A multi-functional reagent suitable for 1-step rapid DNA intercalation fluorescence-based screening of total bacteria in drinking water.

The prerequisites for rapid screening of total bacteria in drinking water are low detection limit and convenience. Inspired by commercial adenosine 5'-triphosphate (ATP) based total bacterial detection kits, we pursued likewise convenience but with much lower detection limit. Existing intercalation fluorescence-based techniques employ multiple reagents to permeate the cell membrane and intercalate dye into the DNA in discrete sequential steps. A simple multi-functional reagent is proposed to do the same within one step. Surfactants (TritonX and SDS), and intercalating dyes (SYBR green, SYBR gold) were examined for their mutual compatibility and augmented with EDTA. Evaluation was performed with Gram negative Escherichia coli K12 (E. coli K12) and Gram positive Bacillus subtilis (B. subtilis) at serial dilution ratios from 10-6 to 10-2. Comparison was made with absorbance (600 nm) measurements and a commercial ATP kit. Using charge integrated photodetection, the proposed 1-step reagent achieved an LOD (1.00 × 10-6, B. subtilis) that is two orders of magnitude lower than that of ATP kit (LOD = 1.06× 10-4). This means it could detect minute quantity of total bacteria that is otherwise undetected by the ATP kit.

Switchable inhibitory behavior of divalent magnesium ion in DNA hybridization-based gene quantification.

Contrary to the understanding that divalent cations only result in under-estimation of gene quantification via DNA hybridization-based assays, we have discovered that Mg2+ could cause either under or over-estimation at different concentrations. Its switchable inhibitory behavior is likely due to its rigid first solvation (hydrated) shell and hence it is inclined to form non-direct binding with DNA. At low concentrations, it caused under-estimation by occupying the hybridization sites. At high concentrations, it caused probe, signaling and target DNA to aggregate non-specifically via Coulomb forces. By quantifying target DNAs at a range of Mg2+ concentrations using a gene quantification assay (NanoGene assay), a Mg2+ inflection concentration of ∼10-3 M was observed for both target ssDNA and dsDNA. Field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDS), and Fourier transform infrared spectroscopy (FT-IR) were employed to observe Mg2+-induced non-specific binding in the complexes that mimicked the presence of target DNA. Together with two other divalent cations Ca2+ and Cu2+, they were further examined via zeta potential measurements as well as NanoGene assay. This study revealed the importance of Mg2+ in achieving accurate gene quantification. Through a better mechanistic understanding of this phenomenon, it will be possible to develop strategies to mitigate the impact of Mg2+ on DNA hybridization-based gene quantification.

Clustered Detection of Eleven Phthalic Acid Esters by Fluorescence of Graphene Quantum Dots Displaced from Gold Nanoparticles.

A gold nanoparticle-quenched graphene quantum dot-based aptasensor was developed to perform clustered detection of 11 phthalic acid esters (PAEs). The binding of the target PAEs to the aptasensor frees the graphene quantum dots that are otherwise quenched by the carrier gold nanoparticle. The resultant fluorescence upon excitation is proportional to the number of freed graphene quantum dots and hence the target PAE concentration. The synthesis of the proposed aptasensor was first verified step-by-step via FT-IR measurement, scanning electron microscopy, and fluorescence measurement. Selectivity was evaluated for individual and combined target PAEs and compared against seven non-PAE endocrine disrupting compounds. The proposed aptasensor successfully quantified 11 PAEs in test samples with varying concentrations of 0.001-50 ng PAEs/mL and demonstrated a limit of detection of ∼4 pg./mL. Finally, the AuNP-gQD aptasensor was employed to detect multiple combinations of commonly regulated PAEs (DBP, DIBP, DEHP, and BBP). The recovery (%) for all four PAEs combination in environmentally relevant concentrations of 0.5, 1, 5, and 10 ng/mL were ∼100%.

Soft Candy as an Electronic Material Suitable for Salivary Conductivity-Based Medical Diagnostics in Resource-Scarce Clinical Settings.

Soft candy was discovered to be an excellent electronic material and was used to fabricate electrodes for salivary conductivity-based diagnostics. Using a simple molding process, a soft candy (Tootsie Roll) was made into 20 × 20 × 5 mm electrodes with a stable frequency response (0.1-100 kHz). The soft candy electrode-liquid interface circuit model was also developed for the first time. Using 0.01, 0.05, and 0.1 M phosphate-buffered saline and artificial saliva of varying conductivities, the performance of the soft candy (Tootsie Roll) electrode was evaluated. The electrode has a low temperature coefficient of ∼0.02 V/C, and the evaporation-induced mass change during measurement (<3 min) was negligible. Using a trenched surface, a limit of detection (LOD) of ∼1630 µS/cm was obtained and was lower than the saliva conductivity of a healthy adult at ∼3500 µS/cm. Thus, it is suitable for monitoring the ovulation cycle for natural family planning as well as chronic kidney disease diagnosis. Given the ubiquity of soft candy, the simplicity of the molding process, and the negligible medical waste stream, it is a more appropriate approach to diagnostics design for resource-scarce clinical settings, such as those in developing countries. The broader impact of this work will be the paradigm shift of soft candy from food to a new class of edible, moldable, high-resistivity, and stable electronic materials.

Chia seed-assisted separation and detection of polyvinyl chloride microplastics in water via gas chromatography mass spectrometry.

Chia seeds were used to significantly improve the separation efficiency of polyvinyl chloride (PVC) microplastics from water samples via centrifugation. Upon hydration, the mucilage of chia seeds were able to capture PVC microplastics with sizes ranging from tens to hundreds of micrometers. Since PVC microplastics contained di-2-etylhexyl phthalate (DEHP) as a plasticizer (verified via Fourier transform infrared spectrometry), DEHP was used as an indicator in the subsequent quantification via gas chromatography - mass spectrometry (GC-MS) analysis. Specifically after verifying the DEHP peak in the GC spectrum using DEHP reference standard as a positive control, the GC spectral area of that peak was used to quantify the amount of DEHP in the sample. Using nominal operation settings at 10 min and 1000 rpm with 100 mg of chia seeds, the separation efficiency could be improved by 5 times (500%) as compared to the absence of chia seeds. Furthermore, chia seeds were also compatible with simulated synthetic wastewater samples. Most importantly, the use of chia seeds did not interfere with GC-MS quantification protocol and accuracy. The result suggested the proposed method can be used as a simple screening tool of microplastics entering wastewater treatment plant, even though a series of follow-up studies are needed in future.

Ozonation enhancement of low cost double-stranded DNA binding dye based fluorescence measurement of total bacterial load in water.

We demonstrated the feasibility of using ozonation to enhance the performance of dsDNA binding dye SYBR Green I in the fluorescence measurement of total bacterial load in water. Unlike its membrane permeable but expensive equivalent such as SYTO82 dye, SYBR Green I is many times cheaper but membrane impermeable. Ozonation allowed SYBR Green I dye to permeate the membrane and bind with the dsDNA within by first breaching it. Using E. coli K12 bacteria at serial dilution ratios from 1/1 (980 CFU mL-1) to 1/200, we achieved corresponding quantification from 618.7 ± 9.4 to 68.0 ± 1.9 RFU (100 to 11.00% normalized RFU). In comparison, plate counting and optical density measurement were only able to quantify up till a serial dilution ratio of 1/50 (40 CFU mL-1 and 0.0421, respectively). Most importantly with ozonation, the sensitivity of SYBR Green I dye based fluorescence measurement was improved by ∼140 to 210% as compared to that without ozonation. Given its low electrical power consumption, lab-on-chip compatibility and reagent-less nature, ozonation is highly compatible with portable fluorimeters to realize low-cost monitoring of total bacterial load in water.

Single-stranded DNA probe paired aptasensor with extra dye binding sites to enhance its fluorescence response in the presence of a target compound.

The purpose of this study is to investigate the possibility of improving the performance of a DNA binding dye water quenching based aptasensor without changing or truncating the aptamer. To demonstrate the possibility of increasing the change in fluorescence of the aptasensor by pairing it with a suitable ssDNA probe, three ssDNA probes (probe 1, 2, and 3) were employed and the fluorescence from the bound dyes was measured. This showed that ssDNA probe 2 created the most additional binding sites. By varying the target compound concentration (0, 0.05, 0.5, 5, 50, and 500 mg L-1 4-n-nonylphenol), the corresponding change in the fluorescence signal of the unpaired and ssDNA probe paired aptasensors were measured and compared over a range of emission wavelengths. The response of all three ssDNA probe paired aptasensors showed good fit (R 2 = 0.88-0.92) to a logarithmic response. The sensitivity of the aptasensor paired with ssDNA probe 2 was improved by ∼60%, whereas that of the aptasensor paired with ssDNA probe 3 was only improved by a marginal ∼3%. This study is a demonstration of using an appropriate ssDNA probe to increase the number of binding sites and hence the performance of a DNA binding dye and water quenched aptasensor. It is a possibility that can be extended to similar aptasensors without having to change or truncate the aptamer.

A simple reagent-less approach using electrical discharge as a substitution for chelating agent in addressing genomic assay inhibition by divalent cations.

Electrical discharge treatment was shown to be a viable substitution for chelating agent in genomic assays. Divalent cation Mg2+ inhibits the performance of DNA hybridization based genomic assays by binding to the DNA and disrupting DNA hybridization. Until now, chelating agents such as ethylenediaminetetraacetic acid (EDTA) was the only option to address the presence of Mg2+ in samples. However, EDTA is a well-known environmental contaminant. In this work, we successfully employed electrical discharge instead of EDTA to render Mg2+ insipid. Its preliminary efficacy was first observed via circular dichroism (CD) and zeta potential analyses. After electrical discharge treatment, the reduction in CD shift at 280 nm was significant for samples with 10-3 and 10-8 M Mg2+. The zeta potential of Mg2+ laden samples were also restored from -4.71 ± 1.38 to -20.59 ± 6.37 mV after electrical discharge treatment. Both CD shift and change in zeta potential suggested that 2 min of electrical discharge treatment could prevent Mg2+ from binding to DNA. The complete efficacy of electrical discharge treatment was demonstrated with the performance recovery (within ∼15% of the control) of a genomic assay variant (NanoGene assay) while analyzing Mg2+ laden samples (10-5-10-3 M). Assuming 10 million samples are analyzed annually, the proposed electrical discharge treatment (∼50 mW per sample) would allow us to trade environmental contamination by ∼50 kg of hazardous EDTA with a single 250 W STC (standard test conditions) solar panel.

Development of non-equilibrium rapid replacement aptamer assay for ultra-fast detection of phthalic acid esters.

In this paper, we developed a non-equilibrium rapid replacement aptamer (NERRA) assay that performed ultra-fast (in 30 s) quantitative detection of phthalic acid esters (PAEs) without waiting for the reaction to reach equilibrium. NERRA assay employed fluorescence PoPo3 dye intercalated in an ssDNA aptamer to selectively detect and quantify the PAEs in water. As the intercalated dye was replaced by the PAEs and quenched in the water, the rate of fluorescence change became proportional to PAEs concentration. The sensitivity of NERRA assay was first evaluated with a commercial spectrofluorometer. The selectivity for PAE mixture, individual PAEs, and non-phthalate compounds were also investigated. NERRA assay was also able to quantitatively detect the PAEs in a common plastic product (picnic mat), and the results were compared with those of gas chromatography mass spectrometry. Finally, a custom analyzer (8.5 cm × 8.5 cm × 16.5 cm) was built to demonstrate the portability of the NERRA assay. Using a commercial spectrofluorometer, NERRA assay was able to quantitatively detect a PAE mixture in 30 min with an LOQ of 0.1 µg/L. Using the portable custom analyzer, the detection time was shortened to 30 s with a tradeoff in the LOQ (1 µg/L). In both cases, the LOQs remain within the environmentally relevant PAE concentrations of 0.1-1472 µg/L.

A self-powered insulin patch pump with a superabsorbent polymer as a biodegradable battery substitute.

Highly popular insulin patch pumps have in-built non-removable batteries. These batteries are routinely disposed of together with the used pumps as medical waste and end up in landfills. This is an environmental contamination conundrum by design. To address this issue, we proposed a self-powered patch pump that uses a biodegradable superabsorbent polymer (SAP) instead of a battery as a power source to drive the infusion. Continuous infusion rates from 6.1 µL min-1 to 49.1 µL min-1 were achieved. Together with valve throttling, basal and bolus infusion rates of ∼10 µL h-1 (1 U h-1) and 100 µL (10 U) in ∼11 min could also be implemented for glycemic control. The generated pressure at ∼0.7 psi is also adequate for infusion as it exceeded an adult's maximum peripheral venous pressure of 0.6 psi. Given the current number of patch pump users, the proposed design could prevent ∼100 000 used batteries from entering the medical waste stream and landfill daily. Most importantly, this work highlights the possibility of addressing environmental contamination without compromising on healthcare standards by using SAP as an alternative means of energy storage.

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.

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.

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 centrifuge's 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.

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

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.

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).

Design, development, and evaluation of a novel microneedle array-based continuous glucose monitor.

The development of accurate, minimally invasive continuous glucose monitoring (CGM) devices has been the subject of much work by several groups, as it is believed that a less invasive and more user-friendly device will result in greater adoption of CGM by persons with insulin-dependent diabetes. This article presents the results of preliminary clinical studies in subjects with diabetes of a novel prototype microneedle-based continuous glucose monitor. In this device, an array of tiny hollow microneedles is applied into the epidermis from where glucose in interstitial fluid (ISF) is transported via passive diffusion to an amperometric glucose sensor external to the body. Comparison of 1396 paired device glucose measurements and fingerstick blood glucose readings for up to 72-hour wear in 10 diabetic subjects shows the device to be accurate and well tolerated by the subjects. Overall mean absolute relative difference (MARD) is 15% with 98.4% of paired points in the A+B region of the Clarke error grid. The prototype device has demonstrated clinically accurate glucose readings over 72 hours, the first time a microneedle-based device has achieved such performance.

Sterilization of Escherichia coli O157:H7 using micro corona ionizer.

We demonstrated in vitro sterilization of Escherichia coli O157:H7 bacteria on agar by a pin-between-planes micro corona ionizer. The gap between the pin and the grid was ~1.1 mm, the length of the grid was ~2.1 mm and the height was ~1.0 mm. The effective pin radius and discharge length were both approximated to be 200 µm. Ozone generation rates of ~2.3 × 10(-3) mg/s, ~2.7 × 10(-3) mg/s and ~3.5 × 10(-3) mg/s at 1,500 V were calculated for relative humidity (RH) of 35 %, 25 % and 10 % respectively. Analytical ozone generation rate increases as RH decreases and it is consistent with experimental observations. Using target and control petri dishes with E. coli plated agar, the sterilization capability of the micro corona ionizer at 37 °C for 24 h was evaluated. A ~60 % reduction in bacterial colony was shown with plate counting and its kill radius could be tuned from ~ 20 mm to ~5 mm by reducing the duty cycle from 100 % to 50 % with 30 min pulse width. The results suggested that the micro corona ionizer might be suitable as a tunable ozone source in wound dressing for chronic wound management.

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).