Tuning of SPR for Colocalized Characterization of Biomolecules Using Nanoparticle-Containing Multilayers

Abstract

In this contribution, we quantify the fine-tuning of surface plasmon resonance via nanoparticle coating of gold SPR chips. We target preparation of atomically flat surface with defined charge, needed for scanning probe characterization of biomolecules, with simultaneous optimization of in situ SPR sensitivity during immobilization of these molecules. Using total internal reflection ellipsometry, we show that the goal can be achieved by combination of charge-stabilized silver nanoparticles over underlying gold chip of reduced thickness. The provided formulas for optimal silver to gold thickness relation are valid for arbitrary analyte and the method was experimentally verified on binding of Rtt103 protein.

Appendix

PVD. The gold layers were prepared by thermal evaporation with thickness gradient along one dimension of 25x25 mm$^2$ BK7 microscopy slides using substrate tilting; in particular, tilt angle 57$^\circ$ was used. The transmissivity across the prepared substrates was measured, see Fig. 8, with optimal SPR of bare gold for $T_{max}= 24$ %.

Fig. 8

Transmissivity of Au layer in air ambient (Chip 1 from Fig. 7) at different positions along the thickness gradient; the thicknesses were calibrated using X-ray reflectivity. The maximum transmissivity $T_{max}$ near $\lambda =500$ nm can be used to characterize the layers

PEM. To create a charged surface coating of the SPR chip we use the PEI-PSS-PAH polyelectrolyte multilayer. In particular, 0.1 mg/ mL poly(ethyleneimine) (PEI, M$_{w}$ 600-1 000 kDa) solution in 150 mM NaCl buffer has been prepared using high purity (>18 M$\Omega$ cm) demineralized water and coated on the gold substrates. Consequently, a polyelectrolyte multilayer can be prepared by alternation of 0.1 mg/mL poly(sodium 4-styrenesulfonate) (PSS, negative charge) and 0.1 mg/mL poly( allylamine hydrochloride) (PAH, positive charge), both in 150 mM NaCl buffer.

Rtt103-CID. The stock solution of the used protein (2KM4 structure at rcsb.org) was 1 mM in ITC buffer (35 mM KH$_2$PO$_4$, 100 mM KCl, 1 mM BME, pH 6.8). Prior to measurement, the stock solution was diluted 1:100 using DI water.

AgNP. The silver nanoparticles were synthesized according to [20]. In brief, using high purity (>18 M$\Omega$ cm) demineralized water (DI), 6 mL of 12.4 mM trisodium citrate dihydrate, 15 mL of 375 $\upmu$M AgNO$_3$, 15 mL of 50 mM H$_2$O$_2$, and 10 $\upmu$l of 1 mM KBr solutions were prepared and mixed in a clean 50 mL beaker. While stirring the solution, 7.5 mL of 5 mM NaBH$_4$ were added (all chemicals were used as purchased from Sigma-Aldrich). Stirring for further 10 minutes, the pale yellow solution changed color from yellow through orange and pink to violet and blue. The vials were kept untied for 24 hours to allow sodium borohydride to decompose safely.

The post-synthesis purification of the nanoparticles is an important step in preparing their immobilization. If used as prepared, the synthesis buffer would screen the nanoparticles from binding to the substrate. To prevent this, we employ centrifugation to decrease significantly the concentration of buffer salts, as the used protocol renders stable nanoparticles even after replacement of synthesis buffer with water. In addition, the centrifugation improves monodispersity of the nanoparticles.

To this end, the nanoparticles were centrifuged prior to use in four steps (10 min at RCF of 5 200 g each). After each step, >99% of supernatant was removed and refluxed with DI to ensure final dilution of the synthesis buffer by at least 1:10$^8$. Both the as-synthesized and centrifuged nanoparticles were kept at 10 $^\circ$C, showing stability for over a month at RT.

The purification was monitored through absorbance measurement: as prepared, the nanoparticles show $\lambda _{LSPR}$ of 608 nm with FWHM 228 nm. After four-step centrifugation, $\lambda _{LSPR}$ moves to 627 nm and FWHM improves to 187 nm. To study the properties of the nanoparticles in detail, we have performed rate-zonal centrifugation [28], which confirmed presence of NPs with fractionated mass (the upper fractions seen in the inset). The situation is depicted in Fig. 9. Apart from slight shift of $\lambda _{LSPR}$ to 638 nm, the bottom centrifugate, containing the targeted NPs, shows absorption spectrum equivalent to the one after four-step centrifugation. Consequently, one can safely adhere to the simpler four-step centrifugation suggested here.

Fig. 9

Absorbance of silver nanoparticles: as prepared, and washed by step centrifugation. The inset shows the result of rate-zonal centrifugation