Microfluidic chip toward cellular ATP and ATP-conjugated metabolic analysis with bioluminescence detection

In this article, a microfluidic platform integrating capillary electrophoresis and bioluminescence (BL) detection that was fabricated in poly(dimethylsiloxane) (PDMS) with lab-on-a-chip technology was demonstrated for cellular metabolic analyses. A microchannels network, "cross combining with Y", was designed to perform on-chip sample preparation, separation, and BL detection of ATP and ATP-conjugated metabolites, using firefly luciferin-luciferase BL system. A dynamic modification of the channel wall of PDMS proved to be crucial to reverse the direction of electroosmotic flow (EOF), which was uniquely achieved by a prewash cycle with a cationic surfactant didodecyldimethylammonium bromide. The influences of surfactant on the EOF and BL reaction were also investigated. Quantitative analyses revealed a dynamic linear range over 2 orders of magnitude for ATP, with a detection limit down to submicromolar (midattomole). The method was validated by measuring cellular ATP of E. coli. with direct on-chip cell lysis. Further work was emphasized on ATP-conjugated metabolite analysis, using galactose as an example. Assays of galactose in human urine samples confirmed the reliability of the protocol, which revealed good prospect of this platform for ATP-conjugated submetabolomic profiling.     

Chemiluminescence imaging immunoassay of multiple tumor markers for cancer screening

A sensitive chemiluminescence (CL) imaging immunoassay method for detection of multiple tumor markers with high throughput, easy operation, and low cost was developed. The immunosensor array was prepared by covalently immobilizing capture antibodies on corresponding sensing sites on a silanized disposable glass chip. Gold nanoparticle-based bioconjugates with a high molar ratio of horseradish peroxidase (HRP) to detection antibodies were used for signal amplification. Under a sandwich immunoassay, the CL signals triggered by HRP captured on each sensing cell were collected by a charge-coupled device for simultaneous measurement of biomarkers and combination diagnosis of certain tumors. As a proof of concept, the immunosensor array was applied to detect α-fetoprotein, carcinoma antigen 125, carbohydrate antigen 153, and carcinoembryonic antigen and to screen patients with liver, breast, or ovarian cancers. This method showed wide linear ranges over 5 orders of magnitude and much lower detection limits than previously reported multiplexed immunoassays. The high throughput and acceptable stability, reproducibility, and accuracy showed good applicability of the proposed multiplex CL imaging immunoassay in clinical diagnosis.     

Sampling-resolution strategy for one-way multiplexed immunoassay with sequential chemiluminescent detection

A sampling-resolution strategy was designed by using a multichannel flow-injection technique for rapid one-way multiplexed immunoassay. The multichannel sampling combined an incubation process in batch with a simple magnetic collection. After incubation for 6 min, free enzyme conjugates could be separated from the formed enzyme-labeled sandwich immunocomplexes with a magnet for simultaneously stopping the immunoreaction. With the help of two valves, the chemiluminescence (CL) substrate was then sequentially mixed with the immunocomplexes in different channels for sequentially triggering the CL reaction in a time interval of 15 s. After triggering for 5 min, the mixtures were sequentially injected into a one-way detection channel in the same interval to form the analyte zones separated with HCl solution and washing buffer for avoiding cross talk. With the use of alpha-fetoprotein, carcinoma antigen 125, carcinoma antigen 199, and carcinoembryonic antigen as proof-of-principle analytes, the sequential CL detection could be completed within 1 min with the linear calibration ranges of 1.0-80 microg/L, 1.0-60 kU/L, 1.0-120 kU/L, and 1.0-100 microg/L, respectively. This system showed acceptable detection and fabrication reproducibility, and the assay results were in acceptable agreement with those from single-analyte tests of clinical sera, showing a promise of automated clinical application.     

Hand-held microanalytical instrument for chip-based electrophoretic separations of proteins

The design, fabrication, and demonstration of a hand-held microchip-based analytical instrument for detection and identification of proteins and other biomolecules are reported. The overall system, referred to as muChemLab, has a modular design that provides for reliability and flexibility and that facilitates rapid assembly, fluid and microchip replacement, troubleshooting, and sample analysis. Components include two independent separation modules that incorporate interchangeable fluid cartridges, a 2-cm-square fused-silica microfluidic chip, and a miniature laser-induced fluorescence detection module. A custom O-ring sealed manifold plate connects chip access ports to a fluids cartridge and a syringe injection port and provides sample introduction and world-to-chip interface. Other novel microfluidic connectors include capillary needle fittings for fluidic connection between septum-sealed fluid reservoirs and the manifold housing the chip, enabling rapid chip priming and fluids replacement. Programmable high-voltage power supplies provide bidirectional currents up to 100 microAlpha at 5000 V, enabling real-time current and voltage monitoring and facilitating troubleshooting and methods development. Laser-induced fluorescence detection allows picomolar (10(-11) M) detection sensitivity of fluorescent dyes and nanomolar sensitivity (10(-9) M) for fluorescamine-labeled proteins. Migration time reproducibility was significantly improved when separations were performed under constant current control (0.5-1%) as compared to constant voltage control (2-8%).     

Sensitive and universal indirect chemiluminescence detection for capillary electrophoresis of cations using cobalt(II) as a probe ion

Highly sensitive and universal indirect chemiluminescence detection for capillary electrophoresis of cations was described. This novel method is based on use of the ultrasensitive cobalt(II) as a probe ion in the running buffer. A strong and stable background chemiluminescent signal can be generated by the luminol-hydrogen peroxide reaction catalyzed by cobalt(II) ion. Displacement of the cobalt(II) probe ion in the running buffer by a migrating sample cation results in a quantifiable decrease in the background signal. The conditions for electrophoresis and the chemiluminescent reaction were systematically investigated using a commercial capillary electrophoresis instrument with an in-house-built chemiluminescence detector. Under the optimal conditions, the detection limits of the concentration for manganese(II), cadmium(II), nickel(II), lead(II), and 14 lanthanides were (3.0-6.0) x 10(-9) mol/L (S/N = 3), which was approximately 3 orders of magnitude better than indirect UV detection and 2 orders better than indirect laser-induced fluorescent detection. A mixture of 18 metal ions including 14 lanthanides was efficiently separated within 3.5 min using lactate to partially complex the metal ions. Our data demonstrated that CE with indirect CL detection was a powerful and universal tool for analysis of inorganic and organic cations.     

High-throughput nanoliter sample introduction microfluidic chip-based flow injection analysis system with gravity-driven flows

In this work, a simple, robust, and automated microfluidic chip-based FIA system with gravity-driven flows and liquid-core waveguide (LCW) spectrometric detection was developed. The high-throughput sample introduction system was composed of a capillary sampling probe and an array of horizontally positioned microsample vials with a slot fabricated on the bottom of each vial. FI sample loading and injection were performed by linearly moving the array of vials filled alternately with 50-microL samples and carrier, allowing the probe inlet to enter the solutions in the vials through the slots sequentially and the sample and carrier solution to be introduced into the chip driven by gravity. The performance of the system was demonstrated using the complexation of o-phenanthroline with Fe(II) as a model reaction. A 20-mm-long Teflon AF 2400 capillary (50-microm i.d., 375-microm o.d.) was connected to the chip to function as a LCW detection flow cell with a cell volume of 40 nL and effective path length of 1.7 cm. Linear absorbance response was obtained in the range of 1.0-100 microM Fe(II) (r2=0.9967), and a good reproducibility of 0.6% RSD (n=18) was achieved. The sensitivity was comparable with that obtained using conventional FIA systems, which typically consume 10,000-fold more sample. The highest sampling throughput of 1000 h-1 was obtained by using injection times of 0.08 and 3.4 s for sample and carrier solution, respectively, with a sample consumption of only 0.6 nL for each cycle.     

Cell chip based monitoring of toxic effects on dopaminergic cell

Oxidative stress has been implicated in pesticide-induced neurotoxicity, base on its role in cascade of biochemical changes that lead to dopaminergic neuronal cell death. The present study examined the role of oxidative stress and the electrochemical detection by polychlorinated biphenyls (PCBs)-induced toxicant in SH-SY5Y cell. The cells were seed in the RED (Arg-Gly-Asp) nanopatterned coating gold substrate and treated with different concentration of PCBs for 24 h in culture, which induced the change of the cyclic voltammetry (CV) current peak. The CV results showed that PCB significantly decreased the current peaks in dose and time-dependent manner. After antioxidant treatment, the CV of the PCB-treated cell chip increased the current peak. Especially, gluthaione and catalase prevent PCB-induced decrease of CV current peak in the cell. The results demonstrated that the current peak decreased by the PCB and recovered by the antioxidant enzyme. In conclusion, results suggest that the electrochemical-based chip provide crucial information to improvement toward a cell chip system for drug screening application.     

Liquid chromatographic arsenic speciation with gas-phase chemiluminescence detection

We present a newly developed gas-phase chemiluminescence (CL) detection method for the separation and quantification of inorganic and organic arsenic species. Arsenite, arsenate, dimethylarsinic acid (DMA), and monomethylarsonic acid (MMA) were separated by anion exchange using carbonate-bicarbonate and NaOH eluents with step-gradient elution. The separated species were passed through a UV photooxidation reactor which decomposed the organic species and converted them to inorganic As(V). Subsequent on-line hydride generation with acid and sodium borohydride produces AsH3 and H2, which are separated from the liquid in a gas-liquid separator. The produced AsH3, driven by H2, reacts with ozone in a small reflective cell located atop a photomultiplier tube, resulting in intense CL. In the present form, the limits of detection (LODs, signal-to-noise = 3), based on peak height, for arsenite, arsenate, MMA, and DMA are 0.4, 0.2, 0.5, and 0.3 microg/L, respectively, for a 100 microL injected sample. This analyzer demonstrates the robustness of the CL detection system for arsenic and provides an affordable alternative to atomic spectrometry for use as a detector after chromatographic speciation. We found no significant practical interferences.     

On-column coaxial flow chemiluminescence detection for underivatized amino acids by pressurized capillary electrochromatography using a monolithic column

A new method for pressurized capillary electrochromatography (pCEC) coupling with chemiluminescence (CL) detection using a modified on-column coaxial flow detection interface was developed. To evaluate the feasibility and reliability of the experimental setup, the typical CL compounds luminol and isoluminol were separated and detected by using this pCEC-CL system. A detailed investigation of CL detection interface and postcolumn CL reagent flow rate parameters was described. The excellent resolution and detection sensitivity was achieved by using 3-microm ODS-C18 packed column with 30% ACN (v/v), 5 mmol/L phosphate buffer (pH 8.0). Moreover, with the presence of Co(II) (1.0 x 10(-4) mol/L) in the mobile phase, the linear range of the concentration for luminol was 2.0 x 10(-9)-2.0 x 10(-6) mol/L with a detection limit (S/N = 3) of 2.0 x 10(-10) mol/L, and 2.5 x 10(4) theoretical plates was achieved. In addition, separation and detection of the underivatized amino acids (l-threonine and l-tyrosine) were accomplished by using a polymerized monolithic column based on the principle of the luminol-H2O2-Cu(II)-amino acid CL system. Under the optimum conditions, the mixture of amino acids was efficiently separated with satisfactory results.     

Luminol chemiluminescence in unbuffered solutions with a cobalt(II)-ethanolamine complex immobilized on resin as catalyst and its application to analysis.

Using a heterogeneous catalyst, Co(II)-ethanolamine complex sorbed on Dowex-50W resin, the chemiluminescence (CL) of luminol in unbuffered or weakly acidic solution was studied in the presence of H2O2. The maximum luminol CL wavelength at pH 5.7 was 448 nm, 23 nm longer than that in a basic solution (pH 10.5). Three different ligands, mono-, di-, and triethanolamine, and six transition metal ions, Co(II), Cu(II), Ni(II), Mn-(II), Fe(II), and Fe(III) were compared by CL measurements. The CL intensity decreased in the order mono- > di- > triethanolamine and Co(II) > Cu(II) > Ni(II) > Fe-(III) > Mn(II) > Fe(II). This heterogeneous CL system was developed as H2O2 and glucose flow-through sensors. Detection limits (S/N = 3) of H2O2 and glucose using Dowex-50W-X4-Co(II)-monoethanolamine as catalyst are 1 x 10(-7) M and 1 x 10(-6) M, respectively. On the basis of the studies of the CL, fluorescence, UV-vis and ESCA spectra and the effect of dissolved oxygen in luminol solution, a mechanism for CL emission in unbuffered solution was considered as the formation of a superoxide radical ion during the decomposition of H2O2 catalyzed by the Co(II)-ethanolamine immobilized resin. Then the superoxide radical ion acted on luminol and the CL was emitted. The applications of the proposed method to determine H2O2 in rainwater without any special pretreatment and glucose in human urine and orange juice samples give satisfactory results.     

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.     

Dynamics and performance of fast linear scan anodic stripping voltammetry of cd, pb, and cu using in situ-generated ultrathin mercury films

The dynamics of fast linear scan (LS) ASV for the simultaneous detection of Cd, Pb, and Cu was investigated at various scan rates (0.5-10 V/s) and at different metal ion concentrations (50-800 nM) utilizing ultrathin mercury films (9 nm) at a conventional size (d(0) = 1 mm) electrode. Results of the investigation show that when the thin films were utilized, diffusion of metals through the mercury film was not the rate-limiting step of the stripping process at moderate to fast scan rates (0.5-10 V/s). A fairly linear relationship between the peak height and scan rate was observed at scan rates (0.5-10 V/s) beyond the upper limit of the theoretical model for the behavior of LS-ASV. In addition, peak width at half-height (b(1/2)) as low as 33 mV was achieved at 0.5 V/s. The behavior of LS-ASV in terms of peak width at these scan rates is thus different from what the theoretical model of LS-ASV would have predicted. For the ultrathin mercury films, at least two additional factors, kinetics and concentration, have significant effects on practical LS-ASV. Experimental results show that the stripping process of Cu was primarily kinetic-controlled for fast scans, while those for Cd and Pb were dependent on both scan rates and concentrations. The ultrathin mercury film resulted in a significant enhancement of the ratio of signal-to-baseline slope (i(p)/Δi(b), a ratio used to measure the effectiveness of discrimination of the peak signal against the steep sloping baseline in LS-ASV) for Cd and Pb stripping peaks, but only a slight enhancement for Cu stripping peaks. The optimal performance of LS-ASV in terms of sensitivity, peak width, and enhancement of the i(p)/Δi(b) ratio for the three metals was achieved at 2 V/s. Because of the high reproducibility of the background currents of the stable in situ MTFs, background subtraction was carried out at 2 V/s with little hysteresis. This feature, combined with the enhancement of the i(p)/Δi(b) ratio at the fast scan rate of 2 V/s, allowed for the detection of sub-ppb levels of Cd, Pb, and Cu at a deposition time of 2 min.     

Dual-electrode electrochemical detection for poly(dimethylsiloxane)-fabricated capillary electrophoresis microchips

The development of a poly(dimethylsiloxane)-based (PDMS-based) microchip electrophoresis system employing dual-electrode electrochemical detection is described. This is the first report of dual-electrode electrochemical detection in a microchip format and of electrochemical detection on chips fabricated from PDMS. The device described in this paper consists of a top layer of PDMS containing the separation and injection channels and a bottom glass layer onto which gold detection electrodes have been deposited. The two layers form a tight reversible seal, eliminating the need for high-temperature bonding, which can be detrimental to electrode stability. The channels can also be temporarily removed for cleaning, significantly extending the lifetime of the chip. The performance of the chip was evaluated using catechol as a test compound. The response was linear from 10 to 500 microM with an LOD (S/N = 3) of 4 microM and a sensitivity of 45.9 pA/microM. Collection efficiencies for catechol ranged from 28.7 to 25.9% at field strengths between 200 and 400 V/cm. Dual-electrode detection in the series configuration was shown to be useful for the selective monitoring of species undergoing chemically reversible redox reactions and for peak identification in the electropherogram of an unresolved mixture.     

Direct detection of biomolecules in a capillary electrophoresis-chemiluminescence detection system

Direct detection of biomolecules, such as alpha-amino acids, peptides, and proteins, was accomplished using a capillary electrophoresis-chemiluminescence detection system, in which a luminol-hydrogen peroxide-Cu(II)-catalyzed chemiluminescence reaction was utilized. Biomolecules migrated in the capillary, where they mixed with luminol and the Cu(II) catalyst included in the running buffer. The capillary outlet was inserted into a batch-type chemiluminescence detection cell with hydrogen peroxide-supplemented electrolyte solution. Chemiluminescence was observed at the tip of the capillary outlet. The chemiluminescence peak from biomolecules appeared due to the enhancement of Cu(II) catalytic activity for luminol-hydrogen peroxide chemiluminescence. The Cu(II) was more catalytically active when it interacted with biomolecules forming Cu(II)-biomolecule complexes. In this study, biomolecules were directly separated and detected in a capillary electrophoresis-chemiluminescence detection system. Twenty alpha-amino acids, 4 peptides, and 11 proteins were examined. Most of them were detected with satisfactory CL intensity response. Glutamic acid, an alpha-amino acid, was detected at concentrations ranging from 2.0 x 10(-7) to 1.2 x 10(-5) M with a detection limit (S/N = 3) of 1.0 x 10(-7) M (0.6 fmol). Glycylglycine, a peptide, was detected at concentrations ranging from 1.7 x 10(-7) to 1.2 x 10(-5) M with a detection limit (S/N = 3) of 1.7 x 10(-7) M (0.9 fmol). Hemoglobin, a heme protein, in which the heme structure was independently catalytically active, was detected at concentrations ranging from 1.2 x 10(-7) to 1.0 x 10(-5) M with a detection limit (S/N = 3) of 1.2 x 10(-7) M (0.6 fmol). Representative mixtures of alpha-amino acids and peptides were well detected with superior separation.     

A porous silicon optical microcavity for sensitive bacteria detection

A porous silicon microcavity (PSM) is highly sensitive to subtle interface changes due to its high surface area, capillary condensation ability and a narrow resonance peak (～10 nm). Based on the well-defined optical properties of a PSM, we successfully fabricated a bacteria detection chip for molecular or subcellular analysis by surface modification using undecylenic acid (UA), and the specific recognition binding of vancomycin to the D-alanyl-D-alanine of bacteria. The red shift of the PSM resonance peak showed a good linear relationship with bacteria concentration ranging from 100 to 1000 bacteria ml( - 1) at the level of relative standard deviation of 0.994 and detection limit of 20 bacteria ml( - 1). The resulting PSM sensors demonstrated high sensitivity, good reproducibility, fast response and low cost for biosensing.     

Influence of wire-EDM textured conventional tungsten carbide inserts in machining of aerospace materials (Ti–6Al–4V alloy)

Importance of this present investigation is to identify the influence of modified tool (tool with texturing) on the process of orthogonal turning of Ti–6Al–4V work material. To achieve the enhanced turning conditions, four different types of textures (plain conventional, cross, perpendicularly textured and parallel textured tool to the chip flow direction) were fabricated on the rake face of the tool insert and the lubricant used during the machining process is molybdenum disulfide (MoS₂). Machining forces (the force of cutting and feed), angle of shear, chip morphology, temperature distribution between tool and chip were measured. Shear strain and strain rate were also computed and compared with all type of cutting tools. Experimental results revealed that the cross-textured cutting tool exhibit an effective reduction in cutting force, friction, shear strain and strain rate. The favorable metal removal condition of curling chip with low diameter was achieved through cross-textured tool.     

Optimization and characterization of a flow injection electrochemical system for pentachlorophenol assay

A flow injection (FI) electrochemical detection system has been developed and optimized for the determination of pentachlorophenol (PCP) in contaminated soil. PCP was oxidized to tetrachloro-1,4-benzoquinone (1,4-TCBQ) with a high yield using bis(trifluoroacetoxy)iodobenzene in 0.1 M tartaric acid, pH 2.0, at ambient temperature. Upon rapid reaction with immobilized glucose oxidase, the detection and amplification scheme was completed as the reduced form of 1,4-TCBQ or tetrachloro-1,4-hydroquinone was reoxidized to 1,4-TCBQ at the surface of the glassy carbon electrode (+ 0.40 V vs Ag/AgCl). Rapid electron exchange between the enzyme and its glucose substrate provided a non-rate-limiting current toward the electrode. The FI electrochemical system was linear up to 1 μM oxidized PCP with a detection limit of 10 nM and exhibited a reproducibility of ±0.6% over 165 repeated analyses during 14 h of continuous operation. When applied to PCP-contaminated soil samples, the results obtained from the FI electrochemical system compared well with those of the HPLC standard method.     

Toward integrated detection and graphene-based removal of contaminants in a lab-on-a-chip platform

A novel miniaturized microfluidic platform was developed for the simultaneous detection and removal of polybrominated diphenyl ethers (PBDEs).The platform consists of a polydimethylsiloxane (PDMS) microfluidic chip for an immunoreaction step,a PDMS chip with an integrated screen-printed electrode (SPCE) for detection,and a PDMS-reduced graphene oxide (rGO) chip for physical adsorption and subsequent removal of PBDE residues.The detection was based on competitive immunoassay-linked binding between PBDE and PBDE modified with horseradish peroxidase (HRP-PBDE) followed by the monitoring of enzymatic oxidation of o-aminophenol (o-AP) using square wave anodic stripping voltammetry (SW-ASV).PBDE was detected with good sensitivity and a limit of detection similar to that obtained with a commercial colorimetric test (0.018 ppb),but with the advantage of using lower reagent volumes and a reduced analysis time.The use of microfluidic chips also provides improved linearity and a better reproducibility in comparison to those obtained with batch-based measurements using screen-printed electrodes.In order to design a detection system suitable for toxic compounds such as PBDEs,a reduced graphene oxide-PDMS composite was developed and optimized to obtain increased adsorption (based on both the hydrophobicity and π-π stacking between rGO and PBDE molecules) compared to those of non-modified PDMS.To the best of our knowledge,this is the first demonstration of electrochemical detection of flame retardants and a novel application of the rGO-PDMS composite in a biosensing system.This system can be easily applied to detect any analyte using the appropriate immunoassay and it supports operation in complex matrices such as seawater.     

DNA detection using a triple readout optical/AFM/MALDI planar microwell plastic chip

A ready-to-spot disposable DNA chip for specific and sensitive detection of DNA was developed. Plastic copolymeric substrate chemistry was optimized to selectively couple the target DNA with the active chip surface. At the same time, the developed substrate limits the unspecific adsorption of probe DNA molecules or additional polar contaminants in the test samples to the chip surface. The combination of glycidyl and n-butyl methacrylates was found to best fit the requirements of the assay. The fabricated DNA microarrays have mechanical properties similar to those of the glass or silicon substrates and, at the same time, provide chemically reactive surfaces that do not require lengthy chemical modification. An additional advantage of the plastic microchip is its compatibility with different analytical readout techniques, such as mass spectrometry (MALDI-TOF/MS), optical detection (fluorescence and enzyme-induced metal deposition), and imaging techniques (atomic force microscopy). These multiple readout techniques have given us the ability to compare the sensitivity, selectivity, and robustness of current state-of-the-art bioanalytical methods on the same platform exemplified by successful DNA-based detection of human cytomegalovirus. The obtained sensitivity for enzymatically enhanced silver deposition (10(-15) M) surpasses that of conventional fluorescence readouts. In addition, the assay's dynamic range (10(-6)-10(-15) M), reproducibility, and reliability of the DNA probe detection speaks for the silver deposition method. At compromised sensitivity (10(-9) M), the length of the DNA probes could be checked and, alternatively, DNA single point polymorphisms could be analyzed.     

Multichannel microchip electrophoresis device fabricated in polycarbonate with an integrated contact conductivity sensor array

A 16-channel microfluidic chip with an integrated contact conductivity sensor array is presented. The microfluidic network consisted of 16 separation channels that were hot-embossed into polycarbonate (PC) using a high-precision micromilled metal master. All channels were 40 microm deep and 60 microm wide with an effective separation length of 40 mm. A gold (Au) sensor array was lithographically patterned onto a PC cover plate and assembled to the fluidic chip via thermal bonding in such a way that a pair of Au microelectrodes (60 microm wide with a 5 microm spacing) was incorporated into each of the 16 channels and served as independent contact conductivity detectors. The spacing between the corresponding fluidic reservoirs for each separation channel was set to 9 mm, which allowed for loading samples and buffers to all 40 reservoirs situated on the microchip in only five pipetting steps using an 8-channel pipettor. A printed circuit board (PCB) with platinum (Pt) wires was used to distribute the electrophoresis high-voltage to all reservoirs situated on the fluidic chip. Another PCB was used for collecting the conductivity signals from the patterned Au microelectrodes. The device performance was evaluated using microchip capillary zone electrophoresis (mu-CZE) of amino acid, peptide, and protein mixtures as well as oligonucleotides that were separated via microchip capillary electrochromatography (mu-CEC). The separations were performed with an electric field (E) of 90 V/cm and were completed in less than 4 min in all cases. The conductivity detection was carried out using a bipolar pulse voltage waveform with a pulse amplitude of +/-0.6 V and a frequency of 6.0 kHz. The conductivity sensor array concentration limit of detection (SNR = 3) was determined to be 7.1 microM for alanine. The separation efficiency was found to be 6.4 x 10(4), 2.0 x 10(3), 4.8 x 10(3), and 3.4 x 10(2) plates for the mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins, respectively, with an average channel-to-channel migration time reproducibility of 2.8%. The average resolution obtained for mu-CEC of the oligonucleotides and mu-CZE of the amino acids, peptides, and proteins was 4.6, 1.0, 0.9, and 1.0, respectively. To the best of our knowledge, this report is the first to describe a multichannel microchip electrophoresis device with integrated contact conductivity sensor array.