FIELD OF THE INVENTION

The present invention relates to a highly sensitive plasmonic substrate for trace level detection of a wide variety of chemicals and biomolecules. In particular, the invention relates to the molecular spectroscopic technique for detection of chemical and biomolecular analytes at trace levels. More particularly, the invention relates to the trace level detection of materials through molecular fingerprinting through Raman spectroscopy. The invention also relates to the fabrication of specialized substrates for enhancing the Raman scattering efficiency of materials. More accurately, the present field of the invention relates to the development of Surface-Enhanced Raman Spectroscopic Substrates for several-fold enhancement of the sensitivity of molecular fingerprinting through Raman Scattering.

BACKGROUND AND PRIOR ART OF THE INVENTION

C.V. Raman, in his seminal paper, described the phenomenon of inelastic scattering of light which has now emerged as a major material characterization tool. Compared to other spectroscopic techniques, its practical application in trace level detection has been impeded by the low scattering efficiency limiting its usage to bulk material identification and for those components in mixtures having concentrations relatively larger than 5%. The accidental discovery of Surface-Enhanced Raman Scattering (SERS), by Fleischmann and co-workers in 1974 and independently by Jeanmaire and Van Duyne, and the subsequent developments in this area has revolutionized the field improving the detection limit down to ppm/attomolar level. For example, as early as in 1977 Albrecht and Creighton, based on the isotherm of Barradas and Conway, theoretically suggested enhancement factors (EFs) of 10 ⁵ -10 ⁶ . Subsequently, the connection of SERS intensities to enhanced fields arising from localized surface plasmons in nanostructured metals was noted by Moskovits. The primarily identified mechanisms for the inelastic scattering enhancement include (i) chemical and (ii) electromagnetic routes. The first mainly involve charge transfer interaction of the analyte with the substrate. While in the second case, enhancement is caused by the generation of a localized electric field by the excitation of surface plasmons. The expected enhancement factor for the former is in the range of 10 ⁴ -10 ⁵ and for the latter is 10 ⁶ -10 ⁷ . In some instances, there can be contributions from both and enhancement factor as high as 10 ⁸ has been observed.

Compared to any other, nanomaterial of coinage metals show better performance which varies in the order Ag>Au>Cu. Also, nanoparticles (NPs) whose size is in the range ~20 - 200 nm produce higher enhancement than those with bigger or smaller sizes. It has been established that NPs in the colloidal state are good enough to produce Raman signal enhancement enabling even single molecular level detection. However, for colloidal NPs dispersed in a liquid medium, the possibility for aggregation which is a dynamic process cannot be ignored. Thus, the SERS spectrum must be recorded within a limited time window, and the reproducibility of the data strongly depends on the precise experimental conditions used. As a result, solid-state substrates are always preferable for practical applications. Some of the reported strategies enable the fabrication of substrates that are not just restricted to flat supports but can also be applied to flexible, curved, or rough supporting materials, thereby leading to many sensing applications. In such cases, it is necessary to reliably engineer hotspot’s size, geometry, and density over large areas. The ideal requirements for SERS substrates are (i) ease and reproducibility of production, (ii) high enhancement activity, (iii) uniformity of performance (iv) stability and (iv) scalability to mass production.

Presently available SERS substrate preparation methodologies can be broadly classified into two. The first one involves the preparation of colloidal nanoparticles and their deposition to selected substrate adopting different strategies. In this case, suspensions of metal nanoparticles are prepared first by either chemical or physical methods. Examples include the preparation of pure colloidal suspensions of gold and silver NPs using pulsed laser ablation method, or the reduction of metal ions in a solution through wet chemical approach, usually medium is aqueous, reducing agents are citrate, sodium borohydride, hydrazine, or hydroxylamine hydrochloride. The NPs thus obtained are then immobilized on solid substrates (e.g. quartz/glass) via the use of chemical linkers such as (3 -mercaptopropyl) trimethoxy silane (MPTMS) or (3-aminopropyl) trimethoxy silane (APTMS). Alternatively, NPs are trapped in paper/alumina filters, via filtration or deposited via dipping or inkjet printing or spray coating. For example, a two-phase colloidal method is used to synthesize octadecanethiolate-passivated silver NPs, and a monolayer of it is formed on substrates such as quartz, indium tin oxide, silicon, polymer by a dip-coating method. (Gwo Shang-JR, Chen Hung-Ying, Lin Meng-Hsein- U.S. Pat. No. 2017/0261434). The EP pat. No.2002/1468054, illustrates the preparation of SERS substrates through inkjet printing wherein the ink contains metal NPs of Ag, Au, Cu or their combinations and the polymeric additives dispersed within water (EP Pat. No. 2002/1468054-Magdassi Shlomo, Kamyshny Alexander, Vinetsky Yelena, Bassa Amal, Abo Mokh Riam). In another publication, fabrication of a disposable SERS substrate by successively spraying of silver and gold NPs onto commercial filter paper and its use in detection of malachite green, methylene blue, and crystal violet in fish, was reported. (Guohai Yang, Xuejiao Fang, Qin Jia, Haixin Gu, Yunpeng Li, Caiqin Han, Lu-Lu Qu, Microchimica Acta, 187:310, 2020)

Second approach involves the preparation of nanostructured surfaces by adopting either chemical or physical routes, including the application of picosecond (ps) or femtosecond (fs) laser pulses and subsequent deposition of a thin layer of SERS active metal. For example, U.S. Pat. No. 2011/7864312, illustrates the generation of micron-sized and submicron-sized, structures on semiconductor (e.g., silicon) surfaces by exposing to short laser pulses such as femtosecond. The micron and submicron-sized structured surfaces thus obtained were then coated with a discontinuous metal layer through the thermal evaporation process. (Eric Mazur, Diebold). Nanosphere lithography (NSL) is yet another strategy, which produces well-ordered 2D periodic arrays of spherical NPs (e.g. latex or silica). The publication entitled ‘Nanosphere Lithography: Size-Tunable Silver Nanoparticles and Surface Cluster Arrays’, focuses on the synthesis of size-tunable silver NP arrays by nanosphere lithography and their structural characterization by atomic force microscopy (AFM), (Hulteen, J.C.; Treichel, D.A.; Smith, M.T.; Duval, M L.; Jensen, T.R.; Van Duyne, R.P. Nanosphere Lithography: Size-Tunable Silver Nanoparticles and Surface Cluster Arrays. J. Phys. Chem. 1999, 103, 3854-3863). Electron-beam lithography technique is another good approach to create Nano patterns with high accuracy and precision. For example, the publication explores new strategies to obtain plasmonic metal nanostructures through the combination of a top- down method, that is electron beam lithography, and a bottom-up technique, that is the chemical electroless deposition. This technique allows tight control over the shape and size of two- and three-dimensional metal patterns at the nanoscale (Maria Laura Carlucci, Francesco Gentile, Marco Francardi, Gerardo Perozziello, Natalia Malara, Patrizio Candeloro and Enzo Di Fabrizio, Electroless Deposition and Nanolithography Can Control the Formation of Materials at the Nano-Scale for Plasmonic Applications, Sensors, 14, 6056-6083, 2014). The nanostructured surface can also be obtained through the chemical etching of the desired surfaces using strong chemicals such as HF, HC1, HNOs etc. The invention disclosed in U.S. Pat. No. 2004/0135997 (Selena Chan, San Jose) describes the preparation of porous silicon substrates by anodic etching with dilute hydrofluoric acid. Subsequently, a thin layer of a Raman active metal, gold or silver, is coated by any known techniques (e.g. cathodic electromigration, thermal evaporation, chemical vapour deposition (CVD), or electrodeposition). The publication entitled ‘Fabrication of Ordered Tubular Porous Silicon Structures by Colloidal Lithography and Metal Assisted Chemical Etching SERS performance of 2D Porous Silicon Structures’ explains the fabrication of well-ordered porous silicon tubular structures using colloidal lithography and metal-assisted chemical etching. The chemical etching process carried out using HF, ethanol and H2O2 dissolved the silicon in contact with the metal nanoparticles, creating a porous tubular array (R. F. Balderas-Valadez, J. O. Estevez-Espinoza, U. Salazar-Kuri, C. Pacholski, W. L. Mochan and V. Agarwal, Applied Surface Science, SO 169, 2018). Alternatively, K. Sieradzki, R. C. Newman reported the fabrication of plasmonic substrate with micro and nanoporous structures from an alloy of one or more noble metals (Au-Ag, Cu-Ag, Au-Cu) through an electrochemical process (U.S. Pat. No. 2011/7864312). In most of these cases, the nanostructures obtained are converted to SERS substrates through the subsequent coating of plasmonic metals using atomic layer deposition, thermal evaporation, sputtering, chemical vapour deposition, electrochemical deposition etc.

Thus, most of the techniques for the fabrication of SERS substrate on solid surface demand high cost and sophisticated setup. For example, laser ablation techniques exploit high energy lasers, which are quite expensive and difficult to maintain. Additionally, most diffused laser sources are not suitable for producing nanostructures on an industrial scale, and also the ablation efficiency decreases with long ablation time because of the significant number of NPs placed along the laser beam. In the case of nanosphere lithography or electron beam lithography techniques are associated with sophisticated arrangements which are not only time-consuming and but also the number of metal particles i.e., the number density, that can be deposited on a planar surface is limited. Roughening of surfaces through chemical etching requires toxic and corrosive chemicals. These are harmful to both the environment as well as human beings. Though SERS substrates can be prepared on plain substrates through the deposition of plasmonic metal using any known techniques such as thermal evaporation, sputtering, the resulting substrates proclaim relatively lower limit of detection (LOD) of analytes. For example, bilayer structure of Ag-Au Nano-islands arrays on the bare glass surface by thermal evaporation deposition exhibits LOD ~ 10' ⁸ M with rhodamine 6G (Wei- Lin Syu, Yu-Hsuan Lin, Abhyuday Paliwal, Kuan-Syun Wang, Ting-Yu Liu., Highly Sensitive and Reproducible SERS Substrates of Bilayer Au and Ag Nano-Island Arrays by Thermal Evaporation Deposition, Surface & Coatings Technology 2018).

Thus, there is a prime demand for a good SERS substrate without using colloidal nanoparticles and complex fabrication techniques, which can be addressed by the method proposed in the present invention. The facile fabrication of high enhancement and reproducible plasmonic substrates by making roughened and sharpened surfaces using abrasive blasting on glass/aluminium sheet/ silicon wafer and subsequent deposition of Raman enhancing materials such as but not limited to Ag, Au, Cu or their combinations using industrially established methods such as thermal evaporation, electroless deposition, etc.

OBJECTIVES OF THE INVENTION

The main objective of the present invention is to establish facile fabrication procedures for robust and scalable nanostructured substrates.

Another objective of the present invention is the application of Raman enhancing materials on these substrates by different techniques to fabricate SERS substrates (sensors) with a high Raman enhancement factor.

Yet, another objective of the present invention is to establish the utility of the fabricated substrates for the trace level detection of chemicals, biomolecules, pesticides etc.

SUMMARY OF THE INVENTION

In view of the foregoing, the present invention relates to the facile and scalable fabrication of novel SERS substrates with a high enhancement factor and sensitivity. In an embodiment of the present invention, the fabrication method combines the processes of nanostructuring through abrasive blasting and making it SERS active through deposition of Raman enhancing materials. Thus, the present invention encompasses ease of production, industrial compatibility facilitating mass-scale production, better reproducibility and Raman signal enhancement. For the successful implementation of SERS substrates in trace level chemical analysis, it should exhibit higher enhancement factor, which in turn depends on the geometry of the underlying nanostructure as well as the density of hotspots. Abrasive blasting generally causes scaling of the surfaces yielding highly roughened surface with many sharpened features having nanoscales dimensions and large surface density. Additionally, the roughened surfaces produced through abrasive blasting will have increased surface area, thereby promoting effective adsorption of more analyte to the surface, which also contributes to the enhancement of sensitivity.

In another embodiment of the present invention, a plasmonic substrate sensor with plasmon resonance in the VIS-NIR region (350 - 800 nm) and with > 10 ⁶ enhancement factor and uniformity (< 2.8 % RSD), for the trace level molecular fingerprinting of a wide variety of chemicals and biochemical, wherein the sensor comprises of a substrate having an area of dense nanoscale wedges causing large enhancement in Raman scattering efficiency of the adsorbed analytes.

In yet another embodiment of the present invention, the substrate of the plasmonic sensor is an insulating, semiconducting or conducting material such as glass, silicon or thin sheet of metal like aluminium.

In yet another embodiment of the present invention, a process for the fabrication of plasmonic substrate sensor comprising of

(i) preparation of an area having nanoscale wedges on the selected substrate via careful surface modification achieved via the process of abrasive blasting with chosen abrasives like Aluminium oxide, glass beads, steel grits etc. having the size in the range of 50-250 microns;

(ii) conversion of the nanostructured substrates to plasmonic substrates via coating with a nanolayer of selected coinage materials such as Au, Ag, or Cu or their combinations via any known technique such as thermal evaporation, electroless deposition.

In yet another embodiment of the present invention, the thickness of the nanolayer is in the range of 20-200 nm.

BRIEF DESCRIPTION OF THE DRAWING

The invention has other advantages and features which will be more readily apparent from the following detailed description of the invention and the appended claims, when taken in conjunction with the accompanying drawings, in which:

Fig.l. Schematic diagram showing the SERS substrate fabrication processes.

Fig.2. Scanning electron microscopic (SEM) images illustrating the surface morphology of the abrasively blasted glass substrate. Fig.3. Representative examples of SERS spectra of 10 mM MBA solution recorded using Raman Enhancing substrate prepared via abrasive blasting of a microscopic glass slide and silicon wafer with 50-micron Aluminium oxide grits and coated with 60 nm thick Ag via thermal evaporation and electroless deposition, respectively.

Fig.4. Plot showing the variation of SERS intensity @ 1085 cm' ¹ for 10 mM MBA recorded from different locations of a single plasmonic substrate prepared via abrasive blasting of microscopic glass slide with 50 micron Aluminium oxide grits and coated with 60 nm thick Ag via thermal evaporation.

Fig.5. Plot showing the variation of SERS intensity @ 1085 cm' ¹ for 10 mM MBA recorded from seven different batches of plasmonic substrates prepared via abrasive blasting of microscopic glass slides with 50-micron Aluminium oxide grits and coated with 60 nm thick Ag via thermal evaporation.

Fig.6. (bottom to top) SERS spectra of solutions of 1 pM MPBA, 1 pM MBA, 1 nM rhodamine, 1 nM crystal violet, 1 nM glucose, 1 pM creatinine, 1 pM urea, 1 pM melamine, 1 nM thiram and 1 pM carbofuran recorded from plasmonic substrate prepared via abrasive blasting of microscopic glass slide with 50 micron Aluminium oxide grits and coated with 60 nm thick Ag via thermal evaporation.

DETAILED DESCRIPTION OF THE INVENTION

While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on." Referring to the drawings, like numbers indicate like parts throughout the views. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein. In line with the above objectives, present invention discloses a highly sensitive plasmonic substrate for the trace level detection of a wide variety of chemicals, biomolecules and a facile fabrication methodology thereof. The identification of trace-level components in the sample of concern is very crucial in chemical and biomolecular analysis. Detection of chemicals such as pesticides, explosives, toxic elements, and adulterants at very low concentrations is important to assure the safety of food as well as the environment. Also, for many of the diseases, the biomarkers may be present in extremely low concentrations and their early, and precise detection helps disease diagnosis, provide apt therapeutics in time, and hence saving many lives. Most of the conventionally used spectroscopic and chromatographic methods for the above-stated purposes are associated with complex instrumental setup, time-consuming experiments, low sensitivity, need for skilled persons, and also the elaborate procedures for sample preparation. In the current scenario, SERS provides an excellent remedy for the problems by its high sensitivity along with the advantages of Raman Spectroscopy, i.e. molecular fingerprinting. The other added benefits include non-destructiveness, no or minimum sample pretreatment/preparation requirements, fast response, ease of acquisition etc. Unlike most of the other spectroscopic techniques (e.g. fluorescence, absorbance, reflectance etc.), which exhibits broad absorption/emission bands, the spectral peaks obtained in SERS are narrow and show multiple characteristic peaks. This molecular fingerprinting capability of SERS spectra facilitates simultaneous multicomponent detection. For practical applications, a suitable SERS substrate must have highly roughened surfaces exhibiting higher sensitivity as well as easy fabrication procedures that are reproducible and scalable.

The present invention will now be further explained in the following examples. However, the present invention should not be construed as limited thereby. One of the ordinary skills in the art will understand how to vary the exemplified preparations to obtain the desired results.

EXAMPLES

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

1. Preparation of SERS substrates

The preparation of Raman signal enhancing plasmonic substrates involves at least two consecutive steps, (i) Preparation of nanostructured surfaces and (ii) subsequent conversion of this nanostructured surfaces into SERS active plasmonic substrates via depositing a nanolayer of selected metals (e.g. Gold/Silver/Copper) or their combinations. The process is schematically represented in figure 1, and the details are described below. (i) Preparation of the Nanostructured substrate

The examples of the base substrate chosen for SERS substrate preparation are selected form the group consisting of glass, silicon or aluminium. The nanostructuring of the selected substrates was typically done through the abrasive blasting technique. The blasting media chosen for the process is of the size ~50 - 250 microns and materials are aluminium oxide, glass, silica, steel grits etc. The blasting medium was applied to the surface through a nozzle of diameter up to ~1.6 mm and the pressure is adjustable with a gauge and a regulator. The distance between the blasting nozzle and substrate is typically maintained at - 5 cm. However, this distance is adjustable, which would leave blasted areas of different diameters. A typical Scanning Electron Microscope (SEM) analysis of the substrate surface obtained through the above process is represented in figure 2. These microscopic studies showed a clear distinction between the original and abrasive blasted surface. The unprocessed glass area is smooth and flat, while the area blasted with abrasives shows highly dense protrusions. The higher magnification images revealed the formation of many sharp and wedged features with nanoscale dimensions in the blasted area.

(ii) Conversion of abrasively blasted nanostructured substrates into SERS active plasmonic substrates.

For converting the nanostructured substrates into SERS active plasmonic substrates, a thin layer of coinage metals (e.g. Ag, Au, Cu) or their combinations are deposited onto them via any known deposition techniques. For best performance, the thickness of such metal layers is to be such that it supports plasmon activity, and typically absorption bands appear in the UV- VIS-NIR range.

2. Metal deposition

(i) Thermal evaporation: Any debris present on the blasted area was thoroughly cleaned by pumping pressurized air, and then transferred to a thermal evaporation unit and a thin layer (20 - 200 nm) of desired metal (Ag/Au/Cu) was coated. During coating, the evaporation rate was typically maintained at - 0.5-1 A/s.

(ii) Electroless deposition: For electroless deposition, the blasted substrates (e.g. silicon wafer) were dipped in 10 mb of an aqueous solution containing an appropriate concentration of silver nitrate (8 mM) and hydrofluoric acid (90 mM) for 3 min yielding AgNP loaded SERS active substrates. Before taken for SERS analysis, these are cleaned using Millipore water and dried.

The SERS activity of these plasmonic substrates was evaluated after drop-casting with 10 mM MBA solution as the Raman reporter molecule. Represented in Figure 3 are examples of SERS spectra recorded using Metrohm Miracal DS spectrometer with 785 nm excitation source (laser power = 21 mW, integration time = 8 s, No. of averages = 1). The major observed peaks are 1592 cm' ¹ , 1410 cm' ¹ , 1184 cm' ¹ , 1148 cm' ¹ , and 1084 cm' ¹ corresponds to aromatic ring breathing mode, COOH stretching, C-H deformation, and aromatic ring vibration/C-S stretching, respectively. Of these highest intensities are noted for 1592 cm' ¹ (Aromatic ring breathing mode) peak. The noted intensities for the peak maximum were ~5.6 x 10 ⁴ and ~3 x 10 ⁴ counts, respectively from the substrate prepared via thermal evaporation and electroless deposition techniques (figure 3).

Calculation of Enhancement Factor

The enhancement factor (EF) for the fabricated substrate was calculated using the equation. where iNormai and ISERS are the intensities for an identified peak obtained through conventional Raman and SERS respectively. NNormai is the number of molecules probed for a normal Raman setting, and NSERS is the number of molecules probed in SERS. For the determination of EF of the substrates fabricated herein, intensity at 1084 cm' ¹ (aromatic ring vibration and C-S stretching) obtained through conventional Raman spectrum of solid mercaptobenzoic acid (MBA) and SERS spectrum of 1 nM mercaptobenzoic acid (MBA)are considered.

For solid samples NNormai was determined by using the following equation:

NNormai = 7ir ² h X pMBA X NA / MMBA (2) where nr ² h is the optical excitation volume; PMBA is the density of MBA (1.50 g/cm ³ ) in the bulk; NA is the Avogadro number, and MMBA is the molecular weight of MBA molecule. The optical excitation volume is estimated as the product of the laser spot (nr ² , r = 0.61 x 1 / N.A.) size and the depth of focus h = 2k/(N.A.) ² . In the present instrumental set up, I = 785 nm, the wavelength of excitation laser source, and N.A.= 0.5 is the numerical aperture of the collection optics. For estimating NSERS, the following assumptions are taken into consideration (i) the reporter molecule, MBA, is present as a monolayer over the plasmonic substrate, (ii) the overall surface area is the product of laser spot area and the ratio of surface area to projected surface area arrived from AFM analysis, (iii) the topological polar surface area of MBA molecule is 38.3 A ² . The substrate exhibits an enhancement factor of ~ 10 ⁶ .

2. Uniformity Studies

To assess the uniformity in SERS activity of the substrates fabricated herein, they were initially dipped in a homogenous solution of 10 mM mercaptobenzoic acid (MBA) for 5 minutes. This process ensures complete surface coverage of the molecules through chemisorption via metal-thiol linkage. The SERS spectral data were collected from different locations of the same substrate by manually positioning them at the focal point of the probe optics. All the spectra were collected with the same laser power, integration time and the number of averages, thus maintaining identical conditions throughout the experiment. The similarity of the obtained data, in terms of spectral profde, peak position and intensities, illustrates the uniformity in SERS performance. Represented in Figure 4 is an example of the graph showing the noted variation in peak intensities at 1084 cm' ¹ (C-S stretching), showing <2.8 % RSD (relative standard deviation) for the highest intense peak.

3. Reproducibility of the process

The reproducibility of the developed process methodology was evaluated by fabricating multiple numbers of SERS substrates through the method outlined here, under identical conditions and comparing their SERS performance. As an example, around seven substrates were prepared by blasting glass slides with 50-micron aluminium oxide grits and coating with 60 nm thick silver nanolayer through thermal evaporation at a rate of~0.5 A/s. To compare the performance, all the seven substrates were dipped in a homogeneous solution of 10 mM mercapto benzoic acid (MBA) for 5 minutes, and the data were acquired using Metrohm Miracal DS spectrometer with 785 nm excitation (laser power = 21 mW, integration time = 8 s and number of acquisitions = 1). The primary analysis of the data thus obtained illustrated little variations in spectral profiles. The data analyzed for the peak intensities at 1080 cm' ¹ yielded <3.9 % RSD (relative standard deviation), illustrating batch to batch reproducibility of the process (fig.5). Examples of Chemical and Biomolecular Sensing

To illustrate the potential of the substrates in chemical and biomolecular sensing, the following category of chemicals are chosen. These include chemicals that are normally used as Raman reporter molecules (e.g. mercaptobenzoic acid (MBA), mercaptophenylboronic acid (MPBA)), important biomarker molecules (e.g. creatinine, glucose), common food adulterants (e.g. urea, melamine), organic dyes (e.g. rhodamine 6G, crystal violet), pesticides (thiram and carbofuran) etc. Concentration ranging from milli to picomolar solutions were tested on the novel SERS substrates fabricated herein by drop-casting as well as dipping methods.

Case study

(i): SERS spectrum of common Raman reporters a) Mercaptophenylboronic acid (MPBA)

Mercaptophenylboronic acid (MPBA) is a commonly used Raman reporter molecule. The SERS spectrum recorded using the plasmonic substrates fabricated herein showed the peaks at 1592 cm’ ¹ , 1491 cm’ ¹ , 1383 cm’ ¹ , 1289 cm’ ¹ , 1197 cm’ ¹ , 1073 cm’ ¹ , 1024 cm’ ¹ , 908 cm’ ¹ , 830 cm' ¹ , 760 cm' ¹ , 696 cm' ¹ , 625 cm' ¹ , and 621 cm' Corresponding C-C stretching, C-C stretching, B-0 stretching, C-H and B-OH bending, C-H and B-OH bending, C-C-C bending /C-S stretching, C-H bending, C-S-H bending, C-H rocking, C-H rocking, C-C-C bending/C- S stretching, C-S stretching, and C-C-C bending vibrations respectively. Out of these, the peaks appearing at 1073 cm''and 1592 cm' ¹ exhibited the highest intensity and ~10 ³ counts are obtained even at picomolar (0.154 pg in 1 mL) concentrations (fig.6) illustrating the high sensitivity of the substrates. b) Mercapto benzoic acid (MBA)

Mercaptobenzoic acid (MBA) is another example of a frequently used Raman reporter molecule where the adsorption is facilitated through the metal -thiol linkage. The SERS spectrum recorded from plasmonic substrates displayed major peaks at 2570 cm' ¹ , 1586 cm' ¹ , 1410 cm' ¹ , 1184 cm' ¹ , 1148 cm' ¹ , and 1084 cm'l and corresponds to S-H stretching, aromatic ring breathing mode, COOH stretching, C-H deformation, C-H deformation, and aromatic ring vibration/C-S stretching respectively. For picomolar (0.15 pg in 1 mL) concentrations, the obtained intensities for major peaks at 1586 cm ^-1 and 1084 cm' ¹ are in the range 10 ³ counts (fig.6) which illustrate the high sensitivity of the substrates. Case study (ii): SERS spectrum of common Biomarkers a) Creatinine

Creatinine is the primary biomarker used to assess the functioning of kidneys and renal clearance tests. The normal creatinine level in a healthy human serum and urine are in the ranges of 40-150 pM and 2.4-27.0 mM, respectively. With the current fabricated plasmonic substrates, the SERS spectrum of the creatinine displayed peaks at 1760 cm' ¹ , 1590 cm' ¹ , 1470 cm' ¹ , 1370 cm' ¹ , 1180 cm' ¹ , 806 cm' ¹ and, 687 cm' ¹ corresponding to the C=O stretching, C=N Stretching, C-N asymmetric stretching, C-H bending, C-H twisting, NH2 wagging and aromatic ring vibrations respectively. For a pico molar solution, the detected count for the 1370 cm' ¹ peak (C-H bending) was ~2.5 x 10 ³ at (fig.6) indicating that the fabricated SERS substrate is sensitive enough to detect creatinine down to 0. 1 pg/mL. b) Glucose

Glucose is the biomarker used for assessment of diabetes. For a healthy human being, the normal blood sugar level are in the range of 80-100 mg/dL. The SERS spectrum of the glucose displayed Raman peaks at 820-950 cm' ¹ (C-C and C-0 stretching), 1000-1180 cm' ¹ (C-O-H stretching), and 1200-1500 cm' ¹ (CH2 vibrations and C-O-H bending). For a nano molar solution, the intensity of the 1360 cm' ¹ (CH2 vibrations) peak is ~ 1.2 x 10 ³ counts indicating sensitive sensing of glucose down to 0.18 ng/mL.

Case study (iii): SERS spectrum of common adulterants a) Urea

Urea constitutes the major portion of non-protein nitrogen in milk. According to FSSAI act 2006 and PFA rules 1955, the maximum allowable limit for urea in milk is 70 mg/100 mb. However, these are intentionally added to milk for faking the nitrogen content and is illegal. The SERS spectra of the urea displayed the Raman peaks at 1664 cm' ¹ , 1647 cm' ¹ , 1612 cm' ¹ , 1461 cm' ¹ and 1010 cm' ¹ and corresponds to C=O stretching, NH2 deformation, NH2 deformation, symmetrical CN stretching, and antisymmetric CN stretching, respectively. Among these, the highly enhanced peaks are 1612 cm' ¹ (NH2 deformation) and 1010 cm' ¹ (Antisymmetric CN). For picomolar (0.06 pg/mL) concentration the obtained intensities for these peaks ~ 1.5 x 10 ³ and ~1.5 x 10 ² counts (fig.6). b) Melamine

The maximum acceptable limit for melamine in imported foods and infant formula are respectively 2.5 mg/kg, and 1 mg/kg. The high dose intake of melamine usually causes renal failure and even infant death in extreme cases. Presently, the presence of melamine is detected as an adulterant in milk powder and many others like wheat gluten, chicken feed, and processed foods. The SERS spectrum recorded using currently fabricated plasmonic substrate displayed the Raman peaks at 1702 cm' ¹ , 1666 cm' ¹ , 1626 cm' ¹ , 1541 cm' ¹ , 1488 cm' ¹ , 1190 cm' ¹ , 1070 cm' ¹ , 1000 cm' ¹ , 705 cm' ¹ , 608 cm' ¹ , 528 cm' ¹ , 408 cm' ¹ , and 345 cm' ¹ and corresponds N-C-N bending, NH2 asymmetry bending, Ring C-N stretching, N-C-N bending, NH2 bending, Ring deformation, Ring breathing, C-N-C bending, Ring breathing, C-N-C bending, NH2 twisting, NH2 wagging, and H-N-C-N twisting, respectively. For micromolar concentration, the intensity appeared as ~ IxlO ³ and ~7xl0 ² counts for the peaks at 710 cm' ¹ (Ring breathing) and 1626 cm' ¹ (Ring C-N stretching), respectively (figure. 6).

Case study (iv): SERS spectrum of common pesticides a) Thiram

Thiram is an example of fungicide used to prevent fungal diseases in seeds and crops. It is moderately toxic by ingestion, but it is highly toxic if inhaled at ppm level. The SERS spectra of the thiram showed the Raman peaks at 1517 cm' ¹ , 1440 cm' ¹ , 1386 cm' ¹ , 1150 cm' ¹ , 928 cm' ¹ , 560 cm' ¹ , and 1023 cm' ^l corrcsponds C=N stretching, CH3 bending, CH3 bending, C-N stretching, CH3N stretching, S-S stretching, and CH3NC bending respectively. LOD of 0.24 ppb (1 nM) of the analyte was achieved using the fabricated plasmonic substrate (figure.6). b) Carbofuran

Carbofuran is one of the most toxic carbamate pesticides and is available in the market under the trade name Furadan. It is being used to control insects in a wide variety of field crops, including potatoes, com and soybeans. Compared to any other widely used insecticides, carbofuran creates the highest acute toxicities in humans, even at lower ppm. The SERS spectra of the carbofiiran showed the Raman peaks at 1590 cm' ¹ , 1494 cm' ¹ , 1410 cm' ¹ , 1333 cm' ¹ , 1245 cm' ¹ , 1201 cm' ¹ , and 1021 cm' ¹ , corresponds C-C stretching within phenol ring, Furan C-H bending, C-H and O-H bending, C-C-C bending, Aromatic C-H bending, C-N stretching, =C-H aromatic in-plane deformation respectively. LOD of 0.221 ppm (1 pM) of the analyte was achieved using the fabricated plasmonic substrate (figure. 6). Case study (v): SERS spectrum of common dyes a) Rhodamine 6G

It is one of the highly fluorescent rhodamine family dyes and is also commonly used as a reporter molecule in several Raman spectral studies. In some instances, it is used as a colouring agent in food items. The SERS spectrum of the rhodamine 6G displayed the Raman peaks at 1650 cm' ¹ , 1575 cm' ¹ , 1510c cm' ¹ , 1360 cm' ¹ , 1310 cm' ¹ , 1180 cm' ¹ , 1125 cm' ¹ , 770 cm' ¹ , and 610 cm' ¹ , corresponds C-C stretching, C-H in-plane bend, C-C stretching, C-C stretching, N-H in-plane bend, C-H in-plane bend, C-H in-plane bend, C-H out of plane bend and C-C-C ring in-plane respectively. Of these, the noted intensities for peaks appearing at 1370 cm ^-1 and 1520 cm' 'were respectively- IxlO ³ and ~5xl0 ² counts - for 1 nM concentration of rhodamine 6G (fig.8). i.e. the fabricated substrate is sensitive to detect rhodamine 6G down to 0.48 ng/mL concentrations. b) Crystal Violet

Crystal violet or gentian violet, also known as methyl violet 10B, is a triarylmethane dye used as a histological stain and in Gram's method of classifying bacteria. It has antibacterial, antifungal, and anthelmintic properties. The SERS spectra of the crystal violet showed the Raman peaks at 1620 cm' ¹ , 1531 cm' ¹ , 1375 cm' ¹ , 1300 cm' ¹ , 1171 cm' ¹ , 915 cm' ¹ , 437 cm' ¹ , and 400 cm' ¹ corresponds ring C-C stretching, ring C-C stretching, N-phenyl stretching, ring C-H deformations, ring C-H bending, ring skeletal vibration of radical orientation, C-phenyl bending and Skeletal ring vibration respectively. With the current plasmonic substrates, for a concentration of 0.101 ng/mL, the intensity appeared as - 4.5xl0 ³ and ~4xl0 ³ counts for the peaks at 1178 cm' ¹ and 1400 cm' ¹ respectively (fig.6).

ADVANTAGES OF THE INVENTION:

• Preparation of SERS substrates with nanoscale wedges is simpler, environmentally friendly and cost-effective.

• Environmentally friendly materials such as aluminium oxide, glass beads, steel grits etc., having the size in the range of 50-250 microns used for preparation of SERS substrates to surfaces with pressurized air.

• Present invention is completely devoid of colloidal nanoparticles and avoids the use of toxic and corrosive chemicals. • The present invention offers flexibility in choosing a wide range of base materials such as insulating (e.g. glass), semiconducting (e.g. silicon) or metals (e.g. aluminium).

• SERS substrates having high surface area, effective adsorption of analyte molecules on the surface, which contributes to the enhancement factor and sensitivity.

• Present invention SERS substrates has good reproducibility (<3.9% RSD), and SERS substrates produced exhibit uniformity in performance with <2.9% RSD).