HHS Public Access Author manuscript Author Manuscript
Expert Rev Mol Diagn. Author manuscript; available in PMC 2017 July 26. Published in final edited form as: Expert Rev Mol Diagn. 2017 April ; 17(4): 351–366. doi:10.1080/14737159.2017.1285228.
Paper-based analytical devices for clinical diagnosis: recent advances in the fabrication techniques and sensing mechanisms Mazhar Shera,b, Rachel Zhuangb, Utkan Demircic,d, and Waseem Asghara,b,e aComputer
Engineering & Electrical Engineering and Computer Science Lab, Florida Atlantic University, Boca Raton, FL, USA
Author Manuscript
bAsghar-Lab,
Micro and Nanotechnology for Medicine, College of Engineering and Computer Science, Boca Raton, FL, USA
cThe
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford School of Medicine, Palo Alto, CA, USA
dDepartment
of Electrical Engineering (by courtesy), Stanford University, Stanford, CA 94305,
USA eDepartment
of Biological Sciences, Florida Atlantic University, Boca Raton, FL, USA
Abstract
Author Manuscript
Introduction—There is a significant interest in developing inexpensive portable biosensing platforms for various applications including disease diagnostics, environmental monitoring, food safety, and water testing at the point-of-care (POC) settings. Current diagnostic assays available in the developed world require sophisticated laboratory infrastructure and expensive reagents. Hence, they are not suitable for resource-constrained settings with limited financial resources, basic health infrastructure, and few trained technicians. Cellulose and flexible transparency paper-based analytical devices have demonstrated enormous potential for developing robust, inexpensive and portable devices for disease diagnostics. These devices offer promising solutions to disease management in resource-constrained settings where the vast majority of the population cannot afford expensive and highly sophisticated treatment options.
Author Manuscript
Areas covered—In this review, the authors describe currently developed cellulose and flexible transparency paper-based microfluidic devices, device fabrication techniques, and sensing technologies that are integrated with these devices. The authors also discuss the limitations and challenges associated with these devices and their potential in clinical settings. Expert commentary—In recent years, cellulose and flexible transparency paper-based microfluidic devices have demonstrated the potential to become future healthcare options despite a few limitations such as low sensitivity and reproducibility.
CONTACT: Utkan Demirci [email protected]; Waseem Asghar [email protected]. Declaration of interest U. Demirci is a founder of, and has an equity interest in: (i) DxNow Inc., a company that is developing microfluidic and imaging technologies for point-of-care diagnostic solutions, and (ii) Koek Biotech, a company that is developing microfluidic IVF technologies for clinical solutions. U. Demerci’s interests were viewed and managed in accordance with the conflict of interest policies. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Sher et al.
Page 2
Author Manuscript
Keywords Point-of-care diagnostics; cellulose paper-based analytical devices; flexible transparency paperbased analytical devices; microfluidics; biosensors; device fabrication
1. Introduction
Author Manuscript
Clinical diagnostics makes up the first and most important step in the treatment of a disease. Around 5.8 billion people worldwide live in settings categorized as low and middle income [1] and do not have access to the expensive medical facilities required for current immunoassays and other disease detection technologies [2,3]. Therefore, it is very important to develop inexpensive and portable point-of-care (POC) devices that can provide rapid and accurate results. The World Health Organization (WHO) has provided essential guidelines for future diagnostic devices using the ASSURED acronym, which stands for affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable [4]. POC diagnostics provide a promising solution that can meet these set parameters with numerous advantages, including affordability, sustainability, portability, disposability, simplicity, and ability to handle very small volumes of unprocessed samples, such as blood, urine, and saliva [5]. The field of microfluidics has witnessed great developments during the last decade and these advancements have played a key role to design various POC devices for resource-constrained settings [6–12].
Author Manuscript
Microfluidic paper-based analytical devices (μPADs) are ideal candidates for POC diagnostic purposes, especially in personalized health care (Figure 1(a,b)). These devices are also well-suited for the food industry, security, and environmental monitoring applications [13,14]. A μPAD consists of paper, which is hydrophilic in nature and allows hydrophobic demarcations to be made with various polymers. A camera-enabled phone can be utilized with μPADs (Figure 1(c,d)), and collected data and images can then be transmitted using the wireless communications networks to centralized laboratories for analyses and obtaining results in real-time [15,16].
Author Manuscript
Paper is one of the promising materials for making bioanalytical devices because of its characteristics like affordability, wide availability, and hydrophilic nature, which allow various solutions to flow through its porous structure via capillary action. In this review, we discuss various device fabrication and development methods currently employed for making μPADs. We also describe the sensing technologies that are integrated with μPADs. The challenges associated with paper-based diagnostic devices that limit their clinical applicability are also considered and future directions are highlighted.
2. Paper-based devices The paper-based dipstick assays are well-known; used to detect glucose in urine [18]. Urine is introduced to dipstick and the resulting color change is compared with standard chart to estimate the glucose levels [19]. Latex agglutination and radio-immunoassays were also developed [20], followed by the introduction of lateral flow technology that was utilized in
Expert Rev Mol Diagn. Author manuscript; available in PMC 2017 July 26.
Sher et al.
Page 3
Author Manuscript
human pregnancy tests. With the introduction of this home diagnostic tests, lateral flow assays (LFA) gained wide popularity [21]. Serological immunoassays based on lateral flow mechanisms are also developed and find applications in many areas including food safety, environmental monitoring, and veterinary diagnostics [19,22]. LFAs show poor detection limits and low sensitivities that confine their applications. To overcome these limitations, efforts are being made. For an instance, wax pillars have been used as delay barriers to improve the sensitivity of LFA (Figure 2(a)) [23]. The fabricated device was utilized for the detection of Human Immunoglobin G (HIgG) and improved the sensitivity by three times as compared to the conventional LFA devices.
3. Paper-based microfluidic devices
Author Manuscript
Paper has been recently utilized as a substrate to fabricate various microfluidic devices [25]. Paper is inexpensive, lightweight, easily available everywhere, and disposable. It is biocompatible with various biological samples [26]. Table 1 provides detailed information about various paper substrates, listing their important characteristics and applications. In one example, chromatography paper was utilized for the detection of proteins and glucose in artificial urine samples by creating hydrophobic lines with photoresist, on hydrophilic paper (Figure 3) [27]. A single μPAD can also be used to conduct various bioassays simultaneously [28]. Examples include the detection of uric acid and nitrate in the saliva [29].
4. Fabrication techniques of μPADS
Author Manuscript
Various μPAD device fabrication methods, such as photolithography [27,37], inkjet printing [33,38], polydimethylsiloxane (PDMS) plotting [39], wax printing [40–42], wax dipping [43], wax screen printing [42,44], and plasma treatment [45], have been proposed for a costeffective, simple, and portable product [25]. The pros and cons of each fabrication method are listed in Table 2. 4.1. Photolithography
Author Manuscript
Photolithography utilizes a photoresist to fabricate paper-based microfluidic devices. The masks required for photolithography can be prepared using an inkjet printer, a photocopying machine, or even hand drawn. The exposure can be accomplished using a UV lamp [37]. Although photolithography is often used in making microfluidic devices, photolithography is still quite expensive and requires access to clean room facilities [58]. A more convenient way of fabricating gold patterns on silicon substrates has been reported [59]. Using this method, patterning and etching of gold particles is quite simple, once a rubber stamp is fabricated. This technique does not require access to cleanroom facilities and other photolithography equipment [59]. Various soft lithography techniques also were developed for the cost-effective fabrication of microfluidic devices such as maskless lithography and phase shift photolithography [60–63]. Soft lithography uses elastomeric materials to fabricate pattern transfer elements and it can be used to pattern complex biochemicals based on embossing, molding, and printing [64].
Expert Rev Mol Diagn. Author manuscript; available in PMC 2017 July 26.
Sher et al.
Page 4
4.2. Inkjet printing
Author Manuscript
Inkjet printing method has been utilized for microfluidic device fabrication for simultaneous detection of glucose, proteins, and pH [38]. Micropatterns were fabricated on paper surfaces using 10 repeated toluene printing cycles. The flow channels were 550 μm wide while the sensing areas were 1.5 mm × 1.5 mm squares. The distinct feature of inkjet printing is that the entire fabrication process relies only on the inkjet printing equipment. A new concept has been demonstrated to create paper-based sensors by generating the hydrophilichydrophobic contrast on paper. This technique is quite useful for the large-scale production of paper-based sensors [33], as the use of polymers to define channels is costly and expensive for large-scale fabrication. 4.3. PDMS plotting
Author Manuscript
PDMS has been utilized over its less flexible counterparts (SU-8) and poly(methyl methacrylate) (PMMA) [39]. A modified plotter is able to fabricate channels of minimum width 1 mm using PDMS dissolved in hexanes. Although this process does not require access to cleanroom, organic reagents, photolithography equipment, and expensive photoresist, it uses custom-built plotters and cannot produce structures at resolutions as high as photolithography. 4.4. Laser cutting
Author Manuscript
The CO2 laser cutter has been widely used to cut the paper, PMMA, and double-sided adhesive (DSA) to fabricate microfluidic devices [2,3,16,49–53]. A simple and inexpensive method was presented for the fabrication of microfluidic devices (Figure 4(a)) [3]. Laser cutter was used to cut the PMMA and DSA as per specific patterns. It is a faster, inexpensive, and efficient process to assemble microfluidic devices. The fabricated device was used to capture CD4 cells from whole blood samples. In another study, a μPAD was developed using laser cutting process [65]. Chromatography paper backed by aluminum foil was used to create small features. The performance of the fabricated device was demonstrated by conducting a glucose test using 2 μL of artificial urine sample. Recently, laser cutting process was used to fabricate a microchip for the diagnosis of tuberculosis (TB) [66]. This proposed microchip-based TB Enzyme-Linked Immunosorbent Assay (ELISA) (MTBE) is a rapid, inexpensive, flow-less, and magnetic actuated platform. It was demonstrated that the assay time can be reduced to almost 15 min while keeping the detection efficiency comparable to standard classical ELISA. 4.5. Laser printing
Author Manuscript
Microfluidic devices can be fabricated on polyester sheets by combining laser printing and laminating processes (Figure 4(b)) [54]. The laser printer deposits toner layers on transparency sheets, with white regions representing the microfluidic channels. The laminating process then seals the microfluidic channels. This process can be utilized to produce multiple devices on a single sheet using a laser printer, paper driller, and laminator. A technique was presented for the fabrication of polyester-toner microfluidic device [67]. The proposed device was utilized for the dynamic solid-phase extraction of DNA. The microfluidic devices prepared using laser printing process have shown great potential for Expert Rev Mol Diagn. Author manuscript; available in PMC 2017 July 26.
Sher et al.
Page 5
Author Manuscript
various clinical assays including dengue diagnosis [68], detection and quantification of glucose in biological samples [69], and PCR amplification of DNA [70]. A toner-based microfluidic device was demonstrated to accurately perform simultaneous detection and quantification of multiple analytes including total proteins, glucose, cholesterol, and triglycerides in biological fluids [71]. 4.6. Wax printing
Author Manuscript
Wax printing for the fabrication of μPADs offers numerous advantages such as lower production costs, disposability, non-toxicity, ease of fabrication, and few steps for mass production. Wax channels can be printed on paper that can confine and direct samples and reagents, so simple paper-based microfluidic devices can be formed. Therefore, this method serves as a better approach for creating paper-based micro-fluidic devices [34]. This process can be accomplished either using a wax pen or printers for large-scale production. However, the utilization of expensive wax printer and heating equipment, along with lower resolution of fabricated devices, are the main drawbacks and limitations of this wax printing technique [43]. A simple and inexpensive process of wax printing was demonstrated utilizing hot plate and printer for the large-scale production of paper-based microfluidic devices [41]. The smallest microfluidic channel with average width of 561 ± 45 μm was fabricated using this technique.
Author Manuscript
A simpler, faster, and more cost-effective wax-patterning method was demonstrated for microfluidic device fabrication [40]. It consists of printing and baking. A nitrocellulose (NC) membrane was used as a paper substrate, which is the most suitable for protein immobilization applications. The distinct features of a NC membrane, such as its smaller and uniform pore size, make the wax penetration process slower and more precisely controllable during baking. This allows preparation of up to 100-μm-wide microchannels, at a resolution comparable to that of photolithography, without the use of harmful organic reagents. The manufactured device was tested for purification of samples and protein immobilization applications. The results proved that wax-patterned NC membrane can be utilized for the purification of micrometer size impurities, such as beads and cells. The fabricated device using this method was also used in dot immunoassay.
Author Manuscript
Recently, wax-ink valves were printed onto cellulose and nitrocellulose membranes using wax-printing technique (Figure 5) [72]. These valves were thermally actuated using thinfilm heater to control the release of fluids. The incorporation of thermally actuated wax-ink valves transformed current existing Lateral Flow Immunoassays (LFIA) into tunable, semiautomated, and multistep LFIA with improved limit of detection (LOD). The enhanced LFIA strips were tested for Escherichia coli (E. coli) assay. The results indicated that both 5.5×105 and 5.5 ×106 E. coli cells/mL were detectable by human eye with 6 times greater intensity than the standard LFIA strip. In one other example, micro-a-fluidics ELISA (mELISA) platform was developed using mineral oil to make valves [52]. The fabricated microfluidic chip was purposely designed to have five circular chambers connected with five elliptical chambers. Elliptical chambers were filled with mineral oil to physically separate stationary liquids/reagents in circular chambers. A droplet of blood along with antibodyfunctionalized magnetic nanoparticles was loaded onto the microfluidic chip. The device
Expert Rev Mol Diagn. Author manuscript; available in PMC 2017 July 26.
Sher et al.
Page 6
Author Manuscript
was placed on a permanent magnet fixed on a motorized stage. The movement of stage was controlled using a software program to perform m-ELISA. The device was used for rapid CD4 cell count at POC settings. Recently, a similar kind of device was proposed for the detection of tuberculosis [66]. 4.7. Wax dipping
Author Manuscript
Wax dipping is also a faster and less expensive technique compared to photolithography for making microfluidic channels on paper. It consists of only wax dipping and channels can be fabricated in less than a minute via successive dipping and ordinary heating arrangements (Figure 6) [43]. The melted wax is utilized to coat hydrophobic barriers, and an iron mold is used to protect the hydrophilic channel. The iron mold is temporarily placed onto the paper using magnetic field of a permanent magnet. When this assembly is dipped into molten wax, it absorbs the wax while the iron mold prevents its penetration. The actual width of the fabricated microfluidic channel is determined by the width of iron mold utilized. 4.8. Movable type wax printing method
Author Manuscript
An equipment free wax printing method was presented for the fabrication of μPADs using a hot plate and movable parts to make various patterns on paper [73]. The proposed method does not require any technical expertise to fabricate a micro-fluidic device with repeatable accuracy and resolution. The process consists of three steps. Initially, moveable parts are assembled in the specific pattern on an iron supporting substrate. This is done when the support’s magnetic field is turned off. Second, these patterned parts are coated with molten wax heated via hot plate. This process is carried out in the presence of the support’s magnetic field. In the third step, this hot stamp is utilized to print wax patterns on the substrate. The molten wax eventually penetrates into the paper, and desired patterns are printed (Figure 7(a)). A hand-held and lightweight stamp has been developed for device fabrication [74]. Initially, the surface of native paper (n-paper) was oxidized. Then, paraffined paper (p-paper) was placed over the surface of n-paper. The final step involved the placement of a preheated stainless steel stamp on p-paper to thermally transfer paraffin to n-paper, and thus 3 mm micro-fluidic channels were formed on n-paper (Figure 7(b)). 4.9. Wax screen printing
Author Manuscript
Wax screen printing is also a simple, cost-effective, and fast method for the fabrication of μPADs. Initially, a mask is designed using computer software programs and printed on a transparency film. This transparency sheet is converted to various screens [44]. Wax is rubbed through the screen, onto the filter paper. Wax can be melted using a hot plate. The molten wax is absorbed by the paper and thus the hydrophobic barriers are printed. After cooling for about 10 s at room temperature, the fabricated device is ready to use. This method forgoes the need for expensive wax printers (~$2500 US) by using very inexpensive wax screens (
Expert Rev Mol Diagn. Author manuscript; available in PMC 2017 July 26. Published in final edited form as: Expert Rev Mol Diagn. 2017 April ; 17(4): 351–366. doi:10.1080/14737159.2017.1285228.
Paper-based analytical devices for clinical diagnosis: recent advances in the fabrication techniques and sensing mechanisms Mazhar Shera,b, Rachel Zhuangb, Utkan Demircic,d, and Waseem Asghara,b,e aComputer
Engineering & Electrical Engineering and Computer Science Lab, Florida Atlantic University, Boca Raton, FL, USA
Author Manuscript
bAsghar-Lab,
Micro and Nanotechnology for Medicine, College of Engineering and Computer Science, Boca Raton, FL, USA
cThe
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford School of Medicine, Palo Alto, CA, USA
dDepartment
of Electrical Engineering (by courtesy), Stanford University, Stanford, CA 94305,
USA eDepartment
of Biological Sciences, Florida Atlantic University, Boca Raton, FL, USA
Abstract
Author Manuscript
Introduction—There is a significant interest in developing inexpensive portable biosensing platforms for various applications including disease diagnostics, environmental monitoring, food safety, and water testing at the point-of-care (POC) settings. Current diagnostic assays available in the developed world require sophisticated laboratory infrastructure and expensive reagents. Hence, they are not suitable for resource-constrained settings with limited financial resources, basic health infrastructure, and few trained technicians. Cellulose and flexible transparency paper-based analytical devices have demonstrated enormous potential for developing robust, inexpensive and portable devices for disease diagnostics. These devices offer promising solutions to disease management in resource-constrained settings where the vast majority of the population cannot afford expensive and highly sophisticated treatment options.
Author Manuscript
Areas covered—In this review, the authors describe currently developed cellulose and flexible transparency paper-based microfluidic devices, device fabrication techniques, and sensing technologies that are integrated with these devices. The authors also discuss the limitations and challenges associated with these devices and their potential in clinical settings. Expert commentary—In recent years, cellulose and flexible transparency paper-based microfluidic devices have demonstrated the potential to become future healthcare options despite a few limitations such as low sensitivity and reproducibility.
CONTACT: Utkan Demirci [email protected]; Waseem Asghar [email protected]. Declaration of interest U. Demirci is a founder of, and has an equity interest in: (i) DxNow Inc., a company that is developing microfluidic and imaging technologies for point-of-care diagnostic solutions, and (ii) Koek Biotech, a company that is developing microfluidic IVF technologies for clinical solutions. U. Demerci’s interests were viewed and managed in accordance with the conflict of interest policies. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Sher et al.
Page 2
Author Manuscript
Keywords Point-of-care diagnostics; cellulose paper-based analytical devices; flexible transparency paperbased analytical devices; microfluidics; biosensors; device fabrication
1. Introduction
Author Manuscript
Clinical diagnostics makes up the first and most important step in the treatment of a disease. Around 5.8 billion people worldwide live in settings categorized as low and middle income [1] and do not have access to the expensive medical facilities required for current immunoassays and other disease detection technologies [2,3]. Therefore, it is very important to develop inexpensive and portable point-of-care (POC) devices that can provide rapid and accurate results. The World Health Organization (WHO) has provided essential guidelines for future diagnostic devices using the ASSURED acronym, which stands for affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable [4]. POC diagnostics provide a promising solution that can meet these set parameters with numerous advantages, including affordability, sustainability, portability, disposability, simplicity, and ability to handle very small volumes of unprocessed samples, such as blood, urine, and saliva [5]. The field of microfluidics has witnessed great developments during the last decade and these advancements have played a key role to design various POC devices for resource-constrained settings [6–12].
Author Manuscript
Microfluidic paper-based analytical devices (μPADs) are ideal candidates for POC diagnostic purposes, especially in personalized health care (Figure 1(a,b)). These devices are also well-suited for the food industry, security, and environmental monitoring applications [13,14]. A μPAD consists of paper, which is hydrophilic in nature and allows hydrophobic demarcations to be made with various polymers. A camera-enabled phone can be utilized with μPADs (Figure 1(c,d)), and collected data and images can then be transmitted using the wireless communications networks to centralized laboratories for analyses and obtaining results in real-time [15,16].
Author Manuscript
Paper is one of the promising materials for making bioanalytical devices because of its characteristics like affordability, wide availability, and hydrophilic nature, which allow various solutions to flow through its porous structure via capillary action. In this review, we discuss various device fabrication and development methods currently employed for making μPADs. We also describe the sensing technologies that are integrated with μPADs. The challenges associated with paper-based diagnostic devices that limit their clinical applicability are also considered and future directions are highlighted.
2. Paper-based devices The paper-based dipstick assays are well-known; used to detect glucose in urine [18]. Urine is introduced to dipstick and the resulting color change is compared with standard chart to estimate the glucose levels [19]. Latex agglutination and radio-immunoassays were also developed [20], followed by the introduction of lateral flow technology that was utilized in
Expert Rev Mol Diagn. Author manuscript; available in PMC 2017 July 26.
Sher et al.
Page 3
Author Manuscript
human pregnancy tests. With the introduction of this home diagnostic tests, lateral flow assays (LFA) gained wide popularity [21]. Serological immunoassays based on lateral flow mechanisms are also developed and find applications in many areas including food safety, environmental monitoring, and veterinary diagnostics [19,22]. LFAs show poor detection limits and low sensitivities that confine their applications. To overcome these limitations, efforts are being made. For an instance, wax pillars have been used as delay barriers to improve the sensitivity of LFA (Figure 2(a)) [23]. The fabricated device was utilized for the detection of Human Immunoglobin G (HIgG) and improved the sensitivity by three times as compared to the conventional LFA devices.
3. Paper-based microfluidic devices
Author Manuscript
Paper has been recently utilized as a substrate to fabricate various microfluidic devices [25]. Paper is inexpensive, lightweight, easily available everywhere, and disposable. It is biocompatible with various biological samples [26]. Table 1 provides detailed information about various paper substrates, listing their important characteristics and applications. In one example, chromatography paper was utilized for the detection of proteins and glucose in artificial urine samples by creating hydrophobic lines with photoresist, on hydrophilic paper (Figure 3) [27]. A single μPAD can also be used to conduct various bioassays simultaneously [28]. Examples include the detection of uric acid and nitrate in the saliva [29].
4. Fabrication techniques of μPADS
Author Manuscript
Various μPAD device fabrication methods, such as photolithography [27,37], inkjet printing [33,38], polydimethylsiloxane (PDMS) plotting [39], wax printing [40–42], wax dipping [43], wax screen printing [42,44], and plasma treatment [45], have been proposed for a costeffective, simple, and portable product [25]. The pros and cons of each fabrication method are listed in Table 2. 4.1. Photolithography
Author Manuscript
Photolithography utilizes a photoresist to fabricate paper-based microfluidic devices. The masks required for photolithography can be prepared using an inkjet printer, a photocopying machine, or even hand drawn. The exposure can be accomplished using a UV lamp [37]. Although photolithography is often used in making microfluidic devices, photolithography is still quite expensive and requires access to clean room facilities [58]. A more convenient way of fabricating gold patterns on silicon substrates has been reported [59]. Using this method, patterning and etching of gold particles is quite simple, once a rubber stamp is fabricated. This technique does not require access to cleanroom facilities and other photolithography equipment [59]. Various soft lithography techniques also were developed for the cost-effective fabrication of microfluidic devices such as maskless lithography and phase shift photolithography [60–63]. Soft lithography uses elastomeric materials to fabricate pattern transfer elements and it can be used to pattern complex biochemicals based on embossing, molding, and printing [64].
Expert Rev Mol Diagn. Author manuscript; available in PMC 2017 July 26.
Sher et al.
Page 4
4.2. Inkjet printing
Author Manuscript
Inkjet printing method has been utilized for microfluidic device fabrication for simultaneous detection of glucose, proteins, and pH [38]. Micropatterns were fabricated on paper surfaces using 10 repeated toluene printing cycles. The flow channels were 550 μm wide while the sensing areas were 1.5 mm × 1.5 mm squares. The distinct feature of inkjet printing is that the entire fabrication process relies only on the inkjet printing equipment. A new concept has been demonstrated to create paper-based sensors by generating the hydrophilichydrophobic contrast on paper. This technique is quite useful for the large-scale production of paper-based sensors [33], as the use of polymers to define channels is costly and expensive for large-scale fabrication. 4.3. PDMS plotting
Author Manuscript
PDMS has been utilized over its less flexible counterparts (SU-8) and poly(methyl methacrylate) (PMMA) [39]. A modified plotter is able to fabricate channels of minimum width 1 mm using PDMS dissolved in hexanes. Although this process does not require access to cleanroom, organic reagents, photolithography equipment, and expensive photoresist, it uses custom-built plotters and cannot produce structures at resolutions as high as photolithography. 4.4. Laser cutting
Author Manuscript
The CO2 laser cutter has been widely used to cut the paper, PMMA, and double-sided adhesive (DSA) to fabricate microfluidic devices [2,3,16,49–53]. A simple and inexpensive method was presented for the fabrication of microfluidic devices (Figure 4(a)) [3]. Laser cutter was used to cut the PMMA and DSA as per specific patterns. It is a faster, inexpensive, and efficient process to assemble microfluidic devices. The fabricated device was used to capture CD4 cells from whole blood samples. In another study, a μPAD was developed using laser cutting process [65]. Chromatography paper backed by aluminum foil was used to create small features. The performance of the fabricated device was demonstrated by conducting a glucose test using 2 μL of artificial urine sample. Recently, laser cutting process was used to fabricate a microchip for the diagnosis of tuberculosis (TB) [66]. This proposed microchip-based TB Enzyme-Linked Immunosorbent Assay (ELISA) (MTBE) is a rapid, inexpensive, flow-less, and magnetic actuated platform. It was demonstrated that the assay time can be reduced to almost 15 min while keeping the detection efficiency comparable to standard classical ELISA. 4.5. Laser printing
Author Manuscript
Microfluidic devices can be fabricated on polyester sheets by combining laser printing and laminating processes (Figure 4(b)) [54]. The laser printer deposits toner layers on transparency sheets, with white regions representing the microfluidic channels. The laminating process then seals the microfluidic channels. This process can be utilized to produce multiple devices on a single sheet using a laser printer, paper driller, and laminator. A technique was presented for the fabrication of polyester-toner microfluidic device [67]. The proposed device was utilized for the dynamic solid-phase extraction of DNA. The microfluidic devices prepared using laser printing process have shown great potential for Expert Rev Mol Diagn. Author manuscript; available in PMC 2017 July 26.
Sher et al.
Page 5
Author Manuscript
various clinical assays including dengue diagnosis [68], detection and quantification of glucose in biological samples [69], and PCR amplification of DNA [70]. A toner-based microfluidic device was demonstrated to accurately perform simultaneous detection and quantification of multiple analytes including total proteins, glucose, cholesterol, and triglycerides in biological fluids [71]. 4.6. Wax printing
Author Manuscript
Wax printing for the fabrication of μPADs offers numerous advantages such as lower production costs, disposability, non-toxicity, ease of fabrication, and few steps for mass production. Wax channels can be printed on paper that can confine and direct samples and reagents, so simple paper-based microfluidic devices can be formed. Therefore, this method serves as a better approach for creating paper-based micro-fluidic devices [34]. This process can be accomplished either using a wax pen or printers for large-scale production. However, the utilization of expensive wax printer and heating equipment, along with lower resolution of fabricated devices, are the main drawbacks and limitations of this wax printing technique [43]. A simple and inexpensive process of wax printing was demonstrated utilizing hot plate and printer for the large-scale production of paper-based microfluidic devices [41]. The smallest microfluidic channel with average width of 561 ± 45 μm was fabricated using this technique.
Author Manuscript
A simpler, faster, and more cost-effective wax-patterning method was demonstrated for microfluidic device fabrication [40]. It consists of printing and baking. A nitrocellulose (NC) membrane was used as a paper substrate, which is the most suitable for protein immobilization applications. The distinct features of a NC membrane, such as its smaller and uniform pore size, make the wax penetration process slower and more precisely controllable during baking. This allows preparation of up to 100-μm-wide microchannels, at a resolution comparable to that of photolithography, without the use of harmful organic reagents. The manufactured device was tested for purification of samples and protein immobilization applications. The results proved that wax-patterned NC membrane can be utilized for the purification of micrometer size impurities, such as beads and cells. The fabricated device using this method was also used in dot immunoassay.
Author Manuscript
Recently, wax-ink valves were printed onto cellulose and nitrocellulose membranes using wax-printing technique (Figure 5) [72]. These valves were thermally actuated using thinfilm heater to control the release of fluids. The incorporation of thermally actuated wax-ink valves transformed current existing Lateral Flow Immunoassays (LFIA) into tunable, semiautomated, and multistep LFIA with improved limit of detection (LOD). The enhanced LFIA strips were tested for Escherichia coli (E. coli) assay. The results indicated that both 5.5×105 and 5.5 ×106 E. coli cells/mL were detectable by human eye with 6 times greater intensity than the standard LFIA strip. In one other example, micro-a-fluidics ELISA (mELISA) platform was developed using mineral oil to make valves [52]. The fabricated microfluidic chip was purposely designed to have five circular chambers connected with five elliptical chambers. Elliptical chambers were filled with mineral oil to physically separate stationary liquids/reagents in circular chambers. A droplet of blood along with antibodyfunctionalized magnetic nanoparticles was loaded onto the microfluidic chip. The device
Expert Rev Mol Diagn. Author manuscript; available in PMC 2017 July 26.
Sher et al.
Page 6
Author Manuscript
was placed on a permanent magnet fixed on a motorized stage. The movement of stage was controlled using a software program to perform m-ELISA. The device was used for rapid CD4 cell count at POC settings. Recently, a similar kind of device was proposed for the detection of tuberculosis [66]. 4.7. Wax dipping
Author Manuscript
Wax dipping is also a faster and less expensive technique compared to photolithography for making microfluidic channels on paper. It consists of only wax dipping and channels can be fabricated in less than a minute via successive dipping and ordinary heating arrangements (Figure 6) [43]. The melted wax is utilized to coat hydrophobic barriers, and an iron mold is used to protect the hydrophilic channel. The iron mold is temporarily placed onto the paper using magnetic field of a permanent magnet. When this assembly is dipped into molten wax, it absorbs the wax while the iron mold prevents its penetration. The actual width of the fabricated microfluidic channel is determined by the width of iron mold utilized. 4.8. Movable type wax printing method
Author Manuscript
An equipment free wax printing method was presented for the fabrication of μPADs using a hot plate and movable parts to make various patterns on paper [73]. The proposed method does not require any technical expertise to fabricate a micro-fluidic device with repeatable accuracy and resolution. The process consists of three steps. Initially, moveable parts are assembled in the specific pattern on an iron supporting substrate. This is done when the support’s magnetic field is turned off. Second, these patterned parts are coated with molten wax heated via hot plate. This process is carried out in the presence of the support’s magnetic field. In the third step, this hot stamp is utilized to print wax patterns on the substrate. The molten wax eventually penetrates into the paper, and desired patterns are printed (Figure 7(a)). A hand-held and lightweight stamp has been developed for device fabrication [74]. Initially, the surface of native paper (n-paper) was oxidized. Then, paraffined paper (p-paper) was placed over the surface of n-paper. The final step involved the placement of a preheated stainless steel stamp on p-paper to thermally transfer paraffin to n-paper, and thus 3 mm micro-fluidic channels were formed on n-paper (Figure 7(b)). 4.9. Wax screen printing
Author Manuscript
Wax screen printing is also a simple, cost-effective, and fast method for the fabrication of μPADs. Initially, a mask is designed using computer software programs and printed on a transparency film. This transparency sheet is converted to various screens [44]. Wax is rubbed through the screen, onto the filter paper. Wax can be melted using a hot plate. The molten wax is absorbed by the paper and thus the hydrophobic barriers are printed. After cooling for about 10 s at room temperature, the fabricated device is ready to use. This method forgoes the need for expensive wax printers (~$2500 US) by using very inexpensive wax screens (
