Multiple detection of proteins by SERS-based immunoassay with core shell magnetic gold nanoparticles

A highly selective and sensitive surface-enhanced Raman scattering (SERS)-based immunoassay for the multiple detection of proteins has been developed. The proposed core shell magnetic gold (Au) nanoparticles allow for successful protein separation and high SERS enhancement for protein detection. To selectively detect a specific protein in a mixed protein solution, we employed the sandwich type SERS immunoassay with core shell magnetic Au nanoparticles utilizing specific antigen–antibody interactions. Based on this proposed SERS immunoassay, we can successfully detect proteins in very low concentrations (∼800 ag/mL of mouse IgG and ∼5 fg/mL of human IgG) with high reproducibility. Magnetically assisted protein separation and detection by this proposed SERS immunoassay would provide great potential for effective and sensitive multiple protein detection. This technique allows for the straightforward SERS-based bioassays for quantitative protein detections.     

Simultaneous immunoassay for the detection of two lung cancer markers using functionalized SERS nanoprobes

A quick and reproducible SERS-based immunoassay, using functionalized hollow gold nanospheres and magnetic beads, has been developed. Here, a simultaneous detection of dual cancer markers in blood serum has been achieved under a single excitation wavelength. The accuracy and sensitivity for clinical sera from five patients confirms their diagnostic feasibility.     

Optofluidic microsystem with quasi-3 dimensional gold plasmonic nanostructure arrays for online sensitive and reproducible SERS detection

Practical applications of chemical and biological detections through surface-enhanced Raman scattering (SERS) require high reproducibility, sensitivity, and efficiency, along with low-cost, straightforward fabrication. In this work, we integrated a poly-(dimethylsiloxane) (PDMS) chip with quasi-3D gold plasmonic nanostructure arrays (Q3D-PNAs), which serve as SERS-active substrates, into an optofluidic microsystem for online sensitive and reproducible SERS detections. The Q3D-PNA PDMS chip was fabricated through soft lithography to ensure both precision and low-cost fabrication. The optimal dimension of the Q3D-PNA in PDMS was designed using finite-difference time-domain (FDTD) electromagnetic simulations with a simulated enhancement factor (EF) of 1.6 × 10⁶. The real-time monitoring capability of the SERS-based optofluidic microsystem was investigated by kinetic on/off experiments through alternatively flowing Rhodamine 6G (R6G) and ethanol in the microfluidic channel. A switch-off time of ∼2 min at a flow rate of 0.3 mL min⁻¹ was demonstrated. When applied to the detection of low concentration malathion, the SERS-based optofluidic microsystem with Q3D-PNAs showed high reproducibility, significantly improved efficiency and higher detection sensitivity via increasing the flow rate. The optofluidic microsystem presented in this paper offers a simple and low-cost approach for online, label-free chemical and biological analysis and sensing with high sensitivity, reproducibility, efficiency, and molecular specificity.     

SERS-based DNA detection in aqueous solutions using oligonucleotide-modified Ag nanoprisms and gold nanoparticles

Oligonucleotide-modified nanoparticle conjugates show highly promising potential for SERS-based DNA detection. However, it remains challenging to carry out the SERS-based DNA detection in aqueous solutions directly using oligonucleotide-modified nanoparticles, because the Raman reporters would exhibit lower signals when they are dispersed in aqueous solutions than laid on “dry” metal nanoparticles. Here, we synthesized stable oligonucleotide-modified Ag nanoprism conjugates, and performed SERS-based DNA detection in aqueous solution directly by using such conjugates in combination with Raman reporter-labeled, oligonucleotide-modified gold nanoparticles. The experimental results indicate that this SERS-based DNA detection approach exhibited a good linear correlation between SERS signal intensity and the logarithm of target DNA concentration ranging from 10^?11～10^?8 M. This sensitivity is comparable to those SERS-based DNA detection approaches with the “dry” process. Additionally, a similar correlation could also be observed in duplex target DNA detection by SERS hybrid probes. Our results suggest that the oligonucleotide-modified Ag nanoprisms may be developed as a powerful SERS-based DNA detection tool.

A high sensitive assay platform based on surface-enhanced Raman scattering for quantification of protease activity

In this study, a new, sensitive, and rapid assay was developed to quantitatively measure the proteolytic enzyme activity using the surface-enhanced Raman scattering (SERS) probe. Two different shapes of gold nanoparticles, gold nanosphere and nanorod particles were produced. SERS label, comprising self-assembled monolayers (SAMs) of Raman reporter molecule (5,5-Dithiobis (2-Nitrobenzoic acid), DTNB), was coated on the surface of the nanoparticles. Two different SERS-based analysis platforms were designed using gold-coated glass slide and polystyrene microtiter plate. The calibration curves were obtained by plotting the intensity of the SERS signal of symmetric NO₂ stretching of DTNB at 1326 cm⁻¹vs. the protease concentration. The effects of nanoparticle geometry and assay platform on the protease assay were investigated and the best working combination of the parameters was selected as rod shaped SERS probe and gold-coated glass slide. The correlation between the protease activity and SERS signal was found to be linear within the range of 0.1-2 mU/mL (R² = 0.979). The limit of detection (LOD) and limit of quantification (LOQ) values of the validated method were found as 0.43 and 1.30 mU/mL, respectively. The intra-day and inter-day precisions of the method, as relative standard deviation (RSD), were determined as 2.5% and 3.6%, respectively. The developed method was successfully applied for quantitative analysis of the commercial enzyme preparate that is used in cheese making process. It was also used for investigation of substrate specificity of protease enzyme towards the casein and bovine serum albumin. The proposed method has a flexibility to try different substrates for the detection of various enzyme activities.     

Highly sensitive trace analysis of paraquat using a surface-enhanced Raman scattering microdroplet sensor

We report a rapid and highly sensitive trace analysis of paraquat (PQ) in water using a surface-enhanced Raman scattering (SERS)-based microdroplet sensor. Aqueous samples of PQ, silver nanoparticles, and NaCl as the aggregation agent were introduced into a microfluidic channel and were encapsulated by a continuous oil phase to form a microdroplet. PQ molecules were adsorbed onto particle surfaces in isolated droplets by passing through the winding part of the channel. Memory effects, caused by the precipitation of nanoparticle aggregates on channel walls, were removed because the aqueous droplets were completely isolated by a continuous oil phase. The limit of detection (LOD) of PQ in water, determined by the SERS-based microdroplet sensor, was estimated to be below 2×10(-9) M, and this low detection limit was enhanced by one to two orders of magnitude compared to conventional analytical methods.     

Magnetic and electrokinetic manipulations on a microchip device for bead-based immunosensing applications

The combination of electrophoretic and magnetic manipulations with electrochemical detection for a versatile microfluidic and bead-based biosensing application is demonstrated. Amperometric detection is performed in an off-channel setup by means of a voltammetric cell built at the microchannel outlet and using a gold working electrode. Superparamagnetic particles are introduced and handled inside the channel by means of an external permanent magnet in combination with the electrogenerated flow which allows reproducible loading. The specific detection of phenol as electroactive alkaline phosphatase product is used in this study as proof of concept for a sensitive protein quantification. Characterizations and optimization of different parameters have been carried out in order to achieve the best detection signal. The applicability of the device has been finally demonstrated by the detection of rabbit IgG as model protein after an immunoassay performed on magnetic particles as immobilization platform. A comparison between the electrochemical detection using the developed device and the optical standard detection revealed similar performances with, however, extremely lower amount of reagent used and shorter analysis time. The developed electrophoretic- and magnetic-based chip may open the way to several other biosensing applications with interest not only for other proteins but also for DNA analysis, cell counting, and environmental control.     

Convenient formation of nanoparticle aggregates on microfluidic chips for highly sensitive SERS detection of biomolecules

Microfluidic chips combined with surface-enhanced Raman spectroscopy (SERS) offer an outstanding platform for rapid and high-sensitivity chemical analysis. However, it is nontrivial to conveniently form nanoparticle aggregrates (as SERS-active spots for SERS detection) in microchannels in a well-controlled manner. Here, we present a rapid, highly sensitive and label-free analytical technique for determining bovine serum albumin (BSA) on a poly(dimethylsiloxane) (PDMS) microfluidic chip using SERS. A modified PDMS pneumatic valve and nanopost arrays at the bottom of the fluidic microchannel are used for reversibly trapping gold nanoparticles to form gold aggregates, creating SERS-active spots for Raman detection. We fabricated a chip that consisted of a T-shaped fluidic channel and two modified pneumatic valves, which was suitable for fast loading of samples. Quantitative analysis of BSA is demonstrated with the measured peak intensity at 1,615 cm⁻¹ in the surface-enhanced Raman spectra. With our microfluidic chip, the detection limit of Raman can reach as low as the picomolar level, comparable to that of normal mass spectrometry.     

Fabrication of immunosensor microwell arrays from gold compact discs for detection of cancer biomarker proteins

A simple method is reported to fabricate gold arrays featuring microwells surrounding 8-electrodes from gold compact discs (CDs) for less than $0.2 per chip. Integration of these disposable gold CD array chips with microfluidics provided inexpensive immunoarrays that were used to measure a cancer biomarker protein quickly at high sensitivity. The gold CD sensor arrays were fabricated using thermal transfer of laserjet toner from a computer-printed pattern followed by selective chemical etching. Sensor elements had an electrochemically addressable surface area of 0.42 mm(2) with RSD <2%. For a proof-of-concept application, the arrays were integrated into a simple microfluidic device for electrochemical detection of cancer biomarker interleukin-6 (IL-6) in diluted serum. Capture antibodies of IL-6 were chemically linked onto the electrode arrays and a sandwich immunoassay protocol was developed. A biotinylated detection antibody with polymerized horseradish peroxidase labels was used for signal amplification. The detection limit of IL-6 in diluted serum was remarkably low at 10 fg mL(-1) (385 aM) with a linear response with log of IL-6 concentration from 10 to 1300 fg mL(-1). These easily fabricated, ultrasensitive, microfluidic immunosensors should be readily adapted for sensitive detection of multiple biomarkers for cancer diagnostics.     

Minireview: Recent Advances in Surface-Enhanced Raman Scattering-Based Nucleic Acid Detection with Application to Pathogen Diagnosis

Surface-enhanced Raman scattering (SERS) is a promising analytical tool in nanoscale detection because of its high sensitivity and selectivity. This review focuses on recent advances in SERS-based detection of DNA and RNA. First, nanostructure-based SERS-active substrates are introduced. Using label-free and labeled SERS, target biomolecules such DNA, RNA and microRNA have been successfully detected. Finally, applications in pathogen diagnosis are discussed. The prospects and challenges of SERS-based bioanalysis are highlighted.     

Microfluidic device for concentration and SERS-based detection of bacteria in drinking water

There is a constant need for the development of easy-to-operate systems for the rapid and unambiguous identification of bacterial pathogens in drinking water without the requirement for time-consuming culture processes. In this study, we present a disposable and low-cost lab-on-a-chip device utilizing a nanoporous membrane, which connects two stacked perpendicular microfluidic channels. Whereas one of the channels supplies the sample, the second one attracts it by potential-driven forces. Surface-enhanced Raman spectrometry (SERS) is employed as a reliable detection method for bacteria identification. To gain the effect of surface enhancement, silver nanoparticles were added to the sample. The pores of the membrane act as a filter trapping the bodies of microorganisms as well as clusters of nanoparticles creating suitable conditions for sensitive SERS detection. Therein, we focused on the construction and characterization of the device performance. To demonstrate the functionality of the microfluidic chip, we analyzed common pathogens (Escherichia coli DH5α and Pseudomonas taiwanensis VLB120) from spiked tap water using the optimized experimental parameters. The obtained results confirmed our system to be promising for the construction of a disposable optical platform for reliable and rapid pathogen detection which couples their electrokinetic concentration on the integrated nanoporous membrane with SERS detection.     

Trace detection of tetrahydrocannabinol (THC) with a SERS-based capillary platform prepared by the in situ microwave synthesis of AgNPs

In the present study, an ultra-sensitive and highly reproducible novel SERS-based capillary platform was developed and utilized for the trace detection of tetrahydrocannabinol (THC). The approach combines the advantages of microwave-assisted nanoparticle synthesis, plasmonics and capillary forces. By employing a microwave-assisted preparation method, glass capillaries were reproducibly coated with silver nanoparticles in a batch fabrication process that required a processing time of 3 min without needing to use any pre-surface modifications or add surfactants. The coated capillaries exhibited an excellent SERS activity with a high reproducibility and enabled the detection of low concentrations of target molecules. At the same time, only a small amount of analyte and a short and simple incubation process was required. The developed platform was applied to the spectroscopic characterization of tetrahydrocannabinol (THC) and its identification at concentration levels down to 1 nM. Thus, a highly efficient detection system for practical applications, e.g., in drug monitoring/detection, is introduced, which can be fabricated at low cost by using microwave-assisted batch synthesis techniques.     

Microfluidic channel with embedded SERS 2D platform for the aptamer detection of ochratoxin A

A selective aptameric sequence is adsorbed on a two-dimensional nanostructured metallic platform optimized for surface-enhanced Raman spectroscopy (SERS) measurements. Using nanofabrication methods, a metallic nanostructure was prepared by electron-beam lithography onto a glass coverslip surface and embedded within a microfluidic channel made of polydimethylsiloxane, allowing one to monitor in situ SERS fingerprint spectra from the adsorbed molecules on the metallic nanostructures. The gold structure was designed so that its localized surface plasmon resonance matches the excitation wavelength used for the Raman measurement. This optofluidic device is then used to detect the presence of a toxin, namely ochratoxin-A (OTA), in a confined environment, using very small amounts of chemicals, and short data acquisition times, by taking advantage of the optical properties of a SERS platform to magnify the Raman signals of the aptameric monolayer system and avoiding chemical labeling of the aptamer or the OTA target.

Immunoassay using surface-enhanced Raman scattering based on aggregation of reporter-labeled immunogold nanoparticles

A one-step homogenous sensitive immunoassay using surface-enhanced Raman scattering (SERS) has been developed. This strategy is based on the aggregation of Raman reporter-labeled immunogold nanoparticles induced by the immunoreaction with corresponding antigens. The aggregation of gold nanoparticles results in a SERS signal increase of the Raman reporter. Therefore, human IgG could be directly determined by measuring the Raman signal of the reporter. The process of aggregation was investigated by transmission electron microscopy (TEM) and UV-Vis absorption spectroscopy. The effects of the temperature, time, and size of gold nanoparticles on the sensitivity of the assay were examined. Using human IgG as a model protein, a wide linear dynamic range (0.1-15 microg mL(-1)) was reached with low detection limit (0.1 microg mL(-1)) under optimized assay conditions. The successful test suggests that the application of the proposed method holds promising potential for simple, fast detection of proteins in the fields of molecular biology and clinical diagnostics.     

Recent strategies toward microfluidic‐based surface‐enhanced Raman spectroscopy

Surface‐enhanced Raman spectroscopy (SERS) is an extremely powerful analytical tool, which not only yields information about the molecular structure of the analyte in the form of characteristic vibrational spectrum but also gives sensitivities approaching those in fluorescence spectroscopy. The SERS measurement on the microfluidic platform provides possibility to manufacture the device with design perfectly fulfilling the needs of the application with minimal sample consumption. This review aims at describing basic strategies for SERS measurement in microfluidic devices published in the last decade and covers current trends in microfluidics with SERS detection in the field of bioanalysis and approaches toward on‐line coupling of liquid‐based separation techniques with SERS detection.     

In situ dynamic measurements of the enhanced SERS signal using an optoelectrofluidic SERS platform

A novel active surface-enhanced Raman scattering (SERS) platform for dynamic on-demand generation of SERS active sites based on optoelectrofluidics is presented in this paper. When a laser source is projected into a sample solution containing metal nanoparticles in an optoelectrofluidic device and an alternating current (ac) electric field is applied, the metal nanoparticles are spontaneously concentrated and assembled within the laser spot, form SERS-active sites, and enhance the Raman signal significantly, allowing dynamic and more sensitive SERS detection. In this simple platform, in which a glass slide-like optoelectrofluidic device is integrated into a conventional SERS detection system, both dynamic concentration of metal nanoparticles and in situ detection of SERS signal are simultaneously possible with only a single laser source. This optoelectrofluidic SERS spectroscopy allows on-demand generation of 'hot spots' at specific regions of interest, and highly sensitive, reliable, and stable SERS measurements of the target molecules in a tiny volume (～500 nL) of liquid sample without any fluidic components and complicated systems.     

SERS as tool for the analysis of DNA-chips in a microfluidic platform

A sequence-specific detection method of DNA is presented combining a solid chip surface for immobilisation of capture DNAs with a microfluidic platform and a readout of the chip based on SERS. The solid chip surface is used for immobilisation of different capture DNAs, where target strands can be hybridised and unbound surfactants can be washed away. For the detection via SERS, short-labelled oligonucleotides are hybridised to the target strands. This technique is combined with a microfluidic platform that enables a fast and automated preparation process. By applying a chip format, the problems of sequence-specific DNA detection in solution phase by means of SERS can be overcome. With this setup, we are able to distinguish between different complementary and non-complementary target sequences in one sample solution.     

Recent advances in surface-enhanced Raman scattering detection technology for microfluidic chips

Microfluidic chip devices and their application to sensitive chemical and biological analyses have attracted significant attention over the past decade. The miniaturization of reaction systems offers practical advantages over conventional benchtop systems. In this case, however, a highly sensitive on-chip detection method is important for the monitoring of chemical reactions as well as for the detection of analytes inside the channel because the detection volume in a micrometer-size channel is extremely small. Recently, a surface-enhanced Raman scattering (SERS) technique is being regarded as a potential candidate for the highly sensitive detection of analytes in a microfluidic chip. This review provides a general survey and an in-depth look at recent developments in SERS techniques for the biological/environmental analysis of minute analytes in a microfluidic chip.     

Metal-polymer nanocomposites for integrated microfluidic separations and surface enhanced Raman spectroscopic detection

The widespread development of microfluidics (microfluidics) has allowed the extension of efficient separations, fluid handling, and hyphenation with many detection modes to a small, portable, highly controllable physico-chemical platform. Surface enhanced Raman spectroscopy (SERS) offers the powerful advantage of obtaining vibrational spectroscopic information about analytes in an aqueous matrix with negligible background. The mating of electrophoretic separations with vibrational spectroscopy on a microfluidic device will allow the chromatographic efficiency of capillary electrophoresis (CE) with the unequivocal analyte "fingerprinting" capability of detailed structural information. By utilizing SERS as a means of detection, this work promises to yield redress for the hindrances of electrophoretic separations, including uncertainty in analyte band identification due to changing migration times as well as compromised detection sensitivity for non-fluorescent analytes. Our work represents the first steps toward developing CE-SERS on a microfluidic platform with a region of novel metal-pliable polymer nanocomposite SERS substrate fabricated directly into the device. The device fabrication material has been extensively employed by the microfluidics community for over five years. SERS detection can be achieved in real time or after the separations, with on-column laser-induced fluorescence employed as a secondary detection mode used for confirmation of efficiencies and band locations.     

Controlled fabrication of nanopillar arrays as active substrates for surface-enhanced Raman spectroscopy

Highly ordered gold nanopillar arrays were fabricated using anodized aluminum oxide (AAO) templates. Nanopillars with a dimension of 110 +/- 15 nm in vertical height and 75 +/- 10 nm in base diameter were formed with a density of 150 microm(-2). The ordered nanopillar arrays give reproducible surface-enhanced Raman scattering (SERS) at a detection limit of 10(-8) M using thionine as probing molecules. The enhancement by the Au nanopillar arrays was comparable with or better than that of dispersed gold nanoparticle SERS substrates. This work demonstrates a new technique for producing highly ordered and reproducible SERS substrates potentially applicable for chemical and biological assay.