Recognition Kinetics of Biomolecules at the Surface of Different-Sized Spheres

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1 Supporting Material Recognition Kinetics of Biomolecules at the Surface of Different-Sized Spheres Jun Hu, Cong-Ying Wen, Zhi-Ling Zhang, Min Xie, Hai-Yan Xie, and Dai-Wen Pang * Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, and State Key Laboratory of Virology, Wuhan University, Wuhan , P. R. China. National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan , P. R. China. School of Life Science and Technology, Beijing Institute of Technology, Beijing , P. R. China. *Corresponding author. Phone: ; Fax: ,

2 S.1 Characterization of Spheres SEM images of the three kinds of spheres were shown in Fig. S1. It could be seen that all of them were monodisperse with uniform size. The hydrodynamic sizes of Pst-AAm and Pst-3 were measured to be nm and 2.97 µm (Fig. S2). Both of them were negatively charged. The size of Pst-25 was too large to be measured with DLS. FIGURE S1 SEM images of Pst-AAm (A, magnification 60K), Pst-3 (B, magnification 6K) and Pst-25(C, magnification 600). FIGURE S2 Hydrodynamic sizes and zeta potentials of Pst-AAm (A, B) and Pst-3 (C, D). A, C: hydrodynamic sizes; B, D: zeta potentials.

3 S.2 Standardization of FITC-SA Stock Solution The primary standard was a 40 µμ biotin stock solution, which was prepared by dissolving 1 mg of 99% pure biotin in 100 ml 0.01 M phosphate buffer solution (ph 7.2). Aliquots were stored at -20 o C and diluted to a suitable concentration before use. Fluorescein isothiocyanate labeled streptavidin (FITC-SA) was dissolved in 0.01 M PBS (ph 7.2) at 1 mg/ml and diluted to a 10 µg/ml stock solution for use. The effective concentration of FITC-SA was determined by titration with biotin. Typically, 0.2 ml of 10 µg/ml FITC-SA was added to 1.8 ml of Buffer A in a stirred cuvette and the fluorescence intensity of FITC was monitored while adding 2.5 µm biotin in 10-µL increments at 10-min intervals. The amount of biotin needed for saturation of all the biotin-binding sites could be calculated from the breakpoint between linear increase and the subsequent platform. The effective concentration of FITC-SA was defined as [biotin-binding sites]/4. S.3 Standardization of SA Stock Solution 2 ml of 10 µg/ml SA was added to a quartz cuvette with continuous stirring and the decrease in tryptophan fluorescence was monitored with 250-nm excitation while adding a 25 µm biotin stock solution in 8-µl increments at 5-min intervals. The breakpoint between linear quenching and the subsequent platform allowed calculating the amount of biotin needed for saturation of all biotin-binding sites. The effective concentration of SA was defined as [biotin-binding sites]/4. From Fig. S3, it was 0.15 µm for the stock solution of 10 µg/ml SA.

4 FIGURE S3 Titration of the biotin-binding sites on SA (10 µg/ml, 2 ml) with 25 µm biotin at 5 min-intervals. (A) Tryptophan fluorescence decrease when SA reacted with increasing amount of biotin (from a to i, biotin was added in 8-µL increments). (B) Plot of fluorescence intensity versus consumption of biotin derived from A. S.4 Standardization of F-biotin Stock Solution F-biotin was dissolved in DMSO at 1 mg/ml and stored at -20 o C. This primary stock solution was diluted to 1 µg/ml with 0.01 M PBS (ph 7.2) to give the working reagent. The effective concentration of F-biotin was determined by titration with SA standardized above, based on the fact that fluorescence of F-biotin would be quenched when it bound to SA (Scheme S1). Typically, 2 ml of Buffer A containing 10 µl of 1 µg/ml F-biotin was added to a stirred cuvette. The fluorescence of fluorescein was monitored while adding 10 µg/ml SA in 5-µl increments at 5-min intervals. The breakpoint between linear quenching and the subsequent platform allowed calculating the amount of SA needed for the binding of all biotin. So, the effective concentration of F-biotin was defined as [SA] 4. From Fig. S4, it was 2.1 µm for the stock solution of 1 µg/ml F-biotin. SCHEME S1 Schematically illustrating the binding of F-biotin to SA.

5 FIGURE S4 Titration of F-biotin (0.005 µg/ml, 2 ml) with standardized SA (10 µg/ml) at 5 min-intervals. (A) Fluorescein fluorescence decrease when F-biotin reacted with increasing amount of SA (from a to i, SA was added in 5-µL increments). (B) Plot of fluorescence intensity versus consumption of SA derived from A. S.5 Equivalence of Biotin-Binding Sites on SA We also assumed that the four binding sites of SA were also equivalent for F-biotin. To verify this hypothesis, 1.6 pmol of F-biotin was respectively reacted with 0.4 pmol, 0.8 pmol, 1.2 pmol, 1.6 pmol of SA and the fluorescence intensity variation of fluorescein with time in each reaction was recorded. From which, the binding rate constants of F-biotin to their binding sites on SA were calculated to be M -1 s -1, M -1 s -1, M -1 s -1 and M -1 s -1 respectively (Fig. S5), with a second-order bimolecular reaction model. So, it could be concluded that the four binding sites on SA were equivalent for F-biotin.

6 FIGURE S5 Linearization of the association time course in terms of a bimolecular reaction between F-biotin and several concentrations of SA, where a and b were the initial concentrations of biotin-binding site on SA and F-biotin, x was the consumption of F-biotin at time t. S.6 Binding Kinetics of F-biotin to SA in Solution The working reagent of F-biotin for measuring the binding kinetics was prepared by diluting the F-biotin stock solution with Buffer A to get a terminal concentration at 0.8 nm. 2 ml of working solution was placed in a stirred quartz cuvette and fluorescence was monitored continuously with 0.1-s signal acquisition at 521 nm. Following an initial acquisition period from solution without any SA present to ensure that fluorescein PL signal was stable with time, 0.2 pmol of SA was added to the biotin solution and the fluorescence intensity at 521 nm was monitored within the reaction process. With the aforementioned method, the association rate constant k of F-biotin to their binding site was calculated to be M -1 s -1.

7 FIGURE S6 Fluorescence measurement and analysis for the binding of F-biotin to SA in solution at 25 o C. (A) 2 ml of Buffer A containing 0.8 nm F-biotin was stirred and the fluorescence was monitored at 521 nm when 0.2 pmol SA was added. (B) Linearization of the association time course in terms of a bimolecular reaction, where a and b were the initial concentrations of biotin-binding site on SA and F-biotin, x was the consumption of F-biotin at time t. S.7 Activation Energy To test the activation energy (Ea) for biotin binding to FITC-SA, binding kinetics of them at different temperatures (T) was investigated. Then, Ea could be calculated from the Arrhenius plot (Fig. S7 A), which displayed the logarithm of kinetic constants (ln k, ordinate axis) plotted against inverse temperature (1/T, abscissa). The activation energy for F-biotin binding to SA could be calculated with a similar method (Fig. S7 B). FIGURE S7 Arrhenius plots of (A) FITC-SA/biotin and (B) SA/F-biotin systems.

8 S.8 Binding Kinetics of F-biotin to SA on Sphere Surface With the same method mentioned in Experimental Section, SA was immobilized on the surface of carboxyl-poly(styrene/acrylamide) copolymer nanospheres. The association process of F-biotin with SA on the sphere surface was studied spectrophotometrically by the fluorescence change at 521 nm. Typically, a certain amount of SA-modified Pst-AAm copolymer nanospheres (Pst-AAm-SA) dispersed in 2 ml of Buffer A was placed into a quartz cuvette. Then far more excessive F-biotin was added and a series of 1-s signal acquisitions at 521 nm were carried out. Apparent binding rate was derived from the 521-nm emitting decrease over time. From Equation 2 and 3, it could be calculated that the binding rate constant of F-biotin to surface-bound SA was about M -1 s -1 (Fig. S8). FIGURE S8 Plot of k app versus F-biotin concentration. k app was obtained from the kinetics of F-biotin binding to SA immobilized on the surface of poly(styrene/acrylamide) copolymer nanospheres, when 10 µl of SA-modified Pst-AAm copolymer nanospheres was dispersed into 2 ml of Buffer A and reacted with varying amount and far more excessive of F-biotin.

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