New biochip technology for label-free detection of pathogens and their toxins

microSERS is a new biochip technology that uses surface-enhanced Raman scattering (SERS) microscopy for label-free transduction. The biochip itself comprises pixels of capture biomolecules immobilized on a SERS-active metal surface. Once the biochip has been exposed to the sample and the capture biomolecules have selectively bound their ligands, a Raman microscope is used to collect SERS fingerprints from the pixels on the chip. SERS, like other whole-organism fingerprinting techniques, is very specific. Our initial studies have shown that the Gram-positive Listeria and Gram-negative Legionella bacteria, Bacillus spores and Cryptosporidium oocysts can often be identified at the subspecies/strain level on the basis of SERS fingerprints collected from single organisms. Therefore, pathogens can be individually identified by microSERS, even when organisms that cross-react with the capture biomolecules are present in a sample. Moreover, the SERS fingerprint reflects the physiological state of a bacterial cell, e.g., when pathogenic Listeria and Legionella were cultured under conditions known to affect virulence, their SERS fingerprints changed significantly. Similarly, nonviable (e.g., heat- or UV-killed) microorganisms could be differentiated from their viable counterparts by SERS fingerprinting. Finally, microSERS is also capable of the sensitive and highly specific detection of toxins. Toxins that comprised as little as 0.02% by weight of the biomolecule-toxin complex produced strong, unique fingerprints when spectra collected from the complexes were subtracted from the spectra of the uncomplexed biomolecules. For example, aflatoxins B(1) and G(1) could be detected and individually identified when biochips bearing pixels of antibody or enzyme capture biomolecules were incubated in samples containing one or both aflatoxins, and the spectra were then collected for 20 s from an area of the biomolecule pixel approximately 1 microm in diameter. In the future, we plan to investigate the use of hyperspectral imaging Raman microscopy for collecting fingerprints from all the pixels on the biochip, individually yet simultaneously, to enable the rapid detection of diverse pathogens and their toxins in a sample, using a single biochip.     

Raman tags: Novel optical probes for intracellular sensing and imaging

Optical labels are needed for probing specific target molecules in complex biological systems. As a newly emerging category of tags for molecular imaging in live cells, the Raman label attracts much attention because of the rich information obtained from targeted and untargeted molecules by detecting molecular vibrations. Here, we list three types of Raman probes based on different mechanisms: Surface Enhanced Raman Scattering (SERS) probes, bioorthogonal Raman probes, and Resonance Raman (RR) probes. We review how these Raman probes work for detecting and imaging proteins, nucleic acids, lipids, and other biomolecules in vitro, within cells, or in vivo. We also summarize recent noteworthy studies, expound on the construction of every type of Raman probe and operating principle, sum up in tables typically targeting molecules for specific binding, and provide merits, drawbacks, and future prospects for the three Raman probes.     

Surface‐enhanced Raman spectroscopy for on‐site analysis: A review of recent developments

On‐site identification and quantification of chemicals is critical for promoting food safety, human health, homeland security risk assessment, and disease diagnosis. Surface‐enhanced Raman spectroscopy (SERS) has been widely considered as a promising method for on‐site analysis due to the advantages of nondestructive, abundant molecular information, and outstanding sensitivity. However, SERS for on‐site application has been restricted not only by the cost, performance, and portability of portable Raman instruments, but also by the sampling ability and signal enhancing performance of the SERS substrates. In recent years, the performance of SERS for on‐site analysis has been improved through portable Raman instruments, SERS substrates, and other combined technologies. In this review, popular commercial portable Raman spectrometers and the related technologies for on‐site analysis are compared. In addition, different types of SERS substrates for on‐site application are summarized. SERS combined with other technologies, such as electrochemical and microfluidics are also presented. The future perspective of SERS for on‐site analysis is also discussed.     

A biomolecule friendly photolithographic process for fabrication of protein microarrays on polymeric films coated on silicon chips

The last years, there is a steadily growing demand for methods and materials appropriate to create patterns of biomolecules for bioanalytical applications. Here, a photolithographic method for patterning biomolecules onto a silicon surface coated with a polymeric layer of high protein binding capacity is presented. The patterning process does not affect the polymeric film and the activity of the immobilized onto the surface biomolecules. Therefore, it permits sequential immobilization of different biomolecules on spatially distinct areas on the same solid support. The polymeric layer is based on a commercially available photoresist (AZ5214) that is cured at high temperature in order to provide a stable substrate for creation of protein microarrays by the developed photolithographic process. The photolithographic material consists of a (meth)acrylate copolymer and a sulfonium salt as a photoacid generator, and it is lithographically processed by thermal treatment at temperatures 相似文献   

Direct Fabrication Route to Plastic-Supported Gold Nanoparticles for Flexible NIR-SERS

Stable gold nanoparticles with surface plasmon resonance tunable from visible (Vis) to near-infrared (NIR) are deposited via a direct sputtering methodology on large area polyethylene terephthalate (PET) to be used as effective, flexible NIR surface-enhanced Raman scattering (SERS) substrates. An O₂ plasma treatment of PET is used to tailor growth dynamics, geometry, and plasmonic properties of nanoparticles. The O₂ plasma treatment of PET results also in effective improvement of nanoparticle anchoring on the plastic substrate, providing more stable, flexible SERS systems. The functionality of fabricated SERS substrates has been tested using benzylthiol, and SERS enhancement factors in the range 10⁴ have been achieved, which are comparable with those reported in literature for gold nanostructures fabricated on silicon substrate. These results attest the great potentiality of this methodology for the production of cost-effective flexible and reusable large-scale SERS substrates.     

Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. A review.

The concept and potentialities of electrochemical procedures of biomolecule immobilization based on electropolymerized films are described. The biomolecule entrapment in conventional electrogenerated polymers such as polypyrrole, polyaniline or polyphenol is compared with an electrochemical procedure involving the adsorption of amphiphilic monomers and biomolecules before the polymerization step. Examples of organic phase enzyme electrode and electrical wiring of immobilized enzymes are presented. Furthermore, the construction of controlled architectures based on spatially segregated multilayers, exhibiting complementary biological activities is described. Then, the use of functionalized polymers bearing functional groups for the covalent binding of biomolecules is reported. Moreover, the attachment of biomolecules to biotinylated polymers through affinity interactions based on avidin-biotin bridge is presented.     

Chemoselective surface immobilization of proteins through a cleavable peptide

Surface immobilization of biomolecules is a fundamental step in several experimental techniques such as surface plasmon resonance analysis and microarrays. Oxime ligation allows reaching chemoselective protein immobilization with the retention of native-like conformation by proteins. Beside the need for chemoselective ligation of molecules to surface/particle, equally important is the controlled release of the immobilized molecules, even after a specific binding event. For this purpose, we have designed and assessed in an SPR experiment a peptide linker able to (i) anchor a given protein (enzymes, receptors, or antibodies) to a surface in a precise orientation and (ii) release the immobilized protein after selective enzymatic cleavage. These results open up the possibility to anchor to a surface a protein probe leaving bioactive sites free for interaction with substrates, ligands, antigens, or drugs and successively remove the probe-ligand complex by enzymatic cleavage. This peptide linker can be considered both an improvement of SPR analysis for macromolecular interaction and a novel strategy for drug delivery and biomaterial developments.     

Spectroscopy on the wing: Naturally inspired SERS substrates for biochemical analysis

We show that naturally occurring chitinous nanostructures found on the wings of the Graphium butterfly can be used as substrates for surface‐enhanced Raman scattering when coated with a thin film of gold or silver. The substrates were found to exhibit excellent biocompatibility and sensitivity, making them ideal for protein assaying. An assay using avidin/biotin binding showed that the substrates could be used to quantify protein binding directly from changes in the surface‐enhanced Raman scattering (SERS) spectra and were sensitive over a concentration range comparable with a typical enzyme‐linked immunosorbent assays (ELISA) assay. A biomimetic version of the wing nanostructures produced using a highly reproducible, large‐scale fabrication process, yielded comparable enhancement factors and biocompatibility. The excellent biocompatibility of the wings and biomimetic substrates is unparalleled by other lithographically produced substrates, and this could pave the way for widespread application of ultrasensitive SERS‐based bioassays. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Rapidly-Deposited Polydopamine Coating via High Temperature and Vigorous Stirring: Formation,Characterization and Biofunctional Evaluation

Polydopamine (PDA) coating provides a promising approach for immobilization of biomolecules onto almost all kinds of solid substrates. However, the deposition kinetics of PDA coating as a function of temperature and reaction method is not well elucidated. Since dopamine self-polymerization usually takes a long time, therefore, rapid-formation of PDA film becomes imperative for surface modification of biomaterials and medical devices. In the present study, a practical method for preparation of rapidly-deposited PDA coating was developed using a uniquely designed device, and the kinetics of dopamine self-polymerization was investigated by QCM sensor system. It was found that high temperature and vigorous stirring could dramatically speed up the formation of PDA film on QCM chip surface. Surface characterization, BSA binding study, cell viability assay and antibacterial test demonstrates that the polydopamine coating after polymerization for 30 min by our approach exhibits similar properties to those of 24 h counterpart. The method has a great potential for rapid-deposition of polydopamine films to modify biomaterial surfaces.     

Drug affinity to immobilized target bio-polymers by high-performance liquid chromatography and capillary electrophoresis

This review addresses the use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as affinity separation methods to characterise drugs or potential drugs-bio-polymer interactions. Targets for the development of new drugs such as enzymes (IMERs), receptors, and membrane proteins were immobilized on solid supports. After the insertion in the HPLC system, these immobilized bio-polymers were used for the determination of binding constants of specific ligands, substrates and inhibitors of pharmaceutical interest, by frontal analyses and zonal elution methods. The most used bio-polymer immobilization techniques and methods for assessing the amount of active immobilized protein are reported. Examples of increased stability of immobilized enzymes with reduced amount of used protein were shown and the advantages in terms of recovery for reuse, reproducibility and on-line high-throughput screening for potential ligands are evidenced. Dealing with the acquisition of relevant pharmacokinetic data, examples concerning human serum albumin binding studies are reviewed. In particular, papers are reported in which the serum carrier has been studied to monitor the enantioselective binding of chiral drugs and the mutual interaction between co-administered drugs by CE and HPLC. Finally CE, as merging techniques with very promising and interesting application of microscale analysis of drugs' binding parameters to immobilized bio-polymers is examined.     

Gold Micro-Flowers: One-Step Fabrication of Efficient, Highly Reproducible Surface-Enhanced Raman Spectroscopy Platform

We present a new method enabling simultaneous synthesis and deposition of gold micro-flowers (AuMFs) on solid substrates in a one-pot process that uses two reagents, auric acid and hydroxylamine hydrochloride, in aqueous reaction mixture. The AuMFs deposited onto the substrate form mechanically stable gold layer of expanded nanostructured surface. The morphology of the AuMFs depends on and can be controlled by the composition of the reaction solution as well as by the reaction time. The nanostructured metallic layers obtained with our method are employed as efficient platforms for chemical and biological sensing based on surface-enhanced Raman spectroscopy (SERS). SERS spectra recorded by such platforms for p-mercaptobenzoic acid and phage lambda exhibit enhancement factors above 10⁶ and excellent reproducibility.     

Probing fundamental film parameters of immobilized enzymes--towards enhanced biosensor performance. Part I--QCM-D mass and rheological measurements

Enzyme immobilization is an ever-growing research-area for both analytical and industrial applications. Of critical importance in this area are the effects of immobilization procedures upon the functionality of the immobilized biomolecules. Both beneficial and detrimental effects can be conferred through the selection and tuning of the immobilization procedure. Quartz-crystal microbalance with dissipation (QCM-D) has been previously used to great effect in tracking alterations to thin films of biomolecules immobilized onto quartz transducers.In this study, we investigate the ability of QCM-D to track and monitor film parameters of a monolayer of laccase immobilized on a series of self-assembled monolayers (SAMs), differing in lateral density of binding residues on the SAM and height of the SAM from the quartz surface. Both mass gains and rheological parameters for these varying surfaces were measured and trends later compared to the apparent enzyme kinetics of the immobilized laccase films, assessed electroanalytically (Paper II in this two part study). For covalent attachment of proteins, both shear and viscosity were increased relative to physically adsorbed proteins. An increase in lateral density of protein-binding surface of the SAM components was shown to increase the shear/viscosity of the resultant film while an increase in distance from the electrode (through incorporation of lysine linkers) was shown to decrease the shear/viscosity while simultaneously increasing the wet mass gain of the films. Shear and viscosity may be indicative of both enzyme denaturation and increased lateral protein packing within the film structure hence it is assumed that less distortion occurs with the inclusion of linkers which allow for more optimal protein immobilization.     

Characterization and application of Raman labels for confocal Raman microspectroscopic detection of cellular proteins in single cells

A method using confocal Raman microspectroscopy for the detection of cellular proteins in single intact cells was developed. Two approaches were used to improve the detection of these cellular components. First, compounds with high Raman scattering were investigated for potential use as Raman labels. Raman labels were conjugated to either biomolecules or biotin and used as markers in the detection of cellular enzymes and receptors. Second, silver colloids were used to increase the surface-enhanced Raman scatter (SERS) of these Raman labels. Cresyl violet and dimethylaminoazobenzene are Raman labels that provide very sensitive SERS detection by a confocal Raman microscope with a HeNe laser at wavelength of 632.8 nm. The detection of 12-lipoxygenase and cyclooxygenase-1 in single bovine coronary artery endothelial cells and the binding of angiotensin II to its receptors in zona glomerulosa cells was demonstrated.     

Simultaneous Photoluminescence and SERS Observation of Nanodiamond at Laser Deposition on Noble Metals

The metal-modified luminescence and surface-enhanced Raman scattering (SERS) occurring near nanostructured surfaces of noble metals recently have been observed for different kinds of nanocrystals associated with the metal nanostructures. In the present work, the photoluminescence and Raman scattering of diamond nanocrystals of sizes 100 and 300 nm patterned on Ag and Au thin nanostructured films via laser accelerated deposition using a femtosecond laser are discussed. The laser accelerated deposition forms ordered periodical nanodiamond–metal nanostructures and allows adjusting the interaction between nanodiamond and metal by varying the laser acceleration parameters as well as by using different metals (Ag and Au), and varying the structure of the metal film. Correspondingly, the spectroscopic properties of the system determined by interaction between nanoparticles and metal are tuned. The enhancement of nanodiamond photoluminescence together with SERS of graphite fraction and disordered carbon of nanodiamonds are observed for nanodiamond–Ag structures at 488- and 532-nm excitations, while for the nanodiamond–Au structure some characteristic SERS effects are observed at 785-nm excitation. The mechanisms of enhancement are discussed considering the nanodiamond–metal interaction and laser acceleration effect on nanodiamond.     

Protein separation and identification using magnetic beads encoded with surface-enhanced Raman spectroscopy

This article presents a prototype of a surface-enhanced Raman spectroscopy (SERS)-encoded magnetic bead of 8 μm diameter. The core part of the bead is composed of a magnetic nanoparticle (NP)-embedded sulfonated polystyrene bead. The outer part of the bead is embedded with Ag NPs on which labeling molecules generating specific SERS bands are adsorbed. A silica shell is fabricated for further bioconjugation and protection of SERS signaling. Benzenethiol, 4-mercaptotoluene, 2-naphthalenethiol, and 4-aminothiophenol are used as labeling molecules. The magnetic SERS beads are used as substrates for protein sensing and screening with easy handling. As a model application, streptavidin-bound magnetic SERS beads are used to illustrate selective separation in a flow cytometry system, and the screened beads are spectrally recognized by Raman spectroscopy. The proposed magnetic SERS beads are likely to be used as a versatile solid support for protein sensing and screening in multiple assay technology.     

Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance

Mutagenesis and immobilization are usually considered to be unrelated techniques with potential applications to improve protein properties. However, there are several reports showing that the use of site-directed mutagenesis to improve enzyme properties directly, but also how enzymes are immobilized on a support, can be a powerful tool to improve the properties of immobilized biomolecules for use as biosensors or biocatalysts. Standard immobilizations are not fully random processes, but the protein orientation may be difficult to alter. Initially, most efforts using this idea were addressed towards controlling the orientation of the enzyme on the immobilization support, in many cases to facilitate electron transfer from the support to the enzyme in redox biosensors. Usually, Cys residues are used to directly immobilize the protein on a support that contains disulfide groups or that is made from gold. There are also some examples using His in the target areas of the protein and using supports modified with immobilized metal chelates and other tags (e.g., using immobilized antibodies). Furthermore, site-directed mutagenesis to control immobilization is useful for improving the activity, the stability and even the selectivity of the immobilized protein, for example, via site-directed rigidification of selected areas of the protein. Initially, only Cys and disulfide supports were employed, but other supports with higher potential to give multipoint covalent attachment are being employed (e.g., glyoxyl or epoxy-disulfide supports). The advances in support design and the deeper knowledge of the mechanisms of enzyme-support interactions have permitted exploration of the possibilities of the coupled use of site-directed mutagenesis and immobilization in a new way. This paper intends to review some of the advances and possibilities that these coupled strategies permit.     

Quantitation of immobilized proteins

A method for the quantitative determination of immobilized proteins based on the binding and subsequent elution of Coomassie Blue R is presented. Also presented is a method for the immobilization of proteins in solution by entrapment in polyacrylamide. These entrapped proteins are then available for use in the assay method presented. Other analytical procedures can also be performed on the entrapped proteins, either alone or in combination with the protein quantitation. The dye binding and elution method presented provides a sensitive and, in most applications, rapid method for the quantitative detection of immobilized proteins. Rather than immobilization being an obstacle to the assay method, this approach utilizes the advantages of immobilization for the removal of excess reagents. Application of this approach to several types of immobilized protein are presented.     

Multiple Protein-immobilized Phospholipid Polymer Nanoparticles: Effect of Spacer Length on Residual Enzymatic Activity and Molecular Diagnosis

We report the development of phosphorylcholine (PC) group-covered nanoparticles for multiple immobilization reactions; the surface of these nanoparticles facilitates bioreactions such as enzymatic reactions and molecular diagnoses. The nanoparticles were covered with a bioconjugate PC group containing a polymer backbone, and their surface properties were as follows: (1) suppression of nonspecific protein adsorption and (2) stabilization of immobilized biomolecules. In this study, biomolecules were immobilized on PC-covered nanoparticles by using different spacer lengths between the polymer backbone and biomolecules. The stability of the immobilized biomolecules was evaluated using horseradish peroxidase-labeled IgG, and the bioconjugate nanoparticles were stored at 4, 25, and 40 °C. The residual enzymatic activity of the peroxidase was monitored at a particular time. On the other hand, to test the role of these nanoparticles in molecular diagnosis, we used IgG-conjugated nanoparticles and the fluorescence resonance energy transfer (FRET) phenomenon. The IgG molecules were labeled with either donor or acceptor molecules, and each labeled IgG was simultaneously immobilized on the PC-covered nanoparticles. These labeled IgG molecules induce the FRET phenomenon upon capture of the target antigen provided they are in close proximity. The resulting fluorescence was readable via the FRET phenomenon. In the present study, C-reactive protein (CRP) was used as the target antigen, and the effect of the spacer length is discussed. The bioconjugated nanoparticles covered with PC groups are promising tools for tuning bioreactions.     

Bioanalytical application of surface‐ and tip‐enhanced Raman spectroscopy

Due to its fingerprint specificity and trace‐level sensitivity, surface‐enhanced Raman spectroscopy (SERS) is an attractive tool in bioanalytics. This review reflects the research in this highly interesting topic of the last 3–4 years. The detection of the SERS signature of biomolecules up to microorganisms and cells is introduced. Labeling using modified nanoparticles (SERS tags) is also introduced. In order to establish biomedical applications, SERS analysis is performed in complex matrices such as body fluids. Furthermore, the SERS technique is combined with other methods such as microfluidic devices for online monitoring and scanning probe microscopy (i.e. tip‐enhanced Raman spectroscopy, TERS) to investigate nanoscaled features. The present review illustrates the broad application fields of SERS and TERS in bioanalytics and shows the great potential of these methods for biomedical diagnostics.