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PubMed2001

Palladium film decoupler for amperometric detection in electrophoresis chips.

Demonstrated in this article is that a palladium metal film can be applied to decouple the electric circuitry of electrochemical detection from that of the electrophoretic separation in an electrophoresis chip. The Pd solid-state field decoupler, as well as the working electrodes, is thermally evaporated onto the plastic chip and oriented vertically across the separation channel. After the sample zones flow over the Pd decoupler, their electrochemical response is measured at working electrodes in the downstream pathway. Because the electrodes are on the separation channel, the electrode channel alignment is no longer a problem. For a separation channel of roughly 200 microm in width and 75 microm in depth in 10 mM phosphate (pH 5.1), the noise level at the working electrode is < 15 pA at an electric field of 570 V/cm.

Journal Article↗

PubMed2000

Electrochemical detectors prepared by electroless deposition for microfabricated electrophoresis chips

Microfabricated capillary electrophoresis (CE) chips with integrated electrochemical detection have been developed on glass substrates. An electroless deposition procedure was used to deposit a gold film directly onto the capillary outlet to provide high-sensitivity electrochemical detection for catechol and several nitroaromatic explosives. Scanning electron microscopy revealed that the electroless gold film contains nanoscopic gold aggregates (100-150 nm) with an average thickness of 79 nm. The electroless deposition procedure can be easily and routinely performed in any wet-chemistry laboratory, and electroless gold can be deposited onto complex and internal surfaces. Intimate coupling of electrochemical detection and CE chips obviates the need for a coupling mechanism or tedious alignment procedures. With nitroaromatic compounds as a working model, microchip capillary electrophoresis equipped with electroless gold has proven to provide high sensitivity and fast response times for sensor applications. The CE microchip system was capable of separation and determination of explosive compounds including TNT in less than 130 s with detection limits ranging from 24 to 36 microg/L, i.e., 4-fold enhancements in detection efficiency in comparison to thick-film technology.

Journal Article↗

PubMed2000

Chip-based optical detection of DNA hybridization by means of nanobead labeling.

A new scheme for the detection of molecular interactions based on optical readout of nanoparticle labels has been developed. Capture DNA probes were arrayed on a glass chip and incubated with nanoparticle-labeled target DNA probes, containing a complementary sequence. Binding events were monitored by optical means, using reflected and transmitted light for the detection of surface-bound nanoparticles. Control experiments exclude significant influence of nonspecific binding on the observed contrast. Scanning force microscopy revealed the distribution of nanoparticles on the chip surface.

Base Sequence↗

PubMed2000

Micromachined separation chips with a precolumn reactor and end-column electrochemical detector.

Glass microchips, integrating chemical derivatization reactions, electrophoretic separations, and amperometric detection, have been developed. The performance of the new integrated microfabricated devices is demonstrated for rapid on-chip measurements of amino acids utilizing precolumn reactions of amino acids with o-phthaldialdehyde/2-mercaptoethanol to generate electroactive derivatives that are separated electrophoretically and detected at the end-column electrochemical detector. The influence of the sample/reagent mixing ratio, reagent concentrations, driving voltage, detection potential, and other variables is explored. The integrated microsystem offers a rapid (6 min) simultaneous measurements of eight amino acids, down to approximately 2.5 x 10(-6) M (5 fmol) level, with linearity up to the 2 x 10(-4) M level examined, and good reproducibility (RSD = 2.2-2.7%). A step of the driving voltage is used for decreasing the migration time of late-eluting components and reducing the overall analysis time. The integrated microfabricated device expands the scope of on-chip electrochemical detection to nonelectroactive analytes and holds promise of being a powerful analytical tool.

Electrochemistry↗

PubMed2001

Elimination of high-voltage field effects in end column electrochemical detection in capillary electrophoresis by use of on-chip microband electrodes.

The influence of the separation voltage on end column electrochemical detection (EC) in capillary electrophoresis (CE) has been investigated using an electrochemical detector chip based on an array of microband electrodes. It is shown, both theoretically and experimentally, that the effect of the CE electric field on the detection can be practically eliminated, without using a decoupler, by positioning the reference electrode sufficiently close to the working electrode. In the present study, this was demonstrated by using an experimental setup in which neighboring microband electrodes on a chip, positioned 30 microns from the end of the CE capillary, were used as working and reference electrodes, respectively. The short distance (i.e., 10 microns) between the working and reference electrode ensured that both of the electrodes were very similarly affected by the presence of the CE electric field. With this experimental setup, no significant influence of the CE voltage on the peak potentials for gold oxide reduction could be seen for CE voltages up to +30 kV. The detector noise level was also found to be reduced.

Journal Article↗

PubMed2001

Velocity measurement of particles flowing in a microfluidic chip using Shah convolution Fourier transform detection.

A noninvasive radiative technique, based on Shah convolution Fourier transform detection, for velocity measurement of particles in fluid flows in a microfluidic chip, is presented. It boasts a simpler instrumental setup and optical alignment than existing measurement methods and a wide dynamic range of velocities measurable. A glass-PDMS microchip with a layer of patterned Cr to provide multiple detection windows which are 40 microns wide and 70 microns apart is employed. The velocities of fluorescent microspheres, which were electrokinetically driven in the channel of the microfluidic chip, were determined. The effects of increasing the number of detection windows and sampling period were investigated. This technique could have wide applications, ranging from the determination of the velocity of particles in pressure-driven flow to the measurement of electrophoretic mobilities of single biological cells.

Algorithms↗

PubMed2002

An integrated fritless column for on-chip capillary electrochromatography with conventional stationary phases.

A new polymer device for use with conventional particulate stationary phases for on-chip, fritless, capillary electrochromatography (CEC) has been realized. The structure includes an injector and a tapered column in which the particles of the stationary phase are retained and stabilized. The chips were easily fabricated in poly(dimethylsiloxane) using deep-reactive-ion-etched silicon masters, and tested using a capillary electrophoretic separation of FITC-labeled amino acids. To perform CEC, the separation channel was packed using a vacuum with 3-microm, octadecylsilanized silica microspheres. The packing was stabilized in the column by a thermal treatment, and its stability and quality were evaluated using in-column indirect fluorescence detection. The effects of voltage on electro-osmotic flow and on efficiency were investigated, and the separation of two neutral compounds was achieved in less than 15 s.

Amino Acids↗

PubMed2003

On-chip contactless four-electrode conductivity detection for capillary electrophoresis devices.

In this contribution, a capillary electrophoresis microdevice with an integrated on-chip contactless four-electrode conductivity detector is presented. A 6-cm-long, 70-microm-wide, and 20-microm-deep channel was etched in a glass substrate that was bonded to a second glass substrate in order to form a sealed channel. Four contactless electrodes (metal electrodes covered by 30-nm silicon carbide) were deposited and patterned on the second glass substrate for on-chip conductivity detection. Contactless conductivity detection was performed in either a two- or a four-electrode configuration. Experimental results confirmed the improved characteristics of the four-electrode configuration over the classical two-electrode detection setup. The four-electrode configuration allows for sensitive detection for varying carrier-electrolyte background conductivity without the need for adjustment of the measurement frequency. Reproducible electrophoretic separations of three inorganic cations (K+, Na+, Li+) and six organic acids are presented. Detection as low as 5 microM for potassium was demonstrated.

Journal Article↗

PubMed2002

A chip-based electrophoresis system with electrochemical detection and hydrodynamic injection.

A simple capillary electrophoresis system in the planar format that uses a new, hydrodynamic injection principle is described. The system was realized with poly(dimethylsiloxane)-glass chips and microdisk electrodes for amperometric detection. Using a double-tee injector, no precise voltage control of the electrolyte reservoirs was needed, thus making the microchip CE system more user-friendly. The analytical characteristics of chip-based CE-EC were evaluated using ascorbic acid as the model analyte. The reproducibility of migration time and signal height was expressed by relative standard deviations of 1.2% and 5.1%, respectively (n = 5). The limit of detection for ascorbic acid was approximately 5 microM at a signal-to-noise ratio of 3. Practical application concerning the determination of physiologically relevant compounds such as noradrenaline and L-dopa are discussed.

Ascorbic Acid↗

PubMed2002

Enzymatic microreactor-on-a-chip: protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices.

Enzymatic microreactors have been prepared in capillaries and on microfluidic chips by immobilizing trypsin on porous polymer monoliths consisting of 2-vinyl-4,4-dimethylazlactone, ethylene dimethacrylate, and acrylamide or 2-hydroxyethyl methacrylate. The azlactone functionalities react readily with amine and thiol groups of the enzyme to form stable covalent bonds. The optimized porous properties of the monoliths lead to very low back pressures enabling the use of simple mechanical pumping to carry out both the immobilization of the enzyme from its solution and the subsequent analyses of substrate solutions. The Michealis-Menten kinetic characteristics of the reactors were probed using a low molecular weight substrate: N-alpha-benzoyl-L-arginine ethyl ester. The effects of immobilization variables such as the concentration of trypsin in solution and percentage of azlactone functionalities in the monolith, as well as the effect of reaction time on the enzymatic activity, and of process variables such as substrate flow velocity and residence time in the reactor, were studied in detail. The proteolytic activity of the enzymatic microreactor on chip was demonstrated at different flow rates with the cleavage of fluorescently labeled casein used as a substrate. The excellent performance of the monolithic microreactor was also demonstrated with the digestion of myoglobin at the fast flow rate of 0.5 microL/min, which affords a residence time of only 11.7 s. The digest was then characterized using MALDI-TOF MS, and 102 out of 153 possible peptide fragments were identified giving a sequence coverage of 67%.

Animals↗

PubMed2003

Homogeneous immunoassay for detection of TNT and its analogues on a microfabricated capillary electrophoresis chip.

A homogeneous immunoassay for TNT and its analogues is developed using a microfabricated capillary electrophoresis chip. The assay is based on the rapid electrophoretic separation of an equilibrated mixture of an anti-TNT antibody, fluorescein-labeled TNT, and unlabeled TNT or its analogue. The band intensities of the free fluorescein-labeled TNT and of the antibody-antigen complex reveal the relative equilibrated concentrations. Titration of the anti-TNT antibody with a fluorescein-labeled TNT derivative yields a binding constant of (3.9 +/- 1.3) x 10(9) M(-1). The dissociation rate constant of the complex is determined by kinetic capillary electrophoresis using a folded channel and a rotary scanner to interrogate the separation at multiple time points. The dissociation rate constant is found to be 0.035 +/- 0.005 s(-1), and the resulting binding rate constant is (1.4 +/- 0.7) x 10(7) M(-1) s(-1). Binding constants of TNT and five of its analogues are determined by competitive assays: TNT (4.3 +/- 2.6) x 10(8) M(-1); 1,3,5-trinitrobenzene (5.1 +/- 3.3) x 10(7) M(-1); picric acid (7.5 +/- 4.4) x 10(6) M(-1); 2,4-dinitrotoluene (7.9 +/- 4.0) x 10(6) M(-1); 1,3-dinitrobenzene (1.0 +/- 0.7) x 10(6) M(-1); and 2,4-dinitrophenol (5.1 +/- 3.0) x 10(4) M(-1). TNT and its analogues can be assayed with high sensitivity (LOD 1 ng/mL) and with a wide dynamic range (1-300 ng/mL) using this chip-based method.

Algorithms↗

PubMed2003

Microfabricated on-chip-type electrochemical flow immunoassay system for the detection of histamine released in whole blood samples.

This paper describes an on-chip-type electrochemical flow immunoassay system with a multichanneled matrix column. The multichanneled matrix column was functionally coated with cation-exchange resin and used for separation of proteins. Antihistamine immunoglobulin G (IgG) antibody conjugated with ferrocenemonocarboxylic acid (Fc) was also prepared and used as a novel analytical reagent. Antibody-antigen complexes were separated from free Fc-conjugated IgG antibody (Fc-IgG) on the basis of differences in isoelectric point (pI) using the multichanneled matrix column coated with cation-exchange resin. The assay yields a good relationship between current and histamine concentration in the range of 200-2000 ng/mL. This simple technique enables the assay of histamine released in whole blood within 2 min. Furthermore, a good correlation was found between the response of the electrochemical immunoassay described in this paper and the conventional RIA (radioimmunoassay). This on-chip-type electrochemical flow immunoassay requires only minute quantities of whole blood samples and generates highly reproducible results.

Electrochemistry↗

PubMed2002

Cell docking and on-chip monitoring of cellular reactions with a controlled concentration gradient on a microfluidic device.

We have developed a microfluidic device for on-chip monitoring of cellular reactions. The device consists of two primary analytical functions: control of cell transport and immobilization, and dilution of an analyte solution to generate a concentration gradient. In this device, a dam structure in parallel to the fluid flow was constructed for docking and alignment of biological cells, which allows the fragile cells to move in the microfluidic channels and to be immobilized with controllable numbers in desired locations. The cells docked on the parallel dam structure are exposed to minimal stress caused by fluidic pressure. Additionally, a network of microfluidic channels was designed to generate a concentration gradient by controlled fluid distribution and diffusive mixing. An analyte solution could be diluted to different gradients as a function of distance along the dam. We used the ATP-dependent calcium uptake reaction of HL-60 cells as a model for on-chip measurement of the threshold ATP concentration that induces significant intracellular calcium signal. The results have demonstrated the feasibility of using the microchip for real-time monitoring of cellular processes upon treatment of a concentration gradient of a test solution. The integration of cell manipulation and solution manipulation on a microchip allows the measurement of concentration-dependent biological responses within a confined microscale feature.

Adenosine Triphosphate↗

PubMed2003

Electrochemical protein chip with arrayed immunosensors with antibodies immobilized in a plasma-polymerized film.

An electrochemical protein chip was microfabricated. A thin-film three-electrode system, including an array of 36 platinum working electrodes, a set of thin-film Ag/AgCl electrodes, and platinum auxiliary electrodes, was integrated on a glass substrate. Capture antibodies were immobilized in a 4.5-nm-thick double layer of a hexamethyldisiloxane plasma-polymerized film. Because of their highly cross-linked network structure, the capture antibodies could be firmly immobilized. No nonspecific adsorption was observed during a series of procedures to detect target proteins, and electrochemical cross talk between neighboring sites was negligible. The sandwich immunoassay was conducted on a single chip using model proteins, alpha-1-fetoprotein and beta2-microglobulin. A distinct current increase following the oxidation of hydrogen peroxide produced by the enzymatic reaction of glucose oxidase was observed, which indicates that the capture proteins could actually bind the target proteins. Two kinds of protein were detected independently on multiple sites with respective capture antibodies.

Antibodies↗

PubMed2002

Electrokinetic injection techniques in microfluidic chips.

The separation efficiency of a microfluidic chip is influenced to a significant degree by the flow field conditions within the injection microchannel. Therefore, an understanding of the physics of the flow within this channel is beneficial in the design and operation of such a system. The configuration of an injection system is determined by the volume of the sample plug that is to be delivered to the separation process. Accordingly, this paper addresses the design and testing of injection systems with a variety of configurations, including a simple cross, a double-T, and a triple-T configuration. This paper also presents the design of a unique multi-T injection configuration. Each injection system cycles through a predetermined series of steps, in which the electric field magnitude and distribution within the various channels is strictly manipulated, to effectuate a virtual valve. The uniquemulti-T configuration injection system presented within this paper has the ability to simulate the functions of the cross, double-T, and triple-T systems through appropriate manipulations of the electric field within its various channels. In other words, the proposed design successfully combines several conventional injection systems within a single microfluidic chip.

Journal Article↗

PubMed2003

Characteristic electrochemical responses of polymer microchannel-microelectrode chips.

Polymer microchannel chips (dimension of the channel, 20 microm (depth) x 100 microm (width)) integrated with dual microband electrodes were fabricated by an imprinting method, and their characteristic electrochemical responses were elucidated in detail. A silicon micromachined template fabricated by photolithography and dry etching techniques was used for imprinting. An aqueous solution of a ferrocene derivative was brought into the microchannel by pressure-driven flow, and the electrode responses were studied on the basis of voltammetry and chronoamperometry. A linear sweep voltammetry of 1-hydroxyethylferrocene (FeCp-OH) in water demonstrated that the electrode responses in the microchannel chip were best characterized by one-dimensional diffusion along the channel length, reflecting the structural dimension of the channel. In generation-collection mode experiments, furthermore, a collection efficiency as high as approximately 90% was attained in the microchannel owing to both restricted space and characteristics of solution flow in the channel. It was confirmed that diffusion and solution flow made molecular transport very efficient in the microchannel. The experimental results were also compared with those predicted by computer simulations.

Journal Article↗

PubMed2003

Solid-phase reversible immobilization in microfluidic chips for the purification of dye-labeled DNA sequencing fragments.

In this manuscript, we discuss the use of photoactivated polycarbonate (PC) for purification of dye-labeled terminator sequencing fragments using solid-phase reversible immobilization (SPRI) prior to gel electrophoretic sorting of these DNAs. An immobilization bed for the DNA purification was produced by exposing a posted microchannel to UV radiation, which induced a surface photooxidation reaction, resulting in the production of carboxylate groups. The immobilization microchannel contained microposts to increase the loading level of DNAs to improve signal intensity without the need for preconcentration. By suspending the sequencing cocktail in an immobilization buffer (TEG/ethanol), the DNA fragments demonstrated a high affinity for this carboxylated surface. The loading density of DNAs to this activated surface was found to be 3.9 pmol cm(-2). The captured DNA could be subsequently released from the surface by incubation with ddH2O. SPRI cleanup of dye-terminator sequencing fragments using the photoactivated PC chip and slab gel electrophoresis produced a read length comparable to the conventional SPRI format, which utilized carboxylated magnetic beads and a magnetic field. The read length for the PC-SPRI format was found to be 620 bases with a calling accuracy of 98.9%. The PC-SPRI cleanup format was also integrated to a capillary gel electrophoresis (CGE) system. The PC-SPRI method was shown to effectively remove excess dye terminator from the CGE tract, but yielded lower plate numbers, as compared to a direct injection method with purification accomplished off-chip. The loss in efficiency was found to result primarily from the extended injection time associated with the microchip purification method.

Base Sequence↗

PubMed2003

Chiral on-chip separations of neurotransmitters.

The fast on-chip determination of a range of compounds of relevance in the study and treatment of neurological disorders is demonstrated. These include dopamine and its metabolites methoxytyramine, homovanillic acid, noradrenaline, adrenaline, normetanephrine, and metanephrine, its artificial precursors DOPA and tyrosine; and the related compounds DOPS and CDOPA. Two runs are needed for the determination of these compounds. The enantiomers of adrenaline, noradrenaline, and dopamine can be separated in a buffer containing the novel combination of a cyclodextrin and a dendrimer. The isomers of homovanillic acid, DOPA, CDOPA, methoxytyramine, metanephrine, and normetanephrine, which were found to interact more weakly with cyclodextrins, could be separated in approximately 3 min with a buffer containing a cyclodextrin and a crown ether. To our knowledge, this is the first report of a fast chiral separation of such a complex mixture on an electrophoresis chip. Detection was carried out amperometrically; derivatization of the analytes is not necessary.

Buffers↗