DNA microarrays on silicon nanostructures: optimization of the multilayer stack for fluorescence detection

To improve the sensitivity of fluorescence detection in DNA microarrays, the use of silicon nanostructures based on chemical vapor deposition (CVD) processes adopted for the growth of rough polycrystalline silicon was investigated. These substrates present advantages of two main properties which could lead to an enhancement of the fluorescence detection, i.e. (i) the increase of the available surface area in order to achieve a high loading capacity of biomolecules and (ii) the optimization of the stack of silicon nanostructures support. Indeed, the structures were elaborated on an initial thermal oxide layer and then covered with a silicon oxide layer, obtained by oxidation and allowing the functionalization for the subsequent grafting of DNA probes. Moreover, these oxide layers play a part in the fluorescence detection. The influence of the silicon oxide layer thickness above and below the silicon grains in close relation with the density of nanostructures on the emitted fluorescence was emphasized. This paper presents an experimental characterization of the fluorescence intensity and the optimization of the different layers that composed the substrate used for DNA microarrays. The performances of the microarrays were investigated by means of hybridization experiments using complementary fluorescent labeled-oligonucleotides targets. Our results indicate that an optimized substrate can be designed and that the use of oxidized silicon nanostructures for support of biochip could be a strategy for improving the sensitivity of fluorescence detection.     

SU-8-carbon composite as conductive photoresist for biochip applications

A composite photoresist has been developed for the direct photopatterning of electrodes useful as biochip substrates. The material is composed of SU-8 polymer added with graphite carbon filler which enables patterning of conductive thin films (22μm) on both glass substrate and transparency flexible film with a standard UV photolithography protocol. The resolution obtained using the conductive composite compared well with the bare resist, with lateral resolutions of 5 and 10μm for bare and conductive resists, respectively. The obtained electrodes, after an electrochemical pre-treatment, exhibited very good electrochemical behaviors, opening the path to various electrochemical detections and grafting possibilities. In order to demonstrate the potentialities of the developed material in the biosensors and biochips field, DNA probes were electrografted, using diazonium chemistry, directly at the composite photoresist surface. Target oligonucleotide interactions were detected using chemiluminescent labeling and a satisfactory detection limit of 0.25nM target sequence was demonstrated with a detection ranging over three orders of magnitude.     

Porous devices derived from co-continuous polymer blends as a route for controlled drug release

In this study we examine the release profile of bovine serum albumin (BSA) from a porous polymer matrix derived from a co-continuous polymer blend. The porosity is generated through the selective extraction of one of the continuous phases. This is the first study to examine the approach of using morphologically tailored co-continuous polymer blends as a template for generating porous polymer materials for use in controlled release. A method for the preparation of polymeric capsules is introduced, and the effect of matrix pore size and surface area on the BSA release profile is investigated. Furthermore, the effect of surface charge on release is examined by surface modification of the porous substrate using layer-by-layer deposition techniques. Synthetic, nonerodible polymer, high-density polyethylene (HDPE), was used as a model substrate prepared by melt blending with two different styrene-ethylene-butylene copolymers. Blends with HDPE allow for the preparation of porous substrates with small pore sizes (300 and 600 nm). A blend of polylactide (PLA) and polystyrene was also used to prepare porous PLA with a larger pore size (1.5 microm). The extents of interconnectivity, surface area, and pore dimension of the prepared porous substrates were examined via gravimetric solvent extraction, BET nitrogen adsorption, mercury porosimetry, and image analysis of scanning electron microscopy micrographs. With a loading protocol into the porous HDPE and PLA involving the alternate application of pressure and vacuum, it is shown that virtually the entire porous network was accessible to BSA loading, and loading efficiencies of between 80% and 96% were obtained depending on the pore size of the carrier and the applied pressure. The release profile of BSA from the microporous structure was monitored by UV spectrophotometry. The influence of pore size, surface area, surface charge, and number of deposited layers is demonstrated. It is shown that an effective closed-cell structure in porous PLA can be prepared, effectively eliminating all short-term BSA release.     

Analysis and Modeling of Lithium Flows in Porous Materials

One of the primary conditions necessary for the success of magnetic fusion reactors is the ability to mitigate damage to the first wall during ELMs and plasma disruptions. A potential solution involves the use of flowing liquid metals such as lithium as a first wall, but ensuring its stability under the extreme environments in the reactor would be imperative. The conditions leading to instabilities on the free surface of flowing liquid lithium (LL) layers on a substrate and in a porous material are investigated using both analytical methods and computational modeling, with consideration for the effects of LL velocity, LL layer thickness, substrate porosity, LL permeability, and hydrogen (H) plasma velocity. Linear stability analysis is used to predict the critical velocity and wavelength-dependence of wave growth, as well as the onset of instability. The modeling of LL flows is performed on a flat substrate and in a porous material for various LL thicknesses, LL and H plasma velocities to analyze the conditions leading to droplet formation and ejection.     

Sucrose hydrolysis by invertase immobilized on functionalized porous silicon

A successful recipe for the production of immobilized invertase/porous silicon layer with appropriate catalytic behavior for the sucrose hydrolysis reaction is presented. The procedure is based on support surface chemical oxidation, silanization, activation with glutaraldehyde and finally covalent bonding of the free enzyme to the functionalized surface. The catalytic behavior of the composite layer as a function of pH, temperature, and the current density applied in the porous silicon (PS) preparation is investigated. Interestingly, V_max undergoes a substantial increase (ca. 30%) upon immobilization. The value of K_m increases by a factor of 1.53 upon immobilization. The initial activity is still preserved up to 28 days while the free enzyme undergoes a 26% loss of activity after the same period. Based on the outcomes of this study, we believe that tailored PS layers may be used for the development of new bioreactors in which the active enzyme is immobilized on the internal walls and is not lost during the process.     

Kinetics of oligonucleotide hybridization to DNA probe arrays on high-capacity porous silica substrates

We have investigated the kinetics of DNA hybridization to oligonucleotide arrays on high-capacity porous silica films that were deposited by two techniques. Films created by spin coating pure colloidal silica suspensions onto a substrate had pores of approximately 23 nm, relatively low porosity (35%), and a surface area of 17 times flat glass (for a 0.3-microm film). In the second method, latex particles were codeposited with the silica by spin coating and then pyrolyzed, which resulted in larger pores (36 nm), higher porosity (65%), and higher surface area (26 times flat glass for a 0.3-microm film). As a result of these favorable properties, the templated silica hybridized more quickly and reached a higher adsorbed target density (11 vs. 8 times flat glass at 22 degrees C) than the pure silica. Adsorption of DNA onto the high-capacity films is controlled by traditional adsorption and desorption coefficients, as well as by morphology factors and transient binding interactions between the target and the probes. To describe these effects, we have developed a model based on the analogy to diffusion of a reactant in a porous catalyst. Adsorption values (k(a), k(d), and K) measured on planar arrays for the same probe/target system provide the parameters for the model and also provide an internally consistent comparison for the stability of the transient complexes. The interpretation of the model takes into account factors not previously considered for hybridization in three-dimensional films, including the potential effects of heterogeneous probe populations, partial probe/target complexes during diffusion, and non-1:1 binding structures. The transient complexes are much less stable than full duplexes (binding constants for full duplexes higher by three orders of magnitude or more), which may be a result of the unique probe density and distribution that is characteristic of the photolithographically patterned arrays. The behavior at 22 degrees C is described well by the predictive equations for morphology, whereas the behavior at 45 degrees C deviates from expectations and suggests that more complex phenomena may be occurring in that temperature regime.     

Microporous silicon and biosensor development: structural analysis, electrical characterisation and biocapacity evaluation

An investigation of the fabrication of microporous silicon (MPS) layers as a material for the development of an electrolyte insulator semiconductor (EIS) capacitance sensor has been performed. The goal was to create a high surface area substrate for the immobilisation of biorecognition elements. Structural analysis of MPS layers as a function of key etch parameters, namely implant type (p or n), implant dose, hydrofluoric acid (HF) etch concentration and current density has been performed using scanning electron microscopy (SEM). It was possible to image porous layers with average pore diameter as low as 4 nm. n-type silicon samples had larger pore networks than p-type samples and reducing the silicon resistivity led to a reduction in the pores per microm2. It was found that increasing the HF etch concentration reduced the average pore diameter and increased the pores per microm2. Increasing the current density at which the etch was performed has the same effect. Understanding the effect of these parameters allows the MPS layer to be tuned to match specifications for optimum biocapacity. Different MPS layers were electrically characterised using capacitance-voltage and capacitance-frequency sweeps, in order to determine the effect of porosity on increases in surface area. The measured capacitance increased with increasing pores per microm2. p-type silicon with a boron implant in the back of the wafer, which had been etched in 25% HF in ethanol at a current density of 75 mA/cm2 yielded the highest capacitance signal per unit area. The effect of porosity and pore size on the biocapacity of the samples was also determined. For avidin immobilisation, with pores sizes above 5 nm, as the porosity increased the biocapacity increased. MPS fabricated in p-type silicon with a front and back implant etched in 25% HF at a current density of 25 mA/cm2 was used for the capacitance detection of synthetic oligonucleotides.     

High capacity substrates as a platform for a DNA probe array genotyping assay

Colloidal silica particles were deposited on a glass substrate to produce high-capacity porous supports for high-density DNA probe arrays. Porous surfaces were used to increase the addressable surface area and number of probes available for hybridization. Surfaces derived from 70-100 nm size particles deposited in films from 0.15 to 2 microns thick exhibited excellent performance in light-directed oligonucleotide synthesis. Evaluation of these substrates in a genotyping assay is reported.     

HIGH CAPACITY SUBSTRATES AS A PLATFORM FOR A DNA PROBE ARRAY GENOTYPING ASSAY

Colloidal silica particles were deposited on a glass substrate to produce high-capacity porous supports for high-density DNA probe arrays. Porous surfaces were used to increase the addressable surface area and number of probes available for hybridization. Surfaces derived from 70–100 nm size particles deposited in films from 0.15 to 2 microns thick exhibited excellent performance in light-directed oligonucleotide synthesis. Evaluation of these substrates in a genotyping assay is reported.     

Micropatterning of porous silicon films by direct laser writing

In this study, we demonstrate that porous silicon films can be ablated by the pulsed nitrogen laser of a commercial MALDI mass spectrometer. The extent of laser-induced ablation was found to depend on the doping level and surface chemistry of the porous silicon film. Using direct laser writing with or without a mask, micropatterns were generated on the porous silicon surface. These micropatterns were subsequently used to guide the growth of mammalian cells including neuroblastoma. Excellent selectivity of cell growth toward the laser-ablated regions was established.     

Development of a multilayered polymeric DNA biosensor using radio frequency technology with gold and magnetic nanoparticles

This study utilized the radio frequency (RF) technology to develop a multilayered polymeric DNA sensor with the help of gold and magnetic nanoparticles. The flexible polymeric materials, poly (p-xylylene) (Parylene) and polyethylene naphtholate (PEN), were used as substrates to replace the conventional rigid substrates such as glass and silicon wafers. The multilayered polymeric RF biosensor, including the two polymer layers and two copper transmission structure layers, was developed to reduce the total sensor size and further enhance the sensitivity of the biochip in the RF DNA detection. Thioglycolic acid (TGA) was used on the surface of the proposed biochip to form a thiolate-modified sensing surface for DNA hybridization. Gold nanoparticles (AuNPs) and magnetic nanoparticles (MNPs) were used to immobilize on the surface of the biosensor to enhance overall detection sensitivity. In addition to gold nanoparticles, the magnetic nanoparticles has been demonstrated the applicability for RF DNA detection. The performance of the proposed biosensor was evaluated by the shift of the center frequency of the RF biosensor because the electromagnetic characteristic of the biosensors can be altered by the immobilized multilayer nanoparticles on the biosensor. The experimental results show that the detection limit of the DNA concentration can reach as low as 10 pM, and the largest shift of the center frequency with triple-layer AuNPs and MNPs can approach 0.9 and 0.7 GHz, respectively. Such the achievement implies that the developed biosensor can offer an alternative inexpensive, disposable, and highly sensitive option for application in biomedicine diagnostic systems because the price and size of each biochip can be effectively reduced by using fully polymeric materials and multilayer-detecting structures.     

Superhydrophobic Properties of Nanostructured–Microstructured Porous Silicon for Improved Surface-Based Bioanalysis

Wettability is a fundamental property of a solid surface, which plays important roles in many industrial applications. The possibility to create well-controlled nonwetting states on silicon surfaces without photolithography-based processing can bring many advantages in the biotechnology and microfluidics areas. In this paper, superhydrophobic properties of macroporous–nanoporous structured silicon are reported. The superhydrophobic porous silicon layers are prepared by electrochemical etching of bulk crystalline silicon wafers. Altered anodization conditions provide surfaces with varying pore morphologies, yielding different wetting properties, ranging from highly wetting (nanoporous morphologies) to water-repellent surfaces (macroporous morphologies). Subsequent surface modification with a fluorocarbon coupling agent can further improve nonwetting properties and stabilize the surface for a long term. Contact angles as high as 176° were achieved on macroporous silicon and superhydrophobicity was maintained for several months without degradation. The porous surfaces have proven to be a very attractive substrate for protein microarrays. Fluorescence-based assay of immunoglobulin G in plasma is reported with a limit of detection of 1 pM, a spot size of 50 μm, and an array density of 15,625 spots per square centimeter. Macroporous surfaces have also been developed for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) applications, where the intrinsic hydrophobic surface properties confine the deposited sample to MALDI spots of less than 200 μm with well-defined MALDI crystals, providing a high-sensitivity readout. Furthermore, a superhydrophobic MALDI-TOF MS target anchor chip composed of nonporous anchor points surrounded by superhydrophobic porous areas for sample deposition and on anchor point confinement is reported. Such anchor chips allowed localized crystallization of large sample volumes (5 μL) improving the hydrophobic spot confinement strategy in terms of final MALDI crystal localization and readout sensitivity.     

Effects of Pillar Height and Junction Depth on the Performance of Radially Doped Silicon Pillar Arrays for Solar Energy Applications

The effects of pillar height and junction depth on solar cell characteristics are investigated to provide design rules for arrays of such pillars in solar energy applications. Radially doped silicon pillar arrays are fabricated by deep reactive ion etching of silicon substrates followed by the introduction of dopant atoms by diffusion from a phosphorus oxide layer conformally deposited by low‐pressure chemical vapor deposition. Increasing the height of the pillars has led to doubling of the efficiency from 6% for flat substrates to 12% for 40 μm high pillars with a 900 nm junction depth because of an increase in the total junction area and lower optical reflection. For higher pillars, the current density and efficiency is decreased, which is attributed to the increasing presence of defect states at the surface introduced during the etching process. This effect can be counteracted by an Al₂O₃ passivation layer on the pillar surface. An optimum efficiency of 13% is found for a junction depth of 790 nm for 40 μm pillar height. At increased junction depths, the efficiency is decreased due to the ever thinner undoped core of the pillars, causing pillars with a large junction depth to become less efficient than flat silicon substrates.     

Reversible and specific interaction of dehydrogenases with a coenzyme-coated surface continuously monitored with a reflectometer

Reversible affinity binding of NAD-dependent dehydrogenase to an NAD-coated silicon surface ("NAD biochip") has been accomplished. The silicon surface, which is favorable for use with optical techniques because of its excellent reflection properties, was precoated with a polymer to prevent nonspecific and irreversible adsorption. Using a new reflectometry technique based on measurement of the polarization change of light reflected upon the biochip, continuous monitoring of the affinity binding and subsequent desorption of alcohol dehydrogenase and lactate dehydrogenase from the NAD surface were possible; allowing repeated use of the same NAD chip--an advantage when the assay was carried out in a continuous reflectometer. With a flow rate of 0.5 ml/min, response times on the order of 30 s were obtained.     

An AFM investigation of oligonucleotides anchored on unoxidized crystalline silicon surfaces

Carboxylic terminated monolayers have been covalently attached on phosphorous doped crystalline (100) silicon surfaces using a cathodic electro grafting technique. The functionalization concentration and efficiency have been evaluated with different techniques. In particular, topographic images, performed with an atomic force microscope, were used to optimize the protocol in order to obtain a surface whose characteristics of uniformity and reproducibility are ideal for a bio-electronic device. Phase lag images of the functionalized surfaces were also performed, and show non-topographic structures that have been interpreted as areas of different molecule self-orientation. Poly-thymine oligonucleotides have been anchored on such a surface to form a nano-biosensing device capable to react selectively with a specific target molecule, a poly-adenine oligonucleotide. AFM images of high density (approximately 3x10(12) mol/cm2) single strand and double strand covered samples show toroidal shaped structures formed by the self-assembly of the oligonucleotides on the silicon surface.     

Towards a label-free optical porous silicon DNA sensor

We report on the fabrication of an optical silicon-based label-free DNA sensor. n-Type crystalline silicon wafers have been electrochemically etched to form porous silicon layers and characterized in terms of porosity, pore distribution, surface composition and photoluminescence. Samples (0.25 cm(2)) have been cut and properly derivatized using trimethoxy-3-bromoacetamidopropylsilane in order to link single strand DNA (ss-DNA). Such a molecule is not commercially available and has been ad-hoc prepared by reacting hydrobromic acid and 3-aminopropyltrimethoxysilane in presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as coupling agent. Trimethoxy-3-bromoacetamidopropylsilane acts as a bridge anchored to the porous silicon surface through the silane group while immobilizing ss-DNA by means of the bromoacetamido moiety. We have found that derivatized samples exhibit a photoluminescence that is stable in time and is not modified after exposure to non-complementary DNA strand. On the other hand, a sensible enhancement of the light emission has been observed when the derivatized samples react with the complementary strand, showing that the specific ss-DNA/complementary DNA (c-DNA) interaction can be optically sensed without using further labeling steps. This strongly strengthens the possible role of silicon as a material for biosensors.     

Selective metabolite and peptide capture/mass detection using fluorous affinity tags

A new and general methodology is described for the targeted enrichment and subsequent direct mass spectrometric characterization of sample subsets bearing various chemical functionalities from highly complex mixtures of biological origin. Specifically, sample components containing a chemical moiety of interest are first selectively labeled with perfluoroalkyl groups, and the entire sample is then applied to a perfluoroalkyl-silylated porous silicon (pSi) surface. Due to the unique hydrophobic and lipophobic nature of the perfluorinated tags, unlabeled sample components are readily removed using simple surface washes, and the enriched sample fraction can then directly be analyzed by desorption/ionization on silicon mass spectrometry (DIOS-MS). Importantly, this fluorous-based enrichment methodology provides a single platform that is equally applicable to both peptide as well as small molecule focused applications. The utility of this technique is demonstrated by the enrichment and mass spectrometric analysis of both various peptide subsets from protein digests as well as amino acids from serum.     

Silicon substrate as a novel cell culture device for myoblast cells

Background

Tissue and organ regeneration via transplantation of cell bodies in-situ has become an interesting strategy in regenerative medicine. Developments of cell carriers to systematically deliver cell bodies in the damage site have fall shorten on effectively meet this purpose due to inappropriate release control. Thus, there is still need of novel substrate to achieve targeted cell delivery with appropriate vehicles. In the present study, silicon based photovoltaic (PV) devices are used as a cell culturing substrate for the expansion of myoblast mouse cell (C2C12 cells) that offers an atmosphere for regular cell growth in vitro. The adherence, viability and proliferation of the cells on the silicon surface were examined by direct cell counting and fluorescence microscopy.

Results

It was found that on the silicon surface, cells proliferated over 7 days showing normal morphology, and expressed their biological activities. Cell culture on silicon substrate reveals their attachment and proliferation over the surface of the PV device. After first day of culture, cell viability was 88% and cell survival remained above 86% as compared to the seeding day after the seventh day. Furthermore, the DAPI staining revealed that the initially scattered cells were able to eventually build a cellular monolayer on top of the silicon substrate.

Conclusions

This study explored the biological applications of silicon based PV devices, demonstrating its biocompatibility properties and found useful for culture of cells on porous 2-D surface. The incorporation of silicon substrate has been efficaciously revealed as a potential cell carrier or vehicle in cell growth technology, allowing for their use in cell based gene therapy, tissue engineering, and therapeutic angiogenesis.     

Porous silicon-based biosensor for pathogen detection

A porous silicon-based biosensor for rapid detection of bacteria was fabricated. Silicon (0.01 ohmcm, p-type) was anodized electrochemically in an electrochemical Teflon cell containing ethanoic hydrofluoric acid solution to produce sponge-like porous layer of silicon. Anodizing conditions of 5 mA/cm2 for 85 min proved best for biosensor fabrication. A single-tube chemiluminescence-based assay, previously developed, was adapted to the biosensor for detection of Escherichia coli. Porous silicon chips were functionalized with a dioxetane-Polymyxin B (cell wall permeabilizer) mixture by diffusion and adsorption on to the porous surface. The reaction of beta-galactosidase enzyme from E. coli with the dioxetane substrate generated light at 530 nm. Light emission for the porous silicon biosensor chip with E. coli was significantly greater than that of the control and planar silicon chip with E. coli (P<0.01). Sensitivity of the porous silicon biosensor was determined to be 101-102 colony forming units (CFU) of E. coli. The porous silicon-based biosensor was fabricated and functionalized to successfully detect E. coli and has potential applications in food and environmental testing.     

Enhanced sensitivity detection of protein immobilization by fluorescent interference on oxidized silicon

We present a silicon chip-based approach for the enhanced sensitivity detection of surface-immobilized fluorescent molecules. Green fluorescent protein (GFP) is bound to the silicon substrate by a disuccinimidyl terephtalate-aminosilane immobilization procedure. The immobilized organic layers are characterized by surface analysis techniques, like ellipsometry, atomic force microscopy (AFM) and X-ray induced photoelectron spectroscopy. We obtain a 20-fold enhancement of the fluorescent signal, using constructive interference effects in a fused silica dielectric layer, deposited before immobilization onto the silicon. Our method opens perspectives to increase by an order of magnitude the fluorescent response of surface immobilized DNA- or protein-based layers for a variety of biosensor applications.