Dithiobissuccinimidyl propionate as an anchor for assembling peroxidases at electrodes surfaces and its application in a H2O2 biosensor

Exposure of gold surfaces to solutions of dithiobis N-succinimidyl propionate (DTSP) gives rise to the modification of the surface with N-succinimidyl-3-thiopropionate (NSTP) which can, in turn, react with amino groups allowing for the covalent immobilization of enzymes such as horseradish peroxidase (HRP). The coverage of NSTP has been estimated to be of the order of 1.3 x 10(-10) from the charge consumed during its reductive desorption. The binding reaction of HRP with NSTP modified gold surfaces has been studied with the quartz crystal microbalance, and the results suggest that the immobilization process involves two steps in which the first (faster) appears to correspond to the rapid incorporation of the enzyme whereas the second is likely due to the slow incorporation of additional enzyme and/or reorganization of the immobilized layer. Spectrophotometric and electrochemical assays indicate that the immobilized HRP retains its enzymatic activity after immobilization onto the DTSP modified gold surface. The amount of immobilized (and active) HRP was estimated from QCM and spectrophotometric measurements to be of the order of 1.5 x 10(-11) mol/cm2. A peroxide biosensor was developed making use of a gold surface modified with DTSP and HRP employing Os and Ru complexes of 1,10-phenanthroline 5,6-dione (phen-dione) of the type [M(phendione)x(L)3-x]+2 (where L = 1,10-phenanthroline or 2,2'-bipyridine, x = 1-3) as mediators with the quinone moieties being the active component. The efficiency of the mediators increased with increasing number of phendione ligands.     

A novel technique to immobilize DNA on surface of a quartz crystal microbalance by plasma treatment and graft polymerization

A quartz crystal microbalance (QCM) is well known to provide mass-sensitive devices in nanogram levels, because of the resonance frequency changes upon the adsorption on the electrode. It offers the possibility of monitoring hybridization in real time and with high selectivity. In this study, a biosensor system was developed for the detection of Vibrio parahaemolyticus via its oligonucleotide probe immobilized on the gold electrodes' surface of QCM. However, because the surface of QCM was an inorganic substance, it was difficult to immobilize the oligonucleotide probe. In this study, the plasma surface modification of QCM through deposition of hexamethyldisilazane (HMDSZ) films as an interlayer was investigated. The interlayer provided good adhesion to the substrate and had a uniform structure. The result indicates that plasma deposition was a useful technique to immobilize the oligonucleotide probe on the gold electrodes' surface via glutaraldehyde (GA) coupling. To improve immobilization, post treatments by surface grafting of acrylamide (AAm) and polyethyleneimine (PEI) treatment onto the electrodes were also performed. The result demonstrates that the shift of resonance frequency of QCM was improved via subsequent graft polymerization of AAm and PEI treatment onto the electrodes. The QCM sensor after plasma deposition and surface modification could provide detection sensitivity up to 86 ng/ml and kept at 88% detecting sensitivity after 19 days of storage at 0 °C. After washing with 0.1 M NaOH solution and 7 times of repeated use in detecting, the regeneration rate of QCM could be up to 60%.     

An amperometric fructose biosensor based on fructose dehydrogenase immobilized in a membrane mimetic layer on gold

A prototype amperometric fructose biosensor based on membrane-bound fructose dehydrogenase (Gluconobacter sp.) and the coenzyme ubiquinone-6 immobilized in a membrane mimetic layer on a gold electrode has been constructed and tested. A bare gold electrode first was modified through chemisorption of a mixture of octadecyl mercaptan and two short-chain disulfides, 3,3'-dithiodipropionic acid and cystamine dihydrochloride. The membrane-bound enzyme, coenzyme, and additional phospholipid were codeposited through a detergent dialysis protocol. The short-chain modifiers may provide electrostatic interactions with enzyme surface charges, while the alkanethiolate and phospholipids enable hydrophobic interaction with the largely lipophilic, membrane-bound enzyme. At oxidizing potentials, the enzyme electrode responded with catalytic current densities up to 45 μA/cm(2) when exposed to fructose at 10 mM. The sensor exhibited a response time of less than 20 s, a sensitivity of 15 μA/cm(2)·mM and a detection limit of less than 10 μM. Biosensor measurements of d-fructose in apple and orange juice agreed to within a few percent with those made with an enzymatic spectrophotometric assay. The membrane mimetic layer effectively blocked access of interfering ascorbic acid to the electrode surface. Only a 4% positive error was observed in the presence of ascorbic acid at 5% of the fructose concentration (2 mM), which indicates that this construct could be particularly useful for quantitation of fructose in citrus juice.     

Study on the disposable urea biosensors based on PVC-COOH membrane ammonium ion-selective electrodes

A potentiometric urea biosensor is prepared by the immobilization of urease directly onto the surface of a solid-state ammonium ion-selective electrode. The enzyme is immobilized by entrapment method onto a nonactin membrane that incorporated carboxylated polyvinylchloride. The same method of immobilization method is adopted to compare the characteristics of urea biosensors based on ammonium ion-selective electrodes with those based on pH-sensitive electrodes, using the same tin-oxide (SnO/sub 2/)/indium tin-oxide glass substrate. Urea biosensors based on ammonium ion-selective electrodes respond quickly and stably to changes in urea concentrations between 0.026 and 10 mM. The slope in the linear range is around 55.56/spl plusmn/3.15 mV/decade and the detection limit is around 5 /spl mu/M. The effect of urea biosensors with different pH values is considered, and the characteristics of urea biosensors based on ammonium ion-selective electrodes are described. Additionally, the experimental results from the determination of the urea using biosensors based on pH-sensitive electrodes and ammonium ion-selective electrodes are compared and discussed.     

Selection of ligands for affinity chromatography using quartz crystal biosensor

This paper described a new strategy for rapid selecting ligands for application in affinity chromatography using a quartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip. The binding interactions of the immobilized KM with various proteins in solution were monitored as the variations of the resonant frequency of the modified sensor. Such a rapid screen analysis of interactions indicated clearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultant sensorgrams were rapidly analyzed by using a kinetic analysis software based on a genetic algorithm to derive both the kinetic rate constants (k(ass) and k(diss)) and equilibrium dissociation constants (K(D)) for LZM-KM interactions. The immobilized KM showed higher affinity to LZM with a dissociation constant on the order of 10(-5) M, which is within the range of 10(-4)-10(-8) M and suitable for an affinity ligand. Therefore, KM was demonstrated for the first time as a novel affinity ligand for purification of LZM and immobilized onto the epoxy-activated silica in the presence of a high potassium phosphate concentration. The KM immobilized affinity column has proved useful for a very convenient purification of LZM from chicken egg white. The purity of LZM obtained was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction. These results confirmed that the selected KM ligand is indeed a valuable affinity ligand for purification of LZM. The new screening strategy based on a QCM biosensor is expected to be a promising way for rapid selecting specific ligands for purifying other valuable proteins.     

Glucose biosensors based on dendrimer monolayers

The peculiarities of glucose biosensors based on different generation of dendrimers (G0, G1 and G4) have been studied by amperometry and QCM techniques. It is shown that stable glucose biosensor can be obtained with low generation of dendrimers. The sensor sensitivity, however considerable, increased with increasing number of generation of dendrimers. This can be due to the increased volume of the dendrimer interior as well as with increased number of binding sites for glucose oxidase (GOX). QCM experiments showed that immobilization of GOX resulted in formation of enzyme multilayers on a dendrimer surface. The enzyme turnover for this system (0.1-0.01 s(-1)) was lower then that for immobilization of GOX onto a supported lipid films by means of avidin-biotin technology (1.1 s(-1)). However, dendrimer based biosensors are more stable in comparison with sBLM based sensors and could be stored in a refrigerator in dry conditions over 15 days without substantial loss of sensitivity.     

Electrocatalytic detection of NADH and glycerol by NAD(+)-modified carbon electrodes

The electrochemical oxidation of the adenine moiety in NAD+ and other adenine nucleotides at carbon paste electrodes gives rise to redox-active products which strongly adsorb on the electrode surface. Carbon paste electrodes modified with the oxidation products of NAD+ show excellent electrocatalytic activity toward NADH oxidation, reducing its overpotential by about 400 mV. The rate constant for the catalytic oxidation of NADH, determined by rotating disk electrode measurements and extrapolation to zero concentration of NADH, was found to be 2.5 x 10(5) M-1 s-1. The catalytic oxidation current allows the amperometric detection of NADH at an applied potential of +50 mV (Ag/AgCl) with a detection limit of 4.0 x 10(-7) M and linear response up to 1.0 x 10(-5) M NADH. These modified electrodes can be used as amperometric transducers in the design of biosensors based on coupled dehydrogenase enzymes and, in fact, we have designed an amperometric biosensor for glycerol based on the glycerol dehydrogenase (GlDH) system. The enzyme GlDH and its cofactor NAD+ were co-immobilized in a carbon paste electrode using an electropolymerized layer of nonconducting poly(o-phenylenediamine) (PPD). After partial oxidation of the immobilized NAD+, the modified electrode allows the amperometric detection of the NADH enzymatically obtained at applied potential above 0 V (Ag/AgCl). The resulting biosensor shows a fast and linear response to glycerol within the concentration range of 1.0 x 10(-6)-1.0 x 10(-4) M with a detection limit of 4.3 x 10(-7) M. The amperometric response remains stable for at least 3 days. The biosensor was applied to the determination of glycerol in a plant-extract syrup, with results in good agreement with those for the standard spectrophotometric method.     

Thin films of polyelectrolyte-encapsulated catalase microcrystals for biosensing

Polyelectrolyte (PE)-encapsulated catalase microcrystals were assembled onto gold electrodes by their sequential deposition with oppositely charged PEs, utilizing electrostatic interactions to form enzyme thin films for biosensing. The PE coating around the microcrystals provided a regular surface charge, thus facilitating the stepwise film growth, and it effectively prevented catalase leakage from the assembled films. The encapsulated catalase was shown to retain both its biological and its electrochemical activity. Direct electron transfer between catalase molecules and the gold electrode was achieved without the aid of any electron mediator. In pH 5.0 phosphate buffer solution, the apparent formal potential (E(o)') of catalase was -0.131 V (vs Ag/AgCl). As a H2O2 biosensor, films consisting of one layer of the encapsulated catalase displayed considerably higher (approximately 5-fold) and more stable electrocatalytic responses to the reduction of H2O2 than did corresponding films made of one layer of nonencapsulated catalase or solubilized catalase. An increase in either the number of "precursor" PE layers between the gold electrodes and the catalase microcrystal layers in the film or the number of PE layers encapsulating the catalase microcrystals was found to decrease the electrocatalytic activity of the electrode. At low precursor PE layer numbers (approximately 2) and PE encapsulating layers (approximately 4), the current response was proportional to the H2O2 concentration in the range 3.0 x 10(-6) to 1.0 x 10(-2) M. The overall electroactivity of the multilayer film increased for the first two layers of encapsulated catalase, after which a plateau was observed. This was attributed to the increasing difficulty of electron transfer and substrate diffusion limitations. The current approach of using immobilized PE-encapsulated enzyme microcrystals for biosensing provides a versatile method to prepare high enzyme content films with high and tailored enzyme activities.     

A hydrogen peroxide biosensor based on peroxidase activity of hemoglobin in polymeric film

A Hydrogen peroxide (H2O2) biosensor, based on hemoglobin (Hb) and ortho-phenylenediamine (o-PD) gold electrode, was fabricated. Hb was immobilized onto the electrode surface by electrochemical polymerize method with o-PD. The designed biosensor showed a well defined redox peak which was attributed to the direct electrochemical response of Hb. The immobilized Hb exhibited an excellent electrocatalytical response to the reduction of hydrogen peroxide, enabling the sensitivity determination of H2O2. Factors and performances such as pH, potential, influencing the designed biosensor, were studied carefully. The amperometric detection of H2O2 was carried out at -300 mV in phosphate buffer solution (PBS) (0.1 M) with pH 6.0. This biosensor showed a fast amperometric response (less then 5 s) to H2O2. The levels of the (Relative standard deviation) RSDs (< 3.5%) for the entire analyses reflected a highly reproducible sensor performance. Using the optimized conditions, the detection limit of the biosensor was 1 x 10(-7) M and linear range was from 5 x 10(-6) to 1.25 x 10(-4) M. In addition, this sensor showed long-term stability and good sensitivity.     

A method to construct a third-generation horseradish peroxidase biosensor: self-assembling gold nanoparticles to three-dimensional sol-gel network

A novel method for fabrication of horseradish peroxidase biosensor has been developed by self-assembling gold nanoparticles to a thiol-containing sol-gel network. A cleaned gold electrode was first immersed in a hydrolyzed (3-mercaptopropyl)-trimethoxysilane (MPS) sol-gel solution to assemble three-dimensional silica gel, and then gold nanoparticles were chemisorbed onto the thiol groups of the sol-gel network. Finally, horseradish peroxidase (HRP) was adsorbed onto the surface of the gold nanoparticles. The distribution of gold nanoparticles and HRP was examined by atomic force microscopy (AFM). The immobilized horseradish peroxidase exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide. The performance and factors influencing the performance of the resulting biosensor were studied in detail. The resulting biosensor exhibited fast amperometric response (2.5 s) to H2O2. The detection limit of the biosensor was 2.0 micromol L(-1), and the linear range was from 5.0 micromol L(-1) to 10.0 mmol L(-1). Moreover, the studied biosensor exhibited high sensitivity, good reproducibility, and long-term stability.     

Conductometric transducers for enzyme-based biosensors

The use of alternating current conductometric transducers in biosensing devices has been investigated for urea and D-amino acid sensors using the enzyme systems urease and D-amino acid oxidase/catalase. Transducers with copper and platinum electrodes were constructed and characterized, and two enzyme immobilization methods were tested. Detection limits of 1 x 10(-6)M and linear ranges of 2 orders of magnitude were routinely achieved for these model sensors with enzymes covalently immobilized on collagen films.     

Design and fabrication of a microimpedance biosensor for bacterial detection

A biosensor for bacterial detection was developed based on microelectromechanical systems, heterobifunctional crosslinkers and immobilized antibodies. The sensor detected the change in impedance caused by the presence of bacteria immobilized on interdigitated gold electrodes and was fabricated from (100) silicon with a 2-/spl mu/m layer of thermal oxide as an insulating layer. The sensor active area is 9.6 mm/sup 2/ and consists of two interdigital gold electrode arrays measuring 0.8 /spl times/ 6 mm. Escherichia coli specific antibodies were immobilized to the oxide between the electrodes to create a biological sensing surface. The impedance across the interdigital electrodes was measured after immersing the biosensor in solution. Bacteria cells present in the sample solution attached to the antibodies and became tethered to the electrode array, thereby causing a change in measured impedance. The biosensor was able to discriminate between different cellular concentrations from 10/sup 5/ to 10/sup 7/ CFU/mL in pure culture. The sample testing process, including data acquisition, required 5 min. The design, fabrication, and testing of the biosensor is discussed along with the implications of these findings toward further biosensor development.     

Fabrication of a novel hydrogen peroxide biosensor based on C@Au composite

A promising hydrogen peroxide (H2O2) biosensor was fabricated by the immobilization of hemoglobin (Hb) on C@Au composite surface. The composite with carbon spheres and gold shell (C@Au) was synthesized via the seed-growth assembly technique. The assembly of the gold shell on carbon sphere surfaces was characterized by scanning electron miscroscopy (SEM). Owing to the unique structure and large surface area of the gold shell, the composite offered an effective interface for the immobilization of hemoglobin to fabricate a H2O2 biosensor. The obtained biosensor showed a wide linear range from 5.0 microM to 135 microM with a detection limit of 1.67 microM at 3sigma, and the apparent Michaelis-Menten constant (Km(app)) of the immobilized Hb was calculated to be 88.6 microM. Moreover, the biosensor also exhibited good reproducibility and long-term stability. Therefore, this kind of composite can provide an ideal matrix for protein immobilization and biosensor fabrication.     

Amperometric thick-film strip electrodes for monitoring organophosphate nerve agents based on immobilized organophosphorus hydrolase

An amperometric biosensor based on the immobilization of organophosphorus hydrolase (OPH) onto screen-printed carbon electrodes is shown useful for the rapid, sensitive, and low-cost detection of organophosphate (OP) nerve agents. The sensor relies upon the sensitive and rapid anodic detection of the enzymatically generated p-nitrophenol product at the OPH/Nafion layer immobilized onto the thick-film electrode in the presence of the OP substrate. The amperometric signals are linearly proportional to the concentration of the hydrolyzed paraoxon and methyl parathion substrates up to 40 and 5 μM, showing detection limits of 9 × 10(-)(8) and 7 × 10(-)(8) M, respectively. Such detection limits are substantially lower compared to the (2-5) × 10(-)(6) M values reported for OPH-based potentiometric and fiber-optic devices. The high sensitivity is coupled to a faster and simplified operation, and the sensor manifests a selective response compared to analogous enzyme inhibition biosensors. The applicability to river water sampling is illustrated. The attractive performance and greatly simplified operation holds great promise for on-site monitoring of OP pesticides.     

Towards nanomedicine with a supramolecular approach: a review

A review dedicated mainly to the results obtained by the authors on the use of cyclodextrin (CD) derivatives on protein (enzyme) stabilization through covalent and non-covalent interactions (host-guest supramolecular interactions) is presented here. This latter procedure served to introduce a new method for enzyme immobilization on metallic surfaces that can be used to prepare biosensors and therapeutic nanodevices. The surfaces of gold (and silver) electrodes and nanoparticles were modified with sulphur-containing cyclodextrin derivatives. The protein (enzyme) was then supramolecularly immobilized on the modified surface when one or more of its bulky hydrophobic moieties was included into the CD cavity. The protein can also be modified with a typical CD guest, such as adamantane, to achieve a more stable immobilization. Different examples are presented, such as a biosensor based on monolayers of adamantane-modified cytochrome c and a bienzymatic nanodevice comprising gold nanoparticles stabilized with CD associated to catalase and superoxide dismutase modified with complementary host-guest residues. The possibilities of this new approach for the development of biosensors and therapeutic nanodevices are analyzed.     

Highly selective detection of single-nucleotide polymorphisms using a quartz crystal microbalance biosensor based on the toehold-mediated strand displacement reaction

Toehold-mediated strand displacement reaction (SDR) is first introduced to develop a simple quartz crystal microbalance (QCM) biosensor without an enzyme or label at normal temperature for highly selective and sensitive detection of single-nucleotide polymorphism (SNP) in the p53 tumor suppressor gene. A hairpin capture probe with an external toehold is designed and immobilized on the gold electrode surface of QCM. A successive SDR is initiated by the target sequence hybridization with the toehold domain and ends with the unfolding of the capture probe. Finally, the open-loop capture probe hybridizes with the streptavidin-coupled reporter probe as an efficient mass amplifier to enhance the QCM signal. The proposed biosensor displays remarkable specificity to target the p53 gene fragment against single-base mutant sequences (e.g., the largest discrimination factor is 63 to C-C mismatch) and high sensitivity with the detection limit of 0.3 nM at 20 °C. As the crucial component of the fabricated biosensor for providing the high discrimination capability, the design rationale of the capture probe is further verified by fluorescence sensing and atomic force microscopy imaging. Additionally, a recovery of 84.1% is obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of employing this biosensor in detecting SNPs in biological samples.     

基于生物素-亲和素系统的酶固定化及纳米金增效的研究

应用石英晶体微天平(QCM)研究了基于生物素一亲和素系统的葡萄糖氧化酶的固定化,探讨了纳米金修饰QCM金基片对酶固定化的一系列过程的影响。在本实验条件下,利用生物素．亲和素系统能较好地固定化葡萄糖氧化酶,经过纳米金颗粒修饰的QCM基片对酶的吸附量比未经修饰的基片可提高1倍以上。     

Immobilization of olfactory receptors onto gold electrodes for electrical biosensor

We investigate the immobilization of native nanovesicles containing functional olfactory receptors onto gold electrodes by means of atomic force microscopy in liquid. We show that nanovesicles can be adsorbed without disrupting them presenting sizes once immobilized ranging from 50 nm to 200 nm in diameter. The size of the nanovesicles shows no dependence on the electrode hydrophobicity being constant in a height/width ratio close to 1:3. Nevertheless, electrode hydrophobicity does affect the surface coverage, the surface coverage is five times higher in hydrophilic electrodes than on hydrophobic ones. Surface coverage is also affected by nanovesicles dimensions in suspension, the size homogenization to around 50 nm yields a further five fold increment in surface coverage achieving a coverage of about 50% close to the hard spheres jamming limit (54.7%). A single layer of nanovesicles is always formed with no particle overlap. Present results provide insights into the immobilization on electrodes of olfactory receptors for further olfactory electrical biosensor development.     

金属与非金属纳米颗粒增强葡萄糖生物传感器

为了提高葡萄糖传感器的灵敏度和抗干扰性,利用纳米增强效应,以Au、Ag、Pt、SiO2纳米颗粒及金属-无机复合纳米颗粒与聚乙烯醇缩丁醛(PVB)构成复合固定酶膜基质,采用溶胶-凝胶法固定葡萄糖氧化酶(GOD),组成葡萄糖生物传感器.研究表明,纳米颗粒可以大幅度地提高固定化酶的催化活性,增加电极的电流响应灵敏度,改进生物传感器的抗干扰性能,使信噪比提高了32倍.     

Thin-film glucose biosensor based on plasma-polymerized film: simple design for mass production

We propose a simple thin-film glucose biosensor based on a plasma-polymerized film. The film is deposited directly onto the substrate under dry conditions. The resulting films are extreme thin, adhere well onto the substrate (electrode), and have a highly cross-linked network structure and functional groups, such as amino groups, which enable a large amount of enzyme to be immobilized. Since this design allows fabrication through a dry process, with the exception of the enzyme immobilization, which is the last stage of the process, the chip fabrication can be designed as a full-wafer process to achieve mass production compatibility. The resulting sensors produced using this film are more reproducible, exhibit lower noise, and reduce the effect of interference to a greater degree than sensors made using conventional immobilization methods, e.g., via 3-(aminopropyl)triethoxysilane. The obtained film is a good interfacial design between enzyme and electrode; enzyme two-dimensionally locates very close to the electrode in a manner that is quite reproducible. Therefore, a wide dynamic range (up to 60 mM) and rapid response time (11.5+/-0.8 s) were obtained. Because of its highly cross-linking network structure, the amperometric response due to interferences such as ascorbic acid and acetaminophen was reduced by size discrimination of plasma-polymerized films.