Seongkyeol Hong

Single-walled carbon nanotube based transparent immunosensor for detection of a prostate cancer biomarker osteopontin

Osteopontin (OPN) is involved in almost all steps of cancer development, and it is being investigated as a potential biomarker for a diagnosis and prognosis of prostate cancer. Here, we report a label-free, highly sensitive and transparent immunosensor based on single-walled carbon nanotubes (SWCNTs) for detection of OPN. A high density of COOH functionalized SWCNTs was deposited between two gold/indium tin oxide electrodes on a glass substrate by dielectrophoresis. Monoclonal antibodies specific to OPN were covalently immobilized on the SWCNTs. Relative resistance change of the immunosensors was measured as the concentration of OPN in phosphate buffer saline (PBS) and human serum was varied from 1 pg mL(-1) to 1 μg mL(-1) for different channel lengths of 2, 5, and 10 μm, showing a highly linear and reproducible behavior (R(2)>97%). These immunosensors were also specific to OPN against another test protein, bovine serum albumin, PBS and human serum, showing that a limit of detection for OPN was 0.3 pg mL(-1). This highly sensitive and transparent immunosensor has a great potential as a simple point-of-care test kit for various protein biomarkers.

Combined application of bacterial predation and carbon dioxide aerosols to effectively remove biofilms

This study evaluated predation with Bdellovibrio bacteriovorous and CO2 aerosol spraying to remove fluorescent Escherichia coli biofilms from silicon chips. Initial tests found that 7.5×105 viable E. coli cells were dispersed into the surrounding environment during aerosol treatment. The total number dispersed per test decreased to only 16 for predated biofilms. This is nearly 50,000-fold lower compared to untreated chips and 1000-fold lower compared to chips soaked in HEPES buffer only. Both scanning electron microscopy (SEM) and fluorescent microscopy analyses confirmed that predation alone did not completely eradicate the biofilm population. When used in conjunction with CO2 aerosols, however, no fluorescent signals remained and the SEM pictures showed a pristine surface devoid of bacteria. Consequently, this study demonstrates these two methods can be used with each other to significantly remove biofilms from surfaces while also significantly reducing the likelihood of human exposure to potential pathogens during their removal.

Label-free Detection of Influenza Viruses using a Reduced Graphene Oxide-based Electrochemical Immunosensor Integrated with a Microfluidic Platform

Reduced graphene oxide (RGO) has recently gained considerable attention for use in electrochemical biosensing applications due to its outstanding conducting properties and large surface area. This report presents a novel microfluidic chip integrated with an RGO-based electrochemical immunosensor for label-free detection of an influenza virus, H1N1. Three microelectrodes were fabricated on a glass substrate using the photolithographic technique, and the working electrode was functionalized using RGO and monoclonal antibodies specific to the virus. These chips were integrated with polydimethylsiloxane microchannels. Structural and morphological characterizations were performed using X-ray photoelectron spectroscopy and scanning electron microscopy. Electrochemical studies revealed good selectivity and an enhanced detection limit of 0.5 PFU mL−1, where the chronoamperometric current increased linearly with H1N1 virus concentration within the range of 1 to 104 PFU mL−1 (R2 = 0.99). This microfluidic immunosensor can provide a promising platform for effective detection of biomolecules using minute samples.

Gas-phase removal of biofilms from various surfaces using carbon dioxide aerosols

The present study evaluated the removal of Escherichia coli XL1-blue biofilms using periodic jets of carbon dioxide aerosols (a mixture of solid and gaseous CO2) with nitrogen gas. The aerosols were generated by the adiabatic expansion of high-pressure CO2 gas through a nozzle and used to remove air-dried biofilms. The areas of the biofilms were measured from scanning electron micrographs before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured with various air-drying times of the biofilms before the treatment, surface materials, and durations of CO2 aerosols in each 8-s aerosol–nitrogen cleaning cycle. Nearly 100% of the fresh biofilms were removed from the various surfaces very reliably within 90 s. This technique can be useful for removing unsaturated biofilms on solid surfaces and has potential applications for cleaning bio-contaminated surfaces.

Mechanical desorption of immobilized proteins using carbon dioxide aerosols for reusable biosensors.

Reusability of a biosensor has recently received considerable attention, and it is closely related with the effective desorption of probe molecules. We present a novel mechanical desorption technique to reuse biosensors by using periodic jets of carbon dioxide (CO2) aerosols (a mixture of solid and gaseous CO2), and demonstrate its feasibility by removing physically adsorbed and covalently bonded fluorescent proteins i.e., Escherichia coli fluorescein isothiocyanate antibody and bovine serum albumin (E. coli FITC-Ab and FITC-BSA) from silicon chips. The proteins on the chip surfaces were measured by fluorescent images before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured for various concentrations (1-20 μg mL(-1)) of E. coli FITC-Ab and FITC-BSA with two different removal cycles (5 and 11 cycles; each cycle: 8s). We observed high removal efficiencies (>93.5% for physically adsorbed Ab and >84.6% for covalently bonded Ab) at 11 cycle aerosol treatment. This CO2 aerosol treatment did not undermine re-functionalization, which was confirmed by the fluorescent images of FITC-Abs for fresh and reused chips. Desorption of the immobilized layers was validated by Fourier transform infrared and X-ray photoelectron spectroscopic analyses. We also conducted an experiment on the regeneration of E. coli sensing chips using this aerosol treatment, and the chips were re-used 5 times successfully. This mechanical desorption technique is a highly effective and novel strategy for reusable biosensors.

Simultaneous position and mass determination of a nanoscale-thickness cantilever sensor in viscous fluids

We report simultaneous determination of the mass and position of micro-beads attached to a nanoscale-thickness cantilever sensor by analyzing wave propagations along the cantilever while taking into account viscous and inertial loading due to a surrounding fluid. The fluid-structure interaction was identified by measuring the change in the wavenumber under different fluid conditions. The predicted positions and masses agreed with actual measurements. Even at large mass ratios (6%–21%) of the beads to the cantilever, this wave approach enabled accurate determination of the mass and position, demonstrating the potential for highly accurate cantilever sensing of particle-based bio-analytes such as bacteria.

Determination of Fluid Density and Viscosity by Analyzing Flexural Wave Propagations on the Vibrating Micro-Cantilever

The determination of fluid density and viscosity using most cantilever-based sensors is based on changes in resonant frequency and peak width. Here, we present a wave propagation analysis using piezoelectrically excited micro-cantilevers under distributed fluid loading. The standing wave shapes of microscale-thickness cantilevers partially immersed in liquids (water, 25% glycerol, and acetone), and nanoscale-thickness microfabricated cantilevers fully immersed in gases (air at three different pressures, carbon dioxide, and nitrogen) were investigated to identify the effects of fluid-structure interactions to thus determine the fluid properties. This measurement method was validated by comparing with the known fluid properties, which agreed well with the measurements. The relative differences for the liquids were less than 4.8% for the densities and 3.1% for the viscosities, and those for the gases were less than 6.7% for the densities and 7.3% for the viscosities, showing better agreements in liquids than in gases.

Effects of Carbon Dioxide Aerosols on the Viability of Escherichia coli during Biofilm Dispersal.

A periodic jet of carbon dioxide (CO2) aerosols is a very quick and effective mechanical technique to remove biofilms from various substrate surfaces. However, the impact of the aerosols on the viability of bacteria during treatment has never been evaluated. In this study, the effects of high-speed CO2 aerosols, a mixture of solid and gaseous CO2, on bacteria viability was studied. It was found that when CO2 aerosols were used to disperse biofilms of Escherichia coli, they led to a significant loss of viability, with approximately 50% of the dispersed bacteria killed in the process. By comparison, 75.6% of the biofilm-associated bacteria were viable when gently dispersed using Proteinase K and DNase I. Indirect proof that the aerosols are damaging the bacteria was found using a recombinant E. coli expressing the cyan fluorescent protein, as nearly half of the fluorescence was found in the supernatant after CO2 aerosol treatment, while the rest was associated with the bacterial pellet. In comparison, the supernatant fluorescence was only 9% when the enzymes were used to disperse the biofilm. As such, these CO2 aerosols not only remove biofilm-associated bacteria effectively but also significantly impact their viability by disrupting membrane integrity.

Removal of different-age biofilms using carbon dioxide aerosols

Many techniques to inactivate or remove biofilms in a wide variety of applications have been developed. Most of these techniques have been applied to biofilms at their initial stage of growth, since they are generally difficult to eradicate once established. The removal of established biofilms has received relatively little attention. In this paper, we report the effectiveness of periodic jets of carbon dioxide aerosols (a mixture of solid and gaseous CO2) to remove Escherichia coli (XL1-blue) biofilms of different ages (up to 3 weeks) on silicon surfaces. The biofilms were not immersed in liquids after growth/rinsing and were treated with the CO2 aerosols. The CO2 aerosols were generated by the adiabatic expansion of high-pressure CO2 gas through a nozzle. The surface area of the biofilms was measured from fluorescent images before and after applying the aerosols for 11, 20, and 30 cycles (each cycle: 8 sec), to compute the removal efficiency. The removal efficiencies decreased with increasing growth time and for the 3-week-old biofilms, they ranged from 91.5 to 99.6% within 4 min. This technique was highly effective for removing both fresh and old biofilms, but some of the biofilm debris such as growth media remained. Further, this CO2 aerosol technique was compared with other removal techniques.