Multiplexed, Patterned-Paper Immunoassay for Detection of Malaria and Dengue Fever.

Multiplex assays detect the presence of more than one analyte in a sample. For diagnostic applications, multiplexed tests save healthcare providers time and resources by performing many assays in parallel, minimizing the amount of sample needed and improving the quality of information acquired regarding the health status of a patient. These advantages are of particular importance for those diseases that present with general, overlapping symptoms, which makes presumptive treatments inaccurate and may put the patient at risk. For example, malaria and dengue fever are febrile illnesses transmitted through mosquito bites, and these common features make it difficult to obtain an accurate diagnosis by symptoms alone. In this manuscript, we describe the development of a multiplexed, patterned paper immunoassay for the detection of biomarkers of malaria and dengue fever: malaria HRP2, malaria pLDH, and dengue NS1 type 2. In areas coendemic for malaria and dengue fever, this assay could be used as a rapid, point-of-care diagnostic to determine the cause of a fever of unknown origin. The reagents required for each paper-based immunoassay are separated spatially within a three-dimensional device architecture, which allows the experimental conditions to be adjusted independently for each assay. We demonstrate the analytical performances of paper-based assays for each biomarker and we show that there is no significant difference in performance between the multiplexed immunoassay and those immunoassays performed in singleplex. Additionally, we spiked individual analytes into lysed human blood to demonstrate specificity in a clinically relevant sample matrix. Our results suggest multiplex paper-based devices can be an essential component of diagnostic assays used at the point-of-care.

"Paper Machine" for Molecular Diagnostics.

Clinical tests based on primer-initiated amplification of specific nucleic acid sequences achieve high levels of sensitivity and specificity. Despite these desirable characteristics, these tests have not reached their full potential because their complexity and expense limit their usefulness to centralized laboratories. This paper describes a device that integrates sample preparation and loop-mediated isothermal amplification (LAMP) with end point detection using a hand-held UV source and camera phone. The prototype device integrates paper microfluidics (to enable fluid handling) and a multilayer structure, or a "paper machine", that allows a central patterned paper strip to slide in and out of fluidic path and thus allows introduction of sample, wash buffers, amplification master mix, and detection reagents with minimal pipetting, in a hand-held, disposable device intended for point-of-care use in resource-limited environments. This device creates a dynamic seal that prevents evaporation during incubation at 65 °C for 1 h. This interval is sufficient to allow a LAMP reaction for the Escherichia coli malB gene to proceed with an analytical sensitivity of 1 double-stranded DNA target copy. Starting with human plasma spiked with whole, live E. coli cells, this paper demonstrates full integration of sample preparation with LAMP amplification and end point detection with a limit of detection of 5 cells. Further, it shows that the method used to prepare sample enables concentration of DNA from sample volumes commonly available from fingerstick blood draw.

From the bench to the field in low-cost diagnostics: two case studies.

Despite the growth of research in universities on point-of-care (POC) diagnostics for global health, most devices never leave the laboratory. The processes that move diagnostic technology from the laboratory to the field--the processes intended to evaluate operation and performance under realistic conditions--are more complicated than they might seem. Two case studies illustrate this process: the development of a paper-based device to measure liver function, and the development of a device to identify sickle cell disease based on aqueous multiphase systems (AMPS) and differences in the densities of normal and sickled cells. Details of developing these devices provide strategies for forming partnerships, prototyping devices, designing studies, and evaluating POC diagnostics. Technical and procedural lessons drawn from these experiences may be useful to those designing diagnostic tests for developing countries, and more generally, technologies for use in resource-limited environments.

Additive manufacturing. Continuous liquid interface production of 3D objects.

Additive manufacturing processes such as 3D printing use time-consuming, stepwise layer-by-layer approaches to object fabrication. We demonstrate the continuous generation of monolithic polymeric parts up to tens of centimeters in size with feature resolution below 100 micrometers. Continuous liquid interface production is achieved with an oxygen-permeable window below the ultraviolet image projection plane, which creates a "dead zone" (persistent liquid interface) where photopolymerization is inhibited between the window and the polymerizing part. We delineate critical control parameters and show that complex solid parts can be drawn out of the resin at rates of hundreds of millimeters per hour. These print speeds allow parts to be produced in minutes instead of hours.

Evaporative concentration on a paper-based device to concentrate analytes in a biological fluid.

We report the first demonstration of using heat on a paper device to rapidly concentrate a clinically relevant analyte of interest from a biological fluid. Our technology relies on the application of localized heat to a paper strip to evaporate off hundreds of microliters of liquid to concentrate the target analyte. This method can be used to enrich for a target analyte that is present at low concentrations within a biological fluid to enhance the sensitivity of downstream detection methods. We demonstrate our method by concentrating the tuberculosis-specific glycolipid, lipoarabinomannan (LAM), a promising urinary biomarker for the detection and diagnosis of tuberculosis. We show that the heat does not compromise the subsequent immunodetectability of LAM, and in 20 min, the tuberculosis biomarker was concentrated by nearly 20-fold in simulated urine. Our method requires only 500 mW of power, and sample flow is self-driven via capillary action. As such, our technology can be readily integrated into portable, battery-powered, instrument-free diagnostic devices intended for use in low-resource settings.

A device architecture for three-dimensional, patterned paper immunoassays.

Diagnostic assays can provide valuable information about the health status of a patient, which include detection of biomarkers that indicate the presence of an infection, the progression or regression of a disease, and the efficacy of a course of treatment. Critical healthcare decisions must often be made at the point-of-care, far from the infrastructure and diagnostic capabilities of centralized laboratories. There exists an obvious need for diagnostic tools that are designed to address the unique challenges encountered by healthcare workers in limited-resource settings. Paper, a readily-available and inexpensive commodity, is an attractive medium with which to develop diagnostic assays for use in limited-resource settings. In this article, we describe a device architecture to perform immunoassays in patterned paper. These paper-based devices use a combination of lateral and vertical flow to control the wicking of fluid in three-dimensions. We provide guidelines to aid in the design of these devices and we illustrate how patterning can be used to tune the duration and performance of the assay. We demonstrate the use of these paper-based devices by developing a sandwich immunoassay for human chorionic gonadotropin (hCG) in urine, a biomarker of pregnancy. We then directly compare the qualitative and quantitative results of these paper-based immunoassays to commercially available lateral flow tests (i.e., the home pregnancy test). Our results suggest paper-based devices may find broad utility in the development of immunoassays for use at the point-of-care.

Field evaluation of a prototype paper-based point-of-care fingerstick transaminase test.

Monitoring for drug-induced liver injury (DILI) via serial transaminase measurements in patients on potentially hepatotoxic medications (e.g., for HIV and tuberculosis) is routine in resource-rich nations, but often unavailable in resource-limited settings. Towards enabling universal access to affordable point-of-care (POC) screening for DILI, we have performed the first field evaluation of a paper-based, microfluidic fingerstick test for rapid, semi-quantitative, visual measurement of blood alanine aminotransferase (ALT). Our objectives were to assess operational feasibility, inter-operator variability, lot variability, device failure rate, and accuracy, to inform device modification for further field testing. The paper-based ALT test was performed at POC on fingerstick samples from 600 outpatients receiving HIV treatment in Vietnam. Results, read independently by two clinic nurses, were compared with gold-standard automated (Roche Cobas) results from venipuncture samples obtained in parallel. Two device lots were used sequentially. We demonstrated high inter-operator agreement, with 96.3% (95% C.I., 94.3-97.7%) agreement in placing visual results into clinically-defined "bins" (<3x, 3-5x, and >5x upper limit of normal), >90% agreement in validity determination, and intraclass correlation coefficient of 0.89 (95% C.I., 0.87-0.91). Lot variability was observed in % invalids due to hemolysis (21.1% for Lot 1, 1.6% for Lot 2) and correlated with lots of incorporated plasma separation membranes. Invalid rates <1% were observed for all other device controls. Overall bin placement accuracy for the two readers was 84% (84.3%/83.6%). Our findings of extremely high inter-operator agreement for visual reading-obtained in a target clinical environment, as performed by local practitioners-indicate that the device operation and reading process is feasible and reproducible. Bin placement accuracy and lot-to-lot variability data identified specific targets for device optimization and material quality control. This is the first field study performed with a patterned paper-based microfluidic device and opens the door to development of similar assays for other important analytes.

A point-of-care paper-based fingerstick transaminase test: toward low-cost "lab-on-a-chip" technology for the developing world.

There is currently great need for high-quality, low-cost, point-of-care diagnostics that can benefit patients in resource-limited settings and correspondingly growing interest in the diagnostic utility of microfluidic platforms that are based on paper. We describe the development, early clinical testing, and potential clinical impact of a novel paper-based, multiplexed microfluidic assay designed for rapid, semiquantitative measurement of aspartate aminotransferase and alanine aminotransferase in a fingerstick specimen. This device ultimately holds promise for providing universal access to affordable point-of-care screening for drug-induced liver injury in resource-limited settings and opens the door to development of similar point-of-care clinical assays for other important analytes.

A paper-based multiplexed transaminase test for low-cost, point-of-care liver function testing.

In developed nations, monitoring for drug-induced liver injury through serial measurements of serum transaminases [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] in at-risk individuals is the standard of care. Despite the need, monitoring for drug-related hepatotoxicity in resource-limited settings is often limited by expense and logistics, even for patients at highest risk. This article describes the development and clinical testing of a paper-based, multiplexed microfluidic assay designed for rapid, semiquantitative measurement of AST and ALT in a fingerstick specimen. Using 223 clinical specimens obtained by venipuncture and 10 fingerstick specimens from healthy volunteers, we have shown that our assay can, in 15 min, provide visual measurements of AST and ALT in whole blood or serum, which allow the user to place those values into one of three readout "bins" [<3× upper limit of normal (ULN), 3 to 5× ULN, and >5× ULN, corresponding to tuberculosis/HIV treatment guidelines] with >90% accuracy. These data suggest that the ultimate point-of-care fingerstick device will have high impact on patient care in low-resource settings.

Reductively responsive siRNA-conjugated hydrogel nanoparticles for gene silencing.

A critical need still remains for effective delivery of RNA interference (RNAi) therapeutics to target tissues and cells. Self-assembled lipid- and polymer-based systems have been most extensively explored for transfection with small interfering RNA (siRNA) in liver and cancer therapies. Safety and compatibility of materials implemented in delivery systems must be ensured to maximize therapeutic indices. Hydrogel nanoparticles of defined dimensions and compositions, prepared via a particle molding process that is a unique off-shoot of soft lithography known as particle replication in nonwetting templates (PRINT), were explored in these studies as delivery vectors. Initially, siRNA was encapsulated in particles through electrostatic association and physical entrapment. Dose-dependent gene silencing was elicited by PEGylated hydrogels at low siRNA doses without cytotoxicity. To prevent disassociation of cargo from particles after systemic administration or during postfabrication processing for surface functionalization, a polymerizable siRNA pro-drug conjugate with a degradable, disulfide linkage was prepared. Triggered release of siRNA from the pro-drug hydrogels was observed under a reducing environment while cargo retention and integrity were maintained under physiological conditions. Gene silencing efficiency and cytocompatibility were optimized by screening the amine content of the particles. When appropriate control siRNA cargos were loaded into hydrogels, gene knockdown was only encountered for hydrogels containing releasable, target-specific siRNAs, accompanied by minimal cell death. Further investigation into shape, size, and surface decoration of siRNA-conjugated hydrogels should enable efficacious targeted in vivo RNAi therapies.

Delivery of multiple siRNAs using lipid-coated PLGA nanoparticles for treatment of prostate cancer.

Nanotechnology can provide a critical advantage in developing strategies for cancer management and treatment by helping to improve the safety and efficacy of novel therapeutic delivery vehicles. This paper reports the fabrication of poly(lactic acid-co-glycolic acid)/siRNA nanoparticles coated with lipids for use as prostate cancer therapeutics made via a unique soft lithography particle molding process called Particle Replication In Nonwetting Templates (PRINT). The PRINT process enables high encapsulation efficiency of siRNA into neutral and monodisperse PLGA particles (32-46% encapsulation efficiency). Lipid-coated PLGA/siRNA PRINT particles were used to deliver therapeutic siRNA in vitro to knockdown genes relevant to prostate cancer.

Micromolding for the fabrication of biological microarrays.

The PRINT(®) (pattern replication in non-wetting templates) process has been developed as a simple, gentle way to pattern films or generate discrete particles in arrays out of either pure biological materials or biomolecules encapsulated within polymeric materials. Patterned films and particle arrays can be fabricated in a wide array of sizes and shapes using Fluorocur(®) (a UV-curable perfluoropolyether polymer) from the nanometer to micron scale.

Scalable, shape-specific, top-down fabrication methods for the synthesis of engineered colloidal particles.

The search for a method to fabricate nonspherical colloidal particles from a variety of materials is of growing interest. As the commercialization of nanotechnology continues to expand, the ability to translate particle-fabrication methods from a laboratory to an industrial scale is of increasing significance. In this feature article, we examine several of the most readily scalable top-down methods for the fabrication of such shape-specific particles and compare their capabilities with respect to particle composition, size, shape, and complexity as well as the scalability of the method. We offer an extensive examination of particle replication in nonwetting templates (PRINT) with regard to the versatility and scalability of this technique. We also detail the specific methods used in PRINT particle fabrication, including harvesting, purification, and surface-modification techniques, with an examination of both past and current methods.

Monolithic photolithographically patterned Fluorocur PFPE membrane valves and pumps for in situ planetary exploration.

Photolithographically defined monolithic membrane valves utilizing Fluorocur perfluoropolyether (PFPE) were fabricated and characterized to be essentially unaltered after one million actuations and exposure to the environmental stresses associated with in situ exploration of Mars.

Molded, high surface area polymer electrolyte membranes from cured liquid precursors.

Polymer electrolyte membranes (PEMs) for fuel cells have been synthesized from easily processable, 100% curable, low molecular weight reactive liquid precursors that are photochemically cured into highly proton conductive solid membranes. The liquid precursors were directly cured into membranes of desired dimensions without the need for further processing steps such as melt extrusion or solvent casting. By employing chemical cross-linking, high proton conductivities can be achieved through the incorporation of significant levels of acidic groups without rendering the material water-soluble, which plagues commonly used non-cross-linked polymers. Fabrication of membrane electrode assemblies (MEAs) from these PEMs resulted in fuel cells that outperformed those based on commercial materials. Moreover, these liquid precursors enabled the formation of three-dimensional, patterned PEMs with high fidelity, micron-scale features by using soft lithographic/micromolding techniques. The patterned membranes provided a larger interfacial area between the membrane and catalyst layer than standard flat PEMs. MEAs composed of the patterned membranes demonstrated higher power densities over that of flat ones without an increase in the macroscopic area of the fuel cells. This can potentially miniaturize fuel cells and promote their application in portable devices.

Direct fabrication and harvesting of monodisperse, shape-specific nanobiomaterials.

A versatile "top-down" method for the fabrication of particles, Particle Replication In Nonwetting Templates (PRINT), is described which affords absolute control over particle size, shape, and composition. This technique is versatile and general enough to fabricate particles with a variety of chemical structures, yet delicate enough to be compatible with sophisticated biological agents. Using PRINT, we have fabricated monodisperse particles of poly(ethylene glycol diacrylate), triacrylate resin, poly(lactic acid), and poly(pyrrole). Monodisperse particle populations, ranging from sub-200 nm nanoparticles to complex micron-scale objects, have been fabricated and harvested. PRINT uses low-surface energy, chemically resistant fluoropolymers as molding materials, which eliminates the formation of a residual interconnecting film between molded objects. Until now, the presence of this film has largely prevented particle fabrication using soft lithography. Importantly, we have demonstrated that PRINT affords the simple, straightforward encapsulation of a variety of important bioactive agents, including proteins, DNA, and small-molecule therapeutics, which indicates that PRINT can be used to fabricate next-generation particulate drug-delivery agents.

Fabrication of ultramicroelectrodes using a "teflon-like" coating material.

A new method was developed for the preparation of ultramicroelectrodes (UMEs). In this method, a photo-curable fluorinated functionalized perfluoropolyether, a liquid v-like polymer at room temperature, is used as the material for the insulation of metal microwires or carbon fibers. The UMEs prepared by this method were usually conical shaped and could be used both in aqueous solutions and with aprotic solvents. The relatively small thicknesses of the insulating sheath of the "Teflon-like"UME tips make them particularly useful for scanning electrochemical microscopy measurements.