Effect of the immobilisation of DNA aptamers on the detection of thrombin by means of surface plasmon resonance

We report a multichannel surface plasmon resonance (SPR) sensor for detection of thrombin via DNA aptamers immobilized on the SPR sensor surface. A detailed investigation of the effect of the immobilisation method on the interaction between thrombin and DNA aptamers is presented. Three basic approaches to the immobilisation of aptamers on the surface of the SPR sensor are examined: (i) immobilisation based on chemisorption of aptamers modified with SH groups, (ii) immobilisation of biotin-tagged aptamers via previously immobilized avidin, neutravidin or streptavidin molecular linkers, and (iii) immobilisation employing dendrimers as a support layer for subsequent immobilisation of aptamers. A level of nonspecific binding of thrombin to immobilized human serum albumin (HSA) for each of the immobilisation methods is determined. Immobilisation of aptamers by means of the streptavidin–biotin system yields the best results both in terms of sensor specificity and sensitivity.     

Aptamer-based biosensors for the detection of HIV-1 Tat protein

Two biosensors have been constructed using an RNA aptamer as biorecognition element. The aptamer, specific for HIV-1 Tat protein, has been immobilised on the gold surface of piezoelectric quartz crystals or surface plasmon resonance (SPR) chips to develop a quartz crystal microbalance (QCM)-based and an SPR-based biosensor, respectively. Both the biosensors were modified with the same immobilisation chemistry based on the binding of a biotinylated aptamer on a layer of streptavidin. The binding between the immobilised aptamer and its specific protein has been evaluated with the two biosensors in terms of sensitivity, reproducibility and selectivity. A protein very similar to Tat, Rev protein, has been used as negative control. The two biosensors both were very reproducible in the immobilisation and the binding steps. The selectivity was high in both cases.     

Progress in Enzyme-Based Biosensors Using Optical Transducers

Enzyme-based biosensors are well developed and relatively mature technique in the biosensing field. Biosensors that utilise enzymes as the recognition elements represent the most extensively studied area. The organisation of an enzyme-based biosensor requires the integration of the biocatalyst with the support or immobilised materials to the extent that the biocatalytic transformation is either optically or electronically transduced. Any optical or electrical changes at the support material as a result of the biocatalytic process, that is, depletion of the reactant or formation of the product, provide routes for the opto/electronic transduction of the biological process occurring at the sensing surface. This review focuses on the discussion of some enzyme immobilisation techniques including physical and chemical immobilisation. Enzyme-based biosensors using various optical detection methods such as absorptiometry, luminometry, chemiluminescence, evanescent wave, and surface plasmon resonance are also included. Finally, different types of enzyme-based optical biosensors for ascorbic acid, bilirubin, cholesterol, choline, ethanol, glucose, glutamate/glutamine, lactate, penicillin, urea, and uric acid determinations are presented.On sabbatical leave at The University of North Carolina at Chapel Hill in July 2004–July 2005     

Present and future of surface plasmon resonance biosensors

Surface plasmon resonance (SPR) biosensors are optical sensors exploiting special electromagnetic waves—surface plasmon-polaritons—to probe interactions between an analyte in solution and a biomolecular recognition element immobilized on the SPR sensor surface. Major application areas include detection of biological analytes and analysis of biomolecular interactions where SPR biosensors provide benefits of label-free real-time analytical technology. This paper reviews fundamentals of SPR affinity biosensors and discusses recent advances in development and applications of SPR biosensors.     

Biosensors for environmental monitoring of endocrine disruptors: a review article

This article provides an overview of the applications of biosensors in analysis and monitoring of endocrine-disrupting compounds (EDCs) in the environment. Special attention is devoted to the various types of physical-chemical signal transduction elements, biological mechanisms employed as sensing elements and techniques used for immobilisation of the bioreceptor molecules on the transducer surface. Two different classes of biosensors for EDCs are considered: biosensors that measure endocrine-disrupting effects, and biosensors that respond to the presence of a specific substance (or group of substances) based on the specific recognition of a biomolecule. Several examples of them are presented to illustrate the power of the biosensor technology for environmental applications. Future trends in the development of new, more advanced devices are also outlined.     

Characterisation of morphology of self-assembled PEG monolayers: a comparison of mixed and pure coatings optimised for biosensor applications

For detection of low concentrations of analytes in complex biological matrices using optical biosensors, a high surface loading with capture molecules and a low nonspecific binding of nonrelevant matrix molecules are essential. To tailor biosensor surfaces in such a manner, poly(ethylene glycols) (PEG) in varying lengths were immobilised covalently onto glass-type surfaces in different mixing ratios and concentrations, and were subsequently modified with three different kinds of receptors. The nonspecific binding of a model protein (ovalbumin, OVA) and the maximum loading of the respective analytes to these prepared surfaces were monitored using label-free and time-resolved reflectometric interference spectroscopy (RIfS). The three different analytes used varied in size: 150 kDa for the anti-atrazine antibody, 60 kDa for streptavidin and 5 kDa for the 15-bp oligonucleotide. We investigated if the mixing of PEG in different lengths could increase the surface loadings of analyte mimicking a three-dimensional matrix as was found using dextrans as sensor coatings. In addition, the effect on the surface loading was investigated with regard to the size of the analyte molecule using such mixed PEGs on the sensor surface. For further characterisation of the surface coatings, polarisation modulation infrared reflection absorption spectroscopy, atomic force microscopy, and ellipsometry were applied. All authors contributed equally to this work.     

Dendrimer-functionalized self-assembled monolayers as a surface plasmon resonance sensor surface

We report here a multistep route for the immobilization of DNA and proteins on chemically modified gold substrates using fourth-generation NH(2)-terminated poly(amidoamine) dendrimers supported by an underlying amino undecanethiol (AUT) self-assembled monolayer (SAM). Bioactive ultrathin organic films were prepared via layer-by-layer self-assembly methods and characterized by fluorescence microscopy, variable angle spectroscopic ellipsometry, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR). The thickness of the AUT SAM base layer on the gold substrates was determined to be 1.3 nm from ellipsometry. Fluorescence microscopy and AFM measurements, in combination with analyses of the XPS/ATR-FTIR spectra, confirmed the presence of the dendrimer/biopolymer molecules on the multilayer sensor surfaces. Model proteins, including streptavidin and rabbit immunoglobulin proteins, were covalently attached to the dendrimer layer using linear cross-linking reagents. Through surface plasmon resonance measurements, we found that sensor surfaces containing a dendrimer layer displayed an increased protein immobilization capacity, compared to AUT SAM sensor surfaces without dendrimer molecules. Other SPR studies also revealed that the dendrimer-based surfaces are useful for the sensitive and specific detection of DNA-DNA interactions. Significantly, the multicomponent films displayed a high level of stability during repeated regeneration and hybridization cycles, and the kinetics of the DNA-DNA hybridization process did not appear to be influenced by surface mass transport limiting effects.     

Modified gas-permeable silicone rubber membranes for covalent immobilisation of enzymes and their use in biosensor development

Novel enzyme membranes are introduced. Modified polymeric gas-permeable layers were developed enabling biological components which have available reactive groups (-NH2, -OH, -SH, -COOH) to couple covalently on to their surfaces. Therefore, gas-permeable two component room temperature vulcanizing (2K-RTV) silicone rubber was modified using additional cross-linking agents. Triethoxysilanes with functional groups on their side chains such as epoxy or amino groups were used. A special attribute of the resulting gas-permeable membranes is that their formation and modification occur simultaneously during one reaction step. IR spectroscopy was used to observe the changes in the polymeric structure due to the reaction with the additional cross-linking agents. Sensors equipped with these layers are suitable to measure dissolved gases such as O2, CO2 and NH3 consumed or produced by enzymes converting their substrates. Determination of glucose, a well investigated enzymatic detection process, was chosen to demonstrate the applicability of the enzyme immobilisation. Glucose oxidase was immobilised on the membranes and glucose was detected by amperometric measurement of oxygen consumption. It is expected that this immobilisation method will also be useful for miniaturised planar biosensors.     

Recent applications of surface plasmon resonance biosensors for analyzing residues and contaminants in food

Abstract  

Optical biosensors based on surface plasmon resonance are increasingly used to detect and (semi)quantify residues and contaminants in food. This review provides an overview of the methods published in this field since the year 2003. Such biosensors have mostly been applied to veterinary drug residues, like aminoglycosides, beta-agonists, cephalexin, chloramphenicol, fluoroquinolones, levamisole, nicarbazin, nitroimidazoles, penicillins, ractopamine, sulfonamides, tetracyclines, and tylosin in milk, egg, honey, prawn, muscle, liver and kidney. Only a few methods have been published on pesticide residues, mycotoxins, phycotoxins, polychlorinated biphenyls and surfactants. These procedures are described with regard to biological recognition element, type of sensor chip, immobilisation procedure, sample extraction and clean-up, crossreactivity, nonspecific binding, matrix interference, chip regeneration, assay formats, calibration, validation and instrumentation.     

A simple method of surface functionalisation for immuno-specific immobilisation of proteins

We present a new and advanced methodology, developed for surface functionalisation of gold and to study immobilisation of an immuno-specific system of proteins. A combination of electrochemical quartz crystal microbalance and Raman spectroscopy techniques allowed a complete understanding of the system starting from surface functionalisation and progressing to the functional structure analysis of immobilised proteins. A simple electrochemical procedure was formulated to prepare sulphonyl chloride terminated gold surfaces that form a strong sulphonamide bond with the receptor protein staphylococcal protein A (SpA). On the SpA grafted surfaces, the immobilisation of a human IgG and consecutive binding of an immuno-specific anti-human IgG was observed. The surface functional groups form a strong interaction with SpA without disturbing its functional properties. The native functional structure of SpA and also the IgGs was found to be retained in their immobilised state.     

DNA-binding small-ligand-immobilized surface plasmon resonance biosensor for detecting thymine-related single-nucleotide polymorphisms

A surface plasmon resonance (SPR) biosensor that carries DNA-binding small ligands has been developed for the detection of single-nucleotide polymorphisms (SNPs). 3,5-Diaminopyrazine derivatives, with a hydrogen-bonding profile fully complementary to the thymine base, were utilized as recognition elements on the sensor surface, and a target single-stranded DNA sequence was hybridized with a DNA probe containing an abasic site to place this site opposite a nucleobase to be detected. In a continuous flow of sample solutions buffered to pH 6.4 (0.25 M NaCl), the 3,5-diaminopyrazine-based SPR sensor can detect an orphan nucleobase in the duplex with a clear selectivity for thymine over cytosine, guanine, and adenine (5'-GTT GGA GCT GXG GGC GTA GGC-3'/3'-CAA CCT CGA CNC CCG CAT CCG-5'; X=abasic site, N=target nucleobase G, C, A, or T). The SPR response was linear in the concentration range 10-100 nM. Allele discrimination is possible based on the combination of different binding surfaces in a flow cell of the SPR system, which is demonstrated for the analysis of the thymine/cytosine mutation present in 63-meric polymerase chain reaction (PCR) amplification products (Ha-ras gene, codon 12, antisense strand). Comparison with a bulk assay based on 3,5-diaminopyrazine/DNA binding shows that the immobilization of 3,5-diaminopyrazine derivatives on the SPR sensor allows more sensitive detection of the target DNA sequence, and binding selectivity can be tuned by controlling the salt concentration of sample solutions. These features of the DNA-binding small-molecule-immobilized SPR sensor are discussed as a basis for the design of SPR biosensors for SNP genotyping.     

Fluorescent nanoparticle adhesion assay: a novel method for surface pKa determination of self-assembled monolayers on silicon surfaces

Since the computer industry enables us to generate smaller and smaller structures, silicon surface chemistry is becoming increasingly important for (bio-)analytical and biological applications. For controlling the binding of charged biomacromolecules such as DNA and proteins on modified silicon surfaces, the surface pK(a) is an important factor. Here we present a fluorescent nanoparticle adhesion assay as a novel method to determine the surface pK(a) of silicon surfaces modified with weak acids or bases. This method is based upon electrostatic interactions between the modified silicon surface and fluorescent nanoparticles with an opposite charge. Silicon slides were modified with 3-aminopropyltriethoxysilane (APTES) and were further derivatized with succinic anhydride. Layer thickness of these surfaces was determined by ellipsometry. After incubating the surfaces with an amine-reactive fluorescent dye, fluorescence microscopy revealed that the silicon surfaces were successfully modified with amine- and carboxyl-groups. Two surface pK(a) values were found for APTES surfaces by the fluorescent nanoparticle adhesion assay. The first surface pK(a) (6.55 ± 0.73) was comparable with the surface pK(a) obtained by contact angle titration (7.3 ± 0.8), and the second surface pK(a) (9.94 ± 0.19) was only found by using the fluorescent nanoparticle adhesion assay. The surface pK(a) of the carboxyl-modified surface by the fluorescent nanoparticle adhesion assay (4.37 ± 0.59) did not significantly differ from that found by contact angle titration (5.7 ± 1.4). In conclusion, we have developed a novel method to determine the surface pK(a) of modified silicon surfaces: the fluorescent nanoparticle adhesion assay. This method may provide a useful tool for designing pH-dependent electrostatic protein and particle binding/release and to design surfaces with a pH-dependent surface charge for (bio-)analytical lab-on-a-chip devices or drug delivery purposes.     

SPR imaging as a tool for detecting mucin – anti-mucin interaction. Outline of the development of a sensor for near-patient testing for mucin

Sensors able to provide ‘yes’/‘no’ answers have become of interest in recent years, especially in the fields of environmental research and healthcare. We describe a procedure based on surface plasmon resonance imaging (SPRI) to investigate the interaction between mucin and anti-mucin antibody, and we outline the development of an alarm sensor for the protein mucin, whose high concentration in saliva, blood or tissue is related to illnesses such as gingivitis, peridontitis or even cancer. Anti-mucin gastric antibodies are immobilised onto a gold surface. The immobilisation is evaluated for neat gold chips and for polymer-modified gold surfaces. We found that two different pHs are required, one for the immobilisation of the antibodies on gold (pH 5.5) and a different one for optimal interaction between the sample and the antibody layer (pH 7.0). Finally, we briefly demonstrate the application of the sensor to real saliva samples, both mucin-less and mucin-containing, evaluating the potential of the sensor to discriminate between healthy and ill.     

Preservation of the biofunctionality of DNA and protein during microfabrication

Microfabrication processes, especially in silicon, are not compatible with biomolecules. Silicon and metal-based materials having crystalline structures are manipulated under harsh conditions with acids, bases, and organic solvents at high temperature. In comparison, organic biomolecules such as DNA and proteins have complex, three-dimensional structures and are sensitive to denaturation, oxidation, hydrolysis, and thermal destruction. Here, we report on the integration of DNA and the biotin-binding protein NeutrAvidin into microfabrication processes by using a novel approach based on a gold passivation mask. Our data show that this passivation method preserves approximately 84% of the biofunctionality of DNA and approximately 30% of that of NeutrAvidin under harsh process conditions. This novel technology enables the integration of DNA, proteins, and potentially other biological molecules into mass scalable microfabrication processes for biomedical devices, biochips, biosensors, and microelectromechanical systems with biomolecules (BioMEMS).     

Selective patterning of Si-based biosensor surfaces using isotropic silicon etchants

Ultra-sensitive, label-free biosensors have the potential to have a tremendous impact on fields like medical diagnostics. For the majority of these Si-based integrated devices, it is necessary to functionalize the surface with a targeting ligand in order to perform specific biodetection. To do this, silane coupling agents are commonly used to immobilize the targeting ligand. However, this method typically results in the bioconjugation of the entire device surface, which is undesirable. To compensate for this effect, researchers have developed complex blocking strategies that result in selective patterning of the sensor surface. Recently, silane coupling agents were used to attach biomolecules to the surface of silica toroidal biosensors integrated on a silicon wafer. Interestingly, only the silica biosensor surface was conjugated. Here, we hypothesize why this selective patterning occurred. Specifically, the silicon etchant (xenon difluoride), which is used in the fabrication of the biosensor, appears to reduce the efficiency of the silane coupling attachment to the underlying silicon wafer. These results will enable future researchers to more easily control the bioconjugation of their sensor surfaces, thus improving biosensor device performance.     

Recent applications of surface plasmon resonance biosensors for analyzing residues and contaminants in food

Abstract  Optical biosensors based on surface plasmon resonance are increasingly used to detect and (semi)quantify residues and contaminants in food. This review provides an overview of the methods published in this field since the year 2003. Such biosensors have mostly been applied to veterinary drug residues, like aminoglycosides, beta-agonists, cephalexin, chloramphenicol, fluoroquinolones, levamisole, nicarbazin, nitroimidazoles, penicillins, ractopamine, sulfonamides, tetracyclines, and tylosin in milk, egg, honey, prawn, muscle, liver and kidney. Only a few methods have been published on pesticide residues, mycotoxins, phycotoxins, polychlorinated biphenyls and surfactants. These procedures are described with regard to biological recognition element, type of sensor chip, immobilisation procedure, sample extraction and clean-up, crossreactivity, nonspecific binding, matrix interference, chip regeneration, assay formats, calibration, validation and instrumentation. Graphical Abstract        

Reversible Immobilization of Peptides: Surface Modification and In Situ Detection by Attenuated Total Reflection FTIR Spectroscopy

A generic method is described for the reversible immobilization of polyhistidine‐bearing polypeptides and proteins on attenuated total reflecting (ATR) sensor surfaces for the detection of biomolecular interactions by FTIR spectroscopy. Nitrilotriacetic acid (NTA) groups are covalently attached to self‐assembled monolayers of either thioalkanes on gold films or mercaptosilanes on silicon dioxide films deposited on germanium internal reflection elements. Complex formation between Ni²⁺ ions and NTA groups activates the ATR sensor surface for the selective binding of polyhistidine sequences. This approach not only allows a stable and reversible immobilization of histidine‐tagged peptides (His–peptides) but also simultaneously allows the direct in situ quantification of surface‐adsorbed molecules from their specific FTIR spectral bands. The surface concentrations of both NTA and His–peptide on silanized surfaces were determined to be 1.1 and 0.4 molecules nm^?2, respectively, which means that the surface is densely covered. A comparison of experimental FTIR spectra with simulated spectra reveals a surface‐enhancement effect of one order of magnitude for the gold surfaces. With the presented sensor surfaces, new ways are opened up to investigate, in situ and with high sensitivity and reproducibility, protein–ligand, protein–protein, protein–DNA interactions, and DNA hybridization by ATR–FTIR spectroscopy.     

Easy and efficient bonding of biomolecules to an oxide surface of silicon

A new method is described to attach biological molecules to the surface of silicon. Semiconductors such as Si modified with surface-bound capture molecules have enormous potential for use in biosensors for which an ideal detection platform should be inexpensive, recognize targets rapidly with high sensitivity and specificity, and possess superior stability. In this process, a self-assembled film of an organophosphonic acid is bonded to the native or synthesized oxide-coated Si surface as a film of the correspondingphosphonate. The phosphonate film is functionalized to enable covalently coupling biological molecules, ranging in size from small peptides to large multi-subunit proteins, to the Si surface. Surface modification and biomolecule coupling procedures are easily accomplished: all reactions can proceed in air, and most take place under ambient conditions. The biomolecule-modified surfaces are stable under physiological conditions, are selective for adhesion of specific cells types, and are reusable.     

SECM characterization of immobilised enzymes by self-assembled monolayers on titanium dioxide surfaces

SECM in generator-collector mode was used to detect the presence of immobilised enzymes on titanium dioxide layers which were chemically or electrochemically generated with possible application as chemical sensors and biosensors. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were immobilised by SAM generation using aminopropyltriethoxysilane (APTES) and ascorbic acid. The enzymes were successfully immobilised on two different TiO(2) surfaces. A simple test of durability of the system was made and a model of SAM organisation is presented.     

Immobilisation of lactate dehydrogenase on poly(aniline)-poly(acrylate) and poly(aniline)-poly(vinyl sulphonate) films for use in a lactate biosensor

The immobilisation of enzymes on an electrode surface, in such a manner that they retain both substrate specificity and high levels of catalytic activity, is of great importance in bioelectrochemistry. This includes areas such as the development of enzyme-catalysed fuel cell electrodes, biosensors and other biotechnological applications. We have investigated the catalytic activity of hexahistidine tagged variants of lactate dehydrogenase (EC 1.1.1.27) from the thermophile Bacillus stearothermophilus both in solution and when immobilised on poly(aniline)-poly(acrylate) (PANi-PAA) or poly(aniline)-poly(vinyl sulphonate) (PANi-PVS) composite films. Both the C- and N-terminally tagged enzymes are readily immobilised on the modified electrode and catalyse the conversion of lactate and NAD⁺ to pyruvate and NADH. The NADH that is generated can be readily oxidised at the PANi-modified electrode surface.In solution, the activity of the C-tagged enzyme (LDH-CHis) was some 30% less that of the wild-type under comparable conditions, whereas the N-tagged enzyme was found to possess essentially the same activity as the wild-type. However, when the enzymes were immobilised on PANi-PAA and PANi-PVS the C-tagged enzyme films showed a higher NADH-dependent current than the wild-type LDH whilst the N-tagged enzyme had the highest of the three. In addition, the C-tagged enzyme film appeared more stable than the wild-type LDH-PANi film. A novel immobilisation chemistry of the enzyme is proposed to account for these observations.