Molecular recognition of avidin on biotin-functionalized gold surfaces detected by FT-IRRAS and use of metal carbonyl probes

Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) was successively used to monitor the covalent immobilization of biotin molecules onto a planar gold substrate covered with a self-assembled monolayer of cystamine and to transduce the molecular recognition of avidin and biotin. This detection was greatly facilitated and made selective by the labeling of avidin and of biotin with various transition metal carbonyl probes. The binding of avidin to the surface was optimized by blocking the nonspecific binding sites by adsorption of an unrelated protein, bovine serum albumin. This work exemplifies the feasibility of detecting biomolecular associations involving molecules of any size at a liquid/solid interface by using a simple and accessible surface analysis technique.     

Microfluidic chip with electrochemiluminescence detection using 2-(2-aminoethyl)-1-methylpyrrolidine labeling

A tertiary amine derivative, 2-(2-aminoethyl)-1-methylpyrrolidine (AEMP) was successfully developed as electrochemiluminescence (ECL) probe within microfluidic chip using ECL detection in this paper. The system was characterized by the interaction between biotin and avidin. In principle, tertiary amine derivatives containing active group can be used as a potential alternative of traditional tris(2,2'-bipyridine)ruthenium(II) [Ru(bpy)3(2+)] label. Firstly, The ECL efficiency of AEMP was characterized via comparing with that of two coreactants enhancing Ru(bpy)3(2+) ECL, TPA and proline. At same condition, AEMP has a similar ECL efficiency to TPA, and much higher than proline. After AEMP reacted with NHS-LC-biotin (succinimidyl-6-(biotinamido) hexanoate), the products and their ECL were analyzed by directly injecting it in the microfluidic chip. A 4.5 cm microchannel was used to separate the mixture of AEMP and biotinylated AEMP. The present works indicated that AEMP has a good reactivity to the analytes containing carboxyl group with a similar ECL efficiency to TPA. Under optimal condition, the detection limits (based on 3 S/N) of AEMP was 2.7 microM. The system was also validated by the reaction between biotin and avidin. The calculated binding ratio between avidin and biotin based on the present method was 4.4.     

A Sandwich‐Type Electrochemical Biosensor for Detection of BCR/ABL Fusion Gene Using Locked Nucleic Acids on Gold Electrode

In this study, a sandwich‐type electrochemical enzyme‐based LNA‐modified DNA biosensor was developed to detect relative gene in chronic Myelogenous Leukemia first. This biosensor is based on a ‘sandwich’ detection strategy, which involves a pair of probes (a capture probe immobilized at the electrode surface and a reporter probe labeled biotin as an affinity tag for avidin‐HRP) modified LNA. Since biotin can be connected with avidin‐HRP, this biosensor offers an enzymatically amplified electrochemical current signal for the detection of target DNA. This new pattern exhibits high sensitivity and selectivity, and this biosensor has been used for an assay of PCR real sample with satisfactory result.     

Comparison by QCM and photometric enzymatic test of the biotin-avidin recognition on a biotinylated polypyrrole

By gravimetric measurements using a quartz cristal microbalance (QCM), we have studied the immobilization of biotinylated glucose oxidase enzymes (B-GOx) bound through on an intermediate avidin layer to a biotinylated polypyrrole film. The aim is to assess the amount of B-GOx specifically anchored on the biotinylated polypyrrole/avidin assembly thank to the biotin/avidin interaction between avidin and B-GOx. Indeed the estimated amount from the QCM measurement corresponds to the specific recognition of avidin/B-GOx added to a non-specific recognition (adsorption) of B-GOx. In order to discriminate these two phenomena, we have carried out a study by QCM of the anchoring of B-GOx on an avidin layer linked by adsorption to a polypyrrole free from biotin units. From QCM measurements we have deduced for the biotinylated polypyrrole/avidin assembly that the amount of B-GOx bound via the biotin/avidin interaction and those due to the avidin adsorption process correspond to 3.9 pmol cm(-2) (1.3 equivalent of B-Gox monolayer) and 1.4 pmol cm(-2) (0.46 equivalent of B-GOx monolayer) respectively. These values have been corroborated by measurements of the enzymatic activity of GOx.     

Thermoresponsive dendronized copolymers for protein recognitions based on biotin-avidin interaction

Thermoresponsive biotinylated dendronized copolymers carrying dendritic oligoethylene glycol(OEG) pendants were prepared via free radical polymerization,and their protein recognitions based on biotin-avidin interaction investigated.Both first(PG1)and second generation(PG2)dendronized copolymers were designed to examine possible thickness effects on the interaction between biotin and avidin.Inherited from the outstanding thermoresponsive properties from OEG dendrons,these biotinylated cylindrical copolymers show characteristic thermoresponsive behavior which provides an envelope to capture avidin through switching temperatures above or below their phase transition temperatures(T_cps).Thus,the recognition of polymer-supported biotin with avidin was investigated with UV/vis spectroscopy and dynamic laser light scattering.In contrast to the case for PG1,the increased thickness for copolymer PG2 hinders partially and inhibits the recognition of biotin moieties with avidin either below or above its T_cp.This demonstrates the significant architecture effects from dendronized polymers on the biotin moieties to shift onto periphery of the collapsed aggregates,which should be a prerequisite for protein recognition.These kinds of novel thermoresponsive copolymers may pave a way for the interesting biological applications in areas such as reversible activity control of enzyme or proteins,and for controlled delivery of drugs or genes.     

Studies on biotin–avidin indirect conjugated technology for a piezoelectric DNA sensor

Because of their high sensitivity, piezoelectric sensor techniques are extremely useful for environmental or clinical analysis. We developed a piezoelectric crystal DNA biosensor for the detection of the hybridization reaction based on the self-assembled monolayer technology and biotin–avidin system. 3,3′-Dithiopropionic acid was applied to form a self-assembled monolayer (SAM) on the gold surface of the quartz crystal. Avidin was coated on the gold electrode conjugated with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and then biotinylated nucleotide acids were immobilized on the gold electrode surface through the specific interaction of biotin and avidin. Our results indicated that, using this immobilization method, the piezoelectric DNA sensor shows a higher sensitivity and specificity in detecting the hybridization reaction. The sensor can be used repeatedly by electrode regeneration.     

Preparation of Surface-enhanced Raman Scattering(SERS)-active Optical Fiber Sensor by Laser-induced Ag Deposition and Its Application in Bioidentification of Biotin/Avidin

A surface-enhanced Raman scattering(SERS) optical fiber sensor was prepared by the laser-induced deposition of Ag nanoparticle membrane on a silica optical fiber tip, which was applied to the real time SERS spectral monitoring on the biorecognition of biotin/avidin. The bioidentification of biotin/avidin was carried out through a indirect method, in which the bioidentification is based on the SERS response signal of a labeled dye(Atto610) after its fluorescence has been quenched totally by the deposited Ag nanoparticle membrane. By SERS monitoring the bioidentification process of biotin/avidin, it has been found that this recognition process is finished in 40 min. The lowest detection concentration of biotin is 1.0×10^-7 mg/mL. This research is promising in the application of immunoassays on line and in vivo.     

Highly sensitive biotin-labelled hybridization probe

A simple chemical method for introducing biotin into nucleic acids has been developed for the synthesis of nonisotopic hybridization probes. The method is based on the reaction of biotin hydrazide with amino residues of nucleic acids by using glutaraldehyde as a bifunctional coupling reagent. Biotin-labelled deoxyribonucleic acid (DNA) was detected by the use of alkaline phosphatase-labelled avidin, and alkaline phosphatase activity was measured by colorimetric and chemiluminescence methods. The chemiluminescence method using the nicotinamide adenine dinucleotide phosphate (NADP)/alcohol/alcohol dehydrogenase/microperoxidase/isoluminol system gave the highest sensitivity. A few picograms of lambda-phage DNA coated on a microtiter plate well could be detected by this method.     

Voltammetric detection of avidin and biotin by biotin labeled with cysteine

An avidin-biotin assay was developed from a voltammetric procedure using biotin labeled with cysteine. Mercury(II) as a marker was used to detect avidin and biotin, because the oxidation wave of mercury decreases when the cysteine part of labeled biotin(LB) complexes with mercury(II).The formation of the mercury(II)-cysteine complex is suppressed when the LB binds to the biotin site of avidin. Accordingly, the concentration of avidin can be estimated from the increasing mercury peak current. Detection of biotin is also carried out by a competitive reaction of biotin and the LB to the binding site on avidin, where the addition of biotin decreases the peak current of mercury. Limits of detection for avidin and biotin were in the 10^–9 mol/L range. The length of the spacer between the cysteine and biotin was investigated. It was observed that the strength of binding increased with increasing length of spacer. Size considerations rules out steric influences, so it is suggested that the binding constant depends on hydrophobic interactions in the binding site.     

A new chemically amplified electrochemical system for the detection of biological affinity reactions: direct and competitive biotin assay

Quantitation of biological affinity reactions by a newly developed chemically amplified electrochemical detection method was demonstrated with the biotin-avidin binding pair. In the method, ruthenium tris(2,2'-bipyridine)(Ru-bipy) was used as an electrochemical signal-generating tag. Its oxidation current on an indium tin oxide (ITO) electrode was amplified with a sacrificial electron donor, oxalate. Because oxalate itself produced negligible current on the electrode, the signal-to-background ratio was greatly enhanced in comparison with other chemical amplification systems. Although the Ru-bipy/oxalate redox couple has been employed previously in electrochemiluminescent and photoelectrochemical detection, its use in a catalytic amperometric detection of biological binding assays has not been reported. To implement the method in the detection of biotin-avidin recognition, avidin was immobilized on an ITO electrode, and was reacted with biotin in solution. Immobilization of avidin by passive adsorption was found to be relatively stable under the condition of the affinity reaction. In the direct assay, biotin labelled with Ru-bipy was recognized by avidin and accumulated on the electrode surface, which was then detected electrochemically in the presence of oxalate. A linear relationship between electrochemical current and biotin concentration was obtained in the range of 1-300 ng mL(-1). In the competitive assay, a mixed solution of unlabelled biotin (the analyte) of various concentrations and 100 ng mL(-1) labelled biotin was reacted with avidin on the surface. As the concentration of the unlabelled biotin increased, less labelled biotin bound to avidin, leading to a reduction in the electro-catalytical response of Ru-bipy. A detection limit of 1 ng mL(-1) biotin was obtained in the competitive assay, which is close to the sensitivity of some enzyme-labelled amperometric assays.     

Quantitative FT-IRRAS spectroscopic studies of the interaction of avidin with biotin on functionalized quartz surfaces

The interaction of avidin with biotin was studied on functionalized quartz surfaces terminated with 3-aminopropyltrimethoxysilane (3-APTMS), 2,2'-(ethylenedioxy)bis(ethylenediamine) (DADOO), and fourth-generation amine-terminated polyamidoamine (G4-NH2 PAMAM) dendrimers with the use of Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS). In particular, the molecular recognition ability of these surfaces was quantified through FT-IRRAS in combination with the use of an alkyne dicobalt hexacarbonyl probe coupled with avidin. The degree of nonspecific adsorption of avidin was determined by exposure of the amine-terminated and/or biotinylated surfaces to solutions of biotin-saturated avidin. The results indicate that the biotinylated 3-APTMS layer exhibits a very low specific binding capacity for avidin (on the order of 0.15 pmol of avidin/cm2) and substantial nonspecific adsorption. Both the binding capacity and the specificity were greatly improved when the 3-APTMS layer on quartz was modified through serial chemisorption of glutaraldehyde (GA), DADOO, and/or G4-NH2 PAMAM dendrimer layers. Among these layers, the biotinylated G4-NH2 PAMAM dendrimer layer exhibited the highest capacity for avidin binding (2.02 pmol of avidin/cm2) with a specificity of approximately 90%. This effect can be attributed to the efficient packing/ordering of the binding dendrimer layer, leading to a more dense and better organized layer of biotin headgroups on the subsequent biotinylated surface.     

FT-IRRAS spectroscopic studies of the interaction of avidin with biotinylated dendrimer surfaces

The interaction of avidin with biotin on a functional Au surface containing fourth generation amine-terminated polyamidoamine (G4-NH₂ PAMAM) dendrimers was investigated through the use of Fourier transform infrared reflection–adsorption spectroscopy (FT-IRRAS). The first step in the fabrication of the functional surfaces used was the construction of an aldehyde-terminated self-assembled monolayer (SAM) through the treatment of Au-coated glass slides with ethanol solutions of self-synthesized 2-hydroxypentamethylene sulfide (HPMS). The as-formed aldehyde-terminated monolayer was subsequently immersed in methanol solutions of G4-NH₂ PAMAM dendrimer to obtain well-organized primary amine-terminated surfaces. Biotinylation of the amine-terminated layers thus obtained was accomplished by use of the N-succinimidyl ester of biotin. Each step of the synthetic process, as well as the performance of final surface for protein recognition was monitored by FT-IRRAS. In particular, the molecular recognition ability was examined and quantified by use of an alkyne dicobalt hexacarbonyl probe coupled with avidin. Non-specific adsorption of avidin was determined by exposure of the amine-terminated and/or biotinylated surfaces to solutions of biotin-saturated avidin. The results indicate that the biotinylated G4-NH₂ PAMAM dendrimer layers formed according to this procedure have a high capacity for binding avidin with relatively high specificity. The performance of these layers (i.e. both binding capacity and specificity) improve substantially when 6-mercapto-1-hexanol (MH) is present as a co-adsorbent during the formation of the initial aldehyde-terminated layers. This effect can be attributed to the dilution of the initial aldehyde-terminated SAM, leading to a more favorable spatial arrangement of the subsequent biotinylated surfaces.     

Electropolymerisable pyrrole-oligonucleotide: synthesis and analysis of ODN hybridisation by fluorescence and QCM

Conducting polymer films, such as polypyrrole, appear particularly attractive for the immobilisation of biological molecules by entrapment or covalent grafting. We describe here a new pyrrole phosphorarnidite building block allowing the synthesis of oligonucleotide (ODN) bearing a pyrrole moiety. The electropolymerisable pyrrole moiety was then introduced on the 5' end of the oligonucleotide. The electrosynthesis of a copolymer, from solutions containing pyrrole and pyrrole-ODN, gives in one step strongly adhesive films containing ODN probes at electrode surfaces. In this contribution, we have used such a methodology to verify its feasibility for the modification of quartz crystal microbalance (QCM) electrodes. The obtained biosensors enable the detection of DNA hybridisation in real time by micro-gravimetric transduction. Finally, as DNA targets were previously modified by biotin, we have used the affinity between biotin and avidin to validate the effectiveness of QCM transduction by fluorescence microscopy and to amplify the recorded micro-gravimetric signal.     

光电化学竞争法检测生物素

建立了光电化学系统竞争性检测生物素(Biotin)小分子浓度的方法。采用联吡啶钌[Tris(2,2’-bipyridine)ruthenium,Ru-bpy)]作为标记物,以氧化锡纳米颗粒为电极,草酸盐为电子供体还原标记物。在470 nm光激发下,联吡啶钌的外层电子吸收能量后由基态变为激发态,注入半导体氧化锡纳米颗粒电极的导带,形成光电流信号;草酸盐还原失去电子的联吡啶钌使其恢复初始状态,从而可以再次作为电子供体受激发产生光电流信号。在竞争性检测生物素(Biotin)浓度时,亲和素(Avidin)吸附到氧化锡纳米颗粒电极表面作为识别元件,在浓度大于0.5 g/L时能够达到最大的电极表面覆盖率。1μmol/L Ru-bpy-biotin与不同浓度Biotin组成的混合溶液与电极表面的Avidin发生亲和反应,光激发后检测光电流大小;当溶液中Biotin的浓度增加时,致使与电极表面Avidin结合的Ru-bpy-biotin量减少,在光照射下光电流信号降低。这一竞争性光电检测方法检测Biotin时,检出限为8μg/L。本方法可进一步扩展,应用于有机化合物的竞争性免疫检测。     

Stimuli-sensitive thin films prepared by a layer-by-layer deposition of 2-iminobiotin-labeled poly(ethyleneimine) and avidin

Layered thin films composed of avidin and 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) were prepared by a layer-by-layer deposition of avidin and ib-PEI on a solid surface, and the disintegration induced by changing environmental pH and adding biotin in the solution was studied. The avidin/ib-PEI layered film could be deposited only from the solutions of pH 10-12. The film did not form in pH 9 or more acidic media because of a low affinity of protonated 2-iminobiotin residues in ib-PEI to avidin. The avidin/ib-PEI layered films were stable in pH 8-12 solutions, while in pH 5-7 media the film decomposed spontaneously as a result of the protonation to 2-iminobiotin residues in ib-PEI. The avidin/ib-PEI films were disintegrated also upon addition of biotin and analogues in the solution owing to the preferential binding of biotin or analogues to the binding site of avidin. The decomposition rate was arbitrarily controlled by changing the type of stimulant (biotin or analogues) and its concentration. The avidin/ib-PEI films were disintegrated rapidly by addition of 10(-)(5) M of biotin or desthiobiotin, while the rate was slower upon adding the same concentration of lipoic acid or 2-(4'-hydroxyphenylazo)benzoic acid. On the other hand, the film was fully decomposed within 1 min in the 10(-)(3) M lipoic acid or 2-(4'-hydroxyphenylazo)benzoic acid solution. Thus, the decomposition rate is highly dependent on the concentration of the stimulants. It was observed that the stimuli-induced decomposition of the films is slow at pH 12, in contrast to a rapid decomposition in pH 8 medium due to a low affinity of the protonated 2-iminobiotin to avidin. The present system may be useful for constructing stimuli-sensitive devices that can release drug or other functional molecules.     

Enhancement of the emission intensity of fluorophore-labeled avidin by biotin and biotin derivatives. Evaluation of different fluorophores for improved sensitivity

Fluorescein, Texas Red, Cascade Blue, 7-amino-methylcoumarin-3-acetic acid, and Lucifer Yellow were evaluated as fluorescent labels for homogeneous fluorophore-linked binding assays. Conjugates of avidin with these fluorophores exhibited an enhancement in fluorescence emission in the presence of biotin or biotin derivatives. This property was used in the development of assays for biotin. The biotin-induced fluorescence enhancement of each labeled avidin were compared. Fluorescein led to the most sensitive calibration (dose-response) curve for biotin with a detection limit of 8 x 10(-10)M.     

Dual Signal Amplification Electrochemical Biosensor for Lead Cation

Herein, a sensitive electrochemical Pb²⁺ sensor was developed which based on DNA‐functionalized Au nanoparticles(AuNPs) and nanocomposite modified electrode. The DNA‐functionalized AuNPs includes two types of DNA, namely a Pb²⁺‐mediated DNAzyme comprising a biotin labeled‐enzyme DNA and a substrate strand DNA with a typical stem‐loop structure, and a ferrocene‐labeled linear signal DNA. Without Pb²⁺, the hairpin loop impeded biotin binding to avidin on the electrode. However,when the goal Pb²⁺ exists, the substratum strand was divided into two fragments that lead to the enzyme strand was substratumed on the electrode and biotin was admited by avidin, bringing about DNA‐functionalized AuNP(AuNPs) deposition on the electrode surface.The differential pulse voltammetry (DPV) was used to measure electrochemical response signals connect to signal DNA.For the amplification characters of the DNA‐functionalized AuNPs and nanocomposite, the electrochemical detection signal of Pb²⁺ was greatly improved and revealed high specificity. Under optimum conditions, the resultant biosensor bringed out a high sensitivity and selectivity for the determination of Pb²⁺. The proposed method was able to detect as low as picomolar Pb²⁺ concentrations.     

Novel rhenium(I) polypyridine biotin complexes that show luminescence enhancement and lifetime elongation upon binding to avidin

While most biotin-fluorophore conjugates suffer from significant emission quenching upon binding to avidin due to resonance energy-transfer, three novel rhenium(I) polypyridine biotin complexes have been designed in view of their characteristic photophysical properties, in particular their large Stokes shifts. In contrast to most biotin-fluorophore conjugates, the (3)MLCT emission intensities and lifetimes of these rhenium(I) complexes are increased upon binding to avidin, rendering them luminescent probes for avidin and biotinylated species.     

Half‐Sandwich Ruthenium(II) Biotin Conjugates as Biological Vectors to Cancer Cells

Ruthenium(II)–arene complexes with biotin‐containing ligands were prepared so that a novel drug delivery system based on tumor‐specific vitamin‐receptor mediated endocytosis could be developed. The complexes were characterized by spectroscopic methods and their in vitro anticancer activity in cancer cell lines with various levels of major biotin receptor (COLO205, HCT116 and SW620 cells) was tested in comparison with the ligands. In all cases, coordination of ruthenium resulted in significantly enhanced cytotoxicity. The affinity of Ru^II–biotin complexes to avidin was investigated and was lower than that of unmodified biotin. Hill coefficients in the range 2.012–2.851 suggest strong positive cooperation between the complexes and avidin. To estimate the likelihood of binding to the biotin receptor/transporter, docking studies with avidin and streptavidin were conducted. These explain, to some extent, the in vitro anticancer activity results and support the conclusion that these novel half‐sandwich ruthenium(II)–biotin conjugates may act as biological vectors to cancer cells, although no clear relationship between the cellular Ru content, the cytotoxicity, and the presence of the biotin moiety was observed.     

Development of an egg-white bioassay for monitoring biotin levels in urine and serum.

This article reports on the development of a simple and cost-effective bioassay for the detection of biotin in urine and serum, based on the very selective binding of avidin and biotin. Avidin was allowed to react without isolating it from egg white. Egg white was treated with the dye HABA, which binds to avidin. Upon subsequent treatment with biotin, HABA is released due to the high affinity of biotin to avidin. The amount of HABA released is proportional to the amount of biotin used.