WO2010073260A1

WO2010073260A1 - Sers active paper substrate, a process and a method thereof

Info

Publication number: WO2010073260A1
Application number: PCT/IN2009/000710
Authority: WO
Inventors: Giridhar Udapi Rao Kulkarni; Anurag Gupta; Balasubramanian Karthick
Original assignee: Jawaharlal Nehru Centre For Advanced Scientific Research; Yashnanotech Ltd
Priority date: 2008-12-26
Filing date: 2009-12-09
Publication date: 2010-07-01

Abstract

The present invention relates to a SERS active paper substrate comprising in- situ synthesized nanoparticles. The paper substrate is used for obtaining Raman spectra of an analyte. The invention further relates to a process for obtaining SERS active paper substrate comprising in-situ synthesized nanoparticles, as well as a method to determine Raman Spectra of an analyte with the help of this SERS active paper substrate.

Description

"SERS ACTIVE PAPER SUBSTRATE, A PROCESS AND A METHOD

THEREOF"

FIELD OF THE INVENTION

The present invention relates to a SERS active paper substrate comprising in- situ synthesized nanoparticles. The paper substrate is used for obtaining Raman spectra of an analyte. The invention further relates to a process for obtaining SERS active paper substrate comprising in-situ synthesized nanoparticles, as well as a method to determine Raman Spectra of an analyte with the help of this SERS active paper substrate.

BACKGROUND AND PRIOR ART OF THE INVENTION

Raman Spectroscopy

When monochromatic light such as a laser is targeted over a sample, a part of it is transmitted, some is absorbed and some is scattered. Most of the scattered light will have the same wavelength as the incident light. However, a small portion of the scattered light approximately 1 in 10⁷ photons is shifted in wavelength because the molecules have experienced vibrations and rotations during the interaction with the light. The spectrum of this wavelength-shifted light is called the Raman spectrum.

Raman spectra consist of sharp bands that are typical of the specific molecule in the sample. Each line of the spectrum corresponds to a specific vibrational mode of the chemical bonds in the molecule. Since each molecule has its own Raman spectrum, this can be used to characterize the molecular structure and identify chemical compounds. A small fraction of light is scattered at energies different than that of the incident photons (Raman Effect). The Raman effect is an inelastic process and was first observed in 1928 by Sir. Chandrasekhara Venkata Raman and was awarded with the Nobel prize in the year 1930. Raman spectroscopy is a non-invasive, non-destructive and fast technique. However since only a small fraction of molecules in any sample gives a Raman signal it is relatively insensitive. Surface Enhanced Raman Spectroscopy (SERS) can increase the weak Raman signal by many orders of magnitude, and can extend the range of applications suitable for Raman spectroscopy.

Surface Enhanced Raman Spectroscopy (SERS)

SERS is a surface sensitive technique that results in the enhancement of Raman scattering by molecules adsorbed on to the rough metal surfaces. The enhancement factor of a molecule that is adsorbed on a rough metal surface can be much more intense in the order of 10⁴ - 10¹⁵, than the signal it would emit if deposited on a plain substrate.

This huge enhancement has enabled this technique to be very sensitive that enables detection of even a single molecule. The mechanism of SERS was first observed in the year 1977 and has ever since generated a huge research interest into Raman spectroscopy being used as a major analytical tool. It has been demonstrated that the huge enhancement in signal arises mainly due to 2 major mechanisms namely, Electromagnetic enhancement - proposed by Jeanmarie and Van Duyne in the year 1977, and Chemical enhancement proposed by Albrecht and Creighton in the year 1977.

Literature reports on using paper as a SERS substrate

Surface Enhanced Raman Spectroscopy (SERS) is a surface sensitive technique that results in the enhancement of Raman scattering by molecules adsorbed on rough metal surfaces. The enhancement factor can be as much as from 10⁴ - 10¹⁴, which allows the technique to be sensitive enough to detect single molecules. The main advantages of SERS are it serves as a good analytical tool for chemical and biomolecular detection; it is a Label-free technique and its ability to detect multi-components. Two mechanisms are responsible for the huge enhancement of Raman signals namely the electromagnetic and the chemical enhancements. The former arises due to the enhancement of local electromagnetic field brought about by the excitation of localized surface plasmon in the noble metal substrate. Whereas, the chemical enhancement arises due to an increase in electron-photon coupling and charge transfer by chemisorption of target molecules onto metal surfaces.

Since the discovery of SERS, a key problem in analytical application of SERS is to develop stable and reproducible SERS active substrates that can provide a large enhancement factor. This chapter presents how SERS substrates are being fabricated, in particular to paper being used as a substrate. Since paper is a substrate that is being used in the present preparation of SERS substrates, this invention is of importance to bring out the novelty in these preparation methods.

The first reports of filter paper being used as substrates (referred to rigid supports) for the identification of compounds using SERS were published in the year in 1984(1 ). Even before this report filter paper was used for the identification of compounds at subnanogram levels mainly by using fluorescence and room temperature phosphorescence (RTP) techniques(2). However, these techniques were severely limited as most of the aromatic hydrocarbons absorb in the same region to that of one component in a mixture. Moreover these techniques are structureless and do not provide much information.

In contrast, a Raman spectrum provides rich information on structure of molecules. It was demonstrated that silver colloidal hydrosols stabilized by filter paper enhance the Raman scattering of dyes adsorbed onto them. The silver colloids were prepared by Creighton & Lee-Meisel methods (3, 4). Various concentrations of aqueous dye solutions (2.5 μL) and silver colloidal (22.5 μL) were applied onto filter paper by a micro syringe, either as premixed mixture or separately. The Raman spectra were taken when they were still wet and it was observed that, the observed spectra are relatively free of any background scattering from the filter paper an important aspect of SERS substrates. It was reported that higher Raman signals and better reproducibility were found when dye and silver hydrosols were introduced onto filter paper as a premixed mixture rather than separately. The limits of detection (LOD) for various dyes were also presented with lowest LOD found for CV at 0.5 nanograms.

Mixtures of three dyes that were dissolved in methanol and a volume of 7μl of this mixture was applied on to a chromatographic paper and dried. Silver colloid that was prepared by (3,4) previous methods were sprayed using a spray atomizer and the wet paper was used to record Raman spectra. The enhancement that has been obtained was 9 to 10 orders of magnitude higher, the other advantage of this method is only a small amount of sample is required, and the excitation laser could be of very small intensity (5).

Thin layer chromatography plates were also used as SERS substrates (6) in which these substrates were spotted with an analyte using a syringe. Silver colloids were prepared by Creighton (3) procedure was sprayed using a atomizer over these plates and the Raman spectra of the analyte was recorded. The background effect of TLC plates was negligible and the limit of detection was estimated to be less than 5 ng/spot. Ion beam sputter deposited silver surfaces on a filter paper has been observed as a SERS active substrate (7). Here the filter paper was immersed in the analyte solution and later silver is deposited over the analyte.

One feature of these sputtered samples are that the SERS signals generally increases over a period of time, reaches to a maximum and remains constant over a period of hours. These substrates are stable for few weeks and the reusability of the SERS substrate was reported for the first time. A sputter deposited silver SERS substrate can be used to record a Raman spectrum of an analyte can be washed with water and a different analyte can be coated and obtain a SER spectrum. The SERS bands of the previous analyte were found to be completely absent. But the main disadvantage of these substrates as it has been also reported, there is an absence of any Raman peaks in the 1400- 1600 cm^"1 that are usually associated with carbon contamination occurring during sputter deposition.

Laserna et. al (8) demonstrated a one stage reduction of filter paper as a SERS substrate. When a filter paper is immersed in a silver nitrate solution and later sprayed with sodium tetrahrdroborate which inturn acts as a reducing agent results in an active SERS substrate. It was also observed that the background arising from a freshly prepared substrate is usually high. Soper et al (9) prepared SERS substrates from TLC plates, the authors prepared silver sol from both previously reported (3, 4) methods and added the analyte to the silver sol. This mixture of the analyte and sol was later deposited to TLC plates and SERS spectra were recorded on them. The limit of detection on these plates was obtained to be 33 picogram of the analyte material.

A photographic paper has also been reported to be an excellent SERS substrate (10); here the photographic material is exposed with high energy light. Under these conditions a large number of small silver clusters are formed. The developer solution used contains a reducing agent such as hydroquinone whish directly reduces the silver ions.

Preparation of SERS substrate using filter paper by immersing in silver nitrate solution and then spraying a sodium tetrahydroborate solution has been reported to be a good SERS substrate ( 1 1 ). To this wet paper the analyte was added and SERS spectra recorded when the substrate is still wet. In this article, it was demonstrated that the combination of SERS with spatially resolved detection could be a analytical tool in paper chromatography. An in depth study on filter paper being active substrates for SERS was reported by Laserna et.al ( 12) in which they have taken different Millipore filter papers varying in their porous composition on to which chemically reduced silver or evaporated silver was deposited. The SERS spectra gives a good description of the surface morphology with respect to the SERS activity.

Fang et al. ( 13) have actively pursued the use of filter paper that is coated with silver nanoparticles as a substrate for surface-enhanced Raman scattering. They have studied the SERS spectra of p- hydroxybenzoic acid (PHBA) as a probe molecule and a high-quality SERS spectra has been obtained, indicating that the silver-coated filter paper is a highly SERS-active substrate. In their investigation, silver (Ag) sol was prepared by the Lee and Meisel method. The Ag sol was drop wise added over the filter paper. As reported previously the sol containing Ag nanoparticles have been prepared first, and then introduced onto the substrate medium. By continuously adding either the Ag nanoparticles or the PHBA probe molecule they have prepared samples having different proportions of both silver nanoparticles and PHBA molecules. The authors have reported the adsorption behavior and discussed the changes in adsorption behavior with respect to different proportions of both silver nanoparticles and PHBA molecules.

Fang et al has reported the use of filter paper that is being coated with gold nanoparticles ( 14) as a substrate for surface-enhanced Raman scattering (SERS). The authors studied the SERS spectra of C₆₀/C₇₀ absorbed on a gold nanoparticle substrate and a high SERS activity spectra has been obtained. They have dropped a solution of fullerene onto a filter paper coated with colloidal gold, after drying, high-quality SERS active substrates have been reported. Colloidal gold was prepared through a redox process with KAuCl₄. The authors have coated the filter paper by increasing the layer of gold coatings. It has been observed that 5 times coating gives the best peaks with high intensity. They have used 2ml volume of C₇o/CS₂ solution to all samples.

Recently there is a revival of interest in nanostructured solid substrates as stable SERS substrates. There are reports of producing silver and gold nanostructures on substrates such as silicon, steel, glass and polymer surfaces. There are also commercial suppliers for these substrates.

OBJECTIVES OF THE INVENTION

The main objective of the present invention is to obtain a SERS active paper substrate comprising in-situ synthesized nanoparticles.

Another objective of the present invention is to provide a process for obtaining SERS active paper substrate comprising in-situ synthesized nanoparticles.

Yet another objective of the present invention is to provide a method to determine

Raman Spectra of an analyte.

STATEMENT OF THE INVENTION

Accordingly, the present invention relates to a SERS active paper substrate comprising in-situ synthesized nanoparticles; a process for obtaining SERS active paper substrate comprising in-situ synthesized nanoparticles, said process comprising steps of: a) dipping the paper in metallic precursor solution to obtain metal precursor loaded paper, and b) immersing the metal precursor loaded paper into boiling solution of reducing agent followed by cooling and drying to obtain the SERS active paper substrate comprising in-situ synthesized nanoparticles; and a method to determine Raman Spectra of an analyte, said method comprising steps of: a) applying an analyte on SERS active paper substrate comprising in-situ synthesized nanoparticles, and b) subjecting the paper substrate with analyte to Surface Enhanced Raman Spectroscopy to determine the Raman Spectra of the analyte.

BRIEF DESCIPTION OF THE ACCOMPANYING FIGURES

Figure 1: SEM image of a plain paper

Figure 2: Raman background on a plain paper Figure 3: Flowchart of SERS substrates preparation

Figure 4: SERS substrate showing the active area and level indicator

Figure S: SERS substrates with dipping and spotting techniques

Figure 6: Raman spectrum of a I mM concentration of thiophenol on the nanoAg SERS substrate compared with that from a neat thiophenol

( l Oμl) on a glass substrate.

Figure 7: Raman spectra of a ImM thiophenol by spotting lOμl of ImM concentration

Figure 8: Raman spectra of a 0.01 mM concentration of thiophenol Spot dropped l Oμl

Figure 9: Raman spectrum of a I mM thiophenol coated on a nanoAg

SERS substrate and a substrate that was dipped in distilled water for nearly 5 hours

Figure 10 (a-b): SEM images of nanoparticles that are embedded along the cellulose matrix of the paper under different magnifications.

Figure 11 (a-b): SEM images of nanoparticles that are embedded along the cellulose matrix of the paper under different magnifications

Figure 12 (a-d): SEM images of nanoparticles obtained after using CTAB along with sodium citrate solution.

Figure 13 (a-b): Raman spectra of I mM concentration of thiophenol obtained on the present substrates with normal preparation and after the addition of CTAB solutions.

Figure 14 (a-b): SEM images of the reduced nanoAg SERS substrates

Figure 15: Raman spectra obtained from a I mM concentration of thiophenol

Figure 16: Raman spectra obtained from a neat thiophenol over a glass substrate

Figure 17: Raman spectra of a neat thiophenol coated on a plain paper

Figure 18: Raman spectra obtained by plotting all three (thiophenol, neat thiophenol over a glass substrate and neat thiophenol coated on a plain paper) together

Figure 19: Raman spectra of a I mM thiophenol by spotting l Oμl of I mM concentration Figure 20: Raman spectra of a 0.01 mM concentration of thiophenol Spot dropped l Oμl

Figure 21: Sample A-C Raman spectrum investigation on the ageing effect of substrates. Raman spectrums of ImM concentration of thiophenol coated on nanoAg SERS substrates according to Table 2.

Figure 22: SERS spectra of R6G.

Figure 23: Raman spectra of pyridine.

Figure 24: Raman spectra of pyridine carboxylic acid

Figure 25: Raman spectra of PMMA

Figure 26: Raman spectra of L Alanine

Figure 27: SERS spectra of Haemoglobin

Figure 28: SERS spectra of Haemoglobin

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a SERS active paper substrate comprising in-situ synthesized nanoparticles.

In another embodiment of the present invention, the paper is Raman blank in nature.

In yet another embodiment of the present invention, the paper is rose colored plain maplitho printing paper.

In still another embodiment of the present invention, the nanoparticles are selected from a group comprising silver and gold nanoparticles.

In still another embodiment of the present invention, size of the nanoparticles ranges from about 10 nm to about 30 nm.

In still another embodiment of the present invention, the nanoparticles are non-uniform in shape with sharp tips.

In still another embodiment of the present invention, the paper comprises a layer of thiophenol.

The present invention relates to a process for obtaining SERS active paper substrate comprising in-situ synthesized nanoparticles, said process comprising steps of: a) dipping the paper in metallic precursor solution to obtain metal precursor loaded paper; and b) immersing the metal precursor loaded paper into boiling solution of reducing agent followed by cooling and drying to obtain the SERS active paper substrate comprising in-situ synthesized nanoparticles. In another embodiment of the present invention, the nanoparticles are selected from a group comprising silver and gold nanoparticles.

In yet another embodiment of the present invention, the metallic precursor solution is selected from a group comprising Silver Nitrate and Chloroauric Acid. In still another embodiment of the present invention, the concentration of metallic precursor solution ranges from about 2M to about 3M.

In still another embodiment of the present invention, the paper is dipped in metallic precursor solution for a period of about 25 to about 35 minutes.

In still another embodiment of the present invention, the reducing agent solution comprises a combination of Sodium Citrate and Cetyl trimethylammonium bromide. In still another embodiment of the present invention, the concentration of Sodium citrate ranges from about 3OmM to about 4OmM, and the concentration of Cetyl trimethylammonium bromide ranges from about 3mM to about 1OmM. In still another embodiment of the present invention, the metal precursor loaded paper is immersed into the boiling solution of reducing agent set to a temperature ranging from about 7O⁰C to less than about 75 ⁰C for a period of about 40 to about 50 minutes. In still another embodiment of the present invention, the paper is optionally coated with a layer of thiophenol having concentration of about 0.5mM to about 1.5mM. The present invention relates to a method to determine Raman Spectra of an analyte, said method comprising steps of: a) applying an analyte on SERS active paper substrate comprising in-situ synthesized nanoparticles; and b) subjecting the paper substrate with analyte to Surface Enhanced Raman Spectroscopy to determine the Raman Spectra of the analyte.

In another embodiment of the present invention, the application of analyte is carried out by dipping or spotting.

In yet another embodiment of the present invention, the SERS active paper substrate enhances the sensitivity of Surface Enhanced Raman Spectroscopy of analytes. In still another embodiment of the present invention, the SERS active paper substrate enhances the intensity of the Raman Spectra of the analyte by an Enhancement factor of about 2 million.

In still another embodiment of the present invention, the concentration and volume of the SERS active paper substrate can be as low as about ImM and about lOμl respectively.

The goal of the present invention is to bring out a highly sensitive, cost effective nanomaterial Raman substrates as a product to the research and industrial community working on Raman spectroscopy as an analytical tool which will be greatly benefited by this product for their Raman related investigation and studies.

All the investigations that have been reported in the literature utilizes the means of first preparing a Ag sol by previously reported methods and then depositing/ spraying them on the filter paper substrate. The novelty in the present preparation method is that the reduction of nanoparticles takes place in-situ on the substrate itself. In contrary, the references mentioned in the literature prepare nano particle solutions either Ag/Au sol and then coat it on to the substrate.

In the present process, the substrate is dipped in a solution containing a particular concentration of AgNO₃ that is dissolved in water. After 30 mins of soaking, the substrate is dried and introduced into boiling distilled water containing sodium citrate and CTAB solutions. The reduction of AgNO₃ into Ag nanoparticles takes place insitu on the paper substrate itself. The paper substrate is later dried and used as an active SERS substrate. Thereby the preparation method in the present invention is completely novel to those reported in literature before. Maplitho colour print paper (70-80 gsm & rose in colour) is being used as a substrate in the present invention whereas all the references discussed previously in literature use a filter paper. Thus, the starting material is also completely different from those previously reported. The paper matrix that has been chosen in the present invention has a distinct advantage of having a plain blank Raman spectrum, which will not interfere with the Raman peaks of an analyte under investigation. A much referred preparation method is ion beam evaporator, which utilizes deposition of silver on the filter paper. However, it has been also observed that this method strongly prevents any Raman band appearing over a particular range, which is not a desired property of an excellent SERS substrate.

The SERS substrates of the present invention provide a Raman intensity count that is much higher than the references discussed above. SERS substrates prepared by the present method are much more ultrasensitive than the substrates mentioned in the references that use filter paper. The substrates could easily detect molecules that have a very low concentration & volume as low as l μM & 10 μl respectively whereas a high volume (few ml) of the analyte is required to detect the Raman peaks in some of the references mentioned.

CHARACTERISTICS OF THE PAPER USED

Plain maplitho printing paper (70-80 gsm) was the substrate that is being used in the preparation of the SERS substrates in the present invention. This class of cellulose-based paper is rose in colour. Figure 1 shows a SEM image of a plain paper having cellulose fibres on its surface. As seen in the image cellulose fibre bundles of approximately 20 μm in diameter are randomly spread over the paper surface. RAMAN NATURE OF PAPER

This paper has a unique feature of having no specific Raman peaks on its own, a desired feature for a Raman substrate. The Raman spectrum of a plain maplitho printing paper is shown below in Figure 2 over the entire range of 0-3000 cm^"1. This is a crucial advantage of this paper substrate, as it will not interfere with any peaks of the analyte molecule under investigation. The Raman spectrum was recorded on a LabRAM HR apparatus (Horiba, USA) with an excitation wavelength of 632.8 nm and 5mWcm^~2. D l filter was used for recording which corresponds to 1/10 of the maximum laser intensity.

RELEVANT CONCENTRATIONS OF THE PRECURSORS AND REDUCING AGENTS

The concentrations of the metal precursor and the reducing agents that were used in the present preparational procedure is presented below:

Silver Nitrate concentration

4.68 gms of silver nitrate was measured on a butter paper over a weighing balance and dissolved in a 10 ml of DD water. This 4.68 gms of silver nitrate corresponds to 20% of the maximum solubility of silver nitrate in distilled water at room temperature 20-25⁰C. The concentration of silver nitrate solution in 10ml of distilled water corresponds to 2.75M.

Concentration of Reducing Agents

A combination of Sodium citrate dihydrate and Cetyl trimethylammonium bromide (CTAB) was used as the reducing agent for the preparation of SERS substrates. First, the two solutions were prepared separately. Sodium citrate (100 mg) and CTAB ( 12.39 mg) were dissolved separately, in two glass containers each containing 10 ml of distilled water to give rise to 34 mM and 3.4 mM solutions respectively. EXAMPLEl

DETAILED PROCEDURE

The detailed preparation procedure for obtaining SERS active paper substrate is discussed below:

Paper size

The standard size of Rose maplitho printing paper (9 x 6 cm) was taken for preparation of the SERS substrates of the present invention. Each time, several such stripes, typically 4 were used and they were cut into 8 strips each of same size in order to achieve uniform coating of the precursor solution.

Preparation of Metal Precursors

Preparation of Silver Nitrate solution (for substrate with silver nanoparticles): Silver nitrate (4.68 gms) was dissolved in 10 ml of DD water to achieve 2.75M solution. This corresponds to ~ 20% of the maximum solubility of silver nitrate in distilled water at room temperature. Since silver nitrate is photosensitive and reduces under exposure to light, the room lighting was maintained at lowest possible working conditions. The reduction process takes place insitu in presence of both sodium citrate and CTAB.

Preparation of Chloroauric Acid (for substrate with gold nanoparticles): Hydrogen tetrachloroaurate (HAuCU) or commonly known as chloroauric acid is a metal precursor that was used as a solution for the preparation of gold nanoparticles for a Nanogold Raman substrate. However, owing to the hygroscopic nature of HAuCl₄, pure gold strip was taken and dissolved in a mixture of HCl and HNO₃ (3 : 1 ). The concentration of the gold solution was maintained at 2.75M (same to that of silver nitrate solution). Here also the reduction process takes place insitu in presence of both sodium citrate and CTAB. Loading paper with precursor

Paper strips were immersed into a 10 ml of metal precursor solution on a plastic container such that all 8 strips were completely soaked. These strips were left undisturbed inside the solution for 30 minutes, during which the colour of the rose coloured maplitho printing paper turns brownish.

Drying of the paper

After 30 mins of soaking, the paper strips were removed and dried at ambient temperature. This enables metal precursors to get completely absorbed into the paper matrix.

Preparation of Reducing Agents

A combination of Sodium citrate and Cetyl trimethylammonium bromide (CTAB) was used as the reducing agent for the preparation of SERS substrates. First, the two solutions were prepared separately. Sodium citrate ( 100 mg) and CTAB (12.39 mg) were dissolved separately in two glass containers each containing 10 ml of distilled water to give rise to 34 mM and 3.4 mM solutions respectively. In the preparation of gold nanoparticles, 4ml of sodium citrate ( 100 mg in 10ml) was added, whereas 0.8 ml of sodium citrate ( 100 mg in 10ml)in the preparation of silver nanoparticles.

Volume and temperature of DD water

100 ml of DD water is taken in a beaker and kept inside a hot water bath. The temperature is set at 73⁰C which was constantly monitored using a thermometer. Once the desired temperature is obtained, the reducing agents were added in succession and the complete solution is left to boil for another 5 minutes. Dipping the silver nitrate loaded paper into boiling solution

After 5 mins, the dry paper strips containing metal precursors are dipped in, one after another. All the strips were completely immersed in hot water containing reducing agents at 73C and left undisturbed for exactly 45 minutes.

Reduction of the paper

The metal precursor that is absorbed on the surface of the paper undergoes reduction with the sodium citrate and CTAB solutions with water as solvent. The reduction process takes place insitu on the surface of the paper itself during which silver or gold nanoparticles (depending on the metal precursors used) are formed and are embedded into the paper matrix. As the boiling continues, the colour of the nanoparticle loaded paper turns to darkish brown. After exactly 45 minutes, boiling is stopped and the beaker is removed from the water bath..

Removal of the substrates and drying

The substrates were later removed and dried again at ambient temperature. For the handling and removal of substrates, clean dry forceps was used every time.

Resizing and fixing of the SERS substrate into a holder

The SERS substrates with active areas containing nanomaterial that has been prepared were punched into small strips using a paper punch and fixed into a firm strip again made up of paper. A white art paper with no dye added to it was chosen, and a slot is introduced to hold the SERS substrates firmly. It's another unique advantage of not using any adhesives to attach the active area to the holder as this may interfere while introducing the analyte. The SERS strip is flexible and easy to handle a key advantage in the usability of substrates. Thiophenol coated SERS substrates

Although SERS is a powerful non destructive technique used for the detection of a wide range of molecules, there are analytes (such as amino acids, saccharides) have a low affinity to nanometal substrates, a fact that is widely acknowledged in SERS literature. In order to overcome this limitation and also to promote the application of the SERS substrates of the present invention, the substrates have been functionalized with a monolayer coating of thiophenol which essentially acts as a capture probe to the molecule under investigation.

The preparation of the present invention thereby involves preparing thiophenol coated substrates. Here the substrates were prepared exactly as mentioned in the previous chapter in addition previous to packing them to paper holders they were dipped in I mM solution of thiophenol for 30mins later dried and packed.

Thus the Raman substrate product of the present invention will be packed with two separate pouches containing SERS substrates. One packed with 5 number of normal SERS substrates and the other packed with 5 number of substrates that are coated with a monolayer coating of thiophenol. The later one is labeled 'Thiophenol coated' for identification. The user depending upon the Raman activity of molecule (strong/weak) under investigation could chose the substrate to use. After Raman recording and subsequently performing the normalization with respect to thiophenol the Raman spectra of the molecule under investigation could be obtained. Process flow for SERS substrates

A set of 5 SERS strips are placed in an airtight pouch filled with nitrogen gas and sealed under inert atmosphere. This is required to increase the shelf life of the substrate beyond few months.

EXAMPLE 2

MICRO STEPS IN THE PREPARATION OF SERS SUBSTRATES

1) Take plain maplitho printing paper box.

2) Use a hand glove always while handling with the paper.

3) Paper is kept in a brown folder that is marked Plain Rosy.

4) Check the colour and softness of the paper with standard paper (70 gsm ) used before.

5) Spread a neat white chart sheet on the working area.

6) The paper has to be marked with the size that it has to be cut using a ruler and a light shade pencil.

7) Mark the sizes on the paper such that it measures 9cm x 6cm.

Rosy paper

8) Mark 4 pieces for the above size mentioned size. checks and

9) Take a scissor and clean them using ethanol and distilled re- sizing water. them for the

10) Dry the scissor completely using a tissue paper. preparation

1 1) Cut the paper sharply on the marked area using this clean, dry scissor.

12) Cut it in sharply with no patches on edges.

13) Make the 4 pieces been cut into into 8 pieces of the paper with standard size. This is to facilitate better coating and handling during preparation.

14) Take a clean sample box, the standard rose box that is marked ' Used for Sample Coating' .

15) The size of this sample boxes will be 9cm x 6cm.

16) Place this sample box in the chemical hood safely.

17) Replace the paper folder into the right place.

18) Take 15 ml plastic/glass vials, clean and dry them.

19) Cover up this glass container with insulation tape that is black in colour.

20) Take a measuring cylinder clean and dry them.

21) Measure 10 ml of DD water in a measuring cylinder using wash bottle that is labeled 'DD water'.

46) Make an entry in the register the amount of chemical used and comments.

47) Pour the silver nitrate solution into the rose coloured box that is labeled 'Used for Sample Coating' . Λ

48) Take the 8 pieces of paper.

49) Using a clean sharp forcep carefully immerse them into the solution one by one.

50) Make sure again that the room lighting is kept minimal.

Soaking the

51 ) All the 8 pieces should be completely immersed into the silver rosy paper nitrate solution. in silver 52) Note the time when the paper was immersed in solution. > nitrate 53 ) Go to the chemical hood and switch off the lights. solution 54) Leave the plastic container having papers dipped in silver nitrate solution inside the hood.

55) After exactly 30 minutes the coating duration is finished and the paper should be removed.

56) Ideal soaking time is 30-35 mins, if exceeds leaves a darkish J colour coat over the paper.

57) • Use a clean forcep to take out the paper one by one.

58) Place them one by one on the edges of the watch glass plate

59) Make sure that the papers are not placed one over the other

60) Switch on the chemical hood fan and let the papers dry in ambient.

61 ) Drying under the fan is also advisable provided lighting is minimal.

62) After 20 minutes check if the papers have dried up one side completely.

63) Using forceps place each of the paper upside down. This is to facilitate complete drying up on both surfaces.

64) After 20 minutes check if the papers are completely dry if not continue for more time till the papers are completely dry.

65) Place them in a fresh clean plastic container and close.

66) Always make sure the instruments and containers used are clean and dry.

67) Measure 10 ml of DD water using a measuring cylinder using a¹ wash bottle.

68) Pour this DD water in a fresh clean glass/plastic vial container ( 15 ml).

69) Take a fresh butter paper. ng balance. owder.

74) Take a small spatula that could help in measuring a few mgms..

75) Clean it using ethanol and dry it.

76) Measure 100 mg of sodium citrate over the weighing balance.

77) Carefully transfer this 100 mg into the 10 ml DD water container.

78) Shake it dynamically.

79) Make sure the sodium citrate has completely dissolved in DD water.

80) Label the container as sodium citrate solution.

93) Make sure the CTAB has completely dissolved in DD water.

94) Label the container as CTAB solution.

95) Take a 250 ml beaker.

96) Clean them using soap solution. ^"λ

97) Dry them completely in a hot air oven.

98) Using ethanol and paper clean it again.

Set up for the 99) Measure 100 ml of DD water using a measuring cylinder. reduction 100) Transfer this DD water into the cleaned 250 ml beaker. process

101 ) Remove the top lid of water bath.

102) Check the water level on it.

103) If it less than marked region fill it up more with DD water.

104) Place the top lid back to its position.

105) Place the beaker into the socket provided in the top lid.

106) Always use either the socket in sets either the first two or the second.

107) Take the thermometer and fix it to tripod stand.

108) Set the thermometer that is fixed to a tripod stand into the beaker.

109) Set the knob reading to '85C .

1 10) This is to set the temperature to 73C inside the beaker.

1 1 1 ) Switch on the water bath and exhauster.

1 12) There will be an average difference of 12C between the water bath and the beaker temperature.

113) Wait till the temperature reaches 73C.

1 14) Check if the temperature remains constant at 73C. J

1 15) Take a micropipette.

Addition of sodium 1 16) Set the measuring reading to 78 μl. citrate & 1 17) Insert a new microtip.

CTAB solutions 1 18) Take the sodium citrate solution.

1 19) Measure 78 μl of solution and drop it into boiling 100 ml of DD water.

120) Transfer such that drop by drop of sodium citrate solution in transferred. 121) Repeat this 10 times such that 0.78 ml is dropped.

122) Mix the sodium citrate solution using microtip into the DD"

131) Replace ,the micropipette back to its original position.

132) Wait for 5 minutes for the mixture to reach the set temperature.

133) Take the papers that were soaked in silver nitrate solution and subsequently dried.

134) Insert the strips one by one so that all of them are immersed completely.

135) Note the time. >

136) Also note the temperature if it remains constant.

137) For 45 mins leave it undisturbed

138) Continuously monitor the temperature for the next 45 mins.

139) Note the colour changes over the paper with respect to time.

140) After 45 mins switch off the water bath.

141) Remove the beaker and drain the solution into wastage can.

142) Use a clean forcep to take out the reduced paper one by one.

143) Place them one by one on the edges of the clean watch glass plate

144) Make sure that the papers are not placed one over the other

145) Switch on the chemical hood fan and let the papers dry in ambient.

146) After 20 minutes check if the papers have dried up one side completely.

147) Using a forceps place each of the paper upside down. This is to facilitate complete drying up on both surfaces.

148) After 20 minutes check if the papers are completely dry if not continue for more time till the papers are completely dry.

149) Place them in a fresh plastic container and close.

150) Collect the used beakers, watch glass and measuring cylinders.

15 1 ) Wash them in a soap solution

152) Dry them in a hot air oven.

153) Place them back inside the cupboard.

154) Take one prepared strip at a time and make sure they are completely dry.

155) A clean paper punching machine dedicated for this preparation has to be taken.

156) Place the prepared strip under the punching area of the machine.

157) Press the punch so that one hole is made up on the strip.

158) Notice the punched strip is been collected by the plastic casing at the bottom.

159) Place the punching area nearest to the punched hole and continue punching till maximum number of round shapes pieces are collected.

160) Once the strip is finished continue with the other strips depending upon the number of samples to be prepared.

161) In one preparation four strips are to be used for plain

substrates and the rest four for thiophenol coated ones.

162) Ideally with four strips of prepared paper 100 neatly punched substrates can be obtained.

163) Punch the remaining 4 reduced papers and keep it separately for thiophenol coating.

164) Take a strip/holder and use the slot puncher and make a slot.

165) Keep 2 bundle i.e 200 strip holder for punched with slots.

166) From this 100 will go for plain substrates and 100 for thiophenol coated ones.

167) Insert one punched piece into the slot using a clean forcep.

168) Make sure that pieces that are not completely circular are not loaded.

169) Continue inserting the SERS punched substrates into fresh strips.

170) Only one SERS substrate has to be inserted into one strip.

171 ) Care should be taken that the active area is handled softly.

172) Five SERS strips with substrates attached to them make one SERS pouch.

173) Make sure that the active area is placed well inside the strip holder.

174) Continue making 100 number of SERS strips.

175) Take the 100 punched active ones inside the hood.

176) Take 40 ml of ethanol in a clean beaker using measuring cylinder.

177) Take 40 μl of thiophenol and dissolve in ethanol.

178) Drop all the 100 active ones into the solution.

179) Leave it undisturbed for 30 mins inside the beaker.

180) Make sure the hood exhauster is switched on.

181 ) All the operations using thiophenol has to be inside the hood only.

182) After 30 mins dry up the solution and let it dry inside the hood.

183) After it has completely dried up follow the same procedure of fixing the strips.

184) For one preparation take 40 pouches, 20 for plain substrates and 20 for thiophenol coated.

185) Place the thiophenol coated stickers to the respective pouches. 186) Paste the silver coated stickers to the box.

187) Paste the spelling correction sticker to the box.

188) Open the nitrogen cylinder in the anticlockwise direction using the spindle provided.

189) Open it slowly so that the pressure gauge reading increases slowly.

190) Set the pressure gauge reading to 95 Kg/cm².

191 ) Slowly increase the knob so that pressure reads 5 Kg/cm² on the second gauge bar on your left.

192) Fill in the pouches till they bulge to their maximum limits.

193) Do not continue filling nitrogen gas as it might tear of the pouches.

194) Similarly do not use a high pressure to fill in pouches as it will damage the pouches.

195) After filling individual pouches close the knob as it will prevent wastage of nitrogen.

196) Continue from step 174- 184 for the filling up of other pouches.

197) After finishing all the desired pouches set the knob to decreased position (pressure approx. 0 Kg/cm²) and close the cylinder using the spindle in clockwise direction.

198) Replace the spindle in its original position.

199) Enter your name in the log book and the number of samples filled

200) Connect the sealing machine to the mains.

201) Set the heat control knob to reading 90.

202) Set the sealing control knob to position B.

203) Wait for 5 minutes.

204) Place the pouch area to be sealed over the knurling provided on the sealing machine.

205) Make sure that the pouch is placed even and follows a straight line.

206) Press the handheld stopper towards the sealing area.

EXAMPLE 3

USE OF HIGHLY SENSITIVE NANOMETAL RAMAN SUBSTRATES

To achieve the maximum advantage of the Highly Sensitive Raman substrates, samples should be deposited onto the active surface and analyzed with a standard Raman instrument (Figure 4). Different sample deposition techniques such as dipping and spotting techniques can be employed (Figure 5).

Level indicator is the point up to which the substrates can be dipped into samples under investigation. SERS active area is the region where silver nanoparticles are coated in a desired size range and distribution in order to achieve maximum enhancement.

Dipping & Spotting techniques

For the dipping technique the Raman substrate has to be dipped into the sample under investigation up to the indicator mark that is provided over the substrates. Incase of spotting and multi spotting a micropipette can be used to dispense sample on the active area. Once the samples are deposited the substrates are to be dried up completely in ambient conditions and taken for Raman recording.

Performance of the substrate

To investigate the performance of the SERS active paper substrate comprising in-situ synthesized nanoparticles, Thiophenol and Rhodamine 6G (R6G) were the analyte molecules chosen in this invention.

The performance of a SERS substrate can be established if the analyte molecule under investigation replicates the Raman spectra that has been already reported and documented in literature. In the instant invention, thiophenol was investigated by making a comparison to Raman peaks as reported in Small 2008². Here all the major peaks corresponding to thiophenol namely 1005 cm^"1 , 1028 cm^"1 and 1079 cm^"1 were observed and these peaks correspond to the ring breathing, C-H bending and C-S stretching modes of thiophenol. Secondly, the enhancement obtained in terms of Raman intensity is nearly 45,000 counts around the 1005 cm^"1 as compared to nearly 14,000 counts reported in the reference. In terms of Rhodamine 6G again all the peaks corresponding to the analyte as reported in literature are identified but with a much higher Raman intensity count³.

5ml of ImM concentration of thiophenol in DD water was prepared and a strip of SERS substrate was dipped in for exactly 30 minutes. After 30 minutes, the substrate was removed and washed in ethanol solution to remove any molecules that are not absorbed onto the surface, later dried in the ambient conditions and taken for Raman measurements. The nanosilver coated SERS substrate gives a huge enhancement under Raman investigation. Figure 6 shows the Raman spectrum recorded with ImM concentration of thiophenol giving an intensity count of nearly 45,000 counts around the 1005 cm^"1 Raman peak typical of thiophenol. The Raman bands typical of a thiophenol at 1005 cm^"1 , 1028 cm^"1 and 1079 cm^"1 were observed and these peaks correspond to the ring breathing, C-H bending and C-S stretching modes of thiophenol (Table 1). As a comparison a drop of neat thiophenol was coated on a plain glass substrate and Raman spectrum was recorded under similar conditions showing not much of spectral features. Thus the nanosilver SERS substrate greatly increases the Raman intensity and also reproduces the spectral features of the analyte molecule under investigation.

The nanoAg coated SERS substrate of the present invention gives a huge enhancement under Raman investigation. Figure 6 shows the Raman spectrum recorded with I mM concentration of thiophenol giving an intensity count of nearly 45,000 counts around the 1005 cm -^" 1 Raman peak typical of thiophenol. The Raman bands typical of a thiophenol at

1005 cm^" , 1028 cm -^" 1 and 1079 cm^" were observed and these peaks correspond to the ring breathing, C-H bending and C-S stretching modes of thiophenol (Table 1)

Table 1 : Ring breathing, C-H bending and C-S stretching modes of thiophenol

® Small 2008, 4, No. 5, 670-676

Dipping vs spotting

The SERS substrates of the present invention could be used for both dipping as well as spotting techniques. The Raman spectra of . I mM concentration of thiophenol by dipping the substrate in a 5ml of solution have been discussed before in Figure 6.

The Raman spectra obtained by spotting l Oμl of the solution (I mM) on a nanoAg SERS substrate is shown below in Figure 7. It is observed that even with a low concentration and a small volume of the analyte, the spectral features of the analyte molecule could be seen with a good enhancement

Dilution of the analyte molecule

A l Oμl of 0.01 mM concentration of thiophenol in DD water was spotted on a nanoAg SERS substrate. Figure 8 shows typical Raman bands of thiophenol with good signal to noise ratio. Even at - such low concentrations and volume of the analyte, the SERS substrate could detect the molecule under investigation.

Substrates that were dipped in DD water 10 consecutive times A nanoAg SERS substrate was taken and immersed in a 10 ml of distilled water solution consecutively 10 number of times. In each case it was allowed to be immersed for 30 minutes and this procedure was repeated 10 times.

After nearly 5 hours of immersion in DD distilled water these substrates were later dried and coated with ImM concentration of thiophenol and Raman spectrum was recorded around the 1079 cm^"1 peak as shown in Figure 9.

It could be observed that even after immersing in DD water for nearly 5 hours the substrates gave a good signal and also the characteristic peak of thiophenol is still observed. It is clear that nanoAg sticks to the paper matrix firmly. This feature is particularly useful in studying reaction pathways.

Thus, the SERS substrates of the present invention provide huge enhancement in Raman intensity under different experimental conditions even with very low volume and concentration, they are stable over a period of time and could withstand even if it were subjected to a water medium. These features are not reported before and thereby the SERS substrates of the present invention are much more efficient and usable under different applications.

In-situ synthesis of the nanoparticles

In the preparation of SERS substrates of the present invention, the nanoparticles are synthesized insitu on the paper matrix itself. Figure lθ(a-b) below shows the SEM images of nanoparticles that are embedded along the cellulose matrix of the paper under different magnifications. It can be observed that these nanoparticles are embedded along the sides, underneath and on top of the cellulose fibers that is present along the paper substrate.

The nanoparticles range from 10- 30 nm and are of different sizes and shapes. These nanoparticles which are non circular having sharp tips and also of different sizes and arranged randomly will have important consequences for SERS spectroscopy. The paper substrate is loaded with the metal precursor and allowed to dry. Then in a boiling water solution having a known concentration of reducing agent, the loaded paper is dipped and allowed to boil. Over a period of time (approximately 45mins) the reduction process took place on the surface of the paper itself.

It could be observed that the nanoparticles that were prepared insitu affix to the fibre bundles that is present all over the surface of paper. Thus the deposition of the nanoparticles over the paper surface is aided by the presence of cellulose fibres. The nanoparticles adhere firmly to the paper matrix as discussed previously in Figure 9.

Surface of the nanoparticles and its relevance to SERS analysis

The SEM images of the surface of the nanoparticles that is being subjected to SERS analysis is shown in Figures l l(a-b). The images show the nanoparticles that were formed insitu are non-uniform in shape and also the size varies between 15-30 nm (Figure l ib). These nanoparticles agglomerate into distinct shapes and are also firmly embedded into the paper matrix. These nanoparticles have sharp tips which in turn aid the huge enhancement obtained under Raman investigation.

Further in preparation of SERS substrates of the present invention, the nanoparticles are synthesized insitu on the paper matrix itself. Figure 11 (a, b) show the SEM images of nanoparticles that are embedded along the cellulose matrix of the paper under different magnifications. It can be observed that these nanoparticles are embedded along the sides, underneath and on top of the cellulose fibers that is present along the paper substrate. It could be seen that the nanoparticles that were prepared insitu sticks to the fibre bundles that is present all over the surface of paper. Thus the deposition of the nanoparticles over the paper surface is aided by the presence of cellulose fibres.

The images show the nanoparticles that were formed insitu are nonuniform in shape and also the size varies between 10-30 nm (Figure l ib). These nanoparticles agglomerate into distinct shapes and are also firmly embedded into the paper matrix. These nanoparticles have sharp tips which in turn aid the huge enhancement obtained under Raman investigation.

Substrate performance after adding CTAB as a reducing agent

In the preparation of SERS substrate of the present invention, adding CTAB as a reducing agent has a great influence in the intensity of the Raman signal that could be obtained. With the addition of CTAB along with sodium citrate solution, a Raman intensity count of approximately 17,500 counts is obtained whereas and without the addition of CTAB its approximately 6000 counts (Figure 12 a-d).

Figure 13 a and b show Raman spectra of I mM concentration of thiophenol obtained on the substrates with normal preparation and after the addition of CTAB solutions. The recording was done on substrates that were prepared independently and a ImM solution of thiohenol coated, dried and later Raman recorded on a 632 nm laser. The addition CTAB has been included in further preparations along with sodium citrate solution.

An important feature of these substrates having nanoparticles embedded into the paper substrate is that the nanoparticles range from 10-30 nm and are of different sizes and shapes. These nanoparticles which are non-circular having sharp tips and of different sizes are arranged randomly will have important consequences for SERS spectroscopy.

EXAMPLE 4

ENHANCEMENT FACTOR 'G' CALCULATION

The SEM images of the reduced nanoAg SERS substrates is given in figure 14, wherein the size of the silver nanoparticles varies from 10-30 nm in size. The enahancement factor G for nanoAg substrates is calculated from the equation :

Enhancement factor G = ( ISERS / Inorm ) (Nβuik/ N_surf )

The gaussian area obtained due to the enhancement arising from the plain paper on its own was deducted while calculating the overall enhancement obtained from the substrates. From the SEM images Figure 14 (a, b) it was calculated that there are 22 nanoparticles per l OOnm¹⁶. The Raman spectra obtained from ImM ( 1000 times diluted in ethanol) concentration of thiophenol (Figure 15), a neat thiophenol (pure) coated over a glass substrate (Figure 16) and over a plain paper (Figure 17) is given. Figure 18 shows the comparative figure of Raman spectra obtained from I mM (1000 times diluted in ethanol) concentration of thiophenol, a neat thiophenol (pure) coated over a glass substrate and over a plain paper plotted together. The Enhancement Factor 'G' is calculated as below:

Enhancement factor G = ( ISERS / Inorm ) (N_Buik / N_surf )•

I_SERS ⁼ Measured intensity for thiophenol at 1005 cm^{" 1}

2.014 x 10⁵ Inorm = Measured intensity of normal scattering from bulk thiophenol on glass substrate under 1005 cm - 1 peak

7.02 x 10²

N_surf 4πr² CAN where, radius of nanoparticle

= 20 nm;

₌ 20 x 10^"9 m

C = surface density of thiophenol 6.8xl O¹⁴ molecule/cm² 6.8x l O¹⁸ molecule/m² A = area of focused laser spot

4x 10^"12 m²

N = surface coverage of Ag nanoparticles (calculated from SEM image)

22 nanoparticles / l OOnm² N = 22 x 10¹⁴ nT²

Substituting the above values Nsurf = 3.008 x 10⁸

Now,

Nbuik ⁼ Ah p/m m molecular weight of thiophenol = 1 l O. l δg/mole^"1

A area of the laser spot = 4μm² h- penetration depth =100μm =100 x 10^"6 p density of thiophenol = 1.0073g/cm³

= 10.073 x 10^"3kg/m³ Nbuik = 2.346 x 10¹²

Substituting all the values in G factor calculation, we get

Enhancement factor G at 1005 cm^"1 = 2.24 x 10⁶ (approx. more than 2 million times) Low volume of the analyte

The Raman spectra obtained by spotting l Oμl of the solution ( I mM) on a nanoAg SERS substrate is shown below in Figure 19. It is observed that even with a low concentration and a small volume of the analyte, the spectral features of the analyte molecule could be seen with a good enhancement.

A lOμl of 0.01 mM concentration of thiophenol in DD water was spotted on a nanoAg SERS substrate. Figure 20 shows typical Raman bands of thiophenol with good signal to noise ratio. Even at such low concentrations and volume of the analyte, the SERS substrate could detect the molecule under investigation and also give a high Raman intensity.

Ageing Effect on Nanoparticle SERS Substrates

The nanosilver (nanoAg) SERS substrates withstand its performance over a time period. The ageing effect on these nanoAg substrates were investigated by taking substrates that were prepared from different time intervals. Table 2 shows the age of the substrates that was taken for this invention. These aged substrates were coated freshly, with I mM concentration of thiophenol solution uniformly and investigated under Raman by keeping all the operational parameters constant.

Table 2: Age of substrates

Ageing effect is seen on the substrate that is 72 days old, produced relatively noisy spectrum and the quality improved with the freshness of the substrate. This has made it necessary to seal the substrate under inert atmosphere. An ageing effect on the substrates that were 45 and 72 days old (Sample B and C) respectively was observed due to surface oxidation. These samples produced relatively less Raman intensity count (600- 1000 counts) and also a noisy spectrum compared to Sample A which gave an intensity count of nearly 3000. It was observed that the quality improved with the freshness of the substrate.

This has made it necessary to seal the substrate under inert atmosphere. Figure 21 shows the Raman spectrum investigation on the ageing effect of substrates.

Rhodamine

Rhodamine 6G (R6G), a dye widely used in luminescent chemical analysis, laser technique, and other fields. It could also enhance faint or indistinct impressions developed by other techniques. Rhodamine 6G has an affinity for adhesion to polymerized latent impressions even at levels below visual observation. In the investigation for the instant invention, R6G was chosen as an analyte to demonstrate the substrate performance. Moreover R6G is an analyte that is routinely reported in literature to demonstrate the performance of a SERS active area.

R6G is a strongly fluorescent xanthene derivative which shows a molecular resonance Raman (RR) effect when excited into its visible absorption band (i 7). Here a ImM concentration of R6G is coated on the nanoAg SERS substrates and investigated under Raman. The Raman spectrum was recorded on a LabRAM HR apparatus (Horiba, USA) with an excitation wavelength of 632.8 nm and 5mWcm^"2. D2 filter was used for recording which corresponds to 1/100th of the maximum laser intensity.

It is observed that all the spectral features of R6G can be identified from the substrates of the present invention with a huge intensity count.

Table 3: Raman Bands of Rhodamine 6G

@-Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1984, 88, 5935-

EXAMPLE 5

SERS ACTIVITY WITH DIFFERENT ANALYTES

The SERS activity of substrates of the present invention were tested with different analytes and the respective spectra are as given below.

Raman spectra of pyridine

Pyridine was dissolved in ethanol and the substrates were dipped in for 30 mins. After 30 mins the substrates were washed with ethanol to remove any molecules that are not absorbed to the surface of the substrate. Later the substrates were dried in ambient conditions and later recorded for Raman at 532nm laser. The exposure time was set to 10 sec and acquisition was done for 2 times. According to literature the Raman spectra of pyridine is dominated by two very intense bands around 1000 and 1030 cm-¹, attributed to the ring breathing and ring trigonal mode, respectively (Phys. Chem. Chem. Phys., 2006, 8, 171-178). A similar Raman spectra as reported in literature was obtained in recording of the present invention with a higher enhancement (Figure 23).

Raman spectra of 4 pyridine carboxylic acid

The Raman spectra of I mM Pyridine carboxylic acid is presented in Figure 24. The bands at 839cm- l , 1375cm- l and 1598cm- l , are ascribed to vibration v8a (unprotonated ring nitrogen) v_sym ^co°^" and to ring vibration viob (Elecvochimica Acta. Vol.40, No.15 pp. 2487-2490, 1995). The ring modes at 1009cm- l (vi) is very strong in the SERS spectrum of 4-PCA. In the recording obtained in the present invention, all the bands (Figure 24) that correspond to 4 Pyridine carboxylic acid were obtained and the spectral band frequencies obtained were at 835, 1001 , 1373, 1597 cm- 1.

Raman spectra of PMMA

The SERS spectrum of I mM concentration of PMMA dip-coated onto the SERS substrates is shown in Figure 25 below. The bands near 982, 1026, 1003 cm- 1 (Chinese Chemical Letters Vol. 13, No. 6, pp 563 - 566, 2002) Raman spectra have been assigned to the OCH3 rock vibration interacting with the stretch vibrations of the COC group. Dybal et al. (J. Dybal, S. Krimm, Macromolecules, 1990, 23, 1301 ) confirmed this assignment by the normal coordinate calculations and also the potential energy distribution (PED) indicating a larger contributions from the C-O-C stretch vibrations.

As seen from the SERS spectra of PMMA, the bands near 1340, and 826 cm^{" 1} show great enhancement. Moreover the bands near 1340 and 826 cm^{' 1} are assigned to -CH3 symmetric bend vibration and CH2 rocking vibration respectively and both involve considerable motion perpendicular to the long axis of the molecules. The band near 1454 cm^' ¹ is assigned to CH2 symmetric bend vibration involving motion mostly parallel to the long axis of the molecules. From the SERS spectra the medium strong band in the normal Raman spectrum, near 1732 cm- 1 , is very weak in the SERS spectrum. This band was assigned to C=O stretching mode. However the band near 1004 cm- 1 , which has been assigned to the C-O-C stretch vibration is enhanced largely in the SERS spectra. Both of the two bands are connected with the ester group of PMMA.

However, it is difficult to assign the band near 1607 cm-1 whereas literature has acknowledged the difficulty in assigning the band at 1602 cm- 1 (Chinese Chemical Letters Vol. 13, No. 6, pp 563 - 566, 2002).

SERS spectra of L Alanine

Typical SERS spectra for the Alanine, is shown in Figure 26 where the main vibrational modes of the analyte are clearly identifiable. The typical skeleton, torsional and stretching modes of L-Alanine in the 400- 1000 cm- 1 range is observed from the spectra. Many of the ring stretch and deformation modes are also clearly visible.

SERS spectra of Haemoglobin

The SERS spectra was recorded on substrates that were coated with I mM of thiophenol. Here the strong affinity of thiophenol is utilized, which is a SERS active molecule to the substrates of the present invention to form a monolayer coating and promote the Raman activity of molecules which may be moderate in SERS.

According to the recent literature (K. Kneipp et al Chem. Rew. 1999, 99, 2957-2975 and Hongxing Xu, et al Phys. Rew. Lett., 83, 21 , 1999, 4357), excitation with the 514.5 nm line leads to a SERRS spectrum characterised by three prominent haemoglobin bands: 1375, 1586, 1640 cm^{' 1}. These bands are called "markers" of the hemic group packed in the polipeptidic chain and are assigned to in plane vibrations of the porphyrine ring. In the recording of the present invention, the dominant SERS bands (Figure 27) are located at 1606, 1434 cm^{" 1} being assigned to the v c₌c and v _C=_N modes. The bands at 1 108, 1028 cm^"1 were assigned to v _{= C}-_N, and C-H bending respectively. The out of plane porphyrine bending vibrations are located at 960, 868, 834 and 681 cm^'1 respectively.

SERS spectra of Human blood

SERS spectrum of human blood was successfully obtained and compared to that one of haemoglobin. A small amount of human blood ( 10 μl) was freshly isolated and spot dropped on to the SERS substrates of the present invention. The substrates were allowed to dry in ambient conditions and recorded for Raman. The main hemoglobin marker bands namely 1610 cm^"1 , 1375 cm^"1 , 1 167 cm^{" 1} , 957 cm^{' 1} , 761 cm^"1 and 674 cm^{" 1} (Cinta et al., Physica Special Issue 2001 ) were observed in the SERS spectra of blood, even if some little changes in spectral positions are observed ( 1620, 1372, 1 158, 950, 769, 694 cm^"1) spectral range. Those changes are probably due to the photo- or thermal-induced coagulation of blood (figure 28).

Hence, the Highly Sensitive SERS substrates of the present invention could be employed as a very important tool for detecting small amounts of haemoglobin (micro-nano molar concentrations and even lower) in complicate biosamples, due to its high selectivity and huge enhancement of the Raman signal.

EXAMPLE 6

RAMAN SUBSTRATE PARAMETERS

Table 4: Some key parameters of the Raman Substrate

Some of the key features and applications of 'Highly Sensitive Nanometal Raman Substrates' are presented below.

Colossal enhancement in Raman signal

Highly Sensitive Nanometal Raman substrates provide a huge enhancement in Raman signal that enables detection of molecules at ultra low concentrations and a very less volume. Exceptional cost effectiveness

These Raman substrates are exceptionally cost effective compared with those available in the international market but the performance is on par with those commercially available.

Compatible with standard Raman spectrometers

These Raman substrates can be used directly with the existing Raman spectrometers.

Easy to handle during investigation

These Raman substrates made on paper matrix are flexible and easy to handle during investigations. Being flexible and easy to use is a key advantage from users point of view.

Use and throw

Highly Sensitive Nanometal Raman substrates can be used and thrown away.

Environment friendly disposability

An incinerator is provided along with these substrates facilitates environment friendly disposability of the active area. The active area is burnt into ashes inside the incinerator with the nanoproduct converted to the bulk form.

Various Applications

• Analytical chemistry

• Chemical and biological detection

• Trace analysis

• Forensics

• Pharmaceutical drug development

• Explosive detection References

1. C. D. Trans, Anal. Chem. 1984, 56, 024-026

2. LeuYen-Bower, E.; Ward, J. L.; Walden, G.; Wlneforner, J. D. Talanta 1983, 27, 380-382.

3. J. A. Creighton, C. G. Blactchford, M. G. Albrech, J. Chem. Soc, Faraday Trans. 2 1979, 75, 790-798.

4. P. C. Lee, D. Meilsel, J. Phys. Chem. 1982, 86, 3391-3395.

5. C. D. Trans, Journal of Chromatography, 292 ( 1984) 432-438

6. C. D. Trans, Anal. Chem. 1987, 59, 525-527

7. J. M. Sequaris.; E. Koglin, Anal. Chem. 1986, 59, 1290- 1294.

8. J. J. Laserna. A. D. Campiglia and J. D. Wineforder, Anal. Chim. Acta 208, 21 ( 1988)

9. S. A. Soper, K. L. Ratzlaff and T. Kuwana Anal. Chem 62, 1438 ( 1990).

10. H. Gliemann, U. Nickel and S. Schneider, J. of Raman Spectroscopy 1998, 29, 1041 - 1046.

1 1. L. M. Cabalin, J. J. Laserna Anal. Chimi. Acta (1995) 337-345.

12. J. J. Laserna. W. S. Sutherland and J. D. Wineforder, Anal. Chim. Acta 237, 439- 450 (1990).

13. D. Wu and Y. Fang, Journal of Colloid and Interface Science 265 (2003) 234-238

14. Z. Luo and Y. Fang, Journal of Colloid and Interface Science 283 (2005) 459-463

15. Surface Enhanced Raman Spectroscopy, Christy L. Haynes, Adam D. McFarland, Richard P. Van Duyne*, September, 2005, Analytical Chemistry 339 A

16. A SERS-Active Nanocrystalline Pd Substrate and its Nanopatterning Leading to Biochip Fabrication, Thiruvelu Bhuvana and Giridhar U. Kulkarni*, Small 2008, 4, No. 5, 670- 676 17. Surface-Enhanced Resonance Raman Spectroscopy of Rhodamine 6G Adsorbed on Colloidal Silver, Peter Hildebrandt and Manfred Stockburger*, J. Phys. Chem. 1984,88, 5935-5944

Claims

We Claim

1) A SERS active paper substrate comprising in-situ synthesized nanoparticles.

2) The SERS active paper substrate as claimed in claim 1, wherein the paper is Raman blank in nature.

3) The SERS active paper substrate as claimed in claim 1, wherein the paper is rose colored plain maplitho printing paper.

4) The SERS active paper substrate as claimed in claim 1, wherein the nanoparticles are selected from a group comprising silver and gold nanoparticles.

5) The SERS active paper substrate as claimed in claim 4, wherein size of the nanoparticles ranges from about 10 nm to about 30 nm.

6) The SERS active paper substrate as claimed in claim 5, wherein the nanoparticles are non-uniform in shape with sharp tips.

7) The SERS active paper substrate as claimed in claim 1, wherein the paper comprises a layer of thiophenol.

8) A process for obtaining SERS active paper substrate comprising in-situ synthesized nanoparticles, said process comprising steps of: a) dipping the paper in metallic precursor solution to obtain metal precursor loaded paper; and b) immersing the metal precursor loaded paper into boiling solution of reducing agent followed by cooling and drying to obtain the SERS active paper substrate comprising in-situ synthesized nanoparticles.

9) The process as claimed in claim 8, wherein the nanoparticles are selected from a group comprising silver and gold nanoparticles.

10) The process as claimed in claim 8, wherein the metallic precursor solution is selected from a group comprising Silver Nitrate and Chloroauric Acid.

11) The process as claimed in claim 10, wherein the concentration of metallic precursor solution ranges from about 2M to about 3M.

12) The process as claimed in claim 8, wherein the paper is dipped in metallic precursor solution for a period of about 25 to about 35 minutes. 13) The process as claimed in claim 8, wherein the reducing agent solution comprises a combination of Sodium Citrate and Cetyl trimethylammonium bromide.

14) The process as claimed in claim 13, wherein the concentration of Sodium citrate ranges from about 3OmM to about 4OmM, and the concentration of Cetyl trimethylammonium bromide ranges from about 3mM to about 1OmM.

15) The process as claimed in claim 8, wherein the metal precursor loaded paper is immersed into the boiling solution of reducing agent set to a temperature ranging from about 70⁰C to less than about 75 ⁰C for a period of about 40 to about 50 minutes.

16) The process as claimed in claim 8, wherein the paper is optionally coated with a layer of thiophenol having concentration of about 0.5mM to about 1.5mM.

17) A method to determine Raman Spectra of an analyte, said method comprising steps of: a) applying an analyte on SERS active paper substrate comprising in-situ synthesized nanoparticles; and b) subjecting the paper substrate with analyte to Surface Enhanced Raman Spectroscopy to determine the Raman Spectra of the analyte.

18) The method as claimed in claim 17, wherein the application of analyte is carried out by dipping or spotting.

19) The method as claimed in claim 17, wherein the SERS active paper substrate enhances the sensitivity of Surface Enhanced Raman Spectroscopy of analytes.

20) The method as claimed in claim 17, wherein the SERS active paper substrate enhances the intensity of the Raman Spectra of the analyte by an Enhancement factor of about 2 million.

21) The method as claimed in claim 17, wherein the concentration and volume of the SERS active paper substrate can be as low as about ImM and about lOμl respectively.