Surface-enhanced Raman scattering spectroscopy as a sensitive and selective technique for the detection of folic acid in water and human serum

Surface-enhanced Raman scattering (SERS) is shown to give linear and sensitive concentration-dependent detection of folic acid using silver nanoparticles created via ethylene-diaminetetraacetic acid (EDTA) reduction. Optical detection by SERS overcomes the primary limitation of photodissociation encountered during the application of other shorter wavelength ultraviolet (UV)/near-UV techniques such as fluorescence based microscopy. The SERS approach in water-based samples was demonstrated and optimized using several longer wavelengths of excitation (514.5, 632.8, and 785 nm). Excitation in the green (514.5 nm) was found to achieve the best balance between photodissociation and SERS efficiency. Linear concentration dependence was observed in the range of 0.018 to 1 microM. The importance of folic acid in a clinical setting and the potential applications of this technique in a biological environment are highlighted. We demonstrate the potential to transfer this technique to real biological samples by the detection of folic acid in human serum samples by SERS.     

Sensitive carbohydrate detection using surface enhanced Raman tagging

Glycomic analysis is an increasingly important field in biological and biomedical research as glycosylation is one of the most important protein post-translational modifications. We have developed a new technique to detect carbohydrates using surface enhanced Raman spectroscopy (SERS) by designing and applying a Rhodamine B derivative as the SERS tag. Using a reductive amination reaction, the Rhodamine-based tag (RT) was successfully conjugated to three model carbohydrates (glucose, lactose, and glucuronic acid). SERS detection limits obtained with a 633 nm HeNe laser were ～1 nM in concentration for all the RT-carbohydrate conjugates and ～10 fmol in total sample consumption. The dynamic range of the SERS method is about 4 orders of magnitude, spanning from 1 nM to 5 μM. Ratiometric SERS quantification using isotope-substituted SERS internal references allows comparative quantifications of carbohydrates labeled with RT and deuterium/hydrogen substituted RT tags, respectively. In addition to enhancing the SERS detection of the tagged carbohydrates, the Rhodamine tagging facilitates fluorescence and mass spectrometric detection of carbohydrates. Current fluorescence sensitivity of RT-carbohydrates is ～3 nM in concentration while the mass spectrometry (MS) sensitivity is about 1 fmol, achieved with a linear ion trap electrospray ionization (ESI)-MS instrument. Potential applications that take advantage of the high SERS, fluorescence, and MS sensitivity of this SERS tagging strategy are discussed for practical glycomic analysis where carbohydrates may be quantified with a fluorescence and SERS technique and then identified with ESI-MS techniques.     

A SERS‐Assisted 3D Barcode Chip for High‐Throughput Biosensing

A surface enhanced Raman scattering (SERS)‐assisted 3D barcode chip has been developed for high‐throughput biosensing. The 3D barcode is realized through joint 2D spatial encoding with the Raman spectroscopic encoding, which stores the SERS fingerprint information in the format of a 2D array. Here, the concept of SERS‐assisted 3D barcode is demonstrated through multiplex immunoassay, where simultaneous detection of multiple targets in different samples has been achieved using a microfluidic platform. First, multiple proteins in different samples are spatially separated using a microfluidic patterned antibody barcode substrate, forming a 2D hybridization array. Then the SERS probes are used to identify and quantify the proteins. As different SERS probes are labeled with different Raman reporters, they could be employed as “SERS tags” to incorporate spectroscopic information into the 3D barcode. In this 3D barcode, the 2D spatial information helps to differentiate the samples and targets while the SERS information allows quantitative multiplex detection. It is found that the SERS‐assisted 3D barcode chip can not only accomplish one‐step multiplex detection within 30 min but also achieve an ultrasensitivity down to 10 fg mL^?1 (≈70 aM), which is expected to provide a promising tool for high‐throughput biomedical applications.     

Monitoring the glycosylation status of proteins using Raman spectroscopy

Protein-based biopharmaceuticals are becoming increasingly widely used as therapeutic agents, and the characterization of these biopharmaceuticals poses a significant analytical challenge. In particular, monitoring posttranslational modifications (PTMs), such as glycosylation, is an important aspect of this characterization because these glycans can strongly affect the stability, immunogenicity, and pharmacokinetics of these biotherapeutic drugs. Raman spectroscopy is a powerful tool, with many emerging applications in the bioprocessing arena. Although the technique has a relatively rich history in protein science, only recently has Raman spectroscopy been investigated for assessing posttranslational modifications, including phosphorylation, acetylation, trimethylation, and ubiquitination. In this investigation, we develop for the first time Raman spectroscopy combined with multivariate data analyses, including principal components analysis and partial least-squares regression, for the determination of the glycosylation status of proteins and quantifying the relative concentrations of the native ribonuclease (RNase) A protein and RNase B glycoprotein within mixtures.     

Bio-imaging, detection and analysis by using nanostructures as SERS substrates

Surface-enhanced Raman scattering (SERS) is a phenomenon that occurs on nanoscale-roughed metallic surface. The magnitude of the Raman scattering signal can be greatly enhanced when the scatterer is placed in the very close vicinity of the surface, which enables this phenomenon to be a highly sensitive analytical technique. SERS inherits the general strongpoint of conventional Raman spectroscopy and overcomes the inherently small cross section problem of a Raman scattering. It is a sensitive and nondestructive spectroscopic method for biological samples, and can be exploited either for the delivery of molecular structural information or for the detection of trace levels of analytes. Therefore, SERS has long been regarded as a powerful tool in biomedical research. Metallic nanostructure plays a key role in all the biomedical applications of SERS because the enhanced Raman signal can only be obtained on the surface of a finely divided substrate. This review focuses on progress made in the use of SERS as an analytical technique in bio-imaging, analysis and detection. Recent progress in the fabrication of SERS active nanostructures is also highlighted.     

Molecular-level description of proteins from saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics

For improved detection of diverse posttranslational modifications (PTMs), direct fragmentation of protein ions by top down mass spectrometry holds promise but has yet to be achieved on a large scale. Using lysate from Saccharomyces cerevisiae, 117 gene products were identified with 100% sequence coverage revealing 26 acetylations, 1 N-terminal dimethylation, 1 phosphorylation, 18 duplicate genes, and 44 proteolytic fragments. The platform for this study combined continuous-elution gel electrophoresis, reversed-phase liquid chromatography, automated nanospray coupled with a quadrupole-FT hybrid mass spectrometer, and a new search engine for querying a custom database. The proteins identified required no manual validation, ranged from 5 to 39 kDa, had codon biases from 0.93 to 0.083, and were primarily associated with glycolysis and protein synthesis. Illustrations of gene-specific identifications, PTM detection and subsequent PTM localization (using either electron capture dissociation or known PTM data stored in a database) show how larger scale proteome projects incorporating top down may proceed in the future using commercial Q-FT instruments.     

Highly sensitive detection of proteins and bacteria in aqueous solution using surface-enhanced Raman scattering and optical fibers

We report the detection of the proteins lysozyme and cytochrome c as well as the live bacterial cells of Shewanella oneidensis MR-1 in aqueous solutions with sensitivities order(s) of magnitude higher than those previously reported. Two highly sensitive surface-enhanced Raman scattering (SERS)-based biosensors using optical fibers have been employed for such label-free macromolecule detections. The first sensor is based on a tip-coated multimode fiber (TCMMF) with a double-substrate "sandwich" structure, and a detection limit of 0.2 μg/mL is achieved in protein detections. The second sensor is based on a liquid core photonic crystal fiber (LCPCF) with a better confinement of light inside the fiber core, and a detection limit of 10(6) cells/mL is achieved for the bacteria detection. Both SERS biosensors show great potential for highly sensitive and molecule-specific detection and identification of biomolecules.     

Microfluidic Designing Microgels Containing Highly Concentrated Gold Nanoparticles for SERS Analysis of Complex Fluids

Surface‐enhanced Raman scattering (SERS) is one of the most promising methods to detect small molecules for point‐of‐care analysis as it is rapid, nondestructive, label‐free, and applicable for aqueous samples. Here, microgels containing highly concentrated yet evenly dispersed gold nanoparticles are designed to provide SERS substrates that simultaneously achieve contamination‐free metal surfaces and high signal enhancement and reproducibility. With capillary microfluidic devices, water‐in‐oil‐in‐water (W/O/W) double‐emulsion drops are prepared to contain gold nanoparticles and hydrogel precursors in innermost drop. Under hypertonic condition, water is selectively pumped out from the innermost drops. Therefore, gold nanoparticles are gently concentrated without forming aggregates, which are then captured by hydrogel matrix. The resulting microgels have a concentration of gold nanoparticles ≈30 times higher and show Raman intensity two orders of magnitude higher than those with no enrichment. In addition, even distribution of gold nanoparticles results in uniform Raman intensity, providing high signal reproducibility. Moreover, as the matrix of the microgel serves as a molecular filter, large adhesive proteins are rejected, which enables the direct detection of small molecules dissolved in the protein solution. It is believed that this advanced SERS platform is useful for in situ detection of toxic molecules in complex mixtures such as biological fluids, foods, and cosmetics.     

Multiplexed microfluidic surface-enhanced Raman spectroscopy

Over the past few decades, surface-enhanced Raman spectroscopy (SERS) has garnered respect as an analytical technique with significant chemical and biological applications. SERS is important for the life sciences because it can provide trace level detection, a high level of structural information, and enhanced chemical detection. However, creating and successfully implementing a sensitive, reproducible, and robust SERS active substrate continues to be a challenging task. Herein, we report a novel method for SERS that is based upon using multiplexed microfluidics (MMFs) in a polydimethylsiloxane platform to perform parallel, high throughput, and sensitive detection/identification of single or various analytes under easily manipulated conditions. A facile passive pumping method is used to deliver Ag colloids and analytes into the channels where SERS measurements are done under nondestructive flowing conditions. With this approach, SERS signal reproducibility is found to be better than 7%. Utilizing a very high numerical aperture microscope objective with a confocal-based Raman spectrometer, high sensitivity is achieved. Moreover, the long working distance of this objective coupled with an appreciable channel depth obviates normal alignment issues expected with translational multiplexing. Rapid evaluation of the effects of anion activators and the type of colloid employed on SERS performance are used to demonstrate the efficiency and applicability of the MMF approach. SERS spectra of various pesticides were also obtained. Calibration curves of crystal violet (non-resonant enhanced) and Mitoxantrone (resonant enhanced) were generated, and the major SERS bands of these analytes were observable down to concentrations in the low nM and sub-pM ranges, respectively. While conventional random morphology colloids were used in most of these studies, unique cubic nanoparticles of silver were synthesized with different sizes and studied using visible wavelength optical extinction spectrometry, scanning electron microscopy, and the MMF-SERS approach.     

Chitosan-coated anisotropic silver nanoparticles as a SERS substrate for single-molecule detection

Surface-enhanced Raman spectroscopy (SERS) is a technique that has become widely used for identifying and providing structural information about molecular species in low concentration. There is an ongoing interest in finding optimum particle size, shape and spatial distribution for optimizing the SERS substrates and pushing the sensitivity toward the single-molecule detection limit. This work reports the design of a novel, biocompatible SERS substrate based on small clusters of anisotropic silver nanoparticles embedded in a film of chitosan biopolymer. The SERS efficiency of the biocompatible film is assessed by employing Raman imaging and spectroscopy of adenine, a significant biological molecule. By combining atomic force microscopy with SERS imaging we find that the chitosan matrix enables the formation of small clusters of silver nanoparticles, with junctions and gaps that greatly enhance the Raman intensities of the adsorbed molecules. The study demonstrates that chitosan-coated anisotropic silver nanoparticle clusters are sensitive enough to be implemented as effective plasmonic substrates for SERS detection of nonresonant analytes at the single-molecule level.     

SERS‐Active‐Charged Microgels for Size‐ and Charge‐Selective Molecular Analysis of Complex Biological Samples

Surface‐enhanced Raman scattering (SERS) provides a dramatic increase of Raman intensity for molecules adsorbed on nanogap‐rich metal nanostructures, serving as a promising tool for molecular analysis. However, surface contamination caused by protein adsorption and low surface concentration of small target molecules reduce the sensitivity, which severely restricts the use of SERS in many applications. Here, charged microgels containing agglomerates of gold nanoparticles (Au NPs) are designed using droplet‐based microfluidics to provide a reliable SERS substrate with molecular selectivity and high sensitivity. The limiting mesh size of hydrogel enables the autonomous exclusion of large proteins and the charged matrix concentrates oppositely charged small molecules through electrostatic attraction. As nanogaps among Au NPs in the agglomerates enhance Raman intensity, Raman spectrum of the adsorbed molecules is selectively measured with high sensitivity in the absence of interruption from adhesive proteins. Therefore, the SERS‐active‐charged microgels can be used for direct analysis of pristine biological samples without the pretreatment steps of separation and concentration, which are commonly a prerequisite for Raman analysis. For the purpose of demonstration, a direct detection of fipronil sulfone with partial negative charges, a metabolite of toxic insecticide, dissolved in eggs using the positively charged microgels without any pretreatment of the samples, is shown.     

In situ surface-enhanced Raman scattering analysis of biofilm

Biofilms represent the predominant form of microbial life on Earth. They are aggregates of microorganisms embedded in a matrix formed by extracellular polymeric substances (EPS). Detailed information about chemical composition and structure of the EPS matrix is relevant e.g. for the optimization of biocides, of antifouling strategies and for biological wastewater treatment. Raman microscopy (RM) is a capable tool that can provide detailed chemical information about biofilm constituents with spatial resolution of optical microscope. However, the sensitivity of RM is limited. Surface-enhanced Raman scattering (SERS), which enables investigations of biomolecules at very low concentration levels, allows overcoming this drawback. To our knowledge, this paper is the first report on reproducible SERS spectra from different constituents of a multispecies biofilm. We believe that the reproducibility is partly owed to the in situ measurement of the biofilm, while up to now SERS measurements of microbiological samples by RM were carried out after sample drying. We employed colloidal silver nanoparticles for in situ SERS measurements by RM. The achieved enhancement factor of up to 2 orders of magnitude illustrates a high potential of SERS for ultrasensitive chemical analysis of biofilms, including the detection of different components and the determination of their relative abundance in the complex biofilm matrix.     

Analytical technique for label-free multi-protein detection based on Western blot and surface-enhanced Raman scattering

We have developed a new analytical procedure for label-free protein detection designated "Western SERS", consisting of protein electrophoresis, Western blot, colloidal silver staining, and surface-enhanced Raman scattering (SERS) detection. A novel method of silver staining for Western blot that uses a silver colloid, an excellent SERS-active substrate, is first proposed in the present study. During the process of silver staining, interactions between proteins and silver nanoparticles result in the emergence of SERS of proteins. In the present study, we use myoglobin (Mb) and bovine serum albumin (BSA) as model proteins. From different protein bands on a nitrocellulose (NC) membrane, we have observed surface-enhanced resonance Raman scattering (SERRS) spectra of Mb and SERS spectra of BSA. The proposed technique offers dual advantages of simplicity and high sensitivity. On one hand, after the colloidal silver staining, we can detect label-free multi-proteins directly on a NC membrane without digestion, extraction, and other pretreatments. On the other hand, the detection limit of the Western SERS is almost consistent with the detection limit of colloidal silver staining, and the SERRS detection limit of Mb is found to be 4 ng/band. This analytical method, which combines the technique of protein separation with SERS, may be a powerful protocol for label-free protein detection in proteomic research.     

Gold and magnetic oxide/gold core/shell nanoparticles as bio-functional nanoprobes

The ability to create bio-functional nanoprobes for the detection of biological reactivity is important for developing bioassay and diagnostic methods. This paper describes the findings of an investigation of the surface functionalization of gold (Au) and magnetic nanoparticles coated with gold shells (M/Au) by proteins and spectroscopic labels for the creation of nanoprobes for use in surface enhanced Raman scattering (SERS) assays. Highly monodispersed Au nanoparticles and M/Au nanoparticles with two types of magnetic nanoparticle cores (Fe(2)O(3) and MnZn ferrite) were studied as model systems for the bio-functionalization and Raman labeling. Comparison of the SERS intensities obtained with different particle sizes (30-100?nm) and samples in solution versus on solid substrates have revealed important information about the manipulation of the SERS signals. In contrast to the salt-induced uncontrollable and irreversible aggregation of nanoparticles, the ability to use a centrifugation method to control the formation of stable small clustering sizes of nanoparticles was shown to enhance SERS intensities for samples in solution as compared with samples on solid substrates. A simple method for labeling protein-capped Au nanoparticles with Raman-active molecules was also described. The functionalized Au and M/Au nanoparticles are shown to exhibit the desired functional properties for the detection of SERS signals in the magnetically separated reaction products. These results are discussed in terms of the interparticle distance dependence of 'hot-spot' SERS sites and the delineation of the parameters for controlling the core-shell reactivity of the magnetic functional nanocomposite materials in bio-separation and spectroscopic probing.     

A general protease digestion procedure for optimal protein sequence coverage and post-translational modifications analysis of recombinant glycoproteins: application to the characterization of human lysyl oxidase-like 2 glycosylation

Using recombinant DNA technology for expression of protein therapeutics is a maturing field of pharmaceutical research and development. As recombinant proteins are increasingly utilized as biotherapeutics, improved methodologies ensuring the characterization of post-translational modifications (PTMs) are needed. Typically, proteins prepared for PTM analysis are proteolytically digested and analyzed by mass spectrometry. To ensure full coverage of the PTMs on a given protein, one must obtain complete sequence coverage of the protein, which is often quite challenging. The objective of the research described here is to design a protocol that maximizes protein sequence coverage and enables detection of post-translational modifications, specifically N-linked glycosylation. To achieve this objective, a highly efficient proteolytic digest protocol using trypsin was designed by comparing the relative merits of denaturing agents (urea and Rapigest SF), reducing agents [dithiothreitol (DTT) and tris(2-carboxyethyl)phophine (TCEP)], and various concentrations of alkylating agent [iodoacetamide (IAM)]. After analysis of human apo-transferrin using various protease digestion protocols, ideal conditions were determined to contain 6 M urea for denaturation, 5 mM TCEP for reduction, 10 mM IAM for alkylation, and 10 mM DTT, to quench excess IAM before the addition of trypsin. This method was successfully applied to a novel recombinant protein, human lysyl oxidase-like 2. Furthermore, the glycosylation PTMs were readily detected at two glycosylation sites in the protein. These digestion conditions were specifically designed for PTM analysis of recombinant proteins and biotherapeutics, and the work described herein fills an unmet need in the growing field of biopharmaceutical analysis.     

Multiplex detection of protease activity with quantum dot nanosensors prepared by intein-mediated specific bioconjugation

We report here a protease sensing nanoplatform based on semiconductor nanocrystals or quantum dots (QDs) and bioluminescence resonance energy transfer (QD-BRET) to detect the protease activity in complex biological samples. These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and protease substrates sandwiched between the two as a sensing group. An intein-mediated conjugation strategy was developed for site-specific conjugation of proteins to QDs in preparing these QD nanosensors. In this traceless ligation, the intein itself is spliced out and excluded from the final conjugation product. With this method, we have synthesized a series of QD nanosensors for highly sensitive detection of an important class of protease matrix metalloproteinase (MMP) activity. We demonstrated that these nanosensors can detect the MMP activity in buffers and in mouse serum with the sensitivity to a few nanograms per milliliter and secreted proteases by tumor cells. The suitability of these nanosensors for a multiplex protease assay has also been shown.     

Rapid and sensitive detection of respiratory virus molecular signatures using a silver nanorod array SERS substrate

A spectroscopic assay based on surface enhanced Raman scattering (SERS) using silver nanorod array substrates has been developed that allows for rapid detection of trace levels of viruses with a high degree of sensitivity and specificity. This novel SERS assay can detect spectral differences between viruses, viral strains, and viruses with gene deletions in biological media. The method provides rapid diagnostics for detection and characterization of viruses generating reproducible spectra without viral manipulation.     

Direct and quantitative detection of bacteriophage by "hearing" surface detachment using a quartz crystal microbalance

We show that it is possible to detect specifically adsorbed bacteriophage directly by breaking the interactions between proteins displayed on the phage coat and ligands immobilized on the surface of a quartz crystal microbalance (QCM). This is achieved through increasing the amplitude of oscillation of the QCM surface and sensitively detecting the acoustic emission produced when the bacteriophage detaches from the surface. There is no interference from nonspecifically adsorbed phage. The detection is quantitative over at least 5 orders of magnitude and is sensitive enough to detect as few as 20 phage. The method has potential as a sensitive and low-cost method for virus detection.     

Optical probes for biological applications based on surface-enhanced Raman scattering from indocyanine green on gold nanoparticles

We report surface-enhanced Raman scattering (SERS) studies on indocyanine green (ICG) on colloidal silver and gold and demonstrate a novel optical probe for applications in living cells. In addition to its own detection by the characteristic ICG SERS signatures, the ICG gold nanoprobe delivers spatially localized chemical information from its biological environment by employing SERS in the local optical fields of the gold nanoparticles. The probe offers the potential to increase the spectral specificity and selectivity of current chemical characterization approaches of living cells and biomaterials based on vibrational information.     

SERS biodetection using gold-silica nanoshells and nitrocellulose membranes

We have developed a rapid, reproducible, easy to execute, surface enhanced Raman scattering (SERS) method for detection of low volumes and total amounts of biological antigens using an analyte capture system derived from methods commonly used in Western blotting. Our method is a "half-sandwich" assay with an antigen detection scheme that employs a nitrocellulose (NC) membrane with 200 nm pore size to capture subnanograms of analyte and concentrate them in a small area from applied volumes as low as one microliter. The SERS probes used for detection consist of gold-silica nanoshells modified with a two-component mixed monolayer system. One component consists of a poly(ethylene glycol) (PEG)-modified Raman-active chromophore bound to the gold surface which allows for SERS detection and imparts particle stability. The second component uses (ortho-pyridyl) disulfide-PEG-succinimidyl ester to couple the recognition antibody to the particle surface. By controlling the reaction time and concentration of thiols, a mixed monolayer is prepared on the nanoshell surface with the ability to recognize low concentrations of analyte and generate reproducible SERS signals. Using this strategy, we have achieved SERS signals that are proportional to antigen present on the membrane allowing detection of total antigen amounts as low as 1.25 ng for some cases. The performance of this new SERS bioassay has been tested with a variety of potential antigens, demonstrating the potential for multiplexed detection of analytes.