An AFM investigation of oligonucleotides anchored on unoxidized crystalline silicon surfaces (original) (raw)
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Nucleic Acids Research, 2006
Unoxidized crystalline silicon, characterized by high purity, high homogeneity, sturdiness and an atomically flat surface, offers many advantages for the construction of electronic miniaturized biosensor arrays upon attachment of biomolecules (DNA, proteins or small organic compounds). This allows to study the incidence of molecular interactions through the simultaneous analysis, within a single experiment, of a number of samples containing small quantities of potential targets, in the presence of thousands of variables. A simple, accurate and robust methodology was established and is here presented, for the assembling of DNA sensors on the unoxidized, crystalline Si(100) surface, by loading controlled amounts of a monolayer DNA-probe through a two-step procedure. At first a monolayer of a spacer molecule, such as 10-undecynoic acid, was deposited, under optimized conditions, via controlled cathodic electrografting, then a synthetic DNA-probe was anchored to it, through amidation in aqueous solution. The surface coverage of several DNA-probes and the control of their efficiency in recognizing a complementary target-DNA upon hybridization were evaluated by fluorescence measurements. The whole process was also monitored in parallel by Atomic Force Microscopy (AFM).
Molecular monolayers on silicon as substrates for biosensors
Bioelectrochemistry, 2010
- silicon surfaces can be controlled down to atomic level and offer a remarkable starting point for elaborating nanostructures. Hydrogenated surfaces are obtained by oxide dissolution in hydrofluoric acid or ammonium fluoride solution. Organic species are grafted onto the hydrogenated surface by a hydrosilylation reaction, providing a robust covalent Si-C bonding. Finally, probe molecules can be anchored to the organic end group, paving the way to the elaboration of sensors. Fluorescence detection is hampered by the high refractive index of silicon. However, improved sensitivity is obtained by replacing the bulk silicon substrate by a thin layer of amorphous silicon deposited on a reflector. The development of a novel hybrid SPR interface by the deposition of an amorphous silicon-carbon alloy is also presented. Such an interface allows the subsequent linking of stable organic monolayers through Si-C bonds for a plasmonic detection. On the other hand, the semiconducting properties of silicon can be used to implement field-effect label-free detection. However, the electrostatic interaction between adsorbed species may lead to a spreading of the adsorption isotherms, which should not be overlooked in practical operating conditions of the sensor. Atomically flat silicon surfaces may allow for measuring recognition interactions with local-probe microscopy.
Covalent attachment of oligodeoxyribonucleotides to amine-modified Si (001) surfaces
Nucleic Acids Research, 2000
A recently described reaction for the UV-mediated attachment of alkenes to silicon surfaces is utilized as the basis for the preparation of functionalized silicon surfaces. UV light mediates the reaction of tbutyloxycarbonyl (t-BOC) protected ω-unsaturated aminoalkane (10-aminodec-1-ene) with hydrogenterminated silicon (001). Removal of the t-BOC protecting group yields an aminodecane-modified silicon surface. The resultant amino groups can be coupled to thiol-modified oligodeoxyribonucleotides using a heterobifunctional crosslinker, permitting the preparation of DNA arrays. Two methods for controlling the surface density of oligodeoxyribonucleotides were explored: in the first, binary mixtures of 10-aminodec-1-ene and dodecene were utilized in the initial UV-mediated coupling reaction; a linear relationship was found between the mole fraction of aminodecene and the density of DNA hybridization sites. In the second, only a portion of the t-BOC protecting groups was removed from the surface by limiting the time allowed for the deprotection reaction. The oligodeoxyribonucleotide-modified surfaces were extremely stable and performed well in DNA hybridization assays. These surfaces provide an alternative to gold or glass for surface immobilization of oligonucleotides in DNA arrays as well as a route for the coupling of nucleic acid biomolecular recognition elements to semiconductor materials.
Assessment of porous silicon substrate for well-characterised sensitive DNA chip implement
Biosensors & Bioelectronics
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 biomolecule 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.
Langmuir, 2001
A single-stranded oligonucleotide whose 5′ end is derivatized with a mercaptohexyl tether group was either directly anchored onto a gold surface or attached to a gold surface as part of a mixed self-assembled monolayer that contains mercaptohexanol. The application of these surface-confined DNA oligomers as heterogeneous probes for the detection of polynucleotides (e.g., M13 phage DNA) is considered, with an emphasis on the elucidation of the relationship between the hybridization efficiency and the surface coverage and orientation of the probe molecules. Atomic force microscopy (AFM) and flow-injection quartz crystal microbalance (FI-QCM) were used in tandem to study the immobilization of the probe, to estimate the extent and efficiency of the hybridization of M13 phage DNA (7249 bases), and to examine the effect of using a different alkanethiol to reorient the preformed film for a higher hybridization efficiency. The surface density and the resultant hybridization efficiency were found to be highly dependent on the morphology and surface structure of the gold substrate as well as on the concentration of the solution used for the probe fabrication but much less dependent on the probe immobilization time. The lower limit of the hybridization efficiency was estimated to be about 1.1% which is an underestimate because only the resolvable circular features were included in the estimation. Although the duplex formed at the gold surface covered with only the thiolated DNA probe adopts exclusively the orientation in which the target loop is parallel with respect to the substrate surface, the predominant duplex orientation at the gold substrate modified with mixed self-assembled monolayers is tethered to the surface with a small tilt angle versus the surface normal. Visualization of the duplex orientation allows one to understand whether the Sauerbrey equation is valid for the interpretation of certain FI-QCM results. Although it is probably valid to use the Sauerbrey equation to calculate the amount of a polynucleotide at a surface covered only by the thiolated DNA probe, the practice might be questionable for that at the surface with the DNA/alkanethiol mixed SAM on the basis of our AFM images of the target orientations.
A short route of covalent biofunctionaliztion of silicon surfaces
Sensors and Actuators B-chemical, 2011
Covalently attached organic monolayers on etched Si(1 1 1) surfaces were prepared by heating solutions of 1-alkenes and 1-alkynes in a refluxing mesitylene. Surface modification was monitored by measurement of the static water contact angle, X-ray photoelectron spectroscopy (XPS), infrared reflection absorption spectroscopy (IRRAS), and atomic force microscopy (AFM). Flat and clean N-hydroxysuccinimide (NHS)ester-terminated/1-decyl mixed monolayers were covalently attached in one step onto a silicon surface. This procedure allows a mild and rapid functionalization of the surface by substitution of the NHSester moieties with amines at room temperature. The NHS-ester groups were shown to be fully intact onto the surface. The surface reactivity of the NHS-ester moieties toward amines was qualitatively and quantitatively evaluated via the reaction with methoxytetraethyleneglycolamine (TEGamine) and finally functionalized with single strand and complete DNA molecules.
Synthesis and characterization of DNA-modified silicon (111) surfaces
Journal of The American Chemical Society, 2000
surfaces are modified by attachment of oligodeoxynucleotides and characterized with respect to DNA surface density, chemical stability, and DNA hybridization binding specificity. Surface functionalization employs the reaction of ω-unsaturated alkyl esters with the Si(111) surface using UV irradiation. Cleavage of the ester using potassium tert-butoxide yields a carboxyl-modified surface, which serves as a substrate for the attachment of DNA by means of an electrostatically adsorbed layer of polylysine and attachment of thiol-modified DNA using a heterobifunctional cross-linker. The resultant DNA-modified surfaces are shown to exhibit excellent specificity and chemical stability under the conditions of DNA hybridization. This work provides an avenue for the development of devices in which the exquisite binding specificity of biomolecular recognition is directly coupled to semiconductor devices.
Journal of Solid State Electrochemistry, 2004
We provide a comprehensive study of single-(ss) and double-strand (ds) oligonucleotides with either 25 or 10 bases or base pairs (bp) immobilized on polycrystalline and single-crystal Au(111) surfaces. The study is based on X-ray photoelectron spectroscopy, cyclic and differential pulse voltammetry, interfacial capacitance data, and electrochemical scanning tunnelling microscopy (in situ STM). The sequences used were the 25-bp sequence from the BRCA1 gene (25-mer), while the 10-bp oligonucleotides contained solely linear adenine and thymine sequences. The oligonucleotides were modified by the dimethoxytrityl group (DMT) via a disulfide group [DMT-S-S-ss25-mer and DMT-S-Sds(AT) 10 ], a pure disulfide group (A 10 -S-S-T 10 ), or a thiol group [HS-ss25-mer and HS-ds-(AT) 10 ], all via a hexamethylene linker. The overall pattern suggests strategies for controlled adsorption of DNA-based molecules and recognition of complementary strands or other molecules.
Silicon and Silicon-Related Surfaces for Biosensor Applications
Environmental Biosensors, 2011
Biosensing systems, such as enzyme, immunosensors, and DNA microarrays, are widely used in the field of medical care and medicine manufacturing [Spochiger-Kuller, 1998]. Recent developments in these devices require a high performance integrated micro-multibiosensing system, which can be employed for the recognition of an individual biomolecule and the analysis of bioreactions at the single molecular level. Constructing a highly sensitive biosensing system, precise fabrication of the electrode parts for molecular recognition is of significant importance. In these context, organic monolayers have self-assemble ability onto surfaces [Ulman, 1991]. Monolayer-modified electrode is suitable as the template for ordered immobilization of biomolecules. On the other hand, it is preferable that the detection system can detect the signal immediately, with a high sensitivity. Formation of covalently-bound organic monolayers has been particularly developed in the last two decades. The main benefit of organic monolayers is to add functionality to inorganic surface via the adaptable tailoring of surface properties. These monolayers keep the bulk features of the material (electrical, optical, magnetic, mechanical and structural), while their surface properties (wetting, passivation, bioresistance, biochemical affinity, etc…) can be tuned through a nanometer-sized grafting. This chapter provides substantial information on modification of silicon and silicon-related surfaces by organic monolayers to get the reader acquainted with the different techniques employed in tailoring the surface properties towards biosensing capability. 2. Silicon and silicon related surfaces 2.1 Silicon surfaces Silicon was discovered by Berzelius in 1824 and isolated as amorphous brown powder. Crystalline silicon was first prepared in 1854 as a grey material with metallic luster. Normally, silicon is prepared by reduction of silica, using different reducing agents. Silicon has a crystal structure similar to diamond, with Si-Si bond length of 2.3 Å [Cotton & Wilkinson, 1999]. Cleavage of a silicon crystal results in a large variety of surfaces. Several investigations on these surfaces have been carried out under ultra high vacuum (UHV) conditions [Hamers & Wang, 1996]. The surfaces are characterized by their Miller indices, which refer the plane thorough which the crystal was originally cleaved. www.intechopen.com Environmental Biosensors 172 2.2 Surface orientations of silicon The most common surface orientations of commercially available silicon are Si(100) and Si(111), See Fig. 1.