Opto-electrokinetic manipulation for high-performance on-chip bioassays.

This communication first demonstrates bio-compatibility of a recently developed opto-electrokinetic manipulation technique, using microorganisms. Aggregation, patterning, translation, trapping and size-based separation of microorganisms performed with the technique firmly establishes its usefulness for development of a high-performance on-chip bioassay system.

Intracellularly grown gold nanoislands as SERS substrates for monitoring chromate, sulfate and nitrate localization sites in remediating bacteria biofilms by Raman chemical imaging.

Understanding the chemical composition of biofilm matrices is vital in different fields of biology such as surgery, dental medicine, synthetic grafts and bioremediation. The knowledge of biofilm development, composition, active reduction sites and remediation efficacy will help in the development of effective solutions and evaluation of remediating approaches prior to implementation. Surface-enhanced Raman spectroscopy (SERS) based imaging is an invaluable tool to obtain an understanding of the remediating efficacy of microorganisms and its role in the formation of organic and inorganic compounds in biofilms. We demonstrate for the first time, the presence of chromate, sulfate, nitrate and reduced trivalent chromium in soil biofilms. In addition, we demonstrate that SERS imaging was able to validate two observations made by previous studies on chromate/sulfate and chromate/nitrate interactions in Shewanella oneidensis MR-1 biofilms. Additionally, we show a detailed Raman mapping based evidence of the existence of chromate-sulfate competition for cellular entry. Subsequently, we use Raman mapping to study the effect of nitrate on chromate reduction. The findings presented in this paper are among the first to report - detection of multiple metallic ions in bacterial biofilms using intracellular SERS substrates. Such a detailed characterization of biofilms using gold nanoislands based SERS mapping substrate can be extended to study cellular localization of other metallic ions and chemical species of biological and toxicological significance and their effect on reduction reactions in bacterial biofilms.

FTIR nanobiosensors for Escherichia coli detection.

Infections due to enterohaemorrhagic E. coli (Escherichia coli) have a low incidence but can have severe and sometimes fatal health consequences, and thus represent some of the most serious diseases due to the contamination of water and food. New, fast and simple devices that monitor these pathogens are necessary to improve the safety of our food supply chain. In this work we report on mesoporous titania thin-film substrates as sensors to detect E. coli O157:H7. Titania films treated with APTES ((3-aminopropyl)triethoxysilane) and GA (glutaraldehyde) were functionalized with specific antibodies and the absorption properties monitored. The film-based biosensors showed a detection limit for E. coli of 1 × 10(2) CFU/mL, constituting a simple and selective method for the effective screening of water samples.

Raman chemical imaging of chromate reduction sites in a single bacterium using intracellularly grown gold nanoislands.

Imaging live molecular events within micro-organisms at single-cell resolution would deliver valuable mechanistic information much needed in understanding key biological processes. We present a surface-enhanced Raman (SERS) chemical imaging strategy as a first step toward exploring the intracellular bioreduction pockets of toxic chromate in Shewanella. In order to achieve this, we take advantage of an innate reductive mechanism in bacteria of reducing gold ions into intracellular gold nanoislands, which provide the necessary enhancement for SERS imaging. We show that SERS has the sensitivity and selectivity not only to identify but also to differentiate between the two stable valence forms of chromate in cells. The imaging platform was used to understand intracellular metal reduction activities in a ubiquitous metal-reducing organism, Shewanella oneidensis MR-1, by mapping chromate reduction.

Surface-enhanced Raman imaging of intracellular bioreduction of chromate in Shewanella oneidensis.

This proposed research aims to use novel nanoparticle sensors and spectroscopic tools constituting surface-enhanced Raman spectroscopy (SERS) and Fluorescence Lifetime imaging (FLIM) to study intracellular chemical activities within single bioremediating microorganism. The grand challenge is to develop a mechanistic understanding of chromate reduction and localization by the remediating bacterium Shewanella oneidensis MR-1 by chemical and lifetime imaging. MR-1 has attracted wide interest from the research community because of its potential in reducing multiple chemical and metallic electron acceptors. While several biomolecular approaches to decode microbial reduction mechanisms exist, there is a considerable gap in the availability of sensor platforms to advance research from population-based studies to the single cell level. This study is one of the first attempts to incorporate SERS imaging to address this gap. First, we demonstrate that chromate-decorated nanoparticles can be taken up by cells using TEM and Fluorescence Lifetime imaging to confirm the internalization of gold nanoprobes. Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells. Distinctive differences in Raman signatures of Cr(VI) and Cr(III) enabled their spatial identification within single cells from the Raman images. A comprehensive evaluation of toxicity and cellular interference experiments conducted revealed the inert nature of these probes and that they are non-toxic. Our results strongly suggest the existence of internal reductive machinery and that reduction occurs at specific sites within cells instead of at disperse reductive sites throughout the cell as previously reported. While chromate-decorated gold nanosensors used in this study provide an improved means for the tracking of specific chromate interactions within the cell and on the cell surface, we expect our single cell imaging tools to be extended to monitor the interaction of other toxic metal species.

Biofunctionalized magnetic nanoparticle integrated mid-infrared pathogen sensor for food matrixes.

Magnetic nanoparticles functionalized with anti-Escherichia coli O157:H7 or anti-Salmonella typhimurium antibodies that can specifically bind to their target organisms were used to isolate E. coli O157:H7 and S. typhimurium separately from a cocktail of bacteria and from food matrixes. The pathogens were then detected using label-free IR fingerprinting. The binding and detection protocol was first validated using a benchtop FT-IR spectrometer and then applied to a portable mid-IR spectrometer to enable this approach as a point-of-detection technology. Highly selective detection was achieved in less than 30 min at both species (E. coli O157:H7 vs S. typhimurium ) and strain (E. coli O157:H7 vs E. coli K12) levels in complex food matrixes (2% milk, spinach extract) with a detection limit of 10(4)-10(5) CFU/mL. The combined approach of functionalized magnetic nanoparticles and IR spectroscopy imparts specificity through spectroscopic fingerprinting and selectivity through species-specific antibodies with an in-built sample extraction step and could be applied in the field for on-site food-borne pathogen monitoring.