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Immobilization of Glucose Oxidase to Nanostructured Films of Polystyrene-block-poly(2-vinylpyridine)
... There are a number of techniques that can be used to characterize the nanomaterial/protein interface [91] and obtain valuable information about the adsorption process. Surface features and topography can be determined using atomic force microscopy (AFM) [89,[92][93][94], scanning electron microscopy (SEM), transmission electron microscopy (TEM) [95], and scanning tunneling microscopy (STM) [6]. AFM can render 3D images of surface topography but can be affected by a series of artifacts [96]. ...
... The measurements are complementary to the information obtained by QCM and can be used to determine the optical properties of a substrate and the thickness of multiple layers on the surface. The technique is simple, nondestructive, provides angstrom resolution, and has the capability of allowing the observation of the adsorption process in real time [94,134,135]. The use of imaging ellipsometry can also provide spatial resolution of protein binding [136]. ...
... The latter is particularly interesting due to its high IEP (about 9.5) which serves as a positively charged substrate for the immobilization of GOx (and other negatively-charged proteins) via electrostatic interactions. With the intention of preserving the native structure of the enzyme, Bhakta et al. [94] recently demonstrated the potential advantages of nanoporous substrates fabricated using block co-polymers of polystyrene-block-poly(2-vinylpyridine) for the immobilization of GOx. ...
... 37 To our knowledge, very few examples are reported in the literature about the possibility of using BCPbased nanoporous thin films as supports to physically absorb proteins and enzymes. 48,49 By immobilizing a biomolecule onto the nanostructured surface of a block copolymer, the presence of pores can enhance the amount of absorbed molecules and facilitate the retention of the native structure of an enzyme and its activity, thus making the approach suitable as a novel supporting platform for biotechnological applications. The controlled introduction of a specific chemical functionality within the pore space is another critical issue in order to design a porous material useful for an efficient immobilization protocol. ...
... The approach has been specifically designed to modify the hydrophilic/hydrophobic characteristics of a given surface, to introduce pores of tailored size at the surface of a support and functional groups inside the pores, with the aim of using them for hosting enzymes without impairing their activity. Although BCPs have been used already for enzyme immobilization, 48,49 our approach is more general, robust, and versatile. In this work, we demonstrate its strength when applied to the physical adsorption of horseradish peroxidase (HRP). ...
We have set-up a facile approach for fabrication of supports with tailored nanoporosity for immobilization of enzymes. To this aim block copolymers self-assembly has been used to prepare nanostructured thin films with well-defined architecture containing pores of tailorable size delimited by walls with tailorable degree of hydrophilicity. In particular, we have employed a mixture of polystyrene-block-poly(L-lactide) (PS-PLLA) and polystyrene-block-poly(ethylene oxide) (PS-PEO) diblock copolymers to generate thin films with a lamellar morphology consisting of PS lamellar do-mains alternating with mixed PEO/PLLA blocks lamellar domains. Selective basic hydrolysis of the PLLA blocks generates thin films, patterned with nanometric channels containing hydrophilic PEO chains pending from PS walls. The shape and the size of the channels and the degree of hydrophilicity of the pores depend on the relative length of the blocks, the molecular mass of the BCPs, and the composition of the mixture. The strength of our approach is demonstrated in the case of physical adsorption of the hemoprotein peroxidase from horseradish (HRP) using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) with H2O2 as substrate. The large surface area, the tailored pore sizes and the functionalization with hydrophilic PEO blocks make the designed nanostructured materials suitable supports for the nanoconfinement of HRP biomolecules endowed with high catalytic performance, no mass-transfer limitations, and long-term stability.
... Advance Publication by J-STAGE Received May 11, 2020; Accepted June 9, 2020; Published online on June 12, 2020 DOI: 10 We acknowledge the support of the Australian Centre for Neutron Scattering, beamtime proposal P7962, in providing the neutron research facilities used in this work. ...
In the present study, the adsorption of glucose oxidase (GOD) to a mesoporous aluminum oxide (MAO) film was examined with in-situ neutron reflectometry (NR) measurements. The MAO film was deposited on a cover glass slip and a Si disc, and its pore structure was characterized by X-ray reflectometry (XRR) and NR. The Si disc with MAO film was applied for an in-situ NR experiment, and its NR profiles before/after adsorption of GOD were continuously measured with a flow cell. The results indicated that the negatively-charged GOD molecules hardly penetrate into the narrow pore channel (pore diameter = ca. 10 nm) with opposite surface charge.
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... Ramström and coworkers reported the preparation of UV crosslinked P4VP thin films that act as responsive coatings that control surface wettability and swelling toward external stimuli such as solvent and pH [65]. P4VP was used as an absorbent to remove organic dyes, pollutants, and heavy metals in waste water treatment [66][67][68][69][70][71][72][73][74][75][76], as a host ligand of metal-containing chromophores [77], the positively charged polyelectrolytes as sensors, electrochemical biosensors and actuators [78][79][80][81][82][83][84], in a chemiresistive glucose sensor [85], in wireless and optical oxygen sensor [86,87], nanostructured humidity sensors [88], colorimetric sensor [89], as a fluorescent enantioselective sensor for phenylalaninol and tryptophanas [90], as a template for the preparation of nanostructured carbon films and TiO 2 nanoparticles [91,92], in the preparation of ion-selective electrodes for the preconcentration of ions [93,94], as hydrophilic P4VP gels in ultrafiltration membranes [95], as membranes with improved hemocompatibility [96], as polymers in preparation glucose oxidase (GOx)-wiring and glucose flux-restricting in selfpowered glucose sensors (SPGSs) [97], in the preparation of shape memory polymers [98], in the preparation of self-assembly copolymer nanospheres for CO 2 capture [99], as the model system to measure the ionization constants of polymers by a number of techniques including ellipsometry, dynamic contact angle goniometry, and surface plasmon resonance imaging (SPRi) [100], as a model to achieve the mechanistic insights into drug loading and lyophilization of polymeric micelles [101], thermos-and pH-responsive membranes and nanocomposites [102][103][104][105][106][107][108][109][110], in solar heat reflective coating [111], as a cathode interfacial layer in inverted organic solar cells [112], as a polymeric ligand of mixed-mode chromatography (MMC) of proteins [113], as a platform for Raman-scattering detecting of the physically absorbed dye molecules [114], and as an antimicrobial material [115]. Polyvinyl-pyridine-N-oxide (PVNO) was widely tested to promote its cytoprotective effects on in vivo and in vitro models of silica-induced fibrosis. ...
The PVP and its derivatives have been broadly applied in polymers, organic syntheses, and catalysis processes. The crosslinked PVP is a well-known polymer support for numerous reagents and catalysts. Cross-linked PVPs are commercially available polymers and have attracted much attention over the past due to their interesting properties such as the facile functionalization, high accessibility of functional groups, being nonhygroscopic, easy to prepare, easy filtration, and swelling in many organic solvents. A brief explanation of the reported applications of PVPs in different fields followed by the discussion on the implementation of methodologies for catalytic efficiency of PVP-based reagents in the organic synthesis is included. The aim is to summarize the literature under a few catalytic categories and to present each as a short scheme involving reaction conditions. In the text, discussions on the synthesis and the structural determination of some typical polymeric reagents are presented, and the mechanisms of some organic reactions are given. Where appropriate, advantages of reagents in comparison with the previous reports are presented. This review does not include patent literature.
... Hydrophobic interactions with the two ethyl groups on each PDEA monomer and hydrophobic amino acids may be responsible for this behavior. 39 If that is the case, secondary structure alterations will most likely occur in GOX, 40 because the hydrophobic regions are located in the interior of the enzyme. ...
Catalysis by enzymes on surfaces has many applications. However, strategies for efficient enzyme immobilization with preserved activity are still in need of further development. In this work we investigate polyelectrolyte brushes prepared by both grafting-to and grafting-from with the aim to achieve high catalytic activity. For comparison, self-assembled monolayers that bind enzymes with the same chemical interactions are included. We use the model enzyme glucose oxidase and two kinds of polymers: anionic poly(acrylic acid) and cationic poly(diethylamino)methyl methacrylate. Surface plasmon resonance and spectroscopic ellipsometry are used for accurate quantification of surface coverage. Besides binding more enzymes, the “3D-like” brush environment enhances the specific activity compared to immobilization on self-assembled monolayers. For grafting-from brushes, multilayers of enzymes were spontaneously and irreversibly immobilized without conjugation chemistry. When the pH was between the pI of the enzyme and the pKa of the polymer, binding was considerable (thousands of ng/cm2 or up to 50% of the polymer mass), even at physiological ionic strength. However, binding was observed also when the brushes were neutrally charged. For acidic brushes (both grafting-to and grafting-from), the activity was higher for covalent immobilization compared to non-covalent. For grafting-from brushes, a fully preserved specific activity compared to enzymes in the liquid bulk was achieved, both with covalent (acidic brush) and non-covalent (basic brush) immobilization. Catalytic activity of hundreds of pmol cm-2 s-1 were easily obtained for polybasic brushes only tens of nm in dry thickness. This study provides new insights for designing functional interfaces based on enzymatic catalysis.
... Recently, multifunctional particles have attracted great interests and have been used to construct various kinds of micro/nano-devices including biosensors [23,24]. A critical consideration in the construction of multifunctional sensors is the method selected for the immobilization of the biorecognition element on the surface of a carrier [13,15,25]. For example, Chaichi et al. [23] reported a novel and efficient glucose biosensor based on the chemiluminescence (CL) detection of the enzymatically generated H 2 O 2 , which was constructed by the effective immobilization of glucose oxidase on the Fe 3 O 4 -chitosan nanoparticles support. ...
In this work, we presented the robust and magnetically recoverable dual sensor particles that are capable of real-time monitoring of dissolved oxygen (DO) and glucose concentrations in aqueous solutions. The dual sensor consists of glucose oxidase-functionalized polyethylenimine-coated Fe3O4 particles (Fe3O4@PEI-GOD) and sub-micro-sized Fe3O4-modified poly(Platinum porphyrin-co-Styrene) ((PtTFPPMA-PS)@Fe3O4) fluorescent spheres. The dual sensing functions originate from the cascade catalysis amplification driven by the recyclable Fe3O4@PEI-GOD particles and glucose oxidase consumption of oxygen in the process of glucose oxidation. Consequently, the (PtTFPPMA-PS)@Fe3O4 fluorescent spheres exhibited higher fluorescence intensity due to the effective alleviation of fluorescence quenching effect raised from the decrease of DO. The remarkable fluorescence intensity-changing characteristics of the solutions facilitate convenient identification of oxygen concentrations and glucose concentrations even with naked eyes. Thanks to the magnetically recoverable superiority, the recycle study proved that the multifunctional particles could be repeatedly utilized without significant catalytic activity loss after 10 cycles. Interestingly, the sensing film comprising the (PtTFPPMA-PS)@Fe3O4 fluorescent spheres can also be applied to the unsteady pressure measurement and unsteady air flow visualization due to its remarkable light intensity-changing features.
... To overcome these shortcomings, this report describes a simple approach for the preparation of colloidal CdSe/ZnS QD and their use in an optical paper-based assay for glucose. Glucose was selected as a model system because it is important for several fields including biology [20,21], biochemistry [22], and food science [23]. ...
The article describes a simple optical assay for glucose. It is based on the use of paper spots loaded with colloidal CdSe/ZnS quantum dots (Q-dots) and the enzyme glucose oxidase (GOx). Circular paper sheets were uniformly loaded with Q-dots and then displayed strong fluorescence under a UV lamp (365 nm exCitation). The action of GOx causes the production of H2O2 which, after a typical exposure time of 20 min, causes fluorescence intensity to be quenched. To obtain a reading, the paper sheets were photographed under 365 nm excitation using a digital camera. Several parameters, including the amount of Q-dots, sample pH, and amount of GOx were optimized to maximize the response to glucose. The paper-based assay showed a sigmoidal-shaped response with respect to the glucose concentration in the 5–200 mg·dL−1 range (limit of detection of 5 μg·dL−1), demonstrating their potential use for biomedical applications.
Graphical abstract
A simple and inexpensive paper-based assay for glucose is reported. Following the optimzation of the amount of quantum dots, sample pH value, and amount of glucose oxidase (GOx), a sigmoidal response function was obtained that covers the 5–200 mg dL−1 glucose concentration range.
... In all tested conditions, increased concentration of collagen resulted in increased accumulation on the OTCE substrates. These results are in agreement with previous literature reports[10,11,27] and can be explained by considering the increased number of collagen type I molecules in the vicinity of the electrode surface. The highest initial adsorption rate (d/dt 1 a solution containing 0.25 mg/mL of collagen type I was used. ...
The present article reports on the effect of electric potential on the adsorption of collagen type I (the most abundant component of the organic phase of bone) onto optically transparent carbon films (OTCE) and its mediation on subsequent adhesion of adult, human, mesenchymal stem cells (hMSCs). For this purpose, adsorption of collagen type I was investigated as a function of the protein concentration (0.01, 0.1, and 0.25 mg/mL) and applied potential (open circuit potential (OCP; control), +400, +800, and +1500 mV). The resulting substrate surfaces were characterized using spectroscopic ellipsometry (SE), atomic force microscopy (AFM), and cyclic voltammetry (CV). Adsorption of collagen type I onto OTCE was affected by the potential applied to the sorbent surface and the concentration of protein. The higher the applied potential and protein concentration, the higher the adsorbed amount (Γcollagen). It was also observed that the application of potential values higher than +800 mV resulted in the oxidation of the adsorbed protein. Subsequent adhesion of hMSCs on the OTCEs (pre-coated with the collagen type I films) under standard cell culture conditions for 2 hours was affected by the extent of collagen pre-adsorbed onto the OTCE substrates. Specifically, enhanced hMSCs adhesion was observed when the Γcollagen was the highest. When the collagen type I was oxidized (under applied potential > +800 mV) however, hMSCs adhesion was decreased. These results provide the first correlation between the effects of electric potential on protein adsorption and subsequent modulation of anchorage-dependent cell adhesion.
Nanoporous/nanocomposite thin films with controlled morphology at nanoscale were prepared onto transparent and conductive indium tin oxide (ITO) supports by exploiting the self-assembly of a lamellar polystyrene-b-poly(methyl methacrylate) (PS-b-PMMA) block copolymer (BCP). A perpendicular orientation of PS and PMMA lamellar nanodomains was achieved by grafting a random PS-r-PMMA copolymer to the ITO supports and successive thermal annealing. Stable and reproducible nanoporous morphologies, characterized by PS lamellar nanodomains of width equal to ≈ 20 nm alternating to nanochannels of width equal to ≈ 8 nm, were obtained by irradiating the samples with an appropriate UV-C dose able to fix the relative arrangement of PS domains through activation of cross-linking reactions and selective removal of PMMA blocks. Nanoporous hybrid composites with a stable morphology were obtained either by applying the UV irradiation protocol to BCP nanocomposites characterized by selective inclusion of zinc oxide (ZnO) nanoparticles (NPs) in the PS domains (nanocomposite first/nanopores after) or by selective infiltration of cadmium selenide (CdSe) NPs in the nanochannels left free by PMMA removal through UV irradiation (nanopores first/ nanocomposite after), demonstating the strenght of the approach.
The adsorption of myoglobin (Mb) onto nanoporous thin films build up using block‐copolymers (BCPs) is analyzed. The nanoporous thin films, fabricated by exploiting self‐assembly of lamellar BCPs and the concept of sacrificial block, are characterized by a well‐defined morphology containing nanochannels of width ≈20 nm delimited by polystyrene (PS) domains, decorated with pendant poly(ethylene oxide) (PEO) chains. The adsorption of Mb onto the nanoporous material is studied by means of UV–visible spectroscopy, quartz crystal microbalance, and neutron reflectometry measurements. In order to determine the role of nanopores, experiments are also conducted by using supports made of nonporous PS thin films and nude glass slides. The results indicate that the BCP‐nanoporous material exhibits a remarkably higher adsorption capability than PS and glass. As PEO exhibits a low degree of protein adsorption, this result may be essentially attributed to the presence of the nanopores. The large surface area, the opened pore structure, and the trapping effect of the pores are the main factors determining the increased Mb adsorption capability of the BCP‐based support. Yet, the presence of PEO chains decorating the PS walls at porous surface does not prevent Mb biomolecules to establish good interactions with the support. Block copolymers‐based nanoporous thin films with increased biomolecules adsorption ability are fabricated. The designed material is patterned with nanometric channels delimited by polystyrene walls covalently linked to poly(ethylene oxide) pendant chains. The large surface area and the tailored pore sizes are the key factors determining the observed increased amount of adsorbed protein.
The present study describes a simple strategy to integrate electrochemical detection with an assembled microchip-capillary electrophoresis platform. The electrochemical cell was integrated with a microfluidic device consisting of five plastic squares interconnected with fused silica capillaries, forming a four-way injection cross between the separation channel and three side-arms (each of 15 mm in length) acting as buffer/sample reservoirs. The performance of the system was evaluated using electrodes made with either carbon ink, carbon nanotubes, or gold and under different experimental conditions of pH, capillary length, and injection time. Using this system it was possible to separate the neurotransmitters dopamine and cathecol and to quantify phenol from a real sample using a linear calibration curve with a calculated LOD of 0.7 µM. A similar concept was applied to determine glucose, by including a pre-reactor filled with beads modified with glucose oxidase (GOx). The latter system was used to determine glucose in a commercial sample, with a recovery of 95.2 %. Overall, the presented approach represents a simple, inexpensive, and versatile approach to integrate electrochemical detection with CE separations without requiring access to microfabrication facilities.
Since its introduction in 1994, the preparation of ordered porous polymer films by the breath figure (BF) method has received a considerable interest. The so-called “honeycomb” (HC) films exhibit a hexagonal array of micrometric pores obtained by water droplet condensation during the fast solvent evaporation performed under a humid flow. The main focus of this feature article is to describe the recent advances in the design of honeycomb polymer films by the BF process. We first review the recent studies related to the honeycomb film formation through the exploration of different parameters such as the relative humidity, the polymer concentration, the drying rate, the substrate or the role of interfacial tension. The influence of the architecture and microstructure of the polymer is examined through examples. In this contribution, a special attention is given to the recent articles focused on the preparation of elaborate functional honeycomb-structured polymer films obtained via the simple BF method. In this context, we review the preparation of hierarchical HC films showing either sub- or super-structure, the formation of hybrid HC films by self-assembly of nanoparticles or in situ generation of the inorganic matter, the fluorescence in HC films introduced either by a fluorescent polymer or by fluorescent chemical groups, the elaboration of biomaterials from HC films decorated by glycopolymer and/or showing sensing ability and finally the design of functional polymeric surfaces with either stimuli-responsive or superhydrophobic properties.
This paper describes a simple and inexpensive procedure to produce thin-films of poly(dimethylsiloxane). Such films were characterized by a variety of techniques (ellipsometry, nuclear magnetic resonance, atomic force microscopy, and goniometry) and used to investigate the adsorption kinetics of three model proteins (fibrinogen, collagen type-I, and bovine serum albumin) under different conditions. The information collected from the protein adsorption studies was then used to investigate the adhesion of human dermal microvascular endothelial cells. The results of these studies suggest that these films can be used to model the surface properties of microdevices fabricated with commercial PDMS. Moreover, the paper provides guidelines to efficiently attach cells in BioMEMS devices.
MicroRNAs (miRNAs) are key posttranscriptional regulators of gene expression involved in diverse biological pathways in bilateral animals and plants. The key to understanding the biological function of a miRNA is to identify its regulatory targets. Although a few miRNA targets have been identified genetically, the rapidly expanding list of miRNAs has necessitated genome-wide tools for identifying target mRNAs, and a number of computational and experimental approaches have consequently emerged. Some of these approaches have also provided insights into the mechanistic aspects of miRNA-mediated regulation, another intensely debated area in the miRNA field. Here, we review several emerging features of miRNA-target interactions in animals and genome-wide approaches for probing those interactions.
The convolution of tip shape on sample topography can introduce significant inaccuracy in an AFM image, when the tip radius is comparable to the typical dimension of the sample features to be observed. The blind estimation method allows one to obtain information on the AFM tip through an unknown characterizer sample and thus to perform the deconvolution of the tip shape from an image. When applying the blind estimation method to determine the AFM tip shape, some apparently trivial issues relating to the experimental operating parameters must be taken into account. In this paper, the effects of the operating parameters, e.g., sampling intervals (resolution) and instrumental noise, have been taken into account for the practical use of blind estimation and the result is that instrumental noise tends to provide a smaller estimation of the tip size, while larger sampling intervals provide a larger value of it. This paper presents guidelines to those effects in AFM and appropriate experimental conditions for applying the blind estimation method to obtain more reliable data on tip radius and therefore on sample topography.
The paper describes a set of simple experiments performed to develop an optical model to describe Si/SiO(2) substrates coated with two transparent films of carbon nanotubes. The final goal is to use such optical model to investigate the interaction of proteins with carbon nanotubes. Experiments were performed to assess light reflection as a function of the wavelength or angle of incidence using two substrates (same material, different amounts) composed of oxidized carbon nanotubes. The experimental results indicate that the selected carbon nanotubes layers are anisotropic and significantly different from each other. Experiments performed by spectroscopic ellipsometry (as a function of the wavelength and incident angle) enabled the development of an Effective Medium Approximation model consisting in a two-fraction phase (arc-evaporated carbon and void space). Furthermore, the model enabled calculating the amount of protein adsorbed on the surface of the carbon nanotubes film.
The interactions of proteins with solid surfaces occur in a variety of situations. Motivated by the many nanoengineering applications of protein-carbon nanotube hybrids, we investigate the conformational transitions of hen egg white lysozyme adsorbed on a carbon nanotube. Using a C(α) structure-based model and replica exchange molecular dynamics, we show how the folding/unfolding equilibrium of the adsorbed protein varies with the strength of its coupling to the surface. The stability of the native state depends on the balance between the favorable entropy and unfavorable enthalpy change on adsorption. In the case of a weakly attractive surface when the former dominates, the protein is stabilized. In this regime, the protein can fold and unfold while maintaining the same binding fraction. With increasing surface attraction, the unfavorable enthalpic effect dominates, the native state is destabilized, and the protein has to extensively unbind before changing states from unfolded to folded. At the highest surface coupling, the entropic penalty of folding vanishes, and a folding intermediate is strongly stabilized. In this intermediate state, the α-domain of lysozyme is disrupted, while the β-sheet remains fully structured. We rationalize the relative stability of the two domains on the basis of the residue contact order.
This paper is the first report on the characterization of the hydrodynamic conditions in a flow cell designed to study adsorption processes by spectroscopic ellipsometry. The resulting cell enables combining the advantages of in situ spectroscopic ellipsometry with stagnation point flow conditions. An additional advantage is that the proposed cell features a fixed position of the "inlet tube" with respect to the substrate, thus facilitating the alignment of multiple substrates. Theoretical calculations were performed by computational fluid dynamics and compared with experimental data (adsorption kinetics) obtained for the adsorption of polyethylene glycol to silica under a variety of experimental conditions. Additionally, a simple methodology to correct experimental data for errors associated with the size of the measured spot and for variations of mass transfer in the vicinity of the stagnation point is herein introduced. The proposed correction method would allow researchers to reasonably estimate the adsorption kinetics at the stagnation point and quantitatively compare their results, even when using different experimental setups. The applicability of the proposed correction function was verified by evaluating the kinetics of protein adsorption under different experimental conditions.
The authors measured ionic current blockages caused by protein translocation through voltage-biased silicon nitride nanopores in ionic solution. By calculating the mean amplitude, time duration, and the integral of current blockages, they estimated the relative charge and size of protein molecules at a single molecule level. The authors measured the change in protein charge of bovine serum albumin (BSA) protein induced by pH variation. They also confirmed that BSA molecules indeed traverse nanopores using an improved chemiluminescent analysis. They demonstrated that a larger protein fibrinogen could be distinguished from BSA by a solid-state nanopore measurement.
Basic aspects and examples of biosensors, including definitions and applications, response of enzyme-based biosensors, examples of biosensor configurations, ferrocene-mediated amperometric glucose sensor, potentiometric biosensor for phenyl acetate, potentiometric immunosensor for digoxin, evanescent-wave fluorescence biosensor for bungarotoxin, optical biosensor for glucose based on fluorescence energy transfer, piezoelectric biosensor for nucleic acid detection, enzyme thermistors, evaluation of biosensor performance.
The adsorption of glucose oxidase (GOx) was studied at the interface between two immiscible electrolyte solutions (ITIES) by interfacial capacitance and surface tension measurements and at the air/water (phosphate buffer) interface by surface tension and neutron reflection measurements. The adsorption at both interfaces was found to be time, enzyme concentration, and ionic strength dependent. There was a switch from one interfacial adsorption state to another, as the enzyme concentration was increased. At the ITIES, there was evidence of an interaction between the adsorbed enzyme and the hydrophobic cation in the organic phase (1,2-dichloroethane). The enzyme adsorbed at the air/water interface was found to dissociate into monomers at the lower buffer total concentration of 2 mM while, at the higher buffer concentration of 0.2 M, the adsorbed enzyme retained its dimer structure. The adsorption mostly formed monolayers and the layer thickness varied with bulk concentration, indicating deformation related to the packing of the enzyme at the interface. For enzyme concentrations above 1 muM, in high ionic strength medium, bilayers of enzyme started to form, and the interlayer interactions resulted in a less densely packed second layer forming on the aqueous side of the first one. The switch in properties of the adsorbed layer observed in interfacial tension and capacitance measurements at the ITIES occurred over the same enzyme concentration range as the formation of a more densely packed layer detected from neutron reflection at the air/water interface.
Recently, a new spotlight has been focused on block copolymers, thoroughly studied for nearly half a century, because of their potential use in numerous nanotechnologies. This renewed interest is a consequence of the self-assembled microdomains characteristic of these materials. The size, shape, and arrangement of these nanoscopic structures are all tunable through synthetic chemistry of the constituent molecules. Capturing the vast technological potential of block copolymers will, in many cases, require precise control over the orientation and alignment of the microdomains. This review summarizes extant applications and alignment techniques and provides an outlook toward the future. In an effort to provide a practical resource for researchers, the article is structured to identify the reported alignment approaches for a given polymer morphology rather than sorting by alignment technique. Specific materials have also been deemphasized because the alignment methods, with few exceptions, are general to a specific morphology or set of morphologies. In addition to a detailed summary of traditional methodologies, some very recent results such as optical alignment of liquid crystalline block copolymers, lithographic chemical patterning, and alignment in pores are highlighted.
Active Langmuir-Blodgett (LB) films of glucose oxidase (GO, Aspergillus niger) can be prepared directly by spreading the enzyme at the air-water interface and transferring to a substrate; no interaction with lipid films is required. Native enzyme LB film activity is poor, however, as determined by electrolysis of H2O2 produced by reaction with glucose and O2. Modified enzyme can be prepared by reaction with glutaraldehyde before spreading on the subphase, and this results in LB films on Pt substrates that are 5 to 15 times more active, depending on reaction conditions. These films exhibit an activity similar to conventional bovine serum albumin immobilized enzyme electrodes and a response time of less than 3 s. Nominally monomolecular layers are formed by using GO modified by reaction with glutaraldehyde for 24 h at 5 °C, followed by ultrafiltration to remove oligomers before spreading on the subphase. Deposition at a surface pressure of 30 mN/m onto Si gives a film thickness of 48 angstrom/layer, compared to 30 angstrom/layer for the native enzyme. Films of ultrafiltered, modified GO appear smooth at 70 000 times magnification using scanning electron microscopy (SEM).
Spectroscopic ellipsometry was used to characterize the optical properties of thin (< 5 nm) films of nanostructured titanium dioxide (TiO2). These films were then used to investigate the dynamic adsorption of bovine serum albumin (BSA, a model protein), as a function of protein concentration, pH, and ionic strength. Experimental results were analyzed by an optical model and revealed that hydrophobic interactions were the main driving force behind the adsorption process, resulting in up to 3.5 mg/m2 of albumin adsorbed to nanostructured TiO2. The measured thickness of the adsorbed BSA layer (less than 4 nm) supports the possibility that spreading of the protein molecules on the material surface occurred. Conformational changes of adsorbed proteins are important because they may subsequently lead to either accessibility or inaccessibility of bioactive sites which are ligands for cell interaction and function relevant to physiology and pathology.
Morphologies of polystyrene-block-poly(2-vinylpyridine) copolymer (S2VP) thin films, which are forming poly(2-vinylpyridine) cylinders in bulk phase, were investigated by atomic force microscopy (AFM) and transmission electron microscopy (TEM) to account for their ordering behavior induced by solvent annealing. Initially, when the copolymer was dissolved in toluene, which is selective solvent for majority polystyrene (PS) blocks, and was spin-coated on Si substrates, dimple-type micellar structures of S2VP were formed. After the film was placed in a solvent-annealing chamber covered with a lid under the existence of chloroform, surface morphologies of S2VP were measured as a function of annealing time. In this study, it was found that the morphologies of S2VP thin film repeated the cycle of the creation and extinction of various morphologies on ordering process. Namely, S2VP exhibited the various transformations between different morphologies, including highly disordered state, cylinders normal to the plane, and cylinders parallel to the plane. Each of the morphologies observed here was employed as a template to synthesize gold (Au) nanoparticles or nanowires. The arrays of Au nano-objects were used to tune a surface plasmon resonance.
This study is the first to focus on the potential use of carbon nanotube (CNT) scaffolds as enzyme immobilization substrates for analytical purposes. Besides all the well-known advantages of CNT, three-dimensional scaffolds can significantly increase the amount of enzymes adsorbed per unit area, preserve the catalytic activity of the adsorbed molecules, and allow effective exposure to substrates present in the adjacent medium. Additionally, our results indicate that the sensitivity of analytical probes based on enzyme-loaded CNT scaffolds is proportional to the thickness of the scaffold providing 3-fold sensitivity improvements with respect to the control surfaces.
The distortion of images by the finite radius of the tip is a significant artifact in atomic force microscopy (AFM) imaging of thin films. This article examines the effect of the finite radius of the AFM tip on the measurement of five surface roughness parameters: R a and R rms roughness, horizontal roughness, bearing ratio, and power spectral density. The effect of the finite size of the AFM tip on the measurement of these roughness parameters was studied using simulations. We found that the vertical roughness measures (R a and R rms ) were the least affected by the tip induced distortion. For a spherical tip, the error in R a and R rms roughness was less than 15% as long as the radius of the major features in the AFM image (R AFM ) was greater than twice the radius of the tip (R tip ). The horizontal roughness of the surface was sensitive to the distortion due to the tip. For distorted AFM images (R AFM /R tip =5) the bearing ratio curve was similar in shape to the curve for the original surface. The bearing ratio curve of the severely distorted profile (R AFM /R tip =1.5) was significantly different from the curve for the original trial surface. The general shape of the power spectral density (PSD) curves for the original and both distorted profiles were similar. However, the lower frequency peaks in the PSD curves for the distorted profiles differed from those in the PSD curve for the original surface. © 1995 American Vacuum Society
Atomic force microscopy (AFM) has been developed as a tool for investigating any type of surface at an atomic scale. Since then, except for particular surfaces exhibiting an atomic roughness, we know that the finite size of the tip does not allow us to access at the whole structure of the surface. Moreover, the tip geometry radically modifies the range of the interactions. Therefore, even an approximate knowledge of the tip geometry is of particular importance. The aim of the present note is to provide a simple way to get the main parameters of a tip — its apex radius of curvature and the cone angle — by using a simple reference sample: latex balls. To do so, simple geometrical arguments are used, assuming that both the tip and the sample behave like hard samples, a reasonable assumption at a mesoscopic scale (tens of nanometres to micrometres). Using this simplifying assumption, we present a formula which could be used to rapidly evaluate the effect of the finite size of the apex of the tip in the formation of AFM images of simple objects: steps, isolated spheres or two-dimensional close-packed lattices of spheres, and cosinusoidal corrugations. Two types of tip geometry are presupposed: a conic tip truncated by a spherical apex or a parabolic tip. It is then shown that, in practice, latex balls can be used as a reference to estimate the radius of curvature of the apex, and the angle of the cone.
The nanometer-scale architectures in thin films of self-assembling block copolymers have inspired a variety of new applications. For example, the uniformly sized and shaped nanodomains formed in the films have been used for nanolithography, nanoparticle synthesis, and high-density information storage media. Imperative to all of these applications, however, is a high degree of control over orientation of the nanodomains relative to the surface of the film as well as control over order in the plane of the film. Induced fields including electric, shear, and surface fields have been demonstrated to influence orientation. Both heteroepitaxy and graphoepitaxy can induce positional order on the nanodomains in the plane of the film. This article will briefly review a variety of mechanisms for gaining control over block copolymer order as well as many of the applications of these materials. Particular attention is paid to the potential of perfecting long-range two-dimensional order over a broader range of length scales and the extension of these concepts to functional materials and more complex architectures.
We report the detection of protein molecules with nanofabricated pores using the resistive pulse sensing method. A 20-nm-thick silicon nitride membrane with a nanofabricated pore measuring about 55 nm in diameter separated an electrolyte cell into two compartments. Current spike trains were observed when bovine serum albumin (BSA) was added to the negatively biased compartment. The magnitude of the spikes corresponded to particles 7-9 nm in diameter (the size of a BSA molecule) passing through the pore. This suggests that the current spikes were current blockages caused by single BSA molecules. The presented nano-Coulter counting method could be applied to detect single protein molecules in free solution, and to study the translocation of proteins through a pore. (c) 2006 American Institute of Physics.
The objective of this investigation was to monitor the adsorption of antibodies to polystyrene surfaces using ellipsometry. Commercial polystyrene slides used for solid state diagnostics were selected as substrates and the adsorption of three different antibodies (human IgG, bovine IgG and goat anti-human IgG) were evaluated. Based on theoretical models describing the ellipsometric data, it was concluded that the adsorption of antibodies should result in layers that are sufficiently thick to be able to monitor the adsorption in terms of adsorbed amount and thickness of the layer with a reasonable precision. The experimental results confirmed this assumption and values of 2.0-2.3 mg/m(2) were detected for the adsorbed amount with a corresponding thickness of 10-16 nm. It was furthermore found that the antibodies bound irreversibly with respect to rinsing with protein-free solutions. In additional experiments, the consecutive incubation of human IgG and anti-human IgG was investigated. These results showed that, on average, approximately half of the surface immobilized anti-human IgG molecules are capable of binding to human IgG during its incubation. From the consecutive binding experiments it could also be concluded that antibodies present in the polyclonal anti-human IgG preparation were capable of binding to around four different epitopes on the human IgG. A final set of experiments addressed the stability of adsorbed human IgG layers with respect to drying and incubation with surfactant. The results revealed that the adsorbed antibody layer is relatively resistant to these treatments.
This paper describes a methodology for the rapid and highly selective detection of cocaine using a membrane protein channel combined with a DNA aptamer. The DNA aptamer recognizes the cocaine molecule with high selectivity. We successfully detected a low concentration of cocaine (300 ng/mL, the drug test cutoff limit) within 60 s using a biological nanopore embedded in a microchip.
Spatial confinement from the nano- to the microscale is ubiquitous in nature. Striving to understand the behavior of nanoscale objects in confined domains we present a nanofluidic silicon device which consists of two stacked nanopores forming the in/outlets to a pyramidal cavity of micrometer dimensions (10 fL volume). Being electrically addressable, charged objects can be actively loaded into, trapped inside, and unloaded from the "pore-cavity-pore" (PCP) device. When operated passively, confined Brownian motion and the entropy barriers of the nanopores govern the behavior of nano-objects within the PCP device. We present measurements with single fluorescent nanoparticles as well as particle-ensembles and analyze their trajectories and residence times. Experimental data are compared to random walk simulations and analytical theories on confined diffusion and the Brownian escape of nano-objects across entropy barriers. Single particle data corroborate analytical solutions of the narrow escape problem, but ensemble measurements indicate crowding effects even at low particle concentrations. The utilization of the device to trap biomolecules is demonstrated for single λ-DNA molecules.
The adsorption conditions used to immobilize catalase onto thin films of carbon nanotubes were investigated to elucidate the conditions that produced films with maximum amounts of active catalase. The adsorption kinetics were monitored by spectroscopic ellipsometry, and the immobilized catalase films were then assayed for catalytic activity. The development of a volumetric optical model used to interpret the ellipsometric data is discussed. According to the results herein discussed, not only the adsorbed amount but also the initial adsorption rates determine the final catalytic activity of the adsorbed layer. The results described in this paper have direct implications on the rational design and analytical performance of enzymatic biosensors.
This communication describes a simple way to improve the sensitivity of spectroscopic ellipsometry, when applied to monitor the adsorption of proteins to solid surfaces. The method described herein is based on the reaction of a commercially available dye (Coomassie brilliant blue G) with the adsorbed proteins and the subsequent analysis by spectroscopic ellipsometry. In order to demonstrate the potential advantages of this method, the adsorption of bovine serum albumin to an antifouling coating was also investigated. According to our results, the modification with the dye significantly affects the optical properties of the adsorbed protein layer, which can be represented using a simple optical model (Lorentz). In general, the proposed modification increases the sensitivity of the detection by 2.5 ± 0.4-fold and enables the analysis of thin layers of adsorbed protein not obtainable by conventional methods. These results particularly reveal the importance of the proposed modification for the evaluation of low adsorbing substrates and antifouling coatings.
We reported the deliberate control on the micelle opening and closing of amphiphilic polystyrene-block-poly(2-vinylpyridine) (PS-b-P2VP) micellar films by exposing them to selective solvents. We first treated the micellar films with polar solvents including ethanol and water (pH = 4, 8, and 12) that have different affinities to P2VP. We observed opening of the micelles in all the cases. Both the size of opened pores and the opening rate are dependent on the solvency of different solvents for P2VP. We then explored the closing behavior of the opened micelles using solvents having different affinities to PS. We found that the opened micelles were recovered to their initial closed micelle forms. The recovery was accompanied by a slow micelle disassociation process which gradually reduced the micelle size. The rates of the micelle closing and disassociation are also dependent on the solvency of different solvents for PS.
The interactions between single protein molecules and nanoporous polycarbonate membranes were investigated at the single molecule level. Entrapment of proteins was shown to be size selective and was dependent on the membrane pore diameter. A pore size that is only slightly larger than the maximum dimension of the proteins was inadequate for intrusion into the pores. For a given protein, the number of molecules found at a given depth decreased as the pore size decreased. In addition, as the depth increased, for a given size pore, the number of molecules decreased rapidly. The depth-dependent histograms nicely fit a one-dimensional diffusion model. However, a highly restricted motion was observed even when the pore diameter was 10 times the size of the protein, resulting in anomalously small diffusion coefficients. We also demonstrated the subtle differences in depth distribution among BSA and hemoglobin that have nearly the same molecular weight but slightly different molecular shapes. These results give unique insights into the detailed mechanism of size-exclusion chromatography and membrane filtration.
We use single silicon nitride nanopores to study folded, partially folded, and unfolded single proteins by measuring their excluded volumes. The DNA-calibrated translocation signals of beta-lactoglobulin and histidine-containing phosphocarrier protein match quantitatively with that predicted by a simple sum of the partial volumes of the amino acids in the polypeptide segment inside the pore when translocation stalls due to the primary charge sequence. Our analysis suggests that the majority of the protein molecules were linear or looped during translocation and that the electrical forces present under physiologically relevant potentials can unfold proteins. Our results show that the nanopore translocation signals are sensitive enough to distinguish the folding state of a protein and distinguish between proteins based on the excluded volume of a local segment of the polypeptide chain that transiently stalls in the nanopore due to the primary sequence of charges.
We have investigated the interaction of d-amino acid oxidase (DAAO) with single-walled carbon nanotubes (CNT) by spectroscopic ellipsometry. Dynamic adsorption experiments were performed at different experimental conditions. In addition, the activity of the enzyme adsorbed at different conditions was studied. Our results indicate that DAAO can be adsorbed to CNT at different pH values and concentrations by a combination of hydrophobic and electrostatic interactions. Considering that the highest enzymatic activity was obtained by adsorbing the protein at pH 5.7 and 0.1 mg x mL(-1), our results indicate that DAAO can adopt multiple orientations on the surface, which are ultimately responsible for significant differences in catalytic activity.
We explored surface-anchored poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) brushes as potential templates for protein immobilization. The brushes were grown using atom transfer radical polymerization from surface-anchored initiators and characterized by a combination of ellipsometry, atomic force microscopy, and X-ray photoelectron spectroscopy. RNase A was immobilized as a model enzyme through the nucleophilic attack of azlactone by the amine groups in the lysines located in the protein. The surface density of RNase A increased linearly from 5 to 50 nm. For 50 nm thick poly(2-vinyl-4,4-dimethyl azlactone) brushes, 7.5 microg/cm2 of RNase A was bound. The kinetics and thermodynamics of RNase A immobilization, the activity relative to surface density, and the pH and temperature dependence were examined. A Langmuir-like model for binding kinetics indicates that the kinetics are controlled by the rate of adsorption of RNase A and has an adsorption rate constant, k(ads), of 2.8 x 10(-8) microg(-1) s(-1) cm3. A maximum relative activity of approximately 0.95, which is near the activity of free RNase A, was reached at 1.2 microg/cm2 (approximately 3.0 monolayers) of immobilized RNase A. The immobilized RNase A had a similar temperature and pH dependence as free RNase A, indicating no significant change in conformation. The PVDMA template was extended to other biotechnologically relevant enzymes, such as deoxyribonuclease I, glucose oxidase, glucoamylase, and trypsin, with relative activities higher than or comparable to those of enzymes immobilized by other means. PVDMA brushes offer an efficient route to immobilize proteins via the ring opening of azlactone without the need for activation or pretreatment while retaining high relative activities of the bound enzymes.
Functionalization of carbon nanotubes (CNTs) with proteins is often a key step in their biological applications, particularly in biosensing. One popular method has used the cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to covalently conjugate proteins onto carboxylated CNTs. In this article, we critically assess the evidence presented in these conjugation studies in the literature. As CNTs have a natural affinity for diverse proteins through hydrophobic and electrostatic interactions, it is therefore important to differentiate protein covalent attachment from adsorption in the immobilization mechanism. Unfortunately, many studies of conjugating proteins onto CNTs using EDC lacked essential controls to eliminate the possibility of protein adsorption. In studies where the attachment was claimed to be covalent, discrepancies existed and the observed immobilization appeared to be due to adsorption. So far, bond analysis has been lacking to ascertain the nature of the attachment using EDC. We recommend that this approach of covalent immobilization of proteins on CNTs be re-evaluated and treated with caution.
The tertiary structure of glucose oxidase (GOD) from Aspergillus niger was determined by x-ray crystallography (to be described elsewhere). The overall folding of the enzyme is described with regard to its application in biosensors, and conclusions are drawn from experiments on electrical communication between the enzyme and the electrodes.
Glucose oxidase (GOD) from Aspergillus niger is an acidic dimeric enzyme having a high degree of localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations on the structure and stability of GOD using various optical spectroscopic techniques, limited proteolysis, size exclusion chromatography, differential scanning calorimetry, and enzymic activity measurements. The monovalent cations were found to influence the enzymic activity and tertiary structure of GOD, but no effect on the secondary structure of the enzyme was observed. The monovalent cation-stabilized GOD was found to have a more compact dimeric structure but lower enzymic activity than the native enzyme. The enzyme's K(m) for D-glucose was found to be slightly enhanced for the monovalent cation-stabilized enzyme (maximum enhancement of about 35% for LiCl) as compared to native GOD. Comparative denaturation studies on the native and monovalent cation-stabilized enzyme demonstrated a significant resistance of cation-stabilized GOD to urea (about 50% residual activity at 6.5 M urea) and thermal denaturation (Delta T(m) maximum of 10 degrees C compared to native enzyme). However, pH-induced denaturation showed a destabilization of monovalent cation-stabilized GOD as compared to the native enzyme. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister series: K(+) > Na(+) > Li(+).
We have carried out a systematic study on the guanidinium chloride- and urea-induced unfolding of glucose oxidase from Aspergillus niger, an acidic dimeric enzyme, using various optical spectroscopic techniques, enzymatic activity measurements, glutaraldehyde cross-linking, and differential scanning calorimetry. The urea-induced unfolding of GOD was a two-state process with dissociation and unfolding of the native dimeric enzyme molecule occurring in a single step. On the contrary, the GdmCl-induced unfolding of GOD was a multiphasic process with stabilization of a conformation more compact than the native enzyme at low GdmCl concentrations and dissociation along with unfolding of enzyme at higher concentrations of GdmCl. The GdmCl-stabilized compact dimeric intermediate of GOD showed an enhanced stability against thermal and urea denaturation as compared to the native GOD dimer. Comparative studies on GOD using GdmCl and NaCl demonstrated that binding of the Gdm(+) cation to the enzyme results in stabilization of the compact dimeric intermediate of the enzyme at low GdmCl concentrations. An interesting observation was that a slight difference in the concentration of urea and GdmCl associated with the unfolding of GOD was observed, which is in violation of the 2-fold rule for urea and GdmCl denaturation of proteins. This is the first report where violation of the 2-fold rule has been observed for a multimeric protein.
Adsorption of chicken egg lysozyme on silica nanoparticles of various diameters has been studied. Special attention has been paid to the effect of nanoparticle size on the structure and function of the adsorbed protein molecules. Both adsorption patterns and protein structure and function are strongly dependent on the size of the nanoparticles. Formation of molecular complexes is observed for adsorption onto 4-nm silica. True adsorptive behavior is evident on 20- and 100-nm particles, with the former resulting in monolayer adsorption and the latter yielding multilayer adsorption. A decrease in the solution pH results in a decrease in lysozyme adsorption. A change of protein structure upon adsorption is observed, as characterized by a loss in alpha-helix content, and this is strongly dependent on the size of the nanoparticle and the solution pH. Generally, greater loss of alpha helicity was observed for the lysozyme adsorbed onto larger nanoparticles under otherwise similar conditions. The activity of lysozyme adsorbed onto silica nanoparticles is lower than that of the free protein, and the fraction of activity lost correlates well with the decrease in alpha-helix content. These results indicate that the size of the nanoparticle, perhaps because of the contributions of surface curvature, influences adsorbed protein structure and function.
We have examined the structure and function of two enzymes, alpha-chymotrypsin (CT) and soybean peroxidase (SBP), adsorbed onto single-walled carbon nanotubes (SWNTs). SBP retained up to 30% of its native activity upon adsorption, while the adsorbed CT retained only 1% of its native activity. Analysis of the secondary structure of the proteins via FT-IR spectroscopy revealed that both enzymes undergo structural changes upon adsorption, with substantial secondary structural perturbation observed for CT. Consistent with these results, AFM images of the adsorbed enzymes indicated that SBP retains its native three-dimensional shape while CT appears to unfold on the SWNT surface. This study represents the first in depth investigation of protein structure and function on carbon nanotubes, which is critical in designing optimal carbon nanotube-protein conjugates.
Novel methods for immobilizing proteins on surfaces have the potential to impact basic biological research as well as various biochip applications. Here, we demonstrate a unique method to pattern proteins with a nanometer periodicity on silicon oxide substrates using microphase-separated diblock copolymer thin films. We developed a straightforward and effective protein immobilization technique using the microphase-separated domains of polystyrene-block-poly(methyl methacrylate) to localize various model protein molecules such as bovine immunoglobulin G, fluorescein isothiocyanate conjugated anti-bovine immunoglobulin G, and protein G. The self-organizing nature of the diblock copolymer was exploited to produce periodically alternating, nanometer-spaced polymeric domains exposing the two chemical compositions of the diblock to surface. We demonstrate that the model proteins selectively self-organize themselves on the microdomain regions of specific polymer components due to their preferential interactions with one of the two polymer segments. This diblock copolymer-based, self-assembly approach represents a step forward for facile, nanometer-spaced protein immobilization with high areal density and could provide a pathway to high-throughput proteomic arrays and biosensors.
X-ray photoemission electron microscopy using synchrotron radiation illumination has been used to measure the spatial distributions of albumin on a phase-segregated polystyrene/poly(methyl methacrylate) (PS/PMMA) polymer thin film following adsorption from unbuffered, deionized aqueous solutions under a range of solution concentrations and exposure times. Chemical mapping of the albumin, PS, and PMMA shows that the distribution of albumin on different adsorption sites (PS, PMMA, and the interface between the PS and PMMA domains) changes depending on the concentration of the albumin solution and the exposure time. The preferred sites of absorption at low concentration and short exposure are the PS/PMMA interfaces. Albumin shows a stronger preference for the PS domains than the PMMA domains. The exposure-time dependence suggests that a dynamic equilibrium between albumin in solution and adsorbed on PS domains is established in a shorter time than is required for equilibrating albumin between the solution and the PMMA domains. The explanation of these preferences in terms of possible adsorption mechanisms is discussed.
A method to immobilize proteins at predefined position using gold-conjugated protein nanoarrays was investigated. Two different proteins, gold nanoparticle-conjugated streptavidin and gold-conjugated protein G molecules were selected as model proteins in the study. The individual protein molecules are immobilized and stabilized with inherent spacing without interfering with the intermolecular interactions. The electrochemical behavior of the exposed gold film was studied as a function of the RIE time to verify whether the ordered pore region is exposed to the underlying surface, which is critical for the immobilization of probe biomolecules. The selective deposition of SA-AuNPs on the biotin-modified surface was further supported by CV analysis, where the voltammetric peak current associated with the redox pair became substantially lower as the surface became blocked by adsorbed streptavidin molecule. This method provides a promising tool for biosensor applications.
Nanofabricated pores in 20 nm-thick silicon nitride membranes were used to probe various protein analytes as well as to perform an antigen-antibody binding assay. A two-compartment electrochemical cell was separated by a single nanopore, 28 nm in diameter. Adding proteins to one compartment caused current perturbations in the ion current flowing through the pore. These perturbations correlated with both the charge and the size of the protein or of a protein-protein complex. The potential of this nanotechnology for studying protein-protein interactions is highlighted with the sensitive detection of beta-human chorionic gonadotropin, a hormone and clinical biomarker of pregnancy, by monitoring in real time and at a molecular level the formation of a complex between hormones and antibodies in solution. In this form, the assay compared advantageously to immunoassays, with the important difference that labels, immobilization, or amplification steps were no longer needed. In conclusion, we present proof-of-principle that properties of proteins and their interactions can be investigated in solution using synthetic nanopores and that these interactions can be exploited to measure protein concentrations accurately.
The alpha-hemolysin (alphaHL) protein pore has many applications in biotechnology. This article describes a single-molecule manipulation system that utilizes the nanocavity enclosed by this pore to noncovalently encapsulate a guest molecule. The guest is the thrombin-binding aptamer (TBA) that folds into the G-quadruplex in the presence of cations. Trapping the G-quadruplex in the nanocavity resulted in characteristic changes to the pore conductance that revealed important molecular processes, including spontaneous unfolding of the quartet structure and translocation of unfolded DNA in the pore. Through detection with Tag-TBA, we localized the G-quadruplex near the entry of the beta-barrel inside the nanocavity, where the molecule vibrates and rotates to different orientations. This guest-nanocavity supramolecular system has potential for helping to understand single-molecule folding and unfolding kinetics.
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