Ahjeong Son

Characterization of denaturation and renaturation of DNA for DNA hybridization

Objectives The present study was designed to systematically characterize the denaturation and the renaturation of double stranded DNA (dsDNA), which is suitable for DNA hybridization. Methods A series of physical and chemical denaturation methods were implemented on well-defined 86-bp dsDNA fragment. The degree of each denaturation was measured and the most suitable denaturation method was determined. DNA renaturation tendency was also investigated for the suggested denaturation method. Results Heating, beads mill, and sonication bath did not show any denaturation for 30 minutes. However probe sonication fully denatured DNA in 5 minutes. 1 mol/L sodium hydroxide (alkaline treatment) and 60% dimethyl sulfoxide (DMSO) treatment fully denatured DNA in 2-5 minutes. Conclusions Among all the physical methods applied, the direct probe sonication was the most effective way to denature the DNA fragments. Among chemical methods, 60% DMSO was the most adequate denaturation method since it does not cause full renaturation during DNA hybridization.

Environmental behavior of engineered nanomaterials in porous media: a review

A pronounced increase in the use of nanotechnology has resulted in nanomaterials being released into the environment. Environmental exposure to the most common engineered nanomaterials (ENMs), such as carbon-based and metal-based nanomaterials, can occur directly via intentional injection for remediation purposes, release during the use of nanomaterial-containing consumer goods, or indirectly via different routes. Recent reviews have outlined potential risks assessments, toxicity, and life cycle analyses regarding ENM emission. In this review, inevitable release of ENMs and their environmental behaviors in aqueous porous media are discussed with an emphasis on influencing factors, including the physicochemical properties of ENMs, solution chemistry, soil hydraulic properties, and soil matrices. Major findings of laboratory column studies and numerical approaches for the transport of ENMs are addressed, and studies on the interaction between ENMs and heavy metal ions in aqueous soil environments are examined. Future research is also presented with specific research directions and outlooks.

Highly Sensitive Detection of Bisphenol A by NanoAptamer Assay with Truncated Aptamer

For the sensitive quantification of bisphenol A (BPA), we have developed NanoAptamer assay, which employs aptamer and complementary signaling DNA, a set of quantum dots (QD), and magnetic beads (MBs). Signaling DNA-QD655 was tethered to MB-QD565 via the aptamer. The affinity of the aptamer to BPA resulted in the release of the signaling DNA-QD655 from the complex and hence the corresponding decrease in the QD655 fluorescence measurement signal. Three new aptamers (23, 58, and 24-mer) were designed via truncation of the reference aptamer (73-mer). The sensitivity and selectivity of each aptamer for BPA detection via NanoAptamer assay were investigated. One of the truncated aptamers (24-mer) has shown a significantly better performance (limit of detection, LOD, 0.17 pg/mL) than the reference 73-mer aptamer (LOD, 570 pg/mL). It has also shown the best selectivity for BPA detection over BPA analogues (i.e., bisphenol B, bisphenol C, and diethylstilbestrol). It corresponded to a normalized fluorescence change of 33.7% at the environmentally relevant concentration of 1 ng/mL (1 ppb) BPA; however, the analogues remained unchanged (2.3-3.9%).

Detection of bisphenol A using palm-size NanoAptamer analyzer

We have demonstrated a palm-size NanoAptamer analyzer capable of detecting bisphenol A (BPA) at environmentally relevant concentrations (<1ng/mL or ppb). It is designed for performing reaction and fluorescence measurement on single cuvette sample. Modified NanoGene assay was used as the sensing mechanism where signaling DNA and QD655 was tethered to QD565 and magnetic bead via the aptamer. Aptamer affinity with BPA resulted in the release of the signaling DNA and QD655 from the complex and hence corresponding decrease in QD655 fluorescence measurement signal. Baseline characterization was first performed with empty cuvettes, quantum dots and magnetic beads under near-ideal conditions to establish essential functionality of the NanoAptamer analyzer. Duration of incubation time, number of rinse cycles, and necessity of cuvette vibration were also investigated. In order to demonstrate the capability of the NanoAptamer analyzer to detect BPA, samples with BPA concentrations ranging from 0.0005 to 1.0ng/mL (ppb) were used. The performance of the NanoAptamer analyzer was further examined by using laboratory protocol and commercial spectrofluorometer as reference. Correlation between NanoAptamer analyzer and laboratory protocol as well as commercial spectrofluorometer was evaluated via correlation plots and correlation coefficients.

Quantitative detection of single walled carbon nanotube in water using DNA and magnetic fluorescent spheres.

Carbon nanotubes (CNTs) possess unique properties that have led to an increase in their research and usage for a wide variety of fields. This growing demand of CNTs poses a major public health risk given its unregulated release into the environment. Unfortunately there is a significant information gap on the actual quantity of CNTs in the environment due to limitation of existing detection methods. This is mainly owing to the ubiquitous carbon chemistry of CNT. In response we developed a method (CNT-capture method) that is able to structurally differentiate CNT from other interference carbon materials in an aqueous medium. The affinity between single walled nanotubes (SWNTs) and specific single stranded DNA (ssDNA) was employed to capture SWNTs in water. SWNT-specific separation was obtained via magnetic separation. Dual fluorescent labels attached to sandwich ssDNA probes were used for quantification. The specific affinity between DNA and SWNTs was verified and no significant side-interactions were observed. With optimized incubation duration (30 min) and buffer composition (10(-7) % sodium dodecyl sulfate and pH 7.9), a calibration curve of quantification (R(2) = 0.90) was obtained with a range of SWNT concentration (0.05-10 μg/mL) against graphene as a planar analog. Comparison to other spectroscopy based methods was carried out to highlight the specificity and sensitivity of the presented method for CNT detection in aquatic sample.

Process control factors for continuous microbial perchlorate reduction in the presence of zero-valent iron

Process control parameters influencing microbial perchlorate reduction via a flow-through zero-valent iron (ZVI) column reactor were investigated in order to optimize perchlorate removal from water. Mixed perchlorate reducers were obtained from a wastewater treatment plant and inoculated into the reactor without further acclimation. Examined parameters included hydraulic residence time (HRT), pH, nutrients requirement, and perchlorate reduction kinetics. The minimum HRT for the system was concluded to be 8 hr. The removal efficiency of 10 mg·L−1 influent perchlorate concentration was reduced by 20%–80% without control to the neutral pH (HRT = 8 hr). Therefore pH was determined to be an important parameter for microbial perchlorate reduction. Furthermore, a viable alternative to pH buffer was discussed. The microbial perchlorate reduction followed the first order kinetics, with a rate constant (K) of 0.761 hr−1. The results from this study will contribute to the implementation of a safe, cost effective, and efficient system for perchlorate reduction to below regulated levels.

Detection of Cyanobacteria in Eutrophic Water Using a Portable Electrocoagulator and NanoGene Assay

We have demonstrated the detection of cyanobacteria in eutrophic water samples using a portable electrocoagulator and NanoGene assay. The electrocoagulator is designed to preconcentrate cyanobacteria from water samples prior to analysis via NanoGene assay. Using Microcystis aeruginosa laboratory culture and environmental samples (cell densities ranging from 1.7 × 105 to 4.1 × 106 and 6.5 × 103 to 6.6 × 107 cells·mL-1, respectively), the electrocoagulator was evaluated and compared with a conventional centrifuge. Varying the operation duration from 0 to 300 s with different cell densities was first investigated. Preconcentration efficiencies (obtained via absorbance measurement) and dry cell weight of preconcentrated cyanobacteria were then obtained and compared. For laboratory samples at cell densities from 3.2 × 105 to 4.1 × 106 cells·mL-1, the preconcentration efficiencies of electrocoagulator appeared to be stable at ∼60%. At lower cell densities (1.7 and 2.2 × 105 cells·mL-1), the preconcentration efficiencies decreased to 33.9 ± 0.2 and 40.4 ± 5.4%, respectively. For environmental samples at cell densities of 2.7 × 105 and 6.6 × 107 cells·mL-1, the electrocoagulator maintained its preconcentration efficiency at ∼60%. On the other hand, the centrifuges preconcentration efficiencies decreased to nondetectable and below 40%, respectively. This shows that the electrocoagulator outperformed the centrifuge when using eutrophic water samples. Finally, the compatibility of the electrocoagulator with the NanoGene assay was verified via the successful detection of the microcystin synthetase D (mcyD) gene in environmental samples. The viability of the electrocoagulator as an in situ compatible alternative to the centrifuge is also discussed.

Quantitative Polymerase Chain Reaction for Microbial Growth Kinetics of Mixed Culture System.

Microbial growth kinetics is often used to optimize environmental processes owing to its relation to the breakdown of substrate (contaminants). However, the quantification of bacterial populations in the environment is difficult owing to the challenges of monitoring a specific bacterial population within a diverse microbial community. Conventional methods are unable to detect and quantify the growth of individual strains separately in the mixed culture reactor. This work describes a novel quantitative PCR (qPCR)-based genomic approach to quantify each species in mixed culture and interpret its growth kinetics in the mixed system. Batch experiments were performed for both single and dual cultures of Pseudomonas putida and Escherichia coli K12 to obtain Monod kinetic parameters (μmax and Ks). The growth curves and kinetics obtained by conventional methods (i.e., dry weight measurement and absorbance reading) were compared with that obtained by qPCR assay. We anticipate that the adoption of this qPCR-based genomic assay can contribute significantly to traditional microbial kinetics, modeling practice, and the operation of bioreactors, where handling of complex mixed cultures is required.

Microorganism-ionizing respirator with reduced breathing resistance suitable for removing airborne bacteria

Abstract In this paper, we have demonstrated the feasibility of using microorganism-ionizing respirators with reduced breathing resistance to remove airborne bacteria. Using a miniaturized corona ionizer and two pairs of separator electrodes, airborne bacteria were ionized and removed from the airflow. Two microorganism-ionizing respirator designs were experimentally evaluated with flow rates ranging from ∼10 to 20 L/min and yielded airborne bacterial removal efficiencies of ∼75%–100%. Further, they were in close agreement with the analytical airborne particle removal efficiencies, at a similar range of flow rates. These flow rates also correspond to the breathing rates of standing and walking adults. More importantly, the breathing resistance could be reduced by more than 50% for flow rates of ∼200 L/min. Using manganese (IV) oxide coated mesh, the ozone concentration in the air outflow was reduced to less than 0.1 ppm, at a flow rate of ∼20 L/min, thus enabling safe use. The power consumption was less than 1 W.

The Implications of Fragmented Genomic DNA Size Range on the Hybridization Efficiency in NanoGene Assay

DNA hybridization-based assays are well known for their ability to detect and quantify specific bacteria. Assays that employ DNA hybridization include a NanoGene assay, fluorescence in situ hybridization, and microarrays. Involved in DNA hybridization, fragmentation of genomic DNA (gDNA) is necessary to increase the accessibility of the probe DNA to the target gDNA. However, there has been no thorough and systematic characterization of different fragmented gDNA sizes and their effects on hybridization efficiency. An optimum fragmented size range of gDNA for the NanoGene assay is hypothesized in this study. Bacterial gDNA is fragmented via sonication into different size ranges prior to the NanoGene assay. The optimum size range of gDNA is determined via the comparison of respective hybridization efficiencies (in the form of quantification capabilities). Different incubation durations are also investigated. Finally, the quantification capability of the fragmented (at optimum size range) and unfragmented gDNA is compared.