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.     

Submicron patterning of DNA oligonucleotides on silicon

The covalent attachment of DNA oligonucleotides onto crystalline silicon (100) surfaces, in patterns with submicron features, in a straightforward, two-step process is presented. UV light exposure of a hydrogen-terminated silicon (100) surface coated with alkenes functionalized with N-hydroxysuccinimide ester groups resulted in the covalent attachment of the alkene as a monolayer on the surface. Submicron-scale patterning of surfaces was achieved by illumination with an interference pattern obtained by the transmission of 248 nm excimer laser light through a phase mask. The N-hydroxysuccinimide ester surface acted as a template for the subsequent covalent attachment of aminohexyl-modified DNA oligonucleotides. Oligonucleotide patterns, with feature sizes of 500 nm, were reliably produced over large areas. The patterned surfaces were characterized with atomic force microscopy, scanning electron microscopy, epifluorescence microscopy and ellipsometry. Complementary oligonucleotides were hybridized to the surface-attached oligonucleotides with a density of 7 × 10¹² DNA oligonucleotides per square centimetre. The method will offer much potential for the creation of nano- and micro-scale DNA biosensor devices in silicon.     

DNA and protein microarray printing on silicon nitride waveguide surfaces

Sputtered silicon nitride optical waveguide surfaces were silanized and modified with a hetero-bifunctional crosslinker to facilitate thiol-reactive immobilization of contact-printed DNA probe oligonucleotides, streptavidin and murine anti-human interleukin-1 beta capture agents in microarray formats. X-ray photoelectron spectroscopy (XPS) was used to characterize each reaction sequence on the native silicon oxynitride surface. Thiol-terminated DNA probe oligonucleotides exhibited substantially higher surface printing immobilization and target hybridization efficiencies than non-thiolated DNA probe oligonucleotides: strong fluorescence signals from target DNA hybridization supported successful DNA oligonucleotide probe microarray fabrication and specific capture bioactivity. Analogously printed arrays of thiolated streptavidin and non-thiolated streptavidin did not exhibit noticeable differences in either surface immobilization or analyte capture assay signals. Non-thiolated anti-human interleukin-1 beta printed on modified silicon nitride surfaces reactive to thiol chemistry exhibited comparable performance for capturing human interleukin-1 beta analyte to commercial amine-reactive microarraying polymer surfaces in sandwich immunoassays, indicating substantial non-specific antibody-surface capture responsible for analyte capture signal.     

准噶尔小胸鳖甲抗冻蛋白基因Mpafp149的生物信息学分析

利用生物信息学方法对准噶尔小胸鳖甲抗冻蛋白基因Mpafp149所编码蛋白MPAFP149进行分析,分别从氨基酸组成、理化性质、二三级结构和功能、进化关系等方面进行预测。结果表明MPAFP149与其他昆虫抗冻蛋白在氨基酸水平上具有79％的相似性,二级结构上预测其为水溶性蛋白,含有信号肽和跨膜区。三级结构上与黄粉虫抗冻蛋白YL-1具有较高的相似性,并且具有昆虫抗冻蛋白所特有的结构域。本研究为准噶尔小胸鳖甲抗冻蛋白功能的研究鉴定了基础。     

黄粉虫不同抗冻蛋白基因家族成员的低温应激表达

黄粉虫Tenebrio molitor L.抗冻蛋白基因家族有多个成员,其氨基酸数量和蛋白结构存在差异。尽管有报道发现冷驯化后这些抗冻蛋白的表达量会升高,但不同家族成员是否存在功能分化尚不清楚。本研究中,检测了冷驯化对低温死亡率的效应和对不同类型的抗冻蛋白家族成员基因表达量的影响。结果表明,冷驯化可以显著降低黄粉虫幼虫的低温死亡率和提高不同类型抗冻蛋白基因的表达量。其中,长的抗冻蛋白和低温死亡率的相关关系最为明显。说明不同的抗冻蛋白家族成员的功能有明显的分化,为进一步理解抗冻蛋白的活性和利用抗冻蛋白提供了新的认识。     

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.     

Humidity dependence of charge transport through DNA revealed by silicon-based nanotweezers manipulation

The study of the electrical properties of DNA has aroused increasing interest since the last decade. So far, controversial arguments have been put forward to explain the electrical charge transport through DNA. Our experiments on DNA bundles manipulated with silicon-based actuated tweezers demonstrate undoubtedly that humidity is the main factor affecting the electrical conduction in DNA. We explain the quasi-Ohmic behavior of DNA and the exponential dependence of its conductivity with relative humidity from the adsorption of water on the DNA backbone. We propose a quantitative model that is consistent with previous studies on DNA and other materials, like porous silicon, subjected to different humidity conditions.     

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.     

A label-free optical sensor based on nanoporous gold arrays for the detection of oligodeoxynucleotides

Interest in using nanoporous materials for sensing applications has increased. The present study reports a method of preparing well-ordered nanoporous gold arrays using a porous silicon (PSi) template. Gold nanolayer could be electrodeposited on the surface of the PSi template at low electrolysis currents in low concentration of chloroauric acid (HAuCl₄) solution. Surface morphology characterizations and optical measurements revealed that a PSi-templated nanoporous gold (Au–PSi) array well replicated the nanoporous structure and retained the optical properties of PSi. Fourier transform reflectometric interference spectra showed that a characteristic blue-shifted effective optical thickness (EOT) was observed due to the low refractive index of the gold film. An optical DNA biosensor was then fabricated via the self-assembly of single-stranded DNA (ssDNA) with a specific sequence on the surface of Au–PSi. The attachment of ssDNA and its hybridization with target oligonucleotides (ODNs) persistently caused the blue shift of the EOT. Consequently, a relationship between the EOT shift and the ODN concentration was established. The mechanism of the optical response caused by DNA hybridization on the Au–PSi surface was qualitatively explained by the electromagnetic theory and electrochemical impedance spectroscopy (EIS). The lowest detection limit for target ODNs was estimated at around 10⁻¹⁴ mol L⁻¹, when the baseline noise, a variation in the value of EOT is around 5 nm. The fabricated Au–PSi based optical biosensor has potential use in the discovery of new ODN drugs because it will be able to detect the binding event between ODNs and the target DNA.     

Inhibition of recrystallization in ice by chimeric proteins containing antifreeze domains

Using synthetic DNA, we assembled a gene encoding a protein identical in sequence to one of the antifreeze proteins produced by the fish Pseudopleuronectes americanus (winter flounder). To address the relationship between structure and function, we also assembled genes encoding proteins varying in sequence and length. The synthetic genes were cloned into a bacterial expression vector to generate translational fusions to the 3' end of a truncated staphylococcal protein A gene; the chimeric proteins encoded by these fusions, varying only in their antifreeze domains, were isolated from Escherichia coli. The antifreeze domains conferred the ability to inhibit ice recrystallization, which is characteristic of naturally occurring antifreeze proteins, on the chimeric proteins. The chimeric proteins varied in their effectiveness of inhibiting ice recrystallization according to the number of 11-amino acid repeats present in the antifreeze moiety. A protein with only two repeats lacked activity, while the inhibitory activity increased progressively for proteins containing three, four, and five repeats. Some activity was lost upon removal of either the salt bridge or the carboxyl-terminal arginine, but surprisingly, not when both features were absent together.     

Improved procedures for immobilisation of oligonucleotides on gold-coated piezoelectric quartz crystals

The high sensitivity and specificity of DNA hybridisation techniques makes them powerful tools for environmental or clinical analysis. This work describes the development of a DNA piezoelectric biosensor for the detection of the hybridisation reaction. Attention was focused on the choice of the coating chemistry that could be used for the immobilisation of oligonucleotides onto the gold surface of the quartz crystal. Four immobilisation procedures were tested and compared considering the amount of immobilised probe, the extent of the hybridisation reaction, the possibility of regeneration and the absence of non-specific adsorption. All the experiments were performed with oligonucleotides of 25 bases (probe, target and non-complementary oligonucleotide). The four coating methods were all based on the use of self-assembled monolayers (SAM). Three of them employed the interaction between streptavidin and biotin for the immobilisation of a biotinylated probe. Results indicated that immobilisation of a biotinylated probe on streptavidin linked to a layer of carboxylated dextran provides higher sensitivity for the detection of the hybridisation reaction, absence of non-specific adsorption and a higher stability with respect to the regeneration step.     

A novel optical biosensor format for the detection of clinically relevant TP53 mutations

The TP53 gene has been the subject of intense research since the realisation that inactivation of this gene is common to most cancer types. Numerous publications have linked TP53 mutations in general or at specific locations to patient prognosis and therapy response. The findings of many studies using general approaches such as immunohistochemistry or sequencing are contradictory. However, the detection of specific mutations, especially those occurring in the structurally important L2 and L3 zinc binding domains, which are the most common sites of TP53 mutations, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. In this study, the TI-SPR-1 surface plasmon resonance system and Texas Instruments Spreeta chips were used to develop a DNA biosensor based on thiolated probes complementary to these domains. The sensors were able to detect these mutations in both oligonucleotides and PCR products with normal and mutant TP53 DNA, but the difference in hybridisation signal was small. Preliminary experiments to enhance the signal using Escherichia coli mismatch repair proteins, MutS and single strand binding protein were carried out. It was found that MutS was unable to bind to mismatch oligonucleotides, but single strand binding protein was able to bind to single stranded probes, which had not hybridised to the target, resulting in a three-fold increase in the sensitivity of the biosensor. While further work needs to be carried out to optimise the system, these preliminary experiments indicate that the TI-SPR-1 can be used for the detection of clinically relevant mutations in the TP53 gene and that the sensitivity can be increased significantly using single strand binding protein. This system has a number of advantages over current mutation detection technologies, including lower cost, ease of sensor preparation and measurement procedures, technical simplicity and increased speed due to the lack of need for gel electrophoresis.     

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.     

Interactions between single-stranded DNA binding protein and oligonucleotide analogs with different backbone chemistries

Chemical modification of backbone structures has been an important strategy in designing oligonucleotides capable of improved antisense effects. However, altered backbone chemistry may also affect the binding of oligonucleotides to key cellular proteins, and thus may impact on the overall biological action of antisense agents. In this study we have examined the binding of oligonucleotides having four different backbone chemistries to single-strand binding protein (SSB), a protein having a key role in DNA repair and replication. The oligomers tested had the same sequence, while the internucleoside linkages were phosphodiester (PO), phosphorothioate (PS), phosphorodithioate (PS2), or methylphosphonate (MP). We found that both PS and PS2 oligomers bound to SSB with higher affinity than PO oligonucleotides, while MP oligonucleotides did not bind appreciably at the concentrations tested. Oligonucleotide length was also an important factor in binding to SSB, but sequence was less critical. These observations indicate that backbone chemistry is an important factor in interactions between oligonucleotides and critical cellular proteins, and thus may be a key determinant of the biological effects of antisense oligonucleotides. © 1997 John Wiley & Sons, Ltd.     

Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR

Site-directed spin labeling and pulsed electron–electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.     

Biosensor detection of triplex formation by modified oligonucleotides

Due to the instability of DNA oligonucleotides in biological solutions, antisense or antigene therapies aimed at modulation of specific gene expression will most likely require the use of oligonucleotides with modified backbones. Here, we examine the use of a surface plasmon resonance biosensor (BIAcore) to compare triplex-directed binding of modified oligonucleotides targeted to a region of the murine c-myc promoter. We describe optimization of experimental conditions to minimize nonspecific interactions between the oligonucleotides and the sensor chip surface, and the limitations imposed by certain backbones and sequence types. The abilities of pyrimidine oligonucleotides with various modified backbones to form specific triple helices with an immobilized hairpin duplex were readily determined using the biosensor. Modification of the third-strand oligonucleotide with RNA or 2(')-O-methyl RNA was found to enhance triplex formation, whereas phosphorothioate or phosphotriester substitutions abrogated it. A comparison of these results to DNase I footprinting experiments using the same oligonucleotides showed complete agreement between the two sets of data.     

Towards a biosensor based on anti resonant reflecting optical waveguide fabricated from porous silicon

Recently, we demonstrated that Anti Resonant Reflecting Optical Waveguide (ARROW) based on porous silicon (PS) material can be used as a transducer for the development of a new optical biosensor. Compared to a conventional biosensor waveguide based on evanescent waves, the ARROW structure is designed to allow a better overlap between the propagated optical field and the molecules infiltrated in the porous core layer and so to provide better molecular interactions sensitivity. The aim of this work is to investigate the operating mode of an optical biosensor using the ARROW structure. We reported here an extensive study where the antiresonance conditions were adjusted just before the grafting of the studied molecules for a given refractive index range. The interesting feature of the studied ARROW structure is that it is elaborated from the same material which is the porous silicon obtained via a single electrochemical anodization process. After oxidation and preparation of the inner surface of porous silicon by a chemical functionalization process, bovine serum albumin (BSA) molecules, were attached essentially in the upper layer. Simulation study indicates that the proposed sensor works at the refractive index values ranging from 1.3560 to 1.3655. The experimental optical detection of the biomolecules was obtained through the modification of the propagated optical field and losses. The results indicated that the optical attenuation decreases after biomolecules attachment, corresponding to a refractive index change Δn(c) of the core. This reduction was of about 2 dB/cm and 3 dB/cm for Transverse Electric (TE) and Transverse Magnetic (TM) polarizations respectively. Moreover, at the detection step, the optical field was almost located inside the core layer. This result was in good agreement with the simulated near field profiles.     

Nanoscale porous silicon waveguide for label-free DNA sensing

Porous silicon (PSi) is an excellent material for biosensing due to its large surface area and its capability for molecular size selectivity. In this work, we report the experimental demonstration of a label-free nanoscale PSi resonant waveguide biosensor. The PSi waveguide consists of pores with an average diameter of 20nm. DNA is attached inside the pores using standard amino-silane and glutaraldehyde chemistry. Molecular binding in the PSi is detected optically based on a shift of the waveguide resonance angle. The magnitude of the resonance shift is directly related to the quantity of biomolecules attached to the pore walls. The PSi waveguide sensor can selectively discriminate between complementary and non-complementary DNA. The advantages of the PSi waveguide biosensor include strong field confinement and a sharp resonance feature, which allow for high sensitivity measurements with a low detection limit. Simulations indicate that the sensor has a detection limit of 50nM DNA concentration or equivalently, 5pg/mm2.     

Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces americanus

The ocean pout (Macrozoarces americanus) produces a set of antifreeze proteins that depresses the freezing point of its blood by binding to, and inhibiting the growth of, ice crystals. The amino acid sequences of all the major components of the ocean pout antifreeze proteins, including the immunologically distinct QAE component, have been derived by Edman degradation. In addition, sequences of several minor components were deduced from DNA sequencing of cDNA and genomic clones. Fifty percent of the amino acids are perfectly conserved in all these proteins as well as in two homologous sequences from the distantly related wolffish. Several of the conserved residues are threonines and asparagines, amino acids that have been implicated in ice binding in the structurally unrelated antifreeze protein of the righteye flounders. Aside from minor differences in post-translational modifications, heterogeneity in antifreeze protein components stems from amino acid differences encoded by multiple genes. Based on genomic Southern blots and library cloning statistics there are 150 copies of the 0.7-kilobase-long antifreeze protein gene in the Newfoundland ocean pout, the majority of which are closely linked but irregularly spaced. A more southerly population of ocean pout from New Brunswick in which the circulating antifreeze protein levels are considerably lower has approximately one-quater as many antifreeze protein genes. Thus, there appears to be a correlation between gene dosage and antifreeze protein levels, and hence the ability to survive in ice-laden seawater. Southern blot comparison of the two populations indicates that the differences in gene dosage were not generated by a simple set of deletions/duplications. They are more likely to be the result of differential amplification.     

Detection of clinically relevant point mutations by a novel piezoelectric biosensor

Piezoelectric sensing is here applied to point mutation detection in human DNA. The mutation investigated is in the TP53 gene, which results inactivated in most cancer types. TP53 gene maps on chromosome 17 (17p13.1). It contains 11 exons and codifies for the relative protein, involved in cell proliferation. The TP53 gene has a wide mutation spectrum that is related to different tumours. In particular, those occurring in the structurally important L2 and L3 zinc-binding domains, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. For this reason, the identification of these mutations represents an important clinical target and biosensors could represent good candidate for fast mutation screening. In this paper, a DNA-based piezoelectric biosensor for the detection of the TP53 gene mutation at codon 248 is reported. A biotinylated probe was immobilised on the sensor surface via dextran-streptavidin modified surfaces. The sensor was optimised using synthetic oligonucleotides. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples of DNA extracted from two cell lines, one normal (wild-type) and one mutated, carrying the mutation at codon 248 of the TP53 gene. The results obtained demonstrate that the DNA-based piezoelectric biosensor is able to detect the point mutations in PCR-amplified samples showing the potentialities of this approach for routine analysis.