Naama Massad-Ivanir

Picking up the pieces: a generic porous Si biosensor for probing the proteolytic products of enzymes.

A multifunctional porous Si biosensor that can both monitor the enzymatic activity of minute samples and allow subsequent retrieval of the entrapped proteolytic products for mass spectrometry analysis is described. The biosensor is constructed by DNA-directed/reversible immobilization of enzymes onto a Fabry-Pérot thin film. We demonstrate high enzymatic activity levels of the immobilized enzymes (more than 80%), while maintaining their specificity. Mild dehybridization conditions allow enzyme recycling and facile surface regeneration for consecutive biosensing analysis. The catalytic activity of the immobilized enzymes is monitored in real time by reflective interferometric Fourier transform spectroscopy. The real-time analysis of minute quantities of enzymes (concentrations at least 1 order of magnitude lower, 0.1 mg mL(-1), in comparison to previous reports, 1 mg mL(-1)), in particular proteases, paves the way for substrate profiling and the identification of cleavage sites. The biosensor configuration is compatible with common proteomic methods and allows for a successful downstream mass spectrometry analysis of the reaction products.

Engineering porous silicon nanostructures as tunable carriers for mitoxantrone dihydrochloride

Nanostructured porous silicon (PSi) thin films, fabricated by the electrochemical anodization of single crystalline Si wafers, are studied as delivery systems for the anticancer drug mitoxantrone dihydrochloride (MTX). The surface chemistry of the PSi carriers was tailored by surface alkylation using thermal hydrosilylation of 1-dodecene and undecylenic acid, followed by physical adsorption or covalent attachment of MTX to the Si scaffold. The nanostructure and the physiochemical properties of the different carriers were characterized by attenuated total reflectance Fourier transform infrared spectroscopy, nitrogen adsorption-desorption and contact angle measurements, demonstrating that surface alkylation results in a pronounced effect on the hydrophobicity/hydrophilicity of the scaffolds and a volumetric gain in pore wall, which in turn results in a decrease in pore diameter (>23%) and available porous volume (>40%). The effect of these key parameters on MTX loading efficacy, release profile, Si scaffold erosion kinetics and in vitro cytotoxicity on human breast carcinoma (MDA-MB-231) cells was studied and compared to the behavior of neat PSi carriers. We show that the chemically modified PSi carriers exhibit sustained release for several days to weeks with minimal to no burst effect, while for the native PSi MTX release was completed within 5h with a substantial burst release of ~40%. Moreover, our in vitro cytotoxicity experiments have clearly demonstrated that the MTX released from all PSi carriers maintained its cytotoxic effect towards MDA-MB-231 cells, in comparison to the low toxicity of the PSi carriers.

Optical biosensing of bacteria and cells using porous silicon based, photonic lamellar gratings

We report on a method to extend the optical sensing capabilities of conventional RIFTS (reflective interferometric Fourier transform spectroscopy) biosensors for real-time detection of large microorganisms, such as bacteria and cells. Using macro porous silicon based 2D arrays of phase (lamellar) grating, we demonstrate that the zero-order optical reflectivity exhibits a similar interference pattern to that obtained for ordinary RIFTS biosensors, which can be Fourier transformed into optical thickness and exploited for biosensing. The sensing capabilities are demonstrated for Escherichia coli bacteria that were captured inside the macro-pores. The entrapment process is monitored and verified by confocal laser scanning microscopy.

DNA-directed immobilization of horseradish peroxidase onto porous SiO2 optical transducers

Multifunctional porous Si nanostructure is designed to optically monitor enzymatic activity of horseradish peroxidase. First, an oxidized PSi optical nanostructure, a Fabry-Pérot thin film, is synthesized and is used as the optical transducer element. Immobilization of the enzyme onto the nanostructure is performed through DNA-directed immobilization. Preliminary studies demonstrate high enzymatic activity levels of the immobilized horseradish peroxidase, while maintaining its specificity. The catalytic activity of the enzymes immobilized within the porous nanostructure is monitored in real time by reflective interferometric Fourier transform spectroscopy. We show that we can easily regenerate the surface for consecutive biosensing analysis by mild dehybridization conditions.

Nanostructured Porous Si Optical Biosensors: Effect of Thermal Oxidation on Their Performance and Properties

The influence of thermal oxidation conditions on the performance of porous Si optical biosensors used for label-free and real-time monitoring of enzymatic activity is studied. We compare three oxidation temperatures (400, 600, and 800 °C) and their effect on the enzyme immobilization efficiency and the intrinsic stability of the resulting oxidized porous Si (PSiO2), Fabry-Pérot thin films. Importantly, we show that the thermal oxidation profoundly affects the biosensing performance in terms of greater optical sensitivity, by monitoring the catalytic activity of horseradish peroxidase and trypsin-immobilized PSiO2. Despite the significant decrease in porous volume and specific surface area (confirmed by nitrogen gas adsorption-desorption studies) with elevating the oxidation temperature, higher content and surface coverage of the immobilized enzymes is attained. This in turn leads to greater optical stability and sensitivity of PSiO2 nanostructures. Specifically, films produced at 800 °C exhibit stable optical readout in aqueous buffers combined with superior biosensing performance. Thus, by proper control of the oxide layer formation, we can eliminate the aging effect, thus achieving efficient immobilization of different biomolecules, optical signal stability, and sensitivity.

Hydrogels synthesized in electrochemically machined porous Si hosts: effect of nano-scale confinement on polymer properties

Imprisonment of polymers and hydrogels in inorganic nanostructures is of growing interest in many areas of materials research and various biomedical devices. In this study, we synthesize the hydrogels polyacrylamide and poly(N-isopropylacrylamide) in situ within nanostructured porous Si (PSi) hosts and characterize their thermal properties by differential scanning calorimetry (DSC) and thermal gravimetry. The PSi hosts are electrochemically machined as thin optical Fabry–Perot films with a well-characterized interconnected nanostructure. Confining hydrogels in these hosts is found to significantly decrease the transition temperatures of the polymers i.e., glass transition temperature (Tg) and temperature-dependent volume phase transition (VPT), in comparison to those of the neat hydrogels. We show that incorporation of a responsive hydrogel, poly(N-isopropylacrylamide), into nanochannels of the PSi thin film permits optical transduction of the hydrogel behavior during its volume phase transition. The “real-time” optical response is correlated with DSC data and provides an alternate means for studying nano-scale confinements of responsive hydrogels. In addition, we found significant differences between the thermal degradation behavior of the confined hydrogels compared with those of neat hydrogels and thin hydrogel films on planar Si surfaces. The confined hydrogels have an inferior thermal stability than that of the neat hydrogels. These findings suggest that the in situ polymerization and the hydrogel confinement conditions have a profound effect on the nanostructure and resulting behavior of the hydrogel phase.

Porous Silicon-Based Biosensors: Towards Real-Time Optical Detection of Target Bacteria in the Food Industry

Rapid detection of target bacteria is crucial to provide a safe food supply and to prevent foodborne diseases. Herein, we present an optical biosensor for identification and quantification of Escherichia coli (E. coli, used as a model indicator bacteria species) in complex food industry process water. The biosensor is based on a nanostructured, oxidized porous silicon (PSi) thin film which is functionalized with specific antibodies against E. coli. The biosensors were exposed to water samples collected directly from process lines of fresh-cut produce and their reflectivity spectra were collected in real time. Process water were characterized by complex natural micro-flora (microbial load of >107 cell/mL), in addition to soil particles and plant cell debris. We show that process water spiked with culture-grown E. coli, induces robust and predictable changes in the thin-film optical interference spectrum of the biosensor. The latter is ascribed to highly specific capture of the target cells onto the biosensor surface, as confirmed by real-time polymerase chain reaction (PCR). The biosensors were capable of selectively identifying and quantifying the target cells, while the target cell concentration is orders of magnitude lower than that of other bacterial species, without any pre-enrichment or prior processing steps.

Antimicrobial LDPE/EVOH Layered Films Containing Carvacrol Fabricated by Multiplication Extrusion

This work describes the fabrication of antimicrobial multilayered polymeric films containing carvacrol (used as a model essential oil) by co-extrusion and multiplication technique. The microlayering process was utilized to produce films, with up to 65 alternating layers, of carvacrol-containing low-density polyethylene (LDPE) and ethylene vinyl alcohol copolymer (EVOH). Carvacrol was melt compounded with LDPE or loaded into halloysite nanotubes (HNTs) in a pre-compounding step prior film production. The detailed nanostructure and composition (in terms of carvacrol content) of the films were characterized and correlated to their barrier properties, carvacrol release rate, and antibacterial and antifungal activity. The resulting films exhibit high carvacrol content despite the harsh processing conditions (temperature of 200 °C and long processing time), regardless of the number of layers or the presence of HNTs. The multilayered films exhibit superior oxygen transmission rates and carvacrol diffusivity values that are more than two orders of magnitude lower in comparison to single-layered carvacrol-containing films (i.e., LDPE/carvacrol and LDPE/(HNTs/carvacrol)) produced by conventional cast extrusion. The (LDPE/carvacrol)/EVOH and (LDPE/[HNTs/carvacrol])/EVOH films demonstrated excellent antimicrobial efficacy against E. coli and Alternaria alternata in in vitro micro-atmosphere assays and against A. alternata and Rhizopus in cherry tomatoes, used as the food model. The results presented here suggest that sensitive essential oils, such as carvacrol, can be incorporated into plastic polymers constructed of tailored multiple layers, without losing their antimicrobial efficacy.

EFFECT OF THERMAL OXIDATION ON THE PERFORMANCE OF NANOSTRUCTURED POROUS SI OPTICAL BIOSENSORS

H peroxide (H2O2) is one of the several reactive oxygen species (ROS) generated as a by-product of many biological processes. While it occurs naturally in relatively low concentrations throughout the human body, deviation from the normal physiological range may be indicative of a number of conditions. As such, hydrogen peroxide may be viewed as a biomarker, allowing it’s concentration to be monitored and thus used to augment diagnosis of critical ailments such as sepsis and oxidative stress. To this end, we have sought to investigate the development of a non-enzymatic sensor capable of quantifying peroxide. The core aim of the approach relates to the provision of a micro scale sensor that could be ultimately be used for in vivo measurements or transdermal sensing. The underpinning methodology pursued involves the design and development of a monofilament carbon fiber probe (10 micron diameter) onto which a nano layer of palladium is electrochemically deposited. Examination of the latter using scanning electron microscopy revealed a forest of Pd nano fibrils and a representative image highlighting the partial formation of the film is shown in Figure 1. These structures have been shown to exhibit exceptional catalytic activity towards the oxidation of peroxide at low operating potentials where there is very little interference from other matrix components. The high sensitivity and selectivity for peroxide can be further exploited through coupling of oxidase enzymes to expand the range of biomarkers that can be quantified. The subsequent modification of the Pd film and the extrapolation of the amperometric approach to the measurement of glucose and lactate (itself an important marker of sepsis) is considered and critically appraised.I view of their facile automation, wide linear range and low limits of detection, conducting polymers have been extensively employed as biosensors. Furthermore, electrochemical synthesis of conducting polymers can be carried out effortlessly on various electrodes leading to the robust adherence of the polymer films. Among various sensing applications of conducting polymers, enzymatic and non-enzymatic sensing of glucose, urea, dopamine, etc., deserves their biological importance. Conducting polymers extensively investigated in this context encompass polyaniline and polypyrrole. The enzymatic sensing of glucose using polyaniline nanofibers has been demonstrated using cyclic voltammetric, amperometric and impedimetric analysis with impressive detection limits and calibration range. The potentiodynamic polymerization of pyrrole on Pt is shown to yield non-enzymatic sensors of urea with satisfactory linear range of calibration. The electrochemical sensing of other compounds such as levothyroxine, dopamine, etc., will be highlighted.