Characterization of peptide adsorption on InAs using X-ray photoelectron spectroscopy

The well-defined structure and high stability of peptides make them attractive biotemplates for low-temperature synthesis of semiconductor nanocrystals. Adsorbed peptide monolayers could also potentially passivate semiconductors by preventing regrowth of the oxide layer. In this work, the adsorption and passivation capabilities of different collagen-binding peptides on InAs surfaces were analyzed by X-ray photoelectron spectroscopy (XPS). Before peptide functionalization, Br(2)- and HCl-based etches were used to remove the native oxide layer on the InAs surfaces. The presence of the N 1s peak for peptide-functionalized samples confirms the adsorption of peptides onto the etched InAs surfaces. Calculated coverages were similar for all peptide sequences and ranged from ～20 to 40% of a monolayer using the deconvoluted C 1s spectra and from ～2 to 5% for the N 1s spectra. The passivation ability of the peptides was analyzed by comparing the ratios of the oxide components to the nonoxide components in the XPS spectra. The thickness of the oxide layer was also approximated by accounting for the attenuation of the substrate photoelectrons through the oxide layer. We find that the oxide layer regrowth still occurs after peptide functionalization. However, the oxide layer thicknesses for peptide-functionalized samples do not reach as received levels, indicating that the peptides do have some passivation ability on InAs.     

Cysteamine-based functionalization of InAs surfaces: revealing the critical role of oxide interactions in biasing attachment

Attaching functional molecules such as thiols and proteins to semiconductor surfaces is increasingly exploited in functional devices such as sensors. Despite extensive research to understand this interface and demonstrate a robust protocol for attachment, the bonding chemistry of thiolates to III-V surfaces has been under great debate in the literature. This study provides a comprehensive chemical model for the attachment of thiols to InAs, an increasingly device-relevant III-V semiconductor, using cysteamine as a model molecule. We examine the attachment of cysteamine to InAs via the thiol group using X-ray photoelectron spectroscopy and spectroscopic ellipsometry and confirm that thiolate bonding to the substrate occurs preferentially to As sites over In sites as a limit. These experiments explore the interplay of the native oxide chemical properties, the cysteamine concentration, and the evolving InAs surface chemistry with functionalization. The thiol-InAs interaction can be framed as a general acid-base reaction, where the nucleophilic and/or electrophilic attack of the surface (i.e., binding to In sites and/or As sites) depends on the acidity of the thiol. The roles of the initial oxide composition, the solvent of the functionalizing solution, and the cysteamine as a limiting reagent in fully displacing the oxide and creating In-S and As-S bonds are highlighted.     

Quantitative characterization of DNA films by X-ray photoelectron spectroscopy

We describe the use of self-assembled films of thiolated (dT)25 single-stranded DNA (ssDNA) on gold as a model system for quantitative characterization of DNA films by X-ray photoelectron spectroscopy (XPS). We evaluate the applicability of a uniform and homogeneous overlayer-substrate model for data analysis, examine model parameters used to describe DNA films (e.g., density and electron attenuation length), and validate the results. The model is used to obtain quantitative composition and coverage information as a function of immobilization time. We find that when the electron attenuation effects are properly included in the XPS data analysis, excellent agreement is obtained with Fourier transform infrared (FTIR) measurements for relative values of the DNA coverage, and the calculated absolute coverage is consistent with a previous radiolabeling study. Based on the effectiveness of the analysis procedure for model (dT)25 ssDNA films, it should be generally valid for direct quantitative comparison of DNA films prepared under widely varying conditions.     

Facile Electrochemical Hydrogenation and Chlorination of Glassy Carbon to Produce Highly Reactive and Uniform Surfaces for Stable Anchoring of Thiolated Molecules

Carbon is a highly adaptable family of materials and is one of the most chemically stable materials known, providing a remarkable platform for the development of tunable molecular interfaces. Herein, we report a two‐step process for the electrochemical hydrogenation of glassy carbon followed by either chemical or electrochemical chlorination to provide a highly reactive surface for further functionalization. The carbon surface at each stage of the process is characterized by AFM, SEM, Raman, attenuated total reflectance (ATR) FTIR, X‐ray photoelectron spectroscopy (XPS), and electroanalytical techniques. Electrochemical chlorination of hydrogen‐terminated surfaces is achieved in just 5 min at room temperature with hydrochloric acid, and chemical chlorination is performed with phosphorus pentachloride at 50 °C over a three‐hour period. A more controlled and uniform surface is obtained using the electrochemical approach, as chemical chlorination is observed to damage the glassy carbon surface. A ferrocene‐labeled alkylthiol is used as a model system to demonstrate the genericity and potential application of the highly reactive chlorinated surface formed, and the methodology is optimized. This process is then applied to thiolated DNA, and the functionality of the immobilized DNA probe is demonstrated. XPS reveals the covalent bond formed to be a C?S bond. The thermal stability of the thiolated molecules anchored on the glassy carbon is evaluated, and is found to be far superior to that on gold surfaces. This is the first report on the electrochemical hydrogenation and electrochemical chlorination of a glassy carbon surface, and this facile process can be applied to the highly stable functionalization of carbon surfaces with a plethora of diverse molecules, finding widespread applications.     

Optimization of DNA hybridization efficiency by pH-driven nanomechanical bending

The accessibility and binding affinity of DNA are two key parameters affecting the hybridization efficiency in surface-based biosensor technologies. Better accessibility will result in a higher hybridization efficiency. Often, mixed ssDNA and mercaptohexanol monolayers are used to increase the hybridization efficiency and accessibility of surface-bound oligonucleotides to complementary target DNA. Here, no mercaptohexanol monolayer was used. We demonstrate by differential microcantilever deflection measurements at different pH that the hybridization efficiency peaks between pH 7.5 and 8.5. At low pH 4.5, hydration and electrostatic forces led to tensile surface stress, implying the reduced accessibility of the bound ssDNA probe for hybridization. In contrast, at high pH 8.5, the steric interaction between neighboring ssDNA strands was decreased by higher electrostatic repulsive forces, bending the microcantilever away from the gold surface to provide more space for the target DNA. Cantilever deflection scales with pH-dependent surface hybridization efficiency because of high target DNA accessibility. Hence, by changing the pH, the hybridization efficiency is adjusted.     

X-ray induced damage in DNA monitored by X-ray photoelectron spectroscopy

In this work, the chemical changes in calf thymus DNA samples were analyzed by X-ray photoelectron spectroscopy (XPS). The DNA samples were irradiated for over 5 h and spectra were taken repeatedly every 30 min. In this approach the X-ray beam both damages and probes the samples. In most cases, XPS spectra have complex shapes due to contributions of C, N, and O atoms bonded at several different sites. We show that from a comparative analysis of the modification in XPS line shapes of the C 1s, O 1s, N 1s, and P 2p peaks, one can gain insight into a number of reaction pathways leading to radiation damage to DNA.     

Quantitative analysis and characterization of DNA immobilized on gold

We describe the complementary use of X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy to quantitatively characterize the immobilization of thiolated (dT)(25) single-stranded DNA (ssDNA) on gold. When electron attenuation effects are accurately accounted for in the XPS analysis, the relative coverage values obtained by the two methods are in excellent agreement, and the absolute coverage can be calculated on the basis of the XPS data. The evolution of chemically specific spectral signatures during immobilization indicates that at lower coverages much of the DNA lies flat on the surface, with a substantial fraction of the thymine bases chemisorbed. At higher immobilization densities, the (dT)(25) film consists of randomly coiled ssDNA molecules each anchored via the thiol group and at possibly one or two other bases. We use two examples to demonstrate how the quantitative analysis can be applied to practical problems: the effects of different buffer salts on the immobilization efficiency; the immobilization kinetics. Buffers with divalent salts dramatically increase the efficiency of immobilization and result in very high surface densities (>5 x 10(13)/ cm(2)), densities that may only be possible if the divalent counterions induce strong attractive intermolecular interactions. In contrast with previous reports of alkanethiol adsorption kinetics on gold, ssDNA immobilization in 1 M phosphate buffer does not occur with Langmuir kinetics, a result attributable to rearrangement within the film that follows the initial adsorption.     

Comparison between X-ray photoelectron and X-ray absorption spectra of an environmental aerosol sample measured by synchrotron radiation

Sulfur X-ray photoelectron spectra (XPS) and X-ray absorption spectra (XAS) of an aerosol sample collected by an Andersen sampler were measured using a synchrotron beam line. While the XPS was more surface sensitive than XAS, the detection limit of XAS was better than that of XPS. It was concluded that the XAS was more suitable for the chemical state analysis of sulfur in aerosol samples than XPS.     

Hybridization in ssDNA films--a multi-technique spectroscopy study

A combination of X-ray photoelectron spectroscopy (XPS), high-resolution XPS, near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and sum-frequency-generation (SFG) spectroscopy was used to monitor two types of ssDNA films on Au(111) before and after hybridization. As probe systems, films of thiolated and block-oligonucleotides were used, taking thiolated thymine d(T) homo-oligonucleotides and thymine-adenine d(A-T) diblock-oligonucleotides as representative examples. In accordance with previous work, hybridization of the shorter and more densely packed thiolated ssDNA films produced fewer (if any) hybrids, whereas the longer and less densely packed layers exhibited a larger hybridization yield. The above effects were less pronounced in the case of the d(A-T) films where the hybridization yield of the less densely packed monolayers was significantly lower. This was presumably due to the formation of internal dimeric hybrids in the immobilization step of the probe molecules, resulting in the generation of fewer probe-target hybrids upon exposure to the target molecules. In all ssDNA films displaying a reasonable number of hybrids present, significant orientational changes were observed and could be monitored in detail. These results suggest that the given combination of spectroscopic techniques can be a valuable tool to gain molecular-level information about hybrids at interfaces.     

单链脱氧核糖核酸在石墨电极表面固定化的研究

用５％（Ｖ／Ｖ）３－氨基丙基三乙氧基硅烷（ＰｒＮＨ２硅烷Ⅱ）在石墨电极表面硅烷化以导入氨基（－ＮＨ２）,然后用乙基－（３－二甲基丙基）碳二亚胺盐要卤）ＥＤＣ）关活化剂,将单链ＤＮＡ（共价固定在石墨电极表面。采用显微分光光度法、红外光谱法和电化学方法对电极表面的ｓｓＤＮＡ层进行了表征,并用紫外－可见光谱法对电极表面固定化ｓｓＤＮＡ的杂交特性进行了研究。结果表明,ｓｓＤＮＡ可以比较均匀地固定在石墨电极     

Hybridization of oligonucleotide by using DNA self-assembled monolayer

The interaction between DNA immobilized on surface and oligonucleotides at the interface is important in detection and diagnostic processes. However, it is difficult to immobilize DNA with maintaining its activity and to realize an efficient hybridization in previous methods. Here, to establish a novel DNA-functionalized surface, the DNA self-assembled monolayer (SAM) was constructed on a gold substrate using thiolated DNA composed of double-stranded (ds) and single-stranded (ss) portion. The DNA SAM was characterized by surface plasmon resonance (SPR), XPS. The hybridization of ss portion of DNA was attempted using the SAM, and in situ monitored by SPR. XPS measurement indicated that the thiolated DNA could form a stable monolayer on a gold substrate through sulfur–gold interaction. SPR measurement implied that the long axis of the DNA standing on the substrate. These results indicated formation of the DNA SAM on the substrate. Hybridization of target DNA containing a complementary sequence for the probe portion was observed by SPR. Moreover, one mismatch of oligonucleotide could be distinguished using the DNA SAM. The SPR result indicates that hybridization of target DNA and probe DNA on the DNA SAM occurs on the DNA SAM.     

A non-oxidative approach toward chemically and electrochemically functionalizing Si(111)

A general method for the non-oxidative functionalization of single-crystal silicon(111) surfaces is described. The silicon surface is fully acetylenylated using two-step chlorination/alkylation chemistry. A benzoquinone-masked primary amine is attached to this surface via Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition ("click" chemistry). The benzoquinone is electrochemically reduced, resulting in quantitative cleavage of the molecule and exposing the amine terminus. Molecules presenting a carboxylic acid have been immobilized to the exposed amine sites. X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), cyclic voltammetry (CV), and contact angle goniometry were utilized to characterize and quantitate each step in the functionalization process. This work represents a strategy for providing a general platform that can incorporate organic and biological molecules on Si(111) with minimal oxidation of the silicon surface.     

Fluorescence, XPS, and TOF-SIMS surface chemical state image analysis of DNA microarrays

Performance improvements in DNA-modified surfaces required for microarray and biosensor applications rely on improved capabilities to accurately characterize the chemistry and structure of immobilized DNA molecules on micropatterned surfaces. Recent innovations in imaging X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) now permit more detailed studies of micropatterned surfaces. We have exploited the complementary information provided by imaging XPS and imaging TOF-SIMS to detail the chemical composition, spatial distribution, and hybridization efficiency of amine-terminated single-stranded DNA (ssDNA) bound to commercial polyacrylamide-based, amine-reactive microarray slides, immobilized in both macrospot and microarray diagnostic formats. Combinations of XPS imaging and small spot analysis were used to identify micropatterned DNA spots within printed DNA arrays on slide surfaces and quantify DNA elements within individual microarray spots for determination of probe immobilization and hybridization efficiencies. This represents the first report of imaging XPS of DNA immobilization and hybridization efficiencies for arrays fabricated on commercial microarray slides. Imaging TOF-SIMS provided distinct analytical data on the lateral distribution of DNA within single array microspots before and after target hybridization. Principal component analysis (PCA) applied to TOF-SIMS imaging datasets demonstrated that the combination of these two techniques provides information not readily observable in TOF-SIMS images alone, particularly in identifying species associated with array spot nonuniformities (e.g., "halo" or "donut" effects often observed in fluorescence images). Chemically specific spot images were compared to conventional fluorescence scanned images in microarrays to provide new information on spot-to-spot DNA variations that affect current diagnostic reliability, assay variance, and sensitivity.     

Surface functionalization and characterization of magnetic polystyrene microbeads

A new approach to the surface functionalization of magnetic polystyrene microbeads with chloroacetyl chloride in the presence of aluminum chloride was reported. Composite microbeads consisting of polymer-coated iron oxide nanoparticles were prepared by spraying suspension polymerization. Functional chloride groups were introduced onto the surface of magnetic polystyrene microbeads by surface chemical reaction without destroying the magnetite nanoparticles within the microbeads. First, a complex was synthesized by a reaction between aluminum chloride and chloroacetyl chloride. Then, the complex was added dropwise to the solution of magnetic polystyrene microbeads, and a surface acylation reaction between complex and polystyrene microbeads was carried out. Subsequently, the amino groups were coupled to the magnetic microbeads via an ammonolysis reaction between ethylenediamine and chloride groups on the acylated magnetic polystyrene microbeads. The chemical composition, surface functional groups, and magnetism of the magnetic polystyrene microbeads before and after surface functionalization were characterized by Fourier transform infrared spectroscopy and vibrating sample magnetometry. The results showed that the surface functionalization reaction had little impact on the magnetism of the microbeads. The content of surface amino groups on the magnetic polystyrene microbeads was found to be 0.2 mmol/g. An affinity dye, Cibacron Blue F3G-A (CB), was then immobilized to prepare a magnetic affinity adsorbent. It was confirmed from X-ray photoelectron spectroscopy spectra that the CB molecules were covalently coupled on the magnetic microbeads.     

Chemical stability of (NH4)2S-passivated InP(001) surfaces – investigations by XPS and XPD

UV/ozone supported surface oxidation of wet chemically cleaned and sulfurized InP(001) was investigated using XPS in order to study the chemical stability of (NH₄)₂S-passivated surfaces. Sulfur coverages of about one monolayer thickness were not sufficient to completely passivate the InP surface against oxidation. Similar oxides of the substrate components were observed at the surfaces. Evidence for surface passivation was found in the chemical stability of incorporated sulfur (In-S bonds), the lower growth rate of the oxide layer and its reduced thickness at comparably large UV/ozone exposures. The oxide layer was found to be amorphous at all stages of the oxidation process, as was proved by X-ray photoelectron diffraction.     

Self-assembled carbon nanotubes on gold: polarization-modulated infrared reflection-absorption spectroscopy, high-resolution X-ray photoemission spectroscopy, and near-edge X-ray absorption fine structure spectroscopy study

Recently we reported noncovalent functionalization of nanotubes in an aqueous medium with ionic liquid-based surfactants, 1-dodecyl-3-methylimidazolium bromide (1) and 1-(12-mercaptododecyl)-3-methylimidazolium bromide (2), resulting in positively charged single-wall carbon nanotube (SWNT)-1,2 composites. Thiolation of SWNTs with 2 provides their self-assembly on gold as well as templating gold nanoparticles on SWNT sidewalls via a covalent -S-Au bond. In this investigation, we studied the electronic structure, intermolecular interactions, and packing within noncovalently thiolated SWNTs and also nanotube alignment in the bulk of SWNT-2 dried droplets and self-assembled submonolayers (SAMs) on gold by high-resolution X-ray photoemission spectroscopy (HRXPS), C K-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). HRXPS data confirmed the noncovalent nature of interactions within the nanocomposite of thiolated nanotubes. In PM-IRRAS spectra of SWNT SAMs on gold, the IR-active vibrational SWNT modes have been observed and identified. According to PM-IRRAS data, the hydrocarbon chains of 2 are oriented with less tilt angle to the bare gold normal in a SAM deposited from an SWNT-2 dispersion than those of 1 deposited from an SWNT-1 dispersion on the mercaptoethanesulfonic acid-primed gold. For both the dried SWNT-2 bulk and the SWNT-2 SAM on gold, the C K-edge NEXAFS spectra revealed the presence of CH-pi interactions between hydrocarbon chains of 2 and the pi electronic nanotube structure due to the highly resolved vibronic fine structure of carbon 1s --> R*/sigma*C-H series of states in the alkyl chain of 2. For the SWNT-2 bulk, the observed splitting and upshift of the SWNT pi* orbitals in the NEXAFS spectrum indicated the presence of pi-pi interactions. In the NEXAFS spectrum of the SWNT-2 SAM on gold, the upshifted values of the photon energy for R*/sigma*C-H transitions indicated close contact of 2 with nanotubes and with a gold surface. The angle-dependent NEXAFS for the SWNT-2 bulk showed that most of the molecules of 2 are aligned along the nanotubes, which are self-organized with orientation parallel to the substrate plane, whereas the NEXAFS for the SWNT-2 SAM revealed a more normal orientation of functionality 2 on gold compared with that in the SWNT-2 bulk.     

Direct electrochemistry and superficial characterization of DNA-cytochrome c-MUA films on chemically modified gold surface

Cytochrome c (Cyt. c) was immobilized on the 11-mercaptohendecanoic acid (MUA)-modified gold electrode. The electrode was stable and sensitive to Cyt. c. Later, DNA was also immobilized on the two-layer modified electrode. Cyclic voltammetry studies show that Cyt. c can interact with dsDNA and ssDNA. The binding site sizes were determined to be 15 base pairs per Cyt. c molecule with dsDNA and 30 nucleotides binding 1 Cyt. c molecule with ssDNA. The modified electrodes were characterized by quartz crystal microbalance (QCM), impedance spectroscopy and atomic force microscope (AFM). The modified electrode can be used for determining DNA.     

Minor groove binding of a novel tetracationic diviologen

Novel tetracationic diviologen compounds of the general formula CH3(CH2)nV2+(CH2)6V2+(CH2)nCH3 (where V2+ = 4,4'-bipyridinium and n = 5 or 11) were investigated as electrochemical reporters of DNA duplex formation. These compounds bind to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) when the DNA is either present in solution or immobilized at electrode surfaces. Binding to thiolated ssDNA and dsDNA immobilized at Au electrodes was characterized using the electrochemical response for the reduction of the V2+ state to the V+ (viologen radical cation) state. An analysis of the charge for this reduction provided isotherms and binding constants for binding of these diviologens to both forms of immobilized DNA. Saturation of the binding is achieved at solution concentrations near 20 microM. For both the n = 5 and 11 diviologens, binding to ssDNA is driven by electrostatic charge neutralization. For the n = 11 case, the binding is cooperative. In the presence of dsDNA, the n = 11 diviologen exhibits a unique reduction potential for the V2+/+ redox couple that is shifted approximately 100 mV negative of that in the presence of ssDNA. This new electrochemical signature is attributed to the reduction of viologen groups bound in the minor groove of the DNA duplex. For dsDNA in solution, an increase in the thermal denaturation temperature (Tm) from 60 to 66 degrees C as a function of the n = 11 diviologen concentration confirmed its interaction with the duplex. Circular dichroism (CD) spectroscopy also was used to investigate the binding of both the V2+ and V+ redox states of the n = 11 diviologen to dsDNA in solution. For the V+ state, a CD signal was observed that is consistent with the presence of face-to-face pi dimers of the viologen groups. This unambiguously demonstrates the binding of this redox state of the diviologen in the dsDNA minor groove and the formation of such dimers in the minor groove.     

Single probe nucleic acid immobilization on chemically modified single protein by controlling ionic strength and pH

In an effort toward determining the feasibility of single molecule analysis, we describe a case whereby the binding of one biotinylated DNA to one streptavidin molecule via electrostatic interactions was controlled by altering in pH 4.0-9.0 and 0.16 of the ion strength. The quantitative analysis of immobilized probe ssDNA was realized in real-time via a quartz crystal microbalance (QCM) and electrochemical (EC) measurement in the range 100 pM to 50 μM of probe oligonucleotide concentration. The variation amount of biotinylated ssDNA immobilized on the streptavidin-modified surface at pH 7.5 was about 0.16 pmol, giving a ratio of streptavidin to biotinylated ssDNA of about 1:1.1. On the other hand, at pH 4.9, it was immobilized about 0.29 pmol. From the shape of the Langmuir plot and QCM, the immobilization efficiency of biotinylated DNA via streptavidin at pH 4.9 was approximately twofold that at pH 7.5. In view points of the reaction velocity, it was increased with decreasing buffer solution pH, indicating a strong interaction of negatively charged probe DNA with the positively charged streptavidin. And also the EC response value of ΔI/I_streptavidin for the immobilized biotinylated ssDNA in pH 4.9 was about 49%, while the corresponding value for the pH 7.5 was approximately 34%. As DNA molecules possess negative charges, electrostatic repulsion occurred between streptavidin and biotinylated ssDNA at pH 7.5. At pH 4.9, the attraction between the biotinylated ssDNA and streptavidin resulted in increased adsorption which has an isoelectric point of about 5.9. It was deduced that the binding of biotinylated ssDNA to one or two of the four binding sites of streptavidin can be controlled by adjusting the pH-controlled electrostatic interaction.