Accepted Manuscript Title: Catalase-Modified Gold Nanoparticles: Determination of the Degree of Protein Adsorption by Gel Electrophoresis Authors: Katarzyna Ranoszek-Soliwoda, Ewa Czechowska, Emilia Tomaszewska, Grzegorz Celichowski, Tomasz Kowalczyk, Tomasz Sakowicz, Janusz Szemraj, Jaroslaw Grobelny PII: DOI: Reference:
S0927-7765(17)30534-9 http://dx.doi.org/10.1016/j.colsurfb.2017.08.019 COLSUB 8775
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
15-11-2016 24-5-2017 12-8-2017
Please cite this article as: Katarzyna Ranoszek-Soliwoda, Ewa Czechowska, Emilia Tomaszewska, Grzegorz Celichowski, Tomasz Kowalczyk, Tomasz Sakowicz, Janusz Szemraj, Jaroslaw Grobelny, Catalase-Modified Gold Nanoparticles: Determination of the Degree of Protein Adsorption by Gel Electrophoresis, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.08.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Catalase-Modified Gold Nanoparticles: Determination of the Degree of Protein Adsorption by Gel Electrophoresis
Katarzyna Ranoszek-Soliwoda†, Ewa Czechowska†, Emilia Tomaszewska†, Grzegorz Celichowski†, Tomasz Kowalczyk§, Tomasz Sakowicz§, Janusz Szemraj‡, Jaroslaw Grobelny†*
Department of Materials Technology and Chemistry, Faculty of Chemistry, University of Lodz, Pomorska 163, 90-236 Lodz, Poland ‡ Department of Medical Biochemistry, Medical University of Lodz, ul. Mazowiecka 6/8, Lodz, Poland § Department of Genetics, Plant Molecular Biology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland *Corresponding author e-mail address: [email protected]
(J. Grobelny) Fax: +48 42 6355832 Tel: +48 42 6355837 Graphical abstract
Gold nanoparticles (AuNPs) with the size of about 13 nm modified with catalase Stable catalase modified-AuNPs colloids obtained Surface coverage of AuNPs with catalase determined Gel electrophoresis presented as a fast and versatile method for determination of surface coverage of NPs with protein
Abstract In this study we present a method to determine the degree to which catalase (CAT) is adsorbed onto gold nanoparticles (AuNPs) using polyacrylamide gel electrophoresis (PAGE) with silver staining. AuNPs (13 nm) were synthesised in water by the chemical reduction method and modified with CAT (AuNPs-CAT). The colloidal stability and NP size before and after the modification were investigated by dynamic light scattering and scanning transmission electron microscopy. Electrophoresis was performed under different conditions (native, with and without SDS, and with and without -mercaptoethanol) to find the optimal conditions for determining the surface coverage of AuNPs with CAT protein. The results clearly indicate that PAGE can be used to determine the amount of protein adsorbed on the NP surface and the use of native PAGE does not alter the colloidal stability of the NPs. These features allowed us to monitor the state of NPs and protein-NP interactions during the electrophoretic process.
Keywords: Catalase-modified nanoparticles, polyacrylamide gel electrophoresis, degree of adsorption 1.
Introduction Proteins can be immobilised on the surface of nanoparticles (NPs) by two main methods: covalent bonding and adsorption. Regardless of the method used, protein immobilisation has several beneficial properties, which makes optimisation of this process very valuable. The immobilisation of proteins, peptides or enzymes on NPs can enhance their stability , improve of their activity [2, 3], help them resist changes in pH , and can allow for the design of multimodal structures that have the synergistic effect of being a proteinparticle hybrid system. Interactions between proteins and nanomaterials can be investigated by several techniques. A typical method and the one most commonly used for the identification and quantification of proteins is electrophoresis [5-7]. Capillary and polyacrylamide gel electrophoresis (PAGE) are widely used to separate and analyse protein mixtures and can also be used to determine the amount of protein adsorbed on the surface of NPs . However, this is typically carried out in a multistep procedure where prior to identification and quantification the proteins by PAGE, they are isolated from the NP surface using denaturing agents according to desorption protocols . Hence, it is not possible to directly monitor interactions between NPs and proteins. Capillary electrophoresis allows for the quantification of protein adsorbed onto NPs without prior desorption, but the detection sensitivity is not 2
always high enough as the proteins can easily adsorb onto the inner surface of the capillary. Changes in the hydrodynamic radius of NPs after protein immobilisation can be determined by dynamic light scattering (DLS). Moreover, the DLS technique can be useful to monitor the binding ratio because NPs increase in size as proteins bind until the surface is saturated [9, 10]. Both DLS and NP tracking analysis can be used to estimate the amount of immobilised protein on NPs. However, these methods do not provide information about the amount of adsorbed protein but rather provide information about the hydrodynamic thickness of the adsorbed layer. X-ray photoelectron spectroscopy or zeta potential measurements can also be used to confirm the presence of a protein layer on NPs, but this data is also not quantitative. Conformational changes in proteins bound to the surface of a NP can be monitored with circular dichroism [11, 12], Fourier transform infrared spectroscopy  or Raman spectroscopy . The protein adsorption capacity of the surface of a NP can be determined with nuclear magnetic resonance (NMR) spectroscopy as amide protons are exquisitely sensitive to their environment [14, 15]. However, NMR spectroscopy cannot be used to detect NPs as their rotational correlation time is too slow. Moreover, once proteins are adsorbed on NPs they become invisible to NMR ; thus, it is not possible to monitor the state of protein-modified NPs. The NMR method is based on calculating the difference between the concentration of free protein in the presence and absence of NPs , and this measurement can be challenging for larger NPs (~ 90 nm) or low concentrations of NP that have a low number of bound proteins. Hence, this method is only suitable for systems with high protein to NP ratios. Another tool that can be used to characterise protein-NP systems is isothermal titration calorimetry (ITC). ITC measures the heat of binding and free energies can be determined . However, thiol oxidation can influence the observed heat and lead to inaccurate calculation of protein concentrations. Suspended microchannel resonators, also referred to as resonant mass measurements, measure the mass of proteins adsorbed to NPs from which the number of proteins on a NP and the surface area occupied by each protein can be determined . However, in this paper polystyrene beads ranged in size from 600–1000 nm and the protein concentration was high (~20,000 molecules per bead) . It is unknown whether suspended microchannel resonators method can be applied to small NPs at low protein concentrations or when only a small fraction of the NP is coated. Microscopic techniques like scanning and transmission electron microscopy (SEM, STEM/TEM) or atomic force microscopy can also confirm protein coating of NPs, but sample preparation for these techniques requires destructive treatments, i.e. sample drying, which may disrupt the protein structure. Although, there are several methods available to investigate NP-protein interactions, quantification of the amount of protein on the NP surface is still challenging. Moreover, selection of the proper method for a specific NP-protein system is not trivial and depends on the NP-protein interactions, protein aggregation as well as on interactions between the protein and other solution components. The most challenging aspect in each of the outlined methods is to accurately identify and quantify the amount of protein attached to a single NP. Therefore, we have elaborated on a simple, reproducible and highly sensitive analytical method to determine the amount of protein adsorbed on a single NP. In this study, we present a simple and versatile PAGE method to quantitatively determine the degree to which catalase (CAT) adsorbs onto well-characterised gold 3
nanoparticles (AuNPs). This method is based on the modification of AuNPs with CAT and subsequent detection of unbound protein by native-PAGE and silver staining. This method does not require any procedures to remove or separate unbound protein from the solution, including centrifugation or chemical treatment. With this method we have confirmed that AuNPs are coated with protein, as only AuNPs coated with protein enter and move in the gel during electrophoresis while non-coated citrate-AuNPs do not. Moreover, these detection conditions do not influence the protein-NP stability; hence, it is possible to monitor the colloidal state of NPs during the electrophoresis process. A drawback of our method is the fact that quantifying the amount of protein on the surface of a single NP requires that the exact number of NPs to be defined. Moreover, this method is suitable for colloids with NPs that are homogenous in size and shape. However, the advantage of this protocol is the fact that it is based on a well-known method typically used to analyse proteins that does not require any sophisticated equipment. 2.
Materials and Methods 2.1. Synthesis of gold nanoparticles (AuNPs) AuNPs (13 nm) were prepared in water by the chemical reduction method as described previously . Briefly, a chloroauric acid water solution (94.25 g, 2.01 × 10-4 % wt) was boiled under reflux and then sodium citrate (5.75 g, 0.877% wt) was added. The reaction was incubated for 15 min and then cooled down to room temperature. The final concentration of AuNPs in the colloid was 100 ppm. The number of particles in 1 mL of colloid was 4.5 × 1012. Deionised water used during the synthesis was obtained from a Millipore-Q water system (18.2 MΩ·cm). 2.2.Catalase preparation (CAT) Construction of the plant expression vector The plant expression vector was constructed based on human catalase cDNA isolated from human blood lymphocytes. The coding sequence of CAT was amplified by PCR (forward: 5’-TAAGCACCATGGATGGCTGACAGCCGGGATC-3, reverse: 5’TGCTTACACGTGTCAGTGTTTGTGACGGTGGTTGT-3’) with the addition of NcoI and EcoRI restriction sites (underlined) generating a 1642 base pair fragment. The amplified sequence was cloned into pCAMBIA 1305.2 with NcoI and EcoRI restriction enzymes, instead of the GUSPlus coding sequence, under control of the CaMV 35S promoter. Agrobacterium tumefaciens transformation A. tumefaciens strain LBA4404 was transformed by the freeze–thaw transformation method . A. tumefaciens LBA4404 carrying the CAMBIA1305.2CAT vector was grown for 2 days at 28oC on YEP media supplemented with 100 µg/mL rifampicin, 200 µg/mL streptomycin and 50 µg/mL kanamycin (for plasmid selection) in a 200 mL Erlenmeyer flask (with rotary shaking at 120 rpm). A. tumefaciens cells were collected by centrifugation at 4000 rpm for 20 min and diluted to OD600 = 0.6 in Murashige and Skoog (MS) media. For leaf discs, Nicotiana tabacum cv. Wisconsin 38 were grown in MS media for five weeks at 26oC, with a photoperiod of 16 h. Healthy, fully expanded leaves were cut into pieces and incubated with A. tumefaciens suspensions for 10 min in petri dish. The leaf discs were 4
transferred onto filter paper to remove the excess bacterial suspension and placed adaxial side down on solid MS medium. Cocultivation was performed for 3 days at 28oC in the dark. After cocultivation with A. tumefaciens cultures, plant explants were washed with distilled water supplemented with cefotaxime (500 µg/mL) and transferred onto solid MS regeneration media with 0.1 mg/L α-naphthaleneacetic acid, 1 mg/L 6-benzylaminopurine, 500 µg/mL cefotaxime and 50 µg/mL hygromycin. Plates were incubated in the light at 26°C, with a 16 h photoperiod and subcultured every two weeks. New shoots were transferred onto MS media supplemented with 500 µg/mL cefotaxime and 50 µg/mL hygromycin, maintained in tissue culture and subcultured every three weeks. Molecular analysis of transgenic plants Total genomic DNA was isolated from regenerated kanamycin resistant plants and untransformed tobacco using the cetyltrimethylammonium bromide method . The presence of a transgene in the transgenic plants was confirmed by PCR with CAT specific primers (forward: 5’-TAAGCACCATGGATGGCTGACAGCCGGGATC-3, reverse: 5’TGCTTACACGTGTCAGTGTTTGTGACGGTGGTTGT-3’). Protein isolation and detection of CAT in transgenic plants Proteins from transgenic and non-transgenic plants were isolated by grinding plant tissue with extraction buffer (PBS, pH 7.8) supplemented with 1% Triton X-100, 2 mM βmercaptoethanol and phenylmethylsulfonyl fluoride (PMSF) as described . SDS-PAGE The protein extract was subjected to standard SDS-PAGE with a Laemmli buffer system (5% stacking gel and 10% resolving gel). Isolated proteins were mixed with sample reducing buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% Bromophenol Blue and 5% β-mercaptoethanol), denatured by heating at 95°C for 5 min and loaded onto a 10% polyacrylamide gel. Electrophoresis was performed at 150 V (constant voltage) for 1 h. Following PAGE, the gel was stained with the colloidal Coomassie brilliant blue (CBB G250). Chromatographic purification of recombinant catalase Recombinant CAT was purified from extracted proteins on a DEAE-cellulose column . The purified protein was analysed by SDS-PAGE (Supplementary Material, Fig. 1S), dialysed and lyophilised. 2.3. Modification of gold nanoparticles with catalase (AuNPs-CAT) AuNPs synthesized in water according to chemical reduction method with sodium citrate (negative charge of synthesized AuNPs - citrate ions adsorbed on NPs surface; Zeta potential = – 37 ± 8 mV) were modified incubation with CAT. The amount of protein used for modification was calculated as 5, 10, 15, 20 and 50 CAT molecules per AuNP. The number of AuNPs was calculated based on the assumption that all NPs were spherical and the mean size of the NPs was 13 nm (as measured by STEM). The concentration of gold in the AuNP colloid was 100 ppm. Hence the calculated number of AuNPs in 1 g of colloid was N = 4.50 × 1012 particles. The exact amount of protein used for modification is shown in Table 1.
The indicated amount of CAT in water was added to 4 mL of a AuNP colloid solution (pH 6.3) at room temperature. Next, colloids with CAT were incubated for at least 2 h to allow the protein to immobilise on the surface of the AuNPs. No post-processing steps were carried out for any of the colloids after the modification process. 2.4. Methods Dynamic Light Scattering The hydrodynamic size of AuNPs and the agglomeration state of colloids before and after CAT immobilisation were measured using DLS (Nano ZS Zetasizer system, Malvern Instruments, laser wavelength 633 nm (He–Ne), scattering angle 173°, measurement temperature 25°C, medium viscosity 0.887 mPa·s, medium refractive index 1.330). All measurements were performed in a quartz microcuvette. Scanning Transmission Electron Microscopy Morphological studies of the AuNPs before and after CAT immobilisation were performed by STEM. Images were recorded using a Nova NanoSEM 450 scanning electron microscope equipped with a Schottky field emission with electron emitter operating at 30 kV and a STEM II detector for transmitted electron detection. Generally, the samples were prepared by placing a droplet of the dispersion onto a carbon-coated copper grid (300 mesh) and leaving it for 60 min to allow the solvent to evaporate. Gel electrophoresis To find the best conditions for determining the degree to which CAT adsorbs onto the surface of AuNPs, PAGE was performed under different conditions: native-PAGE, nonreducing SDS-PAGE, -mercaptoethanol-PAGE, and reducing SDS-PAGE. Native-PAGE Samples were mixed with sample buffer (62.5 mM Tris-HCl, pH = 6.8, 40% glycerol, 0.01% bromophenol blue) in ratio of 1:1. Electrophoresis was performed in 25 mM Tris, 192 mM glycine, pH = 8.3. Non-reducing SDS-PAGE Samples for non-reducing SDS-PAGE were prepared as in the case of native-PAGE but with the addition of SDS (2.1% in the sample buffer and 1% in the running buffer). Prior to electrophoresis the mixtures were heated for 5 min at 90°C in a water bath. -mercaptoethanol-PAGE Samples were mixed with sample buffer (62.5 mM Tris-HCl, pH = 6.8, 40% glycerol 0.01% bromophenol blue, 5% β-mercaptoethanol) at a ratio of 1:1. Electrophoresis was performed in 25 mM Tris, 192 mM glycine, pH = 8.3. Reducing SDS-PAGE Samples were prepared as in the case of -Mercaptoethanol-PAGE but with the addition of SDS (2.1% in the sample buffer and 1% in the running buffer). The mixtures were heated for 5 min at 90°C in a water bath prior to electrophoresis. One-dimensional electrophoresis was performed in a Bio-Rad Mini-Protean Tetra Cell under constant conditions for all four cases (38 min at 200 V, 10% acrylamide gel, 1 mm thick). After electrophoresis the gels were silver stained according to a modified Shevchenko protocol . Briefly, the gel slabs were fixed in 5% acetic acid, 50% methanol in water for 1 h. Next, gels were washed with 50% methanol for 15 min, followed by washing with 6
deionised water for 15 min to remove any remaining acid. The gels were sensitised by incubation in 0.02% sodium thiosulfate for 3 min, and rinsed twice with deionised water for 2 and 1 min. After rinsing, the gels were submerged in 0.15% silver nitrate and incubated for 45 min. After incubation, the silver nitrate was discarded and the gels were rinsed twice with deionised water for 2 and 1 min. The gels were developed in a mixture of 0.04% formalin (35% formaldehyde in water) in 2% sodium carbonate with intensive shaking. After the liquid turned yellow it was discarded and replaced with fresh water. The gels were developed until the desired intensity of staining was achieved (usually not longer than 10 min) and development was terminated by discarding the reagent and rinsing the gels with 5% acetic acid. The intensity of each protein band was estimated digitally by scanning the gel with a densitometer (Molecular Imager Gel Doc XR+) and using an imaging program (ImageLab 2.0, Bio-Rad Lab). Experiments were repeated three times with comparable results, and representative outcomes from such experiments are shown in the figures. 3.
Results and Discussion 3.1. Characterisation of AuNPs-CAT The size of AuNPs before and after modification was investigated by STEM (Fig. 1). STEM images of all CAT -AuNPs conjugates with the increasing amount of CAT per NP are shown in Supplementary Material (Fig. 5S.). The size of the metallic core of the AuNPs before and after the modification process was stable and equal to 13 ± 1 nm. The modification process did not disturb the stability of the NPs. After modification, the size of the metallic AuNPs remained unchanged but the distance between NPs deposited on the carbon-coated copper grid in each cluster increased for AuNPsCAT (Fig. 1b) compared to AuNPs-citrate (Fig. 1a). The changes in NP distances are most likely caused by the size of the modifier molecule adsorbed on the surface of the NPs. Small citrate ions on NPs effectively stabilised and protected them from agglomeration (electrostatic stabilisation in colloidal solution), but the distances between NPs after deposition were quite small. Modification of AuNPs with big protein molecules (such as CAT) also protected these particles from agglomeration (through steric stabilisation) and resulted in an increased distance between single NPs after their deposition onto carbon-copper grids . The colloidal stability of AuNPs before and after CAT modification was monitored by DLS (Fig. 2). The changes of the hydrodynamic size of CAT-modified AuNPs for samples with increasing amount of CAT used for modification versus citrate-AuNPs are shown in Supplementary Material (Fig. 4S.) AuNPs were colloidally stable and did not form any agglomerates or aggregates after CAT modification for all surface coverage. The hydrodynamic size of the NPs after modification increased from 19 ± 2 nm (AuNPs-citrate) to 29 ± 3 nm (AuNPs-CAT). The hydrodynamic size of NPs measured by DLS depends on the size of the metallic core of the particle, substances present on its surface and its solvation shell. Hence, the hydrodynamic size of AuNPs-CAT was larger than the size of AuNPs modified with citrate, which was apparently caused by the different types of modifiers adsorbed on the NP surface: big proteins (CAT) and small ions (citrate), respectively.
3.2. Gel electrophoresis 3.2.1. Optimisation of protein detection conditions To find the best conditions for determining the amount of protein bound to NPs, samples were investigated using four different types of PAGE: native-PAGE, non-reducing SDS-PAGE, -mercaptoethanol-PAGE, and reducing SDS-PAGE. PAGE was performed for AuNPs-citrate (unmodified) and for AuNPs-CAT (CAT-modified) samples with different levels of protein coverage on the NPs (5, 10, 15, 20 and 50 protein molecules per AuNP) (see Supplementary Material Fig. 2S). CAT was detected by PAGE regardless of the chemical conditions of the separation process. However, the protein detection sensitivity depended on the process conditions, i.e. the sample buffer composition (the presence of SDS or mercaptoethanol affected the detection sensitivity) (Fig. 2S b-d). The most sensitive condition was native-PAGE (Fig. 2Sa), where intense brown bands from unbound protein were observed for surface coverages between 15 and 50 protein molecules per NP. The detection limit was 1.5 ng of protein for native-PAGE and about 60 ng of protein for both non-reducing and reducing SDS-PAGE. Thus, the sensitivity of native-PAGE was 40 times higher than in the non-reducing and reducing SDS-PAGE. This clearly indicated that the presence of SDS lowered the detection sensitivity . However, in -mercaptoethanol-PAGE it was also hard to observe the position of modified-AuNPs in the gel during electrophoresis (Fig. 2Sc). This problem was not observed in native-PAGE where modified-AuNPs were easily observed (Fig. 1Sa); hence this method seemed to be preferable for colloids and was selected for the quantification of CAT adsorbed onto AuNPs. To quantify the amount of protein, all protein bands were subject to analysis by native-PAGE. In native-PAGE, protein separation occurs based on changes in the net charge of a protein, while in SDS-PAGE the charge of a protein is screened by SDS molecules that bind to the protein surface at constant ratio (1.4:1), which means that separation is based only on the molecular weight rather than the net charge. In our case, SDS-PAGE had only one band (Fig. 3b and d) which corresponded to CAT (60 kD). No other proteins were detected. Therefore, all of the bands were analysed for our quantification. 3.2.2 Quantification of the degree to which catalase was adsorbed on the surface of NPs by PAGE The degree to which protein adsorbs to NPs (the number of protein molecules adsorbed on the surface of a NP) was based on modification of NPs with an excess of protein and then detecting the amount of unbound protein using PAGE. Briefly, the intensity of the band for unbound protein was measured and compared to the band intensity from CAT not exposed to NPs (taken as 100%). Next, the percent band intensity was converted using the molecular weight of CAT. For each concentration, a reference sample with the same amount of CAT molecules was loaded onto the gel next to samples of CAT-modified AuNPs. Comparison between the reference and sample lanes enabled quantification of the amount of CAT adsorbed onto AuNPs. Native-PAGE not only allowed us to detect the protein but also allowed us to observe the movement of modified NPs during electrophoresis, which depended on the amount of protein present on their surface. A photograph of a gel with AuNPs-CAT following electrophoresis and before silver staining is shown in Fig. 3. 8
The ruby-red colour of the bands in the gel was due to the presence of AuNPs. The position of the band of maximum intensity depended on the amount of CAT used to modify the AuNPs. In the case of 5 CAT molecules per NP, the mobility of AuNPs-CAT was small and NPs were located near the well. Nevertheless, the amount of protein was enough to effectively protect NPs against agglomeration and/or aggregation. Increasing the amount of protein to 10 or 15 molecules per NP resulted in increased mobility of AuNPs-CAT. Most of the AuNPs-CAT were located in one narrow band, which indicated that the surface coverage of AuNPs with CAT was uniform and equal for most NPs. A photograph of the gel with AuNPs-CAT after silver staining is shown in Fig. 4.
Unmodified AuNPs did not enter into the polyacrylamide gel (Fig. 4, AuNPs) while CAT-modified AuNPs moved easily and appeared as violet zones (Fig. 4, AuNPs-CAT-5, 10, 20, 50). After silver staining the unbound CAT was observable in the gel as brown bands. The absence of brown bands for samples with 5 and 10 CAT molecules per NP, but their appearance in samples with 15 indicated that full surface coverage of the 13 nm AuNPs was in the range of 10–15 CAT molecules per NP. To quantify the full surface coverage of AuNPs with CAT, samples with unbound proteins were selected (15, 20, 30, 40 and 50 CAT molecules per NP, which corresponded to 51 ng, 68 ng, 101 ng, 135 ng and 176 ng of CAT in the bands, respectively) (Table 2). Photographs of the gels after electrophoresis and silver staining for AuNPs-CAT and their respective quantity of free CAT (15–50 CAT molecules per NP) are shown in Fig. 3S of the Supplementary Material. The relationship between the amount of CAT used for modification and the amount of unbound CAT detected after AuNP modification is presented in Fig. 5. Three characteristic regions can be easily seen on this plot. The first region (orange colour) was characteristic for AuNPs not fully covered with CAT. All proteins added during the modification step were adsorbed onto the NP surface and there was no unbound protein in the colloidal solution. Increasing the amount of protein used for modification led to further adsorption onto AuNPs until the surface of the particles was saturated and unbound proteins could be detected (second region - red colour). A further increase in the amount of protein resulted in an increase in the amount of unbound CAT detected by PAGE (third region violet colour) while the surface coverage of AuNPs with CAT molecules remained constant within the error of the measurement (2CAT, 3CAT, 4CAT and 5CAT). The mean surface coverage of AuNPs with CAT proteins was calculated as the average distance between the solid line and the dotted line, about 55 ng, which corresponded to 16 CAT molecules per AuNP (average value calculated for samples with unbound proteins, 2CAT - 5CAT). Further increasing CAT above 50 molecules per NP may not be effective at increasing the ratio between the amount of CAT detected in the gel and the amount used for modification and may increase error as the intensity of the bands becomes saturated. 3.
An effective method to determine the degree to which CAT molecules adsorb onto AuNPs using PAGE and silver staining was presented. Determination of CAT adsorption was carried out with different electrophoresis protocols to find the optimal conditions for quantification of the surface coverage of 13 nm AuNPs by CAT. This method is simple and versatile as it does not require the use of any protein isolation procedures. Moreover, the procedure can be universally applied to NPs as reagents typically used in protein electrophoresis (SDS or - mercaptoethanol) that could affect the colloidal stability of NPs modified with protein can be avoided. Moreover, this method allowed for the identification and quantification of the amount of CAT adsorbed on the surface of NPs in a colloidal state. The presented method is versatile and effective, and can be successfully used for the identification and quantification of proteins adsorbed on the surface of different types of colloidal NPs. Acknowledgements This work was supported by the National Science Centre of Poland (project number 2013/09/B/NZ7/01019).
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(a) (b) Fig. 1. STEM images of AuNPs (a) and CAT-modified AuNPs with the surface coverage 50 CAT molecules per 1 NP (b).
25 intensity (%)
30 20 10
20 15 10 5
100 Size (d.nm)
100 Size (d.nm)
(a) (b) Fig. 2. DLS size distribution histograms of citrate-modified AuNPs (a) and CATmodified AuNPs (50 CAT molecules per NP) (b).
Fig. 3. Photograph of a gel of AuNPs-CAT with increasing amounts of protein (5, 10, 15, 20 and 50 molecules per NP) after electrophoresis (but before silver staining).
Fig. 4. Photograph of silver stained native-PAGE gels of unmodified AuNPs and AuNPs-CAT with different amounts of protein (5, 10, 15, 20 and 50 protein molecules per NP) and pure CAT at the same total concentrations.
∆𝑨𝑽 𝑪𝑨𝑻 =
∆𝟏 + ∆𝟐 + ∆𝟑 + ∆𝟒 + ∆𝟓 𝑪𝑨𝑻 = 𝟓𝟓. 𝟒 𝒏𝒈 𝟓
∆𝑨𝑽 𝑪𝑨𝑻 = 𝟏𝟔 𝑪𝑨𝑻 𝒎𝒐𝒍𝒆𝒄𝒖𝒍𝒆/𝑵𝑷 3CAT 2CAT 1CAT
Fig. 5. The relationship between the amount of CAT used for modification and the amount of unbound CAT detected by PAGE (AVCAT - the average amount of CAT adsorbed on the AuNPs).
Table 1. The amount of CAT used for modification of AuNPs. NPs size [nm]
amount of CAT used for modification [molecule/NP]* [g/mL]** 5 2.25 × 10-6 10 4.50 × 10-6 13 15 6.74 × 10-6 20 9.01 × 10-6 50 22.5 × 10-6 *molecule/NP – the amount of CAT molecules per NP **g/mL – the amount of CAT in 1 mL of colloid
Table 2. The amount of CAT used to modify AuNPs and the amount of free CAT detected in electrophoresis experiments was used to determine the surface coverage of AuNPs with CAT. amount of CAT used for modification after modification [molecules per NP] [ng]* unbound [ng]* adsorbed [ng]* 5 17 0 17 10 34 0 34 15 51 4.9 46.1 20 68 12.6 55.4 30 101 48.3 52.7 40 135 56.0 79.0 50 176 99.0 77.0 [ng]*- amount of CAT detected in all bands in each lane of the gel
surface coverage [CAT molecules per NP] 5 10 14 16 14 17 21