A cost-effective identification of tobacco alkaloids using porous Si SERS substrates for forensic and bioanalytical applications
With use of surface-enhanced Raman scattering (SERS) substrate, we are able to identify alkaloids of tobacco leaves. First, porous Si arrays were developed using metal-assisted chemical etching and followed by gold (Au) thin layer (50 nm) coating. Au decorated porous Si arrays provide a strong localized electric field and responsible for the enhancement of weak Raman signals of target molecules. Detection of model molecule rhodamine B at low concentration (10−9 M) confirmed the SERS activity of the developed substrates. The enhancement factor was calculated to be approximate 106. Further, developed substrates were utilized to identify alkaloid in solution of dry tobacco leaves (1 g and 0.25 g) and water (100 Ml). Raman peak 1030 cm−1 corresponding to standard nicotine was observed. Understanding of tobacco composition and their Raman vibration mode could be beneficial to decide the biomarker of various diseases related to tobacco chewing and smoking. This work highlights the level free detection of tobacco alkaloids and confirms that Raman spectroscopy technique can utilize for quality control, forensic and bioanalytical analyses of tobacco alkaloids at trace level.
KeywordsPorous silicon Metal assited chemcial etching (MACE) SERS Tobacco alkaloids
Tobacco consumption in human civilization is one of the main causes of death and disease around globally as well as in India. Among the various products of tobacco, smokeless consumption of tobacco is rapidly increasing and turning a serious public health concern. According to the Global Adult Tobacco Survey (GATS) India, 2010, 206 million Indians are habitual to smokeless tobacco/chewing . In spite of government initiative, use of smokeless tobacco is on rise. Smokeless tobacco causes oral cancers, and a number of other cardiovascular diseases [2, 3]. Almost 90% oral cancers are linked to tobacco uses. Available evidences suggest that the rate of mouth cancer is growing among the younger generation. The rise in popularity of tobacco products like Khaini has created a requirement of a rapid method for determining the nicotine or alkaloid content in tobacco products in the field, ideally at the point of sale or identification of early tobacco user.
Identification and detection of single molecule have significant importance in the field of medicine, chemistry, biology and environmental science. For molecular study, Raman spectroscopy is a powerful tool; however, it is limited to dilute solutions of molecule due to a weak Raman scattering signal. Surface-enhanced Raman spectroscopy (SERS) is another variant of Raman spectroscopy in which incoming laser beam interacts with a target molecule in the vicinity of metallic nanostructures. The coupling between the electromagnetic field of light and plasmon resonance of surface metallic nanostructures exhibits significant enhancement in the local field. This local field leads dramatic increment in weak Raman scattering signal . Meanwhile, charge transfer phenomenon among the metal surface and target molecule also contributes in the enhancement of Raman signal [4, 5]. In fact, electromagnetic mechanism plays its vital role in Raman enhancement due to localized surface plasmons.
Since the first introduction of SERS in 1974, it has gained considerable attention of scientific community due to its several advantages and capabilities to sensitive analysis at trace level. The simplest way to fabricate a SERS substrate is to immobilize gold/silver nanoparticles upon a glass substrate. Notwithstanding, distribution of nanoparticles over the planner substrate limits the hotspot density for optimum enhancement.
Many research groups have been demonstrated various SERS substrates based on various substrate materials, i.e., Si nanowires [4, 5], TiO2 nanorods arrays , ZnO nanorods arrays  and Ag nanorods arrays [8, 9]. Among all these materials, Si nanostructures/metal geometry shows unique advantage as high reflective index of Si nanoscale concentrate light scattering and gives near field electromagnetic enhancement . SERS intensity enhancement depends on size, shape and dielectric constant of the materials. Therefore, SERS platforms with considerable enhancement have been demonstrated through a number of methods, including laser interference lithography , deep UV lithography , galvanic displacement reaction [12, 13], e-beam lithography [10, 11] and evaporation (or magnetron sputtering) [14, 15]. However, these methods are time consuming, complicated and not applicable to large scale production [16, 17]. Moreover, uniformity and reproducibility of SERS devices are highly critical for the effective use of SERS in commercial applications. A comparative alternative simple, facile and cost-effective method is need of the hour.
Herein, we utilized metal-assisted chemical etching (MACE) for the development of porous Si. MACE is a strong candidate for realization of porous Si at low cost, simple equipment and low temperature with high throughput. Au thin film was deliberately deposited on porous Si to produce strong localized surface plasmon resonance to boost Raman intensity of analytes. Furthermore, the detection of rhodamine (RhB) at low concentration (10−9 M) was demonstrated and confirmed the SERS activity of the developed SERS substrates and followed by identification of Alkaloids in tobacco leaves.
2 Experimental detail
2.1 Development of porous Si
Porous silicon was developed using electroless metal deposition methods based on galvanic cell process. Prior to etching, p-type Si boron doped (resistivity 1–10 Ω-cm) was thoroughly cleaned in tri-chloro ethylene (TCE), acetone and isopropanol to remove impurities or dust particles and followed by HF dipped to remove any native oxide. Cleaned Si substrates were dipped in solution containing HF and AgNO3 for one minute at room temperature to deposit Ag nanoparticles (Ag NPs). Further, Ag NPs decorated samples were kept for etching in an etching solution containing HF and H2O2. Etched substrates were transferred in HNO3 acid to dissolve Ag particles present on the etched sample. Finally, etched samples were rinsed with de-ionized water and dried in nitrogen purging.
2.2 SERS active platform preparation
In order to meke SERS active platform, developed porous Si substrates were decorated with gold nanoparticles thickness (≈ 50 nm) by RF-sputtering system. Since the surface of etched substrates was too rough, islands of gold were formed instead of thin film, it was confirmed by continuity test using multimeter. Gold coated SERS substrates offer an inherent advantage of stability over silver coated surfaces as silver makes oxide in atmosphere.
2.3 Samples preparation
In order to examine the SERS functionality of Au coated porous Si and tobacco alkaloids detection, first of all developed substrates were tested using model molecules of rhodamine B (RhB) and calculated the enhancement factor of the platforms. The Au coated samples were submerged in rhodamine solutions of various concentration (10−9 M, 10−6 M and 10−3 M) for 5 min and rinsed thoroughly. Now the samples were ready to capture the Raman spectra of rhodamine on SERS active platforms. For tobacco alkaloids examination, powder of natural tobacco leaves was arranged from the village where people use to smoke daily. Different masses of tobacco leaves in (0.25 g, and 1 g) were mixed with 100 Ml volume of DI water and prepared uniform solution for 10 minutes. Final solutions were cleaned using filter and utilized to examine tobacco alkaloids using developed SERS substrates as previously discussed.
3 Characterization method
A scanning electron microscope was used to observe the surface and morphology of porous Si and gold coated SERS platforms. Raman measurements were carried out on a Renishaw Raman spectroscopy system at room temperature, using 785 nm lasers as an excitation power 30 mW. The exposure time was placed 10 s for all samples. The spectroscopic spectra were recorded in the range of 550–1800 cm−1.
4 Results and discussion
4.1 Surface morphology of porous Si
Figure 2 also reveals variation in intensity counts of RhB Raman signal. Intensity counts increase as the concentration of molecule increases. The highest intensity count was observed to be 12,000 counts for Raman band 1350 cm−1 of 10−3 M RhB samples. At low concentration, the intensity counts drastically decreased around the five fold as the concentration change 10−6 M from 10−3 M and same order was followed for further decrement in concentration (10−9 M) of RhB. The lowest intensity count was 200 for 750 cm−1 peak at 10−9 M RhB. Though, background noise affects Raman signal at low value of target molecules, Raman intensity peaks of RhB (10−9 M) were able to recognize. Here, it is mentioned to worth that the intensity peaks 620, 750 and 1646 cm−1 at low concentration (10−9 M) were quite matched with the prominent peaks of rhodamine at high concentration and confirmed developed SERS platform is capable to detect molecule at trace level. These results confirmed that developed SERS substrates were uniform and reproducible.
Further, developed substrates were utilized for the detection of alkaloids in tobacco leaves. For this, the prepared solution of tobacco leaves in different quantities (1 and 0.25 g) with DI water was dispersed over the SERS active platforms and let the samples dry in atmospheric conditions. The dry samples were scanned to capture Raman spectra of tobacco alkaloids. The recording conditions to capture Raman spectra for the tobacco analysis were kept same as for the RhB in previous experiments.
In summary, ordered and uniform porous Si was developed by a cost-effective MACE process at room temperature and utilized for SERS application with enhancement factor approximate 106. In porous structures, abundant hot spots were formed and electromagnetic coupling occured due to closely separated nano-gap and responsible for the enhancement. The substrate was effectively demonstrated the detection of rhodamine molecule at low concentration (10−9 M) as well as tobacco alkaloids in the solution of tobacco (1 g and 0.25 g) and water (100 mL). The Raman spectra of tobacco alkaloids were confirmed with Raman spectrum of standard nicotine molecule. The developed substrates showed uniform intensity. This fabrication process of SERS platform is very cheap and viable option for large scale production of SERS substrates. Therefore, the platforms are applicable to determine nicotine content at wide scale at the point of sell tobacco product and early identification of tobacco user. Further, developed SERS platforms can be utilized for forensic and bioanalytical analysis as per the requirment.
Authors thank the Process Technology group of CSIR-CEERI Pilani for providing facilities for the fabrication of devices. A. Kumar is grateful to Director, CSIR-CEERI support to carry out this research work. A. Kumar also acknowledges Mr. Prem Kumar, CSIR-CEERI for SEM characterization facility. A. Kumar is thankful to Dr. Kulwant Singh, Associate professor, Department of ECE, Manipal University, Jaipur, for his valuable discussion and suggestion. Authors would also like to thanks CSIR, India for their support through the funding of health mission project HCP0012.
Compliance with ethical standards
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
Statement of human and animal rights
Research does not involve human participants and/or animals.
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