A Widely Applicable Silver Sol for TLC Detection with Rich and Stable SERS Features
© Zhu et al. 2016
Received: 25 February 2016
Accepted: 15 April 2016
Published: 23 April 2016
Thin-layer chromatography (TLC) coupled with surface-enhanced Raman spectroscopy (SERS) has gained tremendous popularity in the study of various complex systems. However, the detection of hydrophobic analytes is difficult, and the specificity still needs to be improved. In this study, a SERS-active non-aqueous silver sol which could activate the analytes to produce rich and stable spectral features was rapidly synthesized. Then, the optimized silver nanoparticles (AgNPs)-DMF sol was employed for TLC-SERS detection of hydrophobic (and also hydrophilic) analytes. SERS performance of this sol was superior to that of traditional Lee–Meisel AgNPs due to its high specificity, acceptable stability, and wide applicability. The non-aqueous AgNPs would be suitable for the TLC-SERS method, which shows great promise for applications in food safety assurance, environmental monitoring, medical diagnoses, and many other fields.
Recently, with the popularization of portable Raman spectrometers, thin-layer chromatography (TLC) coupled with surface-enhanced Raman spectroscopy (SERS) has frequently been applied to analyze various complex systems. Analytes are isolated and preliminarily purified through the classic TLC separation method, and then, the SERS technology is used for the specific detection of trace substances on the TLC plate. Compared to other commonly used techniques, this coupling method has unique advantages such as low cost, less sample pretreatment, and high throughput of TLC separation, along with high specificity and sensitivity of SERS detection, which is suitable for preliminary screening and rapid on-site detection. Since first reported by Hezel , TLC-SERS has been employed for the separation and detection of various complex substances, including substituted aromatic pollutants in water samples , dyestuffs on works of art [3, 4], biomarkers in biological urine , pesticide residues from crops , and adulterants in botanical dietary supplements (BDS) [7, 8]. Furthermore, it has been applied in chemical synthesis analysis [9, 10] and clinical therapeutic drug monitoring (TDM) . The growing number of studies has demonstrated the validity of coupling TLC with SERS and also implied its good prospect in many research fields such as food, drug, and environment.
However, it could be found that nearly all of the reported analytes in the aforementioned applications are hydrophilic and that relatively universal SERS-active sols (which are widely applicable for both hydrophilic and hydrophobic analytes) are urgently needed for the TLC-SERS method. While the aqueous sols show good enhancement for hydrophilic substances, it is difficult to detect hydrophobic analytes. Oriňák et al.  applied TLC-SERS technique to the analysis of hydrophobic diterpenoic acids, but the SERS spectra of the three biologically active diterpenes (gibberellic acid, abietic acid, and kaurenoic acid) showed weak and unsatisfactory Raman signals. According to the theory of dynamic surface-enhanced Raman spectroscopy (DSERS) [13–16], good SERS signals appear during the SERS substrate transformation from a wet state to a dry state. Hydrophobic analytes can hardly be trapped in aqueous nanoparticles for their incompatibility with water. Thus, enhancing SERS signals of hydrophobic analytes with aqueous sols is very difficult. To make matters worse, these SERS signals are unstable and the enhancement keeps for a short time (usually less than 60 s) due to the fast vaporization of water. Besides, SERS feature obtained by aqueous silver sols is not rich which might be overcome by inducing aggregating agents to generate high-enhancing “hot spots” [2, 4, 5, 10], but the optimization is inconvenient and sometimes inapplicable. In a word, aqueous silver sols in the aforementioned TLC-SERS applications cannot provide rich and stable SERS features.
Herein, a new SERS-active silver sol for TLC detection is reported to provide technical support for the world’s first TLC-SERS coupling instrument funded by the Chinese government and still under research for on-site rapid prescreening of adulterated drugs. After the reaction time optimization and morphology characterization, the optimal silver nanoparticles (AgNPs)-DMF sol was obtained rapidly. The newly developed AgNPs were successfully applied to test several analytes including hydrophilic and hydrophobic substances with richer spectral features (more abundant peaks) and more stable intensity than aqueous AgNP sol, which was beneficial for peak assignment and substance’s discrimination. The non-aqueous AgNPs reported herein would be suitable for the TLC-SERS method, which shows great promise to the analysis of complex systems in food safety assurance, environmental monitoring, medical diagnoses, and many other fields.
Materials and Apparatus
Silver nitrate; chloroauric acid; sodium citrate; polyvinylpyrrolidone (PVPK30); and all organic solvents including N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methanol, ethanol, acetonitrile, and acetone of analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd., China. All reference chemicals including rosiglitazone maleate (ROS), pioglitazone hydrochloride (PIO), gliclazide (GLC), glyburide (GLB), and glipizide (GLP) were bought from National Institute for Food and Drug Control, China. While rhodamine 6G (R6G), gibberellic acid (GA), methyl orange (MO), and Sudan III (S III) were purchased from Sinopharm Chemical Reagent Co., Ltd., China. Distilled water was obtained using a Smart-DUV (18 MΩ cm resistivity) filter (Shanghai Hitech Instruments Co., Ltd., China). TLC plates (Yantai E.S.T. Silicone Tech Co., Ltd., China) consist of high-performance silica gel 60-F254 plates (silica gel particle size 8 ± 2 μm ≥80 %, layer thickness 0.2 ± 0.03 mm) with glass back plates.The plate containing a fluorescing additive, F254, was used for easy spot visualization.
Separated spots were located using an ultraviolet analyzer with 254 nm wavelength (WFH-203B, Shanghai Jing Branch Industrial Co., Ltd., China). Ultraviolet–visible (UV–Vis) absorption spectra of silver colloids were obtained with a double beam UV–Vis spectrophotometer (TU 1901, Beijing Purkinje General Instrument Co., Ltd., China). Scanning electron microscope (SEM) images were taken on a ZEISS EVO MA-10 (Carl-Zeiss, Germany). Raman spectra were recorded by a portable Raman spectrometer (BWS415, B&W Tek Inc., USA) at 785 nm, a resolution of 5 cm−1 and a ×20 long working distance microscope objective.
AgNPs of water solution (AgNPs-H2O) were synthesized by the Lee–Meisel method . Briefly, 45 mg of AgNO3 was dissolved in 250 mL of distilled water and heated to boiling. Five milliliters of a 1 % (w/v) sodium citrate tribasic solution was added to the solution under vigorous magnetic stirring and kept boiling for 1 h. AgNPs of DMF solvent (AgNPs-DMF) were prepared as followed; a mixed solution of silver nitrate (17 mg) and PVP (1:1) was added to 100 mL boiling DMF and heated for 1 min, then the AgNPs were obtained. All synthesized nanoparticles (NPs) were kept at room temperature (RT) protected from light. To obtain NPs dispersed in non-aqueous solutions, the original NPs in water were centrifuged at the speed of 9000 rpm for 10 min. The supernatants were discarded carefully, and the precipitate at the bottom was resuspended in different organic solvents.
Analyte stock solutions were prepared by dissolving reference in optimal solvent. ROS and PIO samples were dissolved in methanol; GLB, GLP, and GLC samples were prepared in mixed solvent of methanol–chloroform (1/1, v/v); and the ultimate concentrations were 1 mg/mL. GA, MO, and S III stock solutions were dissolved in ethanol at a concentration of 0.1 mg/mL. Then, each analyte stock solution was ready for detection by the TLC-SERS method.
Analyte stock solutions (1 μL) were applied to a silica gel TLC plate, let dry, and then eluted with CH2Cl2:CH3OH 8:1 (v/v). After the eluent on the TLC plate evaporated naturally, the separated spots were visualized and marked under an ultraviolet illumination at 254 nm. SERS analyses were directly performed on the plate after local deposition of 4 μL NP solution directly on the marked spot. The SERS spectra for the spots were acquired using a Raman spectrometer with a suitable power (200 mW at colorless samples and 90 mW at pigments) and an integration time of 5 s. A continuously recording mode without interval was also applied to investigate the variation discipline of the silver sol. All the measurements were repeated in triplicate. Data were pretreated with the Savitzky–Golay polynomial fitting (9-point smoothing) and baseline correction, with Matlab 7.0 (MathWorks, Massachusetts, USA) and Origin 8.0 software.
Results and Discussion
AgNPs-DMF Preparation and Optimization
Richer Spectral Features Provided by the AgNPs-DMF Sols
More abundant signals obtained by AgNPs-DMF provided more features for peak assignment and discrimination, which also made up for the interference from DMF signals that might cover some SERS peaks of the analyte, and ultimately resulted in higher specificity compared to commonly used silver sols. The ability of distinguishing two close peaks further illustrated its high specificity, and the presence of DMF signals would have little influence with time-dependent detection mode, which will be in the following section.
More Stable Intensity Provided by the AgNPs-DMF Sols
Wider Applicability of the AgNPs-DMF Sols
In the previous TLC-SERS studies [2–8], aqueous silver sols prepared through the classical Lee–Meisel method were the most widely used sols, and most applications are hydrophilic substances. Actually, a lot of analytes in great testing demand were hydrophobic, but this type of aqueous AgNP sols showed poor SERS performance to them, for example, the antidiabetic drug gliclazide (GLC) (Additional file 1: Figure S6). Poor SERS signals of GLC can be obtained by the original aqueous silver sols (Additional file 1: Figure S6a); an aggregating agent (KNO3) which can induce nanoparticle aggregation to improve ameliorating the result  was also employed, but little improvement (a relative intensity of 3030 at 798 cm−1) of SERS signals was observed even though the proportion of AgNPs and KNO3 had been optimized (Additional file 1: Figure S6b), which indicated that aqueous AgNP sols held poor TLC-SERS enhancement to hydrophobic analytes. To expand the application of the TLC-SERS method, SERS-active sols with high universality to not only hydrophilic but also hydrophobic analytes were needed.
In this study, a non-aqueous silver sol for TLC detection with rich and stable SERS features was successfully developed and employed in the analysis of both hydrophobic and hydrophilic analytes. The SERS-active sols prepared herein presented not only richer spectral features for peak assignment which was beneficial for structural analysis and analogue discrimination but also more stable features which would improve the robustness of the TLC-SERS method. What is more is that the applications could be expanded due to the higher universality of the non-aqueous silver sol. To provide better technical support for the TLC-SERS coupling instrument, additional work is currently ongoing in our lab to further optimize the preparation process of the AgNPs-DMF to improve the repeatability and the limits of detection and also to expand the application of the TLC-SERS method in the analysis of complex systems in drug safety, environmental monitoring, and many other fields.
This research was supported by research funds from the School of Medicine, Shanghai Jiao Tong University (Grant no. 14XJ10067); the foundation of Shanghai Third People’s Hospital, School of Medicine, Shanghai Jiao Tong University (Grant no. syz2013-002); the Ministry of Science and Technology of the People’s Republic of China (Grant no. 2012YQ180132); and Science &Technology Commission of Shanghai Municipality (Grant no. 15142201300).
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