Comparison of the performance of the PANArray™ HPV test and DNA chip test for genotyping of human papillomavirus in cervical swabs

송혜정,이정원,김병기,송상용,배덕수,김대식 한국바이오칩학회 2010 BioChip Journal Vol.4 No.3

The aim of this study was to evaluate the recently developed PANArray™ Human papilloma virus (HPV) kit for detection and genotyping of 19 high-risk and 13 low-risk HPV types, and compare it with the commercially available DNA chip kit geno-typing of 24 HPVs. We llected cervical swabs from 741 patients with various stages of invasive cervical carcinoma being treated at the Samsung Medical Center. The overall HPV positivity rate was 73% using PANArray™ HPV and 72.1% with the DNA chip, and no statistically significant differences were found with respect to the cytology grade. Comparing the results of the two chips, concordant results were found in 637/741 samples (85.9%), compatible in 69/741 (9.3%) and discordant in 35/741(4.7%). Type-specific sequencing analysis of all samples revealed a 99.7% confirmation of PANArray™ HPV genotyping results, compared to 91.0% of DNA chip. The PANArray™ HPV test thus proved to be highly sensitive and accurate even when multiple HPV infections were present.

Micro-injection Molded Droplet Generation System for Digital PCR Application

조다애,김소영,강현우,표성한,정남규,배남호,이석재,김용태,이경균 한국바이오칩학회 2022 BioChip Journal Vol.16 No.4

Sensitive, effective, and quantitative analysis of infectious pathogens is an important task for the prevention of human health threats. Herein, we present an advanced approach to producing gene-encapsulated microdroplets for quantitative analysis using a micropatterned metal mold and injection molding technique with an automatically operated system. An injection molded microdroplet generation device was successfully fabricated with a minimum channel width of 30 μm and optimized to produce 100 μm diameter droplets. The optimized microchannel design and flow rate also enable the production of stable numbers of microdroplets (~ 16, 000 droplets). To verify the applicability of our device and system to droplet-based digital PCR analysis, Escherichia coli ( E. coli ) O157:H7 was selected as a model bacterial pathogen, and the stx2 gene was amplified in the microdroplets. The generated microdroplets exhibit both chemical and mechanical stability, and our results are similar to those obtained by a commercially available method. Accordingly, the usefulness of the microdroplet generative device and system is confirmed as a simple, fast, and reliable tool for the quantitative molecular analysis of infectious diseases.

Intein-mediated Protein Engineering for Biosensor Fabrication

전현진,이민형,장원희,권영은 한국바이오칩학회 2016 BioChip Journal Vol.10 No.4

Intein-mediated protein engineering has become a valuable tool for biosensing applications. Expressed protein ligation and protein trans-splicing were used to generate proteins with necessary tags or cyclic peptides that can be used to build various biosensing platforms such as biochips or sensor proteins. Especially the use of split-inteins that carry out conditional protein splicing reaction enabled the fabrication of various genetically-encoded biosensors that can monitor various signaling events in vivo including protein- protein interactions and protein translocalization. Biological targets activated split-inteins to generate functioning reporter molecules, such as luciferase and autofluorescent proteins. We here review various sensing platforms that utilize intein-mediated protein engineering technology mainly focusing on cell-based biosensors.

A Lateral Flow Assay for Nucleic Acid Detection Based on Rolling Circle Amplification Using Capture Ligand-Modified Oligonucleotides

이하늘,이주희,Yoo Kyung Kang,이주훈,양승주,정현정 한국바이오칩학회 2022 BioChip Journal Vol.16 No.4

We introduce a lateral flow assay (LFA) integrated with a modified isothermal nucleic acid amplification procedure for rapid and simple genetic testing. Padlock probes specific for the target DNA were designed for ligation, followed by rolling circle amplification (RCA) using capture ligand-modified oligonucleotides as primers. After hybridization with detection linker probes, the amplified target DNA is flowed through an LFA membrane strip for binding of gold nanoparticles as the substrate for colorimetric detection. We established and validated the “RCA-LFA” method for detection of mecA, the antibiotic resistance gene for methicillin-resistant Staphylococcus aureus (MRSA). The assay was optimized using various concentrations of primers and probes for RCA and LFA, respectively. The sensitivity was determined by performing RCA-LFA using various amounts of mecA target DNA, showing a detection limit of ~ 1.3 fmol. The specificity of the assay was examined using target DNAs for other resistance genes as the controls, which demonstrated positive detection signals only for mecA DNA, when added either individually or in combinations with the control targets. Furthermore, applying the RCA-LFA method using specifically designed probes for RNA-dependent RNA polymerase (RdRp) and receptor binding domain (RBD) gene for SARS-CoV-2, which demonstrated feasibility of the method for viral gene targets. The current method suggests a useful platform which can be universally applied for various nucleic acid targets, allowing rapid and sensitive diagnosis at point-of-care.

Hydrodynamically Focused Particle Filtration Using an Island Structure

이강선,김충,안병욱,강지윤,오광욱 한국바이오칩학회 2009 BioChip Journal Vol.3 No.4

We propose a particle filtration method based on hydrodynamic focusing. Three microchannels associated for the filtration were networked with an island structure to control a boundary to limit particle sizes. By adjusting the ratios of hydraulic resistances of the three microchannels, the boundary distance was controlled. Using hydrodynamic focusing in the microfluidic network, we have successfully designed and tested the device to filtrate particles of 10 μm and 20 μm. The proposed method can continuously separate particles by size. Thus, the proposed separation method can be applicable for many fields in biology and biomedical engineering.

Self-assembled Monolayer Fabrication of Cysteine-modified Ferredoxin

최정우,김영준,오병근,김문일 한국바이오칩학회 2007 BioChip Journal Vol.1 No.1

Rhodobacter sphaeroides ferredoxin is a metalloprotein with ferric ion in its active site. Ferredoxin has redox property and it can transfer the electron. These molecules can be applied to the bioelectronics by fabricating them as a self-assembled bio-film. The significant key of film fabrication is the immobilization method of bio-molecule. In our previous works, it has been reported that metalloprotein film can be fabricated by using chemical linker material that have thiol-group to assemble it on gold substrate. However, the chemical linker can interfere with electron transfer because it is acted as an insulator of the system. So, we used recombinant protein with cysteine functional residue at the end of the protein which can be directly immobilized on the gold (Au) surface. It could be confirmed the immobilization of the protein and surface morphology of thin film by surface plasmon resonance (SPR) and scanning tunneling microscope (STM). These results show that cysteine-modified ferredoxin can be used for making high quality protein film, and applied to the fabrication of nano-scale bioelectronics.

Evaluation of Signal Stability from Electrochemical Immunoanalysis Based on the Enzyme ‘back-filling’ Immobilization to Biorecognition Interfaces

이준황,원병연,윤현철 한국바이오칩학회 2007 BioChip Journal Vol.1 No.1

The signal stability from electrochemical immunosensors based on the enzymatic back-filling strategy was investigated. The presence of an analyte, especially antibody, which is bound to the surfaceimmobilized capture molecule, could cause a significant signal loss during subsequent handling procedures and signaling due to its limited affinity /stability. Compared to the typical method of detection, the signal from the back-filling immunoassay is generated differently from the covalently immobilized (‘back-filled’) glucose oxidase (GOX) to the sensing surface, not from the bound analyte that is usually labeled with signaling molecules. Therefore, an enhancement of signal stability would be expected with the back-filling immunoassay. To evaluate the merits of the enzymatic back-filling method, various antibody dissociation conditions from immunosensor surfaces were compared in terms of signal stability and sensitivity. As a model immunosensing reaction, the 2, 4-dinitrophenyl (DNP) group was functionalized to the biorecognition surface on a thin-film gold electrode, and anti-DNP antibody was employed as the target analyte. Cyclic voltammetry was used for immunosensor signal registration and to trace the protein adsorption/dissociation process. The back-filling method was found to be suitable for the analysis of immune reactions without labeled-antibody molecules, and the signal stability was comparable to the affinity sensor employing biotin/avidin couple.

SUMO(Small Ubiquitin-like Modifier) Chip for Analysis of SUMO-conjugation to a Target Protein

오영희,김학성,김영필 한국바이오칩학회 2007 BioChip Journal Vol.1 No.1

The analysis of protein interactions and modifications in a microarray format has become the focus of great interest in researchers in drug discovery, diagnostics, and cell biology, due to the advent of rapid and high-throughput biological assays requiring only a small amount of sample. Herein, we describe a chip-based analysis of sumoylation, the post -translational dification (PTM) process involving the covalent attachment of the small biquitin-like modifier (SUMO) family protein to a target protein in a mammalian cell. The protein was expressed using an in vitro translation system and mmobilized directly onto a glass slide using epitope tags fused to the protein. In this paper, the microarray-based analysis technique was shown to be a seful strategy for the identification of SUMO target proteins from preexisting protein pools and proteome arrays, in a highthroughput manner.

Immunosensors for Point-of-Care Testing

백세환,조정환,조일훈,오병근,김영기 한국바이오칩학회 2007 BioChip Journal Vol.1 No.1

Although immuno-chromatographic assays on membrane strips have been employed at points of care for more than 20 years, their applications have become limited to qualitative analyses of the analytes present at relatively high concentrations in samples. An evolution of technology in this field will be required in order to achieve the capability of detection sufficient for early diagnosis as well as a degree of quantification sufficient to allow for the monitoring of the sease progress, a digital display of analytical results, and automatic recording and correlation in a database. In order to attain proper sensitivity, the colloidal gold normally used as a tracer for colorimetry may be replaced with different signal generators, including fluorophores, magnetic beads, electrochemiluminescent substances, and enzymes. The means by which ntigen-antibody binding in the assay can be quantified as a measurable signal also varies, depending on the tracer employed and the transduction technology available. Indeed, we have devoted ourselves for more than ten years to the investigation of combinatorial substitutes onsisting of novel immunosensors that fulfill the demands inherent to medical diagnostics. In this review, selected immunosensor technologies developed in our laboratories are introduced, along with their detection principles and analytical characteristics.

Thermoplasmonic Scaffold Design for the Modulation of Neural Activity in Three-Dimensional Neuronal Cultures

Andrea Andolfi,장현수,Sergio Martinoia,남윤기 한국바이오칩학회 2022 BioChip Journal Vol.16 No.4

Neuromodulation has made great strides in recent years, but in vitro studies have been limited to two-dimensional cell cultures, far from in vivo conditions. In this study, we realized a novel thermoplasmonic platform for modulating the neural activity of three-dimensional cell cultures, providing a new tool to bring in vitro neuromodulation studies into a threedimensional environment. The photosensitive scaffold, obtained by covering soda-lime glass microbeads (diameter about 40 μm) with gold nanorods, integrates microbeads’ structural properties, intended to support the development of the neural network in three dimensions, with the photothermal properties of plasmonic nanoparticles. We demonstrate its efficiency in providing support for the construction of three-dimensional cell culture and how, under Near-infrared laser irradiation, their photothermal effect can precisely and non-invasively modulate the activity of the neural network. Our platform is expected to be a useful tool for improving neural network studies to better understand complex brain functions and neural disorders.