Thread- and Capillary Tube-Based Electrodes for the Detection of Glucose and Acetylthiocholine.

An electrochemical sensor for the detection of glucose and acetylthiocholine (ATC) using thread- and capillary tube-based electrodes is described. Three nylon thread-based electrodes were fabricated by painting pieces of trifurcated nylon thread with conductive inks and threading the electrodes into capillary tubes. Two platforms, one paper-based and the other utilizing bubble wrap, were examined. For the glucose detection, a solution containing glucose oxidase (GOx), potassium ferricyanide (K3[Fe(CN)6]), and increasing concentrations of glucose (0-20 mM) in phosphate-buffered saline (PBS) was spotted onto the two platforms. Similarly, increasing concentrations of ATC (0-9.84 mg/mL) in acetylcholinesterase (AChE) (0.08 U/mL) and PBS solution were detected. Using cyclic voltammetry (CV), a scanning voltage was applied to yield a graph of voltage applied (V) vs. current output (A). For both platforms, both glucose and ATC concentrations were observed to be linearly proportional to the current output as demonstrated by the increased height of the oxidation peaks. The three-electrode system was simple to fabricate, inexpensive, and could be used for multiple readings.

Production of a NiO/Al primary battery employing powder-based electrodes.

This paper describes the use of aluminum and zinc as anodic materials for a battery employing nickel (II) oxide (NiO) as cathode. Comparison of both materials resulted in the development of a compact, cost effective, and easy to use primary NiO/Al battery employing an alkaline electrolyte. The system features electrodes composed of powder forms of the active materials on modified paper substrates that are contained in a simple multilayer design utilizing thin laminated plastic materials to provide structure and flexibility to the battery as well as a paper separator. Various concentrations of potassium hydroxide (KOH) electrolyte were examined and maximum performance was observed at 6 M KOH. A maximum current density and power density of 1.94 mA/cm2 and 1 mW/cm2 , respectively was achieved. This user-friendly device was able to produce a maximum capacity of 2.33 mAh/g when 2 mA/g was applied. This work demonstrates the viability of a paper-based battery featuring powder electrodes as a possible power source for microelectronic devices.

Cord-Based Microfluidic Chips as A Platform for ELISA and Glucose Assays.

This paper describes the development and application of microfluidic cord-based analytical devices (µCADs) in two enzyme-linked immunosorbent assays (ELISAs) and glucose assay. In this study, biotinylated goat anti-mouse immunoglobulin (IgG) antibody, rabbit IgG antibody, and glucose are quantitatively detected. In the ELISA systems, the antibody is spotted on the cord at the detection site and a series of washes, followed by streptavidin-alkaline phosphatase (Strep-ALP) or alkaline phosphatase (ALP)-conjugated secondary antibody and colorimetric substrate, completing the experiment. The devices are subsequently scanned and analyzed yielding a correlation between inverse yellow or inverse blue intensity and antibody concentration. For the first ELISA, a linear range of detection was observed at lower concentrations (2.50 × 10-4-1.75 × 10-3 mg/mL) of Strep-ALP with saturation of the enzyme achieved at higher concentrations (>2.50 × 10-4). For the second ELISA, the L50 was demonstrated to be 167.6 fmol/zone. The glucose assay consisted of spotting increasing concentrations of glucose on the analysis sites and transporting, via capillary action, a solution containing glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI) to the detection sites realizing a yellow-brown color indicating oxidation of iodide to iodine. The device was then dried, scanned, and analyzed to show the correlation between yellow inverse intensity and glucose. Glucose in artificial urine showed good correlation using the devices.

Paper-based microfluidic devices for glucose assays employing a metal-organic framework (MOF).

This paper describes the development of two microfluidic paper-based analytical devices (µPADs), one well-based and the other based on a lateral flow assay (LFA) configuration, to detect glucose via a colorimetric assay using the solid metal-organic framework (MOF) Zr-PCN-222(Fe), to encapsulate glucose oxidase (GOx). The well-based platform consisted of laminate sheets and multiple layers of wax-printed chromatography paper. Solutions of KI and glucose placed into the well flowed through the device and reacted with the GOx@MOF species sandwiched between the paper layers realizing a yellow-brown color. The LFA platform consisted of chromatography paper between parafilm and polyvinyl acetate (PVA) layers. GOx@MOFs spotted on the paper subjected to solutions of KI and glucose yielded a brown color. The devices were then dried, scanned, and analyzed yielding a correlation between average inverse yellow intensity and glucose concentrations. The development of these devices employing MOFs as biomimetic catalysts should further expand the applications of microfluidic technologies for sensors a variety of analytes.

A colorimetric assay system for dopamine using microfluidic paper-based analytical devices.

We report the colorimetric detection of dopamine (DA) on microfluidic paper-based analytical devices (µPADs) using an oxidation-reduction method. Here, dopamine reacts with ferric chloride forming reduced Fe2+ that subsequently reacts with phenanthroline to form the red tris(1,10-phenanthroline)iron(II) complex. The devices were fabricated by wax printing and changes in color intensity were recorded using a common cell phone. Subsequent analysis using Photoshop software, yielded a limit of detection (LOD) for DA of 0.37 µmol/L with a linear range of 0.527-4.75 µmol/L and relative standard deviation of 0.11% (inter-day) and 0.15% (intra-day) for n = 15 paper chips. The effects of detection conditions have been investigated and are discussed. Cow serum samples and human blood serum and plasma samples were detected. The work, herein, demonstrates the potential of this method as a low cost and rapid colorimetric technique to detect DA in real samples.

An optimized microfluidic paper-based NiOOH/Zn alkaline battery.

In this paper, an alkaline nickel oxide hydroxide/zinc (NiOOH/Zn) battery featuring a cellulose matrix separator between electrodes is presented. The metallic electrodes and the paper separator are inserted in a layer-by-layer assembly that provides mechanical stability to the system resulting in a lightweight and easy-to-use device. The battery was optimized for the amount of NiOOH-ink used at the cathode (11.1 mg/cm2 ) and thickness of the paper membrane separating the electrodes (360 µm). The battery was able to function using a small volume (75 µL) of 1.5 M potassium hydroxide (KOH) producing a maximum voltage, current density, and power density of 1.35 ± 0.05 V, 10.62 ± 0.57 mA/cm², and 0.56 ± 0.01 mW/cm², respectively. The system displayed a maximum current of 23.9 mA and a maximum power of 1.26 mW. Moreover, four batteries connected in series were able to power a small flameless candle for approximately 22 min. This work has potential in fulfilling the demands for short-term and lightweight power supplies.

Microfluidic Paper-based Analytical Devices (µPADs): Miniaturization and Enzyme Storage Studies.

This paper describes the design and development of miniaturized microfluidic paper-based analytical devices (µPADs) for biological assays and enzyme storage instruments. Here, a glucose assay utilizing glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI) is used as the model system. The efficacy of the miniaturized devices is further examined by assessing the activity of acetylcholinesterase (AChE). Two types of µPADs were developed: one, "strip" chips of detection zones of area 0.5, 0.1 cm2 and, two, "grid" chips of detection zone 0.05 cm2. The devices are easily fabricated via a wax printing process whereby lines of wax are deposited onto chromatographic paper and heated to create rows of hydrophobic barriers. The "strip" chips were subjected to three different temperature environments (-20, 0, and 20°C) over 30 days and glucose assays conducted at intermittent times yielding a correlation between corrected average inverse yellow intensity, days, and glucose concentration. Calculated and experimentally derived color intensity values for 1, 4, and 9 mM glucose concentrations after a 7-day storage study showed a good correlation (0.89 - 15.76% error). Both types of µPADs are effective platforms as potential point-of-care (POC) diagnostic devices and display minimal enzyme denaturation. µPADs of this size show promise as alternative devices for resource-limited regions and especially those areas where materials and instrumentation are not always available.

3D Multilayered paper- and thread/paper-based microfluidic devices for bioassays.

In this paper we describe the fabrication of novel 3D microfluidic paper-based analytical devices (3D-µPADs) and a 3D microfluidic thread/paper-based analytical device (3D-µTPAD) to detect glucose and BSA through colorimetric assays. The 3D-µPAD and 3D-µTPAD consisted of three (wax, heat pressed wax-printed paper, single-sided tape) and four (hole-punched single-sided tape, blank chromatography circles, heat-pressed wax-printed paper, hole-punched single-sided tape containing trifurcated thread) layers, respectively. The saturation curves for each assay were generated for all platforms. For the glucose assay, a solution of glucose oxidase (GOx), horseradish peroxidase, and potassium iodide was flowed through each platform and, upon contact with glucose, generated a yellow-brown color indicative of the oxidation of iodide to iodine. For the protein assay, BSA was flowed through each device and, upon contact with citrate buffer and tetrabromophenol blue, resulted in a color change from yellow to blue. The devices were dried, scanned, and analyzed yielding a correlation between either yellow intensity and glucose concentration or cyan intensity and BSA concentration. A similar glucose assay, using unknown concentrations of glucose in artificial urine, was conducted and, when compared to the saturation curve, showed good correlation between the theoretical and actual concentrations (percent differences <10%). The development of 3D-µPADs and 3D-µTPADs can further facilitate the use of these platforms for colorimetric bioassays.

Microfluidic thread-based electrode system to detect glucose and acetylthiocholine.

A reusable and simple to fabricate electrochemical sensor for the detection of glucose and acetylthiocholine using thread-based electrodes and nylon thread is described. The fabrication of the device consisted of two steps. First, three nylon-based electrodes (reference, working, and counter) were painted with one layer of conductive inks (silver and carbon ink, or silver/silver chloride ink). The electrodes were taped onto parafilm, and a piece of white nylon thread was wrapped around each electrode connecting the three electrodes. For the glucose system, a PBS solution containing glucose oxidase (GOx) (10 mg/mL), and potassium ferricyanide (K3 [Fe(CN)6 ]) (10 mg/mL) as mediator, was dried onto the thread, and increasing concentrations of glucose (0-15 mM) was added to the thread and measured by cyclic voltammetry (CV). The current output from the glucose oxidation was proportional to the concentration of glucose. For the second system, a solution of acetylcholinesterase (AChE) (0.08 U/mL) in PBS was added to the nylon thread, and increasing concentrations of acetylthiocholine (ATC) (0-9.84 mg/mL) was added and measured by CV. The current output from the oxidation of thiocholine (produced by AChE reacting with ATC) was proportional to the concentrations of ATC added to the thread. From both systems, a graph of current output versus substrate concentration was produced and fitted with a linear regression line that gave R2 values of 0.985 (GOX /glucose) and 0.995 (AChE/ATC).

A microfluidic glucose sensor incorporating a novel thread-based electrode system.

An electrochemical sensor for the detection of glucose using thread-based electrodes and fabric is described. This device is relatively simple to fabricate and can be used for multiple readings after washing with ethanol. The fabrication of the chip consisted of two steps. First, three thread-based electrodes (reference, working, and counter) were fabricated by painting pieces of nylon thread with either layered silver ink and carbon ink or silver/silver chloride ink. The threads were then woven into a fabric chip with a beeswax barrier molded around the edges in order to prevent leaks from the tested solutions. A thread-based working electrode consisting of one layer of silver underneath two layers of carbon was selected to fabricate the final sensor system. Using the chip, a PBS solution containing glucose oxidase (GOx) (10 mg/mL), potassium ferricyanide (K3 [Fe(CN)6 ]) (10 mg/mL) as mediator, and different concentrations of glucose (0-25 mM), was measured by cyclic voltammetry (CV). It was found that the current output from the oxidation of glucose was proportional to the glucose concentrations. This thread-based electrode system is a viable sensor platform for detecting glucose in the physiological range.

Thread/paper- and paper-based microfluidic devices for glucose assays employing artificial neural networks.

This paper describes the fabrication of and data collection from two microfluidic devices: a microfluidic thread/paper based analytical device (µTPAD) and 3D microfluidic paper-based analytical device (µPAD). Flowing solutions of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI), through each device, on contact with glucose, generated a calibration curve for each platform. The resultant yellow-brown color from the reaction indicates oxidation of iodide to iodine. The devices were dried, scanned, and analyzed yielding a correlation between yellow intensity and glucose concentration. A similar procedure, using an unknown concentration of glucose in artificial urine, is conducted and compared to the calibration curve to obtain the unknown value. Studies to quantify glucose in artificial urine showed good correlation between the theoretical and actual concentrations, as percent differences were ≤13.0%. An ANN was trained on the four-channel CMYK color data from 54 µTPAD and 160 µPAD analysis sites and Pearson correlation coefficients of R = 0.96491 and 0.9739, respectively, were obtained. The ANN was able to correctly classify 94.4% (51 of 54 samples) and 91.2% (146 of 160 samples) of the µTPAD and µPAD analysis sites, respectively. The development of this technology combined with ANN should further facilitate the use of these platforms for colorimetric analysis of other analytes.

Thread- paper, and fabric enzyme-linked immunosorbent assays (ELISA).

Enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins, and glycoproteins in biological samples. While the procedure is routine and straightforward, there are a number of variables (reagent selection, volume measurement, temperature, and time) that if not carefully considered, can affect the test outcome. Herein, we describe the development of microfluidic thread/paper-based analytical devices (µTPAD), microfluidic fabric-based analytical devices (µFAD), and microfluidic thread-based analytical devices (µTAD) as new platforms for ELISA. The quantitative detection of biotinylated goat anti-mouse IgG (system one) and rabbit IgG (system two) antibodies via colorimetric analysis is detailed. We explain the design and fabrication of the devices and the step-by-step protocol for the ELISA. A comparison between the techniques is described and the results obtained from them elucidated.

Enzyme Chemotaxis on Paper-based Devices.

Microfluidics has served as a technology for the design and development of a myriad of devices owing to their reduced reagent consumption rate and short sampling-to-result time. Chemotaxis is the movement of materials, particularly biological species, in response to the influence of chemical stimulation. Herein, we describe, for the first time, chemotactic behavior on a microfluidic paper-based analytical device (µPAD) to afford a distribution of products not obtainable under other (non-µPAD) experimental conditions using as a model enzyme-substrate system glucose oxidase (GOx) and glucose. µPADs are easily fabricated by patterning hydrophobic materials in hydrophilic paper. They are low cost, compatible with biological samples, and have shown promise as platforms for various applications and in resource-limited settings.

Enzyme-linked immunosorbent assays (ELISA) based on thread, paper, and fabric.

This paper describes enzyme-linked immunosorbent assays (ELISAs) utilizing microfluidic thread/paper-based analytical devices (µTPAD), microfluidic fabric-based analytical devices (µFAD), and microfluidic thread-based analytical devices (µTAD). Here, the quantitative detection of biotinylated goat anti-mouse IgG (system one) and rabbit IgG (system two) antibodies via colorimetric analysis is detailed. In both systems, antibody is spotted on the detection site and subjected to a series of washes, addition of streptavidin-alkaline phosphatase (Strep-ALP) (system 1) or alkaline phosphatase (ALP)-conjugated secondary antibody (system 2), and colorimetric substrate. The devices are scanned and analyzed yielding a correlation between inverse yellow (or purple) intensity. For system one, a linear range of detection at low concentrations of streptavidin-alkaline phosphatase (Strep-ALP) was observed befire the enzyme reached a Vmax . At higher concentrations of Strep-ALP, saturation is achieved for both the µTPAD and µFAD devices. For system two, the IC50 values obtained for the non-trifurcated and trifurcated µTADs were determined to be 180.2 fmol/zone and 133.8 fmol/zone, respectively. The IC50 value was demonstrated to be 1034 fmol/zone and 208.6 fmol/zone for the µTPADs and µFADs, respectively. For all devices the lowest concentration of Strep-ALP or rabbit IgG used in the assay was 3.75 × 10-4  mg/mL and 0.7 fmol/zone, respectively. The development of this technology should further facilitate the use of these platforms for ELISA to detect and quantitate antibodies.

Use of chemometrics to optimize a glucose assay on a paper microfluidic platform.

We describe the use of a chemometrics-based computational platform to optimize a glucose assay on a microfluidic paper-based analytical device (µPAD). Glucose is colorimetrically detected in the presence of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI). Using a Y-shaped paper microfluidic chip, the concentration of glucose, volume of reagents, and the length and width of the microfluidic channel were examined. The responses of the microfluidic chips were analyzed at the halfway point of the channel length. Variables affecting the response were screened by using a 24 factorial design, and among them, volume and concentration of the glucose were optimized by applying a rotatable central composite design (CCD). The optimum and experimental responses are 151.58 and 149.80, respectively, with an absolute error of 1.2%.

Fabric-based alkaline direct formate microfluidic fuel cells.

Fabric-based microfluidic fuel cells (MFCs) serve as a novel, cost-efficient alternative to traditional FCs and batteries, since fluids naturally travel across fabric via capillary action, eliminating the need for an external pump and lowering production and operation costs. Building on previous research with Y-shaped paper-based MFCs, fabric-based MFCs mitigate fragility and durability issues caused by long periods of fuel immersion. In this study, we describe a microfluidic fabric-based direct formate fuel cell, with 5 M potassium formate and 30% hydrogen peroxide as the anode fuel and cathode oxidant, respectively. Using a two-strip, stacked design, the optimized parameters include the type of encasement, the barrier, and the fabric type. Surface contact of the fabric and laminate sheet expedited flow and respective chemical reactions. The maximum current (22.83 mA/cm2 ) and power (4.40 mW/cm2 ) densities achieved with a 65% cotton/35% polyester blend material are a respective 8.7% and 32% higher than previous studies with Y-shaped paper-based MFCs. In series configuration, the MFCs generate sufficient energy to power a handheld calculator, a thermometer, and a spectrum of light-emitting diodes.

Thread-based microfluidic chips as a platform to assess acetylcholinesterase activity.

In this paper, a microfluidic thread-based analytical device (µTAD) to assess the activity of acetylcholinesterase (AChE) via colorimetric analylsis is described. Fabrication of the device consists of two platforms, both with a nylon thread trifurcated into three channels terminating at open analysis sites at the end of the thread. 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) was spotted and dried on the analysis sites. Acetylthiocholine iodide (ATC) (or cysteine, Cys) is transported through an inlet channel of the nylon thread by capillary action due to the hydrophilic nature of nylon. AChE is transported through the other inlet channel and mixes with the ATC (or Cys) as they travel up to the analysis sites. As the solution reaches the analysis sites, an intense yellow color change occurs indicating the reaction of the thiol with DTNB to produce the yellow anion TNB2- . The sites are then dried, scanned, yielding a linear range of inverse yellow mean intensity versus substrate concentration. An IC50 value (1.74 nM) with a known inhibitor, neostigmine bromide (NB), is obtained on the device. The multiplex design enables triplicate data collection in a device that is easy to use. µTADs have great potential to be employed in a myriad of tests including point-of-care diagnostic devices for resource-challenged settings.

An Inexpensive Paper-Based Aluminum-Air Battery.

Paper-based batteries are an alternative to traditional batteries due to their low cost, portability, and simplicity to operate. In the present work, we demonstrate an improved and inexpensive paper-based aluminum-air battery employing KOH as the electrolyte with sufficient energy to power small devices. The dimensions of the device, electrode size, and electrolyte concentration were optimized with respect to amperage and reproducibility. The maximum amperage of 17.4 mA and maximum power of 3.0 mW was achieved with a 9 cm² battery with anode and cathode electrode areas of 5.1 cm² and 3.75 cm² respectively, using 1.5 M potassium hydroxide (KOH). In a series configuration, the batteries generate sufficient energy to power light-emitting diodes (LEDs), a flashlight, a glucometer, and a pregnancy test.

A microfluidic paper-based device to assess acetylcholinesterase activity.

Neurotransmitters play key roles in cell-to-cell communication. These chemical messengers are involved in many functional processes, including growth, reproduction, memory, and behavior. In this communication, we describe a novel microfluidic paper-based analytical device (µPAD) to detect acetylcholinesterase (AChE) activity and inhibitor screening through a colorimetric analysis. The µPAD is easily fabricated via a wax printing process whereby wax is deposited onto the surface of chromatographic paper, and heated to create a hydrophobic barrier. Separate solutions of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and samples containing AChE and acetylthiocholine iodide (ATC) (or cysteine, Cys), respectively, are directly spotted onto the µPAD. DTNB and AChE/ATC (or Cys) flow towards each other where a reaction occurs to form the yellow colored 2-nitro-5-thiobenzoic acid anion (TNB2- ). The device is dried, scanned, and analyzed yielding a linear range of average inverse yellow intensities versus substrate concentration. An IC50 value (0.045 nM) with a known inhibitor, neostigmine bromide (NB), is obtained on the device. µPADs are low cost and easy to fabricate and have great potential to quantify neurotransmitter activity.