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.

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.

Lateral flow tests and hand-held analyzers facilitate diagnostic testing in resource limited settings and at the point-of-care. However, many of these devices require sample preparation such as plasma separation to remove cells and isolate the liquid portion of blood. Specifically, the separation of plasma from blood is necessary for routine health assessments such as comprehensive metabolic panels and chronic HIV viral load monitoring. Away from laboratories, this type of processing has been addressed by unconventional, hand-operated centrifuge devices (high volume) or plasma separation membranes (PSM) coupled with lateral flow tests (low volume). Herein, we describe a device that separates and stores plasma from undiluted blood using only passive filtration in less than 10 min. Integrating a PSM with a prefilter and absorbent material yields a 3-fold increase in separation efficiency compared to similar devices using passive filtration. We demonstrate the reproducibility of our device across the physiological range of hematocrits (20−50%) with an average recovered plasma volume of 61.7 ± 2.6 μL. Maximum separation efficiency (53.8%, 65.6 ± 3.9 μL plasma) was achieved for a sample of whole blood (30% hematocrit) in 10 min. We evaluate the purity of our plasma sample by quantitation of hemoglobin and report hemolysis as either minimal (≤5%) or undetectable (≤1%). Specific recovery of human IgG, IFN-γ, and HIV-1 RNA indicate the diagnostic utility of plasma obtained from our device is unchanged compared to plasma obtained via centrifugation. Finally, we demonstrate the use of recovered plasma, applied via "stamping", to successfully conduct a commercial lateral flow immunochromatographic assay for tetanus antibodies. This device platform is capable of producing pure plasma samples from blood to facilitate tests in resource limited settings to improve access to healthcare.

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