Label-free and high-resolution protein/DNA nanoarray analysis using Kelvin probe force microscopy

Using the scanning probe technique known as Kelvin probe force microscopy it is possible to successfully devise a sensor for charged biomolecules. The Kelvin probe force microscope is a tool for measuring local variations in surface potential across a substrate of interest. Because many biological molecules have a native state that includes the presence of charge centres (such as the negatively charged backbone of DNA), the formation of highly specific complexes between biomolecules will often be accompanied by local changes in charge density. By spatially resolving this variation in surface potential it is possible to measure the presence of a specific bound target biomolecule on a surface without the aid of special chemistries or any form of labelling. The Kelvin probe force microscope presented here is based on an atomic force microscopy nanoprobe offering high resolution (<10 nm), sensitivity (<50 nM) and speed (>1,100 microm s(-1)), and the ability to resolve as few as three nucleotide mismatches.     

Hydrogen detection in metals: a review and introduction of a Kelvin probe approach

Abstract

Hydrogen in materials is an important topic for many research fields in materials science. Hence in the past quite a number of different techniques for determining the amount of hydrogen in materials and for measuring hydrogen permeation through them have been developed. Some of these methods have found widespread application. But for many problems the achievable sensitivity is usually not high enough and ready-to-use techniques providing also good spatial resolution, especially in the submicron range, are very limited, and mostly not suitable for widespread application. In this work this situation will be briefly reviewed and a novel scanning probe technique based method introduced.     

The Kelvin probe method for work function topographies: technical problems and solutions

The study of the physical properties (roughness, optical and electrical properties) of an entire surface or portions of this surface may be done by positioning a probe or using a beam of dimensions smaller than the studied area in front of the surface. The scanning method, often used in microscopy, LEED, etc., is however difficult to set up for measuring work function inhomogeneities by the Kelvin probe method: to be useful, the measurements must be made at a constant probe sample distance.We present here an original apparatus, based on a feedback loop, using the Kelvin probe, which permits us to work at a constant probe sample distance and, therefore, with a constant accuracy. Work function topographies are possible on all surfaces with an area greater than a few mm². It is also possible to find the roughness of the surface, the probe sample distance and to work in varying temperature. Results concerning semiconductor studies are given.     

扫描Kelvin探针力显微镜:工作原理及在材料腐蚀研究中的应用

扫描Kelvin探针力显微镜(SKPFM)是在原子力显微镜(AFM)的基础上应用扫描Kelvin探针(SKP)技术开发的检测表征手段,它能够在获取材料表面纳米级分辨率形貌的同时,原位得到样品表面高分辨率的接触电势差分布图,为揭示腐蚀反应机理提供了崭新的思路,近年来发展迅速。本文介绍了SKPFM两种工作模式的基本原理,总结了SKPFM在应用中的问题,并讨论了SKPFM和传统扫描Kelvin探针技术(SKP)的优缺点,重点综述了SKPFM在腐蚀科学研究中的应用,最后展望了SKPFM的发展方向与应用前景。     

Probing CO on a rutile TiO2(110)surface using atomic force microscopy and Kelvin probe force microscopy

Probing CO at a specific site on a metal oxide surface is essential for characterizing various applications such as CO oxidation,hydrogenation,and water-gas shi...     

Effects of serum protein on ionic exchange between culture medium and microporous hydroxyapatite and silicate-substituted hydroxyapatite

It has been proposed that one of the underlying mechanisms contributing to the bioactivity of osteoinductive or osteoconductive calcium phosphates involves the rapid dissolution and net release of calcium and phosphate ions from the matrix as alternatively a precursor to subsequent re-precipitation of a bone-like apatite at the surface and/or to facilitate ion exchange in biochemical processes. In order to confirm and evaluate ion release from sintered hydroxyapatite (HA) and to examine the effect of silicate substitution into the HA lattice on ion exchange under physiological conditions we monitored Ca²⁺, PO₄ ³⁻ and SiO₄ ⁴⁻ levels in Earl’s minimum essential medium (E-MEM) in the absence (serum-free medium, SFM) or presence (complete medium, C-MEM) of foetal calf serum (FCM), with both microporous HA or 2.6 wt% silicate-substituted HA (SA) sintered discs under both static and semi-dynamic (SD) conditions for up to 28 days. In SFM, variation in Ca²⁺ ion concentration was not observed with either disc chemistry or culture conditions. In C-MEM, Ca²⁺ ions were released from SA under static and SD conditions whereas with HA Ca²⁺ was depleted under SD conditions. PO₄ ³⁻ depletion occurred in all cases, although it was greater in C-MEM, particularly under SD conditions. SiO₄ ⁴⁻ release occurred from SA irrespective of medium or culture conditions but a sustained release only occurred in C-MEM under SD conditions. In conclusion we showed that under physiological conditions the reservoir of exchangeable ions in both HA and SA in the absence of serum proteins is limited, but that the presence of serum proteins facilitated greater ionic exchange, particularly with SA. These observations support the hypothesis that silicate substitution into the HA lattice facilitates a number of ionic interactions between the material and the surrounding physiological environment, including but not limited to silicate ion release, which may play a key role in determining the overall bioactivity and osteoconductivity of the material. However, significant net release of Ca²⁺ and PO₄ ³⁻ was not observed, thus rapid or significant net dissolution of the material is not necessarily a prerequisite for bioactivity in these materials.     

Zinc-substituted hydroxyapatite: a biomaterial with enhanced bioactivity and antibacterial properties

Hydroxyapatite (HA) is a synthetic biomaterial and has been found to promote new bone formation when implanted in a bone defect site. However, its use is often limited due to its slow osteointegration rate and low antibacterial activity, particularly where HA has to be used for long term biomedical applications. This work will describe the synthesis and detailed characterization of zinc-substituted HA (ZnHA) as an alternative biomaterial to HA. ZnHA containing 1.6 wt% Zn was synthesized via a co-precipitation reaction between calcium hydroxide, orthophosphoric acid and zinc nitrate hexahydrate. Single-phase ZnHA particles with a rod-like morphology measuring ~50 nm in length and ~15 nm in width, were obtained and characterized using transmission electron microscopy and X-ray diffraction. The substitution of Zn into HA resulted in a decrease in both the a- and c-axes of the unit cell parameters, thereby causing the HA crystal structure to alter. In vitro cell culture work showed that ZnHA possessed enhanced bioactivity since an increase in the growth of human adipose-derived mesenchymal stem cells along with the bone cell differentiation markers, were observed. In addition, antibacterial work demonstrated that ZnHA exhibited antimicrobial capability since there was a significant decrease in the number of viable Staphylococcus aureus bacteria after in contact with ZnHA.     

Measuring van der Waals and electrostatic forces for an atomic force microscope probe contacting with metal surfaces

A contact force spectrometry technique was used to measure the van der Waals and electrostatic forces acting on platinum-coated silicon probes contacting with metal films on silicon substrates. It is shown that the results of such measurements can be used for determining the geometric characteristics of probes and the Hamaker constant of contacting materials. The experimental data well agree with the theoretical values.     

Heterogeneous nucleation of hydroxyapatite on protein: structural effect of silk sericin.

Acidic proteins play an important role during mineral formation in biological systems, but the mechanism of mineral formation is far from understood. In this paper, we report on the relationship between the structure of a protein and hydroxyapatite deposition under biomimetic conditions. Sericin, a type of silk protein, was adopted as a suitable protein for studying structural effect on hydroxyapatite deposition, since it forms a hydroxyapatite layer on its surface in a metastable calcium phosphate solution, and its structure has been reported. Sericin effectively induced hydroxyapatite nucleation when it has high molecular weight and a beta sheet structure. This indicates that the specific structure of a protein can effectively induce heterogeneous nucleation of hydroxyapatite in a biomimetic solution, i.e. a metastable calcium phosphate solution. This finding is useful in understanding biomineralization, as well as for the design of organic polymers that can effectively induce hydroxyapatite nucleation.     

Nanoscale characterization of the dielectric charging phenomenon in PECVD silicon nitride thin films with various interfacial structures based on Kelvin probe force microscopy

This work presents a novel characterization methodology for the dielectric charging phenomenon in electrostatically driven MEMS devices using Kelvin probe force microscopy (KPFM). It has been used to study plasma-enhanced chemical vapor deposition (PECVD) silicon nitride thin films in view of application in electrostatic capacitive RF MEMS switches. The proposed technique takes the advantage of the atomic force microscope (AFM) tip to simulate charge injection through asperities, and then the induced surface potential is measured. The impact of bias amplitude, bias polarity, and bias duration employed during charge injection has been explored. The influence of various parameters on the charging/discharging processes has been investigated: dielectric film thickness, SiN(x) material deposition conditions, and under layers. Fourier transform infrared spectroscopy (FT-IR) and x-ray photoelectron spectroscopy (XPS) material characterization techniques have been used to determine the chemical bonds and compositions, respectively, of the SiN(x) films being investigated. The required samples for this technique consist only of thin dielectric films deposited over planar substrates, and no photolithography steps are required. Therefore, the proposed methodology provides a low cost and quite fast solution compared to other available characterization techniques of actual MEMS switches. Finally, the comparison between the KPFM results and the discharge current transients (DCT) measurements shows a quite good agreement.     

3D finite element analysis of electrostatic deflection of commercial and FIB-modified cantilevers for electric and Kelvin force microscopy: I. Triangular shaped cantilevers with symmetric pyramidal tips

The investigation of the nanoscale distribution of electrostatic forces on material surfaces is of paramount importance for the development of nanotechnology, since these confined forces govern many physical processes on which a large number of technological applications are based. For instance, electric force microscopy (EFM) and micro-electro-mechanical-systems (MEMS) are technologies based on an electrostatic interaction between a cantilever and a specimen. In the present work we report on a 3D finite element analysis of the electrostatic deflection of cantilevers for electric and Kelvin force microscopy. A commercial triangular shaped cantilever with a symmetric pyramidal tip was modelled. In addition, the cantilever was modified by a focused ion beam (FIB) in order to reduce its parasitic electrostatic force, and its behaviour was studied by computation analysis. 3D modelling of the electrostatic deflection was realized by using a multiphysics finite element analysis software and it was applied to the real geometry of the cantilevers and probes obtained by using basic CAD tools. The results of the modelling are in good agreement with experimental data.     

Adsorption profile of lead on Aspergillus versicolor: a mechanistic probing

The adsorption of lead on Aspergillus versicolor biomass (AVB) has been investigated in aqueous solution with special reference to binding mechanism in order to explore the possibilities of the biomass to address environmental pollution. AVB, being the most potent of all the fungal biomasses tested, has been successfully employed for reducing the lead content of the effluents of battery industries to permissible limit (1.0 mg L(-1)) before discharging into waterbodies. The results establish that 1.0 g of the biomass adsorbs 45.0 mg of lead and the adsorption process is found to depend on the pH of the solution with an optimum at pH 5.0. The rate of adsorption of lead by AVB is very fast initially attaining equilibrium within 3h following pseudo second order rate model. The adsorption process can better be described by Redlich-Peterson isotherm model compared to other ones tested. Scanning electron micrograph demonstrates conspicuous changes in the surface morphology of the biomass as a result of lead adsorption. Zeta potential values, chemical modification of the functional groups and Fourier transform infrared spectroscopy reveal that binding of lead on AVB occurs through complexation as well as electrostatic interaction.     

3D finite element analysis of electrostatic deflection and shielding of commercial and FIB-modified cantilevers for electric and Kelvin force microscopy: II. Rectangular shaped cantilevers with asymmetric pyramidal tips

This paper deals with an application of 3D finite element analysis to the electrostatic interaction between (i) a commercial rectangular shaped cantilever (with an integrated anisotropic pyramidal tip) and a conductive sample, when a voltage difference is applied between them, and (ii) a focused ion beam (FIB) modified cantilever in order to realize a probe with reduced parasitic electrostatic force. The 3D modelling of their electrostatic deflection was realized by using multiphysics finite element analysis software and applied to the real geometry of the cantilevers and probes as used in conventional electric and Kelvin force microscopy to evaluate the contribution of the various part of a cantilever to the total force, and derive practical criteria to optimize the probe performances. We report also on the simulation of electrostatic shielding of nanometric features, in order to quantitatively evaluate an alternative way of reducing the systematic error caused by the cantilever-to-sample capacitive coupling. Finally, a quantitative comparison between the performances of rectangular and triangular cantilevers (part I of this work) is reported.     

Colloidal drug probe: Method development and validation for adhesion force measurement using Atomic Force Microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between a model drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from dry powder inhaler (DPI) formulations. Model silica probes of approximately 4 μm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres pre-attached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.     

Competition-mediated pyrene-switching aptasensor: probing lysozyme in human serum with a monomer-excimer fluorescence switch

Lysozyme (Lys) plays crucial roles in the innate immune system, and the detection of Lys in urine and serum has considerable clinical importance. Traditionally, the presence of Lys has been detected by immunoassays; however, these assays are limited by the availability of commercial antibodies and tedious protein modification and prior sample purification. To address these limitations, we report here the design, synthesis, and application of a competition-mediated pyrene-switching aptasensor for selective detection of Lys in buffer and human serum. The detection strategy is based on the attachment of pyrene molecules to both ends of a hairpin DNA strand, which becomes the partially complementary competitor to an anti-Lys aptamer. In the presence of target Lys, the aptamer hybridizes with part of the competitor, which opens the hairpin such that both pyrene molecules are spatially separated. In the presence of target Lys, however, the competitor is displaced from the aptamer by the target, subsequently forming an initial hairpin structure. This brings the two pyrene moieties into close proximity to generate an excimer, which, in turn, results in a shift of fluorescence emission from ca. 400 nm (pyrene monomer) to 495 nm (pyrene excimer). The proposed method for Lys detection showed sensitivity as low as 200 pM and high selectivity in buffer. When measured by a steady-state fluorescence spectrum, the detection of Lys in human serum showed a strong fluorescent background, which obscured detection of the excimer signal. However, time-resolved emission measurement (TREM) supported the potential of the method in complex environments with background fluorescence by demonstrating the temporal separation of probe fluorescence emission decay from the intense background signal. We have also demonstrated that the same strategy can be applied to the detection of small biomolecules such as adenosine triphosphate (ATP), showing the generality of our approach. Therefore, the competition-mediated pyrene-switching aptasensor is promising to have potential for clinical and forensic applications.     

One-step homogeneous protein detection based on aptamer probe and fluorescence cross-correlation spectroscopy

A new protein assay based on fluorescence cross-correlation spectroscopy (FCCS) and aptamer probe is developed. In this assay, two spectrally distinct fluorophores labeled aptamer probes are used to recognize and detect thrombin through a sandwich reaction. The sandwich complexes are diffused through a confocal detection volume. The cross-correlation signals can be observed only at the presence of the aptamer probes-protein sandwich complexes. Thrombin is selected as a target to validate the assay. The whole detection process can be completed within an hour with low-nanomolar sensitivity and high specificity. The novel aptamer-based FCCS detection offers a simple, rapid and sensitive method for protein analysis in a homogeneous format.     

Nanocrystalline hydroxyapatite doped with magnesium and zinc: Synthesis and characterization

During recent years, there have been efforts in developing nanocrystalline bioceramics, to enhance their mechanical and biological properties for use in tissue engineering applications. In this research, we made an attempt to synthesize nanocrystalline bioactive hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂, HAp) ceramic powder in the lower-end of nano-range (2–10 nm), using a simple low-temperature sol–gel technique and studied its densification behavior. We further studied the effects of metal ion dopants during synthesis on powder morphology, and the properties of the sintered structures. Calcium nitrate and triethyl phosphite were used as precursors for calcium and phosphorous, respectively, for sol–gel synthesis. Calculated quantities of magnesium oxide and zinc oxide were incorporated as dopants into amorphous dried powder, prior to calcination at 250–550 °C. The synthesized powders were analyzed for their phases using X-ray diffraction technique and characterized for powder morphology and particle size using transmission electron microscopy (TEM). TEM analysis showed that the average particle size of the synthesized powders were in the range of 2–10 nm. The synthesized nano-powders were uniaxially compacted and then sintered at 1250 °C and 1300 °C for 6 h, separately, in air. A maximum average sintered density of 3.29 g/cm³ was achieved in structures sintered at 1300 °C, developed from nano-powder doped with magnesium. Vickers hardness testing was performed to determine the hardness of the sintered structures. Uniaxial compression tests were performed to evaluate the mechanical properties. Bioactivity and biodegradation behavior of the sintered structures were assessed in simulated body fluid (SBF) and maintained in a dynamic state.     

Method of fabrication of submicron composite microparticles of hydroxyapatite and ferromagnetic nanoparticles for a protein drug carrier

Composite microparticles of hydroxyapatite (HAp) and ferromagnetic nanoparticles were prepared using algenic acid gel cross-linked by bivalent calcium ions. The resultant size of the composite microparticle could be reduced to the submicron scale using extremely vigorous stirring by a homogenizer at 16 000 r.p.m. The instantaneous gellation induced by the bivalent ion cross-linking was revealed to be advantageous for combining the constituent nanoparticles of HAp and ferromagnetic nanoparticles. Since the structural arrest occurs instantly at the moment of cross-linking by the bivalent calcium ion, the added constituent HAp/ferromagnetic nanoparticles do not have time to be segregated apart from the binding gel; consequently, the submicron-scale composite state of these constituents could be instantaneously immobilized as in the dispersed state in liquid by the applied high shear. Thus, the instantaneous gellation characteristic of the bivalent ion cross-linking is preferable for incorporating different nanoparticles. The retention ability of the test protein (cytochrome c) was examined using the bincinchoninic assay method. Comparison of the protein retention ability with the control sample without HAp nanoparticles revealed that the incorporated HAp predominantly contributes to the retention ability of cytochrome c.