Supporting information Immunoselective cellulose nanospheres â a ...
Supporting information Immunoselective cellulose nanospheres – a versatile platform for nanotheranostics
Christopher Carrick,*a Lars Wågberga,b and Per A. Larsson*a a
KTH Royal Institute of Technology, School of Chemical Science and Engineering, Department of Fibre and Polymer Technology, SE-
100 44 Stockholm, Sweden. E-mail: [email protected]; [email protected]; [email protected] b
KTH Royal Institute of Technology, School of Chemical Science and Engineering, Wallenberg Wood Science Centre, WWSC, SE-
100 44 Stockholm, Sweden.
Size distribution of prepared cellulose spheres Dynamic light scattering (being detected at an angle of 173 and at 25 C) of conjugated and non-conjugated CNSs was used to study the size and change in size following antibody conjugation. The results in Fig. S1 show that the diameter increased due to the antibody conjugation from approximately 160 nm to 190 nm.
Figure S1. The size distribution of anti-BSA-conjugated (crosses and dotted trend line) and non-conjugated cellulose nanospheres (open circles and solid trend line) prepared by the membrane emulsification method in 1 M PBS from DLS measurements. Before the measurements, the solution was filtered through a 5 µm syringe filter to remove large impurities and/or aggregates from the solution.
1
BSA and EGFR model surfaces Prior to the QCM experiment, a silica quartz crystal was oxidized in a plasma oven for 2 min. The experiment was performed by first stabilizing the baseline frequency in MilliQ-water. A 0.01 g/l solution of BSA or EGFR was then pumped through the QCM at a flow rate of 0.1 ml/min (which was held constant during the entire experiment). The pH of the BSA solution was pH 4 and the EGFR had a pH of 5, i.e. about one pH unit below the isoelectric point of the respective proteins. The same protein concentrations, solution pHs and adsorption times were used for preparing model surfaces on oxidized silica wafers used for SEM. Instead of adsorption from a continuous flow, the surfaces were prepared by dipping.
Determination of conjugation efficiency The retention of antibody on the CNSs during the conjugation step was estimated using a Pierce™ BCA Protein Assay (Thermo Scientific). The two assay components were mixed at a ratio of 50:1 and used at a constant volume of 1.5 ml in plastic cuvettes. To these aliquots of assay mixture, 300 µl of a sample of known or unknown concentration was added (i.e. anti-BSA/anti-EGFR standards and/or conjugates with CNSs). The cuvettes were then incubated at room temperature for a minimum of 20 h (due to the low protein concentrations) and scanned at 562 nm in a Shimadzu UV2550 UV-vis spectrophotometer. The retention determinations were performed in two independent conjugation experiments for anti-BSA and anti-EGFR, respectively. Based on the amount of antibody added to the oxidised spheres (assuming that the conjugated protein has an unaffected interaction with the BCA assay reagents), the average retention of added anti-BSA and anti-EGFR was estimated to be about 41% and 21%, respectively. Calibration curves and the absorbance for the conjugated samples from one of the two determinations can be seen in Fig. S2.
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Figure S2. BCA Protein Assay-calibration curve based on unconjugated antibody and samples (of three dilutions: 1:2, 2:1 and undiluted) of antibody-CNS conjugates for (a) anti-BSA and (b) anti-EGFR. Filled symbols indicate calibration measurements and open symbols indicate antibody-CNS conjugates.
Embedded gold nanoparticles The size of the incorporated GNPs was calculated from the X-ray diffraction data according to the Scherrer equation: 1
where
are the dimensionless shape factor (0.9), the X-ray wavelength (1.54 Å), the line broadening at half
the maximum intensity and the Bragg angle respectively (where the peaks at angles of 38.1, 44.3 and 64.6 degrees were used). The calculated size of the incorporated GNPs was, from these values, estimated to be 11.8 ± 1.2 nm. UV-vis was used to further show that the incorporated GNPs were nanometer-sized, and Fig. S3 shows the absorption peaks at ~490 and ~530 nm, which are typical peaks for the surface plasmon resonance absorption of dispersed GNPs.2
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Figure S3. Absorbance of CNS in water with gold (solid line) and without (dashed line) gold nanoparticles. The peaks at ~490 and ~530 nm correspond to the surface plasmon resonance absorption peaks of dispersed gold nanoparticles.
Figure S4 shows the interaction of CNSs with incorporated GNPs with a pre-adsorbed BSA protein layer on a silica crystal. Only a small decrease in frequency can be seen when the pre-adsorbed BSA-layer is exposed to the goldcontaining CNSs, and this can be interpreted as indicating a successful incorporation of GNPs in the CNSs, preventing the expected interaction between gold and protein. The frequency shift was however small compared with the anti-BSA conjugated CNSs seen in Fig. 2a, and when the surfaces were exposed to PBS about half of the change in frequency was lost, showing a desorption of the small amount of adsorbed CNSs.
Figure S4. QCM graph displaying the frequency shift (i.e. mass adsorption) as a function of time. Points of addition of BSA protein, washing buffer (PBS) and CNS with embedded GNPs are indicated by arrows.
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References 1. 2.
Scherrer, P., Bestimmung der Größe und der inneren Struktur von Kolloidteilchen mittels Röntgenstrahlen. Nachrichten von der Gesellschaft der Wissenschaften zu Göttingen, Mathematisch-Physikalische Klasse 1918, 98-100. Zhang, F. X.; Han, L.; Israel, L. B.; Daras, J. G.; Maye, M. M.; K. Ly, N.; Zhong, C.-J., Colorimetric detection of thiolcontaining amino acids using gold nanoparticles. Analyst 2002, 127 (4), 462-465.
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Christopher Carrick,*a Lars Wågberga,b and Per A. Larsson*a a
KTH Royal Institute of Technology, School of Chemical Science and Engineering, Department of Fibre and Polymer Technology, SE-
100 44 Stockholm, Sweden. E-mail: [email protected]; [email protected]; [email protected] b
KTH Royal Institute of Technology, School of Chemical Science and Engineering, Wallenberg Wood Science Centre, WWSC, SE-
100 44 Stockholm, Sweden.
Size distribution of prepared cellulose spheres Dynamic light scattering (being detected at an angle of 173 and at 25 C) of conjugated and non-conjugated CNSs was used to study the size and change in size following antibody conjugation. The results in Fig. S1 show that the diameter increased due to the antibody conjugation from approximately 160 nm to 190 nm.
Figure S1. The size distribution of anti-BSA-conjugated (crosses and dotted trend line) and non-conjugated cellulose nanospheres (open circles and solid trend line) prepared by the membrane emulsification method in 1 M PBS from DLS measurements. Before the measurements, the solution was filtered through a 5 µm syringe filter to remove large impurities and/or aggregates from the solution.
1
BSA and EGFR model surfaces Prior to the QCM experiment, a silica quartz crystal was oxidized in a plasma oven for 2 min. The experiment was performed by first stabilizing the baseline frequency in MilliQ-water. A 0.01 g/l solution of BSA or EGFR was then pumped through the QCM at a flow rate of 0.1 ml/min (which was held constant during the entire experiment). The pH of the BSA solution was pH 4 and the EGFR had a pH of 5, i.e. about one pH unit below the isoelectric point of the respective proteins. The same protein concentrations, solution pHs and adsorption times were used for preparing model surfaces on oxidized silica wafers used for SEM. Instead of adsorption from a continuous flow, the surfaces were prepared by dipping.
Determination of conjugation efficiency The retention of antibody on the CNSs during the conjugation step was estimated using a Pierce™ BCA Protein Assay (Thermo Scientific). The two assay components were mixed at a ratio of 50:1 and used at a constant volume of 1.5 ml in plastic cuvettes. To these aliquots of assay mixture, 300 µl of a sample of known or unknown concentration was added (i.e. anti-BSA/anti-EGFR standards and/or conjugates with CNSs). The cuvettes were then incubated at room temperature for a minimum of 20 h (due to the low protein concentrations) and scanned at 562 nm in a Shimadzu UV2550 UV-vis spectrophotometer. The retention determinations were performed in two independent conjugation experiments for anti-BSA and anti-EGFR, respectively. Based on the amount of antibody added to the oxidised spheres (assuming that the conjugated protein has an unaffected interaction with the BCA assay reagents), the average retention of added anti-BSA and anti-EGFR was estimated to be about 41% and 21%, respectively. Calibration curves and the absorbance for the conjugated samples from one of the two determinations can be seen in Fig. S2.
2
Figure S2. BCA Protein Assay-calibration curve based on unconjugated antibody and samples (of three dilutions: 1:2, 2:1 and undiluted) of antibody-CNS conjugates for (a) anti-BSA and (b) anti-EGFR. Filled symbols indicate calibration measurements and open symbols indicate antibody-CNS conjugates.
Embedded gold nanoparticles The size of the incorporated GNPs was calculated from the X-ray diffraction data according to the Scherrer equation: 1
where
are the dimensionless shape factor (0.9), the X-ray wavelength (1.54 Å), the line broadening at half
the maximum intensity and the Bragg angle respectively (where the peaks at angles of 38.1, 44.3 and 64.6 degrees were used). The calculated size of the incorporated GNPs was, from these values, estimated to be 11.8 ± 1.2 nm. UV-vis was used to further show that the incorporated GNPs were nanometer-sized, and Fig. S3 shows the absorption peaks at ~490 and ~530 nm, which are typical peaks for the surface plasmon resonance absorption of dispersed GNPs.2
3
Figure S3. Absorbance of CNS in water with gold (solid line) and without (dashed line) gold nanoparticles. The peaks at ~490 and ~530 nm correspond to the surface plasmon resonance absorption peaks of dispersed gold nanoparticles.
Figure S4 shows the interaction of CNSs with incorporated GNPs with a pre-adsorbed BSA protein layer on a silica crystal. Only a small decrease in frequency can be seen when the pre-adsorbed BSA-layer is exposed to the goldcontaining CNSs, and this can be interpreted as indicating a successful incorporation of GNPs in the CNSs, preventing the expected interaction between gold and protein. The frequency shift was however small compared with the anti-BSA conjugated CNSs seen in Fig. 2a, and when the surfaces were exposed to PBS about half of the change in frequency was lost, showing a desorption of the small amount of adsorbed CNSs.
Figure S4. QCM graph displaying the frequency shift (i.e. mass adsorption) as a function of time. Points of addition of BSA protein, washing buffer (PBS) and CNS with embedded GNPs are indicated by arrows.
4
References 1. 2.
Scherrer, P., Bestimmung der Größe und der inneren Struktur von Kolloidteilchen mittels Röntgenstrahlen. Nachrichten von der Gesellschaft der Wissenschaften zu Göttingen, Mathematisch-Physikalische Klasse 1918, 98-100. Zhang, F. X.; Han, L.; Israel, L. B.; Daras, J. G.; Maye, M. M.; K. Ly, N.; Zhong, C.-J., Colorimetric detection of thiolcontaining amino acids using gold nanoparticles. Analyst 2002, 127 (4), 462-465.
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