WO2016074653A2

WO2016074653A2 - Method for preparation of silver colloidal particle layers onto glass substrate for surface enhanced raman spectroscopy, substrate and use thereof

Info

Publication number: WO2016074653A2
Application number: PCT/CZ2015/050010
Authority: WO
Inventors: Robert Prucek; Petr Suchomel; Libor Kvitek; Ales Panacek; Radek Zboril
Original assignee: Univerzita Palackeho V Olomouci
Priority date: 2014-11-10
Filing date: 2015-10-29
Publication date: 2016-05-19
Also published as: WO2016074653A3

Abstract

The invention provides a method for preparation of layers of silver colloidal particles deposited on glass substrates for surface enhance Raman spectroscopy, wherein a solution consisting of solvent, a soluble silver salt, ammonia, and a reducing agent is exposed to ultrasound irradiation in the presence of the glass substrate, thereby forming silver colloidal particle layer on glass substrate. The invention further provides glass substrate with layer of silver colloidal particles obtainable by said process and its use for surface enhanced Raman spectroscopy with wide range of laser wavelengths used for excitation.

Description

Method for preparation of silver colloidal particle layers onto glass substrate for surface enhanced Raman spectroscopy, substrate and use thereof

Field of Art

The present invention relates to a sonochemichal method of preparation of silver particle layers on glass substrates for the purposes of surface enhanced Raman spectroscopy which represents a very promising and sensitive analytical method. The invention further relates a substrate obtainable by said method.

Background Art

The discovery of surface enhanced Raman scattering (SERS) on a silver electrode by Fleischmann in 1974, and especially its re-discovery on colloidal silver particles by Creighton in 1977 started the extensive development of a new and very sensitive analytical method that allows the detection of molecules in the concentration range from pico- to femtomols (Doerning W. E. and Nie S. M., J Phys Chem B, 106, 2002). A high enhancement of SERS, reaching the values of up to 10¹⁵, even allowed the detection of individual molecule adsorbed on a single silver nanoparticle (Michaels M. et al. J Am Chem Soc, 121, 1999; Nie S. and Emory S. R., Science, 275, 1997). Some studies have shown that so high a value of the enhancement can be achieved only on particles of a certain sizes which are referred to as "hot particles". These particles' optimum size depends on the wavelength of the laser used for the excitation and ranges approximately from 70 nm to 200 nm for the excitation wavelengths in the range from 488 nm to 647 nm (Emory S. R.et al., J Am Chem Soc 120, 1998). Based on the mentioned dependence of the size of the "hot particles" for a given laser wavelength, it can be expected that when using lasers in the red (785 nm) and near-infrared area (1064 nm), the maximum enhancement of Raman signal should be obtained on silver particles of the size of around 400 nm. Unfortunately, particles of this dimension are unstable in solution and they usually sediment within a few hours. Silver nanoparticles of the size ranging from units of nanometers to tens of nanometers can be stable for several months or years, even without any additional stabilization. However, these small particles themselves usually do not provide surface enhancement of the Raman signal. For this purpose, they must be activated for example by the addition of an inorganic ion solution (Leopold, N. and Lendl, B. J Phys Chem B 107, 2003; Michaels A. et al., J Am Chem Soc 121, 1999; Leng W. N. et al., J Raman Spectrosc 37, 2006; Zhang P. X. et al., J Raman Spectrosc 21, 1990; Campbell M. et al., J Raman Spectrosc 30, 1999; Doering W. E. and Nie S. M., J Phys Chem B 106, 2002). But in these cases, a slow and irreproducible aggregation of silver nanoparticles occurs, resulting in Raman signal irreproducibility and often also in a long activation time.

The disadvantages connected with aggregation or sedimentation of silver nanoparticles can be overcome through the formation of metal particle layers (Van Duyne, R. P., et al., J Chem Phys, 99, 1993) on a suitable substrate such as quartz or glass (Heas, A. J., et al., J Am Chem Soc, 128,

2006) .

The most commonly used materials for surface enhanced Raman spectroscopy are silver and gold. The advantages of using silver include its lower price and better optical properties suitable for surface enhanced Raman spectroscopy (Endo, T. et al., Anal Chem 78, 2006).

The methods for the formation of metal particle layers can be divided into deposition from gas phase and formation by growing up of layers from solution, which is represented by electrochemical deposition, chemical deposition from solution, Langmuir-Blodgett film technique, and self-assembling (Cao, G., Nanostructures and nanomaterials: synthesis, properties and applications, Imperial College Press, London, 2004).

Deposition of particles on the glass substrate can also be performed by lithographic method based on formation of self-assembled layers of polystyrene particles serving as a lithographic mask. After deposition of metal nanoparticles (silver or gold in most cases), the polystyrene particles are removed by organic solvent (Hicks, E. M., J Phys Chem B 109, 2005; Meli, M.V. and Lennox, R.B., J Phys Chem C 111, 2007; Stranik, O., et al., Sensor Actuat B-Chem 107, 2005; Haes, A. J., et al., J Phys Chem B 108, 2004; Hicks, E. M., et al., J Phys Chem C 111,

2007) . The disadvantage of the most of the above mentioned techniques are both high costs for instrumental equipment and long time needed for silver particle layer preparation.

Another available technique of metal layer formation involves deposition of one or more layers using polyelectrolytes such as polydiallyldimethylammonium chloride (PDDA) (Chapman, R. and Mulvaney, P., Chem Phys Lett, 349, 2001) or polyethylenimine (PEI) (Michna, A., et al., J Colloid Interf Sci, 345, 2010). In this approach, the layers of particles are captured between layers of polyelectrolyte through electrostatic interactions. Glass substrate is then immersed into a solution of polyelectrolyte or polymer for several minutes or hours. After careful washing of the glass substrate to remove the excess of polyelectrolyte or polymer, the glass is inserted into a dispersion of silver nanoparticles prepared separately a priori. The whole process takes time on the order of units or tens of hours. Unfortunately, the described methods are usually time consuming, very sensitive to thorough cleaning of the surface, and also require activation of substrate surface prior to deposition of silver particles. Also the presence of polyelectrolyte in the surface of silver particle layers can negatively influence the applicability of such substrate in surface enhanced Raman spectroscopy.

Other way to obtain silver particle layers involves exploitation of 3- aminopropyltriethoxysilane (APTES), which is able to form covalent bond with the activated surface of glass or quartz substrate. When APTES is bonded on the surface, its amino groups can interact through free electron pair with silver nanoparticles (Kim, J., et al., Surf Sci, 602, 2008). This technique requires perfect cleaning of substrate and activation by solutions such as H₂O₂ with NH₄OH or H₂O₂ with H₂SO₄ (piranha solution), for example in the solution of 30% H₂O₂ a 98% H₂SO₄ (1 :3) or 5: 1 : 1 H₂O/28% NH₄OH/30% H₂O₂. Moreover, also in this case the silver nanoparticles must be prepared in a separate step before their subsequent application onto the layer of 3- aminopropyltriethoxysilane residues anchored on the glass or quartz support. The whole process is quite time consuming - takes up several hours.

One of the promising techniques is based on sonochemical preparation of silver layers. Through sonochemical approach, Perkas et al. prepared layers of silver particles stabilized by polyvinylpyrrolidone (PVP) on glass substrates with substantial antibacterial activity. However, application of the reported method in preparation of the effective SERS substrate is questionable due to interfering of the Raman signal originated from the PVP polymer used as stabilizer with Raman signal of analyzed molecules adsorbed on this type of silver nanoparticle layer. The prepared layers using PVP stabilizer consist from polydispersed system of particles (tens to thousands of nanometers) (Perkas N. et al. Nanotechnology 19, 2008). Considerable polydispersity deposited particles substantially decreased reproducibility of exploitation layers in surface enhanced Raman spectroscopy.

The object of US 2014017448 is the preparation of silver layers for surface enhanced Raman spectroscopy (SERS) using imprinting stabilized silver particles onto the appropriate substrate. However, after layer deposition, the residual organic stabilizer must be thermally decomposed in order to remove it from the layer. Also, the generated layer is composed from polydisperse system of particles.

CN 102628809 relates to the formation of noble metal particle layers onto filter paper through physical deposition from gas phase.

The object of the invention CN101566571 is a relatively complicated method for preparation of silver nanoparticle layers. In the first step, polyaniline is dissolved in N-methylpyrrolidone, thereafter the prepared solution is deposited onto glass substrate. In the next step, the gold particle layer is formed, and on top of this layer the silver nanoparticle layer is prepared.

The object of CN101492254 is a time-consuming and complicated technique of particle layer preparation using polyelectrolytes - polydipropylene dimethyl amonnium chloride. The method is similar to the method presented in Chapman, R. and Mulvaney, P., Chem Phys Lett, 349,

2001.

The object of US6406777 is method where in the first step a glass substrate is etched for its roughening. After that an adhesion layer is formed which serves for the anchoring of aggregated silver, gold, or copper particles. The surface of the prepared layers is finally modified by thiols.

Disclosure of the Invention

Object of the present invention is a method for preparation of silver colloidal particle layer onto a glass substrate for use in surface enhanced Raman spectroscopy wherein a solution consisting of a solvent, a soluble silver salt, ammonia, and a reducing agent is exposed to ultrasound irradiation in the presence of the glass substrate, thereby forming silver colloidal particle layer on the glass substrate. Using the sonochemical method, it is possible to prepare within a short time (minutes, preferably up to 10 minutes) a homogeneous layer of silver particles which can be exploited as an effective substrate for surface enhanced Raman spectroscopy purposes.

The method is preferably performed in such a way that a reaction vessel containing the substrate and the solution of the silver salt (preferred solvent is water) is provided, and a reducing agent is added therein. Thereafter, sonication (=ultrasound irradiation) is initiated, and immediately after the initiation of sonication, ammonia is added into the reaction mixture. Preferably, the sonication is interrupted at least once during the course of the reaction. In a preferred embodiment, sonication is carried out for 3 to 7 minutes.

The final concentration of silver ions in the reaction system is typically in the range from 0.005 to 0.1 mol-dm -^"3 , preferably 0.005 to 0.05 mol-dm -^"3 , more preferably 0.005 to 0.02 mol-dm -^"3. Any water-soluble salt containing silver ions can be used as the source of silver ions, provided that its solubility allows to achieve the minimum limits of the silver ion concentrations provided herein above in the solvent, preferably in aqueous solution. The reducing agent is preferably glycerol, ethylene glycol, or reducing saccharides such as glucose or maltose.

Sonochemical preparation of silver particle layers is preferably performed in plastic reaction vessels (and not in glass reaction vessels) in order to achieve a higher yield of the deposited silver on glass substrate in plastic reaction vessels.

A further object of the present invention is a glass substrate provided with a layer of silver colloidal particles obtainable by the method of the present invention, wherein the silver colloidal particle size is in the range from 25 nm to 1000 nm, preferably 25 to 250 nm.

Another aspect of the present invention is use of the substrate with silver colloidal particle layer obtainable by the method of the present invention for surface enhanced Raman spectroscopy using excitation laser wavelength between 532 nm and 1064 nm.

A particular advantage of the substrate with silver colloidal particles according to the present invention over the state of the art is the fact that the silver colloidal particle layers on the glass substrates are usable for application in surface enhanced Raman spectroscopy with a wide range of excitation laser wavelengths, i.e. minimally 532 nm to 1064 nm.

Brief Description of Drawings

Figure 1 shows a digital photo (a), an image from scanning electron microscope (b) and UV-vis absorption spectra (c) of silver colloidal particle layer on glass substrate prepared according to Example 1.

Figure 2 shows a digital photo (a), an image from scanning electron microscope (b) and UV-vis absorption spectra (c) of silver colloidal particle layer on glass substrate prepared according to Example 2.

Figure 3 shows summary of recorded UV-vis absorption spectra of silver colloidal particle layer on glass substrate prepared according to Example 2.

Figure 4 shows a digital photo (a) and an image from scanning electron microscope (b) of silver colloidal particle layer on glass substrate prepared according to Example 4.

Figure 5 shows a digital photo (a) and an image from scanning electron microscope (b) of silver colloidal particle layer on glass substrate prepared according to Example 5. Figure 6 shows a digital photo (a) and image from scanning electron microscope (b) of silver colloidal particle layer on glass substrate prepared according to Example 6.

Figure 7 shows surface enhance Raman spectra of adenine. Application of 20 μΐ adenine solution (concentration 10^"6 mol-dm^"3) onto silver colloidal particle layer on glass substrate prepared according to Example 6 using lasers with excitation wavelengths 532 nm (a), 785 nm (b) a 1064 nm (c).

Examples of Carrying Out the Invention

Silver nitrate (Sigma-Aldrich, p. a.) was used as a precursor of silver particles. Ethylene glycol (Sigma-Aldrich, p. a.), glycerol (Sigma-Aldrich, p. a.), maltose (Sigma-Aldrich, p. a.), glucose (Sigma-Aldrich, p. a.) and lactose (Sigma-Aldrich, p. a.) were used as reducing agents. Polyvinylpyrrolidone (PVP, Sigma-Aldrich, M.W. 40 000) was used as a stabilizer. Ammonia (Sigma-Aldrich, 28 - 30% aqueous solution) was used as a complexing agent. Adenine (Sigma-Aldrich, 99 %) was used for the SERS experiments as a model analyte. All chemicals were used without additional purification. Deionized water (18 ΜΩ-cm, Millipore) was used for preparation of all solutions. The silver particle layers were deposited on glass microscope cover slips (Menzel-Glaser, 18x18 mm) using sonochemical approach. Before deposition, cover slips were thoroughly cleaned by detergent and washed by deionized water. After cleaning, cover slips were carefully inserted vertically in the distance of 1 cm from the sonication probe, using wire holders in a beaker, and then solutions of the reaction precursors were added.

Silver layers on glass slips were prepared by ultrasonic processor Q700 with standard titanium probe 4220 (QSonica LLC, USA, diameter 12.7 mm), 700 W, 20 kHz. Glass slips covered by silver nanoparticles were characterized using scanning electron microscope Hitachi SU6600 (Hitachi, Japan) and UV-Vis spectrometer Specord S600 (Analytic Jena AG, Germany). Silver concentrations were determined by the AAS technique with flame ionization using a ContrAA 300 (Analytik Jena AG, Germany) equipped with a high-resolution Echelle double monochromator (spectral bandwidth of 2 pm at 200 nm) and with a continuum radiation source (xenon lamp). The absorption line used for these analyses was 328.0683 nm.

Surface enhanced Raman spectra with the 532 nm excitation laser were recorded using a DXR Raman Microscope (Thermo Scientific) equipped with a thermoelectrically cooled (-50 °C) charge-coupled device (CCD) camera and a 4x objective, using excitation laser wavelength 532 nm. Surface enhanced Raman spectra with the 785 nm and 1064 nm excitation lasers were recorded using iRaman Plus (BWTEK Inc., USA), scan time 10 s, 6 accumulations were made. The laser light power was 100 mW. For the SERS measurement, 20 μΐ of 10^"6 mol-dm^"3 adenine solution was used.

Example 1

Preparation of silver colloidal particle layer on glass substrate using 0.05 mol-dm^" solution of silver nitrate and 30% intensity of maximal sonication power.

Before deposition, cover slips were thoroughly cleaned by detergent and washed by deionized water. After cleaning, 4 cover slips were carefully inserted vertically using wire holders into 50 ml beaker and then solutions of reaction precursors were added. The 50 ml beaker with glass substrate was immersed using holder into 600 ml beaker with adequate amount of water mixed by magnetic stirrer used to cooling of the reaction mixture. The amount of 5 ml of 0.25 mol-dm^" silver nitrate solution was diluted by 16 ml of deionized water. After that, 2.5 ml of ethylene glycol was added to the reaction mixture. The final volume was 25 ml. After mixing, the sonication tip was immersed into the reaction mixture (1 cm under solution surface). Parameter of sonication was adjusted to the amplitude value equal to 30 % and the sonication begun. Immediately after the start, 1.5 ml of 3% ammonia solution was rapidly injected into the beaker. The process was stopped after 5 minutes for 30 seconds, the sonication was carried out for additional 2 minutes. After that, the sonication tip was pulled out of solution and after 2 minutes, the covered glass substrate was pulled out of the reaction mixture. The glass slips were then pulled out of the holders, washed by deionized water and dried by air flow. The size of the deposited silver particles on glass substrate (Figure la) determined by scanning electron microscopy ranged from 50 nm to 200 nm (average particle size approximately 100 nm) (Figure lb). Homogeneity of the prepared silver particle layer was declared by 4 UV-vis absorption spectra recorded at 4 different sites onto glass substrate covered by silver particles (Figure lc).

Example 2

Preparation of silver colloidal particle layer on glass substrate using 0.05 mol-dm^" solution of silver nitrate and 30% intensity of maximal sonication power

Before deposition, cover slips were thoroughly cleaned by detergent and washed by deionized water. After cleaning, 4 cover slips were carefully inserted vertically using wire holders into 50 ml beaker and then solutions of reaction precursors were added. The 50 ml beaker with glass substrate was immersed using holder into 600 ml beaker with adequate amount of water mixed by magnetic stirrer used to cooling of the reaction mixture. The amount of 5 ml of 0.25 mol-dm^" silver nitrate solution was diluted by 17.2 ml of deionized water. After that, 2.5 ml of glycerol was added to the reaction mixture. The final volume was 25 ml. After mixing, the sonication tip was immersed into the reaction mixture (1 cm under solution surface). Parameter of sonication was adjusted to the amplitude value equal to 30 % and the sonication begun. Immediately after the start, 0.3 ml of 3% ammonia solution was rapidly injected into the beaker. The process was stopped after 4 minutes for 30 seconds, and the sonication was carried out for additional 1 minute. After that, the sonication tip was pulled out of solution and after 2 minutes, the covered glass substrate was pulled out of the reaction mixture. The glass slips were then pulled out of the holders, washed by deionized water and dried by air flow. The size of the deposited silver particles on glass substrate (Figure 2a) determined by scanning electron microscopy ranged from 25 nm to 150 nm (average particle size approximately 120 nm) (Figure 2b). Homogeneity of the prepared silver particle layer was declared by 4 UV-vis absorption spectra recorded at 4 different sites onto glass substrate covered by silver particles (Figure 2c).

Example 3

Reproducibility of preparation the silver colloidal particle layers on glass substrate prepared according to Example 2 demonstrated by recorded UV-vis spectra.

Before deposition, cover slips were thoroughly cleaned by detergent and washed by deionized water. After cleaning, 4 cover slips were carefully inserted vertically using wire holders into 50 ml beaker and then solutions of reaction precursors were added. The 50 ml beaker with glass substrate was immersed using holder into 600 ml beaker with adequate amount of water mixed by magnetic stirrer used to cooling of the reaction mixture. The amount of 5 ml of 0.25 mol-dm^" silver nitrate solution was diluted by 17.2 ml of deionized water. After that, 2.5 ml of glycerol was added to the reaction mixture. The final volume was 25 ml. After mixing, the sonication tip was immersed into the reaction mixture (1 cm under solution surface). Parameter of sonication was adjusted to the amplitude value equal to 30 % and the sonication begun. Immediately after the start, 0.3 ml of 3% ammonia solution was rapidly injected into the beaker. The process was stopped after 4 minutes for 30 seconds, then the sonication was carried out for additional 1 minute. After that, the sonication tip was pulled out of solution and after 2 minutes, the covered glass substrate was pulled out of the reaction mixture. The slips were then pulled out of the holders, washed by deionized water and dried by air flow. The preparation of the silver particle layers on glass substrate was independently repeated 6 times and UV-vis absorption spectra are presented in the Figure 3. Example 4

Preparation of silver colloidal particle layer on glass substrate using 0.005 mol-dm^" solution of silver nitrate and 30% intensity of maximal sonication power.

Before deposition, cover slips were thoroughly cleaned by detergent and washed by deionized water. After cleaning, 4 cover slips were carefully inserted vertically using wire holders into 50 ml plastic beaker and then solutions of reaction precursors were added. The 50 ml plastic beaker with glass substrate was immersed using holder into 600 ml beaker with adequate amount of water mixed by magnetic stirrer used to cooling of the reaction mixture. The amount of 0.5 ml of 0.25 mol-dm^" silver nitrate solution was diluted by 23.9 ml of deionized water. After that, 0.5 ml of glycerol was added to the reaction mixture. The final volume was 25 ml. After mixing, the sonication tip was immersed into the reaction mixture (1 cm under solution surface). Parameter of sonication was adjusted to the amplitude value equal to 30 % and the sonication begun. Immediately after the start, 0.1 ml of 3% ammonia solution was rapidly injected into the beaker. The process was stopped after 4 minutes for 30 seconds, then the sonication was carried out for additional 1 minute. After that, the sonication tip was pulled out of solution and after 2 minutes, the covered glass substrate was pulled out of the reaction mixture. The glass slips were then pulled out of the holders, washed by deionized water and dried by air flow. The size of the deposited silver particles on glass substrate (Figure 4a) determined by scanning electron microscopy ranged from 100 nm to 1000 nm (Figure 4b). Homogeneity of the prepared silver particle layer was declared by 4 UV-vis absorption spectra recorded at 4 different sites onto glass substrate covered by silver particles (Figure 4c). In this case, the prepared silver particle layer could not be characterized by UV-vis absorption spectra because of very high light absorption of the formed silver particle layer.

Example 5

Preparation of silver colloidal particle layer on glass substrate using 0.01 mol-dm^" solution of silver nitrate and 30% intensity of maximal sonication power.

Before deposition, cover slips were thoroughly cleaned by detergent and washed by deionized water. After cleaning, 4 cover slips were carefully inserted vertically using wire holders into 50 ml plastic beaker and then solutions of reaction precursors were added. The 50 ml plastic beaker with glass substrate was immersed using holder into 600 ml beaker with adequate amount of water mixed by magnetic stirrer used to cooling of the reaction mixture. The amount of 1 ml of 0.25 mol-dm^" silver nitrate solution was diluted by 22.7 ml of deionized water. After that, 1 ml of glucose was added to the reaction mixture. The final volume was 25 ml. After mixing, the sonication tip was immersed into the reaction mixture (1 cm under solution surface). Parameter of sonication was adjusted to the amplitude value equal to 30 % and the sonication begun. Immediately after the start, 0.3 ml of 3% ammonia solution was rapidly injected into the beaker. The process was stopped after 3 minutes for 30 seconds, the sonication was carried out for additional 1 minute. After that, the sonication tip was pulled out of solution and after 2 minutes, the covered glass substrate was pulled out of the reaction mixture. The glass slips were then pulled out of the holders, washed by deionized water and dried by air flow. The size of deposited silver particles on glass substrate (Figure 5a) determined by scanning electron microscopy ranged from 40 nm to 200 nm (Figure 5b). In this case, the prepared silver particle layer could not be characterized by UV-vis absorption spectra because of very high light absorption of the formed silver particle layer.

Example 6

Preparation of silver colloidal particle layer on glass substrate using 0.01 mol-dm^" solution of silver nitrate and 30% intensity of maximal sonication power

Before deposition, cover slips were thoroughly cleaned by detergent and washed by deionized water. After cleaning, 4 cover slips were carefully inserted vertically using wire holders into 50 ml plastic beaker and then solutions of reaction precursors were added. The 50 ml plastic beaker with glass substrate was immersed using holder into 600 ml beaker with adequate amount of water mixed by magnetic stirrer used to cooling of the reaction mixture. The amount of 1 ml of 0.25 mol-dm^" silver nitrate solution was diluted by 22.7 ml of deionized water. After that, 1 ml of maltose was added to the reaction mixture. The final volume was 25 ml. After mixing, the sonication tip was immersed into the reaction mixture (1 cm under solution surface). Parameter of sonication was adjusted to the amplitude value equal to 30 % and the sonication begun. Immediately after the start, 0.3 ml of 3% ammonia solution was rapidly injected into the beaker. The process was stopped after 3 minutes for 30 seconds, then the sonication was carried out for additional 1 minute. After that, the sonication tip was pulled out of solution and after 2 minutes, the covered glass substrate was pulled out of the reaction mixture. The glass slips were then pulled out of the holders, washed by deionized water and dried by air flow. The size of deposited silver particles on glass substrate (Figure 6a) determined by scanning electron microscopy ranged from 40 nm to 200 nm (Figure 6b). In this case, the prepared silver particle layer could not be characterized by UV-vis absorption spectra because of very high light absorption of the formed silver particle layer. Example 7

Use of the silver particle layers prepared according to Example 6 in surface enhanced Raman spectroscopy

The measurements of surface enhanced Raman spectra were performed using adenine solution (concentration equal to 10^"6 mol-dm^"3). Adenine served as a model analyte. For the SERS measurement, 20 μΐ of 10^"6 mol-dm^"3 adenine solution was applied on surface of glass substrate with deposited silver particle layers. The measurements were performed for lasers with excitation wavelengths 532 nm, 785 nm a 1064 nm (Figure 7). The enhancement factor was determined by comparing the intensities of the most intensive peak in spectrum of adenine at 738 cm -^"1 of Raman spectra of 0.1 mol-dm -^"3 adenine solution and surface enhanced Raman spectra of 10^"6 mol-dm^"3 adenine solution. The enhancement factor was above the value of 10⁵.

Claims

1. A method for preparation of silver colloidal particles on glass substrate for surface enhanced Raman spectroscopy, characterized in that a solution consisting of solvent, a soluble silver salt, ammonia, and a reducing agent is exposed to ultrasound irradiation in the presence of the glass substrate, thereby forming silver colloidal particle layer on the glass substrate.

2. The method according to claim 1, characterized in that the glass substrate substrate is inserted into a vessel, a solution of the silver salt is provided in said vessel and the reducing agent is added, subsequently ultrasound irradiation is carried out, whereas immediately after starting the ultrasound irradiation, ammonia solution is added to the reaction mixture.

3. The method according to claim 2, characterized in that the ultrasound irradiation is interrupted at least once within the process of ultrasound irradiation.

4. The method according to claim 2 or 3, characterized in that the period of ultrasound irradiation is between 3 and 7 minutes.

5. The method according to any one of the preceding claims, characterized in that the concentration of silver ions in the reaction mixture is in the range 0.005 to 0.1 mol-dm^" .

6. The method according to any one of the preceding claims, characterized in that the reducing agent is selected from glycerol, ethylene glycol and reducing saccharide such as maltose, glucose.

7. A glass substrate with silver colloidal particle layer obtainable according to any one of the preceding claims, wherein the size of silver colloid particles is in the range from 25 nm to 1000 nm, preferably 25 nm to 250 nm.

8. Use of the glass substrate with silver colloidal particle layer according to Claim 7 in surface enhanced Raman spectroscopy with excitation laser wavelengths within the range from 532 nm to 1064 nm.