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1.  Conductometric Sensors for Monitoring Degradation of Automotive Engine Oil† 
Sensors (Basel, Switzerland)  2011;11(9):8611-8625.
Conductometric sensors have been fabricated by applying imprinted polymers as receptors for monitoring engine oil quality. Titania and silica layers are synthesized via the sol-gel technique and used as recognition materials for acidic components present in used lubricating oil. Thin-film gold electrodes forming an interdigitated structure are used as transducers to measure the conductance of polymer coatings. Optimization of layer composition is carried out by varying the precursors, e.g., dimethylaminopropyltrimethoxysilane (DMAPTMS), and aminopropyl-triethoxysilane (APTES). Characterization of these sensitive materials is performed by testing against oil oxidation products, e.g., carbonic acids. The results depict that imprinted aminopropyltriethoxysilane (APTES) polymer is a promising candidate for detecting the age of used lubricating oil. In the next strategy, polyurethane-nanotubes composite as sensitive material is synthesized, producing appreciable differentiation pattern between fresh and used oils at elevated temperature with enhanced sensitivity.
PMCID: PMC3231490  PMID: 22164094
conductometric sensors; engine oil; imprinted polymers; carbon nanotubes; polyurethane
2.  Controlling drug delivery kinetics from mesoporous titania thin films by pore size and surface energy 
The osseointegration capacity of bone-anchoring implants can be improved by the use of drugs that are administrated by an inbuilt drug delivery system. However, to attain superior control of drug delivery and to have the ability to administer drugs of varying size, including proteins, further material development of drug carriers is needed. Mesoporous materials have shown great potential in drug delivery applications to provide and maintain a drug concentration within the therapeutic window for the desired period of time. Moreover, drug delivery from coatings consisting of mesoporous titania has shown to be promising to improve healing of bone-anchoring implants. Here we report on how the delivery of an osteoporosis drug, alendronate, can be controlled by altering pore size and surface energy of mesoporous titania thin films. The pore size was varied from 3.4 nm to 7.2 nm by the use of different structure-directing templates and addition of a swelling agent. The surface energy was also altered by grafting dimethylsilane to the pore walls. The drug uptake and release profiles were monitored in situ using quartz crystal microbalance with dissipation (QCM-D) and it was shown that both pore size and surface energy had a profound effect on both the adsorption and release kinetics of alendronate. The QCM-D data provided evidence that the drug delivery from mesoporous titania films is controlled by a binding–diffusion mechanism. The yielded knowledge of release kinetics is crucial in order to improve the in vivo tissue response associated to therapeutic treatments.
PMCID: PMC4501225  PMID: 26185444
mesoporous titania; controlled drug delivery; release kinetics; alendronate; QCM-D
3.  A Micro Oxygen Sensor Based on a Nano Sol-Gel TiO2 Thin Film 
Sensors (Basel, Switzerland)  2014;14(9):16423-16433.
An oxygen gas microsensor based on nanostructured sol-gel TiO2 thin films with a buried Pd layer was developed on a silicon substrate. The nanostructured titania thin films for O2 sensors were prepared by the sol-gel process and became anatase after heat treatment. A sandwich TiO2 square board with an area of 350 μm × 350 μm was defined by both wet etching and dry etching processes and the wet one was applied in the final process due to its advantages of easy control for the final structure. A pair of 150 nm Pt micro interdigitated electrodes with 50 nm Ti buffer layer was fabricated on the board by a lift-off process. The sensor chip was tested in a furnace with changing the O2 concentration from 1.0% to 20% by monitoring its electrical resistance. Results showed that after several testing cycles the sensor's output becomes stable, and its sensitivity is 0.054 with deviation 2.65 × 10−4 and hysteresis is 8.5%. Due to its simple fabrication process, the sensor has potential for application in environmental monitoring, where lower power consumption and small size are required.
PMCID: PMC4208180  PMID: 25192312
O2 sensors; sol-gel; nanostructured; MEMS (Micro-Electro-Mechanical System); thin films; TiO2
4.  Bactericidal Performance of Visible-Light Responsive Titania Photocatalyst with Silver Nanostructures 
PLoS ONE  2010;5(4):e10394.
Titania dioxide (TiO2) photocatalyst is primarily induced by ultraviolet light irradiation. Visible-light responsive anion-doped TiO2 photocatalysts contain higher quantum efficiency under sunlight and can be used safely in indoor settings without exposing to biohazardous ultraviolet light. The antibacterial efficiency, however, remains to be further improved.
Methodology/Principal Findings
Using thermal reduction method, here we synthesized silver-nanostructures coated TiO2 thin films that contain a high visible-light responsive antibacterial property. Among our tested titania substrates including TiO2, carbon-doped TiO2 [TiO2 (C)] and nitrogen-doped TiO2 [TiO2 (N)], TiO2 (N) showed the best performance after silver coating. The synergistic antibacterial effect results approximately 5 log reductions of surviving bacteria of Escherichia coli, Streptococcus pyogenes, Staphylococcus aureus and Acinetobacter baumannii. Scanning electron microscope analysis indicated that crystalline silver formed unique wire-like nanostructures on TiO2 (N) substrates, while formed relatively straight and thicker rod-shaped precipitates on the other two titania materials.
Our results suggested that proper forms of silver on various titania materials could further influence the bactericidal property.
PMCID: PMC2861596  PMID: 20454454
5.  A non-labeled DNA biosensor based on light addressable potentiometric sensor modified with TiO2 thin film*  
Titanium dioxide (TiO2) thin film was deposited on the surface of the light addressable potentiometric sensor (LAPS) to modify the sensor surface for the non-labeled detection of DNA molecules. To evaluate the effect of ultraviolet (UV) treatment on the silanization level of TiO2 thin film by 3-aminopropyltriethoxysilane (APTS), fluorescein isothiocyanate (FITC) was used to label the amine group on the end of APTS immobilized onto the TiO2 thin film. We found that, with UV irradiation, the silanization level of the irradiated area of the TiO2 film was improved compared with the non-irradiated area under well-controlled conditions. This result indicates that TiO2 can act as a coating material on the biosensor surface to improve the effect and efficiency of the covalent immobilization of biomolecules on the sensor surface. The artificially synthesized probe DNA molecules were covalently linked onto the surface of TiO2 film. The hybridization of probe DNA and target DNA was monitored by the recording of I-V curves that shift along the voltage axis during the process of reaction. A significant LAPS signal can be detected at 10 μmol/L of target DNA sample.
PMCID: PMC2772892  PMID: 19882762
DNA biosensor; Titanium dioxide (TiO2) thin film; Light addressable potentiometric sensor (LAPS); Silanization; Fluorescein label; Gene chip
6.  Enterohaemorrhagic Escherichia coli O157:H7 target Peyer's patches in humans and cause attaching/effacing lesions in both human and bovine intestine 
Gut  2000;47(3):377-381.
BACKGROUND—Enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide, and infections, particularly with serogroup O157:H7, are associated with consumption of a variety of food and water vehicles, particularly food of bovine origin. EHEC cause acute gastroenteritis, bloody diarrhoea, and haemorrhagic colitis; up to 10% of cases develop severe complications, including the haemolytic uraemic syndrome, with a 5% case fatality. A virulence characteristic of enteropathogenic E coli, the attaching/effacing lesion, is considered to be important in EHEC. However, although EHEC produce this lesion on cultured human cells, this has not been demonstrated on human intestinal mucosal surfaces. In addition, the initial site(s) of colonisation of EHEC in humans is not known.
AIMS—To assess the association of EHEC O157:H7 with paediatric and bovine intestine using in vitro organ culture and determine if attaching/effacing lesions occur.
METHODS—Ultrastructural analysis of in vitro intestinal organ cultures of human small and large intestine was used to investigate adhesion of O157:H7 EHEC to intestinal surfaces. Bovine intestinal organ culture was used to examine the pathology produced by the same EHEC strain in cattle.
RESULTS—The study showed that EHEC O157:H7 adhered to human intestinal mucosa. Binding and attaching/effacing lesion formation of O157:H7 in humans was restricted to follicle associated epithelium of Peyer's patches. The same strain caused attaching/effacing lesions on bovine mucosa.
CONCLUSIONS—O157:H7 targets follicle associated epithelium in humans where it causes attaching/effacing lesions. The same human isolate can cause attaching/effacing lesions in cattle, indicating that similar pathogenic mechanisms operate across human and bovine species

Keywords: Escherichia coli; enterohaemorrhagic E coli O157:H7; Peyer's patches; attaching/effacing lesion
PMCID: PMC1728033  PMID: 10940275
7.  Antibacterial performance of nanocrystallined titania confined in mesoporous silica nanotubes 
Biomedical Microdevices  2014;16(3):449-458.
In this paper, we study synthesis and characteristics of mesoporous silica nanotubes modified by titanium dioxide, as well as their antimicrobial properties and influence on mitochondrial activity of mouse fibroblast L929. Nanocrystalized titania is confined in mesopores of silica nanotubes and its light activated antibacterial response is revealed. The analysis of the antibacterial effect on Escherichia coli. (ATCC 25922) shows strong enhancement during irradiation with the artificial visible and ultraviolet light in respect to the commercial catalyst and control sample free from the nanomaterials. In darkness, the mesoporous silica/titania nanostructures exhibited antibacterial activity dependent on the stirring speed of the suspension containing nanomaterials. Obtained micrograph proved internalization of the sample into the microorganism trough the cell membrane. The analysis of the mitochondrial activity and amount of lactate dehydrogenase released from mouse fibroblast cells L929 in the presence of the sample were determined with LDH and WST1 assays, respectively. The synthesized silica/titania antibacterial agent also exhibits pronounced photoinduced inactivation of the bacterial growth under the artificial visible and UV light irritation in respect to the commercial catalyst. Additionally, mesoporous silica/titania nanotubes were characterized in details by means of high resolution transmission electron microscopy (HR-TEM), XRD and BET Isotherm.
Electronic supplementary material
The online version of this article (doi:10.1007/s10544-014-9847-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4009144  PMID: 24676537
Mesoporous silica nanotubes; Titanium dioxide; Antibacterial agent; Nanomaterials bactericidal properties
8.  Single Walled Carbon Nanotube-Based Junction Biosensor for Detection of Escherichia coli 
PLoS ONE  2014;9(9):e105767.
Foodborne pathogen detection using biomolecules and nanomaterials may lead to platforms for rapid and simple electronic biosensing. Integration of single walled carbon nanotubes (SWCNTs) and immobilized antibodies into a disposable bio-nano combinatorial junction sensor was fabricated for detection of Escherichia coli K-12. Gold tungsten wires (50 µm diameter) coated with polyethylenimine (PEI) and SWCNTs were aligned to form a crossbar junction, which was functionalized with streptavidin and biotinylated antibodies to allow for enhanced specificity towards targeted microbes. In this study, changes in electrical current (ΔI) after bioaffinity reactions between bacterial cells (E. coli K-12) and antibodies on the SWCNT surface were monitored to evaluate the sensor's performance. The averaged ΔI increased from 33.13 nA to 290.9 nA with the presence of SWCNTs in a 108 CFU/mL concentration of E. coli, thus showing an improvement in sensing magnitude. Electrical current measurements demonstrated a linear relationship (R2 = 0.973) between the changes in current and concentrations of bacterial suspension in range of 102–105 CFU/mL. Current decreased as cell concentrations increased, due to increased bacterial resistance on the bio-nano modified surface. The detection limit of the developed sensor was 102 CFU/mL with a detection time of less than 5 min with nanotubes. Therefore, the fabricated disposable junction biosensor with a functionalized SWCNT platform shows potential for high-performance biosensing and application as a detection device for foodborne pathogens.
PMCID: PMC4169404  PMID: 25233366
9.  Mechanical, physico-chemical, and antimicrobial properties of gelatin-based film incorporated with catechin-lysozyme 
Microbial activity is a primary cause of deterioration in many foods and is often responsible for reduced quality and safety. Food-borne illnesses associated with E. coli O157:H7, S. aureus, S. enteritidis and L. monocytogenes are a major public health concern throughout the world. A number of methods have been employed to control or prevent the growth of these microorganisms in food. Antimicrobial packaging is one of the most promising active packaging systems for effectively retarding the growth of food spoilage and pathogenic microorganisms. The aim of this study was to determine the mechanical, physico-chemical properties and inhibitory effects of the fish gelatin films against selected food spoilage microorganisms when incorporated with catechin-lysozyme.
The effect of the catechin-lysozyme combination addition (CLC: 0, 0.125, 0.25, and 0.5%, w/v) on fish gelatin film properties was monitored. At the level of 0.5% addition, the CLC showed the greatest elongation at break (EAB) at 143.17% with 0.039 mm thickness, and the lowest water vapor permeability (WVP) at 6.5 x 10−8 g·mm·h-1·cm-2·Pa-1, whereas the control showed high tensile strength (TS) and the highest WVP. Regarding color attributes, the gelatin film without CLC addition gave the highest lightness (L* 91.95) but lowest in redness (a*-1.29) and yellowness (b* 2.25) values. The light transmission of the film did not significantly decrease and nor did film transparency (p>0.05) with increased CLC. Incorporating CLC could not affect the film microstructure. The solubility of the gelatin based film incorporated with CLC was not affected, especially at a high level of addition (p>0.05). Inhibitory activity of the fish gelatin film against E.coli, S.aureus, L. innocua and S. cerevisiae was concentration dependent.
These findings suggested that CLC incorporation can improve mechanical, physico-chemical, and antimicrobial properties of the resulting films, thus allowing the films to become more applicable in active food packaging.
PMCID: PMC3585795  PMID: 23134808
Antimicrobial; Biodegradable packaging; Catechin; Gelatin film; Lysozyme
10.  A rapid and highly sensitive protocol for the detection of Escherichia coli O157:H7 based on immunochromatography assay combined with the enrichment technique of immunomagnetic nanoparticles 
Escherichia coli O157:H7 (E. coli O157:H7) is an important pathogenic bacterium that threatens human health. A rapid, simple, highly sensitive, and specific method for the detection of E. coli O157:H7 is necessary.
In the present study, immunomagnetic nanoparticles (IMPs) were prepared with nanopure iron as the core, coated with E. coli O157:H7 polyclonal antibodies. These IMPs were used in combination with immunochromatographic assay (ICA) and used to establish highly sensitive and rapid kits (IMPs+ICA) to detect E. coli O157:H7. The kits were then used to detect E. coli O157:H7 in 150 food samples and were compared with conventional ICA to evaluate their efficacy.
The average diameter of IMPs was 56 nm and the amount of adsorbed antibodies was 106.0 μg/mg. The sensitivity of ICA and IMPs+ICA was 105 colony-forming units/mL and 103 CFUs/mL, respectively, for purified E. coli O157:H7 solution. The sensitivity of IMPs+ICA was increased by two orders, and its specificity was similar to ICA.
The kits have the potential to offer important social and economic benefits in the screening, monitoring, and control of food safety.
PMCID: PMC3235026  PMID: 22163159
colloidal gold; immunomagnetic nanoparticles; Escherichia coli O157:H7; immunochromatographic assay
11.  Porous silicon functionalization for possible arsenic adsorption 
Nanoscale Research Letters  2014;9(1):508.
Thiol-functionalized porous silicon (PS) monolayer was evaluated for its possible application in As (III) adsorption. Dimercaptosuccinic acid (DMSA) attached to mesoporous silicon via amide bond linkages was used as a chelate for As (III). Two different aminosilanes namely 3-(aminopropyl) triethoxysilane (APTES) and 3-aminopropyl (diethoxy)-methylsilane (APDEMS) were tested as linkers to evaluate the relative response for DMSA attachment. The aminosilane-modified PS samples were attached to DMSA by wet impregnation followed by the adsorption of As (III). Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) have been used to identify the functional groups and to estimate the As (III) content, respectively. FTIR spectroscopy confirmed the covalent bonding of DMSA with amide and R-COOH groups on the nanostructured porous surface. XPS confirms the preferred arsenic adsorption on the surface of PS/DMSA samples as compared to the aminosilane-modified and bare PS substrates.
PMCID: PMC4171714  PMID: 25249826
Arsenic; Porous silicon; DMSA; Adsorption
12.  Development of a Colony Lift Immunoassay To Facilitate Rapid Detection and Quantification of Escherichia coli O157:H7 from Agar Plates and Filter Monitor Membranes 
E. coli O157:H7 is a food-borne adulterant that can cause hemorrhagic ulcerative colitis and hemolytic uremic syndrome. Faced with an increasing risk of foods contaminated with E. coli O157:H7, food safety officials are seeking improved methods to detect and isolate E. coli O157:H7 in hazard analysis and critical control point systems in meat- and poultry-processing plants. A colony lift immunoassay was developed to facilitate the positive identification and quantification of E. coli O157:H7 by incorporating a simple colony lift enzyme-linked immunosorbent assay with filter monitors and traditional culture methods. Polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass.) were prewet with methanol and were used to make replicates of every bacterial colony on agar plates or filter monitor membranes that were then reincubated for 15 to 18 h at 36 ± 1°C, during which the colonies not only remained viable but were reestablished. The membranes were dried, blocked with blocking buffer (Kirkegaard and Perry Laboratories [KPL], Gaithersburg, Md.), and exposed for 7 min to an affinity-purified horseradish peroxidase-labeled goat anti-E. coli O157 antibody (KPL). The membranes were washed, exposed to a 3,3′,5,5′-tetramethylbenzidine membrane substrate (TMB; KPL) or aminoethyl carbazole (AEC; Sigma Chemical Co., St. Louis, Mo.), rinsed in deionized water, and air dried. Colonies of E. coli O157:H7 were identified by either a blue (via TMB) or a red (via AEC) color reaction. The colored spots on the PVDF lift membrane were then matched to their respective parent colonies on the agar plates or filter monitor membranes. The colony lift immunoassay was tested with a wide range of genera in the family Enterobacteriaceae as well as different serotypes within the E. coli genus. The colony lift immunoassay provided a simple, rapid, and accurate method for confirming the presence of E. coli O157:H7 colonies isolated on filter monitors or spread plates by traditional culture methods. An advantage of using the colony lift immunoassay is the ability to test every colony serologically on an agar plate or filter monitor membrane simultaneously for the presence of the E. coli O157 antigen. This colony lift immunoassay has recently been successfully incorporated into a rapid-detection, isolation, and quantification system for E. coli O157:H7, developed in our laboratories for retail meat sampling.
PMCID: PMC95619  PMID: 9665968
13.  Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands 
Applied and Environmental Microbiology  2002;68(10):4853-4862.
Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (CT) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the CT and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10−5 pg of E. coli O157:H7 DNA ml−1 equivalent to approximately 6.4 × 103 CFU of E. coli O157:H7 ml−1 based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 104 CFU g−1. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g−1 with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.
PMCID: PMC126415  PMID: 12324331
14.  Fiber-Optic Biosensor Employing Alexa-Fluor Conjugated Antibody for Detection of Escherichia coli O157:H7 from Ground Beef in Four Hours 
Sensors (Basel, Switzerland)  2006;6(8):796-807.
Fiber optic biosensor has a great potential to meet the need for rapid, sensitive, and real-time microbial detection systems. We developed an antibody-based fiber-optic biosensor to rapidly detect low levels of Escherichia coli O157:H7 cells in ground beef. The principle of the sensor is a sandwich immunoassay using an antibody which is specific for E. coli O157:H7. A polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction that served as a capture antibody. An Alexa Fluor 647 dye-labeled antibody to E. coli O157:H7 was used to detect cells and generate a specific fluorescent signal, which was acquired by launching a 635 nm laser-light from an Analyte-2000. Fluorescent molecules within several hundred nanometers of the fiber were excited by an evanescent wave, and a portion of the emission light from fluorescent dye transmitted by the fiber and collected by a photodetector at wavelengths of 670 to 710 nm quantitatively. This immunosensor was specific for E. coli O157:H7 compared with multiple other foodborne bacteria. In addition, the biosensor was able to detect as low as 103 CFU/ml pure cultured E. coli O157:H7 cells grown in culture broth. Artificially inoculated E. coli O157:H7 at concentration of 1 CFU/ml in ground beef could be detected by this method after only 4 hours of enrichment.
PMCID: PMC3926523
Escherichia coli O157:H7; fiber-optic biosensor; antibodies; detection; ground beef
15.  Quantitative Characterization of the Influence of the Nanoscale Morphology of Nanostructured Surfaces on Bacterial Adhesion and Biofilm Formation 
PLoS ONE  2011;6(9):e25029.
Bacterial infection of implants and prosthetic devices is one of the most common causes of implant failure. The nanostructured surface of biocompatible materials strongly influences the adhesion and proliferation of mammalian cells on solid substrates. The observation of this phenomenon has led to an increased effort to develop new strategies to prevent bacterial adhesion and biofilm formation, primarily through nanoengineering the topology of the materials used in implantable devices. While several studies have demonstrated the influence of nanoscale surface morphology on prokaryotic cell attachment, none have provided a quantitative understanding of this phenomenon. Using supersonic cluster beam deposition, we produced nanostructured titania thin films with controlled and reproducible nanoscale morphology respectively. We characterized the surface morphology; composition and wettability by means of atomic force microscopy, X-ray photoemission spectroscopy and contact angle measurements. We studied how protein adsorption is influenced by the physico-chemical surface parameters. Lastly, we characterized Escherichia coli and Staphylococcus aureus adhesion on nanostructured titania surfaces. Our results show that the increase in surface pore aspect ratio and volume, related to the increase of surface roughness, improves protein adsorption, which in turn downplays bacterial adhesion and biofilm formation. As roughness increases up to about 20 nm, bacterial adhesion and biofilm formation are enhanced; the further increase of roughness causes a significant decrease of bacterial adhesion and inhibits biofilm formation. We interpret the observed trend in bacterial adhesion as the combined effect of passivation and flattening effects induced by morphology-dependent protein adsorption. Our findings demonstrate that bacterial adhesion and biofilm formation on nanostructured titanium oxide surfaces are significantly influenced by nanoscale morphological features. The quantitative information, provided by this study about the relation between surface nanoscale morphology and bacterial adhesion points towards the rational design of implant surfaces that control or inhibit bacterial adhesion and biofilm formation.
PMCID: PMC3180288  PMID: 21966403
16.  Subtractive Inhibition Assay for the Detection of E. coli O157:H7 Using Surface Plasmon Resonance 
Sensors (Basel, Switzerland)  2011;11(3):2728-2739.
A surface plasmon resonance (SPR) immunosensor was developed for the detection of E. coli O157:H7 by means of a new subtractive inhibition assay. In the subtractive inhibition assay, E. coli O157:H7 cells and goat polyclonal antibodies for E. coli O157:H7 were incubated for a short of time, and then the E. coli O157:H7 cells which bound antibodies were removed by a stepwise centrifugation process. The remaining free unbound antibodies were detected through interaction with rabbit anti-goat IgG polyclonal antibodies immobilized on the sensor chip using a BIAcore 3000 biosensor. The results showed that the signal was inversely correlated with the concentration of E. coli O157:H7 cells in a range from 3.0 × 104 to 3.0 × 108 cfu/mL with a detection limit of 3.0 × 104 cfu/mL. Compared with direct SPR by immobilizing antibodies on the chip surface to capture the bacterial cells and ELISA for E. coli O157:H7 (detection limit: both 3.0 × 105 cfu/mL in this paper), the detection limit of subtractive inhibition assay method was reduced by one order of magnitude. The method simplifies bacterial cell detection to protein-protein interaction, which has the potential for providing a practical alternative for the monitoring of E. coli O157:H7 and other pathogens.
PMCID: PMC3231628  PMID: 22163763
subtractive inhibition assay; SPR; E. coli O157:H7
17.  Efficacy of Three Light Technologies for Reducing Microbial Populations in Liquid Suspensions  
BioMed Research International  2014;2014:673939.
The aim of the current study was to evaluate the effectiveness of three nonthermal light technologies (NUV-Vis, continuous UV, and HILP) on their ability to inactivate Escherichia coli K12 and Listeria innocua.  E. coli K12 was selected as a representative microorganism for the enterohaemorrhagic foodborne pathogen E. coli O157:H7 and L. innocua as a surrogate microorganism for the common foodborne pathogen Listeria monocytogenes, respectively. The liquid matrix used for the disinfection experiments was a liquid matrix (MRD solution). The results of the present study show that the HILP treatment inactivated both E. coli and L. innocua more rapidly and effectively than either continuous UV-C or NUV-vis treatment. With HILP at 2.5 cm from the lamp, E. coli and L. innocua populations were reduced by 3.07 and 3.77 log10 CFU/mL, respectively, after a 5 sec treatment time, and were shown to be below the limit of detection (<0.22 log10 CFU/mL) following 30 sec exposure to HILP (106.2 J/cm2). These studies demonstrate the bactericidal efficacy of alternative nonthermal light technologies and their potential as decontamination strategies in the food industry.
PMCID: PMC3960526  PMID: 24724092
18.  Comparative Study of Electroless Copper Film on Different Self-Assembled Monolayers Modified ABS Substrate 
Copper films were grown on (3-Mercaptopropyl)trimethoxysilane (MPTMS), (3-Aminopropyl)triethoxysilane (APTES) and 6-(3-(triethoxysilyl)propylamino)-1,3,5- triazine-2,4-dithiol monosodium (TES) self-assembled monolayers (SAMs) modified acrylonitrile-butadiene-styrene (ABS) substrate via electroless copper plating. The copper films were examined using scanning electron microscopy (SEM) and X-ray diffraction (XRD). Their individual deposition rate and contact angle were also investigated to compare the properties of SAMs and electroless copper films. The results indicated that the formation of copper nuclei on the TES-SAMs modified ABS substrate was faster than those on the MPTMS-SAMs and APTES-SAMs modified ABS substrate. SEM images revealed that the copper film on TES-SAM modified ABS substrate was smooth and uniform, and the density of copper nuclei was much higher. Compared with that of TES-SAMs modified resin, the coverage of copper nuclei on MPTMS and APTES modified ABS substrate was very limited and the copper particle size was too big. The adhesion property test demonstrated that all the SAMs enhanced the interfacial interaction between copper plating and ABS substrate. XRD analysis showed that the copper film deposited on SAM-modified ABS substrate had a structure with Cu(111) preferred orientation, and the copper film deposited on TES-SAMs modified ABS substrate is better than that deposited on MPTMS-SAMs or APTES-SAMs modified ABS resins in electromigrtion resistance.
PMCID: PMC4013637  PMID: 24739812
ABS resin; SAMs; electroless copper film; heterocyclic silane
19.  Luminescence based enzyme-labeled phage (Phazyme) assays for rapid detection of Shiga toxin producing Escherichia coli serogroups 
Bacteriophage  2011;1(2):101-110.
Most diagnostic approaches for Shiga toxin producing Escherichia coli (STEC) have been designed to detect only serogroup O157 that causes a majority, but not all STEC related outbreaks in the United States. Therefore, there is a need to develop methodology that would enable the detection of other STEC serogroups that cause disease. Three bacteriophages (phages) that infect STEC serogroups O26, O103, O111, O145 and O157 were chemically labeled with horseradish peroxidase (HRP). The enzyme-labeled phages (Phazymes) were individually combined with a sampling device (a swab), STEC serogroup-specific immunomagnetic separation (IMS) beads, bacterial enrichment broth and luminescent HRP substrate, in a self-contained test device, while luminescence was measured in a hand-held luminometer.
The O26 and O157 Phazyme assays correctly identified more than 93% of the bacteria tested during this study, the O123 Phazyme assay identified 89.6%, while the O111 and O145 Phazyme assays correctly detected 82.4% and 75.9%, respectively. The decreased specificity of the O111 and O145 assays was related to the broad host ranges of the phages used in both assays. The Phazyme assays were capable of directly detecting between 105 and 106 CFU/ml in pure culture, depending on the serogroup. In food trials, the O157 Phazyme assay was able to detect E. coli O157:H7 in spinach consistently at levels of 1 CFU/g and occasionally at levels of 0.1 CFU/g. The assay detected 100 CFU/100 cm2 on swabbed meat samples and 102 CFU/100 ml in water samples. The Phazyme assay effectively detects most STEC in a simple and rapid manner, with minimal need for instrumentation to interpret the test result.
PMCID: PMC3278647  PMID: 22334866
Shiga toxin producing Escherichia coli (STEC); rapid detection; enzyme-labeled phages (Phazymes); horseradish peroxidase (HRP); swab; hand held luminometer
1. The placental blood film examination is worthy of routine application wherever æstivo-autumnal malaria is endemic. This type of malaria when associated with labor and the early days of the puerperium can be more easily and certainly diagnosed by the use of this film and a polychrome stain than by employing the usual films made from the mother's peripheral blood at the time of labor. The placental film in such an infection offers an abundance of adult parasites and far more evidence of the presence of pigment, while the peripheral blood film frequently offers but a scant number of the small ring or discoid forms of a parasite. The examination of the present series revealed positive placental films in nineteen cases, while but eight of these same cases were positive in the peripheral blood film examination. On the other hand, no peripheral blood films were found positive in which the associated placental films did not reveal a far more abundant evidence of the infection. 2. The early days of the puerperium can by this method be protected many times from a malarial outburst, and, as a rule, puerperal sepsis can be differentiated. 3. The intricate vascular architecture of the mature placenta rivals that of the spleen, liver, and bone marrow as a harbor for adult malarial parasites of this type and as a storage for pigment. 4. The localization of parasites in the placenta is unique. Here is the one vascular system which particularly favors the development of the parasites but which at the same time is so situated that it may be spontaneously discarded by the body at the climax of the attack. By this simple act late in pregnancy the prognosis for both mother and child may be improved. 5. The pregnant state encourages attacks of malaria by lowering bodily resistance and by furnishing an additional harbor for the development of parasites. A tenable theory in regard to most attacks of this nature, occurring in cases under professional care, would appear to be the development of latent malaria (malarial carriers) into acute attacks toward the close of the pregnant state. The women who expose themselves (as the negroes in this series) offer favorable conditions to the introduction of a primary infection. Malaria frequently interrupts the late stages of pregnancy and sometimes causes the death of the mother and the fetus, more often the latter. The records at Ancon indicate that it more frequently exerts a harmful influence than other types of infectious diseases in this locality. 6. Most of the children in this series that were delivered while malaria was present in the mother, were of a race that seems to possess a relative immunity to the ravages of malaria. This may account for the fact that the negro fetus more nearly approximates the full term of development when associated with this disease and is comparatively subjected to a less number of the accidents of pregnancy. Many of them revealed evidence of prematurity and were jaundiced, but, as a rule, they developed rapidly. The commonest mishap is miscarriage late in pregnancy. Occasional still-births occur and sometimes there is a fatal issue to both the mother and child. 7. Cases diagnosed as congenital malaria probably indicate that some accident occurred to the placenta, because it practically never happens that fetal blood is positive at the time of birth, regardless of the degree of infection in the mother. Many of the cases now reported in the literature as congenital malaria suggest immediate postnatal infection as their history, as our pathological and clinical records testify. 8. The size of the intervillous spaces of the placenta and their adaptability in the localization of parasites seem to disprove to a certain extent the old idea that the localization depends on the smallness of the capillary caliber. If this were the case the brain should be more often the seat of an extensive localization than the spleen, bone marrow, and placenta, yet our anatomical records will not support that theory. A sluggish blood sinus with a large endothelial surface, a higher internal body temperature, and red blood cells burdened with parasites of a certain age beyond the ring form seem to be important factors in the localization and development of the æstivo-autumnal parasite. 9. The racial disparity of malarial infections shown in this series is believed to be due to local conditions and a wrong impression is apt to be given by our statistics in regard to the relative immunity of the negro race. The white women on the Canal Zone avail themselves of all the opportunities the sanitary system affords; they live well and place the entire course of their pregnant state under competent professional care, while the negro woman is indifferent to her pregnant state, works as a domestic servant, and lives in the cheapest unprotected quarters that can be rented in the suburban divisions of Panama City where the malarial rate is highest and the sanitary control is difficult. It should be noted that these negro women can carry an infection with little manifestation of its presence that would produce serious results in the white women brought from the temperate zone regions of Europe and the United States.
PMCID: PMC2125361  PMID: 19867927
21.  Same-Day Detection of Escherichia coli O157:H7 from Spinach by Using Electrochemiluminescent and Cytometric Bead Array Biosensors▿  
Applied and Environmental Microbiology  2010;76(24):8044-8052.
Contamination of fresh produce with Escherichia coli O157:H7 and other pathogens commonly causes food-borne illness and disease outbreaks. Thus, screening for pathogens is warranted, but improved testing procedures are needed to allow reproducible same-day detection of low initial contamination levels on perishable foods, and methods for detecting numerous pathogens in a single test are desired. Experimental procedures were developed to enable rapid screening of spinach for E. coli O157:H7 by using multiplex-capable immunological assays that are analyzed using biosensors. Detection was achieved using an automated electrochemiluminescent (ECL) assay system and a fluorescence-based cytometric bead array. Using the ECL system, less than 0.1 CFU of E. coli O157:H7 per gram of spinach was detected after 5 h of enrichment, corresponding to 6.5 h of total assay time. Using the cytometric bead array, less than 0.1 CFU/g was detected after 7 h of enrichment, with a total time to detection of less than 10 h. These results illustrate that both biosensor assays are useful for rapid detection of E. coli O157:H7 on produce in time frames that are comparable to or better than those of other testing formats. Both methods may be useful for multiplexed pathogen detection in the food industry and other testing situations.
PMCID: PMC3008225  PMID: 21037307
22.  Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker 
Applied and Environmental Microbiology  2012;78(15):5297-5304.
The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)–real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 107 dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA–real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.
PMCID: PMC3416439  PMID: 22635992
23.  Gold Nanorod Based Selective Identification of Escherichia coli Bacteria Using Two-Photon Rayleigh Scattering Spectroscopy 
ACS nano  2009;3(7):1906-1912.
The presence of E coli in foodstuffs and drinking water is a chronic worldwide problem. The worldwide food production industry is worth about U.S. $578 billion, and the demand for biosensors to detect pathogens and pollutants in foodstuffs is growing day by day. Driven by the need, we report for the first time that two-photon Rayleigh scattering (TPRS) properties of gold nanorods can be used for rapid, highly sensitive and selective detection of Escherichia coli bacteria from aqueous solution, without any amplification or enrichment in 50 Colony Forming Units (cfu)/mL level with excellent discrimination against any other bacteria. TPRS intensity increases 40 times, when anti E. coli antibody-conjugated nanorods were mixed with various concentrations of Escherichia coli O157:H7 bacterium. The mechanism of TPRS intensity change has been discussed. This bionanotechnology assay could be adapted in studies using antibodies specific for various bacterial pathogens for the detection of a wide variety of bacterial pathogens used as bioterrorism agents in food, clinical samples, and environmental samples.
PMCID: PMC2888863  PMID: 19572619
gold nanorods; Escherichia coli bacteria; two-photon Rayleigh scattering; food pathogens; plasmonics
24.  Colorimetric-Based Detection of TNT Explosives Using Functionalized Silica Nanoparticles 
Sensors (Basel, Switzerland)  2015;15(6):12891-12905.
This proof-of-concept study proposes a novel sensing mechanism for selective and label-free detection of 2,4,6-trinitrotoluene (TNT). It is realized by surface chemistry functionalization of silica nanoparticles (NPs) with 3-aminopropyl-triethoxysilane (APTES). The primary amine anchored to the surface of the silica nanoparticles (SiO2-NH2) acts as a capturing probe for TNT target binding to form Meisenheimer amine–TNT complexes. A colorimetric change of the self-assembled (SAM) NP samples from the initial green of a SiO2-NH2 nanoparticle film towards red was observed after successful attachment of TNT, which was confirmed as a result of the increased separation between the nanoparticles. The shift in the peak wavelength of the reflected light normal to the film surface (λpeak) and the associated change of the peak width were measured, and a merit function taking into account their combined effect was proposed for the detection of TNT concentrations from 10−12 to 10−4 molar. The selectivity of our sensing approach is confirmed by using TNT-bound nanoparticles incubated in AptamerX, with 2,4-dinitrotoluene (DNT) and toluene used as control and baseline, respectively. Our results show the repeatable systematic color change with the TNT concentration and the possibility to develop a robust, easy-to-use, and low-cost TNT detection method for performing a sensitive, reliable, and semi-quantitative detection in a wide detection range.
PMCID: PMC4507664  PMID: 26046595
explosives; functionalized nanoparticles; colorimetric; label-free; self-assembly film; aptamer
25.  Nanostructuring of Biosensing Electrodes with Nanodiamonds for Antibody Immobilization 
ACS Nano  2014;8(2):1419-1428.
While chemical vapor deposition of diamond films is currently cost prohibitive for biosensor construction, in this paper, we show that sonication-assisted nanostructuring of biosensing electrodes with nanodiamonds (NDs) allows harnessing the hydrolytic stability of the diamond biofunctionalization chemistry for real-time continuous sensing, while improving the detector sensitivity and stability. We find that the higher surface coverages were important for improved bacterial capture and can be achieved through proper choice of solvent, ND concentration, and seeding time. A mixture of methanol and dimethyl sulfoxide provides the highest surface coverage (33.6 ± 3.4%) for the NDs with positive zeta-potential, compared to dilutions of dimethyl sulfoxide with acetone, ethanol, isopropyl alcohol, or water. Through impedance spectroscopy of ND-seeded interdigitated electrodes (IDEs), we found that the ND seeds serve as electrically conductive islands only a few nanometers apart. Also we show that the seeded NDs are amply hydrogenated to be decorated with antibodies using the UV-alkene chemistry, and higher bacterial captures can be obtained compared to our previously reported work with diamond films. When sensing bacteria from 106 cfu/mL E. coliO157:H7, the resistance to charge transfer at the IDEs decreased by ∼38.8%, which is nearly 1.5 times better than that reported previously using redox probes. Further in the case of 108 cfu/mL E. coliO157:H7, the charge transfer resistance changed by ∼46%, which is similar to the magnitude of improvement reported using magnetic nanoparticle-based sample enrichment prior to impedance detection. Thus ND seeding allows impedance biosensing in low conductivity solutions with competitive sensitivity.
PMCID: PMC4004312  PMID: 24397797
nanodiamond; biosensing; bioimmobilization; impedance; surface chemistry

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