Binding kinetics of human cellular prion detection by DNA aptamers immobilized on a conducting polypyrrole

We developed a biosensor based on the surface plasmon resonance (SPR) method for the study of the binding kinetics and detection of human cellular prions (PrP^C) using DNA aptamers as bioreceptors. The biosensor was formed by immobilization of various biotinylated DNA aptamers on a surface of conducting polypyrrole modified by streptavidin. We demonstrated that PrP^C interaction with DNA aptamers could be followed by measuring the variation of the resonance angle. This was studied using DNA aptamers of various configurations, including conventional single-stranded aptamers that contained a rigid double-stranded supporting part and aptamer dimers containing two binding sites. The kinetic constants determined by the SPR method suggest strong interaction of PrP^C with various DNA aptamers depending on their configuration. SPR aptasensors have a high selectivity to PrP^C and were regenerable by a brief wash in 0.1 M NaOH. The best limit of detection (4 nM) has been achieved with this biosensor based on DNA aptamers with one binding site but containing a double-stranded supporting part.

An innovative strategy for direct electrochemical detection of microRNA biomarkers

We report an electrochemical method for direct, reagentless, and label-free detection of microRNA, based on a conjugated copolymer, poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-2-carboxyethyl-1,4-naphthoquinone), acting as hybridization transducer. Hybridization between the oligonucleotide capture probe and a microRNA target of 22 base pairs generates an increase in the redox current (“signal-on”), which is evidenced by square wave voltammetry. Selectivity is good, with little hybridization for non-complementary targets, and the limit of detection reaches 650 fM. It is also evidenced that this sensitivity benefits from the high affinity of DNA for RNA.

Sensitive detection of tuberculosis using nanoparticle-enhanced surface plasmon resonance

We show that the antigen CFP-10 (found in tissue fluids of tuberculosis patients) can be used as a marker protein in a surface-plasmon resonance (SPR) based method for early and simplified diagnosis of tuberculosis. A sandwich SPR immunosensor was constructed by immobilizing the CFP-10 antibody on a self-assembled monolayer on a gold surface, this followed by blocking it with bovine serum albumin. Following exposure of the sensor surface to a sample containing CFP-10, secondary antibody immobilized on nickel oxide nanoparticles are injected which causes a large SPR signal change. The method has a dynamic range from 0.1 to around 150 ng per mL of CFP-10, and a detection limit as low as 0.1 ng per mL. This is assumed to be due to the high amplification power of the NiO nanoparticles.

Detection of mismatched caspase-3 DNA oligonucleotides with an SPR biosensor following amplification by Taq polymerase

We demonstrate that base mismatches of caspase-3 DNA sequences can be detected by surface plasmon resonance (SPR) following signal amplification by polymerase from Thermus aquaticus (Taq). The concentration of magnesium ions and the respective dNTPs for polymerase binding to the oligonucleotides on the sensing surface were optimized. Taq polymerase binds to double-stranded DNA that is self-assembled on the gold surface of the biosensor to induce an SPR signal. Experiments are presented on the effect of Mg(II) and dNTP concentrations on the activity of the polymerase on the sensing surface. The detection limits are 50 pM, 0.1 nM, 0.7 nM, 7 nM, and 20 nM for correctly matched, single-base mismatched, two-base mismatched, three-base mismatched and four-base mismatched DNA of caspase-3, respectively. This is attributed to the optimized experimental conditions, with samples containing 2 μM of Mg(II) and 0.3 mM of dNTP.

Direct surface plasmon resonance immunosensing of pyraclostrobin residues in untreated fruit juices

A surface plasmon resonance (SPR) immunoassay for on-line detection of the strobilurin fungicide pyraclostrobin in untreated fruit juices is presented. The analysis of pyraclostrobin residues is accomplished in apple, grape, and cranberry samples by monitoring the recognition events occurring separately in a two-channel home-made SPR biosensor. Covalent coupling of the analyte derivative results in a reversible method, enabling more than 80 measurements on the same sensor surface. Optimization of the immunoassay conditions provides limits of detection as low as 0.16?μg?L^?1. The selectivity and reproducibility of the analysis is ensured by studying both non-specific interactions with unrelated compounds and inter-assay coefficients of variation. Excellent recovery ranging from 98 to 103?% was achieved by a simple 1:5 dilution of fruit juice with assay buffer before the analysis. The lack of previous cleaning and homogenization procedures reduces the analysis time of a single food sample to only 25?min, including the regeneration cycle.

Interaction of mercury(II) ions with immobilized apo-metallothioneins studied by scanning electrochemical microscopy combined with surface plasmon resonance

Scanning electrochemical microscopy (SECM) was combined with surface plasmon resonance (SPR) and applied for in-situ monitoring of the incorporation of Hg²⁺ by apo-metallothionein (apo-MT) immobilized on the SPR substrate. Hg²⁺ was anodically stripped from the Hg-coated SECM Pt tip and sequestered by apo-MT upon its diffusion to the SPR substrate. The high sensitivity of the SPR instrument enabled the detection of the change in the composition and structure of apo-MT molecules that was induced by the metal sequestration of Hg²⁺. The SPR response revealed that the saturation co-ordination number of Hg²⁺ binding to apo-MT was 18. Moreover, an unexpected collapse of the structure of MT was observed when the stoichiometric ratio of Hg²⁺/MT was ~70, and the structure cannot be further altered even by adding a large excess of Hg²⁺. This collapse was also confirmed by Raman spectroscopy. The results are potentially useful for a deeper understanding of the detoxification mechanism of MT to mercury ion.

Improving surface plasmon resonance imaging of DNA by creating new gold and silver based surface nanostructures

The use of nanoparticles (NPs) can substantially improve the analytical performance of surface plasmon resonance imaging (SPRi) in general, and in DNA sensing in particular. In this work, we report on the modification of the gold surface of commercial biochips with gold nanospheres, silica-coated gold nanoshells, and silver nanoprisms, respectively. The NPs were tethered onto the surface of the chip and functionalized with a DNA probe. The effects of tethering conditions and varying nanostructures on the SPRi signals were evaluated via hybridization assays. The results showed that coupling between planar surface plasmons and electric fields, generated by localized surface plasmons of the NPs, is mandatory for signal enhancement. Silver nanoprisms gave the best results in improving the signal change at a target DNA concentration of <50 nM by +50 % (compared to a conventional SPRi chip). The limit of detection for the target DNA was 0.5 nM which is 5 times less than in conventional SPRi.

An indium tin oxide electrode modified with gold nanorods for use in potential-controlled surface plasmon resonance studies

The modification of electrodes with gold nanoparticles results in an increased electrode surface area, enhanced mass transport, and improved catalytic properties. We have extended this approach to indium tin oxide (ITO) electrodes to obtain optically transparent gold nanorod-modified electrodes which display enhanced electrochemical capabilities and have the additional advantage of showing a tunable surface plasmon resonance. The procedures for attaining high surface coverage (15 gold nanorods per square µm) of such electrodes were optimized, and the potential-dependent surface plasmon resonance was studied under controlled electrical potential. In an exemplary sensor application, we demonstrate the detection of mercury via potential-dependent formation of an Au-Hg amalgam.

A surface plasmon resonance study on the optical properties of gold nanoparticles on thin gold films

We report on an investigation of the optical properties of gold nanoparticles assembled as thin films of different thickness. The nanoparticles were linked to the surface of a gold chip by dithiol reagents and studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. There is good correlation between the experimental findings and theoretical simulation, and the respective data reveal the presence of ordered nanostructures in the assemblies. The shift in the SPR angle is linearly dependent on the particle size and the ratio of the different particles. SPR spectroscopy also reveals important information in terms of the optical constants of such films. This shall be further applied to in-situ quality control in the fabrication of optoelectronic, solar cell and semiconductor devices.

Strategies to improve the surface plasmon resonance-based immmunodetection of bacterial cells

We have made a comparison of (a) different surface chemistries of SPR sensor chips (such as carboxymethylated dextran and carboxymethylated C1) and (b) of different assay formats (direct, sandwich and subtractive immunoassay) in order to improve the sensitivity of the determination of the model bacteria Acidovorax avenae subsp. citrulli (Aac). The use of the carboxymethylated sensor chip C1 resulted in a better sensitivity than that of carboxymethylated dextran CM5 in all the assay formats. The direct assay format, in turn, exhibits the best sensitivity. Thus, the combination of a carboxymethylated sensor chip C1 with the direct assay format resulted in the highest sensitivity for Aac, with a limit of detection of 1.6?×?10⁶ CFU mL^-1. This SPR immunosensor was applied to the detection of Aac in watermelon leaf extracts spiked with the bacteria, and the lower LOD is 2.2?×?10⁷ CFU mL^?1.

Molecular beacon based biosensor for the sequence-specific detection of DNA using DNA-capped gold nanoparticles-streptavidin conjugates for signal amplification

We describe a highly sensitive and selective molecular beacon-based electrochemical impedance biosensor for the sequence-specific detection of DNA. DNA-capped conjugates between gold nanoparticles (Au-NPs) and streptavidin are used for signal amplification. The molecular beacon was labeled with a thiol at its 5′ end and with biotin at its 3′ end, and then immobilized on the surface of a bare gold electrode through the formation of Au-S bonds. Initially, the molecular beacon is present in the “closed” state, and this shields the biotin from being approached by streptavidin due to steric hindrance. In the presence of the target DNA, the target DNA molecules hybridize with the loop and cause a conformational change that moves the biotin away from the surface of the electrode. The biotin thereby becomes accessible for the reporter (the DNA-streptavidin capped Au-NPs), and this results in a distinct increase in electron transfer resistance. Under optimal conditions, the increase in resistance is linearly related to the logarithm of the concentration of complementary target DNA in the range from 1.0 fM to 0.1 μM, with a detection limit of 0.35 fM (at an S/N of 3). This biosensor exhibits good selectivity, and acceptable stability and reproducibility.

Surface plasmon resonance, fluorescence, and circular dichroism studies for the characterization of the binding of BACE-1 inhibitors

The mechanism of action underlying β-secretase 1 (BACE-1) inhibition was characterized by a surface plasmon resonance (SPR) method using primary amino groups to immobilize OM99-2, a well-known highly potent peptidic BACE-1 inhibitor, on the carboxyl groups of the dextran layer of a sensor chip. The diluted BACE-1 was mixed with buffer or the test compound and the mixture was flushed through the chip. BACE-1 binding to the immobilized peptide inhibitor was quantified. This SPR method was used to identify BACE-1 inhibitor binding sites and the mechanism of action (competitive/noncompetitive) and to validate findings of fluorescence resonance energy transfer (FRET) inhibition studies. To support this, a multimethodological approach (circular dichroism and fluorescence spectroscopy) was applied in parallel to FRET inhibition studies to characterize the binding modes of peptidic and nonpeptidic BACE-1 inhibitors. Circular dichroism spectroscopy served to correlate the conformation of BACE-1 with enzymatic activity and to monitor secondary structure changes upon ligand binding. In a complementary approach, direct fluorescence spectroscopy was used to characterize different BACE-1 inhibitor binding sites. The influence of pH and inhibitors on BACE-1 secondary structure was also elucidated. This multimethodological approach was applied to identify binding modes of bis(7)-tacrine and myricetin in comparison with well-known peptidic inhibitors.

Comparative SPR study on the effect of nanomaterials on the biological activity of adsorbed proteins

Bioactivity of proteins is evaluated to test the adverse effects of nanoparticles interjected into biological systems. Surface plasmon resonance (SPR) spectroscopy detects binding affinity that is normally related to biological activity. Utilizing SPR spectroscopy, a concise testing matrix is established by investigating the adsorption level of bovine serum albumin (BSA) and anti-BSA on the surface covered with 11-mercaptoundecanoic acid (MUA); magnetic nanoparticles (MNPs) and single-walled carbon nanotubes (SWCNTs), respectively. The immunoactivity of BSA on MNPs and SWCNT decreased by 18?% and 5?%, respectively, compared to that on the gold film modified with MUA. This indicates that MNPs cause a considerable loss of biological activity of adsorbed protein. This effect can be utilized for practical applications on detailed biophysical research and nanotoxicity studies.

A DNA biosensor based on resonance light scattering using unmodified gold bipyramids

We report on a novel biosensor for determining sequence-specific DNA. It is based on resonance light scattering (RLS) caused by the aggregation of gold bipyramids. These display localized surface plasmon resonance and can be used as a bioprobe. The absorption spectra and the transmission electron micrographs provide visual evidence of the aggregation of the gold bipyramids in the presence of DNA. The RLS intensity of the gold bipyramids increases with the concentration of the target DNA. The method was successfully applied to the determination of a 30-mer single-stranded oligonucleotide and works over the 0.1–10?nM concentration range.

Fluorometric determination of paraoxon in human serum using a gold nanoparticle-immobilized organophosphorus hydrolase and coumarin 1 as a competitive inhibitor

A dimeric organophosphorus hydrolase (OPH; EC 3.1.8.1; 72 kDa) was isolated from wild-type bacteria, analyzed for its 16s rRNA sequence, purified, and immobilized on gold nanoparticles (AuNPs) to form the transducer part of a biosensor. The isolated strain was identified as Pseudomonas aeruginosa. The AuNPs were characterized by transmission electron microscopy and localized surface plasmon resonance. Covalent binding of OPH to the AuNPs was confirmed by spectrophotometry, enzymatic activity assays, and FTIR spectroscopy. Coumarin 1, a competitive inhibitor of OPH, was used as a fluorogenic probe. The bioconjugates quench the emission of coumarin 1 upon binding, but the addition of paraoxon results in an enhancement of fluorescence that is directly proportional to the concentration of paraoxon. The gold-OPH conjugates were then used to determine paraoxon in serum samples spiked with varying levels of paraoxon. The method works in the 50 to 1,050 nM concentration range, has a low standard deviation (with a CV of 5.7–11 %), and a detection limit as low as 5?×?10^?11 M.

Label-free detection of gliadin food allergen mediated by real-time apta-PCR

Celiac disease is an immune-mediated enteropathy triggered by the ingestion of gluten. The only effective treatment consists in a lifelong gluten-free diet, requiring the food industry to tightly control the gluten contents of their products. To date, several gluten quantification approaches using antibodies are available and recommended by the legal authorities, such as Codex Alimentarius. However, whilst these antibody-based tests exhibit high sensitivity and specificity, the production of antibodies inherently requires the killing of host animals and is time-consuming and relatively expensive. Aptamers are structured single-stranded nucleic acid ligands that bind with high affinity and specificity to their cognate target, and aiming for a cost-effective viable alternative to the use of antibodies. Herein, we report the systematic evolution of ligands by exponential enrichment (SELEX)-based selection of a DNA aptamer against gliadin from a combinatorial DNA library and its application in a novel detection assay. Taking into account the hydrophobic nature of the gliadin target, a microtitre plate format was exploited for SELEX, where the target was immobilised via hydrophobic interactions, thus exposing aptatopes accessible for interaction with the DNA library. Evolution was followed using surface plasmon resonance, and following eight rounds of SELEX, the enriched DNA pool was cloned, sequenced and a clear consensus motif was identified. An apta-PCR assay was developed where competition for the aptamer takes place between the surface-immobilised gliadin and gliadin in the target sample, akin to an ELISA competitive format where the more target present in the sample, the less aptamer will bind to the immobilised gliadin. Following competition, any aptamer bound to the immobilised gliadin was heat-eluted and quantitatively amplified using real-time PCR, achieving a detection limit of approx. 2 nM (100 ng mL^?1). The specificity of the selected aptamer was demonstrated and no cross-reactivity was observed with streptavidin, bovine serum albumin or anti-gliadin IgG.

Sensitive detection of enteropathogenic E. coli using a bfpA gene-based electrochemical sensor

We have developed a sensitive assay for enteropathogenic E. coli (EPEC) by integrating DNA extraction, specific polymerase chain reaction (PCR) and DNA detection using an electrode modified with the bundle-forming pilus (bfpA) structural gene. The PCR amplified products are captured on the electrode and hybridized with biotinylated detection probes to form a sandwich hybrid containing two biotinylated detection probes. The sandwich hybridization structure significantly combined the numerous streptavidin alkaline phosphatase on the electrode by biotin-streptavidin connectors. Electrochemical readout is based on dual signal amplification by both the sandwich hybridization structure and the enzyme. The electrode can satisfactorily discriminate complementary and mismatched oligonucleotides. Under optimal conditions, synthetic target DNA can be detected in the 1 pM to 10 nM concentration range, with a detection limit of 0.3 pM. EPEC can be quantified in the 10 to 10⁷ CFU mL^?1 levels within 3.5 h. The method also is believed to present a powerful platform for the screening of pathogenic microorganisms in clinical diagnostics, food safety and environmental monitoring.

Luminescent Ru(bpy)3 2+-doped silica nanoparticles for imaging of intracellular temperature

Silica nanoparticles doped with the luminescent temperature probe Ru(bpy)₃ ²⁺ were prepared by a modified Stöber method and are shown to enable optical sensing of intracellular temperatures. Based on the regrowth of silica nanoseeds, the ruthenium probe was easily incorporated and then covered with a shell of pure silica. The resulting nanothermometers were immune to the quenching by oxygen owing to the outer silica layer. The nanoparticles were further coated with poly-L-lysine in order to reduce cytotoxicity and to warrant cellular uptake. The luminescence of these nanosensors is rather sensitive to temperature in the physiological range (25–45 °C), with a decrease of ?1.26 % in intensity per °C increase in temperature. The nanosensors were internalized into living cells of a hepatocellular carcinoma cell line along with gold nanorods. These display longitudinal surface plasmon resonance absorption at ~808 nm that causes a local rise in temperature. The microscopically captured luminescence intensity of the nanosensors after 808 nm irradiation of the gold nanorods decayed with increasing temperature, thereby indicating successful imaging of temperature.

Angle-dependent resonance of localized and propagating surface plasmons in microhole arrays for enhanced biosensing

The presence of microhole arrays in thin Au films is suited for the excitation of localized and propagating surface plasmon (SP) modes. Conditions can be established to excite a resonance between the localized and propagating SP modes, which further enhanced the local electromagnetic (EM) field. The co-excitation of localized and propagating SP modes depends on the angle of incidence (θ _exc) and refractive index of the solution interrogated. As a consequence of the enhanced EM field, enhanced sensitivity and an improved response for binding events by about a factor of 3 to 5 was observed with SPR sensors in the Kretschmann configuration for a set of experimental conditions (λ _SPR, θ _exc, and η). Thus, microhole arrays can improve sensing applications of SPR based on classical prism-based instrumentation and are suited for SP-coupled spectroscopic techniques.

Using a silver-enhanced microarray sandwich structure to improve SERS sensitivity for protein detection

A simple and sensitive method, based on surface-enhanced Raman scattering (SERS), for immunoassay and label-free protein detection is reported. A series of bowl-shaped silver cavity arrays were fabricated by electrodeposition using a self-assembled polystyrene spheres template. The reflection spectra of these cavity arrays were recorded as a function of film thickness, and then correlated with SERS enhancement using sodium thiophenolate as the probe molecule. The results reveal that SERS enhancement can be maximized when the frequency of both the incident laser and the Raman scattering approach the frequency of the localized surface plasmon resonance. The optimized array was then used as the bottom layer of a silver nanoparticle–protein–bowl-shaped silver cavity array sandwich. The second layer of silver was introduced by the interactions between the proteins in the middle layer of the sandwich architecture and silver nanoparticles. Human IgG bound to the surface of this microcavity array can retain its recognition function. With the Raman reporter molecules labeled on the antibody, a detection limit down to 0.1 ng mL^?1 for human IgG is easily achieved. Furthermore, the SERS spectra of label-free proteins (catalase, cytochrome C, avidin and lysozyme) from the assembled sandwich have excellent reproducibility and high quality. The results reveal that the proposed approach has potential for use in qualitative and quantitative detection of biomolecules.