Analytical and Bioanalytical Chemistry Electronic Supplementary Material Spherical Silver Nanoparticles in the Detection of Thermally Denatured Collagens Manuel Ahumada, Sarah McLaughlin, Natalia L. Pacioni, Emilio I. Alarcon S1
Materials and Methods Chemical and reagents Silver nitrate (AgNO 3 ), trisodium citrate, 2-Hydroxy-1-[4-(2-hydroxyethoxy)phenyl]- 2- methyl-1-propanone (I-2959) were purchased from Sigma-Aldrich. Porcine collagen Theracol (Sewon cel contech co., Ltd.), rat tail collagen (Discovery Labware, USA), and type I human recombinant collagen (Fibrogen, USA). Unless otherwise indicated these proteins were used as received. All solutions were prepared using Milli-Q water. Synthesis of citrate capped AgNPs Citrate capped AgNPs were prepared as described in the literature with minor modifications [1-3]. Briefly, a deoxygenated (30 min N 2 ) aqueous solution containing 0.2 mm AgNO 3, 0.2 mm I-2959, and 1.0 mm sodium citrate was irradiated with UVA light (8 lamps, in a Luzchem LZC-4 photoreactor at 25.0 ± 0.5 C) for 30 minutes. Yellow translucent solutions were obtained in all cases and the solutions were kept at room temperature and protected from light. Surface Plasmon band spectra The plasmon absorption band was followed throughout the absorbance spectra in a Libra S50 UV Vis spectrophotometer (Biochrom, Cambridge, UK) at 4 C, room temperature and 37 C, employing 1.0 cm path length cuvettes. Measured solutions correspond to 1:1 v/v mix (AgNPs/collagen type I - 34 μg ml -1 final concentration). For native protein, collagen was used as received, while denatured collagen was obtained by pre-incubation of the protein at 95 C in a heat block for 5 minutes. For citrate capped AgNPs solutions maximum wavelength absorbance was found at 397 nm. Meanwhile, upon adding collagen the SPB maximum was 407±1.0 nm. No appreciable variation was observed for different temperatures or source of collagen (see Fig 1 main text, and Fig S1 in ESM). S2
Lithium dodecyl sulphate gel electrophoresis Mini-PROTEAN TGX precast gels from Bio-rad were employed. To this, 30 μl of a collagen solution (34μg ml -1, same concentration for native, pre-incubated at 37 C and 95 C protein) was mixed with 10 μl of Lithium dodecyl sulphate (LDS, thermo scientific), and denatured at 95 C for 5 minutes. Then, 10 μl of the respective solution was loaded per well. Precision Plus Protein Dual Color was used as standard (10 250 kd). The samples were run at 80 V for one hour and stained with EZBlue gel staining reagent from Sigma. Circular dichroism spectra All the CD spectra were recorded in a Jasco J-815 CD spectrophotometer from Jasco analytical instruments. Collagen solutions (native and denatured) were prepared as mentioned above. Spectra were measured in 0.1 cm path length cuvettes at 25±0.5 C under nitrogen atmosphere. Calibration curve for determining the content of denatured collagen All solutions (AgNPs, native and denatured collagen) were prepared as mentioned above. In 2 ml vials, 1 ml of citrated capped AgNPs was mixed with 1 ml of collagen solution. The collagen solutions were prepared by varying the concentration ratio of dcol/ncol protein (1, 0.875, 0.75, 0.625, 0.5, 0.375, 0.25, 0.125, 0) while keeping the total protein concentration (dcol + ncol)constant at 34 μg ml -1. After incubating for 15 minutes at room temperature, the samples were centrifuged at 10,000 rpm for 15 minutes in a mini Spin microcentrifuge. The resulting supernatant was taken and measured in aspectrophotometer at room temperature. Finally, the maximum absorbance registered at the maximum wavelength ( 407 nm) was plotted as a function of the dcol concentration, present in the sample, see Scheme 1 main text. ANOVA Analysis In order to determine if the SPB shift upon addition of ncol or dcol to AgNPs wassignificant, we ran a one way ANOVA tests (Kaleidah Graph v. 4.1.3). First, we S3
determined that there were nodifferences between batches, and then we grouped all the data together for each treatment (control, ncol and dcol). We show here the results for data taken at 30 minutes. Table Anova 1 Comparison between all three different groups Source Degree Freedom Sum of Squares Mean Square P Total 17 609.11111 35.830065 A 2 582.11111 291.05556 161.69753 <.0001 Error 15 27 1.8 Factor A: 3 Groups (control, ncol, dcol) Table Anova 2 Comparison of ncol and ddcol Source Degree Sum of Mean F P Freedom Squares Square Total 11 28.25 2.5681818 A 1 14.083333 14.083333 9.9411765 0.01028 Error 10 14.166667 1.4166667 Factor A: 2 Groups (ncol, dcol) As P in both cases is smaller than 0.05, we can conclude that there are significative differences between all three groups and also between native and denaturated collagen experiments. In a similar way we performed the same analysis using Absorbance values to corroborate that all batches gave similar results. In all cases we found a P>0.05 value. Then, there were not significant differences between AgNPs batches. Table Anova 3 Comparison of absorbance values between AgNP batches (batch 1 Abs, batch 2 Abs, batch 3 Abs) Source Degree Freedom Sum of Squares Mean Square F P Total 5 0.021687348 0.0043374697 A 2 0.0091863391 0.0045931695 1.10227 0.43763 Error 3 0.012501009 0.0041670031 Factor A: 3 Groups F P S4
410 λ max [nm] 400 390 No COL FWHM [nm] 105 90 No COL Abs SPB 1.2 0.8 No COL 0.4 0 40 80 120 Time [min] Fig. S1 Effect of type I porcine collagen addition, 34 µg ml -1 to a solution of colloidal citrate capped AgNPs at 37 C, ncol (open circles) and dcol (squares). Top, changes in the SPB maximum position before and after adding COL. In the middle, changes in the FWHM of the AgNPs measured before and after adding collagen. Bottom, variation of the SPB absorption maximum for AgNPs measured before and after adding 34 µg ml -1 of type I collagen either native or denatured. Note that the first point after adding collagen was measured within 30 seconds. The plasmon intensity for AgNPs before adding collagen has also been included in the plot, denoted with an arrow, but their absorbance was measured at 397 nm. Error bars correspond to the standard deviation of the experimental data, which was calculated from by performing experiments with three different batches of AgNPs induplicate (N=6) S5
1 2 3 4 250 kda 150 100 Fig. S2 LDS electrophoresis experiment, lane from 1 to 3 corresponds to porcine collagen. 1) preincubated at 95 C; 2) pre-incubated at 37 C; 3) native and 4) Precision Plus Protein Dual Color standard (10 250 kda) 0 CD [mdeg] -20-40 -60 200 220 240 λ [nm] Fig. S3 Circular dichroism spectra for PCOL ( 0.1 μm), in native (squares) and pre-incubated at 95 C (circles) conditions. All measurements were carried out at room temperature S6
Normalized abs 0.8 0.4 0 400 500 600 λ [nm] Fig. S4 Normalized SPB spectra for citrate capped AgNPs with dhcol ( ); drcol ( ) and dpcol ( ) (34 μg ml -1 ) after centrifugation Table S1 Analytical parameters Type I collagen Sensitivity, m [mg -1 ml] 2 R 2 s B (N) LOD [µg ml -1 ] LOQ [µg ml -1 ] dpcol (4.4 ± 0.2) 10-4 0.969 0.029 (21) 14.7 25.6 drcol (6.4 ± 0.3) 10-4 0.980 0.014 (13) 8.5 15.4 dhcol (16.9 ± 0.9) 10-4 0.988 0.038 (13) 8.6 15.0 References 1. Alarcon, E.I., et al., The biocompatibility and antibacterial properties of collagen-stabilized, photochemically prepared silver nanoparticles. Biomaterials, 2012. 33(19): p. 4947-56. 2. Jockusch, S., et al., Photochemistry and Photophysics of α-hydroxy Ketones. Macromolecules, 2001. 34(6): p. 1619-1626. 3. Stamplecoskie, K.G. and J.C. Scaiano, Light Emitting Diode Irradiation Can Control the Morphology and Optical Properties of Silver Nanoparticles. Journal of the American Chemical Society, 2010. 132(6): p. 1825-1827. S7