Surface modification of an acoustic biosensor allowing the detection of low concentrations of cancer markers

Analyte detection with biosensors is strongly influenced by the preparation of the biosensor surface including choice of sensing layers and coupling methods for corresponding capture molecules. We investigated the influence of different coupling procedures, especially considering coupling chemistry and incubation times for reagents, by means of surface acoustic wave (SAW) biosensors. The effect on the signal response was tested in two subsequent protein assays. Our optimized coupling procedure allowed the detection of the breast cancer markers HER-2 (human epidermal growth factor receptor-2) and TIMP-1 (tissue inhibitor of metalloproteinase-1) below the respective clinical cutoff values of only a few nanograms per milliliter.     

Enhanced sensitivity detection of protein immobilization by fluorescent interference on oxidized silicon

We present a silicon chip-based approach for the enhanced sensitivity detection of surface-immobilized fluorescent molecules. Green fluorescent protein (GFP) is bound to the silicon substrate by a disuccinimidyl terephtalate-aminosilane immobilization procedure. The immobilized organic layers are characterized by surface analysis techniques, like ellipsometry, atomic force microscopy (AFM) and X-ray induced photoelectron spectroscopy. We obtain a 20-fold enhancement of the fluorescent signal, using constructive interference effects in a fused silica dielectric layer, deposited before immobilization onto the silicon. Our method opens perspectives to increase by an order of magnitude the fluorescent response of surface immobilized DNA- or protein-based layers for a variety of biosensor applications.     

Specific and selective biosensor for Salmonella and its detection in the environment

The specific and selective detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir-Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated in liquid samples. These biosensors were selective to S. typhymurium in the presence of large concentrations of Escherichia coli O157:H7. They were also specific to S. typhymurium since bacteria preincubated with free antibody produced no signal. Dark-field and electron microscopy showed that two different antibodies, polyvalent somatic O and flagellar H7, were immobilized on the sensor surface producing two distinct attachments of bacteria at the liquid-solid interface. The somatic O antibody exhibits a rigid, binding, while the flagellar H7 antibody forms a flexible connection allowing a large degree of freedom. When the attachment of bacteria was rigid and strong, the responses of the acoustic wave sensors correlated with changes in the mass of bacteria present at the liquid-solid interface. In contrast, when attachment was flexible, the sensor signals were inversely proportional to the additional mass of bound bacteria. This difference is probably determined by the interfacial viscoelasticity and by acoustic and electromagnetic coupling. The signals of environmentally aged sensors with either predominantly rigid or flexible positioning of bacteria were correlated with changes in mass at the liquid-solid interface. Sensors with O or H type of binding could be used for analytical purposes.     

Feasibility study of using the dispersion of surface acoustic wave impulse for viscoelasticity characterization in tissue mimicking phantoms

The aim of this study was to investigate the feasibility of utilizing the phase velocity dispersion of impulse surface acoustic wave (SAW) for viscoelasticity characterization of soft materials. The focused ultrasound transducer and the phase‐sensitive optical coherence tomography were applied as the impulse SAW inducer and tracker, respectively. Three types of liquid‐paraffin‐based cream‐in‐agar phantoms were tested. Phase velocity dispersion curve was extracted using a Fourier transform‐based phase velocity analysis algorithm. Viscoelastic parameters were obtained by fitting the dispersion curve of SAW into Rayleigh wave dispersion equation. The estimated viscoelasticity was compared with that from spherical indenter, ramp‐hold relaxation testing for validation. Both results show an increasing trend in the elasticity and decreasing trend in the viscosity with the concentration of liquid‐paraffin‐based cream increasing in the samples. The proposed method has the capability of evaluating the viscoelastic properties of homogeneous soft tissue. By combining viscoelastic parameters estimated from the proposed method, the dispersive SAW‐impulse‐based viscosity‐compensated elastography could be further developed.      

An interleukin-6 ZnO/SiO(2)/Si surface acoustic wave biosensor

A novel high sensitivity ZnO/SiO(2)/Si Love mode surface acoustic wave (SAW) biosensor for the detection of interleukin-6 (IL-6), is reported. The biosensors operating at 747.7MHz and 1.586GHz were functionalized by immobilizing the monoclonal IL-6 antibody onto the ZnO biosensor surface both through direct surface adsorption and through covalent binding on gluteraldehyde. The morphology of the IL-6 antibody-protein complex was studied using scanning electron microscopy (SEM), and the mass of the IL-6 protein immobilized on the surface was measured from the frequency shift of the SAW resonator biosensor. The biosensor was shown to have extended linearity, which was observed to improve with higher sensor frequency and for IL-6 immobilization through the monoclonal antibody. Preliminary results of biosensor measurements of low levels of IL-6 in normal human serum are reported. The biosensor can be fully integrated with CMOS Si chips and developed as a portable real time detection system for the interleukin family of proteins in human serum.     

Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications

We report the fabrication, characterization and evaluation of three-dimensional (3D) hydrogel thin films used to measure protein binding (antigenicity) and antibody functionality in a microarray format. Protein antigenicity was evaluated using the protein toxin, staphylococcal enterotoxin B (SEB), as a model on highly crosslinked hydrogel thin films of polyacrylamide and on two-dimensional (2D) glass surfaces. Covalent crosslinking conditions were optimized and quantified. Interrogation of the modified 3D hydrogel was measured both by direct coupling of a Cy5-labeled SEB molecule and Cy5-anti-SEB antibody binding to immobilized unlabeled SEB. Antibody functionality experiments were conducted using three chemically modified surfaces (highly crosslinked polyacrylamide hydrogels, commercially available hydrogels and 2D glass surfaces). Cy3-labeled anti-mouse IgG (capture antibody) was microarrayed onto the hydrogel surfaces and interrogated with the corresponding Cy5-labeled mouse IgG (antigen). Five different concentrations of Cy5-labeled mouse IgG were applied to each microarrayed surface and the fluorescence quantified by scanning laser confocal microscopy. Experimental results showed fluorescence intensities 3-10-fold higher for the 3D films compared to analogous 2D surfaces with attomole level sensitivity measured in direct capture immunoassays. However, 2D surfaces reported equal or greater sensitivity on a per-molecule basis. Reported also are the immobilization efficiencies, inter-and intra-slide variability and detection limits.     

Covalent bound sensing layers on surface acoustic wave (SAW) biosensors

This paper reports on the development of immunosensors based on commercially available surface acoustic wave (SAW) devices working at 380 MHz. Approaches for coating the sensor surface with a sensing layer of receptive biomolecules are presented and discussed. It was found that the sensitivity strongly relates to the immobilization method. Additionally, the sensitivity can be influenced by the density of accessible biomolecules on the active sensing area. Usually, by most of the standard immobilization procedures, two-dimensional layers of receptive biomolecules are obtained. We present a three-dimensional layer, which provides a higher absolute amount of recognition molecules. A dextran layer is photoimmobilized to the sensor surface and the recognition molecules are covalently embedded into the dextran matrix. The feasibility of specific immunosensing is investigated using SAW sensors connected to a fluid handling system.     

Point mutation detection with the sandwich method employing hydrogel nanospheres by the surface plasmon resonance imaging technique

We propose a surface modification procedure to construct DNA arrays for use in surface plasmon resonance (SPR) imaging studies for the highly sensitive detection of a K-ras point mutation, enhanced with hydrogel nanospheres. A homobifunctional alkane dithiol was adsorbed on Au film to obtain the thiol surface, and ethyleneglycol diglycidylether (EGDE) was reacted to insert the ethyleneglycol moiety, which can suppress nonspecific adsorption during SPR analysis. Then streptavidin (SA) was immobilized on EGDE using tosyl chloride activation. Biotinylated DNA ligands were bound to the SA surface via biotin-SA interaction to fabricate DNA arrays. In SPR analysis, the DNA analyte was exposed on the DNA array and hybridized with the immobilized DNA probes. Subsequently, the hydrogel nanospheres conjugated with DNA probes were bound to the DNA analytes in a sandwich configuration. The DNA-carrying nanospheres led to SPR signal enhancement and enabled us to discriminate a K-ras point mutation in the SPR difference image. The application of DNA-carrying hydrogel nanospheres for SPR imaging assays was a promising technique for high throughput and precise detection of point mutations.     

Chemical Sensors for Solvent Vapors: Enthalpic and Entropic Contributions to Host-Guest Interactions

Chemical sensors, based on the mass-sensitive quartz micro balance (QMB) or the surface acoustic wave device (SAW), that are coated with thin cyclophane layers allow the detection of harmful organic vapors. The sensor signal of these supramolecular analyte-receptors can be predicted by a method that uses estimated free energies of the host-guest complex formation. From MM3 force field calculations, the reaction enthalpies (H° of host-guest interaction between the macrocycle and the analyte can be calculated, whereas the entropy changes are taken from condensation data. The validity of this condensation model is proven by an excellent linear correlation of the logarithm of the experimental equilibrium constant with the estimated Gibbs energy DG°. In this way promising sensor materials can be selected, even before they are synthesized.     

Oligonucleotide probes applied for sensitive enzyme-amplified electrochemical assay of mercury(II) ions

We developed a novel electrochemical sensor for Hg(2+) detection using two mercury-specific oligonucleotide probes and streptavidin-horseradish peroxidase (HRP) enzymatic signal amplification. The two mercury-specific oligonucleotide probes comprised a thiolated capture probe and a biotinated signal probe. The thiolated capture probe was immobilized on a gold electrode. In the presence of Hg(2+), the thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction between the mismatched T-T base pairs directed the biotinated signal probe hybridizing to the capture probe and yielded a biotin-functioned electrode surface. HRP was then immobilized on the biotin-modified substrate via biotin-streptavidin interaction. The immobilized HRP catalyzed the oxidation of hydroquinone (H(2)Q) to benzoquinone (BQ) by hydrogen peroxide (H(2)O(2)) and the generated BQ was further electrochemically reduced at the modified gold electrode, producing a readout signal for quantitative detection of Hg(2+). The results showed that the enzyme-amplified electrochemical sensor system was highly sensitive to Hg(2+) in the concentration of 0.5 nM to 1 μM with a detection limit of 0.3 nM, and it also demonstrated excellent selectivity against other interferential metal ions.     

Quartz crystal microbalance bioaffinity sensor for biotin based on mixed self-assembled monolayers and metastable molecular complex receptor

A quartz crystal microbalance (QCM) sensor was proposed for the detection of small molecule biotin based on the mixed self-assembled monolayer (SAM) of thiols on gold substrate and the bioaffinity difference between an analyte (biotin) and an analogue compound (HABA) in binding avidin. Avidin formed a metastable complex with 2-[(4-hydroxyphenyl)azo]benzoic acid (HABA) immobilized on the crystal surface. When the sensor contacts a sample solution containing biotin, the avidin was released from the sensor surface to form a more stable complex with biotin in solution. The frequency change recorded is proportional to the desorbed mass of avidin, and there is a clear mathematic relationship between the frequency change and the biotin concentration. The use of mixed SAMs allows the stable attachment of bioreceptor molecules on the QCM, and enhances the amount of the immobilized molecules on the QCM, as a longer "space arm" in the mixed SAMs makes this monolayer membrane more accessible to capture the immobilized molecules. The proposed bioaffinity sensor has nice response to biotin in the range of 0.017-1.67 microg/mL. The sensor could be regenerated under very mild conditions simply by reimmersion of the sensor into a biotin solution to desorb the surplus avidin.     

Three-dimensional hydrogel structures as optical sensor arrays, for the detection of specific DNA sequences

The fabrication and characterization of surface-attached PEG-diacrylate hydrogel structures and their application as sensing platforms for the detection of specific target sequences are reported. Hydrogel structures were formed by a photopolymerization process, using substrate-bound Eosin Y molecules for the production of free radicals. We have demonstrated that this fabrication process allows for control over hydrogel growth down to the micrometer scale. Confocal imaging revealed relatively large pore structures for 25% (v/v) PEG-diacrylate hydrogels, which appear to lie in tightly packed layers. Our data suggest that these pore structures decrease in size for hydrogels with increasing levels of PEG-diacrylate. Surface coverage values calculated for hydrogels immobilized with 21-mer DNA probe sequences were significantly higher compared to those previously reported for 2- and 3-dimensional sensing platforms, on the order of 10(16)molecules cm(-2). Used as sensing platforms in DNA hybridization assays, a detection limit of 3.9 nM was achieved for hybridization reactions between 21-mer probe and target sequences. The ability of these hydrogel sensing platforms to discriminate between wild-type and mutant allele sequences was also demonstrated, down to target concentrations of 1-2 nM. A reduction in the hybridization time down to a period of 15 min was also achieved, while still maintaining confident results, demonstrating the potential for future integration of these sensing platforms within Lab-on-Chip or diagnostic devices.     

Sensitivity enhancement of SPR assay of progesterone based on mixed self-assembled monolayers using nanogold particles

Commercially available nanoparticles have been employed as high mass labels for enhancing the binding signals and improving the detection sensitivity of surface plasmon resonance (SPR) assays. Such a signal enhancement is affected by the size and distance of the nanoparticles from the sensing surface. High signal amplifications are expected with increasing nanoparticle size and as the distance between the sensing surface and the nanoparticle is decreased. This paper describes a new way to improve the SPR assay sensitivity of small molecules using a mixed self-assembled monolayer (mSAM) surface to bring the nanogold particles close to the sensing surface. Progesterone (P4) was conjugated to ovalbumin (OVA) with an oligoethylene glycol (OEG) linker to form protein conjugate (P(4)-OEG-OVA), which was immobilized onto the mSAM surface. Inhibition immunoassays based on this mSAM/P4-OEG-OVA surface have demonstrated that 10nm nanogold dramatically improved the assay sensitivity of progesterone, lowering its limit of detection (LOD) from the original 372.7 to 4.9 ng L(-1). In addition, the high stability of the mSAM/P4-OEG-OVA surface was demonstrated by the use of a single chip for over 400 binding/regeneration cycles without any significant drop in antibody binding capacity and baseline shift.     

Comparison of label-free biosensing in microplate, microfluidic, and spot-based affinity capture assays

Using both experimental assays and fluid-dynamic finite element simulation models, we directly compared the achievable performance limits of four distinct assay configurations for label-free detection of an analyte from a test sample on a biosensor surface. The assay configurations studied in this work included a biosensor incorporated into the bottom surface of a microplate well and a microfluidic channel. For each configuration, we compared assay performance for the scenario in which the entire bottom surface of the fluid-handling vessel is coated with capture ligands with assay performance for the scenario in which the capture ligands are applied in the form of localized spots. As a model system, we used detection of the protein biomarker tumor necrosis factor-alpha (TNF-α) using immobilized TNF-α capture antibody. Results show that the microfluidic assay format dramatically reduces the time required to establish a stable equilibrium. Spot-based assays are advantageous for microplate-based detection for reducing the time required for equilibrium sensor response. The results derived are generally applicable to any label-free biosensor technology and any ligand-analyte system with adjustable variables that include sensor mass density sensitivity, analyte-ligand adsorption/desorption rate constants, immobilized ligand density, flow channel geometry, flow rate, and spot size.     

Peptide biosensor for recognition of cross-species cell surface markers

Biosensors based on phage display-derived peptides as biorecognition molecules were used for the detection of cell surface cross-species markers in tissue homogenates. The peptide selected for murine myofibers was immobilized onto the surface of an acoustic wave sensor by biotin-streptavidin coupling. To detect peptide-receptor interaction, the sensors were exposed to muscle and control (kidney, liver, brain) tissue homogenates. The sensor showed a strong response to murine muscle. The amplitudes of the responses to the feline muscle homogenates were lower compared to those of the murine muscle, while the same K(d) indicated that the peptide has cross-species affinity. In contrast, murine kidney, liver and brain homogenates produced insignificant responses. Specificity of the sensor was shown in a blocking experiment, as reduced signal was detected when muscle preparations were preincubated with free peptide. Additionally, when muscle-specific peptide was replaced with two different random control peptides, the sensors produced no response to murine muscle. Suitability of peptide ligands for a variety of species can be evaluated using this technology.     

DNA microarrays on a dendron-modified surface improve significantly the detection of single nucleotide variations in the p53 gene

Selectivity and sensitivity in the detection of single nucleotide polymorphisms (SNPs) are among most important attributes to determine the performance of DNA microarrays. We previously reported the generation of a novel mesospaced surface prepared by applying dendron molecules on the solid surface. DNA microarrays that were fabricated on the dendron-modified surface exhibited outstanding performance for the detection of single nucleotide variation in the synthetic oligonucleotide DNA. DNA microarrays on the dendron-modified surface were subjected to the detection of single nucleotide variations in the exons 5–8 of the p53 gene in genomic DNAs from cancer cell lines. DNA microarrays on the dendron-modified surface clearly discriminated single nucleotide variations in hotspot codons with high selectivity and sensitivity. The ratio between the fluorescence intensity of perfectly matched duplexes and that of single nucleotide mismatched duplexes was >5–100 without sacrificing signal intensity. Our results showed that the outstanding performance of DNA microarrays fabricated on the dendron-modified surface is strongly related to novel properties of the dendron molecule, which has the conical structure allowing mesospacing between the capture probes. Our microarrays on the dendron-modified surface can reduce the steric hindrance not only between the solid surface and target DNA, but also among immobilized capture probes enabling the hybridization process on the surface to be very effective. Our DNA microarrays on the dendron-modified surface could be applied to various analyses that require accurate detection of SNPs.