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Enriching and locating target analytes into specific "hot spots" are vital for ultrasensitive molecular identification and detection using plasmonic-based techniques. Inspired by mass transportation in lamp wicks, we develop an effective enrichment strategy for highly diluted analytes in which analytes and Au nanoparticles are transported via a solution microflow under the capillarity driving force of glass fiber papers to a heated region. After evaporation, a large volume of a solution containing analytes and Au nanoparticles is condensed into a very limited area, and thus, analyte molecules are effectively enriched and located into surface-enhanced Raman scattering (SERS) hot spots. Using this enrichment strategy, the sensitivity and detection limits of SERS are remarkably improved. Detection levels of crystal violet and anthracene are down to 10 -16 and 10 -10 M, respectively. This enrichment strategy is very robust and easy to implement, and it can potentially be exploited in various plasmonic-based molecular detection and identification techniques.

Semiconductor materials have become competitive candidates for surface-enhanced Raman scattering (SERS) substrates; however, their limited SERS sensitivity hinders the practical applications of semiconductors. Here, we develop a hybrid substrate by integrating anatase/rutile TiO 2 heterostructure with dense plasmonic hotspots of Ag nanoparticle (AgNPs) for efficient photoinduced enhanced Raman spectroscopy (PIERS). The PIERS mechanism is systematically investigated by means of a portable Raman instrument. When ultraviolet (UV) light irradiates the substrate, the TiO 2 -Ag hybrid arrays produce remarkable charge-transfer enhancement, which can be ascribed to the highly efficient charge separation driven by heterojunction and transfer from TiO 2 heterostructure to AgNPs. This platform allows for the rapid detection of multifold organic species, including malachite green (MG), crystal violet (CV), rhodamine 6G (R6G), thiram, and acephate, and as high as 27.8-fold enhancement over the normal SERS is achieved, representing the highest PIERS magnification up to the present time. The intensive PIERS enhancement makes it ultrasensitively detect analyte concentration of an order of magnitude lower than that of SERS method. The improved sensitivity and resolution can be readily realized by simple UV irradiation, which represents a major advantage of our PIERS methodology. Besides, the integration of uniform TiO 2 heterostructure arrays with AgNPs generates superior signal reproducibility with relative standard deviation (RSD) value of less than 14%. In addition, the detected molecules on the substrate can be eliminated by photocatalytic degradation after PIERS measurements by using UV irradiation, which makes the substrate reusable for 15 cycles. The ultrahigh sensitivity, superior reproducibility, and excellent recyclability displayed by our platform may provide new opportunities in field detection analysis coupled with a portable Raman instrument.

Collecting highly diluted target analytes into specific hot spot regions is vital for ultrasensitive surface-enhanced Raman spectroscopy (SERS) applications. In this work, a hydrophobic slippery platform was employed as a concentrator to construct colloidal SERS-active substrates regardless of the diffusion limits during droplet evaporation. Within only 140 s, sufficient absorption between the analytes and colloidal Au nanoparticles (Au NPs) was observed by fluorescence imaging. This effect resulted in excellent SERS sensitivity and stability. Compared with the common metal colloid-based SERS substrates, e.g., drying on a silicon wafer or detection in colloidal solutions, this preconcentrated method showed lower detection limits and the lowest detection concentration of crystal violet molecule down to 10 -12 M with a portable Raman spectrometer. Such high signal enhancement was mainly ascribed to the condensation effect of Au colloids/analytes on the hydrophobic slippery substrate, by which almost all probe molecules were guided into the "hot spot" regions of aggregated Au NPs. Using the SERS platform, various illegal additives in realistic food and health-care products, for example, malachite green (1 ppb) added in fish and morphine (0.1 ppm) added in a chafing dish, could be sensitively detected. Therefore, our protocol is a general SERS platform that may provide a simple, fast, and cost-effective approach for trace molecular sensing.

With the advanced development of miniaturized Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS) has extended its applications into the field of point-of-care testing (POCT) and demonstrated its great significance by virtue of its noninvasive property and capability of fingerprint identification. In the SERS-based analysis and/or sensing system, the preparation of a low-cost, high-performance SERS substrate is critically important. In this manuscript, vacuum filtration is utilized to fabricate the silver nanoparticles (AgNPs)-embedded nylon filter membrane (ANFM) as flexible paper-based SERS chips. By characterizing the typical analytes with a miniaturized smartphone-based Raman analyzer, the proposed SERS chips have successfully demonstrated good sensitivity, repeatability, and stability. The lowest concentration as detected can approach 1 pmol for rhodamine 6G (RH6G) and 10 pmol for both crystal violet (CV) and malachite green (MG), respectively. With the help of the microporous structure of the membrane, the ANFM-based SERS chips can implement the separation of small molecules from a complex mixture and can achieve "purified" SERS signals of targeted molecules. Besides, with the function of antifriction resistance and flexibility, the ANFM can serve as SERS papers to preconcentrate the contaminates by multiple swapping and further enhance the SERS signals for point-of-care analysis. Therefore, we demonstrate multifunctions of the flexible ANFM-based SERS chips, which provide a promising solution for the POCT analysis with the SERS technique on account of their flexibility and low fabrication cost.

It is urgent to develop a rapid, reliable, and in-site determination method to detect or monitor trace amounts of toxic substances in the field. Here, we report an alternative surface-enhanced Raman scattering (SERS) method coupled with a portable Raman device on a plasmonic three-dimension (3D) hot spot sensing surface. Plasmonic Ag nanoparticles (AgNPs) were uniformly deposited on 3D TiO 2 nanopore arrays as a sensitive SERS substrate, and further coated with graphene oxide (GO). We demonstrate the plasmon-induced SERS enhancement (5.8-fold) and the improvement of catalytic activity by incorporation of plasmonic AgNPs into the 3D TiO 2 nanopore arrays. The modification of GO on the TiO 2 -Ag nanopore array further increases by a 6.2-fold Raman enhancement compared to TiO 2 -Ag while maintaining good uniformity (RSD < 10%). The optimized TiO 2 -Ag-GO substrate shows powerful quantitative detection potential for drug residues in fish scales via a simple scrubbing method, and the limit of detection (LOD) for crystal violet (CV) was 10 -8 M. The SERS substrate also showed detection practicability of pesticide residues in banana peel with an LOD of 10 -7 M. In addition, our TiO 2 -Ag-GO substrate exhibits excellent SERS self-monitoring performance for catalytic reduction of multiple organics in NaBH 4 solution, and the substrate shows good recyclability of 6 cycles. Such a 3D TiO 2 -Ag-GO substrate is a promising SERS substrate with good sensitivity, uniformity, and reusability, and may be utilized for further miniaturization for point of analytical applications.

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

We report a novel method for generating magneto-plasmonic carbon nanofilms and nanoscrolls using a combination of two gas-phase synthetic techniques. Ternary Fe@Ag@Si "onion-like" nanoparticles (NPs) are produced by a magnetron sputtering inert gas condensation source and are in situ landed onto the surface of carbon nanofilms, which were previously deposited by a DC arc discharge technique. Subsequently, a polyethylenimine-mediated chemical exfoliation process is performed to obtain carbon nanoscrolls (CNS) with embedded NPs (CNS-NPs). Of note, the carbon nanofilms undergo an interfacial transition upon addition of NPs and become rich in the sp 2 phase. This transformation endows and enhances multiple functions, such as thermal conductivity and the plasmonic properties of the nanocomposites. The obtained two-dimentional (2D) nanocomposites not only exhibit a highly efficient surface-enhanced Raman scattering property, allowing sensitive detection of malachite green isothiocyanate (MGIT) and adenosine-triphosphate (ATP) molecules at concentrations as low as 1 × 10 -10 M, but also show enhanced near-infrared-responsive photothermal activity when forming stable colloidal 1D CNS-NPs. In addition, the CNS-NPs present an enhanced single- and two-photon fluorescence in comparison with pristine CNS and NPs. These results make them suitable for the rational fabrication of "all-in-one" multifunctional nanocomposites with tubular structures toward a wide range of biomedical solutions.

Surface-enhanced Raman scattering (SERS) has been considered as a promising sensing technique to detect low-level analytes. However, its practical application was hindered owing to the lack of uniform SERS substrates for ultrasensitive and reproducible assay. Herein, inspired by the natural cactus structure, we developed a cactus-like 3D nanostructure with uniform and high-density hotspots for highly efficient SERS sensing by both grafting the silicon nanoneedles onto Ag dendrites and subsequent decoration with Ag nanoparticles. The hierarchical scaffolds and high-density hotspots throughout the whole substrate result in great amplification of SERS signal. A high Raman enhancement factor of crystal violet up to 6.6 × 10(7) was achieved. Using malachite green (MG) as a model target, the fabricated SERS substrates exhibited good reproducibility (RSD ∼ 9.3%) and pushed the detection limit down to 10(-13) M with a wide linear range of 10(-12) M to 10(-7) M. Excellent selectivity was also demonstrated by facilely distinguishing MG from its derivative, some organics, and coexistent metal ions. Finally, the practicality and reliability of the 3D SERS substrates were confirmed by the quantitative analysis of spiked MG in environmental water with high recoveries (91.2% to 109.6%). By virtue of the excellent performance (good reproducibility, high sensitivity, and selectivity), the cactus-like 3D SERS substrate has great potential to become a versatile sensing platform in environmental monitoring, food safety, and medical diagnostics.

We report a new paper-based surface enhanced Raman scattering (SERS) substrate platform contributed by a poly(l-lactic acid) (PLLA) nanofibrous paper adsorbed with plasmonic nanostructures, which can circumvent many challenges of the existing SERS substrates. This PLLA nanofibrous paper has three-dimensional porous structure, extremely clean surface with good hydrophobicity (contact angle is as high as 133.4°), and negligible background interference under Raman laser excitation. Due to the strong electrostatic interaction between PLLA nanofiber and cetyltrimethylammonium bromide (CTAB) molecules, the CTAB-coated gold nanorods (GNRs) are efficiently immobilized onto the fibers. Such a hydrophobic paper substrate with locally hydrophilic SERS-active area can confine analyte molecules and prevent the random spreading of molecules. The confinement leads to focusing effect and the GNRs-PLLA SERS substrate is found to be highly sensitive (0.1 nM Rhodamine 6G and malachite green) and exhibit excellent reproducibility (∼8% relative standard deviation (RSD)) and long-term stability. Furthermore, it is also cost-efficient, with simple fabrication methodology, and demonstrates high sample collection efficiency. All of these benefits ensure that this GNRs-PLLA substrate is a really perfect choice for a variety of SERS applications.

To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG), we identified the immunocomplex binding phage-borne peptides for use in the noncompetitive phage anti-immunocomplex assay (PHAIA). An anti-LMG monoclonal antibody (mAb) was used to select immunocomplex binding peptides from a circular random eight-amino-acid phage-displayed library. After three rounds of panning-elution, five peptides that bound the LMG-mAb immunocomplex were obtained. One of the phage-borne peptide clones that resulted in an assay with the highest sensitivity was chosen for further research. The concentration of LMG producing 50% of the saturated signal and the limit of detection of the assay were 7.02 and 0.55 ng/mL, respectively, with a linear range of 1.35 to 21.56 ng/mL. The PHAIA based on the same antibody was 16 times more sensitive compared to the competitive immunoassay. PHAIA was used to analyze LMG, MG, and two mixtures of spiked fish samples, with validation by high-performance liquid chromatography (HPLC) with fluorescence detector. Results showed a good correlation (R(2)LMG = 0.9841; R(2)MG = 0.993; R(2)Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety.

We report a simple and rapid method for fabricating a surface-enhanced Raman scattering (SERS) substrate, which offers good flexibility, excellent optical transparency, and high SERS activity. Specifically, the SERS substrate (AuNPs/PMMA film) was obtained through self-assembly of gold nanoparticles (AuNPs) on newborn poly(methyl methacrylate) (PMMA) template. The UV-vis spectroscopy analysis and scanning electron microscopy observation revealed that the gold nanoparticles were closely assembled on the flexible and transparent PMMA template. The fabricated AuNPs/PMMA film SERS substrate allowed detection of model molecule, malachite green isothiocyanate, at a concentration as low as 0.1 nM, and exhibited good reproducibility in the SERS measurement. The Raman enhancement factor (EF) of the AuNPs/PMMA film was found to be as high as (2.4 ± 0.3) × 10(7). In addition, measure of residual malachite green on fish surface was carried out, and the result indicated that the AuNPs/PMMA film had great potential in the in situ ultrasensitive detection of analyte on irregular objects.

We present herein characteristics of a conjugate in which dL5, a fluorogen activating protein (FAP), and AEAEAKAK, an amphiphilic peptide, are combined to form a solid-phase fluorescence detection platform. The FAP dL5 is a covalently linked dimer of two identical light chain variable fragments which activates the fluorescence of the fluorogen malachite green (MG). The amphiphilic peptide of sequence AEAEAKAK is a building block of stimuli-responsive materials that undergoes sol-gel phase transition at high ionic strengths. We hypothesize that the novel bifunctional protein containing both the FAP and the amphiphile, termed dL5&#95;EAK coassembles with the self-assembling peptide [AEAEAKAK]2 (EAK16-II) to form an insoluble membrane composite whereby the fluorescence enhancement function of the FAP domain remains intact. Denaturing polyacrylamide electrophoresis indicated that greater than 78% of dL5&#95;EAK incorporates into the EAK16-II membrane. Conversely, less than 32% of dL5 without the EAK sequence associates with the insoluble fraction of EAK16-II in buffers. Membranes containing dL5&#95;EAK and EAK16-II exhibited at least 4-fold higher fluorescence intensity compared to mixtures containing dL5 and EAK16-II. Scanning electron microscopy revealed the presence of particulates, presumably FAPs, scattered on the membrane fibrils. The evidence suggests a system of materials that can be developed into in situ forming local sensors by immobilizing dL5 into coacervate, on which MG can be detected. It is envisioned that dL5 membranes can be established in diseased locales to monitor infiltration and migration of inflammatory cells marked with antibodies conjugated to MG.

We have designed a large-scale three-dimensional (3D) hybrid nanostructure as surface-enhanced Raman scattering (SERS) sensor by decorating silver nanoparticles on TiO2 nanorods scaffold (Ag/TiO2). Taking p-mercaptobenzoic acid (PMBA) as the probe molecule, the SERS signals collected by point-to-point and time mapping modes show that the relative standard deviation (RSD) in the intensity of the main Raman vibration modes (1079, 1586 cm(-1)) is less than 10%, demonstrating good spatial uniformity and time stability. This hybrid substrate also exhibits excellent SERS enhancement effect due to the formation of high-density hot spots among the AgNPs, which was proved by finite-difference time-domain (FDTD) simulations. The application of the new nanostructures as SERS sensors was demonstrated with the detection of malachite green (MG). The quantification of MG can be accomplished with the detection limit of 1 × 10(-12) M based on the Raman intensity. The results show that the Ag/TiO2 nanostructure can be a promising candidate for SERS sensor.

An electrochemiluminescence (ECL) inhibition method combined with molecularly imprinted solid phase extraction (MISPE) was developed for quantitative determination of malachite green (MG) residues in fish. It was found that MG could strongly inhibit the ECL signal of luminol. Under the optimized conditions, the quenched ECL intensity versus the logarithm of the concentration of MG was in good linear relationship over a concentration range from 20 to 5000 ppt. The method detection limit was found to be about 6 ppt. Molecularly imprinted polymers (MIPs) were synthesized as solid phase extraction (SPE) sorbents, and MISPE was used for the selective extraction and purification of MG. By carrying out the oxidation reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), which could convert leucomalachite green (LMG) into MG, this method was successfully applied to determine MG residues in fish. A possible mechanism for the quenching effects of MG on luminol was also proposed.

Residues of malachite green (MG), gentian violet (GV), and their leuco metabolites in channel catfish muscle were individually determined by HPLC using diode array and fluorescence detectors and confirmed by tandem mass spectrometry. This detection scheme obviates a PbO(2) reactor that converts leuco forms to chromatic forms for absorbance detection, therefore eliminating uncertainties in oxidant depletion and data integrity. Extraction was performed once in pH 3 McIlvaine buffer and acetonitrile, followed by cleanup using a polymeric strong cation-exchange column. Liquid-liquid extraction was excluded to provide an environmentally responsible and relatively rapid protocol. Spectrometric limits of detection (LOD; S/N = 3) for MG (lambda = 620 nm) and GV (lambda = 588 nm) were 0.38 and 0.26 ng/g with 44.5-49.2% and 92.2-101.4% recoveries (1-10 ng/g, n = 6), respectively. Fluorometric LOD (S/N = 3) for LMG and LGV (lambda(ex) = 266 nm, lambda(em) = 360 nm) were 0.10 and 0.09 ng/g with 74.3-84.5% and 80.6-86.5% recoveries (1-10 ng/g, n = 6), respectively. This simplified protocol saves costs and meets the sensitivity requirements set by the Food and Drug Administration and the European Union.

In the Bradford protein assay, protein concentrations are determined by the absorbance at 595 nm due to the binding of Coomassie brilliant blue G-250 (CBBG) to proteins. In a protein-CBBG liquid mixture, surface-enhanced Raman scattering (SERS) is sensitive to the amount of unbound CBBG molecules adsorbed on silver surfaces, and the bound CBBG amount is directly related to the target protein concentration. Accordingly, a novel method for detecting total protein concentration in a solution has been developed based on SERS of unbound CBBG with an internal standard of silicon. Two obvious advantages of the proposed protein assay over conventional Bradford protein assay are its much wider linear concentration range (10(-5)-10(-9) g/mL) and 200 times lower limit of detection (1 ng/mL), which demonstrates its great potential in rapid, highly sensitive concentration determination of high and low-abundance proteins.

A robust screening assay employing solid phase extraction (SPE) followed by a novel aptamer-based procedure is presented for the rapid detection and semiquantitation of the triphenylmethane dye, Malachite Green (MG) and its primary metabolite Leucomalachite Green (LMG) in fish tissue. To the authors' knowledge, this is the first reported use of an RNA aptamer for the development of a diagnostic assay for the detection of chemical residues in food. The aptamer based screening assay is found to be highly specific for MG; but has negligible affinity for the LMG metabolite. However, because the LMG metabolite is lipophilic and known to be highly persistent in tissues, an oxidation step has been incorporated within the sample cleanup procedure to ensure that all LMG residues are converted to MG prior to measurement. This article provides evidence that an oligonucleotide aptamer can be used as an alternative recognition element to conventional antibodies with application to the detection of residues in food. Furthermore, this finding has the future potential to reduce the number of animals currently being used in the production of antibodies for immunodiagnostic kits.

The safety of imported seafood products because of the contamination of prohibited substances, including crystal violet (CV) and malachite green (MG), raised a great deal of concern in the United States. In this study, a fractal-like gold nanostructure was developed through a self-assembly process and the feasibility of using surface-enhanced Raman spectroscopy (SERS) coupled with this nanostructure for detection of CV, MG, and their mixture (1:1) was explored. SERS was capable of characterizing and differentiating CV, MG, and their mixture on fractal-like gold nanostructures quickly and accurately. The enhancement factor of the gold nanostructures could reach an impressive level of approximately 4 x 10(7), and the lowest detectable concentration for the dye molecules was at approximately 0.2 ppb level. These results indicate that SERS coupled with fractal-like gold nanostructures holds a great potential as a rapid and ultra-sensitive method for detecting trace amounts of prohibited substances in contaminated food samples.

This study provides a practical method for production of the antibodies against malachite green (MG) and its primary metabolite leucomalachite green (LMG). Two ELISA kits are constructed with the MG and LMG antibodies for detection of the residual MG and LMG in fish muscle and fishpond water. The detection limit is established at the level of 0.05 microg/L for both MG and LMG. Our ELISA kits show the advantages of good specificity, high sensitivity, and convenience in rapid screening of MG and LMG residues. The sample of fishpond water, without extraction or prior preparation, is directly assayed by the ELISA kit. More then 80 fish samples can be simultaneously tested in a kit. The toxic crystal violet and its metabolite leucocrystal violet of illegal use in aquaculture are detected by our prepared MG and LMG antibodies, whereas the antibodies do not cross-react with common antibiotics, sulfonamides, and benzene derivatives.

Liquid chromatographic methods are presented for the quantitative and confirmatory determination of malachite green (MG) and leucomalachite green (LMG) for channel catfish, rainbow trout, tilapia, basa, Atlantic salmon, and tiger shrimp. Residues were extracted from tissues with ammonium acetate buffer and acetonitrile and isolated by partitioning into dichloromethane. LMG was quantitatively oxidized to the chromic MG with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Extracts were analyzed for total MG by liquid chromatography with both visible detection (LC-VIS) at 618 nm for routine screening and ion trap mass spectrometry (LC-MSn) with no discharge-atmospheric pressure chemical ionization for residue confirmation. The method was validated in each species fortified with LMG at 1, 2, 4, and 10 ng/g (ppb), and average recoveries ranged from 85.9 to 93.9%. Quantitative data were consistent for the two detection methods, with measured method detection limits of 1.0 ng/g for LC-VIS and 0.25 ng/g for LC-MSn. Incurred tissues from catfish, trout, tilapia, and salmon that had been treated with MG were also extracted and analyzed as part of this study.