We report the first demonstration of electrochemical detection for paper-based microfluidic devices. Photolithography was used to make microfluidic channels on filter paper, and screen-printing technology was used to fabricate electrodes on the paper-based microfluidic devices. Screen-printed electrodes on paper were characterized using cyclic voltammetry to demonstrate the basic electrochemical performance of the system. The utility of our devices was then demonstrated with the determination of glucose, lactate, and uric acid in biological samples using oxidase enzyme (glucose oxidase, lactate oxidase, and uricase, respectively) reactions. Oxidase enzyme reactions produce H2O2 while decomposing their respective substrates, and therefore a single electrode type is needed for detection of multiple species. Selectivity of the working electrode for H2O2 was improved using Prussian Blue as a redox mediator. The determination of glucose, lactate, and uric acid in control serum samples was performed using chronoamperometry at the optimal detection potential for H2O2 (0 V versus the on-chip Ag/AgCl reference electrode). Levels of glucose and lactate in control serum samples measured using the paper devices were 4.9 +/- 0.6 and 1.2 +/- 0.2 mM (level I control sample), and 16.3 +/- 0.7 and 3.2 +/- 0.3 mM (level II control sample), respectively, and were within error of the values measured using traditional tests. This study shows the successful integration of paper-based microfluidics and electrochemical detection as an easy-to-use, inexpensive, and portable alternative for point of care monitoring.

Wax screen-printing as a low-cost, simple, and rapid method for fabricating paper-based microfluidic devices (µPADs) is reported here. Solid wax was rubbed through a screen onto paper filters. The printed wax was then melted into the paper to form hydrophobic barriers using only a hot plate. We first studied the relationship between the width of a hydrophobic barrier and the width of the original design line. We also optimized the heating temperature and time and determined the resolution of structures fabricated using this technique. The minimum width of hydrophilic channel and hydrophobic barrier is 650 and 1300 µm, respectively. Next, our fabrication method was compared to a photolithographic method using the reaction between bicinchoninic acid (BCA) and Cu(1+) to demonstrate differences in background reactivity. Photolithographically defined channels exhibited a high background while wax printed channels showed a very low background. Finally, the utility of wax screen-printing was demonstrated for the simultaneous determination of glucose and total iron in control human serum samples using an electrochemical method with glucose oxidase and a colorimetric method with 1,10-phenanthroline. This study demonstrates that wax screen-printing is an easy-to-use and inexpensive alternative fabrication method for µPAD, which will be especially useful in developing countries.

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The development of simple fluorescent and colorimetric assays that enable point-of-care DNA and RNA detection has been a topic of significant research because of the utility of such assays in resource limited settings. The most common motifs utilize hybridization to a complementary detection strand coupled with a sensitive reporter molecule. Here, a paper-based colorimetric assay for DNA detection based on pyrrolidinyl peptide nucleic acid (acpcPNA)-induced nanoparticle aggregation is reported as an alternative to traditional colorimetric approaches. PNA probes are an attractive alternative to DNA and RNA probes because they are chemically and biologically stable, easily synthesized, and hybridize efficiently with the complementary DNA strands. The acpcPNA probe contains a single positive charge from the lysine at C-terminus and causes aggregation of citrate anion-stabilized silver nanoparticles (AgNPs) in the absence of complementary DNA. In the presence of target DNA, formation of the anionic DNA-acpcPNA duplex results in dispersion of the AgNPs as a result of electrostatic repulsion, giving rise to a detectable color change. Factors affecting the sensitivity and selectivity of this assay were investigated, including ionic strength, AgNP concentration, PNA concentration, and DNA strand mismatches. The method was used for screening of synthetic Middle East respiratory syndrome coronavirus (MERS-CoV), Mycobacterium tuberculosis (MTB), and human papillomavirus (HPV) DNA based on a colorimetric paper-based analytical device developed using the aforementioned principle. The oligonucleotide targets were detected by measuring the color change of AgNPs, giving detection limits of 1.53 (MERS-CoV), 1.27 (MTB), and 1.03 nM (HPV). The acpcPNA probe exhibited high selectivity for the complementary oligonucleotides over single-base-mismatch, two-base-mismatch, and noncomplementary DNA targets. The proposed paper-based colorimetric DNA sensor has potential to be an alternative approach for simple, rapid, sensitive, and selective DNA detection.

A microfluidic paper-based analytical device (μPAD) for the separation of blood plasma from whole blood is described. The device can separate plasma from whole blood and quantify plasma proteins in a single step. The μPAD was fabricated using the wax dipping method, and the final device was composed of a blood separation membrane combined with patterned Whatman No.1 paper. Blood separation membranes, LF1, MF1, VF1 and VF2 were tested for blood separation on the μPAD. The LF1 membrane was found to be the most suitable for blood separations when fabricating the μPAD by wax dipping. For blood separation, the blood cells (both red and white) were trapped on blood separation membrane allowing pure plasma to flow to the detection zone by capillary force. The LF1-μPAD was shown to be functional with human whole blood of 24-55% hematocrit without dilution, and effectively separated blood cells from plasma within 2 min when blood volumes of between 15-22 μL were added to the device. Microscopy was used to confirm that the device isolated plasma with high purity with no blood cells or cell hemolysis in the detection zone. The efficiency of blood separation on the μPAD was studied by plasma protein detection using the bromocresol green (BCG) colorimetric assay. The results revealed that protein detection on the μPAD was not significantly different from the conventional method (p > 0.05, pair t-test). The colorimetric measurement reproducibility on the μPAD was 2.62% (n = 10) and 5.84% (n = 30) for within-day and between day precision, respectively. Our proposed blood separation on μPAD has the potential for reducing turnaround time, sample volume, sample preparation and detection processes for clinical diagnosis and point-of care testing.

The release of metals and metal-containing compounds into the environment is a growing concern in developed and developing countries, as human exposure to metals is associated with adverse health effects in virtually every organ system. Unfortunately, quantifying metals in the environment is expensive; analysis costs using certified laboratories typically exceed $100/sample, making the routine analysis of toxic metals cost-prohibitive for applications such as occupational exposure or environmental protection. Here, we report on a simple, inexpensive technology with the potential to render toxic metals detection accessible for both the developing and developed world that combines colorimetric and electrochemical microfluidic paper-based analytical devices (mPAD) in a three-dimensional configuration. Unlike previous mPADs designed for measuring metals, the device reported here separates colorimetric detection on one layer from electrochemical detection on a different layer. Separate detection layers allows different chemistries to be applied to a single sample on the same device. To demonstrate the effectiveness of this approach, colorimetric detection is shown for Ni, Fe, Cu, and Cr and electrochemical detection for Pb and Cd. Detection limits as low as 0.12 μg (Cr) were achieved on the colorimetric layer while detection limits as low as 0.25 ng (Cd and Pb) were achieved on the electrochemical layer. Selectivity for the target analytes was demonstrated for common interferences. As an example of the device utility, particulate metals collected on air sampling filters were analyzed. Levels measured with the mPAD matched known values for the certified reference samples of collected particulate matter.

We report a comparative study of the structure and chemistry of methyl-terminated n-alkanethiol selfassembling monolayers (SAMs) formed from liquid and vapor phases. Three different SAMs are considered: Au/ HS(CH2)"CH3, n = 5, 11, and 15. Liquid-phase-deposited films were prepared by exposure of Au substrates to dilute ethanol solutions of the n-alkanethiols followed by ethanol rinsing, and vapor-phase-deposited SAMs were prepared by exposure of the Au surface to 10%-of-saturation n-alkanethiol vapors followed by N2 purging, which removes loosely bound n-alkanethiol molecules from the surface. The matrix of six organic surfaces was studied by FTIR external reflectance spectroscopy (FTIR-ERS), ellipsometry, and cyclic voltammetry, which provide information about the average structure of the SAMs, and a newly developed scanning tunneling microscope (STM)-based method, which provides information about individual SAM defect structures. FTIR-ERS and ellipsometry indicate no detectable differences between liquid-and vapor-phase-deposited SAMs. Data obtained using cyclic voltammetry and STM show that the barrier properties of SAMs depend on the ambient phase from which the SAM assembles, the length of the rt-alkanethiol, and the chemical nature of the molecular probe used to evaluate the monolayer structure. For example, vapor-phase-deposited Au/HS(CH2)nCH3 SAMs are better CN mass-transfer barriers than their liquid-phasedeposited analogs. However, Au/HS(CH2)]5CH3 SAMs are better CNbarriers when they are formed from the liquid phase. In contrast, Au/HS(CH2)nCH3 SAMs prepared from either phase present nearly identical barriers to electron exchange between the Au surface and solution-phase Ru(NH3)63+. STM reveals some of the nanostructural details of SAMs and confirms that individual defects govern their barrier properties.