Beelee Chua

Development of first generation in-situ pathogen detection system (Gen1-IPDS) based on NanoGene assay for near real time E. coli O157:H7 detection.

We developed the first generation in-situ pathogen detection system (Gen1-IPDS) based on the NanoGene assay for detecting and quantifying Escherichia coli O157:H7 specific eaeA gene. The NanoGene assay employs the hybridization of target DNA with quantum dot labeled magnetic beads and probe DNAs to detect and quantify the target bacterial gene. The Gen1-IPDS is currently capable of executing four key steps required in the NanoGene assay: sample and reagents introduction, DNA hybridization, magnetic separation of complexes, and sample collection. Operational parameters such as magnet position, hybridization buffer composition, hybridization flow rate, and hybridization temperature were investigated. Using the experimentally determined operational parameters, the target gene was successfully quantified (R(2)=0.97) over a range of six orders of magnitude (10(-12) to 10(-6) mol L(-1)). The limit of detection (LOD) was determined to be 49×10(-15) mol L(-1). The specificity was also demonstrated by the differential discrimination of mismatched target DNAs. The NanoGene assay quantification results via Gen1-IPDS were validated by correlation with its laboratory version (R(2)=0.97).

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.

Detection of airborne bacteria with disposable bio-precipitator and NanoGene assay

We demonstrated the detection of airborne bacteria by a disposable bio-precipitator and NanoGene assay combination. The bio-precipitator employed micro corona discharge at 1960V and at less than 35µA to simultaneously charge, capture and lyse the airborne bacteria. This was enabled by the use of a 15μL liquid anode. Using a custom exposure setup, the target bacterium Bacillus subtilis in the atomization solution was rendered airborne. After exposure, the liquid anode in the bio-precipitator was subsequently measured for DNA concentration and analyzed with the NanoGene assay. As the bacterial concentration increased from 0.0104 to 42.6 g-DCW/L the released DNA concentration in the liquid anode increased from 2.10±1.57 to 75.00±7.15ng/μL. More importantly, the NanoGene assay showed an increase in normalized fluorescence (gene quantification) from 18.03±1.18 to 49.71±1.82 as the bacterial concentrations increased from 0.0104 to 42.6 g-DCW/L. the electrical power consumption of the bio-precipitator was shown to be amenable for portable use. In addition, the detection limit of bio-precipitator and NanoGene assay combination in the context of environmentally relevant levels of airborne bacteria was also discussed.

A disposable bacterial lysis cartridge (BLC) suitable for an in situ water-borne pathogen detection system.

We constructed a disposable bacterial lysis cartridge (BLC) suitable for an in situ pathogen detection system. It had an in-built micro corona discharge based ozone generator that provided ozone for cell lysis. Using a custom sample handling platform, its performance was evaluated with a Gram-positive bacterium of Bacillus subtilis. It was capable of achieving a similar degree of lysis as a commercial ultrasonic dismembrator with a P-1 microprobe in 10 min at an air pump flow rate of 29.4 ml min(-1) and an ozone generator operating voltage of 1600 V. The lysing duration could be significantly reduced to 5 min by increasing the air pump flow rate and the ozone generator operating voltage as well as by the addition of sodium dodecyl sulfate (SDS).

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.

Wideband Mechanical Excitation by a Microcorona-Driven Vibrating Element

We have designed, fabricated, and tested a microcorona driven (MCD) vibrating element. The vibrating element consists of a mass plate at the end of a cantilever. The proof mass is selectively driven by either one or two microcorona ionizers. During the dc negative corona discharge, the build-up of negative space charge electrostatically repelled the cathodes on the mass plate against the mechanical elastic force of the spring, damping force, and electrostatic force. This resulted in the wideband mechanical self-excitation of the MCD vibrating element. Using laser Doppler vibrometry (LDV), two resonance frequencies of out-of-plane modes were measured experimentally at peak values of 896 and 1312 Hz, and it was consistent with the ANSYS finite element modal analysis results at ~823 and 1323 Hz, respectively. The transition from Trichel pulse mode to diffuse glow mode resulted in a discontinuity in the experimental plot of proof mass velocity versus applied voltage. The MCD vibrating element consumed a maximum power of ~100 mW and had a maximum resultant driving force of ~0.45 μN in the first observed driving mode. The maximum out-of-plane oscillation amplitude measured was to be ~2 μm.

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.

Development of quantum dot aptasensor and its portable analyzer for the detection of di-2-ethylhexyl phthalate

We have developed a quantum dot aptasensor (QD-aptasensor) and its accompanying portable analyzer for the detection of di-2-ethylhexyl phthalate (DEHP). This sensor is based on a newly screened aptamer (60-mer) via SELEX and shows a binding affinity of 213 nmol/L with DEHP. The 60-mer aptamer together with its three shorter truncated aptamers (45, 28, and 22-mer) as well as three different DNA probes (12, 9, and 13-mer) were further investigated to form the best combination for the QD-aptasensor. Using a 22-mer-truncated aptamer and a 12-mer DNA probe combination, the QD-aptasensor demonstrated excellent DEHP sensitivity with an LOQ = 0.5 pg/mL as well as good selectivity in the presence of other phthalate analogs. The binding between the truncated aptamers and DEHP was also characterized. Finally, a QD-aptasensor-based portable analyzer was also developed, and its equivalence to the laboratory protocol was established with a correlation coefficient r = 0.86 for DEHP concentrations ranging from 0.0005 to 100 ng/mL.

Portable lysis apparatus for rapid single-step DNA extraction of Bacillus subtilis

To demonstrate and characterize a portable lysis apparatus for rapid single‐step bacterial DNA extraction.