Biomedical subjects

Jeong-Woo Choi

Publications and source records attributed to Jeong-Woo Choi.

At least 19 recordsLinked to original sources

PubMed2006

Drop formation via breakup of a liquid bridge in an AC electric field.

Experimental results are presented for the study of drop formation mechanism in a newly proposed electrohydrodynamic (EHD) method of drop generation in an AC electric field. In the method, a small drop is generated in two stages. A pendant drop is elongated with large oscillation by an electric force in the first stage. Then, it undergoes formation and breakup of a liquid bridge between the upper nozzle and the insulator-coated lower flat plate in the second stage. It is found that there exists a resonant frequency for maximum oscillation, which leads to an efficient drop formation in the latter stage. It is also found that breakup of liquid bridge is accelerated by the electrowetting tension acting on the drop perimeter contacting the insulator-coated flat plate. Thus the whole procedure of drop formation depends heavily on the frequency of AC field and the properties of the insulator such as hydrophilicity, thickness, and the dielectric constant. It is demonstrated that a wide range of drop size, from picoliter to nanoliter, can be obtained by controlling such key parameters without changing the nozzle diameter.

DNA↗

PubMed2006

Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification.

Optimal detection of a pathogen present in biological samples depends on the ability to extract DNA molecules rapidly and efficiently. In this paper, we report a novel method for efficient DNA extraction and subsequent real-time detection in a single microchip by combining laser irradiation and magnetic beads. By using a 808 nm laser and carboxyl-terminated magnetic beads, we demonstrate that a single pulse of 40 seconds lysed pathogens including E. coli and Gram-positive bacterial cells as well as the hepatitis B virus mixed with human serum. We further demonstrate that the real-time pathogen detection was performed with pre-mixed PCR reagents in a real-time PCR machine using the same microchip, after laser irradiation in a hand-held device equipped with a small laser diode. These results suggest that the new sample preparation method is well suited to be integrated into lab-on-a-chip application of the pathogen detection system.

DNA, Bacterial↗

PubMed2006

Application of complement 1q for the site-selective recognition of immune complex in protein chip.

Complement 1q (C1q) was applied for the specific recognition of antibody-antigen complex in antibody-based protein chip. The specific binding of C1q to antibody-antigen complex was investigated by surface plasmon resonance (SPR) with respect to Yersinia entericolitica, Salmonella typimurium, insulin, and bovine serum albumin. The protein chip was fabricated with two different kinds of antibodies a zigzag configuration. When one of antigens and fluorescein-isothiocyanate (FITC)-labeled C1q was applied on the protein chip, the specific binding event of C1q to immune complexes formed on protein chip was observed by fluorescence microscopy. These results implicate that the C1q can be used as an alternative to many antibodies that may be utilized individually on each spot of the protein chip.

Antigen-Antibody Complex↗

PubMed2005

Electrohydrodynamic (EHD) dispensing of nanoliter DNA droplets for microarrays.

In the present paper, we first demonstrated the possibility of electrohydrodynamic (EHD) dispensing method for preparing nanoliter probe DNA droplets on surfaces in DNA microarrays. To study the effect of an electric field on the dynamic behavior of pendent DNA droplet, visualization experiments with three kinds of electrode shapes are performed. In the early stage of droplet dispensing, it is shown that applied electric field assists a gravitational force exerted on DNA droplet. The pendent droplet is elongated in the parallel direction of applied electric field. However, after making fluid bridge between electrodes, it is shown that the electric force accelerates the capillary breaking of droplet by assisting a surface tension force exerted on droplet surface. Specifically, nanoliter dispensing volume (2 nL) is obtained in the needle-type electrode configuration. In addition, for the case of hydrophobic electrode surface, it is shown that the dispensing volume and spot size are remarkably decreased. Under the high relative humidity condition, it is observed that spot size is rapidly increased because of reduction in evaporation rate on droplet surface during the dispensing procedure. On the other hand, it is obtained that the spot size is not changed significantly in the wide range of DNA concentration from 1 to 10,000 nM. To monitor the influence of high electric voltage on DNA stability, we prepared a silicon-based chip with five capture probes for pathogens related with respiratory infectious diseases by EHD dispensing method. From the examination, it is clearly confirmed that pathogens are detected and the effective signal levels of pathogenic bacteria after hybridization are retained. Consequently, it is found that EHD dispensing method can be used to make cost-effective DNA microarrays with no thermal and electrical influences on DNA properties.

DNA↗

PubMed2005

Immunosensor for the detection of Vibrio cholerae O1 using surface plasmon resonance.

An immunosensor for the detection of Vibrio cholerae O1 was developed on the basis of surface plasmon resonance (SPR). A protein G layer was fabricated by means of the chemical coupling between the free amine (-NH2) groups of protein G and the activated carboxyl groups present on a self-assembled monolayer (SAM) consisting of a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2). A monoclonal antibody, which was confirmed to be specific to V. cholera O1 by the Western blotting technique, was immobilized on the protein G layer. The formation of the SAM, the protein G layer and the sequential binding of the antibody against V. cholera O1 were investigated with SPR spectroscopy. As the number of fabricated layers increased, the minimum angle of plasmon resonance was increased accordingly. The target bacteria, V. cholera O1, was measured with the fabricated immunosensor, whose detection range was between 10(5) and 10(9) cells/mL.

Biosensing Techniques↗

PubMed2005

Study on orientation of immunoglobulin G on protein G layer.

A comparative study of immunoglobulin G (IgG) immobilization was performed, both on a thiolated protein G layer, where this immobilization was due to affinity binding with an Fc fragment of IgG, and on 11-mercaptoundecanoic acid (11-MUA), where the immobilization was due to chemical bonding. The change of IgG layer formation on the two base layers as a function of the IgG concentration was investigated by surface plasmon resonance (SPR), atomic force microscopy (AFM) in a non-contact mode, and spectroscopic ellipsometry (SE). It was observed that the IgG layer was immobilized more evenly on the thiolated protein G layer than on the 11-MUA layer, based on the SPR measurements. The surface topology analysis by AFM indicated that the IgG layer was immobilized on the protein G layer according to the envelope profile of the base layer. Based on the SE analysis, it was determined that the IgG layer thickness on the thiolated protein G layer increased with increasing IgG concentration. Based on the above analyses, the scheme for orientation of IgG immobilized on the thiolated protein G layer was proposed.

Biosensing Techniques↗

PubMed2005

Protein array consisting of sol-gel bioactive platform for detection of E. coli O157:H7.

Sol-gel-derived bioactive platform was fabricated for detection of pathogenic microbes, E. coli O157:H7. Design flexibility of sol-gel technique and ease of fabrication can fulfill to create the surfaces with structural and chemical features that are compatible with biomaterials such as antibody, enzymes, etc. In this study, the bioactive platform was prepared based on the silica gels, which were produced by hydrolyzing tetraethylorthosilane (TEOS) in ethanol. The mercaptopropyl triethoxysilane (MPTS) was mixed with the TEOS solution for the surface functionalization of bioactive platform. During TEOS hydrolysis, the modified thin film was prepared by sol-gel dip coating. Antibody against E. coli O157:H7 was immobilized with a configuration of protein array using piezo-type dispensing system. Surface morphology of the prepared bioactive platform was analyzed using atomic force microscopy (AFM). The antibody-antigen interaction was investigated with fluorescence microscopy and sandwich type immunoassay using fluorescein isothiocyanate (FITC)-labeled antibody. The results showed that antibody was sequestered within the sol-gel-derived bio-gel due to physical adsorption. The measurement of E. coli O157:H7 was done using the fabricated antibody surface. The fluorescence intensity was proportional to the concentration of E. coli O157:H7, of which the detection limit was 10(2)CFU/ml.

Antibodies, Monoclonal↗

PubMed2005

Cell immobilization using self-assembled synthetic oligopeptide and its application to biological toxicity detection using surface plasmon resonance.

The immobilized cell using self-assembled synthetic oligopeptide was applied to the biological toxicity detection of environmental pollutant. Thin films based on cysteine-terminated synthetic oligopeptides were fabricated for the immobilization of Escherichia coli O157:H7 on gold (Au) substrate. Layer formation and immobilization of E. coli O157:H7 were investigated with surface plasmon resonance (SPR) and atomic force microscopy (AFM). Experimental results showed that the thin film of cysteine-terminated synthetic oligopeptide was successfully fabricated and it could be applied for the immobilization of E. coli O157:H7. The attached living cell was exposed to toxic chemical such as phenol, which induced the change of SPR angle. As the exposed concentration of phenol was increased, the change of plasmon resonance angle was increased, which indicates the decrease of cell viability. The detection limit based on SPR was determined as 5 ppm. The proposed cell immobilization method using self-assembly technique can be applied to construct the cell microarray for the diagnosis, drug detection, and on-site monitoring.

Biological Assay↗

PubMed2005

Adenoviral p53 effects and cell-specific E7 protein-protein interactions of human cervical cancer cells.

We investigated the time-course tumor growth suppression effects of recombinant adenovirus expressing p53 on human cervical cancer cells and cell-specific E7 protein-protein interactions in cell lysates using surface plasmon resonance (SPR) biosensor. Six HPV-infected human cervical cancer cell lines (HPV 16-positive cells, CaSki and SiHa cells; HPV 18-positive cells, HeLa and HeLaS3 cells; and HPV negative C33A and HT3 cells) were used. After infection with AdCMVp53, the cell-specific growth inhibition was studied in vitro and in vivo. Also, we produced the recombinant E7 oncoprotein of HPV 16 type and tested chip-based protein-protein interactions with each cell lysate. For each cervical cancer cell, differential cell growth inhibitions were shown via cell count assay and MTT assay. Note that the same trend in suppression levels was shown in CaSki, HeLa and in SiHa, HeLaS3, respectively. In contrast, infection with AdCMVLacZ showed increased cell growth in a manner similar to the negative control group. The levels of p53 protein were notably expressed in CaSki and HeLa more than in SiHa and HeLaS3 for 4 days. In contrast, p53 expression was continually maintained in C33A and HT3 for 6 days. After transfection AdCMVp53 into CaSki- and SiHa-xenografted nude mice, the size of tumor was remarkably decreased in SiHa cells as compared to AdCMVLacZ transfection. The SPR sensor surface was successfully modified with the recombinant E7 oncoprotein and showed cell-specific interactions between E7 and its target proteins from cell lysates. The anti-tumor effects were accomplished via differential role of p53-specific apoptotic cell death, which is dependent upon the cervical cancer cell line. Also, a molecular level understanding of cell-dependent protein interaction effects of recombinant E7 was shown.

Adenoviridae↗

PubMed2005

The fabrication of protein chip based on surface plasmon resonance for detection of pathogens.

Protein chip based on surface plasmon resonance (SPR) was developed for detection of pathogens existing in contaminated environment, such as Escherichia coli O157:H7, Salmonella typhimurium, Legionella pneumophila, and Yersinia enterocolitica. Protein G was immobilized to endow the orientation of antibody molecules on the SPR surface. The pathogen binding of the protein chip was investigated by SPR spectroscopy. Consequently, it was found that the four kinds of pathogen could be selectively detected by using SPR-based protein chip.

Bacteria↗

PubMed2005

Fabrication of DNA-protein conjugate layer on gold-substrate and its application to immunosensor.

The fabrication of antibody thin film using both protein G and oligonucleotide was carried out by self-assembly (SA) technique for immunosensor. A mixture of 11-mercaptoundecanoic acid (MUA) and oligonucleotide with thiol (SH) end group was self-assembled of gold (Au) surface for two-dimensional (2D) configuration. Protein G was chemically adsorbed on the 11-MUA surface, and then the antibody was immobilized on the protein G region. On the immobilized single-stranded DNA, the complementary DNA-antibody conjugate was hybridized for the oriented immobilization of antibody. The formation of self-assembled 11-MUA/oligonucleotide layer, protein G immobilization, antibody layer, and antigen binding was investigated using surface plasmon resonance (SPR). The topographies of the fabricated surfaces were observed by atomic force microscopy (AFM). When compared with the amount of antigen binding on the antibody thin film fabricated by protein G only, the proposed biosurface fabricated with both protein G and oligonucleotide showed better binding capacity, which implicates the improvement of the detection limit.

Antibodies↗

PubMed2005

Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance.

Biosurface fabrication using the Fab' fragment of immunoglobulin (IgG) was carried out by self-assembly (SA) technique. The pepsin-digested monoclonal antibody (Mab) against bovine insulin containing the F(ab')(2) fragment and residual proteins was separated using affinity chromatography and dialysis. To prevent the nonspecific binding of F(ab')(2) onto gold (Au) substrate, the native disulfide bridge was reduced using dithiothreitol (DTT) to convert F(ab')(2) into Fab', which made the immobilization to be carried out via the native thiol (-SH) group. The fabricated biosurface using SA technique showed the formation of stable thin film through AFM topography. Through the concentration change of DTT and Fab', the absorption characteristics against the Au surface were investigated using surface plasmon resonance (SPR) with the flow cell. The amount of immobilized antibody fragment and the antigen binding capacity were regulated with respect to the reduction state and concentration of F(ab')(2). Based on the biosurface of the fabricated Fab', the insulin-detection was carried out by the measurement of SPR. The proposed antibody surface could successfully detect the bovine insulin at the concentration from 100 ng/mL to 10 microg/mL.

Animals↗

PubMed2005

Formation of a protein monomolecular layer by a combined technique of LB and SA methods.

This paper describes a technique developed for the formation of the self-assembled protein monomolecular layer. The main idea is a direct transfer of protein molecules consisting of a Langmuir-Blodgett (LB) film onto the surface of another chemically modified metal substrate and induction of the spontaneous formation of a self-assembled monolayer (SAM) by chemisorption without protein aggregates. In the present experiments, a cytochrome c (cyt c) SAM on a gold substrate was prepared by incubating a EDC/MUA-modified gold substrate overlaid with a cyt c LB film in a phosphate buffer solution. Scanning tunneling microscopy (STM) image of a cyt c SAM shows that the size of cyt c clusters in the most part of the substrate is approximately 5 nm, indicating the cyt c monomolecular layer. The rectifying property of a cyt c monolayer was confirmed to remain by an asymmetric I-V curve in the applied bias from -1 V to 1 V.

Animals↗

PubMed2004

Detection of insulin-antibody binding on a solid surface using imaging ellipsometry.

Imaging ellipsometry (IE) was used to detect the binding of insulin to its antibody on a solid surface. The modification of a gold surface with 11-mecaptoundecanoic acid (11-MUA), the adsorption of protein G, and antibody immobilization onto the protein G layer were confirmed by surface plasmon resonance. Ellipsometric images and ellipsometric angles of the surface antibody were acquired using the IE system by off-null ellipsometry. Ellipsometric images of antigen binding to the antibody were acquired, and their mean optical intensities estimated. Changes in mean optical intensity indicated that the detection range for insulin was from 10 ng/ml to 100 microg/ml.

Antigen-Antibody Complex↗

PubMed2004

Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi.

A surface plasmon resonance (SPR) based immunosensor using self-assembled protein G was developed for the detection of Salmonella paratyphi. In order to endow a solid substrate binding affinity to protein G, the free amine (-NH2) of protein G was substituted into thiol (-SH) using 2-iminothiolane. Thus, self-assembled protein G was fabricated on gold (Au) substrate. The formation of protein G layer on Au surface, and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analysis of the protein G layer on Au surface was performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. paratyphi using self-assembled protein G was developed with a detection range of 10(2)-10(7) CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. paratyphi could be applied to construct other immnosensors or protein chips.

Biosensing Techniques↗

PubMed2004

Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium.

An immunosensor based on surface plasmon resonance (SPR) using protein G was developed for the detection of Salmonella typhimurium. A protein G layer was fabricated by binding chemically to self-assembly monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) on gold (Au) surface. The formation of protein G layer on Au surface modified with 11-MUA and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The effect of detergent such as Tween-20 on binding efficiency of antibody and antigen was investigated by SPR. The binding efficiency of antigen to the antibody immobilized on Au surface was improved up to about 85% and 100% by using protein G and Tween-20, respectively. The surface morphology analyses of 11-MUA monolayer on Au substrate, protein G layer on 11-MUA monolayer and antibody layer immobilized on protein G layer were performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. typhimurium using protein G was developed with a detection range of 10(2) to 10(9)CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. typhimurium could be applied to construct other immnosensors or protein chips.

Gold↗

PubMed2004

Immunosensor for detection of Yersinia enterocolitica based on imaging ellipsometry.

An immunosensor for the detection of pathogens was developed using imaging ellipsometry (IE) as a detection method. Yersinia enterocolitica was selected as the target pathogen in this study. A gold surface deposited with a self-assembled layer of 11-mercaptoundecanoic acid (11-MUA) was used as a substrate. For the fabrication of the immunosensor, protein G spots were made on the substrate using an inkjet-type microarrayer, and monoclonal antibody (Mab) was adsorbed onto the protein G spots. Deposition of each layer onto the substrate was confirmed by the measurement of surface plasmon resonance. The ellipsometric image of the protein G spot and the Mab-adsorbed protein G spot were acquired using an off-null ellipsometry type of imaging ellipsometry system. By measuring the ellipsometric angles of the protein layers, the surface concentration of each protein layer was calculated. The change in the mean optical intensity of the protein spot to the various concentrations of Y.enterocolitica was estimated. The immunosensor using imaging ellipsometry could successfully detect Y. enterocolitica in concentrations varying from 10(3) to 10(7) cfu/mL. The proposed immunosensor system has the advantage of allowing label-free detection, high sensitivity, and operational simplicity.

Antibody Affinity↗

PubMed2003

Development of a membrane strip immunosensor utilizing ruthenium as an electro-chemiluminescent signal generator.

A photometric immunosensor that can be used for on-site diagnosis has been constructed. The sensor system was assembled by partially superimposing a nitrocellulose membrane strip (the lower) containing an immobilized antigen on the surface with a glass fiber membrane strip (the upper) including two electrodes on the opposite surfaces. To amplify the signal, we introduced a liposome, containing ruthenium molecules trapped in the core, chemically coupled to an antibody specific to the analyte (e.g. Legionella antigen). In the presence of the analyte, immune complexes were formed by antigen-antibody reactions upon addition of the immuno-liposome into a sample. This mixture was then absorbed by the capillary action from the bottom of the membrane strip. The liposome particles in the complexes were carried by a medium through the antigen pad without interaction, while free immuno-liposome was trapped by immune reactions on the pad surfaces. The aqueous medium influx into the glass pad dissolved a detergent pre-located within the compartment and the liposome rupture thereby released ruthenium molecules into the solution. The molecules were oxidized on the electrode surfaces and produced an electro-chemiluminescence (ECL) in proportion to the analyte concentration. The signal generation based on ECL resulted in an exponential dose-response pattern and the analyte detection limit of 2 ng/ml was approximately 10-fold more sensitive than that obtained from a conventional system.

Adsorption↗