Sang Kyung Kim

Detection of Hepatitis B Virus (HBV) DNA at femtomolar concentrations using a silica nanoparticle-enhanced microcantilever sensor.

We report Hepatitis B Virus (HBV) DNA detection using a silica nanoparticle-enhanced dynamic microcantilever biosensor. A 243-mer nucleotide of HBV DNA precore/core region was used as the target DNA. For this assay, the capture probe on the microcantilever surface and the detection probe conjugated with silica nanoparticles were designed specifically for the target DNA. For efficient detection of the HBV target DNA using silica nanoparticle-enhanced DNA assay, the size of silica nanoparticles and the dimension of microcantilever were optimized by directly binding the silica nanoparticles through DNA hybridization. In addition, the correlation between the applied nanoparticle concentrations and the resonant frequency shifts of the microcantilever was discussed clearly to validate the quantitative relationship between mass loading and resonant frequency shift. HBV target DNAs of 23.1 fM to 2.31 nM which were obtained from the PCR product were detected using a silica nanoparticle-enhanced microcantilever. The HBV target DNA of 243-mer was detected up to the picomolar (pM) level without nanoparticle enhancement and up to the femtomolar (fM) level using a nanoparticle-based signal amplification process. In the above two cases, the resonant frequency shifts were found to be linearly correlated with the concentrations of HBV target DNAs. We believe that this linearity originated mainly from an increase in mass that resulted from binding between the probe DNA and HBV PCR product, and between HBV PCR product and silica nanoparticles for the signal enhancement, even though there is another potential factor such as the spring constant change that may have influenced on the resonant frequency of the microcantilever.

A new combination MALDI matrix for small molecule analysis: application to imaging mass spectrometry for drugs and metabolites

Since the development of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, this procedure has been specifically used for analyzing proteins or high molecular weight compounds because of the interference of matrix signals in the regions of the low mass range. Recently, scientists have been using a wide range of chemical compounds as matrices that ionize small molecules in a mass spectrometer and overcome the limitations of MALDI mass spectrometry. In this study, we developed a new combination matrix of 3-hydroxycoumarin (3-HC) and 6-aza-2-thiothymine (ATT), which is capable of ionizing small molecules, including drugs and single amino acids. In addition to ionization of small molecules, the combination matrix by itself gives less signals in the low mass region and can be used for performing imaging mass spectrometry (IMS) experiments on tissues, which confirms the vacuum stability of the matrix inside a MALDI chamber. The drug donepezil was mapped in the intact tissue slices of mice simultaneously with a spatial resolution of 150 μm during IMS. IMS analysis clearly showed that intact donepezil was concentrated in the cortical region of the brain at 60 min after oral administration. Our observations and results indicate that the new combination matrix can be used for analyzing small molecules in complex samples using MALDI mass spectrometry.

Highly sensitive sandwich-type SPR based detection of whole H5Nx viruses using a pair of aptamers.

In this research, we report highly sensitive and specific sandwich-type SPR-based biosensor for the detection H5Nx whole viruses. A few of aptamers, for the first time, were successfully screened and characterized for whole avian influenza (AI) viruses, H5Nx, by using Multi-GO-SELEX method. The affinities of the aptamers developed in this study were ranged from 8×10(4) to 1×10(4)EID50/ml, and the aptamers IF22, IF23 were found to be specific to H5N1 and H5N8, respectively. In addition, some flexible aptamers IF20, IF15, and IF10 were found to bind to the H5N1 and H5N2, H5N1 and H5N8, or H5N1, H5N2, and H5N8, respectively. Moreover, aptamers IF10 and IF22 were found to bind H5N1 virus simultaneously and confirmed to bind the different site of the same H5N1 whole virus. Therefore, this pair of aptamers, IF10 and IF22, were successfully applied to develop the sandwich-type SPR-based biosensor assay which is rapid, accurate for the detection of AI whole virus from H5N1-infected feces samples. The minimum detectible concentration of H5N1 whole virus was found to be 200 EID50/ml with this sandwich-type detection using the aptamer pair obtained in this study. In addition, the sensitivity of this biosensor was successfully enhanced by using the signal amplification with the secondary aptamer conjugated with gold nanoparticles.

Rapid microfluidic separation of magnetic beads through dielectrophoresis and magnetophoresis

We present the design and fabrication of a new microfluidic device in which the dielectrophoresis and magnetophoresis phenomena were used for the separation of the superparamagnetic microbeads of different sizes. By exploiting the fact that two different particles can exhibit different dielectrophoretic force–frequency spectra, we utilize this device to perform multiplex detection from a single sample solution. We found the transition frequency range for 1, 2.8, and 4.5 μm magnetic beads using our device. Bead‐based analysis revealed that a high separation efficiency (∼90%) could be obtained from a single sample solution containing both 4.5 and 2.8 μm beads. The average flow velocity of the beads was maintained at 9.8 mm/s, enabling fast analysis with a smaller amount of reagents. The magnetic field distribution on the beads and the bead flow at the channel cross section for different dielectrophoretic conditions was obtained using CFD‐ACE+ simulation. Issues relating to the fabrication and operation of the device are discussed in detail. Finally, we demonstrated the feasibility of parallel detection/trapping of different beads on the same chip. This separation approach offers the performance of multiplex analysis in lab‐on‐a‐chip devices.

Facile Method for Development of Ligand-Patterned Substrates Induced by a Chemical Reaction

Patterned substrates have been widely used in various applications, including arrays of biomolecules and cells, highthroughput assays, and direct target sensing. In practice, those demands have been achieved by either of or a combination of two strategies: 1) direct incorporation of biomolecules or functional-group-containing molecules into desired patterns and 2) generation of functional-group-presenting patterns by way of chemical conversions on the surface. The former encompasses microcontact printing (mCP), dip-pen nanolithography (DPN), polymer-pen lithography (PPL), microfluidic networks (mFNs), and microarrays. The latter utilizes the “turning-on” strategy, in which inactive substrates are switched to an active state to reveal organic functional groups, in most cases by electrochemical or photochemical conversions. Patterned functional groups in both strategies are further used as chemical handles for immobilization of biomolecules, such as cell-adhesion ligands, enzyme substrates, proteins, oligosaccharides, and oligonucleotides, to afford patterned substrates. As a typical recent example, Rozkiewicz et al. reported on modified mCP for the preparation of oligonucleotide micropatterns. In their report, oxidized PDMS stamps were first coated with positively charged dendrimers followed by negatively charged oligonucleotides in a layer-by-layer arrangement, and were transferred to a solid support for the generation of microarrays. Smith and co-workers introduced a photo-labile protecting group to a thiol functionality. Various patterns of small molecules and proteins were prepared by using a photolithographic method in combination with thiol-specific conjugation chemistry. Yousaf et al. showed that ligand density and composition influence the rate of stem-cell differentiation by using hydroquinone-based electroactive substrates, which were patterned with a variety of ligands by using microarray technology. Although these two strategies are reliable, well established, and, therefore, widely used, each of the strategies offers limitations on practical use as a general platform for ligand-patterned substrates. For instance, direct contact printing methods, such as mCP, cannot control ligand density on the surface, which can provide important quantitative information for use in experimental design. A concern with regard to the turning-on strategy is that in some cases activated functional groups require specified conjugation chemistry and, therefore, necessitate preparatory steps (tagging steps) to make the ligands compatible with the conjugation reaction. Herein, we describe a simple, efficient, and straightforward method for ligand patterning on a surface, induced by a non-invasive organic chemical reaction—which we have termed a chemical-reaction-induced patterning (CRIP)— and equipped with the capability for control of ligand density. In addition, our method is compatible with common patterning tools and conjugation chemistry. Herein, we demonstrate our strategy by using two popular patterning tools, mCP and microarray, and verify the fidelity of the preparation of ligand-patterned substrates by patterning cell-adhesion ligands and aptamers. Our strategy for preparation of ligand-patterned substrates relies on conversion of a substrate from a dormant (inactive) state to an active state by way of a chemical reaction grafted with a patterning method (Figure 1). The resulting patterned area presents a chemically reactive functional group, that is, a primary amine, which can be harnessed for the immobilization of biomolecules of interest. Our approach utilizes self-assembled monolayers (SAMs) on gold terminated with masked functional groups. Figure 1B shows the structure of the monolayer and the chemical reactions taking place on the surface for the preparation of the amine-functionalized substrate (for the synthesis of the quinone-terminated alkanethiol, see the Supporting information). The quinone-presenting monolayer was treated with a reducing agent by using patterning tools. Upon reduction of the quinone to the corresponding hydroquinone, a cyclization reaction ensues to afford an amine group. The resulting amine can react with linkers or ligands through aminespecific bioconjugation chemistry. The surrounding tri(ethylene glycol) groups provide inertness towards the nonspecific adsorption of proteins and cells, which is the most demanding feature of substrates in biological/biochemical studies at interfaces. Herein, we demonstrate this CRIP strategy by preparing various patterns of RGD (Arg-Gly-Asp) [a] H. Seo, I. Choi, J. Lee, Dr. S. Kim, Prof. D.-E. Kim, Prof. W.-S. Yeo Department of Bioscience and Biotechnology Konkuk University, Seoul, 143-701 (Korea) Fax: (+82) 2-2030-7890 E-mail : wsyeo@konkuk.ac.kr [b] S. K. Kim Nano-Bio Research Center Korea Institute of Science and Technology, Seoul, 136-791 (Korea) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201100084.

Single-carbon discrimination by selected peptides for individual detection of volatile organic compounds

Although volatile organic compounds (VOCs) are becoming increasingly recognized as harmful agents and potential biomarkers, selective detection of the organic targets remains a tremendous challenge. Among the materials being investigated for target recognition, peptides are attractive candidates because of their chemical robustness, divergence, and their homology to natural olfactory receptors. Using a combinatorial peptide library and either a graphitic surface or phenyl-terminated self-assembled monolayer as relevant target surfaces, we successfully selected three interesting peptides that differentiate a single carbon deviation among benzene and its analogues. The heterogeneity of the designed target surfaces provided peptides with varying affinity toward targeted molecules and generated a set of selective peptides that complemented each other. Microcantilever sensors conjugated with each peptide quantitated benzene, toluene and xylene to sub-ppm levels in real time. The selection of specific receptors for a group of volatile molecules will provide a strong foundation for general approach to individually monitoring VOCs.

Extensible Multiplex Real-time PCR of MicroRNA Using Microparticles

Multiplex quantitative real-time PCR (qPCR), which measures multiple DNAs in a given sample, has received significant attention as a mean of verifying the rapidly increasing genetic targets of interest in single phenotype. Here we suggest a readily extensible qPCR for the expression analysis of multiple microRNA (miRNA) targets using microparticles of primer-immobilized networks as discrete reactors. Individual particles, 200~500 μm in diameter, are identified by two-dimensional codes engraved into the particles and the non-fluorescent encoding allows high-fidelity acquisition of signal in real-time PCR. During the course of PCR, the amplicons accumulate in the volume of the particles with high reliability and amplification efficiency over 95%. In a quick assay comprising of tens of particles holding different primers, each particle brings the independent real-time amplification curve representing the quantitative information of each target. Limited amount of sample was analyzed simultaneously in single chamber through this highly multiplexed qPCR; 10 kinds of miRNAs from purified extracellular vesicles (EVs).

Direct Electrical Measurement of Protein–Water Interactions and Temperature Dependence Using Piezoelectric Microcantilevers

Since proteins are important biopolymers that act as the most important functional component of living cells, as well as being related to complex diseases such as cancer, detection tools that measure the dynamics of protein folding, their conformational stability, and specifi c interactions are essential in understanding biological functions. Major studies have provided some answers regarding the protein dynamics related to protein–water interactions and temperature-dependent interactions. [ 1–3 ] Analytical tools used to investigate protein dynamics, such as NMR spectroscopy, X-ray crystallography, neutron scattering, and molecular dynamic (MD) simulations, have been studied. [ 4–6 ] Although these detection methods provide microscopic insight into protein interactions, a real-time electrical measurement system that can be used for quantitative analysis has not yet been demonstrated. Cantilevers have proven to be a valuable tool in the study of biopolymers. The fundamental merit of nanomechanical translation using a cantilever for biological detection is that it can provide a common platform for high-throughput label-free analysis of protein–protein binding, DNA hybridization, and protein–protein interactions. [ 7–10 ] The most commonly used cantilevers rely on optical signals that inevitably lead to diffi culties in utilizing the application in point-of-care diagnostics and have a limited use in turbid or opaque fl uidic environments. [ 7 , 11 ] In order to address these problems, an approach for the electrical detection of cantilevers has been only recently demonstrated, using metal–oxide–semiconductor fi eld-effecttransistor-embedded cantilevers, [ 12 ] piezoresistive detectors, [ 13 , 14 ]

Quantification of disease marker in undiluted serum using an actuating layer-embedded microcantilever

In this study, we describe the application feasibility of a dynamic microcantilever with regard to the detection of a specific protein in human serum or real blood using an end-point analysis. The mechanical response (i.e., resonant frequency) of a functionalized dynamic microcantilever was shown to be altered by molecular interactions, which allowed for the detection of biomolecules present in small quantities without any additional signal enhancements, such as labeling. For the application of the microcantilever sensors to bioassays of serum samples, the mechanical response from the nonspecific adsorption of abundant proteins must be reduced, because it significantly influences the output signal deviation of the microcantilever sensor. We implemented a label-free prostate specific antigen (PSA) detection protocol in standard serum via our established process, which was designed to minimize nonspecific protein adsorption. PSA is a tumor marker for prostate cancer, with a threshold concentration of 2–4 ng...

Microparticle-based RT-qPCR for highly selective rare mutation detection

The quantitative reverse transcription polymerase chain reaction (RT-qPCR) has become one of the most widely used methods in the detection of disease-specific RNAs. The RT-qPCR involves two separate steps, RT and qPCR. In this study, we suggest a new RT-qPCR protocol with the particles of primer-immobilized networks (PINs), performing capture, RT and amplification of a target RNA in one particle. The production of undesired cDNAs was dramatically suppressed by the specific capture of the target RNA within the particle. Afterward, RT and amplification processes are performed without loss of cDNAs as exchanging the reaction solution. The biomarker gene of chronic myeloid leukemia, Bcr-Abl fusion transcript, is detected in the sensitivity of single mutant leukemic cell mixed in 104 normal cell using this protocol with the excellent restraint of non-specific signal. This protocol that whole processes are performed in the particle in a row is preferred for the highly specific detection of target RNAs in complex sample.