Rev. Drug Discov. 2, 369–378 3 Hughes, J.P. et al. (2011) Principles of early drug discovery. Br. J. Pharmacol. 162, 1239–1249 4 Paul, S.M. et al. (2010) How to improve R&D productivity: the pharmaceutical industry’s grand challenge. Nat. Rev. Drug Discov. 9, 203–214 5 Dickson, M. and Gagnon, J.P. (2004) Key factors in the rising cost of new drug discovery and development. Nat. Rev. Drug Discov. 3, 417–429 6 Rawlins, M.D. (2004) Cutting the cost of drug development? Nat. Rev. Drug Discov. 3, 360–364 7 Wang, S. et al. (2012) Paper-based chemiluminescence ELISA: lab-onpaper based on chitosan modified paper device and wax-screenprinting. Biosens. Bioelectron. 31, 212–218 8 Ellerbee, A.K. et al. (2009) Quantifying colorimetric assays in paperbased microfluidic devices by measuring the transmission of light through paper. Anal. Chem. 81, 8447–8452 9 Cheng, C.M. et al. (2010) Millimeter-scale contact printing of aqueous solutions using a stamp made out of paper and tape. Lab Chip 10, 3201–3205 10 Martinez, A.W. et al. (2010) Programmable diagnostic devices made from paper and tape. Lab Chip 10, 2499–2504 11 Vella, S.J. et al. (2012) Measuring markers of liver function using a micropatterned paper device designed for blood from a fingerstick. Anal. Chem. 84, 2883–2891 12 Lo, S.J. et al. (2013) Molecular-level dengue fever diagnostic devices made out of paper. Lab Chip 13, 2686–2692 13 Derda, R. et al. (2009) Paper-supported 3D cell culture for tissue-based bioassays. Proc. Natl. Acad. Sci. U.S.A. 106, 18457–18462 14 Derda, R. et al. (2011) Multizone paper platform for 3D cell cultures. PLoS ONE 6, e18940 15 Lutz, B. et al. (2013) Dissolvable fluidic time delays for programming multi-step assays in instrument-free paper diagnostics. Lab Chip 13, 2840–2847 16 Cheng, C.M. et al. (2010) Paper-based ELISA. Angew. Chem. Int. Ed. Engl. 49, 4771–4774 17 Pollock, N.R. et al. (2012) A paper-based multiplexed transaminase test for low-cost, point-of-care liver function testing. Sci. Transl. Med. 4, 152ra129 18 Wong, A.P. et al. (2008) Egg beater as centrifuge: isolating human blood plasma from whole blood in resource-poor settings. Lab Chip 8, 2032– 2037 19 Tsai, T.T. et al. (2013) Paper-based tuberculosis diagnostic devices with colorimetric gold nanoparticles. Sci. Technol. Adv. Mater. 14, 044404 20 Yetisen, A.K. et al. (2013) Paper-based microfluidic point-of-care diagnostic devices. Lab Chip 13, 2210–2251 21 Hu, J. et al. (2014) Advances in paper-based point-of-care diagnostics. Biosens. Bioelectron. 54, 585–597 22 Martinez, A.W. et al. (2010) Diagnostics for the developing world: microfluidic paper-based analytical devices. Anal. Chem. 82, 3–10
Opinion 23 Carrilho, E. et al. (2009) Paper microzone plates. Anal. Chem. 81, 5990– 5998 24 Martinez, A.W. et al. (2008) Three-dimensional microfluidic devices fabricated in layered paper and tape. Proc. Natl. Acad. Sci. U.S.A. 105, 19606–19611 25 Deiss, F. et al. (2013) Platform for high-throughput testing of the effect of soluble compounds on 3D cell cultures. Anal. Chem. 85, 8085–8094 26 Hsu, M.Y. et al. (2014) Monitoring the VEGF level in aqueous humor of patients with ophthalmologically relevant diseases via ultrahigh sensitive paper-based ELISA. Biomaterials 35, 3729–3735 27 Wang, H.K. et al. (2014) Cellulose-based diagnostic devices for diagnosing serotype-2 dengue Fever in human serum. Adv. Healthc. Mater. 3, 187–196 28 Dungchai, W. et al. (2009) Electrochemical detection for paper-based microfluidics. Anal. Chem. 81, 5821–5826 29 Yamada, K. et al. (2014) An antibody-free microfluidic paper-based analytical device for the determination of tear fluid lactoferrin by fluorescence sensitization of Tb3+. Analyst 139, 1637–1643 30 Hsu, C.K. et al. (2014) Paper-based ELISA for the detection of autoimmune antibodies in body fluid-the case of bullous pemphigoid. Anal. Chem. 86, 4605–4610 31 Yager, P. et al. (2006) Microfluidic diagnostic technologies for global public health. Nature 442, 412–418
Trends in Biotechnology January 2015, Vol. 33, No. 1
32 Klasner, S.A. et al. (2010) Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva. Anal. Bioanal. Chem. 397, 1821–1829 33 Martinez, A.W. (2011) Microfluidic paper-based analytical devices: from POCKET to paper-based ELISA. Bioanalysis 3, 2589–2592 34 Laurie, D. et al. (1989) A rapid, qualitative ELISA test for the specific detection of morphine in serum or urine. Clin. Chim. Acta 183, 183–195 35 Rajkowski, K.M. et al. (1989) A competitive microtitre plate enzyme immunoassay for plasma testosterone using polyclonal antitestosterone immunoglobulins. Clin. Chim. Acta 183, 197–206 36 Nie, J. et al. (2012) Low-cost fabrication of paper-based microfluidic devices by one-step plotting. Anal. Chem. 84, 6331–6335 37 Mu, X. et al. (2014) Multiplex microfluidic paper-based immunoassay for the diagnosis of hepatitis C virus infection. Anal. Chem. 86, 5338–5344 38 Murdock, R.C. et al. (2013) Optimization of a paper-based ELISA for a human performance biomarker. Anal. Chem. 85, 11634–11642 39 Deiss, F. et al. (2014) Flow-through synthesis on Teflon-patterned paper to produce peptide arrays for cell-based assays. Angew. Chem. Int. Ed. Engl. 53, 6374–6377 40 Funes-Huacca, M. et al. (2012) Portable self-contained cultures for phage and bacteria made of paper and tape. Lab Chip 12, 4269–4278 41 Deiss, F. et al. (2014) Antimicrobial susceptibility assays in paperbased portable culture devices. Lab Chip 14, 167–171
9
Paper – a potential platform in pharmaceutical development Yi-Hsun Chen1,2*, Zong-Keng Kuo1,3*, and Chao-Min Cheng1 1
Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 300, Taiwan Pharmacokinetic Technology Department, Center of Excellence for Drug Development, Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu 300, Taiwan 3 Pharmacodynamics Technology Department, Center of Excellence for Drug Development, Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu 300, Taiwan 2
Paper is predominantly composed of cellulose fibers that have an inherent ability to wick fluids by capillary action; it provides an interesting diagnostic platform that is inexpensive, easily obtained, and eco-friendly. Paper has been used in various types of biologically relevant applications including paper-based molecular assays, paper-based ELISA (P-ELISA), paper-based nucleic acid assays, and paper-based cell assays. Based on recent successes with the use of paper as a platform, we contend that paper is not only very suitable for diagnostics but could provide a more advantageous platform than current plastics-based platforms for drug discovery, and would be useful for accomplishing in vitro precompound screening steps while offering a possible solution to several economic obstacles inherent in the pharmaceutical industry. Paper – a potential platform to adjust the resource structure of pharmaceutical development Pharmaceutical companies develop well-established workflows to keep pace with the timelines for unmet medical needs. They usually devote all their resources to developing new drugs for prolonging life, improving quality of life, avoiding the adverse effects of drug treatment, and increasing convenience for people who require medications [1]. Launching a new drug to market is widely known to cost more than a billion dollars from concept to execution. Companies have refined the drug-development process to take a drug from benchtop to the market, while minimizing any waste in funding or resources as a result of trial and error. The drug-development process can usually be divided into six stages: target identification and validation, hit selection, lead identification [2], lead optimization [3], drug nomination, and clinical trials. The early stages of research and development focus on the molecular targets of disease, pre-compound synthesis, and screening, which are costly and very important in the pursuit of a successful product [4–6]. In early drug-screening stages such as hit selection, Corresponding author: Cheng, C.-M. ([email protected]). Keywords: bioassay; diagnostics; paper-based microfluidic device; point-of-care diagnostics; clinically based diagnostics in hospital; translational medicine. * These authors contributed equally. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.11.004
4
Trends in Biotechnology, January 2015, Vol. 33, No. 1
pharmaceutical companies have developed and have long employed a variety of molecular or cell based assays to test amounts of pre-compounds. They have also attempted to minimize cost and economize manpower with the use of automatic, high-throughput systems. However, the automatic high-throughput system is a costly instrument that wastes considerable resources and materials, including but not limited to the use of expensive reagents [e.g., monoclonal antibodies (mAbs), enzymes, and substrates]. These disadvantages make biological assays very expensive and create a high threshold for launching pharmaceutical industry research. Paper has been successfully used for biological assays in recent years [7–19], and many review articles have summarized how well these versatile paper-based platforms perform in biological assays, and several advantages over conventional platforms have been demonstrated [20,21]. For instance, paper-based platforms can replace current in vitro assays including cell assays, and thus can overcome the high cost of microtiter plates. Furthermore, such assays can be implemented not only for hit selection but also for lead selection, lead optimization, candidate nomination, and even clinical trials. Based on these promising results, we will try, in this manuscript, to expand upon the benefits of paper-based assays and provide a perspective on using paper-based assays as an alternative to existing tools in the pharmaceutical industry (Figure 1). While we highlight assay advantages, it is also important to note that paper-based platforms are economically advantageous for use in developing and underdeveloped countries. Benefits of paper-based diagnostics in current translational-medicine applications Paper is affordable, abundant, disposable, and compatible with large-scale manufacturing processes for the production of microfluidic devices. Paper, essentially a thin sheet of cellulose fibers, possesses advantages that are compatible with many bioassays [20,22]. Paper is thin, lightweight, amenable to long-term storage, and of low cost [22]; paper is also easily disposed of by incineration, which is more ecofriendly than plastics. Paper is usually white, and is therefore suitable for colorimetric assays. Paper can be easily modified chemically and can be conjugated with many biomolecules, including peptides and nucleotides, and
Opinion
Trends in Biotechnology January 2015, Vol. 33, No. 1
Plate type
Plascs
Paper
Plaorm cost
High
Low
Eco-friendly
No
Yes
Storage space
Large
Small
Time of tesng
Long
Short
Reagent volume
High
Low
3D in cell culture
No
Yes
Integraon with HTS
Yes
Yes
Potenal of paper-based assay to accelerate drug discovery
New drug approved
Clinical trials Candidate nominaon paper vs plascs
10
5
Lead opmizaon paper vs plascs
8
4 Lead idenficaon paper vs plascs
Target discovery/ validaon
Small molecule/mAb drug
1
0
Ye ars
10
?
100
Paper-based in vitro assays
2 1 10 0 00
3D cell culture in paper
4
2
Com poun d nu mbe r
Hit selecon paper vs plascs
Ye ars
Paper-based cell assays
6
3
Paper-based assays for animal study
Real meframe of drug discovery TRENDS in Biotechnology
Figure 1. Adjusting the resource structure of drug development with paper-based platforms. Paper has distinct advantages over plastic (top left). P-ELISA, paper-based cell assays, and paper-based animal studies (bottom left) have the potential to accelerate the rate of drug discovery and thus decrease the amount of time required to achieve drug approval (right). Abbreviations: HTS, high-throughput screening; mAB, monoclonal antibody; P-ELISA, paper-based ELISA.
can be customized to meet special needs [21]. Patterned barriers (e.g., 96-well and 384-well formats for highthroughput use) can be easily created on paper using wax printing (Box 1) [23], microchannels can easily be fabricated on paper, and some multiplex and labor-consuming steps can be simplified by leveraging the microfluidic advantages of paper (Box 1) [24]. Based on these characteristics and advantages, paper can be used in many bioassays, including molecular assays, P-ELISA, cell culture studies, and more [8–17,19,21,25–27]. Diagnostic assays are the most achievable application for paper-based platforms, and these have already been used to examine clinical samples such as blood, saliva, tears, aqueous humor, seminal fluid, and more [10,11,17,26,28– 32]. Highly versatile, paper-based micro-fluidic devices (mPADs) have several advantages because they require small sample and reagent volumes, and can be rapidly and conveniently integrated into portable instruments that use diverse analytical detection approaches including colorimetry, fluorescence and chemiluminescence, electrochemical methods, or transmittance [33]. We will highlight current studies that use paper bioassay platforms for ELISA (Table 1) and cell based assays, and discuss their benefits as superior replacements for current plastic-based bioassay platforms in the pharmaceutical industry. Paper-based ELISA assays Ligand-binding assays (LBA) have developed for the detection or quantification of molecules based on immunological
affinity. ELISA, the most widely used LBA, employs signal amplification via a specific antibody combined with highturnover catalytic enzymes and an enzymatic substrate that produces a detectable signal. ELISA is an efficient method for the routine assessment of large numbers of samples, for example in the quantification of drugs [34] and hormones [35], and provides a fundamental tool for measuring drugs and biomarkers in in vitro and in vivo samples during compound screening, animal studies, and clinical trials. ELISA assays are routinely used for drug screening of over 100 000 compounds. Although ELISA can provide highthroughput performance with rapid runs, the assays rely on high sample volume and use multiple unique reagents. PELISA provides several distinct advantages over conventional ELISA, such as sample conservation, economical use of reagents, and time and labor savings. P-ELISA was first used to determine immunoglobulin G (IgG) and HIV antigen titers via colorimetric assay [16]. PELISA can reduce reagent requirements to 1/25 of the volume needed for current microtiter plate processes, and can reduce the reaction time to 1/5 of that required for conventional ELISA [16]. P-ELISA is also a powerful tool for quantifying drugs and biomarkers in living samples. For example, P-ELISA using antibody against vascular endothelial growth factor (VEGF) has been used to measure VEGF concentrations in aqueous humor. In humans, only 200 mL of aqueous humor can be collected from the anterior chamber before threat of anterior chamber collapse. For P-ELISA, only 2 mL of aqueous humor is 5
Opinion
Trends in Biotechnology January 2015, Vol. 33, No. 1
Box 1. Paper-based devices Paper and other related membranes composed of chemically modified cellulose or other materials (for example, nitrocellulose) have been used in diverse biochemical assays. Three major platforms have been used: dipsticks, lateral flow assays (LFAs), and microfluidic paper-based analytical devices (mPADs). The simplest platform is the dipstick format, in which analytes are added to paper coated with chemicals, the chemical reaction proceeds, and a color change indicates analyte concentration. This process is illustrated, for example, by pH test strips (Figure IA). LFA is a simple method for detecting analytes and uses capillary flow of samples onto antibodycoated paper or other materials. In this process, specific analytes are captured by an antibody and a test line is revealed, as with pregnancy
test strips that detect hCG (human chorionic gonadotropin; Figure IB). First introduced by the Whiteside group, mPADs employ waxpatterned channels that can be designed easily using graphics software, printed using a wax-printer, and wicked into the paper by applying heat (Figure IC). They take advantage of capillary flow in wax-patterned channels to create a variety of interesting paper-based microfluidic devices that have been successfully applied in the detection of many different analytes (Figure ID). In an example of accommodation to existing technological formats, wax-patterned paper has been used to create a 96-well format that has been successfully used for paper-based ELISA, cell culture assays, and more.
(B)
(A) 4.5
5.0
5.5
5.75
6.0
6.25
(C)
Cross-secon of paper
6.5
Posive Very acidic 6.75
7.0
7.25
Acidic 7.5
8.0
8.5
Test zone
HCG anbody
9.0
Negave Control anbody Opmal
Print paerns on paper Wax Heat paper
Too alkaline
Wax barrier
(D)
Mulplex chip
Heat paper
Perform assays
Channel
Design and print
Wax
Test zone
Mul-well plate TRENDS in Biotechnology
Figure I. Paper-based diagnostic devices. (A) Dipsticks: diagnostic pH test dipsticks. (http://www.phionbalance.com). (B) LFAs: HCG pregnancy test strip. (C) The use of a wax-pattern to create mPADs. Modified from [24]. (D) Different mPADs.
needed for each test zone of a paper-based 96-well plate. Furthermore, considerably less detection probe, for example anti-VEGF mAb, is needed. In ophthalmological research using P-ELISA it was found that mean aqueous VEGF levels differed between patients with proliferative diabetic retinopathy (n = 14), age-related macular degeneration (n = 17), and retinal vein occlusion (n = 10). VEGF increases to 740.1 pg/mL, 383 pg/mL, and 219.4 pg/mL, respectively; these values were higher than in samples from control patients (n = 13). Senile cataract (VEGF levels as low as 14.4 pg/mL) was used as a further control [26]. This platform may elicit a paradigm shift regarding current diagnostics and treatment. Medical practitioners are provided with a useful and convenient tool to monitor ocular diseases, and retinal ischemic patient compliance is increased. Several paper-based platforms have been developed for disease diagnostics that should improve drug discovery of several diseases including, but not limited to, cancer [7,36], infectious viral diseases [12,37], dry eye disease 6
[29], post-traumatic stress disorder [38], and bullous pemphigoid (BP) [30]. Great advances have been made with mAb-based protein therapeutics such as infliximab and adalimumab for rheumatoid arthritis, and bevacizumab, cetuximab, panitumumab, and trastuzumab for cancer. Such mAb therapies can be highly effective and are becoming increasingly important in the treatment of inflammatory syndromes and cancer. The sticking point in the evaluation of mAb pharmacodynamics and pharmacokinetics is ELISA. Specifically, determinations of drug exposure (area under the curve, AUC), half-life (T1/2), Cmax, and anti-drug antibody production are crucial for improving efficacy and safety following mAb treatment, and all are ELISA-focused. Replacing traditional approaches with P-ELISA here could reap great benefits in terms of cost and speed. Cell based assays in paper The potential of every candidate drug must be evaluated in cell based assays before entering in vivo studies. Many aspects of cell based assays could be improved. In particular,
Biomarker
ALT (alanine transaminase) AST (aspartate transaminase) ALP (alkaline phosphatase) Cholesterol Nitrite
Fields in paper-based platform Metabolic assay
Ketone
Glucose Lactate Uric acid Albumin
Neuropeptide Y (NPY) VEGF (vascular endothelial growth factor) Lactoferrin Serotype-2 dengue HCV (hepatitis C virus) a
P-ELISA
Sample
Application
mPAD dynamic range
LoD a
Cut-off value/reference in clinics
Refs
Liver injury
Serum
Point-of-care diagnostics
0–400 U/L
53 U/L
>40 U/L
[17]
Liver injury Liver injury
Serum Serum
44–200 U/L
84 U/L; 44 U/L 44 U/L
>40 U/L 30–120 U/L
[11,17] [11]
Biliary obstruction/ hypophosphatemia Metabolic disease End-stage renal disease Diabetic ketoacidosis/ end-stage renal disease Renal disease/ metabolic disease Metabolic disease Metabolic disease Renal disease/ metabolic disease Hepatocellular carcinoma Ovarian cancer
Serum
15–1000 U/L
15 U/L
30–120 U/L
[11]
Plasma Salivary (artificial) Urine (artificial)
0.8–6.5 mM 5–2000 mM
0.67 mM 5 mM
[18] [32]
5–16 mM
0.5 mM
>5.2 mM 1–40 mM (normal), 40–160 mM (patients) 1.9 mM for ‘low’, between 2.9 and 3.9 mM for ‘moderate’, and 7.8 mM for ‘high’
0–20 mM/ 3–50 mM 0–50 mM 0–32 mM 0.38–60 mM; 0.38–7.5 mM 0.1–35 ng/mL
2.5 mM/2.8 mM
>0.8 mM
[22,32]
0.36 0.03 mM 1.38 0.13 mM 0.38 mM
>3.5 mM Abnormal range, >0.4 mM; 4 mM)
[28] [28] [11,22]
0.06 ng/mL
>25 ng/mL
[7]
Serum
0.5–80 U/mL
0.33 U/mL
>35 U/mL
[7]
Colorectal cancer
Serum
0.1–70.0 ng/mL
0.05 ng/mL
>5 ng/mL
[7]
Prostate cancer
Serum
0.5 50 mg/L
360.2 ng/L
>4 mg/L
[36]
Bullous pemphigoid
Serum or blister fluid Saliva
1–50 mg/mL
Aqueous humor
10
Tear
0.5–3 mg/mL
0.3 mg/mL
Serum Serum
100 pg/mL–14 mg/mL 26.7 fmol–267 amol
100 pg/mL 6.7 amol (1 pg)
Post-traumatic stress disorder (PTSD) Retinal ischemia
Disorders of the corneal epithelium Infectious disease Infectious disease
Urine (artificial) Serum Serum Urine (artificial) Serum
Clinic-based diagnostics
[30]
10 pM–100 nM 14
to 10
[32]
6
g/mL
AMD, age-related macular degeneration; LoD, limit of detection; PDR, proliferative diabetic retinopathy; RVO, retinal vein occlusion.
1 pM 10
14
g/mL
50 100 pM (normal), 400 1400 pM (stress)
[38]
14.4 8.5 pg/mL (normal), PDR (740.1 267.7 pg/mL), AMD (383 155.5 pg/mL), and RVO (219.4 92.1 pg/mL) a
[26]
[29] 4 ng/mL (conventional ELISA)
[12,27] [37]
7
Trends in Biotechnology January 2015, Vol. 33, No. 1
AFP (a-fetoprotein) CA125 (cancer antigen 125) CEA (carcinoembryonic antigen) PSA (prostate-specific antigen) NC16A
Disease/condition
Opinion
Table 1. Applications of clinically related biomarkers in translational medicine through esystems
Opinion it would be desirable to shorten the translational gaps between in vitro and in vivo studies, and to further increase the efficiency and lower the cost of cell based assays. Currently, 96-well plates have been the most useful tool for highthroughput cell based assays, but cells can only grow as a 2D monolayer on these plates, a far cry from physiological conditions, which have 3D structure and nutrient and biosignal gradients. In recent years, paper has been successfully demonstrated as a novel 3D platform for cell culture. By stacking layers of paper and impregnating the layers with cells, 3D structures can be created that allow the examination of cellular responses to oxygen and nutrient gradients in a high-throughput manner, and subsequent destacking has provided further valuable information [13,14,25]. Furthermore, using Teflon-patterned paper has enabled parallel flow-through synthesis of peptides on paper, and significantly ameliorated issues regarding low yield for chemical reactions compared to SPOT synthesis. Automated spotting of different amino acids on paper for peptide synthesis has enabled the production of paper-based peptide arrays which allow cell responses to different peptides to be observed efficiently in a highthroughput manner [39]. In addition to eukaryotic cell culture, prokaryotic cell culture has been performed using a paper-based platform. In such a device, bacterial growth rates, antibiotic responses, and phage amplification were comparable to those observed on agar plates or in shaking cultures [40,41]. In summary, paper not only shows comparable results in cell culture to conventional microplates but also provides a 3D structure for cell culture, demonstrating that paper can act as a viable alternative to plastic-based platforms. Concluding remarks and future perspectives In the period covered by this manuscript there has been increased use of paper to develop low-cost devices dedicated to diagnostic and clinical assays. Moreover, using paperbased platforms in many bioassays has been shown to provide comparable results to traditional platforms, including molecular assays, ELISA, nucleic acid-based assays, and cell based assays. Based on previous successful studies using paper, we feel that paper could provide an advantageous platform for accomplishing in vitro pre-compound screening steps, and could offer a solution to many economic obstacles inherent in the pharmaceutical industry. The high cost of drug development in the pharmaceutical industry impedes progress in generating novel drugs, especially in underdeveloped countries. Even in some developing countries, such as Taiwan, where there are excellent insurance and medical services, pharmaceutical companies are small and cannot always invest heavily in drug-discovery projects. Pharmaceutical companies have attempted to minimize cost and economize manpower with the use of automated high-throughput systems. However, such systems are still unattainable for small companies, especially given the material requirements. Moreover, it is still necessary to enhance accuracy and precision in high-throughput screening. One strategy to overcome such performance problems might be to leverage the hydrophobic and hydrophilic properties of patterned, wax-printed paper to guide a loading sample to the test zone in a high-throughput system. 8
Trends in Biotechnology January 2015, Vol. 33, No. 1
We believe that using paper as an assay platform to substitute for existing assay platforms would open up new possibilities in the pharmaceutical and biotechnological communities. Further, this approach offers great potential for drug development in the pharmaceutical industry, especially in less economically developed nations or in small pharmaceutical offices connected to healthcare centers. This could ultimately impact upon drug development and delivery for rare disorders in local communities, and improve healthcare for those currently suffering from diseases without drugs. Acknowledgments We would like to thank the National Science Council of Taiwan for financially supporting this research under Contract NSC 101-2628-E-007011-MY3 (to C.-M.C.).
References 1 Aronson, J.K. et al. (2012) Defining rewardable innovation in drug therapy. Nat. Rev. Drug Discov. 11, 253–254 2 Bleicher, K.H. et al. (2003) Hit and lead generation: beyond highthroughput screening. Nat. Rev. Drug Discov. 2, 369–378 3 Hughes, J.P. et al. (2011) Principles of early drug discovery. Br. J. Pharmacol. 162, 1239–1249 4 Paul, S.M. et al. (2010) How to improve R&D productivity: the pharmaceutical industry’s grand challenge. Nat. Rev. Drug Discov. 9, 203–214 5 Dickson, M. and Gagnon, J.P. (2004) Key factors in the rising cost of new drug discovery and development. Nat. Rev. Drug Discov. 3, 417–429 6 Rawlins, M.D. (2004) Cutting the cost of drug development? Nat. Rev. Drug Discov. 3, 360–364 7 Wang, S. et al. (2012) Paper-based chemiluminescence ELISA: lab-onpaper based on chitosan modified paper device and wax-screenprinting. Biosens. Bioelectron. 31, 212–218 8 Ellerbee, A.K. et al. (2009) Quantifying colorimetric assays in paperbased microfluidic devices by measuring the transmission of light through paper. Anal. Chem. 81, 8447–8452 9 Cheng, C.M. et al. (2010) Millimeter-scale contact printing of aqueous solutions using a stamp made out of paper and tape. Lab Chip 10, 3201–3205 10 Martinez, A.W. et al. (2010) Programmable diagnostic devices made from paper and tape. Lab Chip 10, 2499–2504 11 Vella, S.J. et al. (2012) Measuring markers of liver function using a micropatterned paper device designed for blood from a fingerstick. Anal. Chem. 84, 2883–2891 12 Lo, S.J. et al. (2013) Molecular-level dengue fever diagnostic devices made out of paper. Lab Chip 13, 2686–2692 13 Derda, R. et al. (2009) Paper-supported 3D cell culture for tissue-based bioassays. Proc. Natl. Acad. Sci. U.S.A. 106, 18457–18462 14 Derda, R. et al. (2011) Multizone paper platform for 3D cell cultures. PLoS ONE 6, e18940 15 Lutz, B. et al. (2013) Dissolvable fluidic time delays for programming multi-step assays in instrument-free paper diagnostics. Lab Chip 13, 2840–2847 16 Cheng, C.M. et al. (2010) Paper-based ELISA. Angew. Chem. Int. Ed. Engl. 49, 4771–4774 17 Pollock, N.R. et al. (2012) A paper-based multiplexed transaminase test for low-cost, point-of-care liver function testing. Sci. Transl. Med. 4, 152ra129 18 Wong, A.P. et al. (2008) Egg beater as centrifuge: isolating human blood plasma from whole blood in resource-poor settings. Lab Chip 8, 2032– 2037 19 Tsai, T.T. et al. (2013) Paper-based tuberculosis diagnostic devices with colorimetric gold nanoparticles. Sci. Technol. Adv. Mater. 14, 044404 20 Yetisen, A.K. et al. (2013) Paper-based microfluidic point-of-care diagnostic devices. Lab Chip 13, 2210–2251 21 Hu, J. et al. (2014) Advances in paper-based point-of-care diagnostics. Biosens. Bioelectron. 54, 585–597 22 Martinez, A.W. et al. (2010) Diagnostics for the developing world: microfluidic paper-based analytical devices. Anal. Chem. 82, 3–10
Opinion 23 Carrilho, E. et al. (2009) Paper microzone plates. Anal. Chem. 81, 5990– 5998 24 Martinez, A.W. et al. (2008) Three-dimensional microfluidic devices fabricated in layered paper and tape. Proc. Natl. Acad. Sci. U.S.A. 105, 19606–19611 25 Deiss, F. et al. (2013) Platform for high-throughput testing of the effect of soluble compounds on 3D cell cultures. Anal. Chem. 85, 8085–8094 26 Hsu, M.Y. et al. (2014) Monitoring the VEGF level in aqueous humor of patients with ophthalmologically relevant diseases via ultrahigh sensitive paper-based ELISA. Biomaterials 35, 3729–3735 27 Wang, H.K. et al. (2014) Cellulose-based diagnostic devices for diagnosing serotype-2 dengue Fever in human serum. Adv. Healthc. Mater. 3, 187–196 28 Dungchai, W. et al. (2009) Electrochemical detection for paper-based microfluidics. Anal. Chem. 81, 5821–5826 29 Yamada, K. et al. (2014) An antibody-free microfluidic paper-based analytical device for the determination of tear fluid lactoferrin by fluorescence sensitization of Tb3+. Analyst 139, 1637–1643 30 Hsu, C.K. et al. (2014) Paper-based ELISA for the detection of autoimmune antibodies in body fluid-the case of bullous pemphigoid. Anal. Chem. 86, 4605–4610 31 Yager, P. et al. (2006) Microfluidic diagnostic technologies for global public health. Nature 442, 412–418
Trends in Biotechnology January 2015, Vol. 33, No. 1
32 Klasner, S.A. et al. (2010) Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva. Anal. Bioanal. Chem. 397, 1821–1829 33 Martinez, A.W. (2011) Microfluidic paper-based analytical devices: from POCKET to paper-based ELISA. Bioanalysis 3, 2589–2592 34 Laurie, D. et al. (1989) A rapid, qualitative ELISA test for the specific detection of morphine in serum or urine. Clin. Chim. Acta 183, 183–195 35 Rajkowski, K.M. et al. (1989) A competitive microtitre plate enzyme immunoassay for plasma testosterone using polyclonal antitestosterone immunoglobulins. Clin. Chim. Acta 183, 197–206 36 Nie, J. et al. (2012) Low-cost fabrication of paper-based microfluidic devices by one-step plotting. Anal. Chem. 84, 6331–6335 37 Mu, X. et al. (2014) Multiplex microfluidic paper-based immunoassay for the diagnosis of hepatitis C virus infection. Anal. Chem. 86, 5338–5344 38 Murdock, R.C. et al. (2013) Optimization of a paper-based ELISA for a human performance biomarker. Anal. Chem. 85, 11634–11642 39 Deiss, F. et al. (2014) Flow-through synthesis on Teflon-patterned paper to produce peptide arrays for cell-based assays. Angew. Chem. Int. Ed. Engl. 53, 6374–6377 40 Funes-Huacca, M. et al. (2012) Portable self-contained cultures for phage and bacteria made of paper and tape. Lab Chip 12, 4269–4278 41 Deiss, F. et al. (2014) Antimicrobial susceptibility assays in paperbased portable culture devices. Lab Chip 14, 167–171
9
