PLATINUM BASED NANOBIOSENSOR FOR CARBOPLATIN-DNA …shodhganga.inflibnet.ac.in/bitstream/10603/13787/10... · nanoparticle based nanobiosensors which monitor the toxicological behavior

CHAPTER-3

PLATINUM BASED NANOBIOSENSOR

FOR CARBOPLATIN-DNA INTERACTION

Chapter 3

Institute of R & D, GFSU, Gandhinagar, Gujarat 58

3.1 ABSTRACT

The interaction of DNA and Carboplatin was studied with platinum (Pt) nanoparticle

based nanobiosensor. Carboplatin, a cytotoxic drug is responsible for producing

nephrotoxicity at effective dose. Thus, we have developed nanobiosensor for monitoring

Carboplatin-DNA interaction by measuring change in electrode potential. Surface of

electrode was modified with platinum nanoparticles to develop an electro-analytical

method, which can monitor Carboplatin-DNA interaction. As the time elapse, the change

in electrode potential gets elevated, while at one time the change in electrode potential

get stopped due to formation of Carboplatin-DNA adduct. This adduct is responsible for

producing toxic effects. The adduct was identified by UV-spectroscopy. The developed

nanobiosensor shows detection limit up to 10 ng/ml, which can be a crucial for drug-

DNA interaction studies while performing lead optimization and toxicological studies.

Chapter 3


3.2 INTRODUCTION

Nanotechnology is playing an increasingly important role in the advancement of

biosensors. The sensitivity and performance of biosensors is being improved by using

nanomaterials for their construction1. Nanoparticles play a key role in adsorption of

biomolecules due to their large specific surface area and high surface free energy. Metal

nanoparticles such as silver, gold, platinum, zinc, etc., are widely applied for

Electrochemical based nanobiosensor. Metal nanoparticles have been used to catalyze

biochemical reactions and this capability can be usefully employed in biosensor design.

Catalysis is the most important and widely used chemical application of metal

nanoparticles and has been studied extensively 2. Thus, we have developed platinum

nanoparticle based nanobiosensors which monitor the toxicological behavior of

Carboplatin via Carboplatin-DNA interaction.

Zhu et al. reported nanobiosensor consisting of Platinum nanoparticles was combined

with Nafion-solubilized MWCNTs, which were used for the electrode surface

modification3. Platinum nanoparticles possess the high catalytic activities for chemical

reactions, so the sensing signal for DNA hybridization is greatly amplified. By

minimizing the background signal, the sensitivity of the DNA biosensors is remarkably

improved. Qiao et al. have developed a Cholesterol Oxidase biosensor based on Pt-

nanoparticle /Carbon Nanotube modified electrode which measure cholestral

concentration in presence of ascorbic acid or uric acid4. H J Wang et al.

5 reported

Glucose biosensor based on platinum nanoparticles supported sulfonated-carbon

nanotubes modified glassy carbon electrode showed very high detection sensitivity.

Platinum nanoparticles-doped sol–gel/carbon nanotubes composite based electrochemical

biosensor was developed by Minghui Yang and his co-workers. This Pt-CNT-silicate

system provides a potential platform to immobilize different enzymes for other

bioelectrochemical applications with improved electrocatalytic activity and surface

renewability6. Hong et al. reported Glucose biosensor based on platinum

nanoparticles/graphene/chitosan nanocomposite film. This biosensor has good

reproducibility, long-term stability and negligible interfering signals due to large surface

area and fast electron transfer properties of Pt nanoparticles7. The above studies suggest

Chapter 3


that Pt nanoparticles have good biocompatibility, large surface area and fast electron

transfer properties. Thus, this nanobiosensor has been designed to monitor interaction of

Carboplatin with DNA.

The electromotive force (EMF) is the maximum potential difference or charge between

two electrodes. This causes electrons to move so that there is an excess of electrons at

one point and a deficiency of electrons at a second point8. The electrochemical signals are

usually generated by redox reactions and changes in ionic composition. Potentiometric

sensors measure the potential of an electrode at equilibrium (i.e. in the absence of the

appreciable currents) by measuring the electrochemical cell potential vs. a reference

electrode potential9. Carboplatin allow extensive stabilization of the intercalated adduct

by hydrogen-bonding interactions with DNA10

. Thus, this nanobiosensor has been

designed to monitor interaction of Carboplatin with DNA.

Figure 3.1: Schematic representation of development of Pt nanoparticles based

nanobiosensor

Chapter 3


In this report, we developed a real-time nanobiosensor by modifying the electrode with Pt

nanoparticle and DNA. This nanobiosensor was immersed in the solution containing

carboplartin to monitor Carboplatin-DNA interaction (Figure 3.1). Anti-cancer classes of

drug are toxic and produce acute and chronic toxicity. Carboplatin, a second generation

cytotoxic drug exhibit significant anti-cancer activity with less side effects than Cisplatin.

The anti-tumor effect is due to interaction with DNA via intrastrand and interstrand

cross-links. Carboplatin interact with DNA, therefore DNA replication, transcription and

repair will be lost11

. These leads Carboplatin-DNA adduct formation, which may

contribute to toxic effects. Therefore, the change in Carboplatin-DNA interaction was

observed by measuring changes in EMF (mV). All experiments were carried out at

neutral pH and at room temperature since double – strand of DNA breaks at neutral pH

and single - strand breaks at high pH12

.

Chapter 3


3.3 EXPERIMENTAL

3.3.1 Chemicals

Highly polymerized calf thymus DNA (MP Biomedicals, US) was used in this study.

DNA dilutions were prepared in Phosphate buffer pH 7. Phosphate buffer was prepared

by dissolving 0.1M disodium hydrogen phosphate in water and adjusting the pH by

adding 0.1M HCl. Ethylene glycol, silver nitrate, poly vinylpyrrolidone, dihydrogen

hexachloroplatinate and ethanol were used to prepare Pt nanoparticles. All chemicals

were purchased from E-Merck (India, Mumbai) and were all of analytical reagent grade.

Carboplatin was obtained from Cipla Ltd (India, Mumbai) and used without purification.

All aqueous solutions were prepared in Milli-Q water from a Millipore purification

system and all experiments were done at room temperature.

3.3.2 Apparatus

The potential measurements were carried out at 25.0±0.1◦C with a digital pH meter

(Model LI120, ELICO, India). Saturated calomel electrode (SCE) was used. UV spectra

were obtained on a JASCO V-630 spectrophotometer. Particle size of Pt nanoparticles

was carried out by Malvern Zeta-sizer (Model- The Zetasizer Nano ZS, UK).

3.3.3 Preparation of Pt nanoparticles

Pt nanoparticles were prepared according to the literature with mere modifications13

. 2.5

ml of ethylene glycol was refluxed at 5000 rpm to obtained homogeneity and heated at

160 C for 5 min. 0.002 M silver nitrate in 2.5 ml of ethylene glycol was added to above

solution and refluxed at 5000 rpm. After that, this mixture was refluxed at 3000 rpm for 1

hour. To, this 1.5 ml of 0.0625 M dihydrogen hexachloroplatinate was added along with

small volume of 0.375 M poly vinylpyrrolidone (3 mL) within one minute. The resultant

mixture heated at 160 C for 2 hour for chemical reduction and the color of the resultant

solution becomes dark brown. After that, the mixture was centrifuge at 5000 rpm for 5

min. Supernatant was separated and precipitated by adding triple volume of acetone to

Chapter 3


remove any impurities. Repeat the centrifugation again at 3000 rpm for 5 min. Collect the

precipitate and redispersed in 3 ml of ethanol with sonication for 15 min.

3.3.4 Fabrication of electrode by Pt nanoparticles

The surface of working calomel electrode was used for modified with Pt nanopaticles.

Before modifying the electrode with Pt, the electrode was cleaned by washing it with

distilled water and was allowed to dry. Then the dry electrode was immersed in solution

containing Pt nanoparticles for 15 min with stirring at room temperature. The electrode

was removed and was left for drying for about 15 min. Potentiometric measurement was

performed at working calomel electrode versus a calomel reference electrode.

3.3.5 Immobilization of DNA on Pt modified electrode

10 ppm DNA solution was prepared in phosphate buffer pH 7. The electrode was

immobilized by drop casting technique. A 10 µL (100 ng) drop of DNA was delivered on

the modified Pt surface of electrode by micropipette and allowed to dry in air. After

drying, this nanobiosensor was used for monitoring toxicological behavior of

Carboplatin.

Chapter 3


3.4 RESULTS AND DISCUSSION

3.4.1 Characterization of Pt nanoparticles

The morphology of Pt nanoparticles were obtained by SEM. Figure 3.2 illustrates that the

particles are predominantly spherical in shape with diameter ranging from 70 to 80 nm.

Larger and uneven shaped particles with diameter 100–140 nm were also obtained.

Figure 3.2: SEM image of Pt nanoparticles

Particle size of Pt was determined by Malvern zeta-sizer , which was found as an average

53.48 nm (Figure 3.3). Particles were ranging from 77.50 nm (96.2%) and 4708 nm

(3.8%).

Chapter 3


Figure 3.3: Particle size distribution of Pt nanoparticles

3.4.2 Carboplatin-DNA interaction in solution

Carboplatin solution of varying concentration (100, 75, 50, 25 and 10 ng/ml) was

prepared in distilled water. The DNA-Carboplatin interaction in solution was carried out

at room temperature. 1 ml of 100 ng/ml of Carboplatin and 10 µL of 10 µg/ml of DNA

DNA was taken to perform interaction study by potentiometry. As shown in Figure 3.4,

the electrode potential shifted to positive direction steadily in all concentration series.

But, at one point, the change in EMF was not increased. This is due to no hydrogen from

guanine and cytosine base can be liberated and oxidized which could lead to the stopped

EMF change. So, EMF change will shows until all amount of Carboplatin get interacted

with DNA. Initially the change in EMF was very fast, but as time elapse, the change in

EMF gets slow in all concentration series. The change in EMF with respect to time

indicates the interacting behavior of Carboplatin with DNA.

In case of 100 ng/ml of Carboplatin concentration, the interaction of Carboplatin with

DNA takes more time (Figure 3.4) as compare to other concentration of Carboplatin. This

is due to higher amount of Carboplatin was available to interact with DNA. Thus, this

study suggests that concentration of drug is directly proportional to interaction with DNA

and shows significant EMF changes. (Table 3.1)

Chapter 3


Table 3.1: Time dependent changes of Carboplatin-DNA interaction determined in

solution (Without nanobiosensor)

Time EMF

100 ng/ml 75 ng/ml 50 ng/ml 25 ng/ml 10 ng/ml

5 3.7 ± 0.6 3.6 ± 1.5 10.3 ± 0.6 9.3 ± 1.5 13 ± 1

10 4.6 ± 0.6 6 ± 1.7 12.3 ± 0.6 10.6 ± 1.5 14 ± 1

20 6.3 ± 0.6 7.6 ± 1.5 14.6 ± 0.5 12.3 ± 1.5 15 ± 1

30 7.3 ± 0.5 9 ± 2 16.6 ± 0.5 14.6 ± 2.1 17 ± 1

40 8.7 ± 0.6 11 ± 2 19.3 ± 0.6 15.3 ± 1.7 17.6 ± 0.6

60 10.6 ± 0.6 12 ± 1 21.6 ± 0.5 16 ± 1.5 18.3 ± 0.6

90 13 ± 1 14.3 ± 0.6 23.6 ± 0.6 17.3 ± 1.1 19.3 ± 0.6

120 15.6 ± 0.5 16.3 ± 0.6 26.3 ±0.5 18.6 ± 1.7 20 ± 1

150 18.6 ± 0.5 20 ± 1 28.6 ± 0.6 19 ± 1.7 20.3 ± 1.5

180 21.3 ± 0.5 23 ± 1 29.6 ± 0.6 20 ± 1.1 20.3 ± 1.5

210 23.6 ± 0.6 25.3 ± 1.5 30.6 ± 0.5 20.6 ± 1.1 20.3 ± 1.5

240 28.6 ± 0.5 27.6 ± 2.3 31.6 ± 0.6 21.6 ± 0.6 20.3 ±1.5

290 34.6 ± 0.6 30.6 ± 1.5 32.6 ± 0.1 22.3 ± 1 20.3 ± 1.5

350 41 ± 1 32.6 ± 1.5 33.3 ± 0.1 22.6 ± 1 20.3 ± 1.5

410 44.7 ± 0.5 34 ± 1 34 ± 1 23 ± 1 20.3 ± 1.5

470 49.6 ± 1.1 35 ± 1 34 ± 1 23 ± 1 20.3 ± 1.5

530 52.6 ± 0.6 35.3 ± 0.6 34 ± 1 23 ± 1 20.3 ± 1.5

590 53.6 ± 0.6 35.3 ± 0.6 - - -

710 54.3 ± 0.6 35.3 ± 0.6 - - -

930 54.3 ± 0.6 - - - -

Chapter 3


Figure 3.4: Carboplatin-DNA interaction at various concentration of Carboplatin in

solution (without nanobiosensor)

3.4.3 Nanobiosensor monitoring Carboplatin-DNA interaction

Carboplatin (100, 75, 50, 25 and 10 ng/ml) and DNA interaction was performed by

developed nanobiosensor. The results obtained from without modified sensor was nearly

similar in all concentration series (Figure 3.5), but change in EMF was improved with

respect to time. The electrode potential shifted toward more positive direction until all the

amount of Carboplatin gets interacted with DNA. In all Carboplatin concentration series,

the Carboplatin-DNA interaction shows more change in electrode potential. (Table 3.2)

The electrode potential increased steadily until all the amount of Carboplatin gets

interacted with DNA. The Carboplatin-DNA interaction performed by developed

nanobiosensor showed more potential shifting than without nanobiosensor. This suggests

that the sensitivity of sensor improves much better due to Pt nanoparticles. Pt

nanoparticles have larger surface area, so it provides more attachment of DNA. Also, it

has good electrochemical properties, so the sensing signal for Carboplatin-DNA

Chapter 3


interaction is greatly amplified, and hence it showed good EMF change at lower

concentration.

Figure 3.5: Carboplatin-DNA interaction at various concentration of Carboplatin

determined by nanobiosensor

Table 3.2: Time dependent changes of Carboplatin-DNA interaction performed by

nanobiosensor

Time EMF

100 ng/ml 75 ng/ml 50 ng/ml 25 ng/ml 10 ng/ml

5 7.3 ± 0.6 9 ± 2 13.3 ± 0.5 14.6 ± 1.5 18 ± 1

10 14 ± 1 13.3 ± 0.6 18 ± 0.6 16.3 ± 0.6 19.6 ± 0.6

20 21.6 ± 0.6 20.3 ± 2.1 22.6 ± 1 19 ± 1 20.6 ± 0.6

30 28 ± 1 24 ± 3.6 25.6 ± 2.5 21.6 ± 2.1 22.3 ± 0.6

40 34.3 ± 0.6 27 ± 4.5 29 ± 3.5 25.3 ± 2.5 24 ±1

60 43 ± 1 29.3 ± 4.0 32 ± 3.6 29 ± 2 26.3 ± 0.5

Chapter 3


At lower concentration i.e., 10 ng/ml of Carboplatin, the change in EMF was remarkable

than without modification. So, sensitivity was also improved at lower concentration.

Hence, this nanobiosensor can play a pivotal role for monitoring toxicological studies

while developing series of new drugs. The linearity and reproducibility of the

nanobiosensor was investigated by performing three different experiments using the same

working calomel electrode.

80 48.6 ± 1.5 33 ± 4.5 37 ± 3.6 33 ± 2 29 ± 1

100 52.6 ± 1.5 37.3 ± 3.1 42 ± 2.6 35.6 ± 1.5 31.3 ± 1.5

120 58 ± 1 44.3 ± 1.5 49 ± 2 39 ± 1.5 33.3 ± 1.1

140 62 ± 1 50.6 ± 2.8 54.3 ± 2.3 42.3 ± 2 36.3 ± 1.1

160 67.3 ± 1.1 53.6 ± 3.1 57.3 ± 1.5 46.3 ± 1.5 38.6 ± 1.1

180 72.7 ± 1.5 59 ± 1.5 61.6 ± 1.1 52 ± 2.1 41.6 ±1.5

200 78 ± 1.5 63.6 ± 2.8 65 ± 2.5 54.3 ± 3 44 ± 1.7

220 82.6 ± 1.5 68 ± 3.1 70.6 ± 2.6 57.3 ± 2.1 45.6 ± 0.6

250 87.6 ± 1.5 74 ± 3 76.6 ± 2.6 61.3 ± 2.1 46.6 ± 0.6

280 93.6 ± 0.6 78.6 ± 2.1 80 ± 2.6 64.3 ± 2.5 48.6 ± 1.5

310 97.3 ± 1 82.6 ± 1 82 ± 2.5 68 ± 2.5 49.3 ± 0.5

340 101.6 ± 1 87.3 ± 1 84.3 ± 3 72.3 ± 3 51 ±1

370 104 ± 1.5 93.6 ± 1.5 86 ± 3.0 75.6 ± 3.1 53.6 ± 1.5

400 106 ± 1 97.3 ± 0.6 88.3 ± 1.5 77.6 ± 3.5 58.3 ±2.5

460 108.6 ± 1.1 102.3 ± 1.5 90.3 ± 1.7 80.3 ± 3.2 62.3 ± 3.1

520 111.3 ± 1.1 104 ± 1.5 92.3 ± 1.5 81 ± 2.0 65.3 ± 3.6

580 113 ± 1.5 104.6 ± 2.1 94 ±1.5 81.6 ± 2.1 67 ± 3.6

700 114.3 ± 1.1 105 ± 3.5 95.3 ± 1 81.6 ±1.5 67 ± 3.6

820 116 ± 2 105 ± 3 95.6 ± 1 81.6 ± 1.5 67 ± 3.5

940 116 ± 2 105 ± 2.3 95.6 ± 1 - -

1060 116 ± 2 - - - -

Chapter 3


3.4.4 Sensitivity and selectivity of Nanobiosensor

To evaluate the performance of the nanobiosensor, potential shift ∆E was calculated, i.e.

the potential change from equilibrium time to the end of the experiment. From Figure 3.6,

it is clearly seen that, the potential shift is directly proportional to Carboplatin

concentration. If, the concentration of Carboplatin is higher, more potential shift was

observed. Nanobiosensor showed more ∆E than without modification of sensor in all

concentration series. This confirms the improvements of electrical signals and thus,

nanobiosensor reveals high sensitivity.

Figure 3.6: Potential difference between nanobiosensor and without nanobiosensor at

various Carboplatin concentration series

On addition of incremental concentrations of Carboplatin to DNA, potential difference

increases in all concentration series. It is evident from the Table 3.3 that interacting

behavior of DNA with the stock concentrations of Carboplatin are as follows:

100 ng/ml > 75 ng/ml > 50 ng/ml > 25 ng/ml > 10 ng/ml.

Chapter 3


Table 3.3: Potential difference at various concentration series

Carboplatin

concentration

ng/ml

Nanobiosensor

∆E

SD Without

Nanobiosensor

∆E

SD

100 108.67 2.081 52 1

75 96 1 30.67 1.52

50 82.33 1.53 23.67 1.154

25 66.67 1.154 13.67 0.58

10 49 4.35 7.33 1.154

3.4.5 Analytical performance of Nanobiosensor

The linearity and reproducibility of the nanobiosensor was investigated by performing

three different experiments using the same working electrode. Figure 3.7 depicts the

calibration curves of Carboplatin concentration ranging from 100 to 10 ng/ml versus the

∆E values. It was observed that the nanobiosensor showed good reproducibility for all

three measurements. The working electrode was water-washed to take away the DNA

residuals from the surface of the electrode after each measurement. The stability of the

nanobiosensor was tested by performing the experiments daily for a period of 30 days

while storing in a suitable environment when not in use. (Table 3.4) Almost 80% of the

initial sensitivity was retained at the end of the period and the biosensor half-life is

estimated to almost 2 month.

Table 3.4: Comparison of the analytical parameters for nanobiosensor and without

nanobiosensor

Method Shelf-

life

Range Reproducibility

Nanobiosensor 30 days 10-100 ng/ml >15 times

Without nanobiosensor - 10-100 ng/ml -

Chapter 3


Figure 3.7: Calibration curves of Carboplatin concentration versus the ∆E values.

The comparison of analytical performances for determining Carboplatin-DNA interaction

by nanobiosensor and without nanobiosensor is given in Table 3.5. For nanobiosensor,

the values of correlation coefficient (R2), slope and intercept were found as 0.997, 0.664

and 114.05 respectively. Limit of detection (LOD) and limit of quantification (LOQ)

were found as 5.89 (µg/mL) and 17.85 (µg/mL) respectively. For Without nanobiosensor,

the values of correlation coefficient (R2), slope and intercept were found as 0.893, 0.449

and 48.81 respectively. LOD and LOQ were found as 8.45 (µg/mL) and 25.61 (µg/mL)

respectively.

Chapter 3


Table 3.5: Comparison of the analytical performance for nanobiosensor and without

nanobiosensor

aLOD =3.3ₓSD/slope

bLOQ = 10ₓSD/slope

3.4.6 Comparative study of Carboplatin-DNA interaction by nanobiosensor

In order to compare the results and hence detect systematic errors between the two

methods, a student t-test was employed to check whether the standard deviations for the

same sample differ significantly (Table 3.6). Since the experimental value of t-test is

higher than the critical, it is concluded that the proposed nanobiosensor technique is more

precise than without nanobiosensor. Table 3.6 shows the statistical comparison between

two methods at various Carboplatin concentrations. Thus, the results obtained from both

the methods were not in agreement, indicating significant difference between two.

Parameters Nanobiosensor Without nanobiosensor

Regression equation (Y)

Slope (b)

Intercept (c)

0.664

0.449

114.05 48.81

Correlation coefficient (r) 0.997 0.893

LODa 5.89 (µg/mL) 8.45 (µg/mL)

LOQb 17.85 (µg/mL) 25.61 (µg/mL)

Chapter 3


Table 3.6: Statistical Comparison between two methods

Carboplatin

concentration

(ng/ml)

Nanobiosensor Without

nanobiosensor

t-test

∆E SD ∆E SD tExperimental tCritical

100 108.67 2.081 52 1 42.60 2.132

75 96 1 30.67 1.52 62.21 2.132

50 82.33 1.53 23.67 1.154 52.84 2.132

25 66.67 1.154 13.67 0.58 58.10 2.132

10 49 4.35 7.33 1.154 16.08 2.132

3.5 Confirmation of Carboplatin-DNA adduct by UV-spectroscopy

The Carboplatin-DNA adduct obtained from potentiometric measurement was validated

by UV spectroscopy. In Figure 3.8, the pure DNA shows absorbance at 255 nm, while

Carboplatin shows peak at 279 nm. The Carboplatin-DNA adduct shows peak at 277.5

nm indicates that the formation of adduct in case of 0.5 ppm of Carboplatin. It is

interesting to note that the shifting of peak of DNA characteristic UV–vis band at 277.5

nm was due to major Carboplatin–DNA interaction.

Chapter 3


Figure 3.8: Carboplatin-DNA adduct for 100 ng/ml of Carboplatin by UV-spectroscopy

UV spectra of 10 ng/ml of Carboplatin were shown in Figure 3.9. Absorbance for pure

DNA was 255 nm, while for Carboplatin the absorbance was 278.5 nm. The Carboplatin-

DNA interaction was confirmed by shifting of peak at 280 nm and shows bathochromic

shift.

Figure 3.9: Carboplatin-DNA adduct for 10 ng/ml of Carboplatin by UV-spectroscopy

Chapter 3


3.5 CONCLUSION

This work has shown experimental evidence of interaction of Carboplatin with DNA and

may contribute to the understanding of the mechanism of action of this drug with DNA. It

was observed that drug-DNA interaction occurring with time which suggests that

Carboplatin intercalates with DNA and slowly interacts with it causing some breaking of

the hydrogen bonds. It is interesting to note that the nanobiosensor experiments suggest

preferential interaction of Carboplatin with DNA at very low concentration. Without

surface modified electrode seems to be less sensitive than developed nanobiosensor for

monitoring interaction of Carboplatin with DNA. The sensor revealed high sensitivity

and selectivity. Formation of Carboplatin-DNA adduct was confirmed by spectroscopic

analysis. Overall, we developed nanobiosensor which allows non-invasive, real-time

monitoring of the Drug-DNA interaction changes by measuring potential at sensor

interface which could be crucial biosensor in molecular toxicology and drug discovery

pipelines.

Chapter 3


3.6 REFERENCES

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PLATINUM BASED NANOBIOSENSOR FOR CARBOPLATIN-DNA …shodhganga.inflibnet.ac.in/bitstream/10603/13787/10... · nanoparticle based nanobiosensors which monitor the toxicological behavior

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