Immobilization Of Biotin Research Articles

Do-It-Yourself Pyramidal Mold for Nanotechnology.

The handcrafted fabrication of a pyramidal mold on a silicon wafer for nanopatterning was investigated. This process started with the manual delivery of an aqueous glycerol solution onto the SiO2/Si wafer using a micropipette and subsequent drying to form a hemisphere whose diameter is in the range of hundreds of micrometers. A coating of polystyrene (PS) onto this wafer generates a circular hole caused by dewetting. Subsequently, anisotropic wet-etching with the PS film as a mask produces a pyramidal trench, whose apex approaches hundreds of nanometers. Various elastomeric materials were casted into this pyramidal mold. A pyramidal tip mounted on a simple micropositioner was used for electrochemistry and patterning of a protein. First, an agarose hydrogel was cast with a hydrogel pen for the electrochemical reaction (HYPER). The redox reaction at the HYPER–electrode interface demonstrated the characteristics of an ultramicroelectrode or bulk electrode based on the contact area. Second, the pyramidal polydimethylsiloxane served as a polymer pen for the contact printing of silane on a glass substrate. After the successive immobilization of biotin and avidin with fluorescence labeling, the resulting fluorescence image demonstrated the successful patterning of the protein. This new process for the creation of a pyramidal mold, referred to as a “do-it-yourself” process, offers advantages to nonspecialists in nanotechnology compared to conventional lithography, specifically simplicity, rapidity, and low cost.

Electrospun Mesoporous Poly(Styrene‐Block‐Methyl‐ Methacrylate) Nanofibers as Biosensing Platform: Effect of Fibers Porosity on Sensitivity

AbstractThe present work demonstrates the use of mesoporous nanofibers for the enhanced analytical performance of electrochemical biosensor. By exploiting the phase separation property of the block copolymers, a simple three‐step process was used to generate the porosity in the nanofibers. Here we present the effect of the porosity on the sensing ability of the electrospun PS‐b‐PMMA nanofibers. The functional groups present on the nanofiber surface were characterized using DPV. The nanofibers modified electrode showed a large decrease in the oxidation current with the increase in the pH from 4.2 to 6.8 for the anionic redox couple whereas the change in the current is negligible for a neutral redox couple, this suggested the presence of ‐COOH groups. A one‐step process was used for the immobilization of biotin. There were about 35.5 % and 66.6 % decrease in the redox current for the as‐spun and porous nanofibers after functionalization respectively which indicate the presence of a high amount of active sites in the porous nanofibers. Finally, the sensor response was studied using streptavidin (1μg/ml–1fg/ml) as a model analyte. CV studies showed a 2.7‐fold increase whereas DPV showed a 6‐fold increase in the sensitivity for the porous nanofibers as compared to the as‐spun nanofibers.

A simple approach for fabrication of optical affinity-based bioanalytical microsystem on polymeric PEN foils.

Herein, we report a novel concept of low-cost flexible platform for fluorescence-based biosensor. The surface of polyethylene naphthalate (PEN) foil was exposed to KrF excimer laser through a photolitographic contact mask. Laser initiated surface modification resulted in micro-patterned areas with surface functional groups available for localized covalent immobilization of biotin. High affinity binding protein (albumin-binding domain (ABD) of protein G, Streptococcus G148) recognizing human serum albumin (HSA), genetically fused with streptavidin (SA-ABDwt), was immobilized on the micro-patterned surface through biotin-streptavidin coupling. Fluorescently labelled HSA analyte was detected in several blocking environments, in 1% bovine serum albumin (BSA) and 6% fetal serum albumin (FBS), respectively. We conclude that the presented novel concept enabled us to micropattern functional biosensing layers on the surface of PEN foil in a fast and easy way. It brings all necessary aspects for continuous roll-to-roll fabrication of low-cost optical bioanalytical devices.

Direct immobilization of biotin on the micro-patterned PEN foil treated by excimer laser

Polymers with functionalized surfaces have attracted a lot of attention in the last few years. Due to the progress in the techniques of polymer micro-patterning, miniaturized bioanalytical assays and biocompatible devices can be developed. In the presented work, we performed surface modification of polyethylene naphthalate (PEN) foil by an excimer laser beam through a photolithographic contact mask. The aim was to fabricate micro-patterned areas with surface functional groups available for localized covalent immobilization of biotin. It was found out that depending on the properties of the laser scans, a polymer surface exhibits different degrees of modification and as a consequence, different degrees of surface biotinylation can be achieved. Several affinity tests with optical detection of fluorescently labeled streptavidin were successfully performed on biotinylated micro-patterns of a PEN foil. The polymer surface properties were also evaluated by electrokinetic analysis, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The results have shown that PEN foils can be considered suitable substrates for construction of micro-patterned bioanalytical affinity assays.

Covalent Immobilization of Biotin on Magnetic Nanoparticles: Synthesis, Characterization, and Cytotoxicity Studies.

A simple protocol for covalent immobilization of biotin onto the surface of Fe3O4 magnetic nanoparticles (MNPs) for improving the biocompatibility of original MNPs has been realized. MNPs were first prepared by co-precipitation method which was subsequently anchored with functionalized biotin. The as-synthesized MNPs were observed to be monocrystalline as evidenced from XRD and TEM images. The covalent grafting of biotin to MNPs was confirmed by FT-IR. The XPS analysis suggested the successful preparation of Biotin-f-MNPs. The as-synthesized Biotin-f-MNPs were found to be superparamagnetic character as recorded by SQUID. Cell viability studies revealed that the biocompatibility of MNPs was improved upon Biotin immobilization.

Towards Maintenance‐Free Biosensors for Hundreds of Bind/Release Cycles

AbstractA single aptamer bioreceptor layer was formed using a common streptavidin–biotin immobilization strategy and employed for 100–365 bind/release cycles. Chemically induced aptamer unfolding and release of its bound target was accomplished using alkaline solutions with high salt concentrations or deionized (DI) water. The use of DI water scavenged from the ambient atmosphere represents a first step towards maintenance‐free biosensors that do not require the storage of liquid reagents. The aptamer binding affinity was determined by surface plasmon resonance and found to be almost constant over 100–365 bind/release cycles with a variation of less than 5 % relative standard deviation. This reversible operation of biosensors based on immobilized aptamers without storage of liquid reagents introduces a conceptually new perspective in biosensing. Such new biosensing capability will be important for distributed sensor networks, sensors in resource‐limited settings, and wearable sensor applications.

Poly(glycidyl methacrylate) grafted CdSe quantum dots by surface-initiated atom transfer radical polymerization: Novel synthesis, characterization, properties, and cytotoxicity studies

A novel approach for the synthesis of poly(glycidyl methacrylate) grafted CdSe quantum dot (QDs) (PGMA-g-CdSe) was developed. The PGMA-g-CdSe nanohybrids were synthesized by the surface-initiated atom transfer radical polymerization of glycidyl methacrylate from the surface of the strategic initiator, CdSe-BrIB QDs prepared by the interaction of 2-bromoisobutyryl bromide (BrIB) and CdSe-OH QDs. The structure, morphology, and optical property of the PGMA-g-CdSe nanohybrids were analyzed by FT-IR, XPS, TGA, XRD, TEM, and PL. The as-synthesized PGMA-g-CdSe nanohybrids having multi-epoxide groups were employed for the direct coupling of biotin via ring-opening reaction of the epoxide groups to afford the Biotin-f-PGMA-g-CdSe nanobioconjugate. The covalent immobilization of biotin onto PGMA-g-CdSe was confirmed by FT-IR, XPS, and EDX. Biocompatibility and imaging properties of the Biotin-f-PGMA-g-CdSe were investigated by MTT bioassay and PL analysis, respectively. The cell viability study suggested that the biocompatibility was significantly enhanced by the functionalization of CdSe QDs by biotin and PGMA.

Sensitivity improved plasmonic gold nanoholes array biosensor by coupling quantum-dots for the detection of specific biomolecular interactions

In this paper, we focused on the large-scale fabrication of gold nanoholes array capable of supporting surface plasmonic resonance (SPR) via the developed nanosphere lithography (NSL) technique, which could be used as high performance biosensor for the detection of specific streptavidin–biotin interactions. Direct UV–vis absorption mode measurement was used to monitor the SPR peak shift. For the better immobilization of biotin, the surface of gold nanoholes array was functionalized with 3-mercaptopropyl trimethoxysilane (MPTS) and 3-aminopropyl triethoxysilane (APTES). After the streptavidin binding to the biotin, the SPR peak position showed an 11nm wavelength shift due to the refractive index change caused by the biotin–streptavidin binding. The sealing treatment was performed by using bovine serum albumin (BSA) to eliminate the influences of nonspecific adsorption for more accurate detection. Interestingly, the detection sensitivity of the gold nanoholes array could be further enhanced by coupling the water-soluble CdSe/ZnS quantum dots (QDs), which showed four-fold improvement in detection sensitivity as compared to the gold nanoholes array biosensor without the coupling of QDs. The mechanisms for the enhancement of detection sensitivity were also discussed. This would provide new capabilities for the highly sensitive measurements of biomolecular binding.

Functionalization of Nanostructured ZnO Films by Copper-Free Click Reaction

The copper-free click reaction was explored as a surface functionalization methodology for ZnO nanorod films grown by metal organic chemical vapor deposition (MOCVD). 11-Azidodecanoic acid was bound to ZnO nanorod films through the carboxylic acid moiety, leaving the azide group available for Cu-free click reaction with alkynes. The azide-functionalized layer was reacted with 1-ethynylpyrene, a fluorescent probe, and with alkynated biotin, a small biomolecule. The immobilization of pyrene on the surface was probed by fluorescence spectroscopy, and the immobilization of biotin was confirmed by binding with streptavidin-fluorescein isothiocyanate (streptavidin-FITC). The functionalized ZnO films were characterized by Fourier transform infrared attenuated total reflectance (FTIR-ATR), steady-state fluorescence emission, fluorescence microscopy, and field emission scanning electron microscopy (FESEM).

3D-nanostructured scaffold electrodes based on single-walled carbon nanotubes and nanodiamonds for high performance biosensors

We report a novel strategy for controlled nanostructuring of electrodes surfaces using single-walled nanotubes and nanodiamonds in order to form highly porous structures for biosensors applications. These nanostructures were obtained by subsequent deposition of nanotubes and nanodiamonds followed by functionalization of this assembly with pyrenenitrilotriacteic acid (NTA) via dip coating. The electropolymerizaton of the pyrene derivative leads to a reinforcement of the structure and allows simultaneously the immobilization of biotin labeled bioreceptor units via coordinative affinity interactions. By using biotin modified glucose oxidase (B-GOx) as model bioreceptor, we could obtain impressive maximum current densities and sensitivities up to 465μAcm−2 and 85.78mAM−1cm−2, respectively.

Discrimination of Differentially Inhibited Cysteine Proteases by Activity-Based Profiling Using Cystatin Variants with Tailored Specificities

Recent research has shown the possibility of tailoring the inhibitory specificity of plant cystatins toward cysteine (Cys) proteases by single mutations at positively selected amino acid sites. Here we devised a cystatin activity-based profiling approach to assess the impact of such mutations at the proteome scale using single variants of tomato cystatin SlCYS8 and digestive Cys proteases of the herbivorous insect, Colorado potato beetle, as a model. Biotinylated forms of SlCYS8 and SlCYS8 variants were used to capture susceptible Cys proteases in insect midgut protein extracts by biotin immobilization on avidin-embedded beads. A quantitative LC-MS/MS analysis of the captured proteins was performed to compare the inhibitory profile of different SlCYS8 variants. The approach confirmed the relevance of phylogenetic inferences categorizing the insect digestive Cys proteases into six functionally distinct families. It also revealed significant variation in protease family profiles captured with N-terminal variants of SlCYS8, in line with in silico structural models for Cys protease-SlCYS8 interactions suggesting a functional role for the N-terminal region. Our data confirm overall the usefulness of cystatin activity-based protease profiling for the monitoring of Cys protease-inhibitor interactions in complex biological systems. They also illustrate the potential of biotinylated cystatins to identify recombinant cystatin candidates for the inactivation of specific Cys protease targets.

Structured biotinylated poly(3,4-ethylenedioxypyrrole) electrodes for biochemical applications

The immobilization of biotin on transducer surfaces is a very important step for the fabrication of biosensors for many applications (immunoassay, DNA-hybridization assays, targeted imaging). Biotinylated polypyrroles have been studied and tested but gave rise to problems of polymerization and stability due to the intrinsic properties of pyrrole. As an alternative, biotinylated pyrroles were often used in a copolymerization with pyrrole or with an amphiphilic pyrrole derivative in a copolymerization to reduce the problems due to the pyrrole substitution. To find a new strategy, this paper presents the homopolymerization, instead of the use of a copolymerization, by replacing pyrrole by 3,4-ethylenedioxypyrrole bearing biotinylated substituent. We report the synthesis, characterization and electrochemical properties of two biotinylated 3,4-ethylenedioxypyrroles differing by the length of the alkyl spacer (ethyl or dodecyl) as well as the characterization of the corresponding polymer films. We successfully show, by cyclic voltammetry, that these monomers polymerize perfectly and give relatively stable polymer films. The increase of the alkyl spacer improves the polymerization and increases the polymer stability. For the first time, we also studied the surface morphology of an electrodeposited biotinylated polymer. The electrodeposition of these biotinylated derivatives gave rise to the ability to modulate the surface microstructuration, which consists of microspheres or cauliflower-like microstructures according to the length of the alkyl spacer.

Functionalized magnetic composites based on block copolymers poly(succinimide)-b-poly(ethylene glycol) with potential applications in blood detoxification

Functionalized magnetic composites based on block copolymers poly(succinimide)-b-poly(ethylene glycol) have been obtained by immobilization of biotin on composites particles via activation with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDAC). Dynamic light scattering measurements showed that hydrodynamic mean diameters of the functionalized particles were about 500nm and indicate their potential to be formulated as injectable suspensions. These particles exhibit a superparamagnetic behavior and good magnetic saturation, characteristics that recommend them for magnetic separation techniques. Blood coagulation assays indicated that functionalized magnetic composites did not induce thrombogenic reactions in contact with blood.

A strategy for signal amplification using an amperometric enzyme immunosensor based on HRP modified platinum nanoparticles

A novel sandwich-type electrochemical enzyme immunosensor for sensitive detection of α-1-fetoprotein (AFP) using a signal amplification strategy based on the enzyme and the catalytic activity of Pt nanoparticles (PtNPs) is investigated in this work. Sensitivity was greatly amplified by the detection mode which was based on dual signal amplification: (1) secondary antibody labeled HRP-functionalized PtNPs were introduced on the electrode surface through biotin–avidin interactions; (2) the biotin-labeled HRP was further attached to HRP-functionalized PtNPs to assemble a dense HRP film, which resulted in significant enhancement of the current response. The adjusted (but not random) orientation of anti-AFP on the nanohybrid film of gold nanoparticles/multi-walled carbon nanotubes (AuNP/MWNT) was accomplished using an avidin–biotin immobilization technique. The nanohybrid film AuNP/MWNT can improve the response of the immunosensors due to its higher exposed surface area, high catalytic activity which facilitates electron transfer and excellent biocompatibility. The performance of the amperometric immunosensor was evaluated and optimized for best performance. The detection limit for AFP was found to be 0.08 ng/mL at S/ N = 3 and the detection concentration were in the range between 0.25 and 100 ng/mL.

Introducing surface-tethered poly(acrylic acid) brushes as 3D functional thin film for biosensing applications

Carboxyl groups of surface-tethered poly(acrylic acid) (PAA) brushes should be able to serve as versatile moieties for a wide range of chemical modifications, including an attachment of bioactive species that can act as sensing probes for biosensors. In this research, poly(tert-butyl acrylate) (Pt-BA) brushes were prepared by surface-initiated atom transfer radical polymerization of tert-butyl acrylate. PAA brushes were then obtained after removal of the tert-butyl groups from the Pt-BA brushes by acid hydrolysis. The carboxyl group density of the PAA brushes can be varied as a function of chain length or molecular weight. The reactivity of the carboxyl groups of PAA brushes towards the immobilization of biotin, a frequently used model bioactive probe in biosensing applications, was evaluated. Qualitative determination of streptavidin (SA) binding to the biotin-attached PAA brushes was verified by fluorescence microscopy. The efficiency of the PAA brushes to act as a three dimensional (3D) precursor layer for biosensing applications was further demonstrated using surface plasmon resonance (SPR), where the biotin-attached PAA brushes showed an enhanced signal for the biospecific binding of SA in comparison with a self-assembled monolayer (SAM) of a carboxyl-terminated alkanethiol, used as a model two-dimensional (2D) conventional precursor layer. The PAA brushes showed very low non-specific interactions with two other tested proteins of a similar pI but different sizes. This desirable feature should be highly beneficial for the development of biosensors.

Localized surface plasmon resonance biosensor integrated with microfluidic chip

A sensitive and low-cost microfluidic integrated biosensor is developed based on the localized surface plasmon resonance (LSPR) properties of gold nanoparticles, which allows label-free monitoring of biomolecular interactions in real-time. A novel quadrant detection scheme is introduced which continuously measures the change of the light transmitted through the nanoparticle-coated sensor surface. Using a green light emitting diode (LED) as a light source in combination with the quadrant detection scheme, a resolution of 10(-4) in refractive index units (RIU) is determined. This performance is comparable to conventional LSPR-based biosensors. The biological sensing is demonstrated using an antigen/antibody (biotin/anti-biotin) system with an optimized gold nanoparticle film. The immobilization of biotin on a thiol-based self-assembled monolayer (SAM) and the subsequent affinity binding of anti-biotin are quantitatively detected by the microfluidic integrated biosensor and a detection limit of 270 ng/mL of anti-biotin was achieved. The microfluidic chip is capable of transporting a precise amount of biological samples to the detection areas to achieve highly sensitive and specific biosensing with decreased reaction time and less reagent consumption. The obtained results are compared with those measured by a surface plasmon resonance (SPR)-based Biacore system for the same binding event. This study demonstrates the feasibility of the integration of LSPR-based biosensing with microfluidic technologies, resulting in a low-cost and portable biosensor candidate compared to the larger and more expensive commercial instruments.

Biotin−Avidin Binding Kinetics Measured by Single-Molecule Imaging

The high affinity of avidin for biotin has made it useful for many bioanalytical applications involving the immobilization of proteins, vesicles, and other biomolecules to surfaces. To understand the formation and stability of the resulting biotin-avidin complex, it is useful to know the kinetics of the binding reaction, especially for situations where the complex is formed at a liquid-solid interface typically used in sensor or separation applications. In this work, a single-molecule fluorescence method is developed for measuring the kinetics and affinity constant for the binding of neutravidin, a deglycosylated variant of avidin, to surface-immobilized biotin. Biotin was immobilized using succinimidyl ester chemistry onto amine sites on glass surfaces. The surface density of biotin was controlled by the extreme dilution of 3-aminopropyltriethoxysilane into a monolayer of 2-cyanoethyltriethoxysilane. The resulting biotin binding sites are spaced apart by micrometer distances, and this avoids crowding effects and makes the resolution of single molecules possible. The binding and unbinding of individual tetramethylrhodamine-labeled neutravidin molecules is measured in situ by total-internal-reflection fluorescence (TIRF) microscopy imaging. Single-molecule detection and counting is readily achieved by this measurement, where quantitative control is established by determining the probabilities of false positive and negative events based on the intensity distributions of background and single-molecule spots and by comparing the bound molecule populations with the independently measured density of binding sites on the surface. The kinetics of binding and unbinding are evaluated by intermittent imaging and counting the number of bound neutravidin molecules versus time, following introduction of a neutravidin solution or its replacement by buffer over the low-density biotinylated surface. The neutravidin binding kinetics were found to be fast, essentially diffusion-controlled, while the stability of the complex and its dissociation rate appear to be influenced by the chemistry of biotin immobilization.

Comparison of different procedures of biotin immobilization on gold for the molecular recognition of avidin: an FT‐IRRAS study

AbstractIn an attempt to build new, sensitive and easy to handle biosensors, we investigated different methods to immobilize biotin molecules at a gold surface and the subsequent molecular recognition of neutravidin. We compared a two‐step procedure: covalent binding of biotin to a previously chemisorbed ω‐functionalized thiolate monolayer; and direct chemisorption of a long‐chain biotinylated thiol. Fourier transform infrared reflection–absorption spectroscopy (FT‐IRRAS) was used to characterize the molecular films at each step. Subsequent binding of the protein neutravidin to each of these biotin layers was readily detected owing to labelling of the protein with an alkyne dicobalt hexacarbonyl complex, yielding characteristic mid‐infrared vC≡O signals that were shown to be sensitive to nanomolar concentrations of proteins in solution.A fully covalent binding of biotin was achieved by first chemically modifying the biotin molecule to yield a long‐chain biotinylated thiol, followed by direct adsorption to the gold surface. The modification of biotin by a thiol bearing a side COOH function enabled full insertion of this molecule into the avidin binding pocket and prevented non‐specific interaction of the protein with the surface. Copyright © 2002 John Wiley & Sons, Ltd.

Immobilized liposome chromatography to study drug–membrane interactions: Correlation with drug absorption in humans

For rapid screening of drug–membrane interactions and predicting drug absorption in vivo, unilamellar liposomes were stably immobilized in the pores of gel beads by avidin–biotin binding. Interactions of a diverse set of well-described drugs with the immobilized liposomal membranes were reflected by their elution profiles. The membrane partitioning coefficients ( K LM) of the drugs were determined from the retention volumes. The drug retentions on egg phosphatidylcholine (EPC)–phosphatidylserine (PS)–cholesterol (chol) and EPC–PS–phosphatidylethanolamine (PE)–chol columns intended to mimic small intestine membranes were similar, although the positively-charged drugs were more strongly retarded on the negatively-charged liposomes than the negatively-charged drugs. The relationship between log K LM with the drug fraction absorbed in humans showed that the log K LM values obtained with unilamellar liposomes can be used to predict drug passive transcellular absorption, similarly to that previously shown for entrapped multilamellar liposomes. The immobilized liposome chromatography method should be useful for screening compounds at an early stage of the drug discovery process. The avidin–biotin immobilization of the liposomes prolongs the lifetime of the columns.

Probing the electrochemical deposition and/or desorption of self-assembled and electropolymerizable organic thin films by surface plasmon spectroscopy and atomic force microscopy

The combination of surface plasmon spectroscopy (SPS) and atomic force microscopy (AFM) with electrochemical methods (cyclic voltammetry, potential step) is used to (i) probe the film/substrate interaction in alkanethiol monolayers formed on gold surfaces and (ii) monitor the electrochemically driven deposition of organic molecules onto a metal surface. The reductive desorption of alkanethiols at single crystal and polycrystalline gold surfaces was investigated by SPS and AFM. These experiments demonstrate the possibility of desorbing a self-assembled monolayer at a well-defined potential with all the consequences for selective (re)-functionalization. The self-assembly of alkanethiols on gold under potential control was also monitored by SPS. The results show that the surface derivatization of gold electrodes can be actively controlled by the manipulation of the electrode potential. Finally, the immobilization of biotin on gold surfaces has been carried out by the electropolymerization of a water-soluble, biotinylated derivative of 3-hydroxyphenylacetic acid. The molecular recognition of the biotinylated polyphenol film by the bacterial protein streptavidin was monitored by SPS. The packing density of the biotin labels in the polymer film leads to a very fast diffusion-controlled docking of the streptavidin to the surface. These studies clearly prove the usefulness of electrochemically controlled deposition to produce ultrathin film organic surfaces with specific function.