Dae-Yeon Cho

Studies on the biosorption of heavy metals onto Chlorella vulgaris

Abstract The metal binding characteristics of Chlorella vulgaris were investigated. Metal uptake by intact algal cells was found to consist of two processes; (1) a fast, metabolism‐independent surface reaction and (2) metabolism‐dependent slow uptake. Results of both the pH and the ionic strength effects on the metal adsorption indicate that the specific interaction accounts for the majority of metal adsorption. Carboxyl and amine surface functional groups of Chlorella vulgaris were modified to assess the role of the surface charge site as the metal binding one. The carboxyl‐modified algae showed major decreases in the adsorption capacity of Cd(II) and Zn(II) binding. The amine‐modified algae also displayed some decreases in metal adsorptions.

Induction of apoptosis by selenite and selenodiglutathione in HL‐60 cells: Correlation with cytotoxicity

Effects of selenite and selenodiglutathione, an initial metabolite of selenite, on the induction of apoptosis and cytotoxicity were investigated in human promyelocytic leukemia HL‐60 cells. Treatment of selenite or selenodigtutathione resulted in concentration‐dependent cytotoxicity, measured by lactate dehydrogenase leakage assay, and by tetrazolium salt reduction assay. Selenodiglutathione has been shown to exert more cytotoxic effect than selenite in both assay systems. Time‐course study of cellular selenium uptake suggests that the higher cytotoxicity of selenodiglutathione be largely due to faster and greater selenium uptake rate. Treatment with selenite or selenodiglutathione also induced apoptosis in a dose‐dependent manner, as detected by enzyme‐linked immunosorbent assay and by DNA fragmentation assay. The dose‐response data of apoptosis induced by selenite or selenodiglutathione were similar to those of cytotoxicity, implicating a relationship between the induction of apoptosis and cytotoxicity. Zn, which is a well‐known inhibitor of apoptosis, dose‐dependently blocked not only the induction of apoptosis, but also the membrane damage induced by selenium, corroborating this hypothesis. It was noted that the inhibition of apoptosis by Zn exerted little protective effect on cytotoxicity at higher concentrations of selenium, compared with a perfect protective effect at low concentration of selenium. These results suggest that cytotoxicity induced by selenium may be partially correlated with apoptosis.

A Polydiacetylene Microchip Based on a Biotin–Streptavidin Interaction for the Diagnosis of Pathogen Infections

A micropatterned polydiacetylene (PDA) chip, utilizing the unique fluorogenic property of PDA and a specific biotin-streptavidin (STA) interaction, is constructed to detect pathogen infections. To construct the PDA chip, biotin-modified diacetylene liposomes are immobilized on aldehyde glass and conjugated with STA, followed by UV irradiation to polymerize the STA-functionalized diacetylene liposomes. Genomic DNA of a model pathogen, Chlamydia trachomatis, is isolated from human samples and biotin-labeled target DNA is obtained through PCR amplification using biotin-11-dUTP. Owing to the stimulus caused by the biotin-STA interaction, the biotinylated DNA induces an intense fluorescence signal on the immobilized PDA. By using this strategy, it is possible to diagnose Chlamydia infections by applying DNA samples from several nonhealthy humans to a single PDA chip. The results of this study serve as the basis for a new strategy for fluorogenic PDA microarray-based diagnosis of pathogen infections.

Colorimetric quantification of galactose using a nanostructured multi-catalyst system entrapping galactose oxidase and magnetic nanoparticles as peroxidase mimetics

A colorimetric method for quantification of galactose, which utilizes a nanostructured multi-catalyst system consisting of Fe(3)O(4) magnetic nanoparticles (MNPs) and galactose oxidase (Gal Ox) simultaneously entrapped in large pore sized mesocellular silica, is described. Gal Ox, immobilized in a silica matrix, promotes reaction of galactose to generate H(2)O(2) that subsequently activates MNPs in silica mesopores to convert a colorimetric substrate into a colored product. By using this colorimetric method, galactose can be specifically detected. Along with excellent reusability via application of simple magnetic capturing, enhanced operational stability was achieved by employing a cross-linked enzyme aggregate (CLEA) method for Gal Ox immobilization. This protocol leads to effective prevention of enzyme leaching from the pores of mesocellular silica. The analytical utility of the new colorimetric biosensor was demonstrated by its use in diagnosing galactosemia, a genetic metabolic disorder characterized by the inability to utilize galactose, through analysis of clinical dried blood spot specimens. A microscale well-plate format was employed that possesses a multiplexing capability. The multi-catalyst system entrapping Gal Ox and MNPs represents a new approach for rapid, convenient, and cost-effective quantification of galactose in human blood and it holds promise as an alternative method for galactosemia diagnosis, replacing the laborious procedures that are currently in use.

A DNA intercalation-based electrochemical method for detection of Chlamydia trachomatis utilizing peroxidase-catalyzed signal amplification

A sensitive electrochemical DNA detection method for the diagnosis of sexually transmitted disease (STD) caused by Chlamydia trachomatis was developed. The method utilizes a DNA-intercalating agent and a peroxidase promoted enzymatic precipitation reaction and involves the following steps. After hybridization of the target C. trachomatis gene with an immobilized DNA capture probe on a gold electrode surface, the biotin-tagged DNA intercalator (anthraquinone) was inserted into the resulting DNA duplex. Subsequently, the polymeric streptavidin/peroxidase complex was applied to the biotin-decorated electrode. Peroxidase catalyzed 4-chloronaphthol to produce insoluble product, which is precipitated on the electrode surface in the presence of hydrogen peroxide. Cyclic voltammograms with the gold electrode exhibited a peak current of ferrocenemethanol in electrolyte, which decreased in a proportional way to increasing concentration of target DNA owing to insulation of electrode surface by the growing insoluble precipitate. Using this strategy, we were able to detect picomolar concentrations of C. trachomatis gene in a sample taken from a real patient.

Colorimetric SNP Genotyping Based on Allele-Specific PCR by Using a Thiol- Labeled Primer

chemistry. [7] However, these methods usually require multiple manipulations, high levels of technical expertise, and expensive instruments, all of which makes them unsuitable for pointof-care testing (POCT) applications. Consequently, a great incentive exists for the development of simple, rapid, and costeffective SNP genotyping methods that are suitable for POCT in facility-limited environments. Colorimetric methods fulfill most of the above requirements as they enable cost-effective and on-site detection without the need for sophisticated equipment. To date, a number of such methods for SNP detection that utilize, for example, metal nanoparticles, [8] peroxidase-mimicking DNAzymes, [9] and peptide nucleic acids (PNAs), [1] have been reported. Among these, gold nanoparticle (AuNP)-based approaches [10–12] have received great attention owing to their unique optical properties, robustness, and high surface areas, which make them ideally suited as signaling probes in colorimetric-detection platforms. [12] However, the requirement for modification of the AuNP surface with oligonucleotide probes and/or the need for precise temperature control during the assay significantly limits applications of AuNP-based methods. In recent studies, we developed a novel strategy based on unmodified AuNPs for the colorimetric detection of target nucleic acids that are amplified by thiol-labeled primers without time-consuming and complicated procedures. [13] We successfully demonstrated its diagnostic utility by reliably identifying Chlamydia trachomatis infection. Here, we have extended our previous strategy for nucleic acid detection by devising a novel method for SNP identification that incorporates a modified allele-specific PCR (ASPCR) method. In principle, ASPCR is a cost-effective procedure that characterizes SNPs based on differences between the PCR efficiency of allele-specific primers. [14] The primers consist of sequences adjacent to the polymorphic site that are complementary to the allelic variant and differ only in the terminal nucleotide at the 3’-end. [2] In the first step of our new assay procedure, ASPCR is performed with a thiolated primer. In the second step, AuNPs are mixed with the products and then subjected to salt-induced aggregation. The resulting color of the solution (i.e., red or blue) indicates the genotype at the SNP site because only the thiolated PCR products provide the AuNPs with a significant resistance to salt-induced aggregation and inhibit the red-to-blue color transition. The strategy we have devised for SNP identification is schematically illustrated in Figure 1 for the detection of a single base mutation. In the first step, ASPCR is performed separately in four different reaction tubes containing each of the four thiolated forward primers, which differ only in the terminal base at their 3’-ends (A, T, C and G). When the base at the 3’end of the forward primer is complementary to the antisense strand of the genomic target DNA, PCR amplification occurs and generates PCR amplicons labeled with a thiol group at the 5’-end of one strand. In contrast, the other three primers, which have a noncomplementary nucleotide at their 3’-ends, cannot be extended and thus they fail to generate PCR amplicons. The existence of the thiol-labeled PCR products is then colorimetrically determined by sequentially adding unmodified AuNPs and NaCl to each tube. When thiolated PCR amplicons are mixed with AuNPs, they bind to the surface of unmodified AuNPs through a strong gold–thiol interaction. The electrostatic repulsion between the AuNPs is enhanced as a consequence of the large number of negative charges on their surfaces due to the negatively charged phosphate backbone of the bound DNA. In addition, steric repulsion between the AuNPs is increased because the longer (compared to the diameter of AuNPs) DNA grafted onto the AuNPs surface forms a thick polymeric barrier that prevents the particles from approaching each other. [15] As a consequence of these “electrostatic/steric stabilization effects”, [16] salt-induced aggregation of AuNPs is significantly inhibited, and the solution remains red when salt is added. In contrast, in the absence of the thiol-labeled PCR amplicon, AuNPs undergo immediate aggregation upon salt addition; this results in a colorimetric transition from red to blue. By employing this strategy, the genotypes at mutation sites can be conveniently determined based on the color of the sample. To prove the conceptual basis of this novel strategy for SNP genotyping and to confirm the crucial role played by thiolated primers in the AuNPs-based colorimetric assay, ASPCR was per

Mismatch DNA-specific enzymatic cleavage employed in a new method for the electrochemical detection of genetic mutations

Utilizing enzymatic mismatched DNA-specific cleavage and electrocatalytic signaling, a new electrochemical method for the detection of DNA mutations was developed and successfully applied to detect various mutations in the BRCA1 gene.

A Sexually Transmitted Disease (STD) DNA chip for the diagnosis of genitourinary infections

The results of an investigation aimed at the development of a DNA chip for the detection of genitourinary infections are described. Through analysis of over 35,000 clinical cases, 14 pathogens which are most abundantly found among Koreans were selected and candidate sequences for capture probes were accordingly chosen by considering their sequences and β-globin house-keeping gene. Among this group, the most suitable capture probe sequences were selected by employing repeated chip tests in which they are immobilized on a glass chip by using a recently developed novel gold nanoparticles-based method. A multiplex PCR method was established to generate fluorescence-labeled sequences for all 14 pathogens along with the β-globin gene. By using optimized hybridization conditions, the final chip was constructed and employed to diagnose reliably both single and multiple infections in clinical human samples for 14 target pathogens. The results show that the novel chip methodology serves as a highly reliable and convenient tool for the diagnosis of Sexually Transmitted Diseases (STDs). Furthermore, this study has its great significance in that it demonstrates the entire process from statistical analysis of a large number of clinical cases to the final development of STD DNA chip just ready to be applied or commercialized in the clinical diagnostic field.

Cell-Based Quantification of Homocysteine Utilizing Bioluminescent Escherichia coli Auxotrophs

A cell-based quantitative assay system for Hcy has been developed by utilizing two Escherichia coli auxotrophs that grow in the presence of methionine (Met) and either homocysteine (Hcy) or Met, respectively. A bioluminescent reporter gene, which produces luminescence as cells grow, was inserted into the auxotrophs, so that cell growth can be readily determined. When the relative luminescence unit (RLU) values from the two auxotrophs immobilized within agarose gels arrayed on a well plate were measured, the amount of Hcy was quantitatively determined on the basis of differences between two RLU values corresponding to cell growth of two auxotrophs with excellent levels of precision and reproducibility. Finally, the diagnostic utility of this assay system was verified by its employment in reliably determining different stages of hyperhomocysteinemia in human plasma samples providing CVs of within and between assays that are less than 2.9% and 7.1%, respectively, and recovery rates of within and between assays that are in the range of 99.1-103.5% and 97.5-105.5%, respectively. In contrast to existing conventional methods, the new system developed in this effort is simple, rapid, and cost-effective. As a result, it has great potential to serve as a viable alternative for Hcy quantification in the diagnosis of hyperhomocysteinemia.

A mass spectrometry-based multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification

We herein describe a new mass spectrometry-based method for multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification (SDA) reaction. In this method, allele-specific ligation is first performed to discriminate base sequence variations at the SNP site within the PCR-amplified target DNA. The primary ligation probe is extended by a universal primer annealing site while the secondary ligation probe has base sequences as an overhang with a nicking enzyme recognition site and complementary mass marker sequence. The ligation probe pairs are ligated by DNA ligase only at specific allele in the target DNA and the resulting ligated product serves as a template to promote the SDA reaction using a universal primer. This process isothermally amplifies short DNA fragments, called mass markers, to be analyzed by mass spectrometry. By varying the sizes of the mass markers, we successfully demonstrated the multiplex SNP genotyping capability of this method by reliably identifying several BRCA mutations in a multiplex manner with mass spectrometry.