Covalent DNA immobilization on polymer-shielded silver-coated quartz crystal microbalance using photobiotin-based UV irradiation.

The use of a commercial, silver-coated quartz crystal microbalance (QCM) as a disposable, low-cost, and reliable DNA sensor is presented. This is an incorporation of polymer-based silver electrode shielding and photochemistry-based surface modification for covalent DNA immobilization. To prevent undesired oxidation, the silver electrodes are coated with thin polystyrene films. The polymer surfaces are then modified by a photoreactive biotin derivative (photobiotin) under UV irradiation. The resulting biotin residues on the polymer-shielded surface react with a tetrameric avidin. Consequently a biotin-labeled DNA probe can be immobilized through a biotin-avidin-biotin bridge. A 14-mer single-stranded biotin-DNA probe and a 70-mer single-stranded DNA fragment containing complementary or noncomplementary sequences are used as a model system for DNA hybridization assay on the proposed sensors. The shielding ability of the polystyrene coatings after photo irradiation is investigated. The DNA probe binding capacity, hybridization efficiency, and kinetics are also investigated.     

Investigation of biotin-streptavidin binding interactions using microcantilever sensors

We report the investigation of biotin-streptavidin binding interactions using microcantilever sensors. A symmetric cantilever construction is employed to minimize the effects of thermal drift and the control of surface chemistry on the backside of the cantilever is demonstrated to reduce the effects of non-specific binding interactions on the cantilever. Three structurally different biotin modified cantilever surfaces are used as a model system to study the binding interaction with streptavidin. The cantilever response to the binding of streptavidin on these biotin sensing monolayers is compared. The lowest detection limit of streptavidin using biotin-HPDP is found to be between 1 and 10nM limited by the optical measurement setup. Surface characterization using quartz crystal microbalance (QCM) and high-resolution atomic force microscope (AFM) is used to benchmark the cantilever sensor response. In addition, the QCM and AFM studies reveal that the surface density of bound streptavidin on biotin modified surfaces was low, thereby implying that effects other than steric hindrance are responsible for defining cantilever response.     

Acoustic and optical transduction of BuChE binding to procainamide modified surfaces

A novel polymer, poly(procainamide), PPA, containing numerous binding sites for cholinesterases was synthesized as a recognition layer for butyryl cholinesterase (BuChE) interaction with the ligand procainamide, utilizing TSM and SPR sensors. The polymer was synthesized by the reaction of methacryloyl chloride and procainamide followed by radical polymerization. Sensor surfaces (Au or SiO(2)) were spin-coated by the polymer solution to form thin layers. Binding of BuChE was found to be sensitive to the drying procedure of the polymer layer. The binding of BuChE to the polymer coated sensors was monitored on-line by following the response of thickness shear mode (TSM) and surface plasmon resonance (SPR) sensors. Binding of BuChE to PPA-coated TSM sensors were shown to follow a Langmuir isotherm giving association constant 3.4x10(6) M(-1).     

Detection of salmonella in poultry using a silicon chip-based biosensor.

Salmonella typhimurium was detected to levels as low as 119 CFUs using the Threshold Immunoassay System. This immunoassay system utilizes solution-based binding of the biotin and fluorescein labeled antibodies to salmonella, followed by filtration-capture of the immunocomplex on a biotin-coated nitrocellulose membrane. Lastly, an anti-fluorescein urease conjugate is bound to the immunocomplex. Detection of the bound immunocomplex is made possible via the silicon chip-based light-addressable potentiometric sensor. In the presence of the urea, urease converts the substrate to ammonia and CO2 and this results in a pH change at the silicon surface. The resultant pH change is monitored with time and the signal output is reported in microV s(-1). An experiment whereby chicken carcass washings were fortified with salmonella showed a recovery of 90%, indicating that the technique can be used to test for salmonella under these conditions. Precautions must be used with this instrument as sample debris will affect sample flow through the membrane and hence the signal output.     

Biotinylated granulocyte/macrophage colony-stimulating factor analogues: effect of linkage chemistry on activity and binding.

Biotinylated granulocyte/macrophage colony-stimulating factor (GM-CSF) analogues with different linkage chemistries and levels of conjugated biotin were synthesized by reacting recombinant human GM-CSF with sulfosuccinimidyl 6-biotinamidohexanoate or biotin hydrazide/1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide. These chemically reactive forms of biotin produced derivatives biotinylated at amine or carboxyl groups, respectively. Amine-derivatized analogues of 1.2 and 3.8 mol of biotin/mol of protein (N1-bGM-CSF and N4-bGM-CSF) and a carboxyl-modified analogue of 4.6 mol of biotin/mol of protein (C5-bGM-CSF) were synthesized. These analogues were compared to determine the effect of biotinylation on biological activity and GM-CSF receptor binding characteristics. The biotinylated proteins migrated with the same molecular weight as the native, unmodified protein as determined by SDS-PAGE and could be detected by Western blotting with alkaline phosphatase conjugated streptavidin, thus demonstrating the biotin linkage. All three analogues retained full agonist activity relative to the native protein (EC50 = 10-15 pM) when assayed for the stimulation of human bone marrow progenitor cell growth. Cell surface GM-CSF receptor binding was characterized by the binding of the analogues to human neutrophils, with detection by fluorescein-conjugated avidin and fluorescence-activated cell sorting. The N-bGM-CSFs demonstrated GM-CSF receptor specific binding that was displaceable by excess underivatized protein, with the detected fluorescence signal decreasing with increasing biotin to protein molar ratio. In contrast, C5-bGM-CSF binding above background fluorescence could not be detected using this system, suggesting that this derivative could bind to and activate the receptor, but not simultaneously bind fluorescein-conjugated avidin. The amine-derivatized biotinylated GM-CSF analogues retained biological activity, could specifically label cell surface receptors, and may be useful nonradioactive probes with which to study GM-CSF receptor cytochemistry and receptor modulation by flow cytometry.     

Miniaturized pH biosensors based on electrochemically modified electrodes with biocompatible polymers

Potentiometric pH sensors based on linear polyethylenimine (L-PEI) and linear polypropylenimine(L-PPI), two synthetic enzymes and biocompatible polymers, films were prepared by electropolymerization of three different monomers: ethylenediamine (EDA), 1,3-diaminopropane (1,3-DAP) and diethylenetriamine (DETA) in order to be used in clinical, dermatological and biological applications, such as in vivo analysis. In a first step a biosensor was tested which consisted in a platinum wire protruded from glass sheath. The polymer film coated on these platinum electrodes showed good linear potentiometric responses to pH changes from pH 3 to 10. Resulting electrodes present both good reversibility and good stability versus time. The effect of the different polymer film thicknesses to potentiometric responses was also studied. This study allowed us to develop a miniaturized pH biosensor in the second step. This sensor was fabricated using photo-lithography, followed by sputtering and lift-off processes, and it included an electronic detection system. We have also successfully studied the potentiometric responses to pH changes of this device over a period of 1 month, and so we propose this new pH micro-biosensor as an alternative to classical pH sensors currently used in dermatology.     

Spatial relationship between SH1 and the actin binding site on myosin subfragment-1 surface

To examine the spatial relationship between SH1 thiol and actin binding site on subfragment-1 surface, we studied the interaction with actin of subfragment-1 whose SH1 was labeled with an iodoacetate derivative of biotin and covered with avidin. Subfragment-1--avidin complex bound F-actin and its Mg2+ ATPase activity was activated by actin. Considering the size and the location of biotin binding site on avidin, our results suggest that SH1 is separated from the actin binding site on subfragment-1 surface by at least 17-20 A.     

Evidence that L1AD3, an apoptosis-inducing cyclic peptide, binds a leukemic T-cell membrane protein receptor

Human leukemic T-lymphocytes undergo extensive and rapid apoptosis in the presence of L1AD3, a small cyclic peptide derivative of cobra cardiotoxin. The first step in this process involves its binding to membranes of susceptible cells. By the use of a biotin "handle" synthetically incorporated at the N-terminus of L1AD3, we show that binding is saturable and selective: normal human peripheral blood lymphocytes do not bind this peptide. Fluorescence resonance energy transfer experiments indicate that the binding sites are separated by at least 55 A. Loss of binding occurs if membrane proteins are enzymatically degraded, suggesting that L1AD3's target is a cell-membrane surface protein receptor. Finally, crosslinking of cyclic BTNL1AD3 peptide to a leukemic T-cell membrane surface receptor, as examined using a biotin-avidin blot, indicated a molecular weight of approximately 34,400.     

Galactosylated N-vinylpyrrolidone-maleic acid copolymers: synthesis,characterization, and interaction with lectins

Water-soluble artificial glycoconjugate polymers were synthesized from poly(N-vinylpyrrolidone-co-maleic anhydride) by amidation with an amine-containing galactose derivative. The glycopolymers having different galactose contents were fully characterized in terms of chemical structure by NMR and potentiometric titrations, and their aqueous behavior was studied by viscometric measurements. Their specific binding properties were examined by enzyme-linked lectin assays using RCA(120) lectin. Whatever the glycopolymer, the grafted galactoses were shown to behave similarly to free galactose.     

Biosensor-based evaluation of liposomal behavior in the target binding process

In this study we evaluated the quartz crystal microbalance (QCM) as a biosensor for a real-time investigation of liposomal binding, under dynamic flow conditions, onto target proteins immobilized at the sensor. The mass-sensitive frequency changes of quartz sensors allow for a quantification of the liposomal binding process. Furthermore, simultaneous damping analysis gives an insight into liposomal behavior, such as the degree of liposomal deformation or spreading at the target surface. In this study a series of liposomes was evaluated, differing in the kind and concentration of ligands interacting with appropriate target proteins. It became evident that an increase in homing device concentration accelerated deformation and flattening of liposomes, triggering a fusion process. Furthermore, liposomal deformation corresponded with the binding affinity of target molecules, comparing biotin/avidin with E-selectin/ligand interactions. Deformation could be emphasized using dioleoylphosphatidylethanolamine (DOPE) as a fusiogenic membrane component, while sterical stabilization by polyethylenglycol (PEG-PE) appeared in a low degree of deformation. Consequently, the online detection of liposomal target binding by QCM is an excellent facility to control and predict the liposomal behavior at the target site for increasing therapeutic potency.     

Detection of nicotinic receptor ligands with a light addressable potentiometric sensor.

The nicotinic acetylcholine receptor, purified from Torpedo electric organ, was coupled to a light addressable potentiometric sensor (LAPS) to form a LAPS-receptor biosensor. Receptor-ligand complexes containing biotin and urease were captured on a biotinylated nitrocellulose membrane via a streptavidin bridge and detected with a silicon-based sensor. Competition between biotinylated alpha-bungarotoxin and nonbiotinylated ligands formed the basis of this assay. This biosensor detected both agonists (acetylcholine, carbamylcholine, succinylcholine, suberyldicholine, and nicotine) and competitive antagonists (d-tubocurarine, alpha-bungarotoxin, and alpha-Naja toxin) of the receptor with affinities comparable to those obtained using radioactive ligand binding assays. Consistent with agonist-induced desensitization of the receptor, the LAPS-receptor biosensor reported a time-dependent increase in affinity for the agonist carbamylcholine as expected, but not for the antagonists.     

Biosensor-Based Evaluation of Liposomal Behavior in the Target Binding Process

In this study we evaluated the quartz crystal microbalance (QCM) as a biosensor for a real-time investigation of liposomal binding, under dynamic flow conditions, onto target proteins immobilized at the sensor. The mass-sensitive frequency changes of quartz sensors allow for a quantification of the liposomal binding process. Furthermore, simultaneous damping analysis gives an insight into liposomal behavior, such as the degree of liposomal deformation or spreading at the target surface. In this study a series of liposomes was evaluated, differing in the kind and concentration of ligands interacting with appropriate target proteins. It became evident that an increase in homing device concentration accelerated deformation and flattening of liposomes, triggering a fusion process. Furthermore, liposomal deformation corresponded with the binding affinity of target molecules, comparing biotin/avidin with E-selectin/ligand interactions. Deformation could be emphasized using dioleoylphosphatidylethanolamine (DOPE) as a fusiogenic membrane component, while sterical stabilization by polyethylenglycol (PEG-PE) appeared in a low degree of deformation. Consequently, the online detection of liposomal target binding by QCM is an excellent facility to control and predict the liposomal behavior at the target site for increasing therapeutic potency.     

Selective isolation of individual cell surface proteins from tissue culture cells by a cleavable biotin label

A method was developed to isolate cell surface proteins by a simple two-step procedure. Hepatocyte cell surface proteins were labeled by a cleavable biotin derivative in a covalent pulse reaction. Under the described conditions, NHS-SS-biotin proved to be an impermeant, cell surface-specific label which does not affect the impermeant, cell surface-specific label which does not affect the viability of rat hepatocytes. Biotinylated cell surface proteins could be selectively separated under non-denaturing conditions from non-biotinylated proteins and biotin-containing carboxylases by avidin affinity chromatography and sulfhydryl-mediated elution. Subsequent to alkylation of the eluted protein, individual cell surface proteins could be isolated by immunoprecipitation as shown for a selected Mr 120,000 glycoprotein gp120 of the hepatocyte plasma membrane. Using this technique, a transit time of gp120 from the endoplasmic reticulum to the cell surface of 2 h was determined. The results show that the combination of labeling with a cleavable biotin derivative, non-denaturing avidin affinity chromatography and immunoprecipitation is a useful method to isolate and study individual cell surface proteins.     

Avidin-biotin interactions at vesicle surfaces: adsorption and binding, cross-bridge formation, and lateral interactions.

Densely packed domains of membrane proteins are important structures in cellular processes that involve ligand-receptor binding, receptor-mediated adhesion, and macromolecule aggregation. We have used the biotin-avidin interaction at lipid vesicle surfaces to mimic these processes, including the influence of a surface grafted polymer, polyethyleneglycol (PEG). Single vesicles were manipulated by micropipette in solutions of fluorescently labeled avidin to measure the rate and give an estimate of the amount of avidin binding to a biotinylated vesicle as a function of surface biotin concentration and surface-grafted PEG as PEG-lipid. The rate of avidin adsorption was found to be four times less with 2 mol% PEG750 than for the unmodified surface, and 10 mol% PEG completely inhibited binding of avidin to biotin for a 2-min incubation. Using two micropipettes, an avidin-coated vesicle was presented to a biotinylated vesicle. In this vesicle-vesicle adhesion test, the accumulation of avidin in the contact zone was observed, again by using fluorescent avidin. More importantly, by controlling the vesicle membrane tension, this adhesion test provided a direct measure of the spreading pressure of the biotin-avidin-biotin cross-bridges confined in the contact zone. Assuming ideality, this spreading pressure gives the concentration of avidin cross-bridges in the contact zone. The rate of cross-bridge accumulation was consistent with the diffusion of the lipid-linked "receptors" into the contact zone. Once adherent, the membranes failed in tension before they could be peeled apart. PEG750 did not influence the mechanical equilibrium because it was not compressed in the contact zone, but it did perform an important function by eliminating all nonspecific adhesion. This vesicle-vesicle adhesion experiment, with a lower tension limit of 0.01 dyn/cm, now provides a new and useful method with which to measure the spreading pressures and therefore colligative properties of a range of membrane-bound macromolecules.     

Characterization and application of a surface modification designed for QCM-D studies of biotinylated biomolecules

The rapid development of surface sensitive biosensor technologies, especially towards nanoscale devices, requires increasing control of surface chemistry to provide reliable and reproducible results, but also to take full advantage of the sensing opportunities. Here, we present a surface modification strategy to allow biotinylated biomolecules to be immobilized to gold coated sensor crystals for quartz crystal microbalance with dissipation monitoring (QCM-D) sensing. The unique feature of QCM-D is its sensitivity to nanomechanical (viscoelastic) properties at the sensing interface. The surface modification was based on mixed monolayers of oligo(ethylene glycol) (OEG) disulfides, with terminal -OH or biotin groups, on gold. Mixtures containing 1% of the biotin disulfide were concluded to be the most appropriate based on the performance when streptavidin was immobilized to biotinylated sensors and the subsequent biotinylated bovine serum albumin (BSA) interaction was studied. The OEG background kept the unspecific protein binding to a minimum, even when subjected to serum solutions with a high protein concentration. Based on characterization by contact angle goniometry, ellipsometry, and infrared spectroscopy, the monolayers were shown to be well-ordered, with the OEG chains predominantly adopting a helical conformation but also partly an amorphous structure. Storage stability was concluded to depend mainly on light exposure while almost all streptavidin binding activity was retained when storing the sensors cold and dark for 8 weeks. The surface modification was also tested for repeated antibody-antigen interactions between BSA and anti-BSA (immobilized to biotinylated protein A) in QCM-D measurements lasting for >10h with intermediate basic regeneration. This proved an excellent stability of the coating and good reproducibility was obtained for 5 interaction cycles. With this kind of generic surface modification QCM-D can be used in a variety of biosensing applications to provide not only mass but also relevant information of the structural properties of adlayers.     

Fabrication of butyrylcholinesterase sensor using polyurethane-based ion-selective membranes.

A simple method of enzyme immobilization was investigated which is useful for fabrication of enzyme sensors based on polymeric ion-selective membranes. The enzyme membrane was built by coating a thin hydrophilic polyurethane (HPU) film directly mixed with an enzyme over an underlying polyurethane (PU)-based ion-selective membrane. This highly simple method of enzyme immobilization was applied to the fabrication of a potentiometric butyrylcholinesterase-based biosensor for the determination of organophosphorus pesticides. The enzyme was well entrapped within the HPU film and the intrinsic potentiometric response of the underlying ion-selective PU membrane was not influenced significantly by the outer HPU/enzyme membrane. The enzyme electrode was optimized by changing systematically the composition of the enzyme membrane to evaluate the effect of the changes on sensor response. The sensor was successfully applied to the analysis of paraoxon, an organophosphorus pesticide.     

Purification of the Fusicoccin-Binding Protein from Oat Root Plasma Membrane by Affinity Chromatography with Biotinylated Fusicoccin

Fusicoccin (FC), a fungal phytotoxin, evokes a number of physiological responses after binding to the FC-binding protein (FCBP). For characterization of this plasma membrane protein and elucidation of the signal transduction pathway, we purified active FCBP from oat (Avena sativa L. cv Valiant) root plasma membranes using avidin-biotin affinity chromatography. For the binding of FCBP to immobilized avidin, a bifunctional FC derivative (FC-biotin, FCBio) was synthesized. FCBio retained high binding affinity for the FCBP (KD = 70 nM), it elicited a biological response comparable to FC, and it was bound by avidin. The purification of the FCBP involved three important steps. First, FCBio was bound to the FCBP in purified plasma membrane vesicles. Next, plasma membrane proteins were solubilized in detergent, and part of the solubilized proteins was precipitated by decreasing the detergent concentration below the critical micelle concentration. The FCBP remained in the soluble fraction, and this fraction was loaded on a "low-affinity" avidin column. Proteins, bound through a biotin moiety to the column, were specifically eluted with excess biotin. This resulted in fractions active in [3H]FC binding and two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 31 and 30 kD. The nonhydrophobic behavior of the FCBP was confirmed by means of phase separation with Triton X-114, wherein the FCBP migrated to the hydrophilic phase. Purification of the FCBP in active form using this novel affinity technique opens the possibility to study other features of the FCBP necessary for inducing physiological responses in plant cells.     

Affinity selection of target cells from cell surface displayed libraries: a novel procedure using thermo-responsive magnetic nanoparticles

Biotinylated thermo-responsive magnetic nanoparticles for use in affinity selection from yeast cell surface display libraries were prepared by coating magnetite nanoparticles with a thermo-responsive polymer consisting of N-isopropyl acrylamide and a biotin derivative. These particles showed a reversible transition between flocculation and dispersion at around the lower critical solution temperature of 30 degrees C, above which the flocculated particles--which absorbed a large amount of avidin due to their large surface area--were quickly separable by magnet. The model library was constructed by mixing control yeast cells with target yeast cells co-displaying IgG binding protein (ZZ) and enhanced green fluorescence protein. Biotinylated IgG and avidin were subsequently added to the model library, and target cells were efficiently enriched with the biotinylated magnetic nanoparticles by avidin-biotin sandwich and ZZ-IgG interaction. The few target cells (0.001%) in the model library were enriched by up to 100% in only 5 days by an affinity selection procedure repeated four times. This novel method based on magnetic nanoparticles and a yeast cell surface display system could fulfill a wide range of applications in the analysis of protein-protein interactions and rapid isolation of novel biomolecules.     

Label-free THz sensing of living body-related molecular binding using a metallic mesh

We have demonstrated label-free THz sensing of living body-related molecular binding using a thin metallic mesh and a polyvinylidene difluoride (PVDF) membrane. Metallic meshes in the THz region are designed for anomalous transmission phenomena derived from a resonant excitation of surface waves. Additionally, they are designed to have a sharp dip in transmittance. The metallic mesh is very sensitive to a change of the refractive index of materials attached to the metallic mesh. In this paper, we report sensing of interactions between lectin and sugar using this technique. We found that the dip frequency shift, transmittance attenuation of the dip frequency, and peak shift of the derivative spectrum of the phase shift depend on the bonding amount of lectin–sugar interactions. We also applied this technique to detect avidin–biotin interactions, leading to the detection of a small amount of biotin (0.17 pg/mm²).     

Temperature control of biotin binding and release with A streptavidin-poly(N-isopropylacrylamide) site-specific conjugate.

The many laboratory and diagnostic applications utilizing streptavidin as a molecular adaptor rely on its high affinity and essentially irreversible interaction with biotin. However, there are many situations where recovery of the biotinylated molecules is desirable. We have previously shown that poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, can reversibly block biotin association as the polymer's conformation changes at its lower critical solution temperature (LCST). Here, we have constructed a streptavidin-PNIPAAm conjugate which is able to bind biotin at room temperature or lower and release bound biotin at 37 degrees C. The conjugate can repeatedly bind and release biotin as temperature is cycled through the LCST. A genetically engineered streptavidin mutant, E116C, which has only one cysteine residue, was conjugated site specifically via the sulfhydryl groups with a PNIPAAm that has pendent sulfhydryl-reactive vinyl sulfone groups. The conjugation site is near the tryptophan 120 residue, which forms a van der Waals contact with biotin that is important in generating the large binding free energy. The temperature-induced conformational change of the polymer at position 116 may lead to structural changes in the region of tryptophan 120 that are responsible for the reversible binding between biotin and the conjugated streptavidin.