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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 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).

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.

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.

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.

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%.

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.

Paper-based microfluidic fuel cells (MFCs) are a potential replacement for traditional FCs and batteries due to their low cost, portability, and simplicity to operate. In MFCs, separate solutions of fuel and oxidant migrate through paper due to capillary action and laminar flow and, upon contact with each other and catalyst, produce electricity. In the present work, we describe an improved microfluidic paper-based direct formate FC (DFFC) employing formate and hydrogen peroxide as the anode fuel and cathode oxidant, respectively. The dimensions of the lateral column, current collectors, and cathode were optimized. A maximum power density of 2.53 mW/cm(2) was achieved with a DFFC of surface area 3.0 cm(2) , steel mesh as current collector, 5% carbon to paint mass ratio for cathode electrode and, 30% hydrogen peroxide. The longevity of the MFC's detailed herein is greater than eight hours with continuous flow of streams. In a series configuration, the MFCs generate sufficient energy to power light-emitting diodes and a handheld calculator.

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

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.

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.

We describe the first direct formate fuel cell on a paper microfluidic platform. In traditional membrane-less microfluidic fuel cells (MFCs), external pumping consumes power produced by the fuel cell in order to maintain co-laminar flow of the anode stream and oxidant stream to prevent mixing. However, in paper microfluidics, capillary action drives flow while minimizing stream mixing. In this work, we demonstrate a paper MFC that uses formate and hydrogen peroxide as the anode fuel and cathode oxidant, respectively. Using these materials we achieve a maximum power density of nearly 2.5 mW/mg Pd. In a series configuration, our MFC achieves an open circuit voltage just over 1 V, and in a parallel configuration, short circuit of 20 mA absolute current. We also demonstrate that the MFC does not require continuous flow of fuel and oxidant to produce power. We found that we can pre-saturate the materials on the paper, stop the electrolyte flow, and still produce approximately 0.5 V for 15 min. This type of paper MFC has potential applications in point-of-care diagnostic devices and other electrochemical sensors.

A novel microfluidic paper-based analytical device (μPAD) utilizing a nitrocellulose (NC) membrane to detect IgG antibodies through a colorimetric analysis is described. The μPAD was constructed using layered polyethylene terephthalate (PET) and pressure-sensitive adhesives (PSA). The biotin labeled Goat Anti-Mouse IgG antibody was spotted and dried on the NC channel prior to subjecting it to a series of wash solutions (Tris-tween), increasing concentrations of alkaline phosphatase conjugated to streptavidin (Strep-ALP), and para-nitrophenyl phosphate (p-NPP) realizing a vibrant yellow color. The reaction proceeds for 10 min before applying the p-NPP stop solution. The device was then dried, scanned, and analyzed yielding a linear range of inverse yellow color intensities versus Strep-ALP concentrations. The development of this simple μPAD should further facilitate the use of NC in colorimetric assays to detect and quantitate antibodies.

Osteocalcin (bone gamma-carboxyglutamate protein; BGLAP) is a highly conserved molecule associated with mineralization of bone matrix. It regulates the dynamics of new bone formation and bone resorption. The synthesis of osteocalcin by osteoblasts is regulated by the active form of vitamin D. In this paper, we report the use of two fluorescent-based assays, one bead-based and a second involving an amino-silane surface pre-treatment, to obtain binding constants of 3.53×10 6 M −1 and 3.19×10 6 M −1 , respectively, for the binding of BGLAP to fluorescently labeled monoclonal-anti BGLAP-clone 2D5. These simple microfluidic techniques demonstrate the feasibility of developing microsphere-based and surface electrostatically-attached binding assays to study peptide–antibody interactions. Both techniques utilize sub-microliter volumes of material an important consideration when studying bone turnover markers (BTMs) like BGLAP.

We describe the use of a computational neural network platform to optimize the fluorescence upon binding 5-carboxyfluorescein-d-Ala-d-Ala-d-Ala (5-FAM(DA)3 ) (1) to the antibiotic teicoplanin covalently attached to a glass slide. A three-level response surface experimental design was used as the first stage of investigation. Subsequently, three defined experimental parameters were examined by the neural network approach: (i) the concentration of teicoplanin used to derivatize a glass platform on the microfluidic device, (ii) the time required for the immobilization of teicoplanin on the platform, and (iii) the length of time 1 is allowed to equilibrate with teicoplanin in the microfluidic channel. Optimal neural structure provided a best fit model, both for the training set (r(2) = 0.961) and test set (r(2) = 0.934) data. Model simulated results were experimentally validated with excellent agreement (% difference) between experimental and predicted fluorescence shown, thus demonstrating efficiency of the neural network approach.

Surface plasmon resonance (SPR) spectroscopy is utilized to study in real-time and, by label-free means, the reversible and quasi-irreversible adsorption of small ionic or neutral molecules, pharmaceuticals, and proteins on poly(dimethylsiloxane) (PDMS) surfaces. The SPR sensor is covered with 0.2% (w/v) PDMS in octane. During the timescale of a typical lab-on-a-chip analysis or an electrophoretic separation, it was found that small neutral components containing a hydrophobic part do not adsorb or absorb onto PDMS, while larger, water-soluble polymer-like materials like proteins generally irreversibly adsorb to PDMS. The technique can be used to monitor the kinetics of adsorption and desorption of the molecules. For the non-specific adsorption of teicoplanin to PDMS, a Langmuir-like adsorption isotherm was obtained (Kd = 32 ± 2 μmol L−1).

This paper demonstrates the use of surface plasmon resonance to study adsorption (either reversible or irreversible) of detergents on PDMS surfaces in real time. The surface plasmon resonance measurements can directly provide information about the adsorption/desorption processes of detergents on the surface revealing the durability of the adsorbed layer and the anticipated degree of the EOF. Hydroxypropyl methylcellulose very strongly adsorbs onto PDMS and can be considered both a semipermanent layer and stable semipermanent coating. Adsorbed SDS or CTAB layers were stable for several minutes upon rinsing the surface with solution not containing the detergent. It was shown that SDS coated onto PDMS in microchips has the potential to afford similar separations in PDMS as found in conventional fused silica capillaries.

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.

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.

A novel microfluidic thread/paper-based analytical device (μTPAD) to detect glucose through a colorimetric assay is described. The μTPAD was fabricated from nylon thread trifurcated into three channels terminating at analysis sites comprised of circular zones of chromatography paper, which have previously been spotted with glucose of different concentrations. A solution of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI) is transported via capillary action to the analysis sites where a yellow-brown color is observed indicating oxidation of iodide to iodine. The device was then dried, scanned, and analyzed yielding a correlation between yellow intensity and glucose concentrations. Both a flat platform constructed mainly of tape, and a cone platform constructed from tape and polyvinyl chloride, are described. Studies to quantitate glucose in artificial urine showed good correlation using the μTPAD.

This paper describes the use of a genetically tuned neural network platform to optimize the fluorescence realized upon binding 5-carboxyfluorescein-D-Ala-D-Ala-D-Ala (5-FAM-(D-Ala)(3) ) (1) to the antibiotic teicoplanin from Actinoplanes teichomyceticus electrostatically attached to a microfluidic channel originally modified with 3-aminopropyltriethoxysilane. Here, three parameters: (i) the length of time teicoplanin was in the microchannel; (ii) the length of time 1 was in the microchannel, thereby, in equilibrium with teicoplanin, and; (iii) the amount of time buffer was flushed through the microchannel to wash out any unbound 1 remaining in the channel, are examined at a constant concentration of 1, with neural network methodology applied to optimize fluorescence. Optimal neural structure provided a best fit model, both for the training set (r(2) = 0.985) and testing set (r(2) = 0.967) data. Simulated results were experimentally validated demonstrating efficiency of the neural network approach and proved superior to the use of multiple linear regression and neural networks using standard back propagation.

We describe a paper microfluidic-based enzyme catalyzed double microreactor assay using fluorescent detection. Here, solutions of lactate dehydrogenase (LDH) and diaphorase (DI) were directly spotted onto the microfluidic paper-based analytical device (μPAD). Samples containing lactic acid, resazurin, and nicotinamide adenine dinucleotide oxidized form (NAD(+) ), potassium chloride (KCl), and BSA, in MES buffer were separately spotted onto the μPAD and MES buffer flowed through the device. A cascade reaction occurs upon the sample spot overlapping with LDH to form pyruvate and nicotinamide adenine dinucleotide reduced form (NADH). Subsequently, NADH is used in the conversion of resazurin to fluorescent resorufin by DI. The μPAD avoids the need of surface functionalization or enzyme immobilization steps. These microreactor devices are low cost and easy to fabricate and effect reaction based solely on buffer capillary action.

This paper describes the design, development and application of microfluidic "thin chips" fabricated from PDMS. Thin chips consist of multiple layers of PDMS chemically bonded onto each other. Unlike thicker PDMS chips that suffer from lack of sensitivity due to PDMS absorption in the VIS and UV range, the thinness of these chips allows for the detection of chromophoric species within the microchannel via an external fiber optics detection system. C18-modified reversed-phase silica particles are packed into the microchannel using a temporary taper created by a magnetic valve and separations using both pressure- and electrochromatographic-driven methods are detailed.

The applicability of micellar electrokinetic capillary chromatography (MEKC) for the analysis of temozolomide (TMZ) and its degradants, 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) and 5-amino-imidazole-4-carboxamide (AIC) has been studied. Using short-end injection, the analysis of TMZ and its degradants could be performed within 1.2 min. The obtained precision of migration times was better than 1.6 RSD%, and the limit of quantitation (LOQ) was 0.31-0.93 microg/mL. The therapeutic concentration of TMZ in blood samples can be determined after direct sample injection and conventional on-capillary UV detection. The proposed MEKC method was applied to study the stability of TMZ in water and serum at different pH values. It was established that the half-life of the TMZ in vitro serum at room temperature was 33 min, close to the half-life (28 min) obtained in water at pH 7.9.

Microchip frontal affinity chromatography was demonstrated to estimate the binding of 5-carboxyfluorescin-(D-Ala)(3) (1) to magnetic microbeads derivatized with teicoplanin (Teic) from Actinoplanes teicomyceticus. In this technique, a cross-chip was used whereby the two side channels contained an identical length (1.5 mm) of derivatized Teic microbeads (affinity column) and underivatized beads (control column), respectively. Cylindrical NdFeB magnets were fabricated into the PDMS chips to retain the magnetic beads. Upon application of a voltage, a sample of 1 was continuously introduced into the affinity column followed by a buffer wash and the same sample from the control channel. The extent of interaction between 1 and the two types of beads in either microchannel resulted in differences in migration time of the ligand as detected by fluorescence. This difference was used to obtain a value for the binding constant between 1 and Teic-beads of 5.4 x 10(4) M(-1). This technique reduces the amount of sample needed for the binding assay as compared with conventional frontal affinity chromatography techniques.

The first detailed examination of flow injection-capillary electrophoresis (FI-CE) active parameters and their interactions via response surface methodology (RSM) is presented. Specifically, RSM in the form of a Box-Behnken design was implemented to effectively predict the significance of capillary length, voltage and injection volume on the optimization of an in-house built FI-CE analyzer. Initial studies were performed assessing peak height and peak shape of the model compound N,N-dimethylformamide. Optimum model conditions were then derived and used in the model separation of two small molecules, nicotinamide adenine dinucleotide, reduced form (NADH) and benzenesulfonamide. By implementing the RSM approach, detailed examination of active FI-CE parameters was possible, including the ability to reveal a significant interactive effect. This work is not only highly significant for advancing FI-CE developments, but instructive for investigators actively exploring other coupled analytical techniques and associated experimental parameters.

Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to carboxy-terminal D-Ala-D-Ala intermediates in the peptidoglycan of the cell wall of gram-positive bacteria. In this paper we report the derivatization of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding properties of the microspheres to two D-Ala-D-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled D-Ala-D-Ala-D-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5 x 10(-6) and 3.0 x 10(-6) mol L(-1), respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial detection was determined to be 30 colon-forming units; cfu) of gram-positive bacteria has been demonstrated. A simple microfluidic experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide-antibiotic interaction.

This work expands the knowledge of the use of chemometric response surface methodology (RSM) in optimizing conditions for competitive binding partial filling ACE (PFACE). Specifically, RSM in the form of a Box-Behnken design was implemented in flow-through PFACE (FTPFACE) to effectively predict the significance of injection time, voltage, and neutral ligand (neutral arylsulfonamide) concentration, [L(o)], on protein-neutral ligand binding. Statistical analysis results were used to create a model for response surface prediction via contour and surface plots at a given maximum response (DeltaRMTR) to reach a targeted K(b) = 2.50 x 10(6) M(-1). The adequacy of the model was then validated by experimental runs at the optimal predicted solution (injection time = 2.3 min, voltage = 11.6 kV, [L(o)] = 1.4 microM). The achieved results greatly extend the usefulness of chemometrics in ACE and provide a valuable statistical tool for the study of other receptor-ligand combinations.

This paper shows the applicability of a disposable and inexpensive microfluidic chip for electrochromatographic separations. The chip, recently developed by us for chip-based LC, was fabricated from PDMS incorporating conventional chromatographic RP silica particles (C18) without the use of frits. Three cephalosporin antibiotics were used to demonstrate the applicability of the chip-based chromatographic packing for electrochromatographic determinations. The used sample injection method utilizes hydrodynamic pressure, thereby, reducing the propensity for sample bias during the injection.

This work presents the first known use of response surface methodology (RSM) in electrophoretically mediated microanalysis. This concept is demonstrated by examining the optimization of reaction conditions for the conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced form by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in the conversion of glucose-6-phosphate to 6-phosphogluconate. Experimental factors including voltage, enzyme concentration, and mixing time of reaction at the applied voltage were selected at three levels and tested in a Box-Behnken response surface design. Upon migration in a capillary under CE conditions, plugs of substrate and enzyme are injected separately in buffer and allowed to react at variable conditions. Extent of reaction and product ratios were subsequently determined by CE. The model predicted results are shown to be in good agreement (7.1% discrepancy difference) with experimental data. The use of chemometric RSM provides a direct relationship between electrophoretic conditions and product distribution of microscale reactions using CE, thereby offering a new and versatile approach to optimizing enzymatic experimental conditions.

Vancomycin (Van) from Streptomyces orientalis has been derivatized with polyethylene glycol [PEG; PEG-550 (1), 750 (2), 1,100 (3), 2,000 (4), 5,000 (5), and 8,000 (6) g mol−1] at the N-terminus of the glycopeptide backbone and their binding to d-Ala-d-Ala terminus peptides assessed using affinity capillary electrophoresis (ACE). Utilizing ACE, a plug of Van-PEG and non-interacting standards are injected and electrophoresed. Analysis of the change in the relative migration time ratio of the Van-PEG species, relative to the non-interacting standards, as a function of the concentration of peptide, yields a value for the binding constant (K b). Values of K b for N-acetyl-d-Ala-d-Ala, 7 to the Van-PEG derivatives are weaker than those for N α,N e-diacetyl-Lys-d-Ala-d-Ala, 8 (for example, values of K b for 7-1 and 8-1 are 1.8 and 47.7 × 103 M−1, respectively). These results demonstrate that derivatization of Van with PEG has little effect on the affinity of d-Ala-d-Ala peptide ligands to it. The findings further prove the versatility of ACE and its ability to estimate binding parameters of ligands to antibiotics.

Multiple-injection affinity capillary electrophoresis (MIACE) was used to determine binding constants ( K b ) between vancomycin, ristocetin, and teicoplanin from Streptomyces orientalis , Nocardia lurida , and Actinoplanes teichomyceticus , respectively, and fluorenylmethoxycarbonyl (Fmoc)-(Gly, Ala, Val, and Phe)- d -Ala- d -Ala peptides. In this technique, separate plugs of sample containing non-interacting standards, peptide one, buffer, and peptide two, were injected into the capillary column and electrophoresed. Peptides migrate through the column at similar electrophoretic mobilities but remain as distinct zones due to the buffer plug between peptides. The electrophoresis is then carried out in an increasing concentration of antibiotic in the running buffer. Continued electrophoresis results in a shift in the migration time of the peptides upon binding to the antibiotic. Analysis of the change in the relative migration time ratio (RMTR) of the resultant complexes relative to the non-interacting standards, as a function of the concentration of antibiotic yields a value for K b . MIACE is a versatile technique that can be used to measure affinity constants between ligands of similar relative molecular mass and charge without the need of separate binding experiments. The findings described, herein, demonstrate the advantages of using MIACE to estimate binding parameters between ligands and receptors.

This work details the determination of the minimal injection time of ligand required in flow-through partial-filling affinity capillary electrophoresis (FTPFACE) to estimate binding constants of ligands to receptors. Two model systems are examined in this study: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, and vancomycin from Streptomyces orientalis and d -Ala- d -Ala peptides. Using CAB, a minimal injection time of 0.07 min at high pressure was determined that provided for the accurate and reproducible measurement of binding constants. In the FTPFACE technique, the capillary is first partially filled with a zone of ligand followed by a sample plug containing receptor and non-interacting standards. Upon application of a voltage the receptor and standards flow into the zone of ligand where a dynamic equilibrium is achieved between receptor and ligand. Continued electrophoresis results in the receptor and standards flowing through the domain of the ligand plug prior to detection. Analysis of the change in the relative migration time ratio (RMTR) of the receptor, relative to the non-interacting standards, as a function of the concentration of ligand, yields a value for the binding constant. In the present study, variable injection times of 4-carboxybenzenesulfonamide (CBSA) were examined to determine the minimal injection time needed to establish an equilibrium between CAB and ligand. A mathematical relationship was derived that correlated injection time and ligand concentration to the change in RMTR and comparisons made between the experimental and calculated values. Binding constants were obtained for a series of arylsulfonamide ligands and d -Ala- d -Ala terminus peptides to CAB and Van, respectively. The results support the use of FTPFACE to estimate affinity constants under variable experimental conditions.

Binding constants were determined for the activator fructose-6-phosphate (F6P) and substrate adenosine 5'-triphosphate (ATP) (in the presence and absence of F6P) to the recombinant wild-type (WT) Rhodobacter sphaeroides adenosine 5'-diphosphate-(ADP)-glucose pyrophosphorylase (ADPGlc PPase) using affinity capillary electrophoresis (ACE). In these binding studies, the capillary is initially injected with a plug of sample containing ADPGlc PPase and noninteracting standards. The sample is then subjected to increasing concentrations of F6P or ATP in the running buffer and electrophoresed. Analysis of the change in the migration times of ADPGlc PPase, relative to those of the noninteracting standards, as a function of the varying concentration of F6P or ATP yields a binding constant. The values obtained were in good agreement with kinetic parameters obtained from steady state activity assays. The method was extended to examine the F6P binding constants for the R33A and R22A enzymes and the ATP binding constants for the R8A enzyme in the presence and absence of F6P. The R33A enzyme has been shown by activity assays to be insensitive to F6P activation, indicating a defect in binding or in downstream transmission of the allosteric signal required for full activation. ACE indicated no apparent binding of F6P, supporting the former hypothesis. The R22A enzyme was shown by activity assays to have a approximately 15-fold decrease in apparent affinity for F6P compared to that of WT while ACE indicated an affinity comparable to that of WT; potential reasons for this discrepancy are discussed. The R8A enzyme as measured by activity assays exhibits reduced fold-activation by F6P compared to that of WT but increased apparent affinity for ATP in the presence of F6P. The ACE results were in good agreement with the activity assay data, confirming the increased affinity for ATP in the presence of F6P. This method demonstrates the quantitative ability of ACE to study different binding sites/ligand interactions in allosteric enzymes.

Binding constants between the glycopeptides teicoplanin (Teic) and ristocetin (Rist) and their derivatives to D-Ala-D-Ala terminus peptides were determined by on-column receptor synthesis coupled to partial-filling affinity capillary electrophoresis (PFACE) or affinity capillary electrophoresis (ACE). In these techniques, the column is first partially filled with increasing concentrations of D-Ala-D-Ala terminus peptides. This is followed by plugs of buffer, antibiotic and two noninteracting standards, and acetic and/or succinic anhydride (and buffer in the case of ACE). The order of the reagent plugs containing the antibiotic and anhydride varies with the charge of the glycopeptide. Upon electrophoresis, the antibiotic reacts with the anhydride yielding a derivative of Teic or Rist. Continued electrophoresis results in the overlap of the derivatized antibiotic and the plug of D-Ala-D-Ala peptide. Analysis of the change in the relative migration time ratio (RMTR) of the new glycopeptide relative to the standards, as a function of the concentration of the D-Ala-D-Ala ligand yields a value for the binding constant K(b). The techniques described here can be used to assess how the derivatization of drugs alters their affinities for target molecules.

Binding constants between the antibiotic ristocetin A (Rist A) and D-Ala-D-Ala terminus peptides were determined using affinity capillary electrophoresis (ACE). In these experiments two techniques are used to obtain binding constants. In the first, a plug of Rist A and non-interacting standards are injected and electrophoresed. Analysis of the change in the relative migration time ratio (RMTR) of Rist, relative to the non-interacting standards, as a function of the concentration of peptide, yields a value for the binding constant (Kb). In the second, samples of peptide and standards are injected and electrophoresed in increasing concentrations of Rist A in the running buffer. Analysis using theRMTR yields aKb. The findings described here demonstrate the advantage of using ACE for estimating binding parameters between antibiotics and ligands.

The use of uncoated fused silica capillary columns and hydroxypropylmethyl cellulose (HPMC) in capillary electrophoresis (CE) to separate DNA ladders and plasmids is described. In these studies HPM...

Binding constants between the glycopeptide antibiotic, Teicoplanin (Teic), and D-Ala-D-Ala terminus peptides were determined by affinity capillary electrophoresis (ACE) and by on-column ligand synthesis coupled to ACE. In the first technique, a plug of Teic and two non-interacting standards are injected and electrophoresed. Analysis of the change in the relative migration time ratio (RMTR) of Teic, relative to the non-interacting standards, as a function of the concentration of D-Ala-D-Ala peptide, yields a value for the binding constant. In the second technique, 9-fluorenylmethoxycarbonyl (Fmoc)-amino acid-DAla-D-Ala species are first synthesized on-column. The initial sample plug contains a D-Ala-D-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions

This paper describes a two-step procedure whereby on-column ligand synthesis and partial-filling affinity capillary electrophoresis (PFACE) are sequentially coupled to each other to determine the binding constants of 9-fluorenylmethoxy carbonyl (Fmoc)-amino acid-D-Ala-D-Ala species to vancomycin (Van) from Streptomyces orientalis. In this technique four separate plugs of sample are injected onto the capillary column and electrophoresed. The initial sample plug contains a D-Ala-D-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions of Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester and running buffer, respectively. The fourth sample plug contains an increasing concentration of Van partially-filled onto the capillary column. Upon electrophoresis the initial D-Ala-D-Ala peptide reacts with the Fmoc-amino acid NHS ester yielding the Fmoc-amino acid D-Ala-D-Ala peptide. Continued electrophoresis results in the overlap of the plugs of Van and Fmoc-amino acid-D-Ala-D-Ala peptide and non-interacting markers. Analysis of the change in the relative migration time ratio of the Fmoc-amino acid-D-Ala-D-Ala peptide relative to the non-interacting standards, as a function of the concentration of Van, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques.

This work demonstrates the use of multiple-step ligand injection affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique a sample plug of receptor and non-interacting standards is injected by pressure and electrophoresed in a buffer containing a given concentration of ligand. The sequence is repeated for all concentrations of ligand generating a single electropherogram containing a series of individual sample plugs superimposed on environments of buffer containing increasing concentrations of ligand. Analysis of the change in the relative migration time ratio, RMTR, relative to the non-interacting standards, as a function of the concentration of the ligand, yields a value for the binding constant. A competitive assay using the technique is also demonstrated using neutral ligands for CAB. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method are presented.

This work demonstrates the use of an in-capillary procedure for derivatization of amino acids, peptides, and alkylamines by anhydrides using capillary electrophoresis (CE). Migrating in an uncoated fused-silica capillary, plugs of substrate and anhydride are injected separately and electrophoresed. Differential transport velocities permit the separate zones to penetrate each other under an applied field, thereby facilitating reaction. In initial experiments the extent of reaction between tryptophan and acetic anhydride was examined and product amounts quantitated by CE. In separate experiments a series of amino acids and peptides were injected into the capillary and reacted with phthalic anhydride on-column to yield the phthalic derivatized species. Finally, on-column derivatization of alkylamines with phthalic anhydride was investigated and electrophoretic mobility related to molecular weight of the derivatized amines. These procedures illustrate the use of the capillary as a microreactor in the facile synthesis of derivatized molecules and ease of quantitation of reaction products under conditions of electrophoresis.

A new approach for estimating binding constants of ligands to receptors, flow-through partial-filling affinity capillary electrophoresis (FTPFACE), is introduced for studying the interaction of carbonic anhydrase B (CAB, EC 4.2.1.1) with arylsulfonamides and vancomycin fromStreptomyces orientalis with D-Ala-D-Ala peptides. In this technique the capillary is first partially-filled with ligand followed by a sample of receptor and non-interacting standards. Upon application of a voltage the receptor and standards flow into the ligand plug where equilibrium is achieved between the receptor and ligand. Continued electrophoresis results in the receptor and standards flowing through the domain of the ligand plug. Analysis of the change in the relative migration time ratio of the receptor, relative to the non-interacting standards, as a function of the concentration of ligand, yields a value for the binding constant. These values are comparable to those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.

This paper describes the use of capillary electrophoresis (CE) to relate electrophoresis conditions of in-capillary enzyme-catalyzed microreactions to product distribution profiles. Migrating in a capillary under electrophoresis conditions, plugs of substrate and enzyme are injected separately, and allowed to react. A mathematical relation is obtained whereby the electrophoresis parameters, voltage (V), enzyme concentration [E], and mixing time of reaction (M) at the applied voltage, are correlated to product ratios. This concept is demonstrated using as a model system the conversion of nicotinamide adenine dinucleotide (NAD) to nicotinamide adenine dinucleotide, reduced form (NADH) in the oxidation of glucose-6-phosphate (glc-6-P) to 6-phosphogluconate by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49). The simulated results are shown to be in good quantitative agreement with experimental data. These procedures provide a direct relationship between electrophoretic conditions and product distribution of microscale reactions using CE. This technique offers a new and versatile approach to analyzing enzymatic reactions on a microscale.

This work evaluates the concept of a double enzyme-catalyzed microreactor using capillary electrophoresis (CE). Migrating in a capillary under electrophoresis conditions, plugs of substrate and two enzymes are injected separately in buffer and allowed to react. Extent of reaction and product ratios were subsequently determined by CE. This concept is demonstrated using two model systems: the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) by hexokinase (HK, EC 2.7.1.1) and apyrase (APY, EC 3.6.1.5), respectively, in the conversion of glucose to glucose-6-phosphate and inorganic phosphate, respectively, and the conversion of nicotinamide adenine dinucleotide, reduced form (NADH), to nicotinamide adenine dinucleotide (NAD) and back to NADH by lactate dehydrogenase (LDH, EC 1.1.1.27) and glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), respectively, in the conversion of pyruvate to lactate and glucose-6-phosphate (glc-6-P) to 6-phosphogluconate, respectively. These procedures illustrate the use of the capillary as a double microreactor and the ease of quantitation of reaction products under conditions of electrophoresis.

This work describes the quantitative analysis of incapillary enzyme-catalyzed microreactions using capillary electrophoresis. Migrating in a capillary under electrophoresis conditions, a plug of substrate and enzyme are injected separately, and allowed to react. Extent of reaction was subsequently determined by capillary electrophoresis. Two model systems were examined: the conversion of nicotinamide adenine dinucleotide (NAD) to nicotinamide adenine dinucleotide, reduced form (NADH) in the oxidation of glucose-6-phosphate (glc-6-p) to 6-phosphogluconate by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), and the conversion of adenosine diphosphate (ADP) to adenosine monophosphate (AMP) by acid phosphatase (AP, EC 3.1.3.2.). These procedures illustrate the use of the capillary as a microreactor and the ease of quantitation of reaction products under conditions of electrophoresis. This technique offers a new and expeditious approach to analyzing enzymatic reactions on a microscale.

Binding constants between adamantane carboxylic acids and β-cyclodextrin derivatives were determined by capillary electrophoresis (CE) using indirect detection. In this procedure, a mixture of adamantane derivative and non-interacting anionic standards, is injected and analysis of the electrophoretic mobility, relative to the noninteracting anionic standards, as a function of the concentration of cyclodextrin, yields values for their binding constants to the adamantane derivative. The usefulness of the method for quantification of binding constants is demonstrated.

This work evaluates the concept of a multiple-plug binding assay to estimate binding constants of proteins to ligands using affinity capillary electrophoresis (ACE). This concept is demonstrated using two model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and vancomycin from Streptomyces orientalis. Multiple plugs of protein, and non-interacting neutral and protein standards, are injected and analysis of the electrophoretic mobilities of the individual protein plugs, relative to the non-interacting neutral standard, as a function of the concentration of ligand yields values for their binding constants to the protein. These values agree well with those estimated using other assay and ACE techniques. This technique offers a new and expeditions approach to estimating binding constants of ligands to proteins.

A miniaturized capillary electrophoresis system coupled to a surface plasmon resonance (SPR) sensor on a microfluidic platform fabricated from PDMS is detailed. A previously described split-flow injection technique is first utilized to manipulate sample into the microfluidic chip, followed by separation within the fused-silica capillary and final off-capillary detection of analytes via SPR. Instead of using commercial SPR flow cells requiring relatively large detection volumes, samples of less than 1 nL volume are utilized. The interface between the CE system and SPR sensor made it possible to detect minute volumes of sample with minimal dispersion. The flow cell has the potential to be applicable to miniaturized flow-injection (FI) systems where submicroliter volumes of sample are frequently only available for analysis. The components present in solution, but not bound to the sensor surface, were also investigated. The sensitivity of the CE-SPR system was similar to that found in UV-spectrometric instruments and nonchromophoric components could also be measured.

This communication reports a promising platform for rapid, simple, direct, and ultrasensitive determination of serotonin. The method is related to integration of vertically aligned single-walled carbon nanotubes (SWCNTs) in electrochemical microfluidic devices. The required microfabrication protocol is simple and fast. In addition, the nanomaterial influenced remarkably the obtained limit-of-detection (LOD) values. Our system achieved a LOD of 0.2 nmol L(-1) for serotonin, to the best of our knowledge one of the lowest values reported in the literature.

The synthesis and characterization of a family of pyrazole-bridged metallacarborane clusters is described. These species serve as nonradioactive models for the corresponding radio-transition-metal carriers which are potentially useful for the antibody-mediated γ-imaging or β-therapy of tumors.