Search

Paper-based microfluidic devices, also known as microPADs, are an emerging analytical platform with the potential to improve point-of-care diagnostics. MicroPADs are fabricated by patterning hydrophobic inks onto sheets of paper to create hydrophilic channels and test zones. One of the main advantages of microPADs is that they are inexpensive and simple to fabricate, making them accessible even to researchers with limited budgets or no prior fabrication expertise. Wax printing, where a solid ink printer is used to pattern wax on paper, has been the most convenient and popular method for fabricating paper-based microfluidic devices. Unfortunately, solid ink printers were discontinued in 2016 and are no longer available commercially. Here we introduce a method for fabricating microPADs using a portable thermal transfer printer that retains the convenience of wax printing. Devices fabricated by thermal transfer printing were comparable to devices fabricated via wax printing and laser printing. The low cost, convenience, and portability of the thermal transfer printer make this approach an exciting prospect for replacing wax printing and facilitating the continued development of paper-based microfluidics.

Shrinking paper money is a captivating and memorable demonstration of chemistry in action that can be carried out using minimal equipment and reagents. Paper bills can be shrunk down to ∼25% of their initial surface area by treating them with an aqueous solution of sodium periodate. The cellulose in the paper bill is oxidized to dialdehyde cellulose via the Malaprade reaction, and the resulting change in size of the paper money provides macroscopic evidence of the reaction. The chemical change that occurs in the cellulose can be confirmed by a Tollen’s test.

Three commercially-available conductive filaments were evaluated for 3D printing flexible circuits on paper. While all three filaments were printed successfully, the resulting conductive traces were found to have significantly different impedances when characterized by electrochemical impedance spectroscopy. Using a graphite-doped polylactic acid filament, the flexibility of paper-based conductive traces was evaluated, methods of integrating common electrical and electronic components with the conductive traces were demonstrated, and the resistive heating of the traces was characterized. The ability to 3D print conductive traces on paper using commercially available materials opens many opportunities for rapid prototyping of flexible electronics and for integrating electronic circuits with paper-based microfluidic devices.

Cellulose-based paper is a versatile material with a diverse array of applications. While paper is not commonly thought of as a material that shrinks, here we present a method for miniaturizing paper via periodate oxidation. Chromatography paper was exposed to varying concentrations of periodate (0.1–0.5 M) over a 96-h period. Following optimization of miniaturization parameters, fourteen different types of paper were miniaturized and reductions in surface area ranging from 60 to 80% were observed. All cellulose paper types, but not cellulose-derivatives, displayed successful miniaturization. Results were highly tunable dependent upon periodate concentration and reaction time. Potential applications of the technique are discussed, including its use as a microfabrication method.

Paper-based microfluidics is the branch of microfluidics involving devices made out of paper, or other porous membranes, that wick fluids by capillary action. Paper-based microfluidic devices have several advantages over conventional microfluidic devices including simpler fabrication, lower cost, easier disposal, and the ability to operate without pumps or other supporting equipment. The most common application of paper-based microfluidic devices is in the development of point-of-care (POC) diagnostic devices, which could eliminate the need for costly and time-consuming laboratory-based analytical procedures. This review provides an overview of current methods of fabricating paper-based microfluidic devices, examples of applications of these devices, a discussion of their current limitations, and an outlook on their future.

The following article summarizes United States Patent Application No. US20180052155A1, titled ‘Assay Devices and Methods’ (filed 16 August 2016, published 22 February 2018). While lateral flow assays (LFAs) have revolutionized point-of-care diagnostics by enabling accurate, inexpensive, and rapid detection of biomarkers, they typically do not provide quantitative results. Hence, there is a significant need for quantitative assays at the point of care. This patent summary describes a novel method of chronometric biomarker quantitation via enzymatic degradation of a metastable gelatin-based biomatrix, principally suited for use in paper-based microfluidic devices (microPADs). This new quantitation mechanism was designed to meet the ASSURED criteria for point-of-care diagnostic devices laid forth by the World Health Organization and may ultimately provide increased access to healthcare, at a significantly reduced cost, around the world.

Microbial staining techniques are widely employed in clinical and academic laboratories for classifying and identifying microorganisms derived from clinical, food and environmental samples. Staining allows for the rapid visualization and determination of many morphological characteristics of microorganisms, used for their identification and classification. Over the past century, staining techniques such as the Gram stain, the Capsule stain, the Acid-fast stain and the Endospore stain, have seen few advances, and manual staining remains the gold standard. Typical instructions for these staining procedures recommend 'flooding' glass slides with milliliter volumes of dye, resulting in large volumes of hazardous waste. Here we present micro-staining, a simple alternative to flooding that utilizes microliter volumes of dye. Micro-staining minimizes the volume of waste generated, leads to significant cost savings for the laboratory, requires limited training, and produces results with equivalent quality to traditional stains.

Microfluidic paper-based analytical devices (microPADs) have emerged as a promising platform for point-of-care diagnostic devices. While the inherent wicking properties of microPADs allow for fluid flow without supporting equipment, this also presents a major challenge in achieving robust fluid control, which becomes especially important when performing complex multi-step assays. Herein, we describe an ideal method of fluid control mediated by wax-printed fluidic time delays. This method relies on a simple fabrication technique, does not utilize chemicals/reagents that could affect downstream assays, is readily scalable, and has a wide temporal range of tunable fluid control. The delays are wax printed on both the top and bottom of pre-fabricated microPAD channels, without subsequent heating, to create hemi-/fully-enclosed channels. With these wax printed delays, we were able to tune the time it took aqueous solutions to wick across a 25 mm-long channel between 3.6 min and 13.4 min. We then employed these fluid delays in the sequential delivery of four dyes to a test zone. Additionally, we demonstrated the automation of two simple enzymatic assays with this fluid control modality. This method of fluid control may allow future researchers to automate more complex assays, thereby further advancing microPADs toward real-world applications.

Microfluidic paper-based analytical devices (microPADs) are emerging as cost-effective and portable platforms for point-of-care assays. A fundamental limitation of microPAD fabrication is the imprecise nature of most methods for patterning paper. The present work demonstrates that paper patterned via wax printing can be miniaturized by treating it with periodate to produce higher-resolution, high-fidelity microPADs. The optimal miniaturization parameters were determined by immersing microPADs in various concentrations of aqueous sodium periodate (NaIO4) for varying lengths of time. This treatment miniaturized microPADs by up to 80% in surface area, depending on the concentration of periodate and length of the reaction time. By immersing microPADs in 0.5-M NaIO4 for 48 hours, devices were miniaturized by 78% in surface area, and this treatment allowed for the fabrication of functional channels with widths as small as 301 µm and hydrophobic barriers with widths as small as 387 µm. The miniaturized devices were shown to be compatible with redox-based colorimetric assays and enzymatic reactions. This miniaturization technique provides a new option for fabricating sub-millimeter-sized features in paper-based fluidic devices without requiring specialized equipment and could enable new capabilities and applications for microPADs.

Custom-made pencils containing reagents dispersed in a solid matrix were developed to enable rapid and solvent-free deposition of reagents onto membrane-based fluidic devices. The technique is as simple as drawing with the reagent pencils on a device. When aqueous samples are added to the device, the reagents dissolve from the pencil matrix and become available to react with analytes in the sample. Colorimetric glucose assays conducted on devices prepared using reagent pencils had comparable accuracy and precision to assays conducted on conventional devices prepared with reagents deposited from solution. Most importantly, sensitive reagents, such as enzymes, are stable in the pencils under ambient conditions, and no significant decrease in the activity of the enzyme horseradish peroxidase stored in a pencil was observed after 63 days. Reagent pencils offer a new option for preparing and customizing diagnostic tests at the point of care without the need for specialized equipment.

Reagent pencils allow for solvent-free deposition of reagents onto paper-based microfluidic devices. The pencils are portable, easy to use, extend the shelf-life of reagents, and offer a platform for customizing diagnostic devices at the point of care. In this work, reagent pencils were characterized by measuring the wear resistance of pencil cores made from polyethylene glycols (PEGs) with different molecular weights and incorporating various concentrations of three different reagents using a standard pin abrasion test, as well as by measuring the efficiency of reagent delivery from the pencils to the test zones of paper-based microfluidic devices using absorption spectroscopy and digital image colorimetry. The molecular weight of the PEG, concentration of the reagent, and the molecular weight of the reagent were all found to have an inverse correlation with the wear of the pencil cores, but the amount of reagent delivered to the test zone of a device correlated most strongly with the concentration of the reagent in the pencil core. Up to 49% of the total reagent deposited on a device with a pencil was released into the test zone, compared to 58% for reagents deposited from a solution. The results suggest that reagent pencils can be prepared for a variety of reagents using PEGs with molecular weights in the range of 2000 to 6000 g/mol.

Paper microzone plates in combination with a noncontact liquid handling robot were demonstrated as tools for studying the stability of enzymes stored on paper. The effect of trehalose and SU-8 epoxy novolac resin (SU-8) on the stability of horseradish peroxidase (HRP) was studied in both a short-term experiment, where the activity of various concentrations of HRP dried on paper were measured after 1 h, and a long-term experiment, where the activity of a single concentration of HRP dried and stored on paper was monitored for 61 days. SU-8 was found to stabilize HRP up to 35 times more than trehalose in the short-term experiment for comparable concentrations of the two reagents, and a 1% SU-8 solution was found to stabilize HRP approximately 2 times more than a 34% trehalose solution in both short- and long-term experiments. The results suggest that SU-8 is a promising candidate for use as an enzyme-stabilizing reagent for paper-based diagnostic devices and that the short-term experiment could be used to quickly evaluate the capacity of various reagents for stabilizing enzymes to identify and characterize new enzyme-stabilizing reagents.

This chapter will provide an overview of existing diagnostic devices made primarily out of paper and then focus on paper-based microfluidic devices, the next generation of paper-based diagnostic devices that promises to extend the use of paper as a material for fabricating diagnostic devices well into the future.

Standard addition assays conducted on paper-based microfluidic devices are introduced as an alternative to external standards for calibrating quantitative tests. To demonstrate this technique, a colorimetric, paper-based, standard addition assay was optimized for the determination of glucose concentrations in the range of 0 to 5 mM. Comparable results were obtained from the assay via digital image colorimetry under three different lighting conditions.

Emerging paper-based diagnostics is an inexpensive yet elegant approach to medical diagnostics and environmental testing in resource-poor regions of the world. Wicking by capillary action distributes a small drop of sample to reagent or assay zones without the need for pumps, refrigeration, or electricity. The advantages of using paper for medical diagnostics (low expense, availability, portability, easy disposal) also apply to the classroom. This article describes a two-session high school activity that employs a paper device to solve a crime scene mystery. On the first day, students role-play either suspects or a detective and make inferences to predict the guilty party. In the second session, students perform presumptive, paper-based forensic testing of “unknown” liquids retrieved from the suspects’ homes and compare these findings with their predictions. The primary goals of this exercise are to demonstrate the similarities between forensic investigation and scientific inquiry and to introduce student...

This article introduces fully enclosed microfluidic paper-based analytical devices (microPADs) fabricated by printing toner on the top and bottom of the devices using a laser printer. Enclosing paper-based microfluidic channels protects the channels from contamination, contains and protects reagents stored on the device, contains fluids within the channels so that microPADs can be handled and operated more easily, and reduces evaporation of solutions from the channels. These benefits extend the capabilities of microPADs for applications as low-cost point-of-care diagnostic devices.

This article describes a prototype system for quantifying bioassays, and for exchanging the results of the assays digitally with physicians located off-site. The system uses paper-based microfluidic devices for running multiple assays simultaneously, camera phones or portable scanners for digitizing the intensity of color associated with each colorimetric assay, and established communications infrastructure for transferring the digital information from the assay site to an off-site laboratory for analysis by a trained medical professional; the diagnosis then can be returned directly to the healthcare provider in the field. The microfluidic devices were fabricated in paper using photolithography, and were functionalized with reagents for colorimetric assays. The results of the assays were quantified by comparing the intensities of the color developed in each assay with those of calibration curves. An example of this system quantified clinically relevant concentrations of glucose and protein in artificial urine. The combination of patterned paper, a portable method for obtaining digital images, and a method for exchanging results of the assays with off-site diagnosticians offers new opportunities for inexpensive monitoring of health, especially in situations that require physicians to travel to patients (e.g., in the developing world, in emergency management, and during field operations by the military) to obtain diagnostic information that might be obtained more effectively by less valuable personnel.

This article describes the development of porous two-ply channels for paper-based microfluidic devices that wick fluids significantly faster than conventional, porous, single-ply channels. The two-ply channels were made by stacking two single-ply channels on top of each other and were fabricated entirely out of paper, wax and toner using two commercially available printers, a convection oven and a thermal laminator. The wicking in paper-based channels was studied and modeled using a modified Lucas–Washburn equation to account for the effect of evaporation, and a paper-based titration device incorporating two-ply channels was demonstrated.

This communication describes a simple method for patterning paper to create well-defined, millimeter-sized channels, comprising hydrophilic paper bounded by hydrophobic polymer. We believe that this type of patterned paper will become the basis for low-cost, portable, and technically simple multiplexed bioassays. We demonstrate this capability by the simultaneous detection of glucose and protein in 5 μL of urine. The assay system is small, disposable, easy to use (and carry), and requires no external equipment, reagents, or power sources. We believe this kind of system is attractive for uses in less-industrialized countries, in the field, or as an inexpensive alternative to more advanced technologies already used in clinical settings.[1-4] The analysis of biological fluids is necessary for monitoring the health of populations,[2] but these measurements are difficult to implement in remote regions such as those found in less-industrialized countries, in emergency situations, or in home health-care settings.[3] Conventional laboratory instruments provide quantitative measurements of biological samples, but they are unsuitable for these situations since they are large, expensive, and require trained personnel and considerable volumes of biological samples.[2] Other bioassay platforms provide alternatives to more expensive instruments,[5-7] but the need remains for a platform that uses small volumes of sample and that is sufficiently inexpensive to be used widely for measuring samples from large populations. We believe that paper may serve as a particularly convenient platform for running bioassays in the remote situations locations. As a prototype for a mthod we believe to be particularly promosing, we patterned photoresist onto chromatography paper to form defined areas of hydrophilic paper separated by hydrophobic lines or “walls”; these patterns provide spatial control of biological fluids and enable fluid transport, without pumping, due to capillary action in the millimeter-sized channels produced. This method for patterning paper makes it possible to run multiple diagnostic assays on one strip of paper, while still using only small volumes of a single sample. In a fully developed technology, patterned photoresist would be replaced by an appropriate printing technology, but patterning paper with photoresist is: i) convenient for prototyping these devices, and ii) a useful new micropatterning technology in its own right. We patterned chromatography paper with SU-8 2010 photoresist as shown in Figure 1a and as described below: we soaked a 7.5-cm diameter piece of chromatography paper in 2 mL of SU-8 2010 for 30 s, spun it at 2000 rpm for 30 s, and then baked it at 95 °C for 5 min to remove the cyclopentanone in the SU-8 formula. We then exposed the photoresist and paper to 405 nm UV light (50 mW/cm2) for 10 s through a photo-mask (CAD/Art Services, Inc.) that was aligned using a mask aligner (OL-2 Mask Aligner, AB-M, Inc). After exposure, we baked the paper a second time at 95 °C for 5 min to cross-link the exposed portions of the resist. The unpolymerized photoresist was removed by soaking the paper in propylene glycol monomethyl ether acetate (PGMEA) (5 min), and by washing the pattern with propan-2-ol (3 × 10 mL). The paper was more hydrophobic after it was patterned, presumably due to residual resist bound to the paper, so we exposed the entire surface to an oxygen plasma for 10 s at 600 millitorr (SPI Plasma-Prep II, Structure Probe, Inc) to increase the hydrophilicity of the paper (Figures 2a and 2b). Figure 1 Chromatography paper patterned with photoresist. The darker lines are cured photoresist; the lighter areas are unexposed paper. (a) Patterned paper after absorbing 5 μL of Waterman red ink by capillary action. The central channel absorbs the sample ... Figure 2 Assays contaminated with (a) dirt, (b) plant pollen, and (c) graphite powder. The pictures were taken before and after running an artificial urine solution that contained 550 mM glucose and 75 μM BSA. The particulates do not move up the channels ... The patterned paper can be derivatized for biological assays by adding appropriate reagents to the test areas (Figures 1b and ​and2b).2b). In this communication, we demonstrate the method by detecting glucose and protein,[8] but the surface should be suitable for measuring many other analytes as well.[7] The glucose assay is based on the enzymatic oxidation of iodide to iodine,[9] where a color change from clear to brown is associated with the presence of glucose.[10] The protein assay is based on the color change of tetrabromophenol blue (TBPB) when it ionizes and binds to proteins;[11] a positive result in this case is indicated by a color change from yellow to blue. For the glucose assay, we spotted 0.3 μL of a 0.6 M solution of potassium iodide, followed by 0.3 μL of a 1:5 horseradish peroxidase/glucose oxidase solution (15 units of protein per mL of solution). For the protein assay, we spotted 0.3 μL of a 250-mM citrate buffer (pH 1.8) in a well separate from the glucose assay, and then layered 0.3 μL of a 3.3 mM solution of tetrabromophenol blue (TBPB) in 95% ethanol over the citrate buffer. The spotted reagents were allowed to air dry at room temperature. This pre-loaded paper gave consistent results for the protein assay regardless of storage temperature and time (when stored for 15 d both at 0 °C and at 23 °C, wrapped in aluminum foil). The glucose assay was sensitive to storage conditions, and showed decreased signal for assays run 24 h after spotting the reagents (when stored at 23 °C); when stored at 0 °C, however, the glucose assay was as sensitive after day 15 as it was on day 1. We measured artificial samples of glucose and protein in clinically relevant ranges (2.5-50 mM for glucose and 0.38-7.5 μM for bovine serum albumin (BSA))[12, 13] by dipping the bottom of each test strip in 5 μL of a pre-made test solution (Figure 2d). The fluid filled the entire pattern within ca. one minute, but the assays required 10-11 min for the paper to dry and for the color to fully develop.[14] In all cases, we observed color changes corresponding roughly in intensity to the amount of glucose and protein in the test samples, where the lowest concentrations define the lower limits to which these assays can be used (Figure 2e). For comparison, commercially-available dipsticks detect glucose at concentrations as low as 5 mM[7, 9] and protein as low as 0.75 μM;[6, 15] these limits indicate that these paper-based assays are comparable in sensitivity to commercial dipstick assays. Our assay format also allows for the measurement of multiple analytes. This paper-based assay is suitable for measuring multiple samples in parallel and in a relatively short period of time. For example, in one trial, one researcher was able to run 20 different samples (all with 550 mM glucose and 75 μM BSA) within 7.5 min (followed by another 10.5 min for the color to fully develop). An 18-min assay of this type—one capable of measuring two analytes in 20 different sample—may be efficient enough to use in high-throughput screens of larger sample pools. In the field, samples will not be measured under sterile conditions, and dust and dirt may contaminate the assays. The combination of paper and capillary action provides a mechanism for separating particulates from a biological fluid. As a demonstration, we purposely contaminated the artificial urine samples with quantities of dirt, plant pollen, and graphite powder at levels higher than we might expect to see in the samples in the field. These particulates do not move up the channels under the action of capillary wicking, and do not interfere with the assay (Figure 3). Paper strips have been used in biomedical assays for decades because they offer an inexpensive platform for colorimetric chemical testing.[1] Patterned paper has characteristics that lead to miniaturized assays that run by capillary action (e.g., without external pumping), with small volumes of fluids. These methods suggest a path for the development of simple, inexpensive, and portable diagnostic assays that may be useful in remote settings, and in particular, in less-industrialized countries where simple assays are becoming increasingly important for detecting disease and monitoring health,[16, 17], for environmental monitoring, in veterinary and agricultural practice and for other applications.

This paper describes a paper-based microfluidic device that measures two enzymatic markers of liver function (alkaline phosphatase, ALP, and aspartate aminotransferase, AST) and total serum protein. A device consists of four components: (i) a top plastic sheet, (ii) a filter membrane, (iii) a patterned paper chip containing the reagents necessary for analysis, and (iv) a bottom plastic sheet. The device performs both the sample preparation (separating blood plasma from erythrocytes) and the assays; it also enables both qualitative and quantitative analysis of data. The data obtained from the paper-microfluidic devices show standard deviations in calibration runs and “spiked” standards that are acceptable for routine clinical use. This device illustrates a type of test useable for a range of assays in resource-poor settings.

Microfluidic paper-based analytical devices (microPADs) began as a simple idea with an ambitious goal. The idea was to make microfluidic devices out of paper instead of plas tic or glass. The goal was to develop low-cost and portable paper-based diagnostic devices to improve healthcare in developing countries. Over the past 6 years, many developments have been made in the emerging field of paper-based microfluidic devices. Reviewing the develop ment of these devices in the Whitesides group at Harvard University (Cambridge, MA, USA) can provide some insight into the future of the field and encourage scientists from a variety of backgrounds to contribute their expertise toward further development of useful microPADs.

This paper describes a portable, three-dimensional (3D) microfluidic paper-based analytical device (3D-µPAD) for performing enzyme-linked immunosorbent assays (ELISA). To run an ELISA, the device requires only the addition of a small drop (2 µL per test zone) of sample and six drops (75 µL in total volume) of buffer solution. All the reagents required for the analysis are stored in dry form within the device, and are dissolved in the buffer and delivered to the test zones during the assay. The innovative feature of the device, which enables the delivery of different reagents and washes to the test zones without cross contamination, is a movable strip containing the test zones that can be moved manually through the device, and stops only at specified points where the test zones come into contact with different fluidic paths that sequentially transfer (i.e., wash) the reagents into the test zones. Using rabbit IgG as a model analyte, we performed an ELISA in 43 minutes, with a detection limit of 330 pM. We also demonstrated the detection of hepatitis B surface antigen (HBsAg) in serum.

This paper describes three-dimensional microfluidic paper-based analytical devices (3-D microPADs) that can be programmed (postfabrication) by the user to generate multiple patterns of flow through them. These devices are programmed by pressing single-use 'on' buttons, using a stylus or a ballpoint pen. Pressing a button closes a small space (gap) between two vertically aligned microfluidic channels, and allows fluids to wick from one channel to the other. These devices are simple to fabricate, and are made entirely out of paper and double-sided adhesive tape. Programmable devices expand the capabilities of microPADs and provide a simple method for controlling the movement of fluids in paper-based channels. They are the conceptual equivalent of field-programmable gate arrays (FPGAs) widely used in electronics.

This technical note describes a detailed study on wax printing, a simple and inexpensive method for fabricating microfluidic devices in paper using a commercially available printer and hot plate. The printer prints patterns of solid wax on the surface of the paper, and the hot plate melts the wax so that it penetrates the full thickness of the paper. This process creates complete hydrophobic barriers in paper that define hydrophilic channels, fluid reservoirs, and reaction zones. The design of each device was based on a simple equation that accounts for the spreading of molten wax in paper.

This paper describes the fabrication and the performance of microfluidic paper-based electrochemical sensing devices (we call the microfluidic paper-based electrochemical devices, microPEDs). The microPEDs comprise paper-based microfluidic channels patterned by photolithography or wax printing, and electrodes screen-printed from conducting inks (e.g., carbon or Ag/AgCl). We demonstrated that the microPEDs are capable of quantifying the concentrations of various analytes (e.g., heavy-metal ions and glucose) in aqueous solutions. This low-cost analytical device should be useful for applications in public health, environmental monitoring, and the developing world.

This paper describes 96- and 384-microzone plates fabricated in paper as alternatives to conventional multiwell plates fabricated in molded polymers. Paper-based plates are functionally related to plastic well plates, but they offer new capabilities. For example, paper-microzone plates are thin (approximately 180 microm), require small volumes of sample (5 microL per zone), and can be manufactured from inexpensive materials ($0.05 per plate). The paper-based plates are fabricated by patterning sheets of paper, using photolithography, into hydrophilic zones surrounded by hydrophobic polymeric barriers. This photolithography used an inexpensive formulation photoresist that allows rapid (approximately 15 min) prototyping of paper-based plates. These plates are compatible with conventional microplate readers for quantitative absorbance and fluorescence measurements. The limit of detection per zone loaded for fluorescence was 125 fmol for fluorescein isothiocyanate-labeled bovine serum albumin, and this level corresponds to 0.02 the quantity of analyte per well used to achieve comparable signal-to-noise in a 96-well plastic plate (using a solution of 25 nM labeled protein). The limits of detection for absorbance on paper was approximately 50 pmol per zone for both Coomassie Brilliant Blue and Amaranth dyes; these values were 0.4 that required for the plastic plate. Demonstration of quantitative colorimetric correlations using a scanner or camera to image the zones and to measure the intensity of color, makes it possible to conduct assays without a microplate reader.

This article describes a method for fabricating 3D microfluidic devices by stacking layers of patterned paper and double-sided adhesive tape. Paper-based 3D microfluidic devices have capabilities in microfluidics that are difficult to achieve using conventional open-channel microsystems made from glass or polymers. In particular, 3D paper-based devices wick fluids and distribute microliter volumes of samples from single inlet points into arrays of detection zones (with numbers up to thousands). This capability makes it possible to carry out a range of new analytical protocols simply and inexpensively (all on a piece of paper) without external pumps. We demonstrate a prototype 3D device that tests 4 different samples for up to 4 different analytes and displays the results of the assays in a side-by-side configuration for easy comparison. Three-dimensional paper-based microfluidic devices are especially appropriate for use in distributed healthcare in the developing world and in environmental monitoring and water analysis.

This article describes FLASH (Fast Lithographic Activation of Sheets), a rapid method for laboratory prototyping of microfluidic devices in paper. Paper-based microfluidic devices are emerging as a new technology for applications in diagnostics for the developing world, where low cost and simplicity are essential. FLASH is based on photolithography, but requires only a UV lamp and a hotplate; no clean-room or special facilities are required (FLASH patterning can even be performed in sunlight if a UV lamp and hotplate are unavailable). The method provides channels in paper with dimensions as small as 200 microm in width and 70 microm in height; the height is defined by the thickness of the paper. Photomasks for patterning paper-based microfluidic devices can be printed using an ink-jet printer or photocopier, or drawn by hand using a waterproof black pen. FLASH provides a straightforward method for prototyping paper-based microfluidic devices in regions where the technological support for conventional photolithography is not available.

Protein-bound solutes are poorly cleared by dialysis. Among the most extensively studied of these solutes is p-cresol, which has been shown to be toxic in vitro. This study examined the form in which p-cresol circulates and quantified its removal by hemodialysis. HPLC analysis of plasma from hemodialysis patients contained a peak whose mobility corresponded to synthetic p-cresol sulfate (PCS) but no detectable unconjugated p-cresol. Treatment with sulfatase resulted in recovery of this peak as p-cresol, confirming its identity. Subsequent studies compared the removal of PCS and another protein-bound solute, indican, to the removal of urea during clinical hemodialysis treatments. PCS and indican were 94 +/- 1% and 93 +/- 2% bound to plasma protein, respectively. Protein-binding caused a predictable decrease in measured dialytic clearance, which averaged 20 +/- 4 ml/min for PCS and 25 +/- 5 ml/min for indican as compared with 260 +/- 20 ml/min for urea. Volumes of distribution for the protein-bound solutes were greater than the plasma volume, averaging 15 +/- 7 L for PCS and 14 +/- 3 L for indican as compared with 37 +/- 7 for urea. Solute reduction ratios were 20 +/- 9% for PCS, 30 +/- 7% for indican, and 69 +/- 5% for urea. We conclude that p-cresol circulates in the form of its sulfate conjugate, PCS. PCS is poorly removed by hemodialysis because its clearance is limited by protein binding and the ratio of its volume of distribution to its clearance is high.

The clearance of protein-bound solutes by hemofiltration and hemodiafiltration. Background Hemofiltration in the form of continuous venovenous hemofiltration (CVVH) is increasingly used to treat acute renal failure. Compared to hemodialysis, hemofiltration provides high clearances for large solutes but its effect on protein-bound solutes has been largely ignored. Methods Standard clinical systems were used to remove test solutes from a reservoir containing artificial plasma. Clearances of the protein-bound solutes phenol red (C PR ) and indican (C IN ) were compared to clearances of urea (C UREA ) during hemofiltration and hemodiafiltration. A mathematical model was developed to predict clearances from values for plasma flow Q p , dialysate flow Q d , ultrafiltration rate Q f , filter size and the extent of solute binding to albumin. Results When hemofiltration was performed with Q p 150mL /min and Q f 17mL /min, clearance values were C PR 1.0 ± 0.1mL /min; C IN 3.7 ± 0.5mL /min; and C UREA 14 ± 1mL /min. The clearance of the protein-bound solutes was approximately equal to the solute-free fraction multiplied by the ultrafiltration rate corrected for the effect of predilution. Addition of Q d 42mL /min to provide HDF while Q p remained 150mL /min resulted in proportional increases in the clearance of protein-bound solutes and urea. In contrast, the clearance of protein-bound solutes relative to urea increased when hemodiafiltration was performed using a larger filter and increasing Q d to 300mL /min while Q p was lowered to 50mL /min. The pattern of observed results was accurately predicted by mathematical modeling. Conclusion In vitro measurements and mathematical modeling indicate that CVVH provides very limited clearance of protein-bound solutes. Continuous venous hemodiafiltration (CVVHDF) increases the clearance of protein-bound solutes relative to urea only when dialysate flow rate and filter size are increased above values now commonly employed.

We present a new method for fabricating three-dimensional paper-based fluidic devices that uses toner as a thermal adhesive to bond multiple layers of patterned paper together. The fabrication process is rapid, involves minimal equipment (a laser printer and a laminator) and produces complex channel networks with dimensions down to 1 mm. The devices can run multiple diagnostic assays on one or more samples simultaneously, can incorporate positive and negative controls and can be programmed to display the results of the assays in a variety of patterns. The patterns of the results can encode information, which could be used to identify counterfeit devices, identify samples, encrypt the results for patient privacy or monitor patient compliance.

This communication describes a simple method for printing aqueous solutions with millimeter-scale patterns on a variety of substrates using an easily fabricated, paper-based microfluidic device (a paper-based "stamp") as a contact printing device. The device is made from inexpensive materials, and it is easily assembled by hand; this method is thus accessible to a wide range of laboratories and budgets. A single device was used to print over 2500 spots in less than three minutes at a density of 16 spots per square centimetre. This method provides a new tool to pattern biochemicals-reagents, antigens, proteins, and DNA-on planar substrates. The accuracy of the volume of fluid delivered in simple paper-to-paper printing is low, and although the pattern transfer is rapid, it is better suited for qualitative than accurate, quantitative work. By patterning the paper to which the transfer occurs using wax printing or an equivalent technique, accuracy increases substantially.