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Study on aggregation behavior of cytochrome C-conjugated silver nanoparticles using asymmetrical flow field-flow fractionation Sun Tae Kim, Yong-Ju Lee, Yu-Sik Hwang, Seungho Lee

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Received date: 22 April 2014 Revised date: 16 May 2014 Accepted date: 20 May 2014 Cite this article as: Sun Tae Kim, Yong-Ju Lee, Yu-Sik Hwang, Seungho Lee, Study on aggregation behavior of cytochrome C-conjugated silver nanoparticles using asymmetrical flow field-flow fractionation, Talanta, http://dx.doi.org/ 10.1016/j.talanta.2014.05.060 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 galley proof before it is published in its final citable 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.

Study on aggregation behavior of Cytochrome C-conjugated silver nanoparticles using asymmetrical flow field-flow fractionation Sun Tae Kima, Yong-Ju Leeb, Yu-Sik Hwangb and Seungho Leea,* a

Department of Chemistry, Hannam University, Daejeon 305-811, South Korea

b

Gyungnam Department of Environmental Toxicology, Korea Institute of Toxicology, Jinju 660-844, South Korea

*

To whom correspondence should be addressed (e-mail: [email protected]; telephone +82-42-

629-8822; fax +82-42-629-8811)

ABSTRACT

In this study, 40 nm silver nanoparticles (AgNPs) were synthesized using citrate reduction method and then the surface of AgNPs was modified by conjugating Cytochrome C (Cyto C) to improve stability and to enhance bioactivity and biocompatibility of AgNPs. It is known that Cyto C may undergo conformational changes under various conditions of pH, temperature, ionic strength, etc., resulting in aggregation of the particles. These parameters also affect the size and size distribution of Cyto C-conjugated AgNPs (Cyto C-AgNP). potential measurement revealed that the adsorption of Cyto C on the surface of AgNPs is saturated at the molar ratio [Cyto C]/[AgNPs] above about 300. Asymmetrical flow fieldflow fractionation (AsFlFFF) analysis showed that hydrodynamic diameter of AgNPs increases by about 4 nm when the particle is saturated by Cyto C. The aggregation behavior of Cyto C-AgNP at various conditions of pH, temperature and ionic strength were investigated using AsFlFFF and UV-VIS spectroscopy. It was found that the aggregation of Cyto C-AgNP increases with decreasing pH, increasing temperature and ionic strength due to denaturation of Cyto C on AgNPs and reduction in the thickness of electrostatic double layer on the surface of Cyto C-AgNP.

Keywords: Asymmetrical flow field-flow fractionation (AsFlFFF), Cytochrome C-conjugated AgNPs, aggregation, -potential, UV-VIS spectroscopy

1. Introduction

Metallic nanoparticles are of interest in the field of life science due to their unique chemical, physical and biological properties [1]. Surface modification of metallic nanoparticles with polymer or protein is an approach to stabilize the nanoparticles. Surface modifications with a suitable agent such as organic molecules, surfactant, protein or polymer prevent aggregation, and improve biocompatibility and stability of the metallic nanoparticles in suspensions [2-4]. Among metallic nanoparticle, silver nanoparticles (AgNPs) are of particular interest because of their unique characteristics including antimicrobial activities, which often depend on their size and shape. They can be used as biosensor materials, fibers, superconducting materials, cosmetic products, and electronic components [5-8]. AgNPs are often coated with various types of anions, polymers or proteins to improve their biocompatibility and stability and also to control toxicity [9, 10]. Cytochrome C (Cyto C) is a highly water soluble protein which is often used to coat nanoparticles to improve their biocompatibility.

Sometimes Cyto C undergoes conformational changes as it becomes

partially unfolded upon adsorption onto the surface of nanoparticles. The unfolded Cyto C adsorbed on a nanoparticle interacts with other Cyto C in suspension causing protein aggregation on nanoparticle surface, which results in nanoparticle aggregation [11, 12]. The unfolding of Cyto C induced by presence of denatures or change in pH, temperature or ionic strength has been studied using light scattering [13]. It has been reported that aggregation of Cyto C-AgNP induces a coupling of the localized surface plasmon resonance of AgNPs, and may cause color change at various pH [12]. The effects of pH and ionic strength on aggregation behavior of nanoparticles in aqueous suspensions have been studied in terms of the change in the hydrodynamic diameter

of particles [14, 15]. It has been reported that the combined effect of pH, ionic strength, capping agent, ionic composition and other characteristics of the suspending liquid play a significant role in formation of aggregates or stability of the nanoparticles [16]. Appropriate analytical tool is thus required to detect and quantify aggregates. Various analytical techniques such as light scattering, separation, spectroscopy and microscopy are employed to monitor aggregation of bioconjugated nanoparticles including dynamic light scattering (DLS) [17, 18], size-exclusion chromatography (SEC) [19, 20], UV/VIS spectroscopy [21, 22], electron microscopy (EM) [23, 24]. Each technique has its own merits and limitations for characterization of bioconjugated nanoparticles. DLS provides hydrodynamic diameter and its distribution using Stokes-Einstein equation, and is fast and simple to operate. However its application to polydisperse samples or quantification of aggregates is often limited [25]. SEC provides size-based separation of particles, and yields useful information on size distribution of particles. However the resolution of SEC may be hampered by nonspecific interaction of the sample with stationary phase, and its application to larger particles is rather limited due to lower resolution.

UV-VIS spectroscopy provides

information on aggregation in suspension, but requires fairly concentrated sample for measurement. EM allows one to view the size and shape of individual nanoparticles. During EM measurement, however, the sample suspension is dried and put on a high-vacuum, which may cause shrinkage and agglomeration of particles. Field-flow fractionation (FFF) is a family of size-based separation techniques capable of providing separation and characterization of macromolecules or particles ranging in size from 1 nm up to about 100 m [26]. Among the members of FFF family, asymmetrical flow fieldflow fractionation (AsFlFFF) is particularly useful for separation and characterization of bioconjugated nanoparticle [27, 28] and protein aggregates [28-31]. In this study, 40 nm AgNPs were synthesized using the citrate reduction method [32].

Then the surface of the nanoparticles were modified by conjugating Cyto C at various molar ratios of Cyto C to AgNPs, [Cyto C]/[AgNPs]. AsFlFFF was then used for separation and quantification of Cyto C-conjugated AgNPs (Cyto C-AgNP) obtained at various pH, temperature and ionic strengths.

Also, the aggregation behavior of Cyto C-AgNP was

studied using AsFlFFF and UV-VIS spectroscopy. Aim of this study is to determine the size and size distribution of Cyto C-AgNP and to study stability of Cyto C-AgNP in suspension at various conditions.

2. Theory

In AsFlFFF, the hydrodynamic diameter (dH) of a particle is related with its retention time (tr) by [33]

dH

2V o kTtr SKw2 Fct o

(1)

, where Vo is the void volume of the AsFlFFF channel, k is the Boltzmann constant, T is the absolute temperature (k),  is the viscosity of the carrier liquid, w is the channel thickness, Fc is the volumetric cross-flow rate, and to is the void time (the elution time of the species that are not retained). Using Eq. (1), an AsFlFFF fractogram of the particles can be converted directly to a size distribution.

2. Experimental

2.1. Chemicals

Silver nitrate (AgNO3), trisodium citrate, sodium azide and Cyto C were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium phosphate buffer solution (PBS) was prepared by adjusting the concentrations of sodium dihydrogen phosphate and disodium hydrogen phosphate to achieve desired pH and ionic strength. Cyto C was prepared in 20 mM PBS at pH 7.2.

2.2. Synthesis of citrate-silver nanoparticles and preparation of Cyto C-AgNP

Silver nanoparticles were prepared by the citrate reduction of silver nitrate, first reported by Lee and Meisel with some modifications [32]. A 200 mL of 0.0001 M AgNO3 solution was heated to boil at 100° C.

7.5 mL of 1 % (w/v) sodium citrate solution was then added

and the solution was refluxed for 35 minutes. The temperature, stirring rates were held constant during preparation. In other to obtain the Cyto C-AgNP, pH of AgNPs suspension was adjusted to 11 by adding 0.1 M NaOH. The isoelectric point of Cyto C is near 10, and Cyto C is stable and no aggregation occurs at pH 11 [11]. At pH above the isoelectric point, Cyto C molecules become negatively charged, and produce Cyto C-AgNP via electrostatic interaction.

Then, 0.01 mg/mL or 0.1 mg/mL solutions of Cyto C in PBS buffer was added

at desired molar ratio of [Cyto C]/[AgNPs]. The suspension was kept at 4° C for 1 day.

2.3. Determination of concentration of silver nanoparticles

The concentration of silver nanoparticles was determined by the method which has been previously reported by Liu et al. for gold nanoparticle analysis [34]. The average number of atoms per nanoparticle is defined as

N=

d 3 N 6M A

(2)

, where N is the number of atoms per nanoparticle,  is the density of silver (10.5 g/cm3), d is the average diameter of nanoparticles, M is the atomic mass of silver (107.868 g), NA is the number of atoms per mole (Avogadro’s number). The molar concentration C of the nanoparticles in suspension was determined by C=

NT NVN A

(3)

, where NT is the total number of silver atoms added as AgNO3, N is the number of atoms per nanoparticle in Eq. (2), and V is the volume of the reaction solution.

The concentration of

silver nanoparticles synthesized in this study was calculated to be 4.55 × 10-11 mol/L from Eqs. (1) and (2). 

2.4. Asymmetrical Flow Field-Flow Fractionation (AsFlFFF)

The AsFlFFF system used in this study was the Eclipse AF4 (Wyatt Tech., Europe GmbH, Dernbach, Germany) assembled with a 350 m-thick Mylar spacer and a regenerated cellulose membrane (Millipore, Bedford, MA, USA) having the cut-off molecular weight of 10 kDa. The channel geometry was trapezoidal with the tip-to-tip length of 26.5 cm and breadths at the inlet and the outlet of 2.2 and 0.6 cm, respectively. To measure the channel

thickness, 52 ± 2 nm polystyrene beads having a narrow size distribution were injected with the carrier solution of water containing 0.02% FL-70 and 0.02% NaN3. The channel thickness determined from the retention time of 52 ± 2 nm polystyrene beads was 238 m. A HPLC pump (Futecs P-6000, Daejeon, Korea) was used to deliver the carrier liquid into the AsFlFFF channel. The channel and the cross-flow rates were measured using liquid flow meters (Optiflow 1000, Agilent Technologies, Palo Alto, CA, USA). Eluted particles were monitored using a Model-500 UV detector (Chrom tech Inc., MN, USA) with the wavelength set at 410 nm. The detector signal was collected by using FFF analyzer software supplied by Quantum Soft. (Daejeon, Korea). The 20 L particle suspension was injected into the AsFlFFF channel using a 20 L loop injector (Rheodyne, Cotati, CA, USA). A syringe pump (Model 100, KD Scientific, USA) was used to inject the sample suspension into the AsFlFFF channel at the flow rate of 0.2 mL/min for 60 s.

2.6. Other instruments

DLS and ]-potential measurements were made at the scattering angle of 90° (for DLS measurement) and 17° (for ] -potential measurement), respectively, with a Zetasizer Nano ZS90 (Malvern instrument, Worcestershire, UK) equipped with a He–Ne laser (633 nm) as the light source. All measurements were repeated three times for a DLS analysis and ten times for a ] -potential measurement, respectively. The results were presented as the mean size or ] -potential ± one standard deviation. UV-3101-PC spectrophotometer (Shimadzu, Kyoto, Japan) was used for UV-VIS spectroscopic analysis of AgNPs.

3. Results and discussion 

3.1. Formation of Cyto C-AgNP

The -potential measurement of nanomaterial provides not only information on the electrical potential on the surface (‘surface charge’), yielding an insight into stability of nanometerials in suspension, but also the thickness of the electrical double layer on the surface of bioconjugated nanometerials [11, 35]. As the amount of protein adsorbed on the nanoparticle increases, the surface charge varies, and eventually reaches a stable value, indicating the nanoparticle surface is saturated with the protein. In order to obtain saturated Cyto C-AgNP, -potential was measured by varying the molar ratio, [Cyto C]/[AgNPs], at pH 11. Fig. 1 shows -potential measured for Cyto CAgNP prepared at various molar ratios of [Cyto C]/[AgNPs] ranging from 0 up to 500. As shown in Fig. 1, -potential of AgNPs increases as [Cyto C]/[AgNPs] increases at the beginning, which suggests that the surface charge of AgNPs is neutralized upon adsorption of Cyto C. -potential of the bare AgNPs was -61 ± 4.5 mV and increases rather quickly with increasing [Cyto C]/[AgNPs] until the molar ratio reaches about 100.

At [Cyto C]/[AgNPs]

higher than about 100, -potential still increases with [Cyto C]/[AgNPs], but at much slower rate.

Further increase in [Cyto C]/[AgNPs] above about 300 did not lead to significant

changes in -potential. It seems Cyto C-AgNP is formed by electrostatic attraction of the negatively charged carboxylate groups on AgNPs and the positively charged Cyto C.

3.2. Characterization of Cyto C-AgNP

In order to determine the size of Cyto C adsorbed on AgNPs, hydrodynamic diameter was determined using AsFlFFF and DLS. Fig. 2 shows AsFlFFF fractograms (Fig. 2(a)) and size distributions (Fig. 2(b)) of three types of AgNPs (bare AgNPs and Cyto C-AgNP obtained at [Cyto C]/[AgNPs] of 300 and 20,000). Both Cyto C-AgNP were prepared at pH 11. The AsFlFFF channel flow rate and the cross-flow rate were 0.49 and 1.05 mL/min, respectively. As shown in Fig. 2(a), the elution time of AgNPs at peak was increased by about 0.5 min when the particles were treated with Cyto C at [Cyto C]/[AgNPs] of 300 due to an increase in hydrodynamic diameter of the particles. This increase in the elution time of about 0.5 min is translated to an increase in the hydrodynamic diameter of about 4 nm (from 40.4 ± 0.4 to 44.5 ± 0.2 nm) as shown in Fig. 2(b). Each run was repeated 2 or more times. The size increase of the Cyto C-AgNP was clearly supported by UV/VIS spectroscopy, as shown in Fig. 3(a). The maximum absorbance of bare AgNPs appeared at 410 nm, and the full width at half maximum (FWHM) was 77 nm, indicating the particles have a narrow size distribution [36]. When AgNPs was conjugated with the Cyto C at [Cyto C]/[AgNPs] of 300, the UV/VIS absorption band was red-shifted to 416 nm, and FWHM was increases to 79 nm. It has been reported that the red shift is caused by alteration of the refractive index of AgNPs by the Cyto C layer, resulting particle size increase [11]. Fig. 3(b) shows size distributions of the same two AgNPs obtained from DLS. It can be seen that the hydrodynamic diameter of AgNPs was increased from 47 ± 0.5 to 55 ± 1.1 nm due to adsorption of Cyto C molecules on the AgNPs surface.

It is seen that the

hydrodynamic diameters from DLS (Fig. 3(b)) are somewhat larger than those from AsFlFFF (Fig 2(b)) for the same sample. bias toward large particles [37].

DLS measurements may have been overestimated due to

As shown in Fig 2(b), addition of an excess of Cyto C ([Cyto C]/[AgNPs] of 20,000) did not change the particle size significantly as compared to that of Cyto C-AgNP obtained at the molar ratio of 300. This indicates the AgNPs particle surface is already saturated by Cyto C at the molar ratio of 300, and there will exist a large amount of free Cyto C in solution. The earlier eluting band that appears between the void time and about 2 min in Fig. 2(a) corresponds to those free Cyto C and their aggregates. When Cyto C was injected alone at the same condition, it was eluted together with the void peak as expected. At the molar ratio of 300, all (or most) of Cyto C molecules are adsorbed on AgNPs forming a uniform layer on the surface of the particles. On the other hand, at the molar ratio of 20,000, many of free Cyto C molecules are present between particles, resulting in reduction in repulsive force between particles.

This may explain why the -potential measured at the molar ratio of

20,000 (-23.6 ± 1.6 mV) was much lower than that measured at 300.

3.3 Quantification of aggregates in Cyto C-AgNP suspension determined by UV-VIS spectroscopy and AsFlFFF

UV-VIS spectroscopy can be employed to characterize bioconjugated nanoparticles in terms of the size, aggregation behavior and the change in refractive index [38]. Fig. 4(a) shows variation of UV-VIS spectra of Cyto C-AgNP with pH. As pH decreases from 11.0 to 2.9, the absorbance of the first band (at 416 nm) gradually decreases, and the second band is red shifted from around 520 to 670 nm. The red shift was observed by the change in color of the suspension from yellow to red and then finally to a grey-blue as aggregation of Cyto CAgNP proceeds as reported previously [12]. The ratio of the absorbance at 600 nm to that at 416 nm (A600/A416) can be used as a measure of particle aggregation as it will increase proportionally as aggregation increases [11,

12].

Fig 4(b) shows variation of A600/A416 with pH, which shows gradual decrease of

A600/A416 with increasing pH.

This indicates aggregation of AgNPs decreases as pH

increases. Fig. 5 shows variation of UV-VIS spectra and AsFlFFF fractogram of Cyto C-AgNP with storage time at pH 10. As shown in Fig. 5(a), UV-VIS absorbance of Cyto C-AgNP at 416 nm gradually decreases with storage time, while a new band at around 600 grows. This indicates aggregation of Cyto C-AgNP increases with increasing storage time. In order to quantify the aggregation behavior, the particles were analyzed by AsFlFFF as shown in Fig. 6(b). Experimental conditions were same as those in Fig. 2(a), except that the cross-flow was turned off after 10 min to allow complete elution of the aggregates. It can be seen that the area of first peak gradually decreases, while that of the second peak (which corresponds to aggregates) gradually increases with increasing storage time.

It seems the Cyto C

molecules adsorbed on AgNPs become increasingly unfolded and interact with Cyto C molecules on other particles that are protonated in the solution with increasing storage time [12, 13, 39]. Sample recovery R of Cyto C-AgNP in AsFlFFF can be determined by

R

§ A· ¨¨ ¸¸ u 100% © Ao ¹

(4)

, where A and Ao are the AsFlFFF peak area of unaggregated Cyto C-AgNP in the elution time range of 0 to 10 min with and without the cross-flow, respectively. The peak area A decreases with increasing storage time, and thus R decreases. Fig. 6 shows plots of A600/A416 of Cyto C-AgNP and the sample recovery, R, in AsFlFFF as a function of storage time. It can be seen that the sample recovery in AsFlFFF decreases rapidly by about 50% after 60 min of storage, and then decreases slowly down by about 20% after300 min.

On the other hand, the absorption ratio slowly increases from about 0.04 to

0.085 after 120 min of storage, and then rapidly increases up to 0.27 after 300 min. It is noted that, while UV/VIS spectroscopy provides qualitative information on the existence of aggregation of Cyto C-AgNP in suspension, AsFlFFF provides quantitative information from the sample recovery data.

3.4 Effect of temperature and ionic strength on stability of Cyto C-AgNP in solution

Fig. 7 shows variation of AsFlFFF fractogram of Cyto C-AgNP with storage temperature (Fig. 7(a)) and with the ionic strength of the suspending liquid (Fig. 7(b)) at pH 11. In both Fig. 7(a) and (b), the external field (cross-flow) was turned off after 10 min. In Fig. 7(a), Cyto C-AgNP suspensions were stored at seven different temperatures of 25, 50, 55, 60, 65, 70, and 80 °C in preheated water bath for 1 hour on a horizontal shaker (shaken at 200 cycles/min). At 25 °C, no elution was observed after the external field was turned off as shown in Fig. 7(a), and the sample recovery, R, was determined to be 100% from Eq. (4). As the storage temperature increases, the peak area decreases gradually, and, at the same time, the elution band area observed after 10 min gradually increases. As mentioned earlier, partially unfolded Cyto C molecules adsorbed on an AgNPs interacts with Cyto C on other AgNPs, and results in aggregation of the AgNPs.

Results shown in Fig.

7(a) indicate that higher storage temperature promotes unfolding of Cyto C’s, and causes more aggregation of Cyto C-AgNP. In Fig. 7(b), Cyto C-AgNPs were stored at 25 °C for 1 h in suspension liquids of six different ionic strengths. The ionic strength was adjusted by adding appropriate amount of sodium chloride.

It can be seen that, as the ionic strength of the suspension liquid increases,

the peak area decreases gradually, and, at the same time, the elution band area observed after 10 min gradually increases. This suggests aggregation of Cyto C-AgNP increases with

increasing ionic strength of the suspension liquid. It seems that, as the ionic strength increases, the thickness of the electrical double layer on the surface of the AgNPs decreases, and the electrostatic repulsion between particles decreases, promoting aggregation of the particles.

4. Conclusion

In this study, AsFlFFF was employed to determine the average size and size distribution of AgNPs, and to study the aggregation behavior of Cyto C-AgNP at various conditions of pH, temperature, ionic strength and storage time.

Results showed that adsorption of Cyto C

on the surface of AgNPs is saturated at the molar ratio ([Cyto C]/[AgNPs]) above about 300, and, from AsFlFFF analysis, the hydrodynamic diameter of the particles increases by about 4 nm. Results suggest AsFlFFF is a useful tool not only for the analysis of the size distribution of nanoparticles, but also for quantitative monitoring of aggregation behavior of bioconjugated nanoparticles.

Acknowledgment

This research was also supported by Korea Research Foundation (KRF) and Korea Institute of Toxicology (KIT).

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Figure captions. Fig. 1. Variation of ]-potential of CytoC-AgNPs with molar ratio of [Cyto C]/[AgNPs]. Fig. 2. AsFlFFF fractograms (a) and size distributions (b) of bare AgNPs and Cyto C-AgNP

obtained at [Cyto C]/[AgNPs] of 300 and 20,000. The channel and cross-flow rate were 0.49 and 1.05 mL/min, respectively Fig. 3. UV/VIS spectra (a) and DLS data (b) of bare AgNPs and Cyto C-AgNP obtained at

[Cyto C]/[AgNPs] of 300. Fig. 4. Variation of UV/VIS absorption spectra (a) and absorption ratio (b) of Cyto C-AgNP

with pH. Fig. 5. Variation of UV/VIS spectra (a) and AsFlFFF fractogram of Cyto C-AgNP with

storage time at pH 10. Fig. 6. Variation of UV/VIS absorption ratio and sample recovery of Cyto C-AgNP with

storage time. Fig. 7. Variation of AsFlFFF fractogram of Cyto C-AgNP with storage temperature (a) and

ionic strength at pH 11.

Graphical abstract Asymmetrical flow field-flow fractionation (AsFlFFF) of Cytochrome Cconjugated silver nanoparticles

Highlights { AsFlFFF was employed for size analysis of Cyto C-AgNP, and to study aggregation

behavior. { Hydrodynamic diameter of AgNP increases by ~5 nm when saturated by Cytochrome

C. { Aggregation of AgNP increases with decreasing pH and increasing ionic strength.



] -potential (mV)

-40 -45 -50 -55 -60 -65 -70 0

100

200

300

400

[Cyto C]/[AgNP]

500

600 

Fig. 1. Variation of ]-potential of Cyto C-AgNP with molar ratio of [Cyto C]/[AgNP].

(a)



UV/Vis response

Bare AgNP Cyto C-AgNP 300 Cyto C-AgNP 20,000

Void time

0

2

4

6

8

10

Elution time (min)

Rel. Mass

(b)

20

40

60

80

Diameter (nm) Fig. 2. AsFlFFF fractograms (a) and size distributions (b) of bare AgNP and Cyto C-AgNPs

obtained at [Cyto C]/[AgNP] of 300 and 20,000. The channel and cross-flow rate were 0.49 and 1.05 mL/min, respectively



(a) 1.2 Bare AgNP Cyto C-AgNP300

Absorbance

1.0 0.8 0.6 0.4 0.2 0.0 300

350

400

450

500

550

600

Wavelength (nm) (b)

14

Intensity (%)

12

Bare AgNP Cyto C-AgNP 300

10 8 6 4 2 0 10

100

Diameter (nm) Fig. 3. UV/VIS spectra (a) and DLS data (b) of bare AgNP and Cyto C-AgNP obtained at

[Cyto C]/[AgNP] of 300.



(a) 1.2 pH 11.0 9.9 8.9 7.9 7.3 6.0 4.5 2.9

Absorbance

1.0 0.8 0.6 0.4 0.2 0.0 300

400

500

600

700

800

Wavelength (nm) 

(b) 1.2

A600/A416

1.0 0.8 0.6 0.4 0.2 0.0 2

4

6

8

10

12

pH Fig. 4. Variation of UV/VIS absorption spectra (a) and absorption ratio (b) of Cyto C-AgNP

with pH.



(a) 1.2 Storage time 5 min 30 60 90 120 150 240 300

Absorbance

1.0 0.8 0.6 0.4 0.2 0.0 300

400

500

600

700

800

wavelength (nm) 

UV/Vis response

(b)

Void time

0

Field off

5

10

Storage time 5 min 30 60 120 180 240 300

15

20

Elution time (min) Fig. 5. Variation of UV/VIS spectra (a) and AsFlFFF fractogram of Cyto C-AgNP with

storage time at pH 10.

A600 /A416

0.25

100 Sample Recovery

0.20

80 A600/A416

0.15

60

0.10

40

0.05

20

0.00 0

50

100

150

200

250

Storage Time (min)

300

Sample recovery (%)

120

0.30

0 350 

Fig. 6. Variation of UV/VIS absorption ratio and sample recovery of Cyto C-AgNP with

storage time.



UV/Vis response

(a)

Void time

Field off

0

5

10

Storgae temp o 25 C 50 55 60 65 70 80

15

20

Elution time (min) 

UV/Vis response

(b) Ioinc strength 0.01 M 0.02 0.05 0.1 0.15 0.2

Void time

Field off

0

5

10

15

20

Elution time (min) Fig. 7. Variation of AsFlFFF fractogram of Cyto C-AgNP with storage temperature (a) and

ionic strength at pH 11.