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Biomedical subjects

Haesik Yang

Publications and source records attributed to Haesik Yang.

12 recordsLinked to original sources
PubMed2004

Protein patterning based on electrochemical activation of bioinactive surfaces with hydroquinone-caged biotin.

We report a protein attachment and patterning method based on a hydroquinone-caged biotin surface that generates bioactive biotin by mild electrochemical perturbation. The electrochemical activation proceeds under the buffered aqueous environment at neutral pH. It also allows site-selective generation of bioactive biotin for the immobilization of target protein by using prepatterned electrode arrays.

Bacterial Proteins↗
PubMed2004

Electrochemically induced and controlled one-step covalent coupling reaction on self-assembled monolayers.

We report on a novel covalent coupling method using electrochemical activation of hydroquinone monoester self-assembled monolayers. The reaction generates benzoquinone as a good leaving group, followed by nucleophilic acyl substitution with a primary amine to form an amide in high yield. The method allows the site-selective and the reaction-controlled positioning of biotin on the individually addressable microelectrode array and, subsequently, density-differentiated patterning of streptavidin on the biotin surfaces. Because the electrochemical coupling method provides a very rapid, mild, and quantitatively controllable reaction pathway for covalent bond formation on organic surfaces, it will be used as a versatile molecular anchoring tool in fields such as molecular electronics and biochip technology.

Letter↗
PubMed2004

Bulk-micromachined submicroliter-volume PCR chip with very rapid thermal response and low power consumption.

The current paper describes the design, fabrication, and testing of a micromachined submicroliter-volume polymerase chain reaction (PCR) chip with a fast thermal response and very low power consumption. The chip consists of a bulk-micromachined Si component and hot-embossed poly(methyl methacrylate)(PMMA) component. The Si component contains an integral microheater and temperature sensor on a thermally well-isolated membrane, while the PMMA component contains a submicroliter-volume PCR chamber, valves, and channels. The micro hot membrane under the submicroliter-volume chamber is a silicon oxide/silicon nitride/silicon oxide (O/N/O) diaphragm with a thickness of 1.9 microm, resulting in a very low thermal mass. In experiments, the proposed chip only required 45 mW to heat the reaction chamber to 92 degrees C, the denaturation temperature of DNA, plus the heating and cooling rates are about 80 degrees C s(-1) and 60 degrees C s(-1), respectively. We validated, from the fluorescence results from DNA stained with SYBR Green I, that the proposed chip amplified the DNA from vector clone, containing tumor suppressor gene BRCA 1 (127 base pairs at 11th exon), after 30 thermal cycles of 3 s, 5 s, and 5 s at 92 degrees C, 55 degrees C, and 72 degrees C, respectively, in a 200 nL-volume chamber. As for specificity of DNA products, owing to difficulty in analyzing the very small volume PCR results from the micro chip, we vicariously employed the larger volume PCR products after cycling with the same sustaining temperatures as with the micro chip but with much slower ramping rates (3.3 degrees C s(-1) when rising, 2.5 degrees C s(-1) when cooling) within circa 20 minutes on a commercial PCR machine and confirmed the specificity to BRCA 1 (127 base pairs) with agarose gel electrophoresis. Accordingly, the fabricated micro chip demonstrated a very low power consumption and rapid thermal response, both of which are crucial to the development of a fully integrated and battery-powered instrument for a lab-on-a-chip DNA analysis.

Calibration↗
PubMed2004

An independent, temperature-controllable microelectrode array.

Rapid, localized temperature control and negligible power consumption are key requisites for realizing effective parallel and sequential processing in the miniaturized, integrated biomedical microdevices where temperature-dependent biochemical reactions and fluid flow occur. In this study, an independent, temperature-controllable microelectrode array, with excellent temperature control rates and minimal power consumption, has been developed using microelectromechanical systems technology. The microfabricated array consists of Pt microelectrodes (100-microm diameter), with n-doped polysilicon microheaters (1.4-k Omega resistance), and vacuum-sealed cavities of depth 6.2 microm and diameter 200 microm. The thermal characteristics of each microelectrode were evaluated electrochemically through surface temperature measurements. The large heater power coefficient (2.1 +/- 0.1 degrees C mW(-1)) and the short heating and cooling times (less than 0.2 s for T(0.95)) are consequences of the vacuum-sealed cavities, which facilitate good thermal isolation and low thermal mass. The temperature of each microelectrode is independently controlled by a dedicated microheater, without thermally influencing the adjacent microelectrodes significantly.

Journal Article↗
PubMed2004

Ferritin immunosensing on microfabricated electrodes based on the integration of immunoprecipitation and electrochemical signaling reactions.

A signal registration strategy from micropatterned immunosensors that converts antigen-antibody binding reactions into electrochemical signals was demonstrated. An array-type micropatterned gold electrode on a silicon wafer was fabricated, containing two electrode geometries of rectangular (100 microm x 500 microm) and circular (r. 50 microm) types, exhibiting electrochemical characteristics of bulk and micro-electrodes, respectively. Ferritin was employed as a model analyte for immunosensing because it has an advantageous molecular structure for functionalization to the sensing interface, and is regarded as a general marker protein for tumors and cancer recurrence. With the fabricated and ferritin-functionalized immunosensors, biospecific interactions were performed with antiferritin antiserum and secondary antibody samples, followed by electrochemical signaling via an immunoprecipitation reaction by the label enzyme. Under the optimized affinity-surface construction steps and reaction conditions, both types of microfabricated electrodes exhibited well-defined calibration results as a function of the protein concentration in antiserum samples. Furthermore, circular-type micropatterned immunoelectrodes exhibited voltammetric characteristics of microelectrodes, which is advantageous in terms of sensor operation under a fixed potential and low signal drift during the signaling reaction compared with the bulk-type electrodes. The results support that the employed signaling method with the proposed immunosensor configuration is fit for sensor miniaturization and integration to future biomicrosystems.

Antigen-Antibody Reactions↗
PubMed2003

An iridium oxide reference electrode for use in microfabricated biosensors and biochips.

In this paper we argue for the use of iridium oxide (IrO(x)) electrodes as quasi-reference electrodes in microfabricated biosensors and biochips that operate in buffered solutions. The simple microfabrication of these electrodes consists of a one-step electrodeposition of IrO(x) onto a microfabricated platinum (Pt) electrode. The IrO(x) electrode potential was found to vary less than 20 mV over 9 days after stabilization for 1 day in a phosphate-buffered saline (PBS) solution; this behavior of the electrode potential was found to be easily reproduced. Moreover, the electrode potential was found to vary by less than 15 mV in the initial hour of its use; this behavior of the electrode potential was also found to be reproducible. The performance of a microfabricated glucose sensor employing an IrO(x) reference electrode is characterized in this paper in order to evaluate the usefulness of this new IrO(x) electrode as a quasi-reference electrode. The glucose sensor consists of a recessed microfabricated Pt electrode array, an electrodeposited IrO(x) film, an inner layer composed of an electropolymerized poly(m-phenylenediamine)/glucose oxidase (PMPD/GOx) film, and an outer or protective layer composed of Teflon and polyurethane (PU) films. The response of this sensor was found to be equivalent to the response of the same sensor employing a commercial Ag/AgCl reference electrode. These results show that a microfabricated IrO(x) electrode can be used as a quasi-reference electrode in microfabricated biosensors and biochips operating in buffered solutions.

Biosensing Techniques↗
PubMed2003

Enzyme-amplified electrochemical detection of DNA using electrocatalysis of ferrocenyl-tethered dendrimer.

We have developed a sandwich-type enzyme-linked DNA sensor as a new electrochemical method to detect DNA hybridization. A partially ferrocenyl-tethered poly(amidoamine) dendrimer (Fc-D) was used as an electrocatalyst to enhance the electronic signals of DNA detection as well as a building block to immobilize capture probes. Fc-D was immobilized on a carboxylic acid-terminated self-assembled monolayer (SAM) by covalent coupling of unreacted amine in Fc-D to the acid. Thiolated capture probe was attached to the remaining amine groups of Fc-D on the SAM via a bifunctional linker. The target DNA was hybridized with the capture probe, and an extension in the DNA of the target was then hybridized with a biotinylated detection probe. Avidin-conjugated alkaline phosphatase was bound to the detection probe and allowed to generate the electroactive label, p-aminophenol, from p-aminophenyl phosphate enzymatically. p-Aminophenol diffuses into the Fc-D layer and is then electrocatalytically oxidized by the electronic mediation of the immobilized Fc-D, which leads to a great enhancement in signal. Consequently, the amount of hybridized target can be estimated using the intensity of electrocatalytic current. This DNA sensor exhibits a detection limit of 20 fmol. Our method was also successfully applied to the sequence-selective discrimination between perfectly matched and single-base mismatched target oligonucleotides.

Alkaline Phosphatase↗
PubMed2003

A polymer-based microfluidic device for immunosensing biochips.

This paper describes the design, fabrication, and test of a PDMS/PMMA-laminated microfluidic device for an immunosensing biochip. A poly(dimethyl siloxane)(PDMS) top substrate molded by polymer casting and a poly(methyl methacrylate)(PMMA) bottom substrate fabricated by hot embossing are bonded with pressure and hermetically sealed. Two inlet ports and an air vent are opened through the PDMS top substrate, while gold electrodes for electrochemical biosensing are patterned onto the PMMA bottom substrate. The analyte sample is loaded from the sample inlet port to the detection chamber by capillary force, without any external intervening forces. For this and to control the time duration of sample fluid in each compartment of the device, including the inlet port, diffusion barrier, reaction chamber, flow-delay neck, and detection chamber, the fluid conduit has been designed with various geometries of channel width, depth, and shape. Especially, the fluid path has been designed so that the sample flow naturally stops after filling the detection chamber to allow sufficient time for biochemical reaction and subsequent washing steps. As model immunosensing tests for the microfluidic device, functionalizations of ferritin and biotin to the sensing surfaces on gold electrodes and their biospecific interactions with antiferritin antiserum and streptavidin have been investigated. An electrochemical detection method for immunosensing by biocatalyzed precipitation has been developed and applied for signal registration. With the biochip, the whole immunosensing processes could be completed within 30 min.

Antibodies↗
PubMed2002

Glucose sensor using a microfabricated electrode and electropolymerized bilayer films.

A new type miniaturized glucose sensor with good selectivity and stable current response has been developed. The structure consists of a recessed rectangular microfabricated platinum electrode, inner layer of two electropolymerized nonconducting films, and outer bilayer of poly(tetrafluoroethylene) (Teflon) and polyurethane (PU) films. Glucose oxidase (GOx) is entrapped during the electropolymerization of a poly(m-phenylenediamine) (PMPD) film in an acetate buffer (AB) solution, on which a highly interference-resistive PMPD film is deposited in a phosphate buffered saline (PBS) solution. The second PMPD film causes no significant decrease in accessibility of glucose to GOx. The inner layer maintains less than 1% permeability to acetaminophen for 12 days. The fairly adhesive outer layer allows stable current response. Due to high permeability, the information about enzyme activity can be obtained without serious error in spite of outer layer intervening between enzymes and solution. The apparent Michaelis-Menten constant and the maximum steady-state current density were 24 mM and 80 microA cm(-2), respectively.

Acetaminophen↗
PubMed2002

Biocatalytic precipitation induced by an affinity reaction on dendrimer-activated surfaces for the electrochemical signaling from immunosensors.

We have developed a strategy of signal generation for immunosensors that transduces biospecific affinity recognition reactions into electrochemical signals. The cyclic voltammetric method, tracking the precipitation of insoluble products onto the sensing surface and the subsequent decrement in the electrode area, was chosen for signal registration. Precipitation of insolubilities was induced by the catalytic reaction of enzymes, which were labeled to the biospecifically attached protein or antibody molecules. As a model system for affinity recognition, we have investigated the functionalization of biotin groups to the sensing monolayer and their biospecific interactions with anti-biotin antibody molecules. The immunosensing interface was developed onto the dendrimer-activated self-assembled monolayers (SAMs), as the base template for the functionalization of the antigen moiety and signal generation. The advantages of using dendrimer-activated SAMs in comparison to the plain modified thiolate SAMs for the sensing surface were shown in terms of sensing performances, and the analytical characteristics of the resulting immunosensor were examined. Additionally, the sensing system was applied for biotin/(strept)avidin couples, extending the applicability of the developed strategy.

Anti-Bacterial Agents↗
PubMed2002

Stepwise surface regeneration of electrochemical immunosensors working on biocatalyzed precipitation.

A new strategy of stepwise surface regeneration for electrochemical immunosensors, working on a biocatalyzed precipitation reaction, has been developed. The strategy is based on the combination of deposited product thin-film dissolution and bound-protein displacement reactions from the modified sensor surfaces. As a model system, surfaces functionalized with biotin groups and their affinity recognition/ displacement reactions with antibiotin antibody molecules were chosen and investigated for affinity-sensing and stepwise regeneration reactions.

Antibodies↗