Analysis of recombinant protein expression using localized surface plasmon resonance (LSPR)

The localized surface plasmon resonance (LSPR)-based optical biosensor using nano-structures of noble metals has been considered as a useful tool for label-free detection of DNA hybridization and protein-protein interactions. We fabricated LSPR-based optical biosensors using gold nano-islands (nominal thickness; 75 A) on glass substrates that were easily made using the conventional fabrication methods. The formation of gold nano-islands on glass substrates was realized by heat treatment of thin gold film deposited with a low deposition rate (approximately 0.05 A/s). The morphologies of sensor surfaces composed of gold nano-islands were observed using an atomic force microscope (AFM) with a non-contact mode. To investigate the sensing capacity of the gold nano-island sensor for the binding of proteins by affinity interactions, the streptavidin and biotin interaction was used as a model system. In addition, detection of recombinant glutathione-S-transferase (GST)-tagged human interleukin-6 (hIL6) expressed in Escherichia coli was carried out by LSPR. It is expected that the LSPR sensors composed of gold nano-islands can be an alternative to traditional methods such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for fast analysis of protein expression.     

Versatile bioelectronic interfaces on flexible non-conductive substrates.

Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. These interfaces are commonly formed on gold films deposited using physical vapor deposition (PVD) or chemical vapor deposition (CVD). PVD and CVD require deposition of a primer layer, such as titanium or chromium, and require the use of expensive equipment and cannot be used on a wide range of substrates. This paper describes a versatile new bench-top method to form bioelectronic interfaces containing a gold film, electron mediator, cofactor, and dehydrogenase enzyme (secondary alcohol dehydrogenase, and sorbitol dehydrogenase) on nonconductive substrates such as polystyrene and glass. The method combines layer-by-layer deposition of polyelectrolytes, electroless metal deposition, and directed molecular self-assembly. Cyclic voltammetry, chronoamperometry, field emission X-ray dispersive spectroscopy, scanning electron microscopy, and atomic force microscopy were used to characterize the bioelectronic interfaces. Interfaces formed on flexible polystyrene slides were shown to retain their activity after bending to a radius of curvature of 18mm, confirming that the approach can be applied on cheap and flexible substrates for applications where traditional wafer-scale electronics is not suitable, such as personal or structural health monitors and rolled microtube biosensors.     

Metallization and Biopatterning on Ultra-Flexible Substrates via Dextran Sacrificial Layers

Micro-patterning tools adopted from the semiconductor industry have mostly been optimized to pattern features onto rigid silicon and glass substrates, however, recently the need to pattern on soft substrates has been identified in simulating cellular environments or developing flexible biosensors. We present a simple method of introducing a variety of patterned materials and structures into ultra-flexible polydimethylsiloxane (PDMS) layers (elastic moduli down to 3 kPa) utilizing water-soluble dextran sacrificial thin films. Dextran films provided a stable template for photolithography, metal deposition, particle adsorption, and protein stamping. These materials and structures (including dextran itself) were then readily transferrable to an elastomer surface following PDMS (10 to 70∶1 base to crosslinker ratios) curing over the patterned dextran layer and after sacrificial etch of the dextran in water. We demonstrate that this simple and straightforward approach can controllably manipulate surface wetting and protein adsorption characteristics of PDMS, covalently link protein patterns for stable cell patterning, generate composite structures of epoxy or particles for study of cell mechanical response, and stably integrate certain metals with use of vinyl molecular adhesives. This method is compatible over the complete moduli range of PDMS, and potentially generalizable over a host of additional micro- and nano-structures and materials.     

Specific and selective probes for Pseudomonas aeruginosa from phage-displayed random peptide libraries

The design of novel biosensors for the detection of biological threats, such as Pseudomonas aeruginosa, requires probes that specifically bind biological agents and insure their immediate and efficient recognition. Advanced bio-selective sensors may meet the requests for isolation, concentration of the agents and their real-time detection. There is a need for robust and inexpensive affinity probes alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we identified from two phage-displayed random peptide libraries phage clones displaying peptides capable of specific and strong binding to P. aeruginosa cell surface. The ability of the phage clones to interact specifically with P. aeruginosa was demonstrated by using enzyme-linked immunosorbent assay (ELISA). We assessed selectivity of phage-bacteria-binding by comparing the binding ability of the selected clones to the selector bacterium and a panel of other bacterial species; we also demonstrated by dot spot and immunoblotting that the most reactive and selective phage peptide bound with high avidity the bacterial cell surface. In addition, as proof-of-concept, we tested the possibility to immobilize the affinity-selected phage to a putative biosensor surface. The quality of phage deposition was monitored by ELISA, and phage-bacterial-binding was confirmed by high-power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including clinical-based diagnostics and possibly biological warfare applications.     

Analytical potential of gold nanoparticles in functional aptamer-based biosensors

Gold nanoparticles (AuNPs) exhibit many predominant capabilities such as high biocompatibility, chemical stability, strong localized surface plasmon resonance absorption, and high extinction coefficient in the visible region. These properties have enabled the extensive use of AuNPs in optical and electrochemical biosensors. As a kind of functional nucleic acids, aptamers can be considered as a valid alternative to antibodies or other bio-receptors and have been widely employed to develop novel biosensors. We are summarizing here the state of the art of AuNP-based biosensors that use functional aptamers as molecular recognition elements. In many cases, AuNPs themselves can be used as a probe for detection, such as various colorimetric aptasensors and fluorescent aptasensors. They also can be used as probe vectors to enlarge detection signals and to increase the number of conceivable substrates in electrochemical aptasensors.     

In vitro assessment of the biocompatibility of chemically modified GaAs surfaces

GaAs has excellent optical, electrical, and mechanical properties and shows promise to be used in the fabrication of novel devices. However, the unprotected GaAs surface can release heavy metal compounds such as AsOx, which are toxic to living cells. A promising approach to reduce or eliminate this release relies on the passivation of the GaAs surface using different chemical approaches. In this work, we compared three different passivation methods aimed at enhancing the viability of cells on GaAs. Protective layers composed of self-assembled alkyl thiols, polypeptides, thick polymer layers, and shells of polyelectrolytes were tested. We confirmed that the GaAs surface can be made biocompatible for several days based on in vitro tests with HeLa and KB cells. In addition, we compared the cell spreading behavior on the GaAs substrates modified by different chemical approaches. Our results suggest that when the toxicity of the GaAs surface is reduced or eliminated, the cells’viability and spreading depend on the chemical and topographical nature of the surface.     

Immobilization of native membrane-bound rhodopsin on biosensor surfaces

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.     

Electrochemical molecular analysis without nucleic acid amplification

Electrochemical biosensors have revolutionized glucose monitoring but have not yet fulfilled their promise of a low cost, direct detection replacement for genetic amplification tests such as PCR [K. Kerman, M. Kobayashi, E. Tamiya, Recent trends in electrochemical DNA biosensor technology, Meas. Sci. Technol. 15 (2004) R1-R11; A. Chaubey, B.D. Malhotra, Mediated biosensors. Biosens. Bioelectron. 17 (6-7) (2002) 441-456]. It has been anticipated that the integration of nanoscale chemical structures such as self-assembled monolayers with electrochemical biosensors would increase sensitivity by decreasing inherent system noise. We have designed a novel biosensing approach incorporating such integration and achieved rapid, ultra-low concentration sensitivities without target amplification. Raw samples are mixed with lysis buffer to allow hybridization of nucleic acid targets with anchor and signal probes before immobilizing a signaling enzyme proximate to the biosensor surface. A bias potential is subsequently applied and the secondary byproduct of a cyclic peroxidase reaction measured. Further studies have demonstrated the application of our approach in protein, clinical chemistry, and ionic assays.     

Biomolecule-embedded metal-organic frameworks as an innovative sensing platform

Technological advancements combined with materials research have led to the generation of enormous types of novel substrates and materials for use in various biological/medical, energy, and environmental applications. Lately, the embedding of biomolecules in novel and/or advanced materials (e.g., metal-organic frameworks (MOFs), nanoparticles, hydrogels, graphene, and their hybrid composites) has become a vital research area in the construction of an innovative platform for various applications including sensors (or biosensors), biofuel cells, and bioelectronic devices. Due to the intriguing properties of MOFs (e.g., framework architecture, topology, and optical properties), they have contributed considerably to recent progresses in enzymatic catalysis, antibody-antigen interactions, or many other related approaches. Here, we aim to describe the different strategies for the design and synthesis of diverse biomolecule-embedded MOFs for various sensing (e.g., optical, electrochemical, biological, and miscellaneous) techniques. Additionally, the benefits and future prospective of MOFs-based biomolecular immobilization as an innovative sensing platform are discussed along with the evaluation on their performance to seek for further development in this emerging research area.     

Effects of surface functionalization on the surface phage coverage and the subsequent performance of phage-immobilized magnetoelastic biosensors

One of the important applications for which phage-immobilized magnetoelastic (ME) biosensors are being developed is the wireless, on-site detection of pathogenic bacteria for food safety and bio-security. Until now, such biosensors have been constructed by immobilizing a landscape phage probe on gold-coated ME resonators via physical adsorption. Although the physical adsorption method is simple, the immobilization stability and surface coverage of phage probes on differently functionalized sensor surfaces need to be evaluated as a potential way to enhance the detection capabilities of the biosensors. As a model study, a filamentous fd-tet phage that specifically binds streptavidin was adsorbed on either bare or surface-functionalized gold-coated ME resonators. The surface functionalization was performed through the formation of three self-assembled monolayers with a different terminator, based on the sulfur-gold chemistry: AC (activated carboxy-terminated), ALD (aldehyde-terminated), and MT (methyl-terminated). The results, obtained by atomic force microscopy, showed that surface functionalization has a large effect on the surface phage coverage (46.8%, 49.4%, 4.2%, and 5.2% for bare, AC-, ALD-, and MT-functionalized resonators, respectively). In addition, a direct correlation of the observed surface phage coverage with the quantity of subsequently captured streptavidin-coated microbeads was found by scanning electron microscopy and by resonance frequency measurements of the biosensors. The differences in surface phage coverage on the differently functionalized surfaces may then be used to pattern the phage probe layer onto desired parts of the sensor surface to enhance the detection capabilities of ME biosensors.     

Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.     

Optimized Electroless Silver Coating for Optical and Plasmonic Applications

Electroless metal deposition is a simple and convenient technique to fabricate metallic films and to provide isotropic metal functionalization of 3D structures with complex geometries. In this work, we describe the synthesis of silver coatings by means of a modified Tollens reaction and their use as optical coating. The chemical composition of the metallization bath is here addressed to optimize the metal coating deposition. The synthesis parameters have been tailored in order to deposit very smooth films which were characterized by scanning electron microscopy, atomic force microscopy, and optical spectroscopy. 2D diffraction gratings and sinusoidal plasmonic gratings were produced with the proposed method. Optical characterization confirmed the plasmonic activities of the resultant structures, proving the efficiency of the described method for optical applications. Thermal annealing was found to improve the surface roughness of the coating and therefore the optical properties of the plasmonic gratings.     

Atomic Layer Deposition of CdS Quantum Dots for Solid‐State Quantum Dot Sensitized Solar Cells

Functioning quantum dot (QD) sensitized solar cells have been fabricated using the vacuum deposition technique atomic layer deposition (ALD). Utilizing the incubation period of CdS growth by ALD on TiO₂, we are able to grow QDs of adjustable size which act as sensitizers for solid‐state QD‐sensitized solar cells (ssQDSSC). The size of QDs, studied with transmission electron microscopy (TEM), varied with the number of ALD cycles from 1‐10 nm. Photovoltaic devices with the QDs were fabricated and characterized using a ssQDSSC device architecture with 2,2',7,7'‐tetrakis‐(N,N‐di‐p methoxyphenylamine) 9,9'‐spirobifluorene (spiro‐OMeTAD) as the solid‐state hole conductor. The ALD approach described here can be applied to fabrication of quantum‐confined structures for a variety of applications, including solar electricity and solar fuels. Because ALD provides the ability to deposit many materials in very high aspect ratio substrates, this work introduces a strategy by which material and optical properties of QD sensitizers may be adjusted not only by the size of the particles but also in the future by the composition.     

Surface Modification and Characterisation of Silk Fibroin Fabric Produced by the Layer-by-Layer Self-Assembly of Multilayer Alginate/Regenerated Silk Fibroin

Silk-based medical products have a long history of use as a material for surgical sutures because of their desirable mechanical properties. However, silk fibroin fabric has been reported to be haemolytic when in direct contact with blood. The layer-by-layer self-assembly technique provides a method for surface modification to improve the biocompatibility of silk fibroin fabrics. Regenerated silk fibroin and alginate, which have excellent biocompatibility and low immunogenicity, are outstanding candidates for polyelectrolyte deposition. In this study, silk fabric was degummed and positively charged to create a silk fibroin fabric that could undergo self-assembly. The multilayer self-assembly of the silk fibroin fabric was achieved by alternating the polyelectrolyte deposition of a negatively charged alginate solution (pH = 8) and a positively charged regenerated silk fibroin solution (pH = 2). Finally, the negatively charged regenerated silk fibroin solution (pH = 8) was used to assemble the outermost layer of the fabric so that the surface would be negatively charged. A stable structural transition was induced using 75% ethanol. The thickness and morphology were characterised using atomic force microscopy. The properties of the self-assembled silk fibroin fabric, such as the bursting strength, thermal stability and flushing stability, indicated that the fabric was stable. In addition, the cytocompatibility and haemocompatibility of the self-assembled silk fibroin fabrics were evaluated. The results indicated that the biocompatibility of the self-assembled multilayers was acceptable and that it improved markedly. In particular, after the self-assembly, the fabric was able to prevent platelet adhesion. Furthermore, other non-haemolytic biomaterials can be created through self-assembly of more than 1.5 bilayers, and we propose that self-assembled silk fibroin fabric may be an attractive candidate for anticoagulation applications and for promoting endothelial cell adhesion for vascular prostheses.     

Characterization of a new maleimido functionalization of gold for surface plasmon resonance spectroscopy

Para‐maleimidophenyl (p‐MP) modified gold surfaces have been prepared by one‐step electrochemical deposition and used in surface plasmon resonance (SPR) studies. Therefore, a FITC mimotope peptide (MP1, 12 aa), a human mucin 1 epitope peptide (MUC, 9 aa) and a protein with their specific antibodies were used as model systems. The peptides were modified with an N‐terminal cysteine for covalent and directed coupling to the maleimido functionalized surface by means of Michael addition. The coupling yield of the peptide, the binding characteristics of antibody and the unspecific adsorption of the analytes were investigated. The results expand the spectrum of biosensors usable with p‐MP by widely used SPR and support its potential to be versatile for several electrochemical and optical biosensors. This allows the combination of an electrochemical and optical read‐out for a broad variety of biomolecular interactions on the same chip. Copyright © 2014 John Wiley & Sons, Ltd.     

Label-Free LSPR Prostate-Specific Antigen Immune-Sensor Based on GLAD-Fabricated Silver Nano-columns

Label-free detection of biomarkers has been recently noticed and optical biosensors showed great potential to be the method of choice in such situation. Here, we used glancing angle deposition (GLAD) method in which silver nano-columns stabilized by a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) and 6-mercaptohexanol to investigate the capability of localized surface plasmon resonance (LSPR)–based silver nanochips to detect prostate-specific antigen (PSA). Using different standard solutions of PSA, limit of detection (LOD) of the nano-sensors has been calculated to be 850 pg/ml. The selectivity of the nano-sensors has also been evaluated. We showed that these nano-sensors could detect PSA in clinically acceptable sensitivity and specificity without any complicated laboratory equipment.

     

“Bottom-up” approach for implementing nano/microstructure using biological and chemical interactions

The “Bottom-up” approach for implementing nano/microstructure using biological self-assembled systems has been investigated with tremendous interest by many researchers in the field of medical diagnostics, material synthesis, and nano/microelectronics. As a result, the techniques for achieving these systems have been extensively explored in recent years. The developed or developing techniques are based on many interdisciplinary areas such as biology, chemistry, physics, electrical engineering, mechanical engineering, and so on. In this paper, we review the fundamentals behind the self-assembly concepts and describe the state of art in the biological and chemical self-assembled systems for the implementation of nano/microstructures. These structures described in the paper can be applied to the implementation of hybrid biosensors, biochip, novel bio-mimetic materials, and nano/microelectronic devices.     

A scanning near-field optical microscope approach to biomolecule patterning

In view of future generations of biosensors and advanced biomaterials, photochemistry in the near field using scanning near-field optical microscopy is investigated. The potential of direct near-field-induced photoactivation is demonstrated on standard photoresist. Photoimmobilization of maleimidoaryldiazirine on silicon substrates and bovine serum albumin on glass substrates is achieved, opening the way to a controlled biopatterning of surfaces with submicrometer feature size. The obtained patterns are characterized using atomic force microscopy, time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and near-field fluorescence microscopy.     

Fabrication of patterned DNA surfaces.

Two photolithographic methods are described for the formation of patterned single or multiple DNA species on SiO2 substrates. In the first approach, substrates are treated with a photochemically labile organosilane monolayer film. Irradiation of these surfaces with patterned deep UV (193 nm) light results in patterned chemically reactive groups which are then reacted with heterobifunctional crosslinking molecules. Covalent attachment of modified synthetic DNA oligomers to the crosslinker results in stable DNA patterns. Alternatively, a photoresist is spin-coated over a silane film which had been previously modified with the heterobifunctional crosslinker. Upon patterned irradiation and subsequent development, the underlying crosslinker-modified layer is revealed, and is then reacted with a chemically modified DNA. Feature dimensions to 1 micron are observed when a single fluorescent DNA is attached to the surface. By performing sequential exposures, we have successfully immobilized two distinguishable DNA oligomers on a single surface. Synthetic DNA immobilized in this manner retains the ability to hybridize to its complementary strand, suggesting that these approaches may find utility in the development of miniaturized DNA-based biosensors.     

Glutamate optical biosensor based on the immobilization of glutamate dehydrogenase in titanium dioxide sol-gel matrix

A simple and novel titania sol-gel derived optical biosensor coupled with carboxy seminaphthorhodamine-1-dextran (SNARF-1-dextran) as the fluorescent dye was fabricated for the determination of glutamate in water and biological samples. The NADH-dependent glutamate dehydrogenase (GLDH) was trapped in titania sol-gel derived matrix prepared by vapor deposition method. In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to characterize the surface morphology of the spots. SEM and AFM images showed that the deposition of titania precursor at 27 degrees C for 6.5h was found to be suitable to form transparent titania sol-gel matrix to encapsulate GLDH and fluorescent probe. AFM images showed that the roughness of TiO(2) surface increased from 2.16 nm in the absence of GLDH and SNARF to 37.8 nm after the immobilization. The developed titania biosensor has good analytical performance with water samples. A dynamic range between 0.04 and 10mM with the detection limit of 5.5 microM were observed. The responses to glutamate in biological samples also showed good performances, and the dynamic range and detection limit were 0.02-10mM and 6.7 microM, respectively. High precision with relative standard deviations of 4.2 and 10.7% in water and biological samples, respectively, were also demonstrated. In addition, the biosensor showed a relatively high storage stability over more than 1 month. Results obtained in this study clearly demonstrate that this simple vapor deposition method can be successfully used to form transparent titania sol-gel film for the fabrication of glutamate biosensors that are suitable for optical detection of glutamate in water and biological samples.