Controlled Clustering of Gold Nanoparticles using Solid-support for Surface-enhanced Raman Spectroscopic Probes

Experimental

Synthesis of Gold Nanoparticles. A 50 mg of HAuCl4was dissolved in DI water (50 mL) followed by adding 1 mL of citrate aqueous (1 w/w%). The temperature during the reaction was maintained at 100 °C for 30 min, and then the colloidal solution was kept in room temperature for 24 h with stirring, followed by storage at 4 °C for long term stability.

Pretreatment of Si Wafer. Silicon wafers were ultrasonicated for 20 minutes in acetone and in DI water serially to clean the surface. And the surface of the Si wafer was activated by soaking in piranha solution (a mixture of H2SO4 and 30 v/v% H2O2 (3:1)) for 15 minutes, and then rinsed with DI water several times, and then dried by blowing nitrogen gas. The mixed self-assembled monolayer (SAM) was formed by dipping the pretreated Si wafer in a mixed solution of 3-aminopropyldimethylethoxysilane (3-APDMS) and and ethoxytrimethylsilane (ETMS) in anhydrous ethanol (total 1 v/v%) with the ratio of 1:30 for 2 h. The excess chemicals were removed by ultrasonicating the Si wafer in ethanol for 20 minutes followed by rinsing with ethanol and DI water several times, and then the substrate were dried with nitrogen gas. Additional spacing linkers composed of β-alanine (β-Ala; B) and ε-aminocaproic acid (ε-ACA; E) pairs was used for easier immobilization of gold nanoparticles from solution to substrate. Fmoc-β-alanine (Fmoc- β-Ala) and Fmoc-6-aminocaproic acid (Fmoc-ε-ACA) were coupled three times in turn to the Si surface through the Fmoc strategy to introduce an ε-ACA-β-Ala-ε-ACA-β-Ala- ε-ACA-β-Ala (BEBEBE) spacer.8 Briefly, the Silane-coated Si wafer was immersed into the preactivated solution (with 3 equiv. of 1-hydroxybenzotriazole (HOBt), benzotriazol-1- yloxy-tris(dimethylamino) phosphonium hexafluorophosphate (BOP), diisopropylethylamine (DIPEA)) of each Fmocamino acid in N-methylpyrrolidone (NMP) for 3 h. And then Si wafer was washed by immersing in NMP with shaking for 15 minutes and then rinsed with NMP. Fmoc-deprotection to reveal the amine-functionality was performed with 20 v/v% piperidine in NMP for 30 min at 25 °C.

Fabrication of Gold Nanoclusters on Si Wafer. The BEBEBE spacer-grafted Si wafer was submerged in a gold colloidal solution for attaching Au NPs (~19 nm) to the surface of the Si wafer for 90 minutes at room temperature. And then the Si wafer was immersed in a solution of 4-ABT in methanol (1 mM) for 1 h at 25 °C followed by 90 minutes dipping the Si wafer into a gold colloidal solution again for formation of Au NP clusters. The substrate was washed sufficiently by the solvent used in that step and dried with blowing nitrogen gas after the each processes.

Measurements and Characterization. UV-Visible extinction spectrum of gold colloid was measured with a UV-Vis spectrometer (Varian, Cary 300 Bio). Atomic Force Microscope (Park systems, XE-100) was utilized to confirm the self-assembly of additional spacer and gold nanoparticles. Nano-scale images also were obtained with field-emission scanning electron microscope (Carl Zeiss, SUPRA 55VP) operated at 2.0 kV and transmission electron microscope (JEOL, JEM1010) operated at 80.0 kV. For the Raman measurement, a micro-Raman system (JY-Horiba, LabRam 300) was utilized to characterize the SERS activity of the prepared gold nanoclusters. The signal was collected by a × 100 objective lens (Olympus, 0.90 NA) with the back-scattering geometry equipped with a thermoelectrically cooled (−70 °C) CCD detector. The 647 nm laser line from Kr ion laser (Coherent, Innova 300C) was used for an excitation source. The laser spot diameter and penetration depth of the focused laser beam were ~1.1 μm and ~6 μm, respectively. At each measurement, acquisition time was 1 s, and the sample power was about 2 mW.