WO2010094106A1 - Substrate for surface-enhanced raman scattering - Google Patents

Abstract

A simple and robust platform for SERS ultradetection involves a SERS-active composite of Au(O) nanoparticles and polyethylene glycol-based microparticles.

Description

SUBSTRATE FOR SURFACE-ENHANCED RAMAN SCATTERING

Cross-reference to Related Applications

This application claims the benefit of United States Provisional Patent Application USSN 61/207,911 filed February 18, 2009, the entire contents of which is herein incorporated by reference

Field of the Invention

The present invention relates to Surface-enhanced Raman Scattering (SERS), more particularly to substrates for SERS

Background of the Invention

Surface-enhanced Raman Scattering (SERS) is recognized as a powerful tool for biodiagnostics, for example ultrasensitive chemical and biochemical analysis (Cao 2002; Kasili 2004; Kneipp 2006; Moore 2004; Stuart 2005, Alvarez-Puebla 2007a, Alvarez- Puebla 2007b). Among common substrates for SERS₁ the most important are colloidal nanocrystal suspensions of gold, silver and their alloys, produced by a variety of physical and chemical reduction methods (Kneipp 1999; Kelly 2003; Aroca 2005). In a colloidal suspension, the closer interaction between the adsorbent and adsorbate allows the analyte to be naturally retained on the colloid's surface. These mixed systems may later be studied directly by bulk SERS or cast and air-dried onto an appropriate substrate (glass or silicon wafers) and analyzed by microspectroscopic SERS (Pieczonka 2005; Adamson 1997; Evans 1999). The main disadvantage of this method is the low stability of colloidal suspensions, which limits their broad application. Physical (i.e , shape and size) and chemical (i.e., surface charge) properties often vary within days of preparation. Thus, in order to extend the shelf life of the colloidal suspensions, the addition of stabilizers to prevent aggregation and flocculation by increasing the electrostatic potential around the nanoparticles was reported. However, while this process stabilizes the particles, it hinders the interaction between the adsorbent and adsorbate, thus decreasing the SERS output (Farah 2008)

In recent years, many attempts have been made to deposit optically active nanoparticles on polymer microbeads in order to produce composite materials suitable for SERS. Unfortunately, such materials tend to have low nanoparticle loading and weak

SERS because of limited nanoparticle accessibility in the polymer matrix (Zhang 2004a;

Zhang 2004b; Larsson 2004; Skirtach 2005; Giesfeldt 2005). Recently, Kinoshita et al. (Kinoshita 2006a Kinoshita 2006b) established the extraordinary capability of polyethylene glycol (PEG) to selectively retain gold ions

There remains a need for SERS suitable substrates having good stability

Summary of the Invention

In accordance with the present invention, there is provided a SERS-active composite material comprising nanoparticles of Au(O), and, microparticles comprising polyethylene glycol

Microparticles comprising polyethylene glycol (PEG) are preferably in the form of microbeads The microparticles preferably have a homogeneous average diameter in a range of from about 1 μm to about 500 μm Preferably, the microparticles comprise graft copolymers in which polyethylene glycol is grafted to a polymeric scaffold The polymeric scaffold comprises any polymeric material having sufficient porosity to allow penetration of gold ions and reducing agent for the gold ions Thermoplastic and elastomeπc polymeric scaffolds are preferred Examples of suitable polymeric scaffolds include, for example, olefinics, vinylics, styrenics, acrylonitπlics, acrylics, cellulosics polyamides, thermoplastic polyesters, thermoplastic polycarbonates, sulfone polymeres, imide polymers, ether-oxide polymers, ketone polymers, fluoropolymers, thermoplastic elastomers, polyisoprene polybutadiene polychloroprene butyl rubbers, styrene- butadiene rubber and the like Particularly preferred polymeric scaffolds are styrenics, which include, for example, polystyrene (PS), polyparamethylstyrene (PPMS), polyalphamethylstyrene (PAS), rubber-toughened impact polystyrene (HIPS) styrene- butadiene (P(S-B)), styrene-acrylonitrile (P(S-AN)), other polystyrene derivatives and the like Polystyrene (PS) is of particular note Such microparticles may be purchased from a supplier or produced by adapting methods generally known in the art (e g Fenniri 2003) Suppliers of PEG-based polymers suitable for this application include, for example, Rapp Polymere GmbH, Novabiochem, and Polymer Laboratories lnc

The polymeric scaffold has sufficient porosity and swelling properties in a suitable solvent to allow penetration of gold ions and reducing agent into the microparticle Pore sizes are preferably from a few nanometers to a few micrometers (e g from about 2 nm to about 5 μm) depending on the size of the beads and the polymeric scaffold

Composites of the present invention may be produced by chemically modifying the microparticles with gold nanoparticles To form the composite, the microparticles may be suspended in a solution of Au cations, preferably Au(III) ions, allowing the gold ions to diffuse into PEG-rich regions of the microparticles, where they may then be reduced to the 0 oxidation state with a reducing agent. Gold ions may be supplied in the form of a solution, preferably an aqueous solution, of a gold salt. Gold salts include, for example, MAuCI₄ where M is a monovalent cation (e.g H⁺, Li⁺, Na⁺, K⁺). Au ions are preferably used at a concentration in a range of about 1 mM to about 5 mM, more preferably at about 2 5 mM. Any suitable reducing agent may be used, for example NaBH₄, LiAIH₄, diisobutylaluminium hydride (DIBAL), hydrazine, citric acid, ascorbic acid, or mixtures thereof. The reducing agent is preferably used in an amount that maximizes the amount of gold that is reduced, for example, about 1-10 mole equivalents based on the moles of gold ions.

Gold nanoparticles in the composite preferably have an average diameter in a range of from about 50 nm to about 80 nm.

Composites of the present invention are useful as substrates for SERS-based ultradetection of substances. Substances include chemical substances (e.g. drugs, pesticides, explosives) or biological substances (e.g. amino acids, peptides, proteins, nucleotides, nucleic acids, whole microorganisms). The composite advantageously demonstrates excellent chemical and optical stability, shelf life, reproducibility, and synthetic accessibility making it an inexpensive and convenient platform for SERS-based ultradetection. Notably, once the composites are fabricated, they can be stored for long periods of time without deterioration of their optical properties. The tremendous simplicity of fabrication and low cost makes these composites a valuable tool for many potential users with no prior knowledge or experience in SERS-active composite materials.

Further features of the invention will be described or will become apparent in the course of the following detailed description.

Brief Description of the Drawings

In order that the invention may be more clearly understood, embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings, in which:

Fig 1 depicts A) SEM images of Au-PEG/PS bead, B) SEM close up Fig. 1A showing gold nanostructures attached to the surface, and C) cross-sectional TEM with XRD pattern of the bead crust of the Au-PEG/PS bead of Fig. 1A. Fig 2 depicts A) XPS of Au-4f, B) SPR of Au-4f, C) C-1S XPS bands of the Au- PEG/PS composite microbeads, and D) Raman spectra (λ_dx 532 nm) of PS, PEG/PS and Au-PEG/PS microbeads

Fig 3 depicts A) SERRS spectrum of R6G (Inset absorption/emission electronic spectra), B) SERRS mapping of one microbead showing the distribution of R6G (at 1650 cm ¹), C) variation of the SPR, and D) SERRS signal of R6G on Au-PEG/PS microbeads with the number of cycles of drying and re-suspension in water

Fig 4 depicts A) Raman spectra of serotonin (5-hydroxytryptamιne, 5-HT), B) SERS spectra of 5-HT on gold colloids and on Au-PEG/PS microbeads (Raman spectrum of PEG/PS with the same concentration of 5-HT is also shown), C) ultradetection of 5-HT on Au-PEG/PS microbeads wherein the last spectrum corresponds to 5-HT where PEG/PS vibrations had been subtracted, and D) SERS mapping of one Au-PEG/PS microbead, showing the distribution of 5-HT (at 1531 cm ¹)

Description of Preferred Embodiments

Examples

Chemicals were purchased from Aldrich and Fluka and were used as received unless otherwise stated

Example 1 Preparation of PEG/PS microbeads and Au-PEG/PS microbeads

Polystyrene-polyethylene glycol grafted copolymer microbeads, with sizes ranging from 106-212 μm, were prepared as previously reported (Fenniπ 2003) They can also be purchased from Rapp Polymer or Novabiochem

Nanostructured Au-PEG/PS microbeads were prepared as follows 0 100 g of dry PEG/PS microbeads were suspended in a 2 5 mM aqueous solution of HAuCI₄ (50 mL deionized water) under stirring at 298 K After 24 h (Kinoshita 2006a, Kinoshita 2006b), samples were centrifuged filtered and washed with deionized water (3 x 50 mL) to remove residual gold ions The microbeads were then suspended in deionized water (25 mL) and an aqueous solution of NaBH₄ (4 mM, 25 mL) was added under vigorous stirring Reduction was carried out at 298 K for 30 mm, giving rise to red colored microbeads, indicating the formation of nanostructured gold on their surfaces The resulting Au- PEG/PS microbeads were then centrifuged, filtered air-dried and stored Other HAuCI₄ concentrations (1 and 5 mM) were tested However, the 2 5 mM concentration provided better material for SERS. The same process was carried out with Ag⁺ ions, but the retention yields were very low (see Table 1 below).

Example 2: Characterization Au-PEG/PS microbeads

The amount and oxidation state of gold (and silver) within the microbeads were studied by X-ray photoelectron spectroscopy (XPS) (Axis 165 XPS, Kratos Analytical). Distribution of gold on the outer surfaces of the microbeads was carried out by means of high resolution scanning electron microscopy SEM (FE-SEM, Hitachi S4800) operating at a voltage of 10-25 kV. For transmission electron microscopy (TEM), the microbeads were mixed with L. R. white resin medium in a gelatin capsule and placed in the oven overnight at 60⁰C. The resulting blocks were sectioned with a Reichert Jung ultramicrotome to obtain slices of approximately 50-70 nm thickness for the TEM study. TEM images and selected area electron diffraction (SAED) patterns were recorded on a JEOL 2010 microscope working at 200 keV. Ultraviolet-visible (UV-Vis) and surface plasmon resonance (SPR) spectra were collected on an Agilent 8453 UV-Vis Spectrophotometer. All the vibrational experiments (Raman and surface-enhanced Raman scattering) were carried out with 532 and 780 nm excitation laser lines. The inelastic scattered radiation was collected with a Nicolet Almega micro-Raman system. Spectra were collected using high resolution gratings with accumulation times of 10 seconds. All measurements were made in a backscattehng geometry using a 10x microscope objective, providing scattering areas of about 3.2 μm². Power at the sample was varied between 1.6 mW to 1 μW.

SEM images of the Au-PEG/PS microbeads showed microspheres of 100-200 μm in diameter characterized by the presence of about 10 μm macropores (Fig. 1A). More detailed images showed gold nanoparticles (50 to 80 nm) directly attached onto the surface of a microbead (Fig. 1 B). Transmission electron microscopy (TEM) images confirmed gold nanostructures forming a homogeneous crust of approximately 20 nm in thickness (Fig. 1C).

Selected area electron diffraction (SAED) patterns (inset in Fig. 1C) revealed a set of diffraction rings corresponding to the [111], [200], [220], [311], and [222] reflections, indicative of metallic gold nanoparticles with a face-centered cubic polycrystalline structure. This is in agreement with both X-ray photoelectron spectroscopy (XPS) and SPR. The XPS (Fig. 2A) clearly confirms the presence of metallic gold (4^ = 87.7 and 4f_7/2 = 84.0 eV) (Boyen 2002), and the SPR (Fig. 2B) shows an absorption band between 400 and 650 nm with a maximum centered at 527 nm These values are consistent with literature values for solid Au nanostructures of similar size (Daniel 2004, Burda 2005)

XPS results for the Au-PEG/PS microbeads also substantiates the PEG interaction (C-1s and Au-4f bands) (Fig 2A, Fig 2C) Deconvolution of the C-1s signal for the Au-PEG/PS microbead revealed two peaks at 286 2 and 288 6 eV due to C-C/C- H C-O bonds and a very weak signal at 290 eV due to the π-π* shake-up satellite peaks from the PS phenyl groups (Jannasch 1998) The C-1s signal (due to the composite material) showed the same bands but with a notable decrease in intensity for the C-O binding energy, suggesting a strong interaction between the Au nanostructures and the PEG domains of the nanocomposite Deconvolution of Au-4f bands showed the presence of a significant contribution (24%) from bands at 85 8 and 89 5 eV, assigned to Au bonded to oxygen (Boyen 2002), further supporting a strong interaction between the AuNPs and PEG Moreover, chemical analysis of the nanocomposite surface (Table 1) established C and O as main compositional elements, with detectable amounts of Au (5 8%) and Na, and traces of Cl Comparison of PEG/PS and Au-PEG/PS surface chemical analysis shows that while C and O content remain similar, the composite material features decreased levels of sodium with increased levels of gold This result is in agreement with the established affinity of PEG for gold (Kinoshita 2006a, Kinoshita 2006b) Attempts to fabricate Ag-PEG/PS microbeads using silver salts instead of gold failed to yield SERS active platforms because of the poor affinity of Ag for the PEG/PS matrix (Table 1)

Table 1 - XPS chemical analysis of microbead surfaces⁸

³ Atomic % obtained by XPS measurements

The Raman spectrum of PEG/PS microbeads (Fig 2D) shows vibrational features characteristic of each compositional polymer, PS and PEG in agreement with literature values (Reynolds 1990, Yang 1997, Harder 1998) The spectrum of the composite material attests that the gold nanostructures did not have any enhancing effect on the PS Raman signals However, a weak but clear enhancement of bands due to the PEG polymer is observed with the appearance of additional vibrational modes not seen in the PEG/PS microbead 1351 cm^'1 (out-of-plane H-C-H deformation), 1141 , 1126 and 1063 cm^"1 (C-C and C-O stretching), 822 cm^"1 (out-of-plane H-C-H deformation) and 362 cm^"1 (C-O-C bending) (Yang 1997; Harder 1998). The enhancement of the signal intensity resulting from PEG moieties suggests that the gold nanostructures are confined to the PEG domains of the nanocomposite.

Example 3- Optical stability of the Au-PEG/PS microbeads

The potential of the Au-PEG/PS microbeads as an ultrasensitive SERS/SERRS analytical platform, and the establishment of their optical stability were investigated using Rhodamine 6G (R6G) as a molecular probe. Stability was studied by means of SPR and SERRS using R6G as an analyte. Dried microbeads were re-suspended in deionized water (50 ml_), sonicated for 2 min and the SPR spectrum of the suspension was directly acquired in transmission mode. For SERRS experiments, 10 μl_ of the microbead suspension was added to a solution of R6G (10^"7 M, 1 mL) After 1 h, 10 μL of the Au- PEG/PS suspension was cast onto a glass slide and air-dried Surfaces were studied using a 532 nm laser line. The stock microbead suspension was centrifuged, filtered, air- dned and stored. This process was repeated 5 times with time intervals of one week

The absorption spectrum of R6G (inset Fig 3A) exhibited a band in the visible region from 440 to 560 nm, with a maximum at 527 nm, in nearly perfect resonance with the 532 nm excitation laser line. The SERRS spectrum of a dilute solution of R6G (10^"7 M, Fig. 3A) on Au-PEG/PS microbeads was very strong and dominated by bands at 1650 cm ¹, 1572 cm^"1, 1511 cm^"1, 1363 cm^"1, 1181 cm^"1, and 771 cm^"1 (Hildebrandt 1984). Raman mapping (Fig. 3B) showed a homogeneous optical enhancing surface throughout the entire Au-PEG/PS microbead This is paramount for SERS application as it ensures reproducibility, reliability, and low cost since the measurement can be carried out on a single bead. In addition, the optical enhancing properties of this platform were exceptionally stable, both in suspension and as powder As shown in Fig 3C and Fig 3D, no notable changes in shape or in intensity of the SPR and SERRS spectra of R6G (averaged measurements on ten different microbeads) were recorded over a 5 week period, even after 5 cycles of drying and re-suspension in water.

Example 4: Ultradetection of serotonin

The potential of the platform of the present invention in bioanalytical ultradetection was established using serotonin (5-HT). Serotonin is an important neurotransmitter related to mood, sleep, emesis, sexuality, appetite, and many other disorders (Ciranna 2006; Lesurtel 2006; Nobler 1999, Svenningsson 2006; Dumont 2006; Nilsson 1986).

To study the detection limits of 5-hydroxytryptamine (serotonin) using Au- PEG/PS, 10 μL of the Au-PEG/PS microbead suspension was added to a 1 mL solution of 5HT, with concentrations ranging from 10⁵ to 10¹⁰ M. After 1 h, 10 μL of these suspensions were cast on glass slides and mapped with a 780 nm laser line.

Fig 4A shows the Raman spectra for 5-HT acquired with 532 and 780 nm laser lines Both Raman spectra are characterized by the same vibrational features and a similar relative intensity. The Raman spectra are dominated by vibrational stretching from rings and amino groups and by in-plane deformation bands (Bayari 2005) The SERS spectra of 5-HT were acquired with the infrared laser line to avoid photodegradation. Fig 4B shows the SERS spectra of 5-HT obtained on gold colloids (as reference) (Camafeita 1995) and Au-PEG/PS microbeads. Both spectra are similar with comparable intensity The vibrational pattern of the SERS spectra is dominated by an appreciable enhancement of ring stretching (1606 and 1529 cm ¹) and in-plane deformations (1424, 1345, 1221 and 820 cm ¹) together with those due to the secondary amine modes (1485, 1299, 1196, 1104 and 932 cm^"1) This is consistent with the analyte being chemisorbed on gold surfaces through coordination with the secondary amine. The SERS spectra also showed a weak band due to PS ring breathing (1001 cm^"1), which grew as the 5-HT concentration decreased (Fig. 4C). However after subtraction of the PEG-PS spectra, 5- HT can still be seen at concentrations reaching 10^"9 M, two orders of magnitude lower than normal serum levels (5.7-16 10^'6 M) (Harenberg 2000). SERS imaging of the microbeads (Fig. 4D) demonstrated homogeneous enhancement over the entire microbead surface.

Thus, a new composite material based on Au-PEG/PS microbeads was fabricated and tested as a platform for SERS. Its chemical and optical stability, shelf life, reproducibility, and synthetic accessibility make it an inexpensive and convenient platform for SERS-based ultradetection

References: The contents of the entirety of each of which are incorporated by this reference.

Adamson AW, Gasp AP (1997) Physical chemistry of surfaces. (Wiley. New York).

Alvarez-Puebla RA, Bravo-Vasquez J-P, Cui B, Veres T, Fenniri H (2007a) ChemMedChem 2, 1165-1167. Alvarez-Puebla RA, Cui B, Bravo-Vasquez J-P, Veres T, Fenniri H (2007b) J Phys Chem C 1 6720-6723

Aroca RF Alvarez-Puebla RA, Pieczonka N, Sanchez-Cortez S, Garαa-Ramos JV (2005) Adv Colloid Interface Sci 116 45-61

Bayari S Saglam S, Ustundag HF (2005) J MoI Struct Theochem 726, 225-232

Boyen H-G, Kastle G, Weigl F, Koslowski B Dietrich C, Ziemann P, Spatz JP, Riethmuller S, Hartmann C Moller M, Schmid G, Gamier MG, Oelhafeπ P (2002) Science 297, 1533-1536

Burda C, Chen X, Narayanan R, El-Sayed MA (2005) Chem Rev 105, 1025-1102

Camafeita L, Sanchez-Cortes S, Garcia-Ramos J (1995) J Raman Spectrosc 26, 149- 153

Cao YC, Jm R, Mirkin CA (2002) Science 297, 1536-1540

Ciranna L (2006) Curr Neuropharmacol 4, 101-114

Daniel M-C, Astruc D (2004) Chem Rev 104, 293-346

Dumont GJH, Verkes RJ (2006) J Psychopharmacol 20, 176-187

Evans DF, Wennerstrom H (1999) The colloidal domain, where physics chemistry and biology meet (VCH New York)

Farah AA Alvarez-Puebla RA Fenniri H (2008) J Coll lnterf Sci 319 572-576

Fenniri H, Chun S, Ding L, Zyπanov Y, Hallenga K (2003) J Am Chem Soc 125, 10546- 10560

Giesfeldt KS, Connatser RM, De-Jesus MA, Dutta P, Sepaniak MJ (2005) J Raman Spectrosc 36, 1134-1142

Harder P Grunze M Dahint R, Whitesides GM Laibinis PE (1998) J Phys Chem B 102 426-436

Harenberg J, Huhle G Giese C, Wang LC, Feuring M Song XH, Hoffmann U (2000) Brit J Haematol 109, 182-186 Hildebrandt P, Stockburger M (1984) J Phys. Chem. 88, 5935-5944.

Jannasch P (1998) Macromol. 31 , 1341-1347

Kasili PM, Song JM, Vo-Dmh T (2004) J. Am Chem Soc. 126 2799-2806

Kelly KL, Coronado E, Zhao LL, Schatz GC (2003) J. Phys. Chem. B. 107, 668-677.

Kiπoshita T, lshigaki Y, Nakano K, Yamaguchi K, Akita S, Nil S, Kawaizumi F (2006a) Sep Puπf Technol. 49, 253-257

Kinoshita T, lshigaki Y, Yamaguchi K, Akita S, Yamada Y, Nii S, Takahashi K, Kawaizumi F (2006b) Sep. Purif. Technol 52, 357-362.

Kneipp K, Kneipp H, Itzkan I, Dasaπ RR, FeId MS (1999) Chem. Rev. 99, 2957-2976.

Kneipp H, Kneipp J, Kneipp K (2006) Anal. Chem 78, 1363-1366

Larsson M, Lu J, Lindgren J (2004) J. Raman Spectrosc. 35, 826-834.

Lesurtel M, Graf R, Aleil B, Walther DJ, Tian YH, Jochum W, Gachet C, Bader M, Clavien PA (2006) Science 312, 104-107.

Moore BD, Stevenson L, Watt A, Flitsch S, Turner NJ, Cassidy C, Graham D (2004) Nat Biotech. 22, 1133-1138.

Nilsson O, Ahlman H, Ericson L, Skolnik G, Dahlstrom A (1986) Cancer. 58, 676-684

Nobler MS, Mann JJ, Sackeim HA (1999) Brain Res. Rev. 30, 250-263.

Pieczonka NPW, Aroca RF (2005) ChemPhysChem. 6, 2473-2484. Reynolds NM, Hsu SL (1990) Macromol 23, 3463 - 3472.

Skirtach AG, Dejugnat C, Braun D, Susha AS, Rogach AL, Parak WJ, Mohwald H, Sukhorukov GB (2005) Nano Lett. 5, 1371-1377.

Stuart DA, Yonzon CR, Zhang X₁ Lyandres O, Shah NC, Glucksberg MR, Walsh JT, Van Duyne RP (2005) Anal. Chem 77, 4013-4019

Svenningsson P, Chergui K, Rachleff I, Fiajolet M, Zhang XQ, El-Yacoubi M, Vaugeois JM, Nomikos GG, Greengard P (2006) Science. 311 , 77-80 Yang X, Su Z, Wu D, Hsu SL, Stidham HD (1997) Macromol. 30, 3796-3802.

Zhang J, Xu S, Kumacheva E (2004a) J. Am. Chem. Soc. 126, 7908-7914.

Zhang J, Liu Z, Han B, Liu D, Chen J, He J, Jiang T (2004b) Chem. Eur. J. 10, 3531- 3536.

Other advantages that are inherent to the structure are obvious to one skilled in the art. The embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims.

Claims

Claims'

1. A SERS-active composite material comprising: nanoparticles of Au(O); and, microparticles comprising polyethylene glycol

2 The material of claim 1 , wherein the microparticles comprise a graft copolymer of polyethylene glycol and a polymeric scaffold.

3. The material of claim 2, wherein the polymeric scaffold comprises a styrenic polymer

4. The material of claim 2, wherein the polymeric scaffold comprises polystyrene.

5 The material of claim 1 , wherein the microparticles comprise a graft copolymer of polyethylene glycol and polystyrene.

6. The material of any one of claims 1 to 5, wherein the nanoparticles are diffused into polyethylene glycol-rich regions of the microparticles.

7. The material of any one of claims 1 to 6, wherein the microparticles have pore sizes in a range of from about 2 nm to about 5 μm.

8 The material of any one of claims 1 to 7, wherein the microparticles are microbeads