Covalent biofunctionalization of cellulose acetate with thermostable chimeric avidin

A stable, bioactive cellulose acetate (CA) surface was developed by functionalizing the surface with highly thermostable avidin form. The CA films were first functionalized with a mixture of 3-aminopropyltrimethoxysilane and tetraethoxysilane to introduce free amino groups onto the surface of CA films. Free amino groups were functionalized with glutaraldehyde to obtain an activated surface for covalent biomolecule immobilization. A genetically engineered, high-affinity biotin-binding protein chimeric avidin, ChiAVD(I117Y), was used for biofunctionalization of the surface. The chimeric avidin protein has an increased stability in chemically harsh conditions and at high temperature when compared to wt (strept)avidin. The biological activity, i.e., biotin-binding capacity, of the immobilized protein was probed by [(3)H]-biotin. The activity of the chimeric avidin on functionalized CA films was fully retained over the three months' study period. The biotin-binding capacity of the immobilized chimeric avidin was compared to that of immobilized streptavidin, chicken avidin, and rhizavidin and significant differences between proteins were detected. The developed material offers a valuable platform for the development of inexpensive in vitro diagnostics and also supports biosensing applications that are required to operate under demanding conditions.     

Comparison of the energetics of avidin, streptavidin, neutrAvidin, and anti-biotin antibody binding to biotinylated lipid bilayer examined by second-harmonic generation

A comparison of the binding properties of avidin, streptavidin, neutrAvidin, and antibiotin antibody to a biotinylated lipid bilayer was studied using second-harmonic generation. Protein binding assays were performed on a planar supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) containing 4 mol?% biotinylated-cap-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (biotin-cap-DOPE). The equilibrium binding affinities of these biotin-protein interactions were determined, revealing the relative energetic contributions for each protein to the biotinylated lipid ligand. The results show that the binding affinities of avidin, streptavidin, and neutrAvidin for biotin were all strengthened by protein-protein interactions but that the stronger protein-protein interactions observed for streptavidin and neutrAvidin make their binding more energetically favorable. It was also shown that neutrAvidin has the highest degree of nonspecific adsorption to a pure DOPC bilayer, compared to avidin and streptavidin. In addition, the biotin-binding affinity of the antibiotin antibody was found to be of the same order of magnitude as that of avidin, streptavidin, and neutrAvidin. These findings provide important new insights into these biotin-bound protein complexes commonly used in several bioanalytical applications.     

The imaging of streptavidin and avidin using scanning tunnelling microscopy

Streptavidin and avidin were adsorbed onto graphite surfaces and images of their structures were obtained using a scanning tunnelling microscope. Individual protein molecules and their subunits were observed, and their sizes were found to agree closely with measurements from a subunit model based upon the crystal structure of streptavidin. The process of adsorption of streptavidin-FITC and avidin succinylfluorescein isothiocyanate onto the graphite surface was also investigated using fluorescence microscopy.     

The imaging of streptavidin and avidin using scanning tunnelling microscopy

Streptavidin and avidin were adsorbed onto graphite surfaces and images of their structures were obtained using a scanning tunnelling microscope. Individual protein molecules and their subunits were observed, and their sizes were found to agree closely with measurements from a subunit model based upon the crystal structure of streptavidin. The process of adsorption of streptavidin-FITC and avidin succinylfluorescein isothiocyanate onto the graphite surface was also investigated using fluorescence microscopy.     

Osmium tetroxide, 2,2'-bipyridine: electroactive marker for probing accessibility of tryptophan residues in proteins

A complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) has been applied as a chemical probe of DNA structure as well as an electroactive DNA label. The Os,bipy has been known to form covalent adducts with pyrimidine DNA bases. Besides the pyrimidines, electrochemically active covalent adducts with Os,bipy are formed also by tryptophan (W) residues in peptides and proteins. In this paper we show that Os,bipy-treated proteins possessing W residues (such as avidin, streptavidin, or lysozyme) yield at the pyrolytic graphite electrode (PGE) a specific signal (peak alphaW) the potential of which differs from the potentials of signals produced by free Os,bipy or by Os,bipy-modified DNA. No such signal is observed with proteins lacking W (such as ribonuclease A or alpha-synuclein). Subpicomole amounts of W-containing proteins modified with Os,bipy can easily be detected using adsorptive transfer stripping voltammetry with the PGE. Binding of biotin to avidin interferes with Os,bipy modification of the protein, in agreement with the location of W residues within the biotin-binding site of avidin. These Ws are accessible for modification in the absence of biotin but hidden (protected from modification) in the avidin-biotin complex. The Os,bipy-modified avidin is unable to bind biotin, and its quarternary structure is disrupted. Analogous effects were observed with another biotin-binding protein, streptavidin. Our results demonstrate that modification of proteins with Os,bipy under conditions close to physiological, followed by a simple electrochemical analysis, can be applied in the microanalysis of protein structure and interactions.     

Construction of pegylated multilayer architectures via (strept)avidin/biotin interactions

Pegylated multilayer architectures were fabricated as films on planar substrates, as shells on colloidal particles, or as free-standing hollow capsules using layer-by-layer (LbL) self-assembly of biotinylated poly-l-lysine (PLL) and (strept)avidin. Poly(ethylene glycol) (PEG) was incorporated into the multilayer architectures by assembly with biotin-derivatized poly(l-lysine)-g-poly(ethylene glycol)(PPB). Stepwise growth of multilayers was followed by UV–vis spectroscopy and the formation of core–shells and hollow capsules characterized by means of confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Both absorbance and TEM data suggest that approximately two layers of FITC–avidin were adsorbed with each surface deposition. In contrast, use of unmodified PLL did not lead to formation of multilayer coatings, confirming that (strept)avidin–biotin interactions were responsible for film growth even in the presence of electrostatic repulsive forces between PLL and avidin and the steric hindrance of associated PEG chains. This technique provides new opportunities for the generation of robust films with tailored interfacial binding and transport properties.     

Kinetics and thermodynamics of biotinylated oligonucleotide probe binding to particle-immobilized avidin and implications for multiplexing applications

In this work, the kinetics and dissociation constant for the binding of a biotin-modified oligonucleotide to microparticle-immobilized avidin were measured. Avidin has been immobilized by both covalent coupling and bioaffinity capture to a surface prefunctionalized with biotin. The measured rate and equilibrium dissociation constants of avidin immobilized by these different methods have been compared with those for nonimmobilized avidin. We found that immobilization resulted in both a decrease in the rate of binding and an increase in the rate of dissociation leading to immobilized complexes having equilibrium dissociation constants of 7 ± 3 × 10(-12) M, higher than the value measured for the complex between biotin-modified oligonucleotide and nonimmobilized avidin and approximately 4 orders of magnitude larger than values for the wild-type avidin-biotin complex. Immobilized complex half-lives were found to be reduced to 5 days, which resulted in biotin ligands migrating between protein attached to different particles. Different immobilization methods showed little variation in complex stability but differed in total binding and nonspecific biotin-modified oligonucleotide binding. These findings are critical for the design of multiplexed assays where probe molecules are immobilized to biosensors via the avidin-biotin interaction.     

Electrochemical synthesis of gold nanoparticles onto indium tin oxide glass and application in biosensors

A simple one-step method for the electrochemical deposition of gold nanoparticles (GNPs) onto bare indium tin oxide film coated glass substrate without any template or surfactant was investigated. The effect of electrolysis conditions such as potential range, temperature, concentration and deposition cycles were examined. The connectivity of GNPs was analyzed by UV-Vis absorption spectroscopy and scanning electron microscopy. The nanoparticles were found to connect in pairs or to coalesce in larger numbers. The twin GNPs display a transverse and a longitudinal localized surface plasmon resonance (LSPR) band, which is similar to that of gold nanorods. The presence of longitudinal LSPR band correlates with high refractive index sensitivity. Conjugation of the twin-linked GNPs with albumin bovine serum-biotin was employed for the detection of streptavidin as a model based on the specific binding affinity in biotin/streptavidin pairs. The spectrophotometric sensor showed concentration-dependent binding for streptavidin.     

Electrode surface confinement of self-assembled enzyme aggregates using magnetic nanoparticles and its application in bioelectrocatalysis

Self-assembled enzyme aggregates, prepared from magnetic iron oxide nanoparticles, avidin, and a biotinylated redox enzyme, were shown particularly useful for the simple, fast, and efficient construction of highly enzyme-loaded electrodes with the help of a magnet. The approach was illustrated in the case of the bioelectrocatalytic oxidation of NADH by a diaphorase oxidoreductase in the presence of a ferrocene mediator. Two different self-assembling procedures were tested, taking advantage of the spontaneous aggregation of the nanoparticles in the presence of avidin and also of the multivalency binding of biotinylated diaphorase toward avidin. Activities of the bound and unbound diaphorase were systematically controlled allowing determination of the number of active biotinylated diaphorase per nanoparticle incorporated within each magnetic enzyme aggregate. An active enzyme loading capacity of up to 2.35 nmol mg-1 was found for the best nanostructured enzyme assembly, which is 200 times better than for commercialized magnetic micrometer-sized beads coated with streptavidin and saturated with diaphorase. With the help of a permanent magnet, the magnetic enzyme aggregates were finally magnetically collected as a film on the surface of a small screen-printed carbon electrode and the catalytic currents recorded by cyclic voltammetry. From the analysis of the steady-state catalytic current responses and the kinetic rate constants of biotinylated diaphorase, it was possible to determine the enzyme concentration within the magnetic films. Owing to the high enzyme loading in the aggregates of nanoparticles (i.e., 130 microM), the catalytic current responses were definitely higher than the ones measured at an electrode coated with a closed-packed monolayer of diaphorase or at an electrode covered with a film of magnetic micrometer-sized streptavidin beads saturated with diaphorase.     

Targeted labeling of cancer cells using biotin tagged avidin functionalized biocompatible fluorescent nanocrystals

The present study details the development of biotin tagged avidin functionalized Zinc Sulphide [ZnS] nanocrystals through a simple aqueous chemistry route at room temperature for targeted imaging applications. Surface functionalization of Manganese doped ZnS nanocrystals with L-cysteine provided functional groups that facilitated its conjugation to avidin. Further biotinylation of these particles through the strong non-covalent interaction between biotin and avidin enabled highly specific labeling of the biotin receptors on human hepatocellular carcinoma (HepG2) cells. The nanobioconjugates thus developed exhibited stable and brilliant fluorescence upon labeling the biotin receptors on cells as observed through fluorescence microscopy. Characterization studies using X-ray diffraction, dynamic light scattering as well as Fourier transform infrared spectroscopy revealed the bioconjugated particles to be appropriately functionalized and stable, with size ranging from 50 to 80 nm. Cytotoxicity of this material system evaluated using MTT, LDH leakage and apoptosis assay revealed its non-toxic nature even for high concentrations extending upto 250 microM and 48 hours of incubation. Our results confirmed that biotinylated ZnS nanocrystals offer great potential for highly specific labeling and targeted imaging of cancer cells.     

Microwave-accelerated ultrafast nanoparticle aggregation assays using gold colloids

In this paper, the proof of principle of microwave-accelerated aggregation assay technology, which shortens the solution-based aggregation assays' run time to seconds (>100-fold increase in kinetics) with microwave heating, was demonstrated using a model aggregation assay based on the well-known interactions of biotin and avidin. Biotinylated gold colloids were aggregated in solution with the addition of streptavidin, which takes 20 min at room temperature to reach >90% completion and only 10 s with microwave heating. The initial velocity (after 1-s microwave heating) of the biotinylated gold colloids reaches up to 10.5 m/s, which gives rise to greater sampling of the total volume but not a large increase in bulk temperature. The room-temperature, steady-state velocity of the colloids was <0.5 microm/s. In control experiments, where streptavidin preincubated with d-biotin in solution is added to biotinylated gold colloids and microwave heated, gold colloids did not aggregate, demonstrating that nonspecific interactions between biotinylated gold colloids and streptavidin were negligible.     

Patterning enzymes inside microfluidic channels via photoattachment chemistry

We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.     

Quartz crystal microbalance study of DNA immobilization and hybridization for nucleic Acid sensor development

The immobilization of two 30-mer oligonucleotides, one biotinylated (biotin-DNA) and the other having a mercaptohexyl group at the 5'-phosphate end (BS1-SH), onto modified gold surfaces has been examined using a quartz crystal microbalance (QCM). Both single-layer and multilayer DNA films were prepared. The single-layer films of biotin-DNA were constructed by binding to a precursor layer of avidin, which had been attached to the QCM either covalently using a water-soluble carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) or via electrostatic interaction with poly(allylamine hydrochloride) (PAH). Single-layer films of BS1-SH were also formed on PAH via the electrostatic attraction between the amine groups on PAH and the negatively charged phosphate backbone of DNA. Multilayer films of DNA were fabricated by the successive deposition of avidin and poly(styrenesulfonate) (PSS), up to a total of nine avidin/PSS layers, followed by DNA adsorption. DNA immobilization and hybridization of the immobilized DNAs was monitored in situ from QCM frequency changes. Hybridization was induced by exposure of the DNA-containing films to complementary DNA in solution. Equal frequency changes were observed for the DNA immobilization and hybridization steps for the single-layer films, indicating a DNA probe-to-hybridized DNA target ratio of 1:1. The multilayer DNA films also exhibited DNA hybridization, with a greater quantity of DNA hybridized compared with the single-layer films. The multilayer films provide a novel means for the fabrication of DNA-based thin films with increased capacity for nucleic acid detection.     

Voltammetric sensor for oxidized DNA using ultrathin films of osmium and ruthenium metallopolymers

Films containing [Os(bpy)2(PVP)10Cl]+ and [Ru(bpy)2(PVP)10Cl]+ metallopolymers were assembled layer by layer on pyrolytic graphite electrodes to make sensors that selectively detect oxidized DNA. These films showed reversible, independent electrochemistry for electroactive Os3+/Os2+ and Ru3+/Ru2+ centers, with formal potentials of 0.34 and 0.76 V vs SCE, respectively. The combination of ruthenium and osmium metallopolymers in the films provided a catalytic Os square wave voltammetry (SWV) peak that is mainly selective for 8-oxoguanine and the detection of other oxidized nucleobases from the Ru peak. The method is applicable to measurements on DNA in solution or DNA incorporated into films. Using the Os SWV peak, 1 oxidized nucleobase in 6000 was detected. The sensor is simple and inexpensive, and the approach may be useful for the detection of oxidized DNA as a clinical biomarker for oxidative stress.     

Detection of abasic sites on individual DNA molecules using atomic force microscopy

We have developed an atomic force microscopy-based method for detecting abasic sites (AP sites) on individual DNA molecules. By using uracil and uracil DNA glycosylase, we first prepared a 250-bp DNA template consisting of two AP sites at specific locations. We then detected the AP sites by marking them with biotinylated aldehyde-reactive probes and monomeric avidin. We demonstrate here that (i) the location of monomeric avidin bound on a single DNA molecule was detectable by atomic force microscopy; (ii) the observed location of avidin was in good agreement to the predicted AP sites at a few nanometer resolution; and (iii) by end-labeling the 5'-terminus of one DNA strand, the AP sites were determined without directional ambiguity. The technique described here will provide a sensitive way of locating AP sites and contribute to screen DNA damages from individual molecules.     

High-performance liquid chromatography-electrospray ionization-mass spectrometry ligand fishing assay: a method for screening triplex DNA binders from natural plant extracts

A novel ligand fishing assay was established to screen triplex DNA binders from complicated samples by a combination of immobilization of triplex DNA on agarose beads and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). The biotinylated oligodeoxynucleotides were first bound to the streptavidin agarose beads and then incubated with the duplex DNA as the baits for ligand fishing. This assay was validated by the testing ligand library consisting of coralyne, ethidium bromide, vitexin, and formononetin. The binding affinities of ligands to target DNA were also obtained based on the calibration curves of ligands. Two components (berberine and palmatine) in the extract of Phellodendron chinense Schneid cortexes were fished out as triplex DNA binders by this assay, which indicated its feasibility for screening triplex DNA binders from complicated samples. This preliminary assay can be used for not only screening binders of triplex DNA from natural products extracts but also can obtain their binding affinity information.     

聚苯胺/氧化石墨的合成及其在DNA识别上的应用

采用层离/吸附和原位聚合相结合的方法合成了聚苯胺/氧化石墨复合材料（PAn/GO）.利用TEM、AFM、XRD、FTIR等方法对PAn/GO的结构和电化学性能进行了研究.以PAn/GO修饰炭糊电极为工作电极,采用方波伏安法（SWV）检测了单链小牛胸腺DNA（CTssDNA）和双链小牛胸腺DNA（CTdsDNA）.研究结果表明：PAn/GO为当量直径约60 nm～70 nm的扁球状纳米颗粒,这些纳米颗粒呈链状聚集,并具有电化学活性;聚苯胺（PAn）以双层平行排列的方式嵌入氧化石墨层间和包覆在氧化石墨表面两种形式与氧化石墨结合;PAn/GO修饰炭糊电极识别单链小牛胸腺DNA（CTssDNA）和双链小牛胸腺DNA（CTdsDNA）时的峰电位分别为90.99 mV和18.00 mV.     

Recombinant Gaussia luciferase. Overexpression,purification, and analytical application of a bioluminescent reporter for DNA hybridization

The cDNA for Gaussia luciferase (GLuc), the enzyme responsible for the bioluminescent reaction of the marine copepod Gaussia princeps, has been cloned recently. GLuc (MW = 19 900) catalyzes the oxidative decarboxylation of coelenterazine to produce coelenteramide and light. We report the first quantitative anaytical study of GLuc and examine its potential as a new reporter for DNA hybridization. A plasmid encoding both a biotin acceptor peptide-GLuc fusion protein as well as the enzyme biotin protein ligase (BPL) is engineered by using GLuc cDNA as a starting template. BPL catalyzes the covalent attachment of a single biotin to the fusion protein in vivo. Purification of GLuc is then accomplished by affinity chromatography using immobilized monomeric avidin. Moreover, the in vivo biotinylation enables subsequent complexation of GLuc with streptavidin (SA), thereby avoiding chemical conjugation reactions that are known to inactivate luciferases. Purified GLuc can be detected down to 1 amol with a signal-to-background ratio of 2 and a linear range extending over 5 orders of magnitude. The background luminescence of coelenterazine is the main limiting factor for even higher detectability of GLuc. Furthermore, the GLuc-SA complex is used as a detection reagent in a microtiter well-based DNA hybridization assay. The analytical range extends from 1.6 to 800 pmol/L of target DNA. Biotinylated GLuc produced from 1 L of bacterial culture is sufficient for 150,000 hybridization assays.     

One-step electrochemically deposited gold nanoparticles interface grafted with avidin for acetylcholinesterase biosensor design

In this study, an interface embedded in situ gold nanoparticles (AuNPs) and biotin in chitosan hydrogel was constructed by one-step electrochemical deposition in solution containing tetrachloroauric (III) acid, biotin and chitosan. This deposited interface acts as biosensing platforms and provides specific binding sites for avidin, which are further capable of attaching any biotinylated bimolecular for biosensor design. Atomic force microscopy (AFM), A.C. impedance and surface plasmon resonance were used to characterize this interface. The immobilized acetylcholinesterase (AChE), as a model, showed excellent activity to its substrate and provided a quantitative measurement of organophosphate pesticide. Under the optimal experimental conditions, the inhibition of dimethoate was proportional to its concentrations in the range of 0.05 to 15 microg mL(-1) with detection limit of 0.001 microg mL(-1). The simple method showed good fabrication reproducibility and acceptable stability, which provided a new avenue for electrochemical biosensor design.     

Covalent binding of streptavidin on gold magnetic nanoparticles for bead array fabrication

In this study, we report the chemical synthesis and functionalization of streptavidin coated gold magnetic nanoparticles (GMNPs) and the immobilization of single-stranded biotinylated oligonucleotides onto these particles. By using covalent interaction or physical adsorption, two kinds of streptavidin coated GMNPs (SA-GMNPs) were prepared. The quantity and stability of streptavidin bound to the GMNPs using different methods were determined by UV-Vis spectrometer. The results indicated that by physical absorption the GMNPs can capture more streptavidin, the SA-GMNPs with either physical adsorption or covalent reaction were both stable in PBS buffer. In contrast, SA-GMNPs with covalent reaction was stable in SDS buffer, while most of the SA-GMNPs by physical adsorption would be eluted from the particles in SDS buffer. Therefore, the SA-GMNPs by covalent immobilization were more suitable for fabrication of bead array. To evaluate the binding efficiency and capacity of DNA on SA-GMNPs, the capture of biotinylated oligonucleotide or PCR products on SA-GMNPs at different concentrations were examined. A magnetic beads array was fabricated by immobilizing DNA-MNPs complexes onto a glass slide using a magnetic field. The synthesized DNA targets with different concentrations were detected with a detection limit of approximately 0.05 nM, indicating the potential application of this MNPs array to high-throughput DNA detection.