Oriented surface immobilization of antibodies at the conserved nucleotide binding site for enhanced antigen detection

The conserved nucleotide binding site (NBS), found on the Fab variable domain of all antibody isotypes, remains a not-so-widely known and unutilized site. Here, we describe a UV photo-cross-linking method (UV-NBS) that utilizes the NBS for oriented immobilization of antibodies onto surfaces, such that the antigen binding activity remains unaffected. Indole-3-butyric acid (IBA) has an affinity for the NBS with a K(d) ranging from 1 to 8 μM for different antibody isotypes and can be covalently photo-cross-linked to the antibody at the NBS upon exposure to UV light. Using the UV-NBS method, antibody was successfully immobilized on synthetic surfaces displaying IBA via UV photo-cross-linking at the NBS. An optimal UV exposure of 2 J/cm(2) yielded significant antibody immobilization on the surface with maximal relative antibody activity per immobilized antibody without any detectable damage to antigen binding activity. Comparison of the UV-NBS method with two other commonly used methods, ε-NH(3)(+) conjugation and physical adsorption, demonstrated that the UV-NBS method yields surfaces with significantly enhanced antigen detection efficiency, higher relative antibody activity, and improved antigen detection sensitivity. Taken together, the UV-NBS method provides a practical, site-specific surface immobilization method, with significant implications in the development of a large array of platforms with diverse sensor and diagnostic applications.     

Surface enhanced Raman scattering for multiplexed detection

The multiplexed detection of biological analytes from complex mixtures is of crucial importance for the future of intelligent management and detection of disease. This review focuses on recent advances in the use of surface enhanced Raman scattering (SERS) spectroscopy as an analytical technique that can deliver multiplexed detection for a variety of biological target in increasingly complex media. The use of SERS has developed from the multipelxed detection of custom dye molecules to biomolecules such as DNA and proteins. Recent work has also shown the capability of SERS multiplexing for in vivo as well as in vitro applications.     

Surface enhanced resonance Raman scattering detection by fluorimeter

A commercially available fluorimeter with a white light source is used to detect surface enhanced resonance Raman scattering (SERRS). This approach allows facile tunability of the excitation source for SERRS.     

Gold nanoparticle-labeled detection antibodies for use in an enhanced electrochemical immunoassay of hepatitis B surface antigen in human serum

The application of gold nanoparticle-based electrochemical immunoassays have been extensively studied for the detection of hepatitis B surface antigen (HBsAg), but most often they exhibit low sensitivity. We describe the fabrication of a new electrochemical immunoassay for signal amplification of the antigen-antibody reaction combined with the nanogold-based bio-barcode technique. Hepatitis B surface antibody (HBsAb) was initially immobilized on a nanogold/thionine/DNA-modified gold electrode, and then a sandwich-type immunoassay format was employed for the detection of HBsAg using nanogold-codified horseradish peroxidase-HBsAb conjugates as secondary antibodies. Under optimal conditions, the current response of the sandwich-type immunocomplex relative to the H₂O₂ system was proportional to HBsAg concentration in the range from 0.5 to 650 ng·mL^?1 with a detection limit of 0.1 ng·mL^?1 (S/N?=?3). The precision, reproducibility and stability of the immunosensor were acceptable. Subsequently, the immunosensors were used to assay HBsAg in human serum specimens. Analytical results were in agreement with those obtained by the standard chemiluminescence enzyme-linked immunosorbent assay.     

A novel Surface Plasmon Resonance enhanced Total Internal Reflection Ellipsometric application: electrochemically grafted isophthalic acid nanofilm on gold surface

The scope of this study is to modify a Surface Plasmon Resonance (SPR) sensor slide with isophthalic acid to evaluate the possible application on the detection of copper(II) ions in aqueous media by total internal reflection ellipsometry. A gold sensor surface was modified by an electrochemical diazonium reduction modification method. The modified surfaces are characterized with cyclic voltammetry (CV) and ellipsometry. Isophthalic acid monolayer modified gold slides were used for in situ detection of aqueous Cu(2+) solution with the SPR enhanced total internal reflection ellipsometry (SPRe-TIRE) technique. Layer formation, pH dependency of adsorption, sensor response of the SPRe-TIRE and isothermal kinetic parameters were examined. A high dependency on the number of CV cycles in the monolayer-multiple layer transition was observed. The suggested sensor gave a linear response over a wide range of Cu(2+) concentrations. It was also reported that adsorption on the SPRe-TIRE sensor gave Langmuir adsorption model behavior.     

Surface plasmon resonance sensors for real-time detection of cyclic citrullinated peptide antibodies

Surface plasmon resonance (SPR) sensors have been used for detection of various biomolecules because of their simplicity, high specificity and sensitivity, real-time detection, low cost, and no requirement of labeling. Recently, molecularly imprinted polymers that are easy to prepare, less expensive, stable, have talent for molecular recognition and also are used for creation selective binding sites for target molecule on the SPR sensors. Here, we show that preparation of cyclic citrullinated peptide antibody (anti-CCP) imprinted SPR sensor to detect CCP antibodies. For this purpose, anti-CCP/AAm pre-complex was synthesized by interacting acrylamide (AAm) monomer with anti-CCP. Then, anti-CCP imprinted (anti-CCP/PAAm) SPR sensor was obtained by reacting with anti-CCP/AAm pre-complex in the presence of the crosslinker, and initiator/activator pair. Besides this, non-imprinted (PAAm) SPR sensor was also prepared without using anti-CCP template. The SPR sensors were characterized and then adsorption-desorption studies were performed with pH 7.0 phosphate buffer (10 mM) and acetic acid (10%) with Tween 20 (1%) in pH 7.0 phosphate buffer. Selectivitiy of sensors was investigated by using immunoglobulin M (IgM) and bovine serum albumin (BSA). To determine the adsorption model of interactions between anti-CCP solutions and anti-CCP/PAAm SPR sensor, different adsorption models were performed. The calculated maximum reflection, detection limit, association and dissociation constants were 1.079 RU/mL, 0.177 RU/mL, 0.589 RU/mL and 1.697 mL/RU, respectively. Repeatability experiments of anti-CCP/PAAm SPR sensor was performed four times with adsorption-desorption-regeneration cycles without any performance losing. Results showed that anti-CCP/PAAm SPR sensor had high selectivity and sensitivity for detection of CCP antibodies.     

PEGylated polyethyleneimine grafted silica nanoparticles: enhanced cellular uptake and efficient siRNA delivery

The present paper reports the utilization of hybrid nanocomposite particles consisting of PEI25k-PEG5k copolymer grafted silica nanoparticles (SiO₂NPs) for enhanced cellular uptake and siRNA delivery. High-resolution transmission electron microscopy and dynamic light scattering measurements ensured the average particle size of the final hybrid component as 45 nm (core SiO₂, 28–30 nm and shell PEI25k-PEG5k, 12–15 nm). Surface morphology from atomic force microscopy analysis showed the significant relationship between the particle size and shape. ²⁹Si and ¹³C cross-polarization–magic angle spinning solid state nuclear magnetic resonance (NMR), ¹H-NMR, and Fourier transform infrared spectroscopy were used to obtain the relevant structural information (such as Q3, silanol; Q4, siloxane functional groups of SiO₂NPs; resonance shifts and bending vibrations of PEI25k, –CH₂–CH₂–NH–; and PEG5k, –CH₂–CH₂–O–) from copolymer nanoparticle. Stable complexation of siRNA and nanocomposite particle (wt.%:wt.%) was achieved from 1:5 to 1:15 ratio. Nanocomposite particle (N/P) ratio and siRNA concentration determine the stability and knockdown efficiency of the PEI25k-PEG5k-graft-SiO₂NPs–siRNA complexes. It was shown that highly positively charged (zeta potential, +66 mV) PEI25k-PEG5k-graft-SiO₂NPs result in strong affinity with negatively charged siRNA. Confocal microscopy showed intensified cellular uptake of siRNA into cytoplasm of A549 cancer cell utilized for in vitro study. In conclusion, the coherence, graft density of copolymer-SiO₂NPs, and siRNA concentration were found to strongly influence the stability, and hence determine the knockdown efficiency, of PEI25k-PEG5k-graft-SiO₂NPs–siRNA complexes.     

Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte

Pendent nitrile groups of multifilamentous polyacrylonitrile (PAN) fibers were reduced to amino groups using lithium aluminum hydride for different time of reduction and amine content was estimated by performing acid-base titrations. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC) were used for the characterization of the generated amino groups and thermal properties of the reduced fibers, respectively. The surface morphology of the fibers after reduction and immobilization was characterized using Scanning Electron Microscope (SEM). The newly formed amino groups of the fibers were activated by using glutaraldehyde for the covalent linking of Goat anti-Rabbit IgG-HRP (GAR-HRP) antibody enzyme conjugate. Modified PAN fibers were evaluated as a matrix for sandwich ELISA by using Goat anti-Rabbit antibody (GAR-IgG), Rabbit anti-Goat (RAG-IgG) as analyte and enzyme conjugate GAR-HRP. The fibers reduced for 24 h were able to detect the analyte RAG-IgG at a concentration as low as 3.75 ng mL⁻¹ with 12% skimmed milk as blocking reagent for the optimized concentration of primary antibody GAR-IgG 3 μg mL⁻¹ and peroxidase conjugate GAR-HRP dilution of 8000 fold. The sensitivity, specificity and reproducibility of the developed immunoassay was further established with antibodies present in human blood using Rabbit anti-Human (RAH-IgG) antibody and the corresponding HRP enzyme conjugate. As low as 0.1 μL of human blood was sufficient to perform the assay with the modified fibers.     

Amperometric detection of antibodies in serum: performance of self-assembled cyclodextrin/cellulose polymer interfaces as antigen carriers

A bifunctionalised carboxymethyl-cellulose polymer bearing adamantane units and an antigenic fragment forms a highly stable interfacial complex with a βCD-containing surface. This allows the highly sensitive detection of antibodies using an amperometric immunosensor.     

Whispering-gallery mode resonators: Surface enhanced Raman scattering without plasmons

Calculations based on the Mie theory are performed to determine the locally enhanced electric fields due to whispering-gallery mode resonances for dielectric microspheres, with emphasis on electromagnetic "hot spots" that are located along the wavevector direction on the surface of the sphere. The local electric field enhancement associated with these hot spots is used to determine the surface enhanced Raman scattering enhancement factors for a molecule, here treated as a classical dipole, located near the surface of the sphere. Both incident and Raman emission enhancements are calculated accurately using an extension of the Mie theory that includes interaction of the Raman dipole field with the sphere. The enhancement factors are calculated for dielectric spheres in vacuum with a refractive index of 1.9 and radii of 5, 10, and 20 microm and for wavelengths that span the visible spectrum. Maximum Raman scattering enhancement factors on the order of 10(3)-10(4) are found at locations slightly off the propagation axis when the incident excitation but not the Stokes-shifted radiation is coincident with a whispering-gallery mode resonance. The enhancement factors are found to vary inversely with the resonance width, and this determines the influence of the mode number and order on the results. Additional calculations are performed for the case where the Stokes-shifted radiation is also on-resonance and Raman enhancement factors as large as 10(8) are found. These enhancement factors are typically a factor of 10(2) smaller than would be obtained from /E/4 enhancement estimates, as enhancement of the Raman dipole emission is significantly reduced compared to the local field enhancement for micron size particles or larger. Conditions under which single-molecule or few-molecule measurements are feasible are identified.     

Surface enhanced Raman scattering of benzotriazole: a molecular orientational study

Surface enhanced Raman scattering of benzotriazole in aqueous silver sol at pH approximately 9 has been investigated. The spectra of the molecule at various concentrations were recorded and the maximum enhancement was observed with a 10(-5) M solution. From the comparison of the surface enhanced Raman spectra with the conventional Raman spectra and applying "surface selection rule", it is inferred that the molecular ion interacts with the silver sol via the nitrogen atoms of the benzotriazole ring and the molecule assumes a near "end-on" orientation.     

Tailoring macromolecular architecture with imidazole functionality: A perspective for controlled polymerization processes

Controlled radical polymerization (CRP) allows for the design and synthesis of functional polymers with tailored composition and unique macromolecular architectures. Synthetic methods that are readily available for controlled radical polymerization include nitroxide-mediated polymerization, reversible addition–fragmentation chain transfer polymerization, and atom transfer radical polymerization. N-Vinyl monomers that are typically amenable to free radical methods are often difficult to synthesize in a controlled manner to high molecular weight due to the lack of resonance stabilization of the propagating radical. However, recent advances in the field of CRP have resulted in successful controlled polymerization of various N-vinyl heterocyclic monomers including N-vinylcarbazole, N-vinylpyrrolidone, N-vinylphthalimide, and N-vinylindole. The incorporation of the imidazole ring into homopolymers and copolymers using conventional free radical polymerization of N-vinylimidazole monomer is particularly widespread and advantageous due to facile functionalization, high thermal stability, and the relevance of the imidazole ring to many biomacromolecules. Copolymers prepared with methyl methacrylate displayed random incorporation according to differential scanning calorimetry and amorphous morphologies according to X-ray scattering. Imidazole- and imidazolium-containing monomers have shown recent success for CRP; however, the controlled polymerization of N-vinylimidazole has remained relatively unexplored. Future efforts focus on the development of tailored imidazole-containing copolymers with well-defined architectures for emerging biomedical, electronic and membrane applications.     

Picomolar detection of carcinoembryonic antigen in whole blood using microfluidics and surface‐enhanced Raman spectroscopy

Carcinoembryonic antigen (CEA) is a wide‐spectrum biomarker. Clinically, we generally use serum sample to detect CEA, which needs to be centrifuged to pretreat the raw blood sample. In this study, we realized direct CEA detection in raw blood samples exploiting microfluidics. The LOD was as low as 10^?12 M.     

Surface enhanced Raman spectroscopic detection of polycyclic aromatic hydrocarbons (PAHs) using a gold nanoparticles-modified alginate gel network

This work aims at the development of a fast scanning method for organic pollutants with low-cost small Raman spectrometers. A gold nanoparticles-embedded alginate gel is prepared and applied as the substrate for surface enhanced Raman spectrometric detection of polycyclic aromatic hydrocarbons (PAHs). The target molecules are captured by the three-dimensional network of the alginate gel, and brought close to the hot spots generated by gold nanoparticles embedded in the gel. Significant Raman enhancement effects are observed in the analysis of four typical PAH compounds including pyrene, anthracene, fluorene and benzo(a)pyrene. Quantitative analysis of BaP shows a limit of detection of 0.365 nM using a low-cost small Raman spectrometer, which is comparable to published values. The practicability of the method is tested by analyzing PAHs in different water samples, offering a fast scanning method for PAHs.     

Thermooptical detection in microchips: from macro- to micro-scale with enhanced analytical parameters

In this paper, we compared the methods of photothermal spectroscopy used in different spatial scales, namely thermal-lens spectrometry (TLS) and thermal-lens microscopy (TLM) to enhance the performance parameters in analytical procedures. All of the experimental results were confirmed by theoretical calculation. It was proven that the design for both TLM and TLS, despite a different scale for the effect, is governed by the same signal-generating and probing conditions (probe beam diameter at the sample should be equal to the diameter of the blooming thermal lens), and almost does not depend on the nature of the solvent. Theoretical and experimental instrumental error curves for thermal lensing were coincident. TLM obeys the same law of instrumental error as TLS and shows better repeatability for the same levels of thermal-lens signals or absorbances. TLS is more advantageous for studying low concentrations in bulk, while TLM shows much lower absolute LODs due to better repeatability for low amounts. The behavior of the thermal-lens signal with different flow rates was studied and optimum conditions, with the minimum contribution to total error, were found. These conditions are reproducible, are in agreement with the existing theory of the thermal response in thermal lensing, and do not significantly affect the design of the optimum scheme for setups. TLM showed low LODs in solvent extraction (down to 10(-8) M) and electrokinetic separation (10(-7) M), which were shown to be governed by discussed instrumental regularities, instead of by microchemistry.     

Sono-electroanalysis of copper: enhanced detection and determination in the presence of surfactants

Surfactant adsorption has been shown to have a passivating effect on the electrode surface during anodic stripping voltammetric measurements. In the present work the feasibility of sono-anodic stripping analysis for the determination of copper in aqueous media contaminated with surfactant has been studied at an unmodified bare glassy carbon electrode. We illustrate the deleterious effect of three common surfactants, sodium dodecyl sulfate (SDS), dodecyl pyridinium chloride (DPC) and Triton-X 100 (TX-100) on conventional electroanalysis. The analogous sono-electroanalytical response was investigated for each surfactant at ultrasound intensities above and below the cavitation threshold. The enhancement in the stripping signal observed is attributed to the increased mass transport due to acoustic streaming and above the cavitation threshold the intensity of cavitational events is significantly increased leading to shearing of adsorbed surfactant molecules from the surface. As a result accurate analyses for SDS concentrations up to 100 ppm are possible, with analytical signals visible in solutions of SDS and TX-100 of 1000 ppm. Analysis is reported in high concentration of surfactant with use of sono-solvent double extraction. The power of this technique is clearly illustrated by the ability to obtain accurate measurements of copper concentration from starting solutions containing 1000 ppm SDS or TX-100. This was also exemplified by analysis of the low concentration 0.3 microM Cu(II) solution giving a percentage recovery of 94% in the presence of 1000 ppm SDS or TX-100.     

Thermally-responsive surfaces comprising grafted poly(N-isopropylacrylamide) chains: Surface characterisation and reversible capture of dispersed polymer particles

In this study we investigate thermally-responsive surfaces prepared by grafting PNIPAm from a cationic macroinitiator (MI) that was adsorbed onto a range of anionic substrates. The substrates used were mica, glass, quartz and high surface area carbon foam. The carbon foam was rendered thermally responsive by first coating it with a layer of calcined laponite particles. PNIPAm brushes were grown from the substrates using surface-initiated atom transfer radical polymerisation. The thermally-responsive PNIPAm layers were characterised in detail at room temperature and 50 °C using atomic force microscopy (AFM) and contact angle measurements. The surfaces changed from being non-adhesive to adhesive when the temperature was increased to 50 °C. Young’s modulus values and adhesive force values are reported. Particle capture experiments involving dispersed polystyrene or poly(BD/MAA) (butadiene and methacrylic acid) particles were conducted. High extents of particle capture were observed. It was shown that the highest extents of thermally-triggered particle capture at 50 °C occurred for surfaces that exhibited the largest increases in contact angle upon increasing the temperature. Importantly, thermally-triggered capture for both anionic polystyrene and poly(BD/MAA) particles was shown to be partially reversible with up to 30% of the captured particles released during cooling. This is the first time that significant reversibility of thermally-triggered capture of polymer particles has been reported.     

Novel fused silica capillary columns: Surface modification through controlled doping of the preform-tube

A method has been developed for fabricating fused silica capillary columns which have specific surface properties but still retain the excellent strength, flexibility, and resilience of pure fused silica. By using the Modified Chemical Vapor Deposition process (MCVD), typically used for the production of optical fiber lightguides, inorganic dopants such as Al, Nd, Ge, and P can be introduced into the preform-tube by MCVD. Doped columns have a wide range of specific surface properties, and columns with undoped fused silica prepared by MCVD are more chemically inert and less acidic than columns prepared by conventional methods. This paper describes the method for fabricating capillaries and the initial studies to characterize them.     

Surface enhanced Raman spectroscopy analysis of the adsorption of 2-thiouracil to Au,Ag and Cu electrodes: Surface potential dependence

Surface enhanced Raman spectroscopy has been used to study the potential controlled behavior of 2-thiouracil adsorbed on silver, gold and copper electrodes. Comparison of the normal Raman and SER spectra shows that the molecule adsorbs chemically to the metallic surfaces through its deprotonated sulfur atom. The position of the molecular aromatic ring in respect to the surface normal under potential modulation was analyzed using the electromagnetic theory's selection rules. Our data indicate that for all electrodes the surface potential induces a molecular reorientation leading to a more upright or tilted position.     

Surface enhanced Raman optical activity of molecules on orientationally averaged substrates: theory of electromagnetic effects

We present a model for electromagnetic enhancements in surface enhanced Raman optical activity (SEROA) spectroscopy. The model extends previous treatments of SEROA to substrates, such as metal nanoparticles in solution, that are orientationally averaged with respect to the laboratory frame. Our theoretical treatment combines analytical expressions for unenhanced Raman optical activity with molecular polarizability tensors that are dressed by the substrate's electromagnetic enhancements. We evaluate enhancements from model substrates to determine preliminary scaling laws and selection rules for SEROA. We find that dipolar substrates enhance Raman optical activity (ROA) scattering less than Raman scattering. Evanescent gradient contributions to orientationally averaged ROA scale to first or higher orders in the gradient of the incident plane-wave field. These evanescent gradient contributions may be large for substrates with quadrupolar responses to the plane-wave field gradient. Some substrates may also show a ROA contribution that depends only on the molecular electric dipole-electric dipole polarizability. These conclusions are illustrated via numerical calculations of surface enhanced Raman and ROA spectra from (R)-(-)-bromochlorofluoromethane on various model substrates.