Using a silver-enhanced microarray sandwich structure to improve SERS sensitivity for protein detection

A simple and sensitive method, based on surface-enhanced Raman scattering (SERS), for immunoassay and label-free protein detection is reported. A series of bowl-shaped silver cavity arrays were fabricated by electrodeposition using a self-assembled polystyrene spheres template. The reflection spectra of these cavity arrays were recorded as a function of film thickness, and then correlated with SERS enhancement using sodium thiophenolate as the probe molecule. The results reveal that SERS enhancement can be maximized when the frequency of both the incident laser and the Raman scattering approach the frequency of the localized surface plasmon resonance. The optimized array was then used as the bottom layer of a silver nanoparticle–protein–bowl-shaped silver cavity array sandwich. The second layer of silver was introduced by the interactions between the proteins in the middle layer of the sandwich architecture and silver nanoparticles. Human IgG bound to the surface of this microcavity array can retain its recognition function. With the Raman reporter molecules labeled on the antibody, a detection limit down to 0.1 ng mL^?1 for human IgG is easily achieved. Furthermore, the SERS spectra of label-free proteins (catalase, cytochrome C, avidin and lysozyme) from the assembled sandwich have excellent reproducibility and high quality. The results reveal that the proposed approach has potential for use in qualitative and quantitative detection of biomolecules.

Label-free electrochemical DNA biosensor based on a glassy carbon electrode modified with gold nanoparticles, polythionine, and graphene

We describe the fabrication of a sensitive label-free electrochemical biosensor for the determination of sequence-specific target DNA. It is based on a glassy carbon electrode (GCE) modified with graphene, gold nanoparticles (Au-NPs), and polythionine (pThion). Thionine was firstly electropolymerized on the surface of the GCE that was modified with graphene by cyclic voltammetry. The Au-NPs were subsequently deposited on the surface of the pThion/graphene composite film by adsorption. Scanning electron microscopy and electrochemical methods were used to investigate the assembly process. Differential pulse voltammetry was employed to monitor the hybridization of DNA by measuring the changes in the peak current of pThion. Under optimal conditions, the decline of the peak current is linearly related to the logarithm of the concentration of the target DNA in the range from 0.1 pM to 10 nM, with a detection limit of 35 fM (at an S/N of 3). The biosensor exhibits good selectivity, acceptable stability and reproducibility.

Coating sulfonated polystyrene microspheres with highly dense gold nanoparticle shell for SERS application

The fabrication of highly dense gold nanoparticles (NPs)-coated sulfonated polystyrene (PS) microspheres and their application in surface-enhanced Raman spectroscopy (SERS) were reported. After the preparation of PS microsphere using dispersion polymerization and subsequent sulfonation, [Ag(NH₃)₂]⁺ ions were adsorbed on the surfaces of the sulfonated PS microspheres and then reduced to silver nanoseeds for further growth of gold NPs shell by seeded growth approach. Reaction conditions such as the concentration of the growth solution and growth time were adjusted to achieve nonspherical gold NPs-coated PS microspheres with different coverage degree. The application of the as-prepared spiky gold NPs-coated PS microsphere hybrid composite in SERS was finally investigated by using 4-aminothiophenol as probe molecules. The results showed that as-prepared gold NPs-coated PS microspheres could be used as functional hybrid materials to exhibit excellent enhancement ability in SERS.

Detection of mRNA from Escherichia coli in drinking water on nanostructured polymeric surfaces using liquid crystals

In this study, we demonstrate the detection of mRNA from Escherichia coli in drinking water using thermotropic liquid crystals (LCs). After hybridization of complementary mRNA with the single-stranded DNA immobilized on a polymer substrate containing periodic sinusoidal wave patterns, the orientation of LCs transits from a uniform to a non-uniform state, thereby inducing a change in the optical response of LCs. The periodic sinusoidal features of the polymer substrate are obtained through buckling the poly-(dimethylsiloxane) slide on a cylindrical surface, followed by replicating the associated relief structures on a poly-(urethaneacrylate) surface, where a film of gold was deposited. Then, thiol-modified single-stranded DNA was functionalized on the gold film as an mRNA receptor. The formation of mRNA–single-stranded DNA complex, which covers the sinusoidal nanostructures on the surface, induces the orientational transition of LCs. This result indicates that LCs can be used to report the specific hybridization of mRNA with single-stranded DNA, which holds promise for the sensitive and label-free detection of viable bacterial pathogens in drinking water.

Temperature-assisted ionic liquid dispersive liquid-liquid microextraction combined with high performance liquid chromatography for the determination of PCBs and PBDEs in water and urine samples

We report on silver–gold core-shell nanostructures that contain Methylene Blue (MB) at the gold–silver interface. They can be used as reporter molecules in surface-enhanced Raman scattering (SERS) labels. The labels are stable and have strong SERS activity. TEM imaging revealed that these nanoparticles display bright and dark stripe structures. In addition, these labels can act as probes that can be detected and imaged through the specific Raman signatures of the reporters. We show that such SERS probes can identify cellular structures due to enhanced Raman spectra of intrinsic cellular molecules measured in the local optical fields of the core-shell nanostructures. They also provide structural information on the cellular environment as demonstrated for these nanoparticles as new SERS-active and biocompatible substrates for imaging of live cells.

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.

DNA biosensors based on metallo-intercalator probes and electrocatalytic amplification

Strategies for electrochemical sensing of DNA can be classified into label-free and label-based approaches, categories of which include enzyme-, nanomaterial- and redox labels that are attached to DNA either by covalent or non-covalent means. Metallointercalators represent one group of small molecule redox labels that non-covalently enter the groove of a DNA. The metallointercalator plays a dual-role in acting as a structure indicator (for hybridization) and a signal generator. Labeling is not needed, and electrochemical measurements can be carried out in a label-free solution of an electrolyte. However, such metallointercalators lack the option of catalytic signal generation as in the case of enzyme- and nanomaterial-based labels. Therefore, signal amplification becomes crucial. We first survey here recent progress in this area. A signal-amplifying system is presented that relies on the electroatalytic oxidation of a metallointercalator ruthenium(II)bipyridine/phenoxazine complex in the presence of electron donor species such as oxalate, DNA bases, or tripropylamine. Recent work on such DNA sensors is discussed. Results suggest that such metallointercalator-based DNA sensors represent a viable platform for developing high-throughput and automated PCR/lab-on-a-chip devices as well as visualized multifunctional DNA sensors.

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.

Recent progress in surface enhanced Raman spectroscopy for the detection of environmental pollutants

Surface enhanced Raman spectroscopy (SERS) has emerged as one of the most promising analytical tools in recent years. Due to advantageous features such as sensitivity, specificity, ease of operation and rapidity, SERS is particularly well suited for environmental analysis. We summarize here some considerations with respect to the detection of pollutants by SERS and provide an overview on recent achievements in the determination of organic pollutants, heavy metal ions, and pathogens. Following an introduction into the topic and considering aspects of sensitivity, selectivity, reproducibility and portability, we are summarizing applications of SERS in the detection of pollutants, with sections on organic pollutants (pesticides, PAHs and PCBs, explosives), on heavy metal ions, and on pathogens. In addition, we discuss current challenges and give an outlook on applications of SERS in environmental analysis. Contains 174 references.

Gold nanoparticle-coated multiwall carbon nanotube-modified electrode for electrochemical determination of methyl parathion

We report on an electrochemical sensor for the determination of methyl parathion. It is based on an electrode modified with multi-walled carbon nanotubes that were covered with gold nanoparticles (Au-NPs). The vertically aligned array of MWCNTs on a tantalum substrate was coated with Au-NPs by overhead magnetron sputtering deposition. Scanning and transmission electron microscopy and XRD were used to characterize the Au-NP-MWCNTs composite. Cyclic voltammetry and differential pulse voltammetry were employed to evaluate the suitability of the new electrode for the determination of methyl parathion. Under the optimal conditions, the current response of the electrode to methyl parathion is linear in the range from 0.50 to 16.0 mg mL^-1, with a detection limit of 50 ??g mL^-1 (signal/noise = 3), and the sensitivity is 4.5 times better than that of the plain MWCNTs electrode. We conclude that this method represents a simple, rapid, effective and sensitive approach for the detection of methyl parathion.

Rapid on-site TLC–SERS detection of four antidiabetes drugs used as adulterants in botanical dietary supplements

A novel facile method has been established for rapid on-site detection of antidiabetes chemicals used to adulterate botanical dietary supplements (BDS) for diabetes. Analytes and components of pharmaceutical matrices were separated by thin-layer chromatography (TLC) then surface-enhanced Raman spectroscopy (SERS) was used for qualitative identification of trace substances on the HPTLC plate. Optimization and standardization of the experimental conditions, for example the method used for preparation of silver colloids, the mobile phase, and the concentration of colloidal silver, resulted in a very robust and highly sensitive method which enabled successful detection when the amount of adulteration was as low as 0.001 % (w/w). The method was also highly selective, enabling successful identification of some chemicals in extremely complex herbal matrices. The established TLC–SERS method was used for analysis of real BDS used to treat diabetes, and the results obtained were verified by liquid chromatography–triple quadrupole mass spectrometry (LC–MS–MS). The study showed that TLC–SERS could be used for effective separation and detection of four chemicals used to adulterate BDS, and would have good prospects for on-site qualitative screening of BDS for adulterants.

Polymer-based devices for the label-free detection of DNA in solution: low DNA concentrations yield large signals

Poly (N-isopropylacrylamide-co-N-(3-aminopropyl) methacrylamide hydrochloride) microgel-based optical devices (etalons) have been shown to change their optical properties in the presence of single-stranded DNA. We hypothesize that this is due to the negatively charged DNA penetrating through the Au overlayer of the etalon, resulting in cross-linking and collapse of the positively charged microgels. We have shown that this technology is capable of detecting micromolar concentrations of target DNA in solutions containing two and four base pair mismatch sequences without the use of labels. Furthermore, the device’s response increases as the concentration of DNA decreases, which is unique for sensing strategies. We point out that coupling this transduction mechanism to DNA amplification strategies could result in extremely low detection limits.

An innovative strategy for direct electrochemical detection of microRNA biomarkers

We report an electrochemical method for direct, reagentless, and label-free detection of microRNA, based on a conjugated copolymer, poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-2-carboxyethyl-1,4-naphthoquinone), acting as hybridization transducer. Hybridization between the oligonucleotide capture probe and a microRNA target of 22 base pairs generates an increase in the redox current (“signal-on”), which is evidenced by square wave voltammetry. Selectivity is good, with little hybridization for non-complementary targets, and the limit of detection reaches 650 fM. It is also evidenced that this sensitivity benefits from the high affinity of DNA for RNA.

Glucose biosensor based on glucose oxidase immobilized on unhybridized titanium dioxide nanotube arrays

A glucose biosensor has been fabricated by immobilizing glucose oxidase (GOx) on unhybridized titanium dioxide nanotube arrays using an optimized cross-linking technique. The TiO₂ nanotube arrays were synthesized directly on a titanium substrate by anodic oxidation. The structure and morphology of electrode material were characterized by X-ray diffraction and scanning electron microscopy. The electrochemical performances of the glucose biosensor were conducted by cyclic voltammetry and chronoamperometry measurements. It gives a linear response to glucose in the 0.05 to 0.65 mM concentration range, with a correlation coefficient of 0.9981, a sensitivity of 199.6 μA mM^?1 cm^?2, and a detection limit as low as 3.8 µM. This glucose biosensor exhibited high selectivity for glucose determination in the presence of ascorbic acid, sucrose and other common interfering substances. This glucose biosensor also performed good reproducibility and long-time storage stability. This optimized cross-linking technique could open a new avenue for other enzyme biosensors fabrication.

Microextraction by packed sorbents combined with surface-enhanced Raman spectroscopy for determination of musk ketone in river water

Microextraction by packed sorbents (MEPS) combined with Surface-enhanced Raman spectroscopy (SERS) was investigated, and applied to the determination of musk ketone (MK) in river water samples. The full MEPS–SERS method includes analyte enrichment by MEPS preconcentration with C₁₈ sorbent followed by SERS detection supported by silver nanoparticles. An eluent drop containing the analyte is deposited directly from the MEPS syringe on a CaF₂ glass plate. When the drop has dried, a specific volume of silver nanoparticles solution is added on it before each SERS measurement. Several experimental variables were studied in depth; under the optimum experimental conditions MK can be extracted from a 500 μL sample with recoveries in the range 47–63 %. The limit of detection was 0.02 mg L^?1 and the relative standard deviation 15.2 % (n?=?4). Although not investigated in this work, the proposed method might be suitable for in-situ monitoring, because of the portability of the Raman spectrometer used.

Determination of mercury(II) by surface-enhanced Raman scattering spectroscopy based on thiol-functionalized silver nanoparticles

Silver nanoparticles (Ag NPs) modified with sodium 2-mercaptoethanesulfonate (mesna) exhibit strong surface-enhanced Raman scattering (SERS). Their specific and strong interaction with heavy metal ions led to a label-free assay for Hg(II). The covalent bond formed between mercury and sulfur is stronger than the one between silver and sulfur and thus prevents the adsorption of mesna on the surface of Ag NPs. This results in a decrease of the intensity of SERS in the presence of Hg(II) ions. The Raman peak at 795?cm^?1 can be used for quantification. The effect of the concentration of mesna, the concentration of sodium chloride, incubation time and pH value on SERS were optimized. Under the optimal conditions, the intensity of SERS decreases with increasing concentration of Hg(II). The decrease is linear in the 0.01 and 2?μmol L^?1 concentration range, with a correlation coefficient (R²) of 0.996 and detection limit (S/N?=?3) is 0.0024?μmol L^?1. The method was successfully applied to the determination of the Hg(II) in spiked water samples.

Plasma stencilling methods for cell patterning

In this paper we describe plasma stencilling techniques for patterning 10 mammalian cell lines on hydrophobic and cell repellent poly(dimethylsiloxane) (PDMS), methylated glass and bacterial grade polystyrene surfaces. An air plasma produced with a Tesla generator operating at atmospheric pressure was used with microengineered stencils for patterned surface oxidation, selectively transforming the surface to a hydrophilic state to enable cell adhesion and growth. Plasma stencilling obviates the need for directly patterning cell adhesion molecules. Instead, during cell culture, adhesion proteins from the media assemble in a bioactive form on the hydrophilic regions. Critically, the removal of protein patterning prior to cell culture provides the option to also use PDMS–PDMS plasma bonding to incorporate cell patterns within microfluidic systems. Linear patterns were generated using PDMS microchannel stencils, and polyimide stencils with through holes were used for the production of cellular arrays. For the production of smaller cellular arrays, a novel microcapillary-based dielectric barrier discharge system was developed. A numerical method to characterise the cell patterns is also introduced and was used to demonstrate that plasma stencilling is highly effective, with complete patterns confined during long term cell culture (>10 days). In summary, plasma stencilling is simple, rapid, inexpensive, reproducible and a potentially universal cell line patterning capability.

Synthesis of p-aminothiophenol-embedded gold/silver core-shell nanostructures as novel SERS tags for biosensing applications

We describe a novel surface-enhanced Raman scattering (SERS) tag that is based on Au/Ag core-shell nanostructures embedded with p-aminothiophenol. The Au/Ag core-shell sandwich nanostructures demonstrate bright and dark stripe structure and possess very strong SERS activity. Under optimum conditions, the maximum SERS signal was obtained with a 10?nm thick Ag nanoshell, and the enhancement factor is 3.4?×?10⁴ at 1077?cm^?1. After conjugation to the antibody of muramidase releasing protein (MRP), the Au/Ag core-shell nanostructures were successfully applied to an SERS-based detection scheme for MRP based on a sandwich type of immunoassay.

A new label-free approach for the determination of reaction rates in oxidative footprinting experiments

Hydroxyl radical-mediated oxidative footprinting coupled to mass spectrometric analysis is an attractive technique for protein surface mapping, conformational changes monitoring, and protein–ligand interfaces mapping in solution. In this technique, a protein is oxidized by in situ-generated hydroxy radicals and the site and rate of oxidation can be determined by proteolysis followed by mass spectrometric analysis. Changes in peptide oxidation rate can then be correlated to the changes in solvent exposure, and information about conformational changes or interaction domains can be obtained. The method relies, therefore, on the accurate measurements of peptide oxidation rate. Here, we describe a new label-free method to determine the oxidation rate of peptides that is based on the consumption of the unoxidized peptide instead of measuring the formation of oxidized peptides. The reaction rate thus obtained presents a better linearity and lower variation when compared to the traditional method. The label-free method is also simpler to implement and automation can be achieved through label-free quantitation software.