In vitro cell response to a polymer surface micropatterned by laser interference lithography

This presentation will introduce laser interference lithography to prepare a periodic line and point micropatterns for study of cell-surface interactions. This process provides a straightforward micropatterning technique based on selective laser ablation of polymers utilizing the periodic energy distribution of two or more beam interference patterns. The micropatterns were characterized by atomic force microscopy, while the surface chemical modification was analyzed using X-ray photoelectron spectroscopy. Human pulmonary fibroblasts cultured on the surface of polycarbonate bearing line micropatterns were elongated, spindlelike, and oriented themselves along the line patterns with all different groove widths. In contrast, cells cultured on point patterns were also bipolar but showed no orientation. Further investigations demonstrated that human pulmonary fibroblast cells cultured on line and point micropatterns showed inflammatory response.     

Preparation of photolithographically patterned inverse opal hydrogel microstructures and its application to protein patterning

Protein pattern has played an important role in biosensors, bioMEMS, tissue engineering, fundamental studies of cell biology, and basic proteomics research. Here, we developed a straightforward and effective protein patterning technique using macroporous poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel micropatterns as a three-dimensional (3D) template for protein immobilization. Micropatterns of macroporous hydrogels with inverse opal structures were prepared on poly(ethylene glycol) (PEG)-coated silicon substrates by combining a colloidal crystal templating method with photopatterning. The resultant inverse opal hydrogel (IOH) micropatterns were modified with 3-aminopropyltriethoxysilane using the hydroxyl groups in PHEMA for the covalent immobilization of proteins. Proteins were selectively immobilized only on the hydrogel micropatterns, while the PEG regions served as an effective barrier to protein adsorption. Because of their highly ordered and interconnected 3D macroporous structures and large internal surface areas, protein loading in the IOH micropattern was about six times greater than that on a non-porous hydrogel micropattern, which consequently improved the protein activity. The porosity of the hydrogel micropatterns could be controlled using different sizes of colloidal nanoparticles, and using smaller nanoparticles produced hydrogel micropatterns with higher protein loading capacities and activities. To demonstrate the potential use of IOH micropatterns in biosensor systems, biotin was micropatterned on the hydrogels and the specific binding of streptavidin was successfully assayed using IOH micropatterns with better fluorescence signals and sensitivity than that of the corresponding non-porous hydrogel micropatterns.     

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.     

Enhancement of analyte ionization in desoprtion/ionization on porous silicon (DIOS)-mass spectrometry (MS)

Desorption/ionization on silicon mass spectrometry (DIOS-MS) is a relatively new laser desorption/ionization technique for mass spectrometry without employing an organic matrix. This present study was carried to survey the experimental factors to improve the efficiency of DIOS-MS through electrochemical etching condition in structure and morphological properties of the porous silicon. The porous structure of silicon structure and its properties are crucial for the better performance of DIOS-MS and they can be controlled by the suitable selection of electrochemical conditions. The fabrication of porous silicon and ion signals on DIOS-MS were examined as a function of silicon orientation, etching time, etchant, current flux, irradiation, pore size, and pore depth. We have also examined the effect of pre- and post-etching conditions for their effect on DIOS-MS. Finally, we could optimize the electrochemical conditions for the efficient performance of DIOS-MS in the analysis of small molecule such as amino acid, drug and peptides without any unknown noise or fragmentation.     

Assessment of porous silicon substrate for well-characterised sensitive DNA chip implement

A biochip approach based on porous silicon as substrate is presented. The goal is to enhance the sensitivity of the biochip by increasing the specific surface area on the support. The elaboration of porous silicon layers has been optimized to guarantee good accessibility for large bio-molecule targets. Oligonucleotide probes are synthesised directly on the surface using phosphoramidite chemistry. The high specific surface area of porous silicon allows the direct characterisation, by infrared spectroscopy, of the porous layer formation and the functionalisation steps. The monolayer grafting and derivatisation protocol is additionally characterized by wettability and fluorescence microscopy. The surface modification of porous layers (i.e. thermal oxidation and chemical derivatisation) ensures the stability of the structure against strong chemical reagents used during the direct oligonucleotide synthesis. Finally the protocol is successfully transferred to a flat Si/SiO(2) substrate, and validated by biological target specific recognition during hybridisation tests. In particular, radioactive measurements show a 10-fold enhancement of the oligonucleotide surface density on the porous silicon substrate compared to the flat thermal silica.     

多孔硅Bragg反射镜适配子生物传感器检测心肌肌钙蛋白I

通过脉冲腐蚀法制备多孔硅Bragg反射镜,将心肌肌钙蛋白I（cTnI）适配子共价固定到多孔硅Bragg反射镜的孔洞中,发现适配子能与cTnI分子特异性结合。定量分析不同浓度的cTnI与适配子结合后多孔硅Bragg反射镜的反射谱峰位的红移情况。结果表明：基于多孔硅Bragg反射镜适配子生物传感器的光学检测具有良好的特异性,且具有免标记及检测时间短等优异性能。传感器的线性检测范围0．05-4nmol／L,最低检测限为0．05nmol／L。     

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.     

Porous silicon biosensor for detection of viruses

There is a growing need for virus sensors with improved sensitivity and dynamic range, for applications including disease diagnosis, pharmaceutical research, agriculture and homeland security. We report here a new method for improving the sensitivity for detection of the bacteriophage virus MS2 using thin films of nanoporous silicon. Porous silicon is an easily fabricated material that has extremely high surface area to volume ratio, making it an ideal platform for surface based sensors. We have developed and evaluated two different methods for covalent bioconjugation of antibodies inside of porous silicon films, and we show that the pore penetration and binding efficiency depend on the wettability of the porous surface. The resulting films were used to selectively capture dye-labeled MS2 viruses from solution, and a viral concentration as low as 2 x 10(7) plaque-forming units per mL (pfu/mL) was detectable by measuring the fluorescence from the exposed porous silicon film. The system exhibits sensitivity and dynamic range similar to the Luminex liquid array-based assay while outperforming protein micro-array methods.     

The Diatom Staurosirella pinnata for Photoactive Material Production

A native isolate of the colonial benthic diatom Staurosirella pinnata was cultivated for biosilica production. The silicified cell walls (frustules) were used as a source of homogeneous and structurally predictable porous biosilica for dye trapping and random laser applications. This was coupled with the extraction of lipids from biomass showing potential to fabricate photoactive composite materials sustainably. The strain was selected for its ease of growth in culture and harvesting. Biosilica and lipids were obtained at the end of growth in indoor photobioreactors. Frustules were structurally characterized microscopically and their chemistry analyzed with Fourier Transform Infrared Spectroscopy. Frustule capacity of binding laser dyes was evaluated on a set of frustules/Rhodamine B (Rho B) solutions and with respect to silicon dioxide and diatomite by Fluorescence Spectroscopy demonstrating a high affinity for the organic dye. The effect of dye trapping property in conveying Rho B emission to frustules, with enhancement of scattering events, was analyzed on Rho B doped polyacrylamide gels filled or not with frustules. Amplified spontaneous emission was recorded at increasing pump power indicating the onset of a random laser effect in frustule filled gels at lower power threshold compared to unfilled matrices.     

Porous silicon-based optical microsensor for the detection of L-glutamine

The molecular binding between the glutamine-binding protein (GlnBP) from Escherichia coli and L-glutamine (Gln) is optically transduced by means of a biosensor based on porous silicon nano-technology. The sensor operates by the measurement of the interferometric fringes in the reflectivity spectrum of a porous silicon Fabry-Perot layer. The binding event is revealed as a shift in wavelength of the fringes. Due to the hydrophobic interaction with the Si-H terminated surface of the porous silicon, the GlnBP protein, which acts as a molecular probe for Gln, penetrates and links into the pores of the porous silicon matrix. We can thus avoid any preliminary functionalization process of the porous layer surface, which is also prevented from oxidation, at least for few cycles of wet measurements. The binding of Gln to GlnBP has also been investigated at different concentration of GlnBP.     

Control of nanobiointerfaces generated from well-defined biomimetic polymer brushes for protein and cell manipulations

To better understand protein/material and cell/material interactions at the submolecular level, well-defined polymer brushes consisting of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) on silicon wafers were prepared by atom transfer radical polymerization (ATRP). Silicon wafers were treated with 3-(2-bromoisobutyryl)propyl dimethylchlorosilane (BDCS) to form a monolayer that acts as initiators for ATRP. Silicon-supported BDCS monolayers were soaked in a methanol/water mixture solution containing Cu(I)Br, bipyridine, and a sacrificial initiator. After MPC was added to the solution, ATRP was carried out for 18 h. The molecular weight and thickness of the PMPC brush layer on the silicon surface increased with an increase in the polymerization time. The dense polymer brushes were obtained by the "grafting from" system. By selective decomposition of the BDCS monolayer by UV light-irradiation, the PMPC brush region and the sizes were well controlled, resulting in fabricating micropatterns of the PMPC brushes. When the thickness of the PMPC brush layer was greater than 5.5 +/- 1.0 nm (3 h polymerization), serum protein adsorption and fibroblast adhesion were effectively reduced, i.e., proteins and cells could recognize such thin polymer brushes on the surface. In addition, the density of the adherent cells on the patterned PMPC brush surface could be controlled by changing the size of the pattern.     

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.     

Desorption/ionization on silicon for small molecules:a promising alternative to MALDI TOF

A method has been developed for laser desorption/ionization of catecholamines from porous silicon. This methodology is particularly attractive for analysis of small molecules. MALDI TOF mass spectrometry, although a very sensitive technique, utilizes matrices that need to be mixed with the sample prior to their analysis. Each matrix produces its own background, particularly in the low-molecular mass region. Therefore, detection and identification of molecules below 400 Da can be difficult. Desorption/ionization of samples deposited on porous silicon does not require addition of a matrix, thus, spectra in the low-molecular mass region can be clearly readable. Here, we describe a method for the analysis of catecholamines. While MALDI TOF is superior for proteomics/peptidomics, desorption/ionization from porous silicon can extend the operating range of a mass spectrometer for studies on metabolomics (small organic molecules and their metabolites, such as chemical neurotransmitters, prostaglandins, steroids, etc.).     

P450-based porous silicon biosensor for arachidonic acid detection

A porous silicon biosensor based on P450 enzyme for arachidonic acid detection was developed. A new transduction method is presented with a simultaneous measurement of refractive index and fluorescence intensity changes when the analyte is binding to an enzyme on the porous silicon surface. A fluorophore bound to a cysteine residue in an allosteric position of the haem domain (BMP) of cytochrome P450 BM3 enhances its fluorescence intensity upon interaction with its substrate arachidonic acid, involved in diseases such as Alzheimer's, liver cancer and cellular inflammation processes. BMP has been anchored on porous silicon surface and the new transduction method has been successfully exploited to develop a biosensor for arachidonic acid, reaching a detection limit of 10 μM arachidonic acid in a dynamic range of 10-200 μM. Moreover, the change of the refractive index has been also monitored at the same time, displaying a higher detection limit of 30 μM. Preliminary test were also conducted in plasma proving the high specificity and selectivity of the sensor even in presence of interferents in the range of 50-100 μM. Here we suggest these two detection systems could be used simultaneously to increase the accuracy and the dynamic range of the sensor avoiding a false positive response.     

Effect of biolinker on the detection of prostate specific antigen in an interferometry

Prostate-specific antigen (PSA) has been identified as a significant biomarker for prostate cancer screening. Heavily-doped porous silicon, etched to form a Fabry-Perot fringe pattern, can be applied to an interferometric sensing for detecting PSA bound with PSA-antibody. In the previous works, a calyx crown derivative (Prolinker-A) was used as an alternative biolinker on the porous silicon surface for interferometric biosensing of DNA-damaging chemical instead of employing the conventional biomolecular affinity method using biotin, which resulted in a denser linker formation. In this study, G5 amineterminated PAMAM (poly amidoamine) dendrimer as a biolinker, was applied to an interferometric sensing of PSA by using porous silicon, which shows the enhancement of adhesion capability and increase of functional groups more than those with Prolinker-A. Considerably low level down to 1 ng/mL of PSA could be detected by this sensing system.     

Porous silicon affinity chips for biomarker detection by MALDI-TOF-MS

B-type natriuretic peptide (BNP) is an important biomarker in early diagnosis of congestive heart failure. Many efforts have been made previously to evaluate the BNP level in human plasma. We developed a porous silicon (PSi) affinity chip to detect BNP present at low concentrations in human plasma by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) directly. The PSi surface immobilized with antibodies captured and concentrated BNP through antibody-antigen interaction specifically and sensitively. A detection limit as low as 10 pg/mL BNP in human plasma was demonstrated by mass analysis. This effective on-chip recognition, enrichment, and detection strategy could be employed in identification of biomarkers in complex body fluids in diagnoses.     

Estimation of ROS in the presence of biologically active substances by porous silicon fluorescence

The fluorescence spectra of the porous silicon modified by water solutions of biologically active materials and materials of biological origin are recorded as well as the fluorescence spectra of the porous silicon modified by lecithin monolayers grown on the surface of water solutions of the biologically active materials. The analysis of the obtained spectra made it possible to conclude on the effect of the studied materials on the content of ROS.     

Using continuous porous silicon gradients to study the influence of surface topography on the behaviour of neuroblastoma cells

The effects of surface topography on cell behaviour are the subject of intense research in cell biology. These effects have so far only been studied using substrate surfaces of discretely different topography. In this paper, we present a new approach to characterise cell growth on porous silicon gradients displaying pore sizes from several thousands to a few nanometers. This widely applicable format has the potential to significantly reduce sample numbers and hence analysis time and cost. Our gradient format was applied here to the culture of neuroblastoma cells in order to determine the effects of topography on cell growth parameters. Cell viability, morphology, length and area were characterised by fluorescence and scanning electron microscopy. We observed a dramatic influence of changes in surface topography on the density and morphology of adherent neuroblastoma cells. For example, pore size regimes where cell attachment is strongly discouraged were identified providing cues for the design of low-fouling surfaces. On pore size regimes more conducive to cell attachment, lateral cell-cell interactions crosslinked the cell layer to the substratum surface, while direct substrate-cell interactions were scarce. Finally, our study revealed that cells were sensitive to nanoscale surface topography with feature sizes of <20 nm.     

Optimizing of Gold Nanoparticles on Porous Silicon Morphologies for a Sensitive Carbon Monoxide Gas Sensor Device

A set of carbon monoxide (CO) gas sensors based on porous silicon (PSi)/gold nanoparticle (AuNP) hetro structures were fabricated. Different forms of PSi surface morphologies were studied as a substrate for growth of AuNPs. Simple dipping process of PSi in hydrogen tetrachloroaurate (III) solution (HAuCl₄) at fixed concentrations of 10⁻² M/3.5 HF was used to synthesize AuNPs. The n-type PSi was equipped through photo-electrochemical etching process at current density value of 10 mA/cm² under illumination condition of 530-nm wavelength and laser illumination intensity of 20 to 80 mW/cm². Three different forms of PSi morphology, meso, macro, and double layers with pore shapes and sizes, were prepared. The structural and surface morphology properties of PSi-based substrate before and after deposition of AuNPs were investigated through studying of scanning electron microscopy (SEM), photoluminescence (PL), and X-ray diffraction (XRD). The electrical property (J-V) was carried out in primary vacuum and CO at low pressure. The results show that PSi surface morphologies strongly influenced the AuNP sizes and hence the sensor performance. It was found that decrease the AuNP sizes could be occasioned in high and fast current response.

     

Micropatterns of cell adhesive proteins with poly(ethylene oxide)-block-Poly(4-vinylpyridine) diblock copolymer

Control of cell shape and behavior through the micropattern technique by spatial immobilization of adhesive proteins on a surface has provided novel insights in several aspects of cell biology, such as tissue morphogenesis, cell growth and cell differentiation, and apoptosis. In this work, we present the use of poly(ethylene oxide-block-poly(4-vinylpyridine) (PEO-b-P4VP) as a non-adhesive background to construct micropatterns of cell adhesive proteins. In the method presented, PEO-b-P4VP is used for its antifouling properties and at the same time, as a photosensitive material to define the micropatterns. The irradiation of PEO-b-P4VP with a short wavelength UV light through photolithographic mask, causes the polymer to crosslink and immobilize in the areas exposed. In the areas non-exposed the polymer can be removed. These areas can be subsequent back filled with the adhesive protein of interest to produce the final micropatterned cell chips.