Sang Woo Joo

DNA hybridization detection in a microfluidic channel using two fluorescently labelled nucleic acid probes.

A conceptually new technique for fast DNA detection has been developed. Here, we report a fast and sensitive online fluorescence resonance energy transfer (FRET) detection technique for label-free target DNA. This method is based on changes in the FRET signal resulting from the sequence-specific hybridization between two fluorescently labelled nucleic acid probes and target DNA in a PDMS microfluidic channel. Confocal laser-induced microscopy has been used for the detection of fluorescence signal changes. In the present study, DNA hybridizations could be detected without PCR amplification because the sensitivity of confocal laser-induced fluorescence detection is very high. Two probe DNA oligomers (5-CTGAT TAGAG AGAGAA-TAMRA-3 and 5-TET-ATGTC TGAGC TGCAGG-3) and target DNA (3-GACTA ATCTC TCTCT TACAG GCACT ACAGA CTCGA CGTCC-5) were introduced into the channel by a microsyringe pump, and they were efficiently mixed by passing through the alligator teeth-shaped PDMS microfluidic channel. Here, the nucleic acid probes were terminally labelled with the fluorescent dyes, tetrafluororescein (TET) and tetramethyl-6-carboxyrhodamine (TAMRA), respectively. According to our confocal fluorescence measurements, the limit of detection of the target DNA is estimated to be 1.0 x 10(-6) to 1.0 x 10(-7)M. Our result demonstrates that this analytical technique is a promising diagnostic tool that can be applied to the real-time analysis of DNA targets in the solution phase.

Colorimetric genotyping of single nucleotide polymorphism based on selective aggregation of unmodified gold nanoparticles

We have developed a colorimetric method for genotyping of single nucleotide polymorphism (rs2131877) in 15 human DNA samples using selective aggregation of unmodified gold nanoparticles. First, we designed two different types of oligonucleotide probes with either thymine or cytosine at the end that are perfect complementary to the target allele sequence. After hybridization of the probe and target DNAs, the double-stranded DNA was added to the unmodified gold nanoparticle suspensions. By adjusting the salt concentration, we could induce aggregation of gold nanoparticles exclusively for the samples with the perfectly matched double-stranded DNAs, which resulted in a distinct color change of the suspension. This enabled us to discern samples with three different genotypes of the target sequences by naked eye: (i) the genotype with only T (thymine) alleles; (ii) that with only C (cytosine) alleles; (iii) that with both T and C alleles. We also confirmed these results by an independent direct sequencing method. These results suggest that the selective aggregation of unmodified gold nanoparticles can successfully be used to discern genotypes of single nucleotide polymorphisms.

Fluorescence-based detection of point mutation in DNA sequences by CdS quantum dot aggregation.

We present a novel method for the detection of single base mismatch based on fluorescence quenching that unmodified CdS quantum dots exhibit upon aggregation. Target DNA sequences of interest are breast cancer 2 (BRCA2) and signal-induced proliferation-associated gene 1 (Sipa1) sequences. We monitor aggregation of CdS quantum dots upon addition of double-stranded DNAs at different salt concentration using quasi-elastic light scattering (QELS), transmission electron microscopy (TEM), photoluminescence spectroscopy, and zeta potential measurement. Our results indicate that the double-stranded DNA with a perfectly matched sequence can easily be discerned by naked eye from the single base mismatched one due to the fluorescence quenching phenomenon caused by selective aggregation of the CdS quantum dots.

Raman detection of localized transferrin-coated gold nanoparticles inside a single cell.

AbstractWe investigated the cellular uptake behavior of non-fluorescent metal nanoparticles (NPs) by use of surface-enhanced Raman scattering (SERS) combined with dark-field microscopy (DFM). The uptake of Au NPs inside a single cell could also be identified by DFM first and then confirmed by z-depth-dependent SERS at micrometer resolution. Guided by DFM for the location of Au NPs, an intracellular distribution assay was possible using Raman dyes with unique vibrational marker bands in order to identify the three-dimensional location inside the single cell by obtaining specific spectral features. Au NPs modified by 4-mercaptobenzoic acid (MBA) bearing its –COOH surface functional group were used to conjugate transferrin (Tf) protein using the 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) reaction. The protein conjugation reaction on Au surfaces was examined by means of color change, absorption spectroscopy, and SERS. Our results demonstrate that DFM techniques combined with SERS may have great potential for monitoring biological processes with protein conjugation at the single-cell level.n Figurez-Depth-dependent Raman detection of localized transferrin-coated gold nanoparticles inside a single cell

Colorimetric detection of mutations in epidermal growth factor receptor using gold nanoparticle aggregation.

We have detected mutations in the epidermal growth factor receptor (EGFR) of non-small cell lung cancer cells using the selective aggregations of gold nanoparticles. Mutations in exon 19 and exon 21 of EGFR gene were detected in non-amplified genomic DNAs that were isolated from both the lung cancer cell lines and the cancer tissues of non-small cell lung cancer patients. At the optimal salt concentration, addition of the mutant DNA that was hybridized with the complementary probe into the suspension of unmodified gold nanoparticles caused the substantial aggregation of the gold nanoparticles and the color change of solution. Gold nanoparticles, however, did not exhibit significant aggregation, and the solution color remained unchanged with the addition of the wild type DNA that was hybridized with the probe. In the eight specimens from non-small cell lung cancer patients, we could detect the in-frame deletion mutant form in exon 19 and the L858R point mutation in exon 21 by selective aggregation with gold nanoparticles. These results were also confirmed by an independent direct sequencing method using a DNA analyzer. We found that selective aggregation with gold nanoparticles could be successfully applied to direct detection of EGFR mutations in non-amplified genomic DNAs.

Interfacial Behavior of Benzoic Acid and Phenylphosphonic Acid on Nanocrystalline TiO2 Surfaces

The interfacial behavior of self-assembled thin films of benzoic acid (BA) and phenylphosphonic acid (PPOA) anchored on TiO(2) surfaces was studied by using temperature-dependent diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. On the basis of the disappearance of the OH band from the infrared spectra at room temperature, BA and PPOA appear to adsorb onto TiO(2) surfaces through carboxylate and phosphonate groups, respectively. Above 420 degrees C, DRIFT spectra indicated that both BA and PPOA desorb from TiO(2) surfaces; however, dissimilar desorption behavior could be inferred for BA and PPOA from their temperature-dependent spectral changes. The benzene ring modes of PPOA remained above 420 degrees C, whereas those of BA disappeared. Density functional theory calculations showed that the adsorption of BA and PPOA on TiO(2) surfaces corresponded to bidentate bridging geometry on TiO(2) surfaces, and the adsorption of PPOA is stronger than that of BA. The monodentate structures with energy differences of 4.9 and 9.1 kcal mol(-1) from the most stable bidentate structures of BA and PPOA, respectively, from the DFT calculations appeared to be possible, particularly at the high temperatures above 420 degrees C, as indicated by the intensified OH bands. The geometry of PPOA was also estimated to be more upright standing than that of BA on TiO(2) surfaces, which may lead a rather straight detachment from the TiO(2) surfaces based on the presence of in-plane ring modes in the DRIFT spectra at the higher temperature.

Preparation of new polyelectrolyte/ silver nanocomposites and their humidity-sensitive properties

A simple strategy was developed based on polyelectrolyte/silver nanocomposite to obtain humidity-sensitive membranes. The major component of a humid membrane is the polyTEAMPS/silver nanocomposite obtained by thermal heating the mixture of a polyelectrolyte and silver isopropylcarbamate complex. Humidity sensors prepared from polyTEAMPS/silver (w/w=100/0 and 100/6) nanocomposites had an average impedance of 292, 8.83 and 0.86 kΩ, and 5,327, 140 and 0.93 kΩ at 30, 60 and 95% relative humidity (RH), respectively. Hysteresis, temperature dependence and response time were also measured. Activation energies and complex impedance spectroscopy of the various components of the polyelectrolyte/silver nanocomposite films were examined for the humiditysensing membrane.

Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

Hydrogen bonding-induced color recovery of gold nanoparticles upon conjugation of amino acids

Hydrogen bonding-induced redispersion of the aggregated Au nanoparticles upon N-hydroxysuccinimide ester bioconjugations may provide a simple and colorimetric tool as an optical sensor to detect a trace amount of amino acids as low as approximately 10(-6) M in an aqueous solution.

Colloidal gold nanoparticle conjugates of gefitinib.

Gefitinib (GF) is a US Food and Drug Administration-approved epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor for treating the lung cancers. We fabricated colloidal gold nanoparticle (AuNP) conjugates of the GF anticancer drug by self-assembly to test their potency against A549, NCI-H460, and NCI-H1975 lung cancer cells. GF adsorption on AuNP surfaces was examined by UV-vis absorption spectra and surface-enhanced Raman scattering. Density functional theory calculations were performed to estimate the energetic stabilities of the drug-AuNP composites. The N1 nitrogen atom of the quinazoline ring of GF was calculated to be more stable than the N3 in binding Au cluster atoms. The internalizations of GF-coated AuNPs were examined by transmission electron and dark-field microscopy. A cell viability test of AuNP-GF conjugates with the EGFR antibody exhibited much higher reductions than free GF for A549, NCI-H460, and NCI-H1975 lung cancer cells after treatment for 48.