Paper diagnostics in biomedicine

Simple 2D PADs

The advent of patterned paper to create micro-channel designs has enabled multidirectional flow paper diagnostics. Martinez et al. (2007) patterned a simple device with three detection zones comprising indicators for glucose and protein, as well as a control zone (Figure 5). The “walls”, emplaced by hydrophobic boundaries patterned using photolithography, allowed fluid to be directed into the three different zones without cross-contamination, thus adding a spatial advantage against the conventional 1D-μPAD dipsticks.

Figure 5

Prototype design of a 2D paper-based microfluidic device that tests multiple analytes simultaneously; showing separate testing zones (T) connected to a single sample zone (S). [Redrawn from Martinez et al. (2007).]

Previous reviews have summarized patterning methods for diagnostic design. Such methods include: photolithography, plotting, inkjet or plasma etching, cutting, and wax printing (Martinez et al. 2010a). A summary of the patterning techniques analysis is presented in Table 3. The concept is to create hydrophobic barriers onto or within the paper structure and to rely on paper capillarity for liquid flow. While the original techniques created rigid and brittle barriers, technology has nicely progressed, allowing channels as narrow as 250 μm width to be created (Khan et al. 2010a). Common 2D-PAD test designs still lack the ability to perform multi-step assays for more complex applications and remain prone to contamination and fluid evaporation. However, 2D-PADs are very cheap (especially when manufactured by printing), easy to use, versatile, and robust.

Partially and fully enclosed PADs

A major disadvantage of the basic 2D-μPAD design is its exposed nature. Both sides of the paper devices are uncovered, resulting in the evaporation of reagents and samples, and risking contamination from the support beneath. Contact with the support can also result in loss of reagent and sample fluids. Early attempts to avoid such loss and contamination investigated adding samples and solutions onto the device held in mid-air, which is simply impractical (Schilling et al. 2012).

Fenton et al. (2008) proposed a method to avoid imbibing paper with hydrophobic/hydrophilic patterns. Instead, the paper was shaped into one of three desired 2D designs using a computer-controlled x-y knife plotter. Type 1 consisted of a single sheet of paper cut into the desired shape; for type 2, paper was mated with one layer of polyester cover tape before being cut; and for type 3, pre-cut paper was sandwiched between two layers of cover tape. Type 3 was the first example of a fully enclosed μPAD. Partially or fully enclosed PADs are reported to decrease the rates of operator error; however, this may simply be that paper cut into clearly labeled sections leaves no room for error interpretation (Fenton et al. 2008). The design is cheaper to fabricate and impervious to external contaminants.

Olkkonen et al. (2010) presented a partially enclosed device which flexographically printed a polystyrene xylene/toluene ink onto the back of the device, while the front was printed with the microfluidic channel design. The hydrophobic back layer provided a protective layer that prevented fluid from escaping through the back via contact with the underlying support and protected against contamination from the support. This method allows direct roll-to-roll production well suited for high throughput manufacture. The enclosed backing contributed to full penetration of the hydrophobic barrier, while protecting from contaminants and loss of fluids.

More recently, Schilling et al. (2012) investigated printing toner to produce fully enclosed μPADs. A thermally bonded thin plastic protective layer was printed onto paper. Similar laser printers and toners are commonly purchased for offices, and have been used extensively in the fabrication of μPADs. It is a cheap and convenient method of manufacturing μPADs. The toner had no effect on the microfluidic channels, since it does not diffuse into paper, nor does it come off when wetted. Printing four layers of toner could enclose the μPADs and resulted in faster wicking rates.

However, the heat required (180°C) during the laser printing method can affect the biological reagents that are affixed prior to printing and enclosed beneath the toner layer (Figure 6). At extreme heats, proteins undergo denaturation, lose their ability to bind to other molecules, and become inactive. Schilling et al. addressed this problem and reported a 90% decrease in enzymatic function using glucose oxidase for testing glucose concentration (Schilling et al. 2012). The decreased function was reported not to affect test sensitivity and to allow detection at concentrations as low as 1 mm. However, this raises serious concerns on optimization and economics, and questions as to why a 90% decrease in enzyme activity did not affect sensitivity. An alternative was explored where a 1 mm diameter hole was designed in the surface to act as a reagent addition port, therefore allowing the addition of reagents after enclosing the μPAD, allowing wicking to the reagent-storage zone (Figure 6). This resulted in no loss of reagent or enzyme function, and with the exception of the area of the port, the reagents were protected from the surrounding environment.

Figure 6

Example of a fully enclosed 2D paper diagnostic; showing separate testing zones (T) connected to a single sample zone (S). (Redrawn from Schilling et al. (2012).

PAD networks

A disadvantage of the “traditional” single sheet μPADs is their limitation to single-step processes. A single-step process is ideal for the user, but restricts its applicability, since most laboratory-based diagnostic assays involve multi-step processes. This is the case for multi-step assays, such as ELISA, which improve sensitivity and specificity with signal amplification and washing steps. 2D paper networks (2D-PNs) attempt to emulate multi-step assays, while using a single activation step, thus retaining the simplicity and affordability of the single-step μPADs.

Fu et al. (2010) developed a 2D-PN that retains the autonomous nature of μPADs, while allowing the complexity of multiple reagents to be delivered sequentially to a detection zone (Figure 7). The first reported 2D-PN used three methods that allowed multiple inlets to simultaneously converge toward a single point (Fu et al. 2010). All methods focused on varying the delivery time of the fluid by: 1) varying the length of paper, 2) varying its width, or 3) creating a dissolvable barrier (trehalose) to slow the liquid in its tracks. Factors, such as paper composition, pore size, and surface chemistry can also affect the fluid flow rate, but were not explored. Paper was patterned using a laser cutter to fabricate the device, much like Fenton et al. (2008), and was supported by double-sided tape on a glass substrate (Fenton et al. 2008, Fu et al. 2010). Absorbent pads were used at the inlets for reagent application, allowing the solutions to wick to the detection zone. Using dye and pH as examples, the 2D-PN design staggered the delivery of each component to a common site. Later studies used the same design for chemical signal amplification (Fu et al. 2010, Fu et al. 2011a). The transport mechanisms of fluid through the paper networks were also explored (Fu et al. 2011a). However, the effects of paper composition, pore size, and surface chemistry remained unaddressed. The added complexity of the 2D-PN design also increases the possibility of errors.

Figure 7

Example of a 2D paper network used for the sequential testing of multi-step analysis with a single activation point. [Redrawn from Fu et al. (2012).]

A method using a single fluid source, rather than an individual source for each of the reagents, was explored Fu et al. (2011b). It involved “programming” the device to disconnect each reagent in a particular order. The design used a single source well and shaped the 2D-PN by varying the length of each reagent segment. Isolated strips of different lengths were used to show the overall method. The paper strips were housed in a poly (methyl methacrylate) (PMMA) plastic casing to reduce evaporation and provide a support from which to mount the device into the source well. Plastic cartridges and wells were designed to receive a paper cartridge/strip inserted by the user. The paper strips are immersed at different depths into the fluid. The well depletes as fluid wicks paper, thereby disconnecting strips from the source well. This was controlled by: 1) the fluid depletion rate, 2) the immersion depths, and 3) the cross-sectional area of the fluid source. The design also relies on a large and thick paper strip used as a regulator; without it, the lengths of paper varied only slightly. The test is very sensitive to the cross-sectional area of paper. Thin paper strips of small cross-sections are often used, which increases variability due to the high heterogeneity of paper structure at different cross sections. Using these basic principles of managing fluid flow, the 2D-PNs can be programmed to deliver fluids to a common point and disconnect from the source well in a timed, sequential manner. This series of experiments used colored dyes, previously dried onto paper.

Origami

3D-μPADs can be constructed using origami, the art of paper folding. Liu and Crooks (2011) report a method using a single sheet of folded paper to design a 3D-μPAD (Figure 9). A piece of chromatography paper is patterned using a single photolithographic step. The design consists of micro-channels, reservoirs, and a frame. The frame provides the template for the paper folding and ensures correct alignment. When folded in a particular sequence, the micro-channels are matched to allow flow in both the lateral and vertical directions. The four corners are trimmed and fit in an aluminum clamp. Samples and solutions are injected through one of the four holes drilled into the clamp’s top plate. An advantage origami-PADs (o-PADs) have over the previous designs is the elimination of double-sided tape, which can diffuse into the paper over long periods of time and decrease the hydrophilicity of the micro-channels, along with assembly tools, such as the laser cutter. However, despite these advantages, origami PADs require an aluminum clamp to ensure alignment, which increases the cost of the device and decreases disposability.

Figure 9

Redrawn examples of origami microfluidic paper diagnostic for multi-step analysis designed by: (A) Govindarajan et al. (2011, 2012); and (B) Liu and Crooks (2011).

Govindarajan et al. (2011, 2012) described a type of 3D-μPAD which utilizes the origami principles (Figure 9) (Govindarajan et al. 2011, Govindarajan et al. 2012). However, it is used functionally rather than to simplify fabrication. The origami serves to sequence the complex processing steps, by folding the layers in succession to prepare collected DNA samples for molecular diagnosis. The design uses stacked layers of cellulose paper and double-sided tape for assembly, but also uses single-sided tape, a repositionable adhesive layer, and card paper to allow for bidirectional folding. Another difference is the way in which the device is constructed. Rather than shaping each layer individually before stacking, a laser cutter is used after stacking instead. The cutting step during fabrication serves as a method for patterning the fluid channels. This is achieved by a combination of full-pass and partial cuts in the device, before peeling away the adhesive layer, leaving fluid channels on a non-wicking substrate.

Methods of reporting

Detecting the presence of an analyte is half the challenge. Accurate and successful diagnosis also requires a reliable and simple method of relaying the result to the user. Currently, most PADs report results visually; some have attempted to incorporate electronics into the reporting process. This section provides an overview of the development and issues of the main technologies.

Biorecognition in paper diagnostics

Accurate diagnosis of a disease is not solely reliant on the reporting method, but also the ability to selectively detect the presence of specific biomolecules, also known as biorecognition. The human body has an abundance of distinct analytes which can be used for the diagnosis of not only pathogenic diseases, but also physiological disorders. It all hinges on finding the correct biomolecular target and developing methodologies to detect its presence. Biomolecules, such as nucleic acids, enzyme proteins, and antibodies, can be immobilized onto paper, and can reportedly be dried without denaturation for this purpose. This process allows for storage and use in remote areas, without access to laboratory facilities, while retaining the thermal stability that is often lost with analyte solutions not properly stored at low temperature. This ability to immobilize bimolecular targets upon paper thus provides the other crucial half of the detection process. There are four main methods of biomolecule immobilization on paper: 1) physical, 2) chemical, 3) biochemical couple, and 4) bioactive pigments; Pelton provided a detailed description of these techniques (Pelton 2009). A summary is provided in Table 4.

Perhaps the greatest challenge of achieving biorecognition is choosing the correct target that will detect a specific marker in the body. The job of this marker is to unequivocally detect the presence of a disease to avoid misdiagnosis of a patient. Whilst the principles of detection have been modeled after current diagnostic methods, the behavior of biomolecules on paper is not always congruent with the behavior exhibited in current laboratory methods.

There is no distinct advantage of a specific biomolecule over another, but is rather a function of the current methods of detection available. For example, utilizing the specificity of antibody-antigen interactions is desired for applications, such as blood typing, while recognition of a specific protein is required when diagnosing malaria. The type of biomolecular target will therefore differ from application to application.

Because of the wide array of types and properties of analytical targets, each must be examined separately to develop a robust detection method for the purpose of each device. The source of the analyte is an important factor in the diagnostic design. Samples can be primarily taken from urine, blood, and saliva for PADs, as they are already present in liquid form, but fecal matter is also a potential analytical source.

Paper micro-zone plates

Many medical applications rely on paper micro-zone plates (Carrilho et al. 2009b). Conventional micro-zone plates are usually made from plastic and consist of 96 or 384 wells, in which analytes can be deposited. The result is then determined quantitatively via absorbance or fluorescence measurements using a micro-plate reader. Paper micro-zone plates can be functionally similar to its plastic predecessor, but with all the familiar benefits of using a paper substrate: cheap, easy storage and disposal, small volume requirements, etc. Like plastic plates, the paper version can be designed with 96 or 384 detection zones using photolithography. Many paper types were found suitable for the micro-zone plate format (Carrilho et al. 2009b). The main limiting factor was paper thickness. If the thickness was too great, the entire depth of the paper could not be properly hydrophobized. Paper thickness defines the required volume of samples and the capacity for the hydrophilic zones. It can be improved through plasma oxidation (Carrilho et al. 2009b). In comparison to the plastic micro-zone plates, paper showed a 40-fold increased sensitivity when read using fluorescence. However, under absorbance mode, detection was limited by light scattering caused by paper. The addition of mineral oil or similar liquids matching the refraction index of cellulose allows accurate measurements by absorbance (Carrilho et al. 2009b). The 2D format of the paper micro-zone plates was ideal for telemedicine reporting methods and can perform serial dilutions – a benefit unachievable with the 1D-μPAD design. A unique advantage paper has over plastic is the ability to perform serial concentrations. A sample can be added, and then a known volume of the solution can be evaporated, thus removing the excess liquid and increasing the sample concentration.

Paper for sample preparation and storage

Rather than testing for a particular disease, paper has also been explored as a platform for preparing samples at POC. While paper for sample preparation has been mostly investigated for urine samples, it is applicable to any biofluid (Parker and Cubitt 1999). Parker and Cubitt (1999) developed a dried blood spot (DBS) device where blood samples could be taken, stored, and transported on paper (Parker and Cubitt 1999). The DBS card is an older concept and can collect blood samples using finger or heel pricks, which can then be reconstituted in the laboratory for examination. This is of interest for epidemiological studies in remote regions. Even when kept in conditions varying from cold storage to humid and tropical conditions, the samples were found to remain stable after many years on paper. Additional benefits are the significantly reduced infection risk, ease of use with minimal training, the elimination of needles and syringes, and storage at ambient temperature, removing refrigerated storage and transport requirements. This method was investigated for the surveillance and detection method of HIV (Parker and Cubitt 1999, Ayele et al. 2007). The interaction between paper and blood is poorly understood, but is critical for improving blood sample aging and preservation.

Sample separation

Not all μPADs report using colorimetry, one of the simplest methods. An alternative strategy is to separate analytes for detection (Carvalhal et al. 2010a). Carvalhal et al. (2010a) reported an electrochemically-coupled device that separates the analytes, uric acid (UA) and ascorbic acid (AA), and amperometrically detects their presence (Carvalhal et al. 2010a,b). Much like a chromatography column, eluent wicks paper by capillary action to dissolve the sample. The solubility is dependent on ionic strength. At an optimal pH lying between the pKa of AA and UA, the AA becomes ionized and more soluble. The type, thickness, and length of the paper control separation efficiency. Electrodes (gold) can be printed on paper for detection. The paper-based device compared to typical high pressure liquid column (HPLC) instrumentation has a slightly longer run time, 16 min compared to 5 min; however, the benefits of paper could outweigh the extended test time. This method could be developed not only for clinical diagnostics, but also for forensics, agriculture, and environment applications.

A potential μPAD application is the separation of red blood cells (RBC) from untreated whole blood, and integration with a colorimetric diagnostic test (Yang et al. 2012). Yang et al. (2012) described a method utilizing RBC agglutination principles using antibody-A,B to form aggregates too large to flow through the porous structure of the substrate (e.g., chromatography paper). However, the plasma constituents can still flow through the pores to adjacent testing terminals functionalized with reagents. Colorimetric detection for various compositional properties of the plasma can be achieved. This principle was demonstrated for measuring glucose concentration in blood. However, this test did not account for the blood group O population.

Blood group typing

The accurate, rapid and reliable blood typing of human blood, particularly during medical emergencies, is important (Hillyer 2007, Daniels and Bromilow 2007, Contreras and Daniels 2010a,b). Mismatched blood typing can lead to a hemolytic transfusion reaction which can be fatal. Traditionally, an individual’s blood type is determined by detecting the presence or absence of antigens on the surface of their RBCs, as well as the antibodies present within the blood serum, more specifically the blood plasma. The most commonly used methods for blood typing include the slide test, tube test, micro-plate method, gel column agglutination systems, thin layer chromatography (TLC)-immunostaining, fiber optic-microfluidic device, and spin tube method, etc. (Harmening 1999). The gel column test is the most prevalent in industry, however, it requires centrifugation, and each of these methods requires professionally trained personnel to be reliably analyzed. Developing a low-cost alternative for remote regions or home care which is user-friendly, portable, and equipment free, could provide a great alternative to the blood typing options currently available (Khan et al. 2010b).

In recent years, several studies have explored the possibility of using paper as a blood typing diagnostic tool (Khan et al. 2010b, Al-Tamimi et al. 2011, Li et al. 2012). Much like the traditional blood typing tests, the paper diagnostic also relies on the principles of hemagglutination.

When an aqueous analyte such as blood is tested using paper, the fluid is driven by capillary actions created by the structure of the cellulose fibers, wicking through the inter-fiber space of the paper (Khan et al. 2010b). This is known as capillarity microfluidics and allows for the passive transport of the analyte without any further equipment (Rivet et al. 2011). However, when blood agglutinates due to a specific antibody/antigen interaction, the resultant RBC complexes become too large to adequately transport through the paper structure and retain on paper; in the absence of agglutination, RBCs are easily removed by elution. This contrast in RBC retention or elution allows for the differentiation between when agglutination does occur (specific) and when it does not (non-specific) to directly report typing (Khan et al. 2010b). This has been the primary basis for the investigation of a paper-based diagnostic tool for blood typing.

Khan et al. (2010b) developed a method soaking paper strips in antibody solutions of either A, B or D antibodies. Droplets of blood were then added to the center of the paper strip and the resultant wicking behavior of the blood was observed. A distinct difference was discernible between agglutinated and non-agglutinated blood, the former presenting a chromatographic separation of the RBCs and plasma (Figure 10A). When RBCs agglutinate with the corresponding antibody, very little wicking is observed, while the plasma does wick along the paper strip. In contrast, non-agglutinated blood forms no separation along the paper strips, but travels uniformly, thus creating a distinction between a positive and negative result.

Figure 10

Use of paper for determining blood groups relying on antibody-antigen interactions to show agglutinated and non-agglutinated RBCs: (A) via wicking (Khan et al. 2010a); (B) chromatographically (Al-Tamimi et al. 2011); (C) using a blood spot test (Al-Tamimi et al. 2011); and (D) using the text-reporting paper substrate method (Li et al. 2012).

Al-Tamimi et al. (2011) next developed another paper-based diagnostic for blood typing using the same principles, but presenting two different testing methodologies. The first described an elution-based method using the chromatographic behavior of blood when added to paper (Figure 10B). This was achieved by spotting blood on the surface of the paper where known antibodies had previously been added. A TLC tank was used to elute the blood spot in saline buffer. The capillary action of the paper allowed for the buffer to travel through the paper structure, thus creating a chromatography-like test. Much like Khan et al. (2010b), the fixed agglutinated blood spots showed a positive result, while non-agglutinated elution paths of unbound RBC were negative. Visually, there was a distinct difference between the two results, using both the density of the blood spot and the presence or absence of an elution path to make an informed analysis.

A second method, known as the spot test (Figure 10C), also involved spotting the blood to stock antibodies upon the paper substrate; however they were not eluted in the TLC tank. Instead, saline solution was used to wash the spot directly by pipetting. Separation occurred by filtration through the paper thickness; RBC aggregated by matching antibodies remain on the top of paper, forming an intense red dot, while non-agglutinated cells (non-specific antibody) simply wash through paper and disappear. The results of both tests accurately detected the ABO and RhD blood groups of 100 samples, including four weak AB and four weak RhD samples, thus supporting the use of paper as a robust bio-diagnostic for blood typing. The effect of paper structure on blood typing performance was also investigated in a different study (Su et al. 2012).

Li et al. (2012) recently developed a text-reporting method. The detection principle is an adaptation of Al-Tamimi’s method (Al-Tamimi et al. 2011), relying on filtration through the paper thickness to retain RBC aggregated and coagulated by specific antibody interaction. By patterning the paper substrate with hydrophobic boundaries to surround a hydrophilic channel in the shape of text or symbols, “invisible writing” is present on the paper and dotted with antibodies. The addition of antibodies remains invisible to the naked eye until blood is added, when the shape of the channel is formed, for example an A or a B, and once washed with a saline solution – if a hemagglutination reaction has occurred – the resultant blood type will be directly printed on paper and can be “read” straight from the device (Figure 10D). This test successfully reported the ABO and RhD blood types of 99 samples including weak groups.

DNA extraction and detection

The genetic material of all living organisms is unique. There are well-established DNA detection techniques used for medical diagnosis of pathological diseases, such as the polymerase chain reaction (PCR) and rolling circle amplification (RCA) (Ali et al. 2009). While sensitive, these techniques require complex methodology and elaborate equipment infrastructure. Ali et al. (2009) investigated paper to fabricate a PAD capable of RCA, a technique used for DNA amplification and detection. Despite the well-established PCR technique for DNA amplification, RCA is more appealing as a PAD. It can be carried out under isothermal conditions, at room temperature, or 30°C, using a simple biochemical procedure involving Phi29 DNA polymerase. The Phi29 DNA polymerase enzyme can displace short DNA strands to amplify a DNA primer into long single-stranded DNA products. This increases the detection capabilities. Immobilization of a DNA oligonucleotide on strips of paper containing poly(N-isopropylacrylamide) microgels creates a platform for which target DNA could ligate and undergo RCA-mediated amplification, enhancing DNA detection. The advantage of identifying a specific DNA sequence, rather than other biomarkers, is the specificity of the DNA itself. It provides an ultrasensitive diagnosis tool for disease. If achievable, it can be applied to any specific pathogen, including bacteria. However, preliminary results have identified a decreased sensitivity compared with other strategies; methods for improving sensitivity are being explored.

Govindarajan et al. (2011) used 2D-oPADs to demonstrate POC cell lysis and bacterial DNA extraction from raw viscous samples of Escherichia coli-spiked pig mucin. The sequential folding of 2D surfaces created temporary circuits, which dictated capillary flow without external pumping or power. The ability to detect DNA using 2D-oPADs can be used for diagnosis of tuberculosis. However, lysis of the Mycobacterium tuberculosis bacteria is difficult and requires further investigation.

Diabetes

One of the most common validation tests used for paper diagnostics is the detection of glucose in urine (Martinez et al. 2007, Fenton et al. 2008, Abe et al. 2008). In fact, dipstick diagnostics to detect glucose were the first functionalization application (Free et al. 1957). While diagnosis and detection of diabetes in patients is well established, paper diagnostics can provide an alternative testing method. The dipstick assay relies on enzymatic reactions of glucose with glucose oxidase to produce gluconic acid and hydrogen peroxide. The hydrogen peroxide would in turn react with orthotolidine which results in a colorimetric change to a deep blue color (Fenton et al. 2008). Later tests involved the addition of red dye and resulted in shades of purple at different intensities to estimate concentration.

Cancer markers

Recently, cancer biomarkers have gained interest as potential analytes for paper diagnostics. An example is a platform for miRNA detection, which does not require the use of equipment for lung cancer diagnosis (Yildiz et al. 2012). miRNA regulates the mRNA function in gene transcription. The miRNA assay detects the formation of duplex and triplex species which show unique and varying optical signals to the naked eye. An miRNA sequence specifically associated with lung cancer is mir21. Analyses showed a linear correlation between concentrations 10 nm to 10 mm. It is selective, displaying a purple color for the duplex and orange color for the triplex. While a good indicative process for lung cancer treatment, further testing is required prior to clinical testing, including an investigation into the sample pretreatment and a sensitivity analysis compared to current standard tests (Yildiz et al. 2012).

Liver function

Paper diagnostics have been considered to monitor liver function (Vella et al. 2012). Overmedication of patients can induce liver toxicity and can also lead to drug resistance, rendering the treatment ineffective. This commonly occurs in developing countries, since tests for monitoring liver function are expensive, and require trained equipment and personnel. Vella et al. (2012) developed a finger-prick paper diagnostic to measure the levels of two enzymatic markers which are indicative of liver function: alkaline phosphatase (ALP) and aspartate aminotransferase. Total serum protein can also be measured. The design comprises a patterned paper-chip, a filter, and self-adhesive laminating sheets. It accomplishes four primary functions: 1) separation of RBC from plasma, 2) distributing the plasma into three regions within the paper, 3) conducting three simultaneous colorimetric assays, and 4) displaying the results for quantitative analysis using telemedicine. The results reported were consistent with the colors seen in tests for all three analytes in artificial blood; however, a sensitivity analysis was not provided. Calibration curves were measured, but failed to take into account the background interference from using whole blood. Additionally, further investigation regarding stability in warmer climates and selectivity from other potential biomarkers are required.

Malaria

Malaria is caused by the Plasmodium parasite, which is transmitted by infectious mosquito bites, displaying symptoms of fever, vomiting, and/or headaches (World Health Organization 2013). It is endemic in 104 countries, most of which are remote with humid weather conditions.

Successful identification of the disease can be achieved by detection of the Plasmodium falciparum histidine rich protein 2 found in malaria (Dineva et al. 2005, Fu et al. 2012). The 2D-PN described by Fu et al. (2012) demonstrated a fully automated system for detection using a sandwich format immunoassay. The 2D-PN diagnostic had two additional processing steps for rinsing and signal amplification using a gold enhancement reagent. The LOD for the amplified assay was similar to that reported for ELISA: 2.9±1.9 ng/ml and 4.0 ng/ml, respectively. This demonstrates the applicability of the automated 2D-PNs in a POC setting for malaria.

HIV-1

The retrovirus, HIV, infects cells within the immune system by destroying or impairing their function (World Health Organization 2013). The immune system is progressively weakened over time, until the patient becomes easily susceptible to infections. While there are antiretroviral drugs which slow down the disease, there is currently no cure for HIV-1. Efficient and accurate detection may not stop the illness, but early detection can help slow progression and patient deterioration, while preventing the spread of the disease.

The dipstick assay uses RT-PCR, which tests for specific nucleic acid sequences. For HIV-1, this means detection and identification of the HIV-1 genome (Dineva et al. 2005). However, the HIV-1 RNA must be prepared before testing. While the instrumentation is relatively inexpensive, it still limits application in remote areas and developing countries where equipment-free diagnostics are preferred.

While the dipstick device relied on signal amplification of nucleic acid hybridization, detection of HIV-1 provides an example for the applicability of the P-ELISA (Cheng et al. 2010). The HIV-1 envelope antigen gp41 successfully detected specific antibodies in human serum by using indirect P-ELISA methodology. Serum samples from HIV-1-positive patients were tested at different concentrations against control samples of human serum without anti-gp41. The colorimetric results indicated decreased intensity signals with serum dilution, but could still distinguish a positive result, thereby showing that a complex mixture, such as human serum, could be analyzed. Additionally, Ayele et al. (2007) described a method to collect and store samples for HIV-1 testing in field conditions.

Hepatitis B and C

Both hepatitis B and C (HBV and HCV, respectively) are viral infections transmitted through contact with blood or other bodily fluids, infecting the liver and causing hepatic diseases. Both vary in severity; however, HCV is known to potentially lead to liver cirrhosis or cancer. A vaccine for HBV is currently available but not for HCV (World Health Organization 2013).

Dineva et al. (2005) described a dipstick method to detect HBV DNA and HCV RNA. However, much like with HIV-1, the procedures and equipment used for preparation are not ideal for POC situations.

Successful HBV analysis showed the applicability of the 3D-ELISA paper device to detect HepB surface antigens (HBsAg) in rabbit serum. However, the protocol varied from the indirect ELISA methodology (Liu et al. 2011). A primary antibody was used combined with an ALP-conjugated secondary antibody to label the HBsAg. The additional steps were achievable thanks to the ease of fabrication and flexibility of the μPADs. Serum samples of HBsAg-positive hosts were compared to serum controls without HBsAg, and showed that a 10-fold dilution was still discernible, thus supporting its potential for detecting hepatitis B and other infectious diseases.