Application of integrated SECM ultra-micro-electrode and AFM force probe to biosensor surfaces

The integration of scanning electrochemical ultra-micro-electrode (UME) with atomic force microscope cantilever probe have been achieved by using a homemade photolithography system. A gold-film-coated AFM cantilever was insulated with photo resist coating and a pointed end of the AFM probe was opened by illuminating with maskless arbitrary optical micro-pattern generator. To realize precise control of probe sample distance constantly, the resulting scanning electrochemical microscopy (SECM)-AFM probe was operated using a dynamic force microscopy (DFM) technique with magnetic field excitation. From a steady-state voltammetric experiment, the effective electrode diameters of the probes thus prepared were estimated to be from 0.050 to 6.2 microm. The capability of this SECM-AFM probe have been tested using gold comb in the presence of Fe(CN)(6)(3-). The simultaneous imaging of the topography and electrochemical activity of the strip electrode was successfully obtained. We also used the SECM-AFM to examine in situ topography and enzymatic activity measurement. Comparison of topography and oxidation current profiles above enzyme-modified electrode showed active parts distribution of biosensor surface.     

Parylene insulated probes for scanning electrochemical-atomic force microscopy

Scanning electrochemical-atomic force microscopy (SECM-AFM) is a powerful technique that can be used to obtain in situ information related to electrochemical phenomena at interfaces. Fabrication of probes to perform SECM-AFM experiments remains a challenge. Herein, we describe a method for formation of microelectrodes at the tip of commercial conductive AFM probes and demonstrate application of these probes to SECM-AFM. Probes were first insulated with a thin parylene layer, followed by subsequent exposure of active electrodes at the probe tips by mechanical abrasion of the insulating layer. Characterization of probes was performed by electron microscopy and cyclic voltammetry. In situ measurement of localized electrochemical activity with parylene-coated probes was demonstrated through measurement of the diffusion of Ru(NH)(6)(3+) across a porous membrane.     

Sequential electrochemical oxidation and site-selective growth of nanoparticles onto AFM probes

In this work, we reported an approach for the site-selective growth of nanoparticle onto the tip apex of an atomic force microscopy (AFM) probe. The silicon AFM probe was first coated with a self-assembled monolayer (SAM) of octadecyltrichlorosilane (OTS) through a chemical vapor deposition (CVD) method. Subsequently, COOH groups were selectively generated at the tip apex of silicon AFM probes by applying an appropriate bias voltage between the tip and a flat gold electrode. The transformation of methyl to carboxylic groups at the tip apex of the AFM probe was investigated through measuring the capillary force before and after electrochemical oxidation. To prepare the nanoparticle terminated AFM probe, the oxidized AFM probe was then immersed in an aqueous solution containing positive metal ions, for example, Ag+, to bind positive metal ions to the oxidized area (COOH terminated area), followed by chemical reduction with aqueous NaBH 4 and further development (if desired) to give a metal nanoparticle-modified AFM probe. The formation of a metal nanoparticle at the tip apex of the AFM probe was confirmed by scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDXA).     

Bubble colloidal AFM probes formed from ultrasonically generated bubbles

Here we introduce a simple and effective experimental approach to measuring the interaction forces between two small bubbles (approximately 80-140 microm) in aqueous solution during controlled collisions on the scale of micrometers to nanometers. The colloidal probe technique using atomic force microscopy (AFM) was extended to measure interaction forces between a cantilever-attached bubble and surface-attached bubbles of various sizes. By using an ultrasonic source, we generated numerous small bubbles on a mildly hydrophobic surface of a glass slide. A single bubble picked up with a strongly hydrophobized V-shaped cantilever was used as the colloidal probe. Sample force measurements were used to evaluate the pure water bubble cleanliness and the general consistency of the measurements.     

Cleaning and hydrophilization of atomic force microscopy silicon probes

The silicon surface of commercial atomic force microscopy (AFM) probes loses its hydrophilicity by adsorption of airborne and package-released hydrophobic organic contaminants. Cleaning of the probes by acid piranha solution or discharge plasma removes the contaminants and renders very hydrophilic probe surfaces. Time-of-flight secondary-ion mass spectroscopy and X-ray photoelectron spectroscopy investigations showed that the native silicon oxide films on the AFM probe surfaces are completely covered by organic contaminants for the as-received AFM probes, while the cleaning methods effectively remove much of the hydrocarbons and silicon oils to reveal the underlying oxidized silicon of the probes. Cleaning procedures drastically affect the results of adhesive force measurements in water and air. Thus, cleaning of silicon surfaces of the AFM probe and sample cancelled the adhesive force in deionized water. The significant adhesive force values observed before cleaning can be attributed to formation of a bridge of hydrophobic material at the AFM tip-sample contact in water. On the other hand, cleaning of the AFM tip and sample surfaces results in a significant increase of the adhesive force in air. The presence of water soluble contaminants at the tip-sample contact lowers the capillary pressure in the water bridge formed by capillary condensation at the AFM tip-sample contact, and this consequently lowers the adhesive force.     

AFM study of hippocampal cells cultured on silicon wafers with nano-scale surface topograph

The rat hippocampal cells were selected as model to study the interaction between the neural cells and silicon substrates using atomic force microscopy (AFM). The hippocampal cells show tight adherence on silicon wafers with nano-scale surface topograph. The lateral friction force investigated by AFM shows significant increase on the boundary around the cellular body. It is considered to relate to the cytoskeleton and cellular secretions. After ultrasonic wash in ethanol and acetone step by step, the surface of silicon wafers was observed by AFM sequentially. We have found that the culture leftovers form tight porous networks and a monolayer on the silicon wafers. It is concluded that the leftovers overspreading on the silicon substrates are the base of cell adherence on such smooth inert surfaces.     

Alternative method for fabricating chemically functionalized AFM tips: silane modification of HF-treated Si3N4 probes

An alternative method for fabricating functionalized, atomic force microscopy (AFM) tips is presented. This technique is simple and requires only minimal preparation and tip modification to generate chemically sensitive probes that have a robust organic monolayer of flexible terminal chemistry attached to the surface. Specifically, commercially microfabricated Si3N4 AFM tips were modified with self-assembled monolayers (SAMs) of octadecyltrichlorosilane and (11-bromoundecyl)trichlorosilane after removing the native silicon oxide surface layer with concentrated hydrofluoric acid. The structure of these SAM films on solid silicon nitride surfaces was studied using contact angle goniometry and Fourier transform infrared spectroscopy. Pull-off force measurements on various bare (mica, graphite, and silicon) and SAM-functionalized substrates confirm that mechanically robust, long-chain organic silane SAMs can be formed on HF-treated Si3N4 tips without the presence of an intervening oxide layer. Adhesion experiments show that the integrity of the organic film on the chemically modified tips is maintained over repeated measurements and that the functionalized tips can be used for chemical sensing experiments since strong discrimination between different surface chemistries is possible.     

In situ estimation of the chemical and mechanical contributions in local adhesion force measurement with AFM: the specific case of polymers

The atomic force microscope (AFM) was used to perform surface force measurements in contact mode to investigate surface properties of model systems at the nanoscale. Two types of model systems were considered. The first one was composed of a rigid substrate (silicon plates) which was chemically modified by molecular self-assembling (SAMs) to display different surface properties (hydroxyl, amine, methyl and ester functional groups). The second system consists of model polymer networks (cross-linked polydimethylsiloxane or PDMS) of variable mechanical properties, whose surfaces were chemically modified with the same groups as before with silicon substrates. The comparison of the force curves obtained from the two model systems shows that the viscoelastic or mechanical contribution dominates in the adhesion on polymer substrates. Finally, a relationship, which expresses the separation energy at a local scale as a function of the energy dissipated within the contact zone, on one hand and the surface properties of the polymer on the other, was proposed.     

AFM colloidal forces measured between microscopic probes and flat substrates in nanoparticle suspensions

Colloidal forces between atomic force microscopy probes of 0.12 and 0.58 N/m spring constant and flat substrates in nanoparticle suspensions were measured. Silicon nitride tips and glass spheres with a diameter of 5 and 15 mum were used as the probes whereas mica and silicon wafer were used as substrates. Aqueous suspensions were made of 5-80 nm alumina and 10 nm silica particles. Oscillatory force profiles were obtained using atomic force microscope. This finding indicates that the nanoparticles remain to be stratified in the intervening liquid films between the probe and substrate during the force measurements. Such structural effects were manifested for systems featuring attractive and weak repulsive interactions of nanoparticles with the probe and substrate. Oscillation of the structural forces shows a periodicity close to the size of nanoparticles in the suspension. When the nanoparticles are oppositely charged to the probes, they tend to coat the probes and hinder probe-substrate contact.     

Mechanisms of single-walled carbon nanotube probe-sample multistability in tapping mode AFM imaging

When using single-walled carbon nanotube (SWNT) probes to create AFM images of SWNT samples in tapping mode, elastic deformations of the probe and sample result in a decrease in the apparent width of the sample. Here we show that there are two major mechanisms for this effect, smooth gliding and snapping, and compare their dynamics to the case when a conventional silicon tip is used to image a bare silicon surface. Using atomistic and continuum simulations, we analyze in detail the shape of the tip-sample interaction potential for three model cases and show that in the absence of adhesion and friction forces, more than two discrete, physically meaningful solutions of the oscillation amplitude are possible when snapping occurs (in contrast to the existence of one attractive and one repulsive solution for conventional silicon AFM tips). We present experimental results indicating that a continuum of amplitude solutions is possible when using SWNT tips and explain this phenomenon with dynamic simulations that explicitly include tip-sample adhesion and friction forces. We also provide simulation results of SWNT tips imaging Si(111)-CH3 surface step edges and Au nanocrystals, which indicate that SWNT probe multistability may be a general phenomenon, not limited to SWNT samples.     

In situ hydrodynamic lateral force calibration of AFM colloidal probes

Lateral force microscopy (LFM) is an application of atomic force microscopy (AFM) to sense lateral forces applied to the AFM probe tip. Recent advances in tissue engineering and functional biomaterials have shown a need for the surface characterization of their material and biochemical properties under the application of lateral forces. LFM equipped with colloidal probes of well-defined tip geometries has been a natural fit to address these needs but has remained limited to provide primarily qualitative results. For quantitative measurements, LFM requires the successful determination of the lateral force or torque conversion factor of the probe. Usually, force calibration results obtained in air are used for force measurements in liquids, but refractive index differences between air and liquids induce changes in the conversion factor. Furthermore, in the case of biochemically functionalized tips, damage can occur during calibration because tip-surface contact is inevitable in most calibration methods. Therefore, a nondestructive in situ lateral force calibration is desirable for LFM applications in liquids. Here we present an in situ hydrodynamic lateral force calibration method for AFM colloidal probes. In this method, the laterally scanned substrate surface generated a creeping Couette flow, which deformed the probe under torsion. The spherical geometry of the tip enabled the calculation of tip drag forces, and the lateral torque conversion factor was calibrated from the lateral voltage change and estimated torque. Comparisons with lateral force calibrations performed in air show that the hydrodynamic lateral force calibration method enables quantitative lateral force measurements in liquid using colloidal probes.     

Preparation of superhydrophobic silicon oxide nanowire surfaces

The paper reports on the preparation of superhydrophobic amorphous silicon oxide nanowires (a-SiONWs) on silicon substrates with a contact angle greater than 150 degrees by means of surface roughness and self-assembly. Nanowires with an average mean diameter in the range 20-150 nm and 15-20 microm in length were obtained by the so-called solid-liquid-solid (SLS) technique. The porous nature and the high roughness of the resulting surfaces were confirmed by AFM imaging. The superhydrophobicity resulted from the combined effects of surface roughness and chemical modification with fluorodecyl trichlorosilane.     

Supported bilayer lipid membrane arrays on photopatterned self-assembled monolayers

This work demonstrates the use of photocleavable cholesterol derivatives to create supported bilayer lipid membrane arrays on silica. The photocleavable cholesteryl tether is attached to the surface by using the reaction of an amine-functionalized self-assembled monolayer (SAM) and the N-hydroxysuccinimide-based reagent 9. The resultant SAM contains an ortho-nitrobenzyl residue that can be cleaved by photolysis by using soft (365 nm) UV light regenerating the original amine surface, and which can be patterned using a mask. The photoreaction yield was approximately 75 % which was significantly higher than previously found for related ortho-nitrobenzyl photochemistry on gold substrates. The SAMs were characterized by means of contact angle measurements, ellipsometry and X-ray photoelectron spectroscopy. Patterned surfaces were characterized with SEM and AFM. After immersing the patterned surface into a solution containing small unilamellar vesicles of egg phosphatidylcholine (PC), supported lipid membranes were formed comprised of lipid bilayer over the amine functionalized "hydrophilic" regions and lipid monolayer over the cholesteryl "hydrophobic" regions. This was confirmed by fluorescence microscopy and AFM. FRAP studies yielded a lateral diffusion coefficient for the probe molecule of 0.14+/-0.05 microm(2) s(-1) in the bilayer regions and approximately 0.01 microm(2) s(-1) in the monolayer regions. This order of magnitude difference in diffusion coefficients effectively serves to isolate the bilayer regions from one another, thus creating a bilayer array.     

Modified electrodes based on lipidic cubic phases

The lipidic cubic phase can be characterized as a curved bilayer forming a three-dimensional, crystallographical, well-ordered structure that is interwoven by aqueous channels. It provides a stable, well-organized environment in which diffusion of both water-soluble and lipid-soluble compounds can take place. Cubic phases based on monoacylglycerols form readily and attract our interest due to their ability to incorporate and stabilize proteins. Their lyotropic and thermotropic phase behaviour has been thoroughly investigated. At hydration over 20%, lipidic cubic phases Ia3d and Pn3m are formed. The latter is stable in the presence of excess water, which is important when the cubic phase is considered as an electrode-modifying material. Due to high viscosity, the cubic phases can be simply smeared over solid substrates such as electrodes and used to host enzymes and synthetic catalysts, leading to new types of catalytically active modified electrodes as shown for the determination of cholesterol, CO(2), or oxygen. The efficiency of transport of small hydrophilic molecules within the film can be determined by voltametry using two types of electrodes: a normal-size electrode working in the linear diffusion regime, and an ultramicroelectrode working under spherical diffusion conditions. This allows determining both the concentration and diffusion coefficient of the electrochemically active probe in the cubic phase. The monoolein-based cubic phase matrices are useful for immobilizing enzymes on the electrode surface (e.g., laccases from Trametes sp. and Rhus vernicifera were employed for monitoring dioxygen). The electronic contact between the electrode and the enzyme was maintained using suitable electroactive probes.     

Integration of an electrochemical-based biolithography technique into an AFM system

An ordinary atomic force microscopy (AFM) was functionalized and applied to electrochemically draw micropatterns of biomolecules. To fabricate an electrochemical AFM probe having an electrode at the tip, a metal-coated AFM probe was first insulated with Parylene C, and then the apex of the tip was ground mechanically to expose the electrode. The effective electrode diameter was estimated to be ca. 500 nm. The electrode probe was positioned close to a heparin-coated antibiofouling substrate and used to locally generate hypobromous acid from a dilute Br⁻ solution to render the substrate surface protein-adhesive. In situ topographical imaging after the electrochemical treatment suggested the heparin layer became detached to allow the adsorption of proteins, in this case fibronectin. The diameter of the drawn fibronectin pattern was 2 μm, which is one order of magnitude smaller than we achieved previously using a microdisk electrode (tip diameter 10 μm). Figure AFM configuration integrated with the electrochemical-based surface modification and resultant micropatterns of fluorescence-labeled fibronectin Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA imaged on a HOPG electrode surface by AFM with controlled potential

Single-molecule AFM imaging of single-stranded and double-stranded DNA molecules self-assembled from solution onto a HOPG electrode surface is reported. The interaction of DNA with the hydrophobic surface induced DNA aggregation, overlapping, intra- and intermolecular interactions. Controlling the electrode potential and using the phase images as a control method, to confirm the correct topographical characterization, offers the possibility to enlarge the capability of AFM imaging of DNA immobilized onto conducting substrates, such as HOPG. The application of a potential of +300 mV (versus AgQRE) to the HOPG enhanced the robustness and stability of the adsorbed DNA molecules, increasing the electrostatic interaction between the positively charged electrode surface and the negatively charged DNA sugar-phosphate backbone.     

Deformation and adhesion of elastomer poly(dimethylsiloxane) colloidal AFM probes

We report on the synthesis and characterization of elastomer colloidal AFM probes. Poly(dimethylsiloxane) microparticles, obtained by water emulsification and cross-linking of viscous prepolymers, are glued to AFM cantilevers and used for contact mechanics investigations on smooth substrates: in detail cyclic loading-unloading experiments are carried on ion-sputtered mica, the deformation rate and dwell time being separately controlled. We analyze load-penetration curves and pull-off forces with models due respectively to Zener; Maugis and Barquins; and Greenwood and Johnson and account for bulk creep, interfacial viscoelasticity, and structural rearrangements at the polymer-substrate interface. A good agreement is found between experiments and theory, with a straightforward estimation of colloidal probes' material parameters. We suggest the use of such probes for novel contact mechanics experiments involving fully reversible deformations at the submicrometer scale.     

AFM study of the behavior of polystyrene and glass particles during the electrodeposition of copper

In this paper, the behavior of polystyrene and glass particles on a copper electrode during the electrodeposition of copper was studied using an atomic force microscope (AFM). Polystyrene or glass particles glued to the tip of the AFM cantilever were kept in contact with the surface of the electrode. The surface forces between the polystyrene or glass particle and the copper electrode were measured before, during, and after electrodeposition. These experiments revealed that glass particles do not make contact with the electrode, probably due to the repulsive hydration force. Polystyrene particles, on the other hand, make contact with the electrode, due to the attractive hydrophobic force. The AFM experiments were correlated with sedimentation co-deposition experiments of polystyrene and glass particles with copper. It was found that 80% of the polystyrene particles added to the plating solution incorporated with copper, while only 0.25% of the glass particles co-deposited under the same conditions.     

AFM investigation of silicon substrates for chemical vapour deposition of diamond films

AFM has been used to study surface modifications on silicon (100) substrates for CVD diamond deposition during bias pretreatment in a hot-filament reactor under various conditions. Both topographical images, force-distance measurements and chemical etching with HF have been implemented to obtain information on the processes involved. The results show, that the observed roughening, which strongly depends on the gas phase composition, is caused by chemical etching of the surface dominated by removal of elemental silicon via formation of silicon hydride.     

Determination of solid surface tension from particle-substrate pull-off forces measured with the atomic force microscope

Atomic force microscopy (AFM) is capable of solid surface characterization at the microscopic and submicroscopic scales. It can also be used for the determination of surface tension of solids (gamma) from pull-off force (F) measurements, followed by analysis of the measured F values using contact mechanics theoretical models. Although a majority of the literature gamma results was obtained using either Johnson-Kendall-Roberts (JKR) or Derjaguin-Muller-Toporov (DMT) models, re-analysis of the published experimental data presented in this paper indicates that these models are regularly misused. Additional complication in determination of gamma values using the AFM technique is that the measured pull-off forces have poor reproducibility. Reproducible and meaningful F values can be obtained with strict control over AFM experimental conditions during the pull-off force measurements (low humidity level, controlled and known loads) for high quality substrates and probes (surfaces should be free of heterogeneity, roughness, and contamination). Any probe or substrate imperfections complicate the interpretation of experimental results and often reduce the quality of the generated data. In this review, surface imperfection in terms of roughness and heterogeneity that influence the pull-off force are analyzed based upon the contact mechanics models. Simple correlations are proposed that could guide in selection and preparation of AFM probes and substrates for gamma determination and selection of loading conditions during the pull-off force measurements. Finally, the possibility of AFM measurements of solid surface tension using materials with rough surfaces is discussed.