Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. ...

Ulsan National Institute of Science and Technology (UNIST)

This study evaluated predation with Bdellovibrio bacteriovorous and CO(2) aerosol spraying to remove fluorescent Escherichia coli biofilms from silicon chips. Initial tests found that 7.5×10(5) 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 CO(2) 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.

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

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 CO(2)) with nitrogen gas. The aerosols were generated by the adiabatic expansion of high-pressure CO(2) 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 CO(2) 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.

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

The influenza virus has received extensive attention due to the recent H1N1 pandemics originating from swine. This study reports a label-free, highly sensitive, and selective electrical immunosensor for the detection of influenza virus H1N1 based on dielectrophoretically deposited single-walled carbon nanotubes (SWCNTs). COOH-functionalized SWCNTs were deposited on a self-assembled monolayer of polyelectrolyte polydiallyldimethyl-ammonium chloride (PDDA) between two gold electrodes by dielectrophoretic and electrostatic forces, which resulted in reproducible, uniform, aligned, and aggregation-free SWCNT channels (2-10 μm in length). Avidin was immobilized onto the PDDA-SWCNT channels, and viral antibodies were immobilized using biotin-avidin coupling. The resistance of the channels increased with the binding of the influenza viruses to the antibodies. These immunosensors showed linear behavior as the virus concentration was varied from 1 to 10(4) PFU ml(-1) along with a detection time of 30 min. The immunosensors with a 2 μm channel length detected 1 PFU ml(-1) of the influenza virus accurately (R(2) = 0.99) and selectively from MS2 bacteriophages. These immunosensors have the potential to become an important component of a point-of-care test kit that will enable a rapid clinical diagnosis.

Electrical immunosensor based on dielectrophoretically-deposited carbon nanotubes for detection of influenza virus H1N1.

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.

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

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.

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

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.

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

Measurements of airborne viruses via sampling have been critical issues. Most electrostatic samplers have been assessed for bacterial aerosols or micrometer-sized viral particles; however, sampling of submicrometer-sized airborne viruses is necessary, especially because of the high probability of their staying airborne and their deposition in the lower respiratory tract. Here, we present a novel personal electrostatic particle concentrator (EPC) for gentle sampling of submicrometer airborne virus particles. Owing to the enhanced electric field designed in this EPC, the collection efficiencies reached values as high as 99.3-99.8% for 0.05-2 μm diameter polystyrene particles at a flow rate of 1.2 L/min. Submicrometer-sized MS2 and T3 virus particles were also collected in the EPC, and the concentrations relative to their respective initial suspensions were more than 10 times higher than those in the SKC BioSampler. Moreover, the recovery rate of T3 was 982 times higher in the EPC (-2 kV) than in the BioSampler at 12.5 L/min because of the gentle sampling of the EPC. Gentle sampling is desirable because many bioaerosols suffer from significant viability losses during sampling. The influence of ozone generated, applied electrostatic field, and the flow rate on the viability of the viruses will also be discussed.

Seongkyeol Hong

School of Mechanical and Nuclear Engineering

Ulsan National Institute of Science and Technology (UNIST)

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Seongkyeol Hong

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Mechanical and Nuclear Engineering

Ulsan National Institute of Science and Technology (UNIST)

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

School of Mechanical and Nuclear Engineering