High Signal Reproducibility Research Articles

Development of three-enzyme lactose amperometric biosensor modified by nanosized poly (meta-phenylenediamine) film

New three-enzyme lactose amperometric biosensor based on platinum disc electrode modified by nanosized semipermeable poly (meta-phenylenediamine) film was developed in this work. It was studied the influence of different conditions of meta-phenylenediamine electropolymerization on the parameters (sensitivity and selectivity) of the amperometric transducers. The most optimal parameters were achieved after performing the electropolymerization of 5 mM meta-phenylenediamine applying 10–13 cyclic voltammograms. The bioselective element of lactose biosensor consisted of three enzymes (β-galactosidase, mutarotase, glucose oxidase) that were immobilized on the surface of modified working electrode. The analytical characteristics of the developed biosensor were studied. The linear range of lactose determination extended from 0.01 mM to 1.25 mM. The response time of the biosensor was 30 s. The biosensor showed high signal reproducibility (RSD = 1.16%), high sensitivity (LOD = 0.005 mM) and high selectivity. The proposed biosensor was tested for measurement of lactose concentration in milk samples. The high correlation between results obtained with lactose biosensor and HPLC was shown (R = 0.96%). Thus, the developed biosensor is suited for rapid, inexpensive, sensitive, selective and simple determination of lactose in milk samples.

Ag-coated 3D Cu(OH)2 nanowires on the woven copper mesh as a cost-effective surface-enhanced Raman scattering substrate

The commercial development of surface-enhanced Raman scattering (SERS) technique is extremely limited to the high-cost and complex fabrication of desired SERS substrate with high SERS sensitivity and good signal reproducibility. Herein, Ag-coated three-dimensional (3D) Cu(OH)2 nanowire arrays on the woven copper mesh were fabricated by a simply in-situ chemical engraving and sputtering deposition as the promising SERS substrate. Importantly, ascribed to the large surface area, the 3D hierarchical micro-nanostructures containing dense needle-like Cu(OH)2 nanowires offer an excellent platform to load the Ag nanoparticles and enrich target molecules, which is favorable for enhancing the Raman signals of adsorbates. As a result, a limit concentration of probe molecule Rhodamine 6G (R6G) down to 0.1 pM was identified, corresponding to an average enhancement factor of 7 orders. The relative signal deviation was tested to be 17.5% for a single sample and 19.3% among 10 samples. Furthermore, thiram fungicide (a sort of pesticide) residue was also analyzed, showing a linear detection range between 0.1 mM and 50 nM. This simple, large-scale and low-cost fabrication approach offers a promising candidate of SERS chips to the practical detection of pesticide residues, food safety inspection and organic pollutants in the environment.

Vertically aligned nanostructures for a reliable and ultrasensitive SERS-active platform: Fabrication and engineering strategies

Surface-enhanced Raman spectroscopy (SERS) is a powerful cross-disciplinary analytical technique allowing ultrasensitive detection of various analytes with a fingerprint-like characteristic, even down to the single-molecule level. One of the key issues for SERS-based trace applications is the rational design and fabrication of structurally uniform substrates with high sensitivity, stability, and good signal reproducibility. Low-dimensional SERS-active nanomaterials are increasingly facing many challenges and cannot meet these requirements. In recent years, due to developments in nanofabrication and an improved knowledge of plasmonic properties of nanomaterials, significant progress has been made in the area of complex nanostructures with ordered architectures, elemental compositions, and controlled plasmonic properties, which are critical for ultrasensitive, uniform, reliable, and reproducible measurements. In this review, the fabrication methodologies and classification of vertically structured nanoarchitectures are outlined. Subsequently, the morphological and geometrical effects on SERS performance are discussed based on the substrate materials. Insight into advanced engineering strategies towards some SERS-active nanostructures is supplied in this context. Finally, the challenges and future directions are pointed out for the development of vertically nanostructured SERS substrates and their ultrasensitive sensing applications.

Plasmonic Microgels for Raman-Based Molecular Detection Created by Simultaneous Photoreduction and Photocross-linking

Molecular detection in complex mixtures is of great importance in biomedical diagnosis, food safety, and environmental monitoring. Although surface-enhanced Raman scattering serves as one of the most promising detection methods, metal surfaces are prone to contamination, making the direct detection of small molecules in mixtures elusive. Metal nanoparticle-loaded hydrogels have been used for the exclusion of large adhesive molecules and direct detection of small molecules. Here, we design microgels containing highly concentrated gold nanoparticles through the simultaneous formation of hydrogel and gold nanoparticles in emulsion droplets. Monodisperse water-in-oil droplets are microfluidically prepared to contain a gold precursor, hydrogel precursor, and photoinitiator. Upon ultraviolet irradiation, a hydrogel is gradually formed in the drop by photocross-linking at which gold nanoparticles are synthesized and grown by photo and thermal reduction. The in situ synthesis provides the uniform distribution of gold nanoparticles at very high concentrations without aggregation, which is otherwise very difficult to achieve. Using the microgels, small molecules in albumin solutions can be detected by Raman measurement with high signal sensitivity and reproducibility in the absence of interruption from albumin. As a proof of concept, we demonstrate the direct detection of pyocyanin, a biomarker for Pseudomonas infection spiked in unpurified saliva.

Monolayer ZnS@Ag Nanospheres SERS Substrate for Highly Sensitive Dye Molecules Detection

Surface-enhanced Raman scattering (SERS) substrates with low cost, high sensitivity and good reproducibility are still challenging in practical application. Herein, we propose a facile method to prepare monolayer ZnS@Ag nanospheres (NSs) by sputtering Ag nanoparticles (NPs) on the surfaces of the monolayer ZnS NSs produced by self-assembly. The monolayer ZnS@Ag NSs have rough surface and nanoscale gaps, which can produce large SERS effect. The dye molecules, Rhodamine 6G (R6G) and Rhodamine B (RhB), were used as probe to evaluate the SERS performance on the monolayer ZnS@Ag NSs. It was found that the monolayer ZnS@Ag NSs showed the high SERS sensitivity in the detection of R6G and RhB, the limit of detection (LOD) down to 9.12×10−13 M and 8.55×10−11 M, respectively. The corresponding enhancement factors (EF) are 3.01×108 and 8.2×106, respectively. Furthermore, the ordered structure makes the monolayer ZnS@Ag NSs substrate with high signal reproducibility and stability, and the relative standard deviation (RSD) values are less than 15%. Therefore, the monolayer ZnS@Ag NSs is a candidate for detecting organic dyes in the environment.

Ag nanoparticles decorated Ag@ZrO2 composite nanospheres as highly active SERS substrates for quantitative detection of hexavalent chromium in waste water

A novel highly active surface enhanced Raman scattering (SERS) substrate of Ag nanoparticles decorated Ag@ZrO2 composite nanosphere was prepared and characterized. A uniform ZrO2 shell was firstly coated over Ag cores by sol-gel process using zirconium butoxide as Zr precursor to achieve Ag@ZrO2 nanospheres and then Ag nanoparticles were in situ deposited on the Ag@ZrO2 through the reduction of AgNO3 to produce Ag@ZrO2@Ag composite nanospheres. The cover density of Ag nanoparticles on Ag@ZrO2@Ag nanospheres could be controlled by changing the concentration of AgNO3. TEM, FE-SEM, EDX, UV–vis and XRD analyses were used to characterize their morphology, structure and composition. The SERS property of Ag@ZrO2@Ag was evaluated using 4-mercaptobenzoic acid (4-MBA) as Raman probe molecules. The experimental results showed Ag@ZrO2@Ag nanospheres presented remarkably SERS activity, high signal reproducibility (RSD = 6.39%) and exceptional storing stability. Subsequently, the Ag@ZrO2@Ag nanospheres acted as SERS substrates to sense Cr (VI) in water at various concentrations, and the SERS results showed great linear correlation between SERS intensities and the concentration of Cr (VI) (R2 = 0.97) with the detection limit of 0.5 μM, which is below the safety level (1 μM) in drinking water of United States Environmental Protection Agency. The Ag@ZrO2@Ag nanospheres were also successfully used to confirm the concentration of Cr (VI) in real industrial wastewater. These results demonstrated the extraordinary potential of Ag@ZrO2@Ag composite nanospheres serving as SERS substrates for trace and quantitative determination of Cr (VI) in environmental field.

Direct Optical and Ultrasensitive Probing of Nonequilibrium Dynamics of Carbon Monoxide in an Aqueous Phase during Biochemical Reactions.

Detection of trace carbon monoxide (CO) dissolved in an aqueous phase is key for monitoring and optimizing biological and chemical gas conversions. So far, irrespective of the nonequilibrium nature of these conversion processes, because of low water solubility of CO, such detection has been performed indirectly, under the assumption of thermodynamic equilibrium, by the combination of chromatographic measurement of relatively abundant CO in a gas phase and Henry's law. Direct and sensitive detection of dissolved CO under nonequilibrium has not been explored yet. Here, we report the direct, ultrasensitive, and real-time monitoring of nonequilibrium dynamics of CO in an aqueous phase during biochemical conversions by devising miniaturized fluidic reactors with built-in CO-specific optical probes via surface-enhanced Raman spectroscopy. As the sensitive and selective probes, we fabricate ligand-free Au@Pd core-shell nanoparticle monolayers to maximize the Raman signal of single CO in the aqueous phase. We confirm that under equilibrium conditions, aqueous and gaseous CO concentrations estimated by our method are in good agreement with those measured directly and indirectly by gas chromatography (GC). We show that our probe can detect the aqueous CO concentrations as low as ca. 0.01% with high signal reproducibility, which is 200-fold more sensitive than that achieved by infrared spectroscopy. Finally, we successfully observe the nonequilibrium dynamics of the aqueous CO during biochemical reactions, which cannot be sensed by other detection methods including even indirect measurement by GC. We anticipate that our method can be widely applied not only for monitoring of biochemical gas reactions on multiple scales from a large reactor to a single-molecule level but also for molecular imaging of biological systems.

Surface-Enhanced Raman Scattering and Fluorescence on Gold Nanogratings.

Surface-enhanced Raman scattering (SERS) spectroscopy is a sensitive sensing technique. It is desirable to have an easy method to produce SERS-active substrate with reproducible and robust signals. We propose a simple method to fabricate SERS-active substrates with high structural homogeneity and signal reproducibility using electron beam (E-beam) lithography without the problematic photoresist (PR) lift-off process. The substrate was fabricated by using E-beam to define nanograting patterns on the photoresist and subsequently coat a layer of gold thin film on top of it. Efficient and stable SERS signals were observed on the substrates. In order to investigate the enhancement mechanism, we compared the signals from this substrate with those with photoresist lifted-off, which are essentially discontinuous gold stripes. While both structures showed significant grating-period-dependent fluorescence enhancement, no SERS signal was observed on the photoresist lifted-off gratings. Only transverse magnetic (TM)-polarized excitation exhibited strong enhancement, which revealed its plasmonic attribution. The fluorescence enhancement showed distinct periodic dependence for the two structures, which is due to the different enhancement mechanism. We demonstrate using this substrate for specific protein binding detection. Similar periodicity dependence was observed. Detailed theoretical and experimental studies were performed to investigate the observed phenomena. We conclude that the excitation of surface plasmon polaritons on the continuous gold thin film is essential for the stable and efficient SERS effects.

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.

Fabrication of Remarkably Bright QD Densely‐Embedded Silica Nanoparticle

We developed a synthesis method for silica‐coated quantum dot (QD)‐densely‐embedded silica nanoparticle (Si@D‐QD@Si NP) with remarkably brighter fluorescence by increasing the number of QDs on silica NPs. Si@D‐QD@Si NPs exhibited a narrow size distribution and a high signal reproducibility. Red, green, and blue Si@D‐QD@Si NPs were successfully fabricated and characterized. The Si@D‐QD@Si NP carried 2.4 times more QDs than previously reported silica‐coated QD‐embedded silica NP (Si@QD@Si NP). The quantum yield of Si@D‐QD@Si NPs was slightly higher than Si@QD@Si NPs. The red Si@D‐QD@Si NPs showed approximately 26.6%, the green Si@D‐QD@Si NP 36.2%, and the blue Si@D‐QD@Si NP 39.3% higher fluorescence intensity compared to the corresponding Si@QD@Si NPs. This result implied that the particles can be used at bio‐field with a higher sensitive detection.

Laser Processing of Low Optical Reflection Micro/Nano-patterned Si Substrates for SERS

ABSTRACTLight collection efficiency and specific molecular detection are crucial factors for the performance of bio-chemical molecule sensors. In this paper, the low optical-reflection silicon (Si) substrates which combine reduced optical reflectivity by light trapping effect and high Raman enhancement ability of gold nanoparticles (Au-NPs) coated textured Si substrates are investigated for surface-enhanced Raman scattering (SERS). A fast, single-step and highly controllable nanosecond (ns) laser processing technique is employed to fabricate textured Si substrates under ambient conditions. Parallel arrays of micro-pyramids are fabricated on Si surface by direct laser writing two-dimensional structures. SEM micrographs clearly show well-ordered surface features in the form of micro-pyramid shape with well-defined sharp tips on the laser processed Si substrates. The aggregation of Si micro/nanoparticles on Si surface forms nanocavities and nanogaps and further enhances the surface roughness in order to minimize the optical reflection. The low optical reflection Si substrates exhibit optical reflection below 15% over a broad wavelength range from 300 nm to 1200 nm. The textured Si substrates with high signal reproducibility are successfully applied as SERS substrates to detect a very small concentration of Rhodamine B molecules with an average enhancement factor of the order of ∼107. The low optical reflection and SERS signal amplification are also altered by the variation of laser pulse energy resulting into low optical reflection and high SERS signal intensity over the entire laser-patterned area. The effect of surface roughness on water contact angle was studied after the modification with Polydimethylsiloxane (PDMS), the surfaces show perfect superhydrophobicity with almost no water adhesion. This approach provides a novel high-speed and cost-effective method for fabricating SERS substrate with micro/nano-scale surfaces roughness and low optical reflection for high Raman signal enhancement.

A highly sensitive, flexible SERS sensor for malachite green detection based on Ag decorated microstructured PDMS substrate fabricated from Taro leaf as template

A bio-inspired flexible surface enhanced Raman spectroscopy (SERS) sensor for malachite green (MG) detection has been fabricated by coating Ag on a structured surface of polydimethylsiloxane (PDMS) substrate using textured nano/microstructured Taro leaf as template. The Ag-coated microcavity structured PDMS exhibits high adhesion, hydrophobic behavior (water contact angle ∼128±1°) and serves as a concentrator for the analyte/probe molecule responsible for enhanced SERS intensity. The SERS activity of the fabricated Ag/PDMS flexible sensor having MG as probe molecules shows an enhancement factor of ∼2.06×105 over bulk MG molecules. The fabricated SERS sensor is showed the high sensitivity and good signal reproducibility. Sensitivity for very low MG concentration (∼10−11M) is reported which is attributed to the presence of high local field produced by Ag granular film and nanovoids onto PDMS surface. AFM and Raman characterizations have revealed the formation of nanovoids on Ag/PDMS structures to create plasmonic hotspots that results in enhanced SERS activity and high sensitivity. Further, the SERS signal under bending strain (tested between 180° and 100° bending), is also detectable for very low concentration of MG (as low as 10−7M) even at substrate bending angles of 100° that establishes the proposed SERS sensor system as an exceptional candidate for flexible MG SERS sensor applications.

Improving Sensitivity and Reproducibility of SERS Sensing in Microenvironments Using Individual, Optically Trapped Surface-Enhanced Raman Spectroscopy(SERS) Probes.

Surface-enhanced Raman spectroscopy (SERS) sensors offer many advantages for chemical analyses, including the ability to provide chemical specific information and multiplexed detection capability at specific locations. However, to have operative SERS sensors for probing microenvironments, probes with high signal enhancement and reproducibility are necessary. To this end, dynamic enhancement of SERS (i.e., in-situ amplification of signal-to-noise and signal-to-background ratios) from individual probes has been explored. In this paper, we characterize the use of optical tweezers to amplify SERS signals as well as suppress background signals via trapping of individual SERS active probes. This amplification is achieved through a steady presence of a single "hot" particle in the focus of the excitation laser. In addition to increases in signal and concomitant decreases in non-SERS backgrounds, optical trapping results in an eightfold increase in the stability of the signal as well. This enhancement strategy was demonstrated using both single and multilayered SERS sub-micron probes, producing combined signal enhancements of 24-fold (beyond the native 106 SERS enhancement) for a three-layered geometry. The ability to dynamically control the enhancement offers the possibility to develop SERS-based sensors and probes with tailored sensitivities. In addition, since this trapping enhancement can be used to observe individual probes with low laser fluences, it could offer particular interest in probing the composition of microenvironments not amenable to tip-enhanced Raman spectroscopy or other scanning probe methods (e.g., intracellular analyses, etc.).

Creatinine Deiminase Adsorption onto Silicalite-Modified pH-FET for Creation of New Creatinine-Sensitive Biosensor.

In the work, silicalite particles were used for the surface modification of pH-sensitive field-effect transistors (pH-FETs) with the purpose of developing new creatinine-sensitive biosensor. Creatinine deiminase (CD) adsorbed on the surface of silicalite-coated pH-FET served as a bioselective membrane. The biosensor based on CD immobilized in glutaraldehyde vapor (GA) was taken as control. The creatinine-sensitive biosensor obtained by adsorption on silicalite was shown to have better analytical characteristics (two- to threefold increased sensitivity to creatinine, three- to fourfold lesser response and recovery times, a decrease of the detection limit of creatinine determination to 5 μM, etc.).Additionally, the biosensor based on CD adsorbed on silicalite (Sil/CD) was characterized by high signal reproducibility (relative standard deviation (RSD) for creatinine measurement = 2.6 %) and stability during storage (over 13 months). It was experimentally confirmed that the proposed biosensor was not sensitive either to high concentrations of sodium chloride or to the macromolecular protein fractions and can be used for direct quantitative analysis of creatinine in the blood serum.It was concluded that the method of CD adsorption on silicalite is well-suited for the creation of creatinine-sensitive biosensor with improved working characteristics.

Determination of water-soluble forms of oxalic and formic acids in soils by ion chromatography

Carboxylic acids (CA) play an important role in the chemical composition origin of soils and migration of elements. The content of these acids and their salts is one of the important characteristics for agrochemical, ecological, ameliorative and hygienic assessment of soils. The aim of the article is to determine water-soluble forms of same carboxylic acids — (oxalic and formic acids) in soils by ion chromatography with gradient elution. For the separation and determination of water-soluble carboxylic acids we used reagent-free gradient elution ion-exchange chromatography ICS-2000 (Dionex, USA), the model solutions of oxalate and formate ions, and leachates from soils of the Kola Peninsula.The optimal gradient program was established for separation and detection of oxalate and formate ions in water solutions by ion chromatography. A stability indicating method was developed for the simultaneous determination of water-soluble organic acids in soils. The method has shown high detection limits such as 0.03 mg/L for oxalate ion and 0.02 mg/L for formate ion. High signal reproducibility was achieved in wide range of intensities which correspond to the following ion concentrations: from 0.04 mg/g to 10 mg/L (formate), from 0.1 mg/g to 25 mg/L (oxalate). The concentration of formate and oxalate ions in soil samples is from 0.04 to 0.9 mg/L and 0.45 to 17 mg/L respectively.

Plasmon enhanced fluorescence studies from aligned gold nanorod arrays modified with SiO2 spacer layers

Here, we demonstrate that quasi self-standing Au nanorod arrays prepared with plasma polymerisation deposited SiO2 dielectric spacers support surface enhanced fluorescence (SEF) while maintaining high signal reproducibility. We show that it is possible to find a balance between enhanced radiative and non-radiative decay rates at which the fluorescent intensity is maximized. The SEF signal optimised with a 30 nm spacer layer thickness showed a 3.5-fold enhancement with a signal variance of <15% thereby keeping the integrity of the nanorod array. We also demonstrate the decreased importance of obtaining resonance conditions when localized surface plasmon resonance is positioned within the spectral region of Au interband transitions. Procedures for further increasing the SEF enhancement factor are also discussed.

Silver nanoparticles deposited inverse opal film as a highly active and uniform SERS substrate

Ag-decorated TiO2 inverse opal films (ATIO) with high surface-enhanced Raman scattering (SERS) enhancement were prepared using an electroless deposition process. The Ag nanoparticles (NPs) are well-dispersed and deposited on the edge of macroporous walls. The structure and optical properties of the sample ATIO have been characterized. The Ag-loading cycles and pore sizes of TiO2 inverse opal are the key factors determining the magnitude of SERS signal enhancement. The optimized ATIO samples exhibit high SERS signal enhancement ability and reproducibility. The enhancement factor about 104 and the detection limit of 10−10M for R6G were achieved. The further application in detecting malachite green is demonstrated. A limit of detection approximately 10−9M was achieved. The results show that the ATIO nanostructure is promising for using as sensor substrates in SERS applications.

Application of silicalite-modified electrode for the development of sucrose biosensor with improved characteristics.

The application of silicalite for improvement of working characteristics of conductometric enzyme biosensors for determination of sucrose was studied in this research. Biosensors based on different types of silicalite-modified electrodes were studied and compared according to their analytical characteristics. Polyethylenimine/glutaraldehyde/silicalite-modified biosensors showed higher sensitivity compared with others type of biosensors. Moreover, the polyethylenimine/glutaraldehyde/silicalite sucrose biosensors were characterized by high selectivity and signal reproducibility (relative standard deviation (RSD) = 2.78% for glucose measurements and RSD = 3.2% for sucrose measurements). Proposed biosensors were used for determination of sucrose in different samples of beverages. The obtained results had good correlation with results obtained by HPLC. Thus, polyethylenimine/glutaraldehyde/silicalite-modified biosensors have shown perspective characteristics for the development of effective conductometric enzyme biosensors.

Ag-nanoparticle-decorated Ge nanocap arrays protruding from porous anodic aluminum oxide as sensitive and reproducible surface-enhanced Raman scattering substrates.

We report on the fabrication of Ag nanoparticle (Ag NP) decorated germanium (Ge) nanocap (Ag-NPs@Ge-nanocap) arrays protruding from highly ordered porous anodic aluminum oxide (AAO) template as highly sensitive and uniform surface-enhanced Raman scattering (SERS) substrates. The hybrid SERS substrates are fabricated via a combinatorial process of AAO template-assisted growth of Ge nanotubes with each tube having a hemispherical nanocap on the AAO pore bottom, wet chemical etching of the remaining aluminum and the AAO barrier layer to expose the Ge nanocaps, and sputtering Ag NPs on the Ge nanocap arrays. Because sufficient SERS "hot spots" are created from the electromagnetic coupling among the Ag NPs on the Ge nanocap and the highly ordered Ge nanocap arrays also have semiconducting chemical supporting enhancement, the hybrid SERS substrates have high SERS sensitivity and good signal reproducibility. Using the hybrid SERS substrates, Rhodamine 6G with a concentration down to 10(-11) M is identified, and one congener of highly toxic polychlorinated biphenyls with a concentration as low as 10(-6) M is also recognized, showing great potential for SERS-based rapid detection of organic pollutants in the environment.

Superhydrophobic Ag nanostructures on polyaniline membranes with strong SERS enhancement.

We demonstrate here a facile fabrication of n-dodecyl mercaptan-modified superhydrophobic Ag nanostructures on polyaniline membranes for molecular detection based on SERS technique, which combines the superhydrophobic condensation effect and the high enhancement factor. It is calculated that the as-fabricated superhydrophobic substrate can exhibit a 21-fold stronger molecular condensation, and thus further amplifies the SERS signal to achieve more sensitive detection. The detection limit of the target molecule, methylene blue (MB), on this superhydrophobic substrate can be 1 order of magnitude higher than that on the hydrophilic substrate. With high reproducibility, the feasibility of using this SERS-active superhydrophobic substrate for quantitative molecular detection is explored. A partial least squares (PLS) model was established for the quantification of MB by SERS, with correlation coefficient R(2) = 95.1% and root-mean-squared error of prediction (RMSEP) = 0.226. We believe this superhydrophobic SERS substrate can be widely used in trace analysis due to its facile fabrication, high signal reproducibility and promising SERS performance.