RESULTS AND DISCUSSION 4.1 Screening of Urease...

RESULTS AND DISCUSSION

4.1 Screening of Urease producing Microbes

Bacillus badius MTCC 8082, Bacillus sphaericus MTCC 5100 and Cornybacterium

sp MTCC 8143 were screened for maximum urease activity by Nesslerization method

(Kayastha et al., 1995). The urease enzyme was found to be intracellular in all the

three isolates and the maximum enzyme activity found at respective incubation period

from 200 ml broth is given in table 4.1.

Table 4.1: Comparison of enzyme activity (IU) of isolates

Micro-Organism Enzyme Activity

(IU)

Incubation

Period (hrs)

Bacillus badius MTCC 8082 13.80 ± 1.120 21

Bacillus sphaericus MTCC 5100 53. 87 ± 1.1.41 24

Cornybacterium sp MTCC 8143 0.53 ± 0.932 18

The enzyme activity was calculated taking NH4+ as the standard curve as shown in

figure 4.1


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Figure 4.1: Standard curve of NH4+ ions (μg ml

-1)

4.2 Kinetic Characterization

As the enzyme activity of Cornybacterium sp MTCC 8143 was found to be very low

as compared to the other two isolates the further work was proceeded with Bacillus

badius MTCC 8082 and Bacillus sphaericus MTCC 5100. The Km and Vmax value of

both the isolates were evaluated in free and immobilized state in the absence and

presence of Pb2+

ions and was found as given in table 4.2 and 4.3.

Table 4.2: Kinetic parameters for Bacillus badius

Parameters Free Enzyme Immobilized Enzyme

Without Pb2+

With 96.6

nanomoles

Pb2+

Without Pb2+

With 96.6

nanomoles

Pb2+

Km (mM) 7.69 ± 0.6 7.69 ± 0.4 5.0 ± 0.7 5.0 ± 1.0

Vmax (µM/min) 110 ± 3.2 76.9 ± 2.9 75.0 ± 3.4 20 ± 3.5


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As per the change in Km and Vmax values in the absence and presence of lead it was

observed that the lead is a non- competitive inhibitor of urease as it does not cause

any change in Km but reduces Vmax drastically to half the value. Also the

immobilization by the hydrosol- gel method decreased the Km indicating a higher

enzyme – substrate affinity after the immobilization. As the studies with lead in the

assay reaction showed non- competitive inhibition of urease, a constant concentration

of 100mM urea was selected for biosensor development. The urease containing

membranes were stored at 4ºC and a decrease of 14.29 % enzyme activity was

observed after 2 months.

Table 4.3: Kinetic parameters for Bacillus sphaericus

Parameters Free Enzyme Immobilized Enzyme

Without Pb2+

With 4.8 X

10-2

nanomoles

nM Pb2+

Without Pb2+

With 4.8 X

10-2

nanomoles

Pb2+

Km (mM) 8.12 ± 0.112 5.83 ± 0.163 5.1 ± 0.141 2.90 ± 0.431

Vmax (µM/min) 139.64 ±8.980 96.45 ±7.850 55.0 ± 7.071 25.6 ± 6.222

As observed the immobilization of the enzyme in hydrosol - gel matrix reduced the

Km from 8.12 ± 0.112 mM to 5.1 ± 0.141 mM suggesting that this immobilization

matrix is favorable for urease immobilization and may be positioning the enzyme’s

active site at a more substrate accessible orientation. The lead was found to be

uncompetitive inhibitor of urease in case of Bacillus sphaericus as it decreased the Km


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and Vmax of the enzyme with the same factor (Loeppky, 2001). The Bacillus

sphaericus is a novel isolate with better kinetic properties than jack bean and Bacillus

pasteurii (Verma and Singh, 2003), hence gives lower limit of detection for heavy

metals (Verma and Singh, 2006). The uncompetitive inhibition of urease by lead

suggests that unlike Cu2+

and Ag2+

which bind to nitrogen of histidine and possibly

oxygen of aspartic and glutamic acid residues in the active site of urease (Saboury et

al., 2010), lead binds to thiol group of cysteine residues present in the flap which

causes modification in cysteine residues due to which the flap loses its mobility and

activity of urease is inhibited (Krajewska and Zaborska, 2007).

4.3 Lead Inhibition Studies

The inhibition effect of lead on urease activity was studied by incubating the enzyme

with urea along with different concentrations of lead in standard Nesslerization

method. It was observed that the Baciilus badius urease was inhibited by lead at the

lowest concentration of 0.22 nanomoles (1.2 µM) in free system (table 4.4) and the

linear range of inhibition was from 0.22 to 22 nanomoles (1.2 – 120 µM) in a

logarithmic pattern (figure 4.2).

Table 4.4: Inhibition affect of lead on Bacillus badius urease

Lead Conc.

(Nanomoles )

% Inhibition

0.22 27.63 ± 2.78

0.87 38.00 ± 1.93

2.2 42.11 ± 1.96


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8.7 55.24 ± 2.19

22 66.67 ± 2.12

Figure 4.2: Linear range of inhibition of lead on Bacillus badius urease

Since the lowest inhibition against lead (using Bacillus badius urease) was found to

be 0.22 nanomoles (1.2 µM), the isolate was further used to develop simple

colorimetric and ISE based potentiometric biosensor for lead in a hope to lower down

the detection limit of lead in milk to permissible levels (96.6 nM or 20 ng ml-1

) using

sensitive detection systems.

The inhibition studies were also conducted on Bacillus sphaericus using the same

Nesslerization method. The inhibition was observed upto 0.87 picomoles (4.83 nM) of

lead concentration with this isolate which was a very promising signal for developing

lead biosensor. The linear range of inhibition was observed from 0.87 to 87 picomoles


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(4.83 – 483 nM) as shown in figure 4.3. So much sensitivity towards lead could be

attributed to its better kinetic properties as compared to other microbial urease (Verma

and Singh, 2003) and the efficiency of the microbe to detect heavy metals like Cu2+

and Ni2+

to nanomolar levels as observed by Verma and Singh (2006).

Figure 4.3: Linear range of inhibition of lead on Bacillus sphaericus urease

The inhibition affect of other heavy metals was also studied on both the isolates and it

was observed that lead has low inhibition efficiency as compared to other highly toxic

metals like Hg2+

and Cu2+

. The pattern of heavy metal inhibition found in B.badius

and B. sphaericus was Hg2+

˃ Cd2+

˃ Ni2+

˃ Cu2+

˃ Zn2+

˃ Co2+

˃ Fe2+

˃ Pb2+

and Hg2+

˃ Cu2+

˃ Cd2+

˃ Zn2+

˃ Co2+

˃ Fe2+

~ Pb2+

respectively. This inhibition

pattern shows the need of a step to nullify the affect of all other heavy metals on

urease.

4.4 Development of Colorimetric Biosensor for lead

The urease enzyme (Bacillus badius) was immobilized by hydrosol - gel method on

nylon membrane and a simple colorimetric method was devised for lead detection.


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Phenol red was used as the pH indicator, and the change in color after 10 mins

incubation of the membrane in lead containing urea solution was observed. There was

a clear distinction in the color of the sample before and after the reaction as shown in

figure 4.4 and the difference in color production with different lead concentrations

was detected spectrophotometrically by taking phenol red absorbance at 508 nm.

A B

Figure 4.4: Color of the reaction mixture before and after the reaction. A- Before

reaction, B- After reaction

In synthetic system with immobilized enzyme linear range detection from 48 – 96.6

nanomoles (48.3 – 96.6 µM) was observed (figure 4.5) and the lower limit of

detection was found to be 38.6 nanomoles (38.6 µM).


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Figure 4.5: Linear range of detection of lead in synthetic system

The same system was applied for the dection of lead in acid exracted spiked milk

samples. The linear range of detection was found to be 38.6 to 96.6 nanomoles (38.6 –

96.6 µM) as shown in figure 4.6 and lower limit of detection achieved in milk

samples was 38.6 nanomoles (38.6 µM).

Figure 4.6: Linear range of detection of lead in spiked milk


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The reliability of the sensor was checked by recovery studies on spiked milk samples

as shown in Table 4.5. It was observed that the lead found to the sample was close to

the added amount as detected by the developed system.

Table 4.5: Percentage recovery in spiked milk samples

Milk Samples Lead Added

(nanomoles)

Lead Found

(nanomoles)

% Recovery

Milk Sample 1 48 58 120.83

Milk Sample 2 68 77 113.23

Milk Sample 3 87 97 111.49

The present biosensor is based on easily extractable, cost effective urease enzyme

having lower limit of detection for lead as compared to the expensive enzymes used

by other workers. Fennouh et al. (1998) developed an amperometric biosensor with

L- lactate dehydrogenase (LDH) and L - lactate oxidase. The biosensor had a Pb2+

detection limit of 0.2 µM and the enzyme membranes were stable for more than 2

months at 4ºC. Kremleva et al. (1999) developed an electrochemical method for

determining Pb2+

using an amperometric cysteine desulfhydrolase tissue biosensor,

detection limit for lead was 20 nM for this system. The present developed biosensor

has a lower detection limit than the optical sensors developed by Kuswandi (2003)

and Tsai et al. (2003) with a detection limit of 10 µM and 100 µM respectively. The

present biosensor, developed by the authors has an application in milk samples as

worked by Babkina and Ulakhovich (2004) using ssDNA (detection limit 0.1nM).

Although the detection limit of our biosensor is higher than the above one, it has an


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advantage of low cost and easy pretreatment of the sample. The present biosensor

has far more better detection limit than the one developed by Rodriguez et al.

(2004a) which was based on urease-glutamic dehydrogenase amperometric assay for

heavy metal screening in polluted samples. The biosensor detected Pb level more

than 2.4 X 10-1

mM. Ogonczyk et al. (2005) developed a screen printed disposable

urease based biosensor for detection of heavy metals and reported a detection limit of

1 mM of lead in 20 min response time. Haron and Ray (2006) developed an optical

biosensor with very low detection limit of 4.83 nM, but have been applied on

drinking water samples only. The detection range obtained by the developed

biosensor is economical and comparable to Bagal – Kestwal et al. (2008). They

obtained a detection limit of 30 nM in 10 min contact time by studying the inhibition

of invertase and glucose oxidase on electrochemical transducer. Recently DNAzyme

based sensors has attained a detection limits ranging from 10 nM to 500 nM (Liu and

Lu, 2000; Wei et al., 2008; Wang and Irudayaraj, 2011). The low detection limit of

the present biosensor could be attributed to the high sensitivity of the urease form

Bacillus badius (Verma et al., 2010) towards the heavy metal ions.

A very simple enzyme inhibition based biosensor has been constructed for the

estimation of lead in milk samples after a brief treatment of milk samples. The

developed biosensor detects lead specifically in the presence of other metal cations in

milk sample, after preconcentration with lead specific column. The present biosensor

could be used for the estimation of lead in alloys.

4.5 Development of Ion Selective Electrode (ISE) based Potentiometric Biosensor

A potentiometric biosensor was successfully developed for lead detection in milk

samples. The study was first conducted with free enzyme in synthetic system and a


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detection limit of 1.93 nanomoles (1.93 μM) was obtained as shown in figure 4.7. A

linear range of lead inhibition was obtained from 1.93 to 4.83 nanomoles (1.93 – 4.83

μM) deduced by change in potential caused by different lead concentrations.

Figure 4.7: Lead inhibition studied on ISE in synthetic system with free enzyme

The method was then applied to nylon immobilized enzyme membrane which was

tied close to the ISE and same method of detection as free system was used.

Hydrosol - gel method of immobilization as mentioned before was carried out and a

detection limit of 9.66 nanomoles (9.66 μM) was obtained in this case. The

percentage inhibition at 9.66 nanomoles (9.66 μM) was 8.27 % and a linear range of

inhibition from 9.66 to 966 nanomoles (9.66 – 966 μM) was observed (figure 4.8).

The relation of percentage inhibition with the lead concentration was found

logarithmic in this case also.


96

Figure 4.8: Lead inhibition studied on ISE in synthetic system with Immobilized

enzyme

The developed potentiometric biosensor was then applied to detect lead in milk

samples without any treatment. The detection limit obtained in this case was also

9.66 nanomoles (9.66 μM) with an inhibition of 9.04 % and linear range of inhibition

from 9.66 to 966 nanomoles (9.66 – 966 μM) as shown in figure 4.9. No data

supporting or contradictory to the present findings is available to validate the results,

still the developed potentiometric biosensor is competent to detect lead equivalent

contamination upto 9.66 nanomoles (9.66 μM). The specificity study was also carried

out in the presence of some masking agents to achieve lead specificity.


97

Figure 4.9: Lead inhibition studied on ISE in milk with Immobilized enzyme

4.5.1 Masking Agent study

The application of masking agents to invalidate the effect of other heavy metals in

the milk was carried out and it was found that EDTA was able to bring the potential

of the milk sample back to the original potential (approximately) in the presence of

all heavy metals by masking as shown by potential shift in table 4.6.

Table 4.6: Urease inhibition study on ISE and EDTA as masking agent for all

heavy metals

Sample Final Potential (mV) Change in potential

(ΔmV)

Control (Milk + 1mM EDTA) -113.6 ± 0.6

Milk + Lead (97 nanomoles) +

All other metals (10

nanomoles ) + EDTA

-114.7 ± 0.3 1.1 ± 0.2

Milk + Lead (97 nanomoles) -116.1 ± 0.4 2.5 ± 0.2


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Milk + Lead (97 nanomoles) +

All other metals (10

nanomoles)

-118.5 ± 0.2 4.9 ± 0.4

It is evident from the table that 96.6 nanomoles (96.6 μM) of lead decreases the

potential from -113 .6 ± 0.6 mV to -116.1 ± 0.4 mV which further got decreased to -

118.5 ± 0.2 mV in the presence of other heavy metal ions, depicting the increased

inhibition of urease in their presence. It also shows that the inhibition effect of all

heavy metals is more than that of lead alone. The addition of EDTA in the system

lead to masking of all heavy metals as evident from the potential change (-114.7 ±

0.3 mV) which is close to control reaction. Now to mask the affect of all other metals

on urease except lead 1, 10 – Phenanthroline and Sodium potassium tartarate were

used and found to be successful in masking the effect of all other metals present in

the system (table 4.7).

Table 4.7: Masking Agent affect on urease in acid extracted milk

Sample Final Potential

(mV)

Change in

potential (ΔmV)

Control (Milk + 1, 10 – Phenanthroline

and Sodium potassium tartarate)

-98.4 ± 0.4

Milk + Lead (97 nanomoles) + All

other metals (10 nanomoles)

-116.1 ± 0.5 17.7 ± 0.1

Milk + Lead (97 nanomoles) + All

other metals (10 nanomoles) + 1, 10 –

Phenanthroline and Sodium potassium

tartarate

-111.5 ± 0.7 13.1 ± 0.3

Milk + Lead (97 nanomoles) -111.0 ± 0.3 11.6 ± 0.1


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The table shows that the lead and other heavy metal ions caused the inhibition of

urease and decreased the potential from -98.4 ± 0.4 mV to -116.1 ± 0.5 mV creating

the potential shift of 17.7 ± 0.1 mV which got decreased to 13.1 ± 0.3 mV in the

presence of 1, 10 – Phenanthroline and Sodium potassium tartarate. The potential

drop in the presence of these masking agents was -111.5 ± 0.7 mV which was close

to that caused by lead alone (-111.0 ± 0.3 mV). This similar potential suggest that 1,

10 – Phenanthroline and Sodium potassium tartarate were able to mask the effect of

all other heavy metals except lead and showed the same potential drop as caused by

lead alone.

4.6 Electrochemical Studies

The electrochemical studies were based on direct oxidation or reduction of Pb or Pb2+

respectively on electrode surface.

At low pH Pb 2+

Pb Reduction

pH > 3.5 Pb Pb 2+

Oxidation

4.6.1 Lead detection on platinum working electrode

The lead detection on platinum electrode was done at low pH (0.9 – 1.2) as well as

on neutral pH. The low pH studies were done to configure whether the developed

method could be applied to acid extracted milk samples directly (without adjusting

the pH to neutral) or not. It was first optimized on synthetic system and then applied

to acid extracted milk samples. The parameters of the study are mentioned in the

figure 4.11. In case of synthetic system at low pH the linear range of lead detection

was found to be from 4.8 X 10-2

nanomoles to 4.8 micromoles (4.83 nM to 483 μM)

as shown in figure 4.10 with lower limit detection of 4.8 X 10-2

nanomoles (4.83


100

nM). Tsai et al. (2001) has achieved a detection limit of 0.1 µM using boron doped

diamond electrode and a detection limit of 48.3 nM has been achieved by Babyak

and Smart (2004) using the same electrode. Goubert – Renaudin et al. (2009) used

amide – cyclan –functionalized silica modified CPE to attain 2.7 nM detection limit.

Figure 4.10: Lead detection in synthetic system ( pH 0.9-1.2). b1.bin - 0.1 M KCl,

p1.bin - 4.8 X 10-2

nanomoles Pb2+

, p2.bin – 4.8 X 10-1

nanomoles Pb2+

, p3.bin -

4.8 nanomoles Pb2+

, p4.bin - 48 nanomoles Pb2+

, p5.bin – 480 nanomoles Pb2+

,

p6.bin - 4.8 micromoles Pb2+

Locally available verka milk samples were used for lead spiking studies. In case of

acid extracted milk the linear range was observed from 4.8 X 10-2

nanomoles to 4.8

micromoles (4.83 nM – 483 μM) Pb2+

as shown in figure 4.11 with detection limit of

4.8 X 10-2

nanomoles (4.83 nM). The method was successfully applied to the milk

samples but the linear range was narrowed down to 48 nanomoles (4.83 μM).


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Figure 4.11: Lead detection in acid extracted milk (pH 0.9-1.2). b2.bin – 0.1 M

KCl, p1.bin - 4.8 X 10-2

nanomoles Pb2+

, p2.bin – 4.8 X 10-1

nanomoles Pb2+

,

p3.bin - 4.8 nanomoles Pb2+

, p4.bin - 48 nanomoles Pb2+

From the current values of different concentration of lead in synthetic and acid

extracted milk sample (table 4.8), a comparative relation between the two systems

was developed which could be applied to detect lead concentration in unknown milk

samples.

Table 4.8: Comparison of results of synthetic system and milk samples at low pH

Lead Conc

(nanomoles)

Current in

Synthetic

system (

10-1

mA)

Change in

current in

synthetic

system (∆I)

Current in

milk

samples (

10-1

mA)

Change in

current in

milk

samples

(∆I)

Change

difference

in synthetic

and milk

samples

Blank (0.1 M

KCl)

1.079 - 1.925 (milk

w/o spiking)

- 0.846


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4.8 X 10-2

1.134 0.055 2.109 0.184 0.975

4.8 X 10-1

1.195 0.116 2.258 0.333 1.063

4.8 1.297 0.218 2.350 0.425 1.053

48 1.386 0.307 2.442 0.517 1.056

From the table it could be furnished that in case of milk an addition of 4.8 X 10-2

nanomoles

(4.83 nM) Pb2+

leads to a difference of 0.129 units in current (0.975 –

0.846 = 0.129) with respect to control which corresponds to a contamination of

approximately 4 X 10-1

nanomoles (48.3 μM ).

Lead detection at neutral pH

Lead detection on neutral pH was done on synthetic samples first and it was observed

that, only the detection of 0.48 micromoles (48.3 μM) lead and above was possible as

shown in figure 4.12. The detection limit (0.48 micromoles or 4.83 μM) and linear

range (0.48 – 4.8 micromoles Pb2+

or 48.3 – 483 μM) was found very much higher

than the permissible limit of lead in milk (96.6 nM) so the study was further not

applied to milk samples. The neutral pH also leads to fast fouling of the platinum

electrode surface and hence loss of sensitivity. So the electrode surface needs to be

regularly polished after few reactions. This polishing step adds time to the whole

process and still was not able to decrease down the detection limit below 0.48

micromoles Pb2+

(48.3 μM).


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Figure 4.12: Lead detection in synthetic system at neutral pH. b2.bin- 0.1 M

KCL, Pb10.bin- 0.48 micromoles Pb2+

, p100.bin- 4.8 micromoles Pb2+

4.6.1.1 Preconcentration study on platinum working electrode

For preconcentration of lead on platinum electrode an alginate- sol gel hybrid

membrane was developed. The alginate-TEOS hybrid was able to form a thin

membrane capable to be used as adsorption matrix on electrode tip. The SEM studies

of the membrane surface (figure 4.13) reveal microstructures showing alginate-

silicate interactions that results in small vesicles with increased surface area which

forms the platform for metal adsorption.

Figure 4.13: SEM image of alginate- TEOS hybrid membrane


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Sodium alginate – sol gel membrane was used for the preconcentration of lead on the

platinum electrode. It was observed that the membrane was able to preconcentrate the

lead as the current was found to be decreased (suppression of current) after applying

the membrane (figure 4.14) but the release of lead complex from the membrane was

not achieved.

a

b

Figure 4.14: Suppression of Lead peak after preconcentration. A- 0.48

micromoles lead without membrane, b- 0.48 micromoles lead with membrane

Promising results were obtained in heavy metal preconcentration studies of the

membrane. As per the electrochemical studies, the membrane applied at the tip of the

electrode was able to preconcentrate almost 99% lead from the solution. The

oxidation peak of 0.48 micromoles (48.3 μM) lead got completely suppressed after

the application of the membrane as shown in the figure 4.15 and as visualized after

reaction the membrane’s morphology changed after complexation with the metal.

Affinity of alginic acid towards metal cations like lead has already been postulated by

Jeon et al. (2002a) and Jeon et al. (2002b). The lead adsorption efficiency has been

improved using magnetically modified alginic acid by Jeon et al. (2007). The

observed interaction of the membrane with lead is also advantageous in developing


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urease immobilized membrane for heavy metal detection as the membrane will be

able to concentrate cations close to the enzyme and hence enhanced inhibition could

be observed towards heavy metal cations. Although this postulated theory was not

further extended in practical work as the release of lead from the membrane was not

achieved successfully. Polarization of Lead- EDTA complex studied on all kind of

electrodes – Platinum, Glassy carbon, screen printed was studied but no distinction in

different concentration of lead was observed. As per the studies conducted, it is

suggested that the prepared alginate-TEOS sol gel membrane is a very promising

matrix for heavy metal preconcentration and its removal from the wastewaters.

4.6.2 Lead detection on Glassy Carbon Electrode (GCE)

Reduction studies of lead were also carried out on Glassy carbon electrode as the

working electrode and a similar pattern of reduction as platinum electrode was

observed as shown in figure 4.15.

Figure 4.15: lead detection on GCE. KCL5- 0.1 M KCl, g3- 4.8 X 10-2

nanomoles

Pb2+

, g6- 4.8 X 10-1

nanomoles Pb2+

, g9- 4.8 nanomoles Pb2+

, g12- 48 nanomoles

Pb2+

, g14- 480 nanomoles Pb2+

, g16- 4.8 micromoles Pb2+


106

The linear range for lead was found to be from 4.8 X 10-2

nanomoles to 4.8

micromoles (4.83 nM – 483 μM) Pb2+

, but the curves were not sharp and as smooth as

on platinum electrode, so platinum electrode was assumed to be better for direct

estimation of lead by reduction.

4.6.3 Lead detection on mercury coated Glassy Carbon Electrode (GCE)

After getting a very low detection limit on platinum electrode and unmodified glassy

carbon electrode, the study was extended to mercury coated GCE to make the study

possible at neutral pH with low detection limits. For this the bare GCE was coated

with mercury and then the polarization of lead (483 μM) was done. It was observed

that the lead polarization took place at specific potential with a very distinct lead peak

at -0.6 V. To sharpen the peak and to make it more stable different parameters were

optimized.

4.6.3.1 Effect of pH on lead polarization

Lead polarization studies from pH 1 to 7 were done and it was observed that pH 1 to 3

does not result in any lead peak at - 0.6V (figure 4.16). There was a very prominent

peak at pH 4 which got reduced at higher pH values. So pH 4 was taken as to be the

optimum pH for lead reduction on mercury coated GCE. A pH range of 4 -5 was also

optimized by Kadara and Tothill, 2004 for lead detection on bismuth film screen

printed electrode.


107

Figure 4.16: Effect of different pH on lead reduction

An electrochemical sensor was developed by Bouwe et al. (2011), for sub- nanomolar

detection of lead (0.4 nM). A 1,10- phenanthroline – montmorillonite intercalate was

formed at acidic and neutral pH and was used as Pb2+

sensor via CPE by adsorptive

stripping voltametry. The optimal pH found in this case was 6.

4.6.3.2 Effect of different buffers on lead polarization

0.1 M KCL, 0.1 M Acetate buffer and 0.05 M Tartarate buffer were used for lead

reduction. No peak corresponding to 483 µM lead was observed in acetate and

tartarate buffer and only KCl buffer was able to deliver a sharp peak at -0.6 V

potential (figure 4.17). So for further study KCl buffer was used for carrying out lead

reduction.


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Figure 4.17: Effect of different buffers on lead reduction. p15.bin – 0.1 M KCl,

p6.bin – Acetate buffer, p8.bin – Tartarate buffer

4.6.3.3 Effect of KCl concentration on lead polalization

From the different buffers used for lead reduction it was very clear that KCl was the

most excellent buffer for carrying out lead reduction. So the effect of KCL

concentration was studied on current enhancement of lead peak as shown in figure

4.18. It was observed that there was an increase in current value of lead from 0.1 to 1

M KCl. At 2 M KCL the current got drastically dropped as compared to the 1M KCL.

So as an optimum value 1 M KCL was used in further study.

Babyak and Smart 2003, had used 0.1 M KCl as supporting electrolyte for lead

detection on boron doped diamond electrode using square wave anodic stripping

voltametry. Hassan et al. (2008) had optimized 0.1 M KCl as supporting electrolyte

for anodic stripping voltametric detection of Pb2+

using carbon paste electrode

modified with chitosan.


109

Figure 4.18: Effect of different concentrations of KCl buffer on lead reduction

After the optimization of all the parameters lead detection was done on mercury

coated GCE and a linear range from 0.96 to 3.8 micromoles (96.6 – 386 μM) lead was

obtained (figure 4.19). A drastic increase in current at 4.8 micromoles (483 μM) lead

was observed and the lowest limit of detection obtained in this case was 0.48

micromoles (48.3 μM).

Figure 4.19: Lead detection on mercury coated GCE. p2.bin – 0.97 micromoles

Pb2+

, p5.bin – 1.93 micromoles Pb2+


, p9.bin – 3.8

micromoles Pb2+


A detection limit of 1.93 X 10-1

nM was achieved by Liu et al. (2005), using carbon

nantotubes- nanoelectrodearrays (CNTs-NEAs) coated with bismuth film. In a similar


110

study as ours, Loh et al. (2006), achieved a detection limit of 4.83 µM using screen

printed electrode (SPE) with poly(vinyl) chloride (PVC) as printed matrix. Lead was

deposited with mercury on working electrode at a pH of 2. The various parameters

optimized were, deposited time = 15sec, deposited potential = 1000 mV, scanning

range = 20mV sec-1

and amplitude = 25. Linearity obtained in this case was from 4.83

to 242 µM of Pb2+

. Hassan et al. (2008), has achieved a detection limit of 9.66 nM

using carbon paste electrode modified with chitosan. Later Senthilkumar and

Saraswathi (2009) developed an electrochemical sensor for lead detection using a

zeolite NH4- Y modified carbon paste electrode (ZYMCPE) with a detection limit of

17.3 µM. Recently enhanced anodic stripping Voltammetry method has been used for

blood lead determination (Zyoud and A-Subu, 2011)

4.6.4 Development of Electrochemical Biosensor using Carbon Paste Electrode

(CPE)

The electrochemical study was conducted on CH Instruments Electrochemical

workstation, Model 660. The working electrode used was the self fabricated carbon

paste electrode with Ag/AgCl2 as reference electrode and platinum wire as the counter

electrode. The oxidation of NADPH was studied in the reaction catalyzed by urease

and glutamate dehydrogenase GLDH present in the whole cell of B. sphaericus. The

study was based on the detection of unspent NADPH after the reaction of urease on

urea and GLDH on α- Ketoglutarate as shown in the Eq 1 & 2.

Urea + H2O HCO3- + 2 NH4

+ (1)

2 NH4+ + α- Ketoglutarate + NADPH + H

+ L-Glutamate + NADP

+

(2)

GLDH

Urease


111

The ammonia produced by action of urease on urea was taken up by α- Ketoglutarate

added exogenously in the system in the presence of NADPH and resulted in the

production of NADP+. As the reaction would not be able to oxidize whole of the

NADPH present in the system, the oxidation of unoxidised NADPH left after the

reaction was studied electrochemically and this was correlated with the lead

concentration. The increase in lead concentration caused the inhibition of urease and

resulted in more amount of unspent NADPH. This caused increase in the current of

the NADPH oxidation as compared to the lead deficient system and formed the basis

of the study. To achieve proper NADPH oxidation various parameters were optimized

and then the lead inhibition studies were conducted on milk as such and after

preconcentration through lead specific column.

4.6.4.1 Optimization of NADPH concentration

The first step in the study was to optimize the minimum amount of NADPH required

to obtain the detectable NADPH oxidation peak. Oxidation of NADPH was studied

amperometrically in a range of - 0.2 to 0.8 V and an oxidation peak was obtained at

0.1V- 0.15V. The current value of different CPE carrying same enzyme units and

prepared at the same time was - 4.4662 mA, with standard deviation of ± 0.784 A. A

significant shift in potential towards higher side was observed with increase in

NADPH concentration. There was a linear increase in current with increasing

NADPH concentration from 4.3 x 10-2

mM to 1.3 x 10-1

mM (figure 4.20), beyond

which fouling of the electrode was observed. The optimized NADPH which has been

added exogenously is 4.3 x 10-2

mM. In a similar study by Rodriguez et al. (2004a), a

fixed concentration of 0.283 mM NADH has been used, but the required amount has

been reduced to very low levels in the present study, owing to the use of whole cell


112

instead of enzyme. Azmi et al. (2009) has also showed that a level of 5.0 x 10-2

mM

NADH is detectable using simple UV-Vis spectrophotometer, which could be further

lowered down by sensitive detecting system as obtained in the present study using

electrochemical workstation.

Figure 4.20: Cyclic voltametric studies on electrochemical oxidation of NADPH.

c7.bin: 4.3 x 10-2

mM NADPH, c8.bin: 8.6 x 10-2

mM NADPH, c9.bin: 1.3 x 10-1

mM NADPH

4.6.4.2 Reaction time optimization

In case of reaction time, an oxidation peak at 0.15 V was observed at 3 and 5 minutes

incubation with enhanced current at 5minutes incubation (figure 4.21). The 10 & 20

minutes incubation gave less and almost same current along with potential shift

towards higher side as compared to the 5 minutes incubation. So the response time

optimized for the reaction is 5 minutes, preceded by an incubation of electrode with

lead for 10 minutes as done by other workers (Ilangovan et al., 2006) to increase the

inhibition efficiency. The same microbe has been shown to give response against

nickel ions in 1.5 minutes (Verma and Singh, 2006).


113

Figure 4.21: NADPH oxidation after different reaction times

4.6.4.3 Temperature effect on NADPH oxidation

The temperature range from 20˚C to 45 ˚C was studied. It was observed that, almost

same amount of current was generated at 20 & 25 ˚C, showing same amount of

oxidizable NADPH availability (figure 4.22). At higher temperatures (30˚C, 37 ˚C &

45 ˚C) there was an increase in current and a potential shift towards lower side was

observed, depicting the inability of the system to oxidize the excess NADPH

completely. At 37 ˚C & 45 ˚C, the oxidation peak also got broadened, depicting the

non- suitability of this temperature for the reaction. So a temperature range of 20 – 25

˚C was optimized for the present study.

Figure 4.22: Temperature effect on NADPH oxidation


114

4.6.4.4 Biomass Optimization

The optimum biomass loading was obtained by immobilizing different biomass

concentrations, with different enzyme units at the tip of the electrode. A study was

conducted in which biomass containing 0.975, 1.95 & 2.915 enzyme units of urease

was immobilized in the CPE and NADPH oxidation was recorded. The increase in

biomass increases the amount of intracellular NADPH available, giving the increase

in current. A sequential higher side potential shift with increasing enzyme units was

observed (figure 4.23). The oxidation peak got broadened at 2.915 U. Inhibition

studies of lead was conducted on all the biomass concentrations and it was observed

that the inhibition affect of lead was less pronounced at higher biomass loading,

which could be because of overloading of biomass. The biomass containing 1.95 U of

urease having a current value of - 4.800 ± 0.313 A, was optimized for oxidation

studies, as inhibition effect of lead was less pronounced at 0.975 and 2.915 U. These

enzyme units are much less as compared to 5 U used by Rodriguez et al. (2004a), in

different immobilization methods for urease. Lesser enzyme units used in the study

indicate better kinetic properties of the immobilized biomass within the carbon paste

without much loss in enzyme activity. The whole cell immobilized CPE was found to

be disposable in nature.


115

Figure 4.23: Biomass optimization for NADPH oxidation. c2.bin: 0.975 U, c3.bin:

1.95 U, c4.bin: 2.915 U

4.6.4.5 Lead Inhibition and monitoring studies

The inhibitory studies for lead were conducted in a concentration range of 4.8 X 10-4

to 0.48 nanomoles (4.83 nM – 4.83 μM) Pb2+

. The linear range of inhibition was

observed from 4.8 X 10-4

to 4.8 X 10-2

nanomoles (4.83 – 483 nM) lead in synthetic

samples (figure 4.24), with shift in potential with increasing lead concentration. The

current at 0.48 nanomoles (4.83 μM) Pb2+

got drastically reduced owing to the

electrode fouling due to excessive NADP+ production. A logarithmic relationship was

observed between current value and lead concentration (figure 4.25). Lower limit of

detection achieved was 4.8 X 10-4

nanomoles (4.83 nM) Pb2+

.


116

Figure 4.24: Inhibition study of lead in synthetic system. c1.bin: 4.8 X 10-4

nanomoles Pb2+

, c2: 4.8 X 10-3

nanomoles Pb2+

, c3: 4.8 X 10-2

nanomoles Pb2+

, c4:

4.8 X 10-1

nanomoles Pb2+

Figure 4.25: Calibration curve of Pb2+

ions

Milk as such and spiked milk samples were analyzed for lead contamination by two

different ways. One is milk samples without any pretreatment and the other, after acid

extraction pretreatment. In direct milk samples there was a large shift in potential at

all lead concentrations in comparison to synthetic samples that could be because of

delayed NADPH oxidation as a result of interference by milk proteins and other milk

constituents. Linear range of detection of 4.8 X 10-3

nanomoles to 4.8 X 10-1

nanomoles (4.83 nM – 4.83 μM) Pb2+

was obtained in this case (figure 4.26) and

lower limit of detection was 4.8 X 10-4

nanomoles (4.83 nM) Pb2+

.


117

Figure 4.26: Linear range detection of Pb2+

ions in Milk. c2.bin: 4.8 X 10-3

nanomoles Pb2+

, c3.bin: 4.8 X 10-2

nanomoles Pb2+

, c4.bin: 4.8 X 10-1

nanomoles

Pb2+

In case of acid extracted milk, lead specificity was achieved by passing the milk

sample through lead selective column (Pb. SpecTM

), which showed a percentage

loading of 79.26 ± 0.8 and a recovery of 85.44% ± 0.4 Pb2+

as detected through

Sodium dithiocarbamate method using the following calibration curve (figure 4.27).

Figure 4.27: Calibration curve of lead using Sodium Dithiocarbamate

In case of acid extracted milk, the linear detection range was from of 4.8 X 10-4

nanomoles to 4.8 X 10-1

nanomoles (4.83 nM – 4.83 μM) Pb2+

(figure 4.28) and a


118

lower limit detection of 2.4 X 10-4

nanomoles (2.4 nM) was achieved. The percentage

inhibition in milk was less as compared to the synthetic samples. It could be defended

by two reasons, firstly the intricate matrix of the milk complexes with the lead and

reduces it inhibition efficiency, secondly the passage of the lead containing milk

sample through the lead column leads to some loss of the lead as the percentage

loading and the recovery from the column is not 100 %. The reliability of the

developed biosensor was checked by studying the change in current (ΔI) of lead

spiked synthetic sample and acid extracted milk samples. The ΔI values obtained for

synthetic and milk sample was 0.949 ± 0.109 and 1.011 ± 0.021 A respectively, which

are comparable. Lead analysis was done in spiked milk samples with the developed

biosensor and the results obtained were as given in table 4.9.

Figure 4.28: Linear range detection of Pb2+

ions in Acid extracted Milk. c1.bin:

4.83 X 10-4

nanomoles Pb2+

, c3.bin: 4.83 X 10-2

nanomoles Pb2+

, c4.bin: 4.83 X 10-

1 nanomoles Pb

2+

Table 4.9: Reliability studies of the electrochemical biosensor

Lead added (nanomoles) Lead found (nanomoles)

4.8 X 10-4

4.7 ± 0.113 X 10-4

48 X 10-3

4.76 ± 0.483 X 10-3


119

The developed biosensor was applied for lead analysis in various milk samples from

different places and a maximum of 48 X 10-3

nanomoles (48.3 nM) lead

contamination was found, most of the samples were found to be safe for human

consumption.

Finally, lower limit of detection (LOD) for lead ions has been compared with

biosensors developed by other workers in table 4.10. It appears that the present

biosensor has far more better detection limit for lead than developed so far. Fungal

and Plant ureases are hexameric (α6) whereas bacterial ureases are heterotrimeric

(αβγ)3. The Bacillus sphaericus is a novel isolate with better kinetic properties than

jack bean and Bacillus pasteurii (Verma and Singh, 2003), hence gives lower limit of

detection for heavy metals (Verma and Singh, 2006). The uncompetitive inhibition of

urease by lead suggests that unlike Cu2+

and Ag2+

which bind to nitrogen of histidine

and possibly oxygen of aspartic and glutamic acid residues in the active site of urease

(Saboury et al., 2010), lead binds to thiol group of cysteine residues present in the flap

which causes modification in cysteine residues due to which the flap loses its mobility

and activity of urease is inhibited (Krajewska and Zaborska, 2007). Biosensors

developed so far had an application of monitoring lead ions in water samples only, but

the present biosensor developed has an application in drinking water as well as in

milk samples, which is a first endeavor of its kind for milk lead analysis at such a low

levels. Babkina and Ulakhovich (2004) had used ssNDA to develop an amperometric

biosensor for heavy metal analysis in food samples and attained a detection limit of in

milk, the authors work was able to achieve the detection limit of 2.4 X 10-4

nanomoles

in milk which is under the permissible limit of lead in milk.. Moreover, the present

biosensor is also very simple, economical and more sensitive than the DNAzyme

based biosensors (Li and Lu, 2000; Xiao et al., 2007; Shen et al., 2008) and the lead


120

specificity has been achieved using the lead specific column, making it a complete

lead specific biosensor.

Table 4.10: Comparison between limits of detection (LOD) of Pb2+

ions

with different biosensors

Biocomponent used Transducer Pb (II) LOD Reference

L-Lactate

dehydrogenase

Amperometrer 0.2 µM Fennouh et al. (1998)

Cysteine

desulfhydrolase

Amperometer 20 nM Kremleva et al. (1999)

Alkaline phosphatase Optical 0.1 nM Veselova and

Shekhovtsova (2000)

DNAzyme Optical 10 nM Liu and Lu (2000)

Antibody Optical 6 nM Blake et al. (2001)

Urease Conductometer 0.9 mM Lee and Lee, (2002)

Cyanobacteria Amperometer IC50 : 1.6 µM Tay et al. (2003)

Urease Optical 1 X 10-2

mM Kuswandi (2003)

Urease Optical 0.1 mM Tsai et al. (2003)

ssNDA Amperometric 0.1 nM Babkina and Ulakhovich

(2004)

Urease Electrochemical 1 mM Ogonczyk et al. (2005)

Human Angiotensin I Electrochemical 1.9 nM Chow et al. (2005)

Urease and

Acetylcholineesterase

Optical 4.83 nM Haron and Ray (2006)

Urease Conductometer 1 mM Ilangovan et al. (2006)


121

DNAzyme Electrochemical 0.3 µM Xiao et al. (2007)

Invertase Electrochemical 30 nM Bagal-Kestwal et al.

(2008)

Alkaline phoaphatase Conductometer

1.93 x 10-2

µM

Berezhetskyy et al.

(2008)

dsNDA Electrochemical 2 mM Oliveira et al. (2008)

DNAzyme Electrochemical 1 nM Shen et al. (2008)

Urease (Bacillus

sphaericus)

Electrochemical 2.4 nM Present study

4.7 Development of DNAzyme based Optical Biosensor

The DNAzyme interaction with lead which results in breakdown of substrate

strand has been taken as the basis of this study. To carry out the fluorescence read

microarray plate reader was first calibrated using fluorescein as the fluorescent

indicator. Different protocols were tried to construct a standard curve for known

concentrations of fluorescein. The final protocol developed was as follows:

Protocol for fluorescence read

Incubate at 30 ˚C for 30 sec

Plate read

Go to line 1 for 2 more times

END

Lid Temperature- Off

Lid off temperature – 30 ˚C

Volume set as used


122

The standard curve for fluorescein obtained shown in figure 4.29

Figure 4.29: Standard curve of Fluorescein obtained at plate reader

4.7.1 DNAzyme study in free system

In initial study 17 E (donor) labeled with fluorescein (Ex = 494 nm, Em = 521

nm) and 17DS labeled with TAMRA (Ex = 542 nm, Em = 568 nm ) were first

hybridized according to Swearingen et al. (2005). The donor (17 E) was excited at

494 nm (λ1) and read against sybergreen (Ex = 497 nm, Em = 520 nm) in the plate

read. The acceptor (17 DS) was excited at 542 nm (λ2) and its emission was read at

568 nm (λ3) against HEX (Ex = 535 nm, Em = 556 nm) in the plate reader as shown

in the table 4.11. The hybridization was confirmed by taking the fluorescence read of

both the strands separately and after the hybridization, shown in table 4.12.


123

Table 4.11: Donor – Acceptor Couple

Designation Wavelength Relation

λ1 494 nm Excitation of donor (fluorescein)

λ2 542 nm Emission of donor/ Excitation of

acceptor (TAMARA)

λ3 568 nm Emission of acceptor (TAMARA)

Table 4.12: Fluorescence read for hybridization

Reagents Fluorescence Wavelengths

Donor 17 E -

Fluorescein

0.812 ± 0.107 Ex at λ1, Em at λ2

Acceptor 17 S - TAMRA 0.134 ± 0.035 Ex at λ2, Em at λ3

Hybrid 0.468 ± 0.037 Ex at λ1, Em at λ3

As the 17 E was acting as donor and 17DS as acceptor, it was observed that

after hybridization the fluorescence of acceptor got enhanced from 0.134 ± 0.035 to

0.468 ± 0.037 supporting the phenomenon of FRET as occurrence of FRET causes

transfer of energy from donor to acceptor thus increasing its fluorescence. After the

successful attainment of hybridization DNAzyme interactions with Pb2+

was studied.

It was observed that the addition of Pb2+

leads to decrease in fluorescence intensity of

acceptor (17 DS) suggesting that it is catalyzing the cleavage of substrate strand by

the enzyme strand and hence the break of FRET also (table 4.13).


124

Table 4.13: DNAzyme - Pb2+

interaction

Reagents Fluorescence

(λ3)

Hybrid + water 0.490 ± 0.011

Hybrid + Lead (0.48 nanomoles) 0.417 ± 0.022

Hybrid + Lead (0.72 nanomoles) 0.402 ± 0.012

Till 2005 the DNAzyme based lead biosensors were constructed using a

fluorophore and a quencher combination (mainly TAMRA as fluorophore and Dabcyl

as quencher) in which the hybridization leads to quenching of the fluorescence and

cleavage of substrate strand leads to increase in fluorescence (Liu, 2002; Chang et al.,

2005; Swearingen et al., 2005; Wernette et al., 2006; Shen et al., 2007). The

efficiency has been increased by using two quenchers, one at the 3’substrate strand

and other at 3’enzyme strand. A fluorophore at 5’ substrate strand gets quenched by

both inter and intra- molecular quenchers decreasing the backgroung fluorescence

(Liu and Lu, 2003). Zhang et al. (2010a) has utilized the potential of DNAzyme as

catalytic and molecular beacon to achieve a detection limit of 0.6 nM. The substrate

strand has been modified to form an intermolecular beacon which has a fluorophore at

one end and a quencher at the other. The cleavage of the substrate strand leads to the

separation of the quencher and the fluorophore and hence increase in the fluorescence.

Now a new era of DNAzyme based sensors has arisen in which new molecular probes

(G rich oligonucleotides and G quadruplex) has been optimized with some

modifications to give better signals with Pb2+

ions. Li et al. (2010a) has developed a

Pb2+

ion detection system based on peroxidase like activity of K+ stabilized G

quadruplex DNAzyme named PS2.M with two detection means. The K+ stabilized


125

PS2.M binds to hemin with high efficiency forming a complex that mimics

horseradish peroxidase (HRP) and catalyzes H2O2 mediated oxidation of 2,2’ – azino

– bis (3 – ethylbenzothiazoline-6 – sulfonic acid) diammonium salt (ABTS) and

luminal to generate color change or chemiluminiscence (CL) emission. But the

addition Pb2+

ion leads to some conformational change in the PS2.M quadruplex

which then does not bind to hemin. This conformational transition is accompanied by

decrease in DNAzyme activity and therefore less change in color and CL emission.

Pb2+

ion could be detected upto 33.8 nM colorimetrically and 9.66 X 10-4

nM using

the CL emission method. Later the same workers, Li et al. (2010b) constructed a

novel turn – on fluorescent sensor based on cleavage of T30695 and X duplex in the

presence of Pb2+

ions and formation of a G4 structure stabilized by Pb2+

ions. The

Pb2+

ions stabilized quadruplex could be detected using Zn protoporphyrin IX

fluorescent probe. This duplex – quadruplex exchange system has a limit of detection

of 20 nM. In an ease to develop a more sensitive sensor Li et al. (2011) has now

developed a sensor in which Pb2+

ions induces formation of G4 quadruplex of

AGRO100 molecule that can bind to hemin to form complexes. This G4 AGRO100 –

hemin complex can effectively catalyze the H2O2 mediated oxidation of amplex

UltraRed (AUR), resulting in increase in fluorescence. The detection limit achieved

by this device was 0.4 nM. The present study is first one which has used a donor

acceptor based FRET method for constructing the lead specific biosensor. Recently,

Ma et al. (2011) developed an electrogenerated chemiluminiscence (ECL) biosensor

based on the use of ruthenium complex tagged 5’- amino -17 E’ as an ECL probe. The

modified Ru 1-17 E’ and substrate strand were covalently coupled on a graphite

electrode modified with 4 – aminobenzoic acid. Pb2+

ions induced cleavage of


126

substrate strand leads to dissociation of double stranded DNA complex and hence

high ECL signals. The detection limit achieved by the workers is 1.4 pM.

4.7.2 DNAzyme study in hydrosol- gel immobilization matrix

The immobilization of hybridized DNAzyme in hydrosol- gel matrix was achieved

using TMOS and TEOS solvents. It was observed that immobilization of DNAzyme

in TMOS leads to better gelatin of the mixture in the well and more fluorescence

intensity compared to control (system without DNAzyme) and TEOS as shown in

table 4.14.

Table 4.14: Fluorescence with different immobilization matrices

Solvent Fluorescence Intensity

(λ3)

TMOS + Water 0.406 ± 0.024

TEOS + Water 0.331 ± 0.050

But the fluorescence change of hybridized DNAzyme in immobilized state was low

compared to the free system as compared to table 4.13, similar to the observations of

Shen et al. (2007). The signal development efficiency of the DNAzyme couple is also

dependent on the sequence of both the strands and the various modifications done to

them (Shen et al., 2007). Also the production of methanol as a byproduct of silane

hydrolysis do not lead to any loss of DNAzyme function (Shen et al., 2007), making it

a desirable method of immobilization for DNAzyme based systems. The detection

limit obtained in immobilized system was 0.12 nanomoles (24 μM) and an appropriate

decrease in fluorescence was observed with higher amount of Pb2+

ions due to break


127

in FRET phenomenon (table 4.15). As the detection limit achieved was higher further

application of the developed method for milk sample was not studied.

Table 4.15: Lead interaction with Hydrosol- gel immobilized DNAzyme

Reagents Fluorescence

(λ3)

Hybrid + TMOS + Water 0.406 ± 0.024

Hybrid + TMOS + 0.12

nanomoles (24 μM) lead

0.294 ± 0.004

Hybrid + TMOS + 0.24

nanomoles (483 μM) lead

0.265 ± 0.025

4.7.3 DNAzyme study on gold chip

DNAzyme was successfully immobilized on gold chip according to Ananthanawant et

al. (2000). The usual protocol for florescence read was modified to carry out the

initial 70 ˚ C incubation of the 17 E strand on the gold chip as mentioned below.

Protocol for high temperature incubation and

fluorescence read

Incubate at 70 ˚C for 20 mins

Plate read

Go to line 1 for 2 more times

END

Lid Temperature- 70 ˚C

Lid off temperature – 30 ˚C

Volume set as used


128

The immobilization of 17 E strand on gold surface was confirmed by first taking the

fluorescence of bare gold chip and then after each step, immobilization of 17 E strand

and during hybridization of 17 E and 17 DS (table 4.16).

Table 4.16: Fluorescence read after each step of immobilization and

hybridization on gold surface

Steps Fluorescence read

(λ3)

Bare gold chip 0.075 ± 0.010

After 17 E - TAMRA immobilization

(acceptor) and fixation with mercaptoethanol

0.146 + 0.015

After hybridization with 17 DS –Fluorescein

(donor)

0.271 ± 0.043

In this case 17 DS was labeled with fluorescein which acted as donor and 17 E strand

with TAMRA which acted as acceptor to create FRET phenomenon. To take the

fluorescence of both the strands separately HEX (Em = 556 nm) was used as the read

out dye for 17 E and Sybergreen (Em = 520 nm) for 17 DS. The increase in

fluorescence of bare gold surface after 17 E immobilization indicates the presence of

fluorescently labeled moiety on gold surface. The treatment with mercaptoethanol

fixes the enzyme strand and then washing lead to the removal of unbound enzyme

strands as evident from the fluorescence read after washing (table 4.16). The

hybridization was achieved successfully as it leads to the increase in fluorescence of

the 17 E (acceptor) strands to almost double.


129

After hybridization DNAzyme was interacted with Pb2+

ions and volume was

miniaturized upto 5µl volume on the gold chip. The change in fluorescence observed

with Pb2+

ions in this system was very low as shown in the table 4.17.

Table 4.17: Lead interaction with DNAzyme immobilized on gold surface

Reagents Fluorescence (F)

At λ3

Change in

Fluorescence (ΔF)

Control 0.166 ± 0.113

0.06 nanomoles Lead (5µl

of 12 μM)

0.124 ± 0.110 0.042 ± 0.003

0.12 nanomoles Lead (5µl

of 24 μM)

0.112 ± 0.109 0.054 ± 0.004

As the change in fluorescence observed was very low enzyme and substrate

strand concentration was optimized to get more signal enhancement and lower limit of

detection. Firstly the 17 DS was optimized for maximum concentration and it was

observed that 1 µM and 2 µM 17 DS had almost same fluorescence (table 4.18), so 1

µM 17 DS was optimized for further experiments.

Table 4.18: Substrate (17 DS) strand optimization

17 DS Concentration

(µM)

Fluorescence at λ3

1 1.338 ± 0.009

2 1.321 ± 0.005

3 1.670 ± 0.177


130

Secondly different concentrations of enzyme strand (17 E) were used for

hybridized DNAzyme and change in fluorescence was observed (table 4.19).

Table 4.19: Enzyme (17 E) strand optimization

17 E Conc.

(µM)

Fluorescence

with water at λ3

Fluorescence

with 0.24

nanomoles Pb2+

ions at λ3

Change in

Fluorescence (ΔF)

1 0.326 ±0.018 0.263 ± 0.026 0.063 ± 0.008

2 0.715 ± 0.023 0.109 ± 0.142 0.606 ± 0.119

3 0.660 ± 0.045 0.315 ± 0.146 0.315 ± 0.101

As maximum change in fluorescence was observed with 2 µM 17 E, it was

optimized for further experiments. Also the effect of Pb2+

ions was also more

pronounced at 2 µM. A decrease in fluorescence at 3 µM 17 E concentrations could

be attributed to higher dye content which failed to follow the Lambert’s Beer Law.

As indicated by the observations 1 µM 17 DS and 2 µM 17 E was finally

optimized based upon their fluorescence signal to carry out lead interaction study. In a

similar study a concentration of 1 µM 17 E and 17 DS was used by Swearingen et al.

(2005) to develop lead biosensor after immobilization on gold surface. The present

work has utilized the advantage of DNAzyme immobilization on gold surface through

thiolation of enzyme strand as done by Swearingen et al. (2005). This allowed better

sensitivity and lead analysis on solid surface which decreases background signals.

Wernette et al. (2006) also immobilized DNAzyme in a Au coated polycarbonate

track – etched (PCTE) nanocapillary array tube (NCAM) through thiolated enzyme.

In this case the cleaved fluorophore at the substrate strand has been analyzed in

solution from only as the cleaved moieties move on through the nanocapillary and


131

hence follow aqueous state chemistry instead solid surface chemistry. Shen et al.

(2008) has developed an electrochemical biosensor for lead using DNAzyme and a

concentration of 1 µM 17 E was immobilized on the gold electrode and hybridized

with 2 µM 17 DS. In a recent study by Wang and Irudayaraj, (2011) DNAzyme based

lead biosensor has been constructed using surface enhanced raman scattering (SERS)

effect of gold nanopartical (AuNPs) in which enzyme strand was immobilized on gold

surface through thiolation followed by hybridization with AuNP labeled substrate

strand. The DNAzyme and raman reporters has been conjugated with AuNPs to

achieve a detection limit of 19.3 nM.

Finally the optimized enzyme and substrate strands were used to develop a

competent biosensor for lead detection. A linear range of detection was observed for

lead from 0.024 – 0.24 nanomoles (4.83 – 48.3 μM) as depicted in table 4.20 and

lower limit of detection achieved was 6 x 10-3

nanomoles (1.2 μM).

Table 4.20: Lead interaction with DNAzyme immobilized on gold surface after

17 E and 17 DS optimization

Lead Concentration

(nanomoles)

Fluorescence

(w.r.t acceptor against

HEX)

Change in fluorescence

(ΔF)

Control 0.777 ± 0.116

0.024 0.413 ± 0.129 0.364 ± 0.013

0.06 0.317 ± 0.112 0.460 ± 0.004

0.24 0.116 ± 0.134 0.661 ± 0.018


132

As evident from the observations, the increase in lead concentration caused decrease

in fluorescence of acceptor as compared to the control. This was because the lead

catalyzed cleavage of the substrate strand which was acting as the donor in causing

FRET phenomenon and its cleavage resulted in break of FRET, also causing the

fluorescence of acceptor to decrease and change in fluorescence to increase. The

developed system was used to analyze lead in spiked milk samples and a detection

limit of 6 x 10-3

nanomoles (1.2 μM) was achieved in milk samples also (table 4.21),

but the change in fluorescence was low as compared to the synthetic system which

could be attributed to the complex matrix of milk which may interfere in fluorescence

signal.

Table 4.21: DNAzyme application on milk samples

Lead Conc.

(nanomoles)

Fluorescence Change in Fluorescence

(ΔF)

Control 1.705 ± 0.025

6 x 10-3

1.601 ± 0.038 0.104 ± 0.012

2.4 X 10-2

1.475 ± 0.022 0.230 ± 0.003

The advantage of using this DNAzyme based method for lead analysis in milk is that

there is no need of any pretreatment of milk to attain lead specificity and the complex

matrix of milk was still able to give detectable fluorescence signal unlike other optical

methods where removal of milk protein is a necessary step before analysis. DNAzyme

has been used to detect lead by many means either through gold nanoparticals (Liu

and Lu, 2003; Wei et al., 2008) or Fluorescent probes with or without quenchers (Liu


133

and Lu, 2003; Chang et al., 2005). Single or both strands could be labeled with

fluorescent dyes to study the quenching or FRET phenomenon caused by Pb (II)

ions.

This DNAzyme approach provides high sensitivity and specificity towards Pb (II)

ions. DNAzyme has also been combined with network of microfluidic channels

coupled via a nanocapillary array to achieve a detection limit of 11 nM (Chang et al.,

2005). Wernette et al. (2005) had incorporated DNAzyme into PCTE-NCAMs and

obtained a detection limit of 16.9 nM. An electrochemical DNAzyme biosensor has

also been developed with detection limit of 0.3 µM (Xiao et al., 2006). DNAzyme has

also been entrapped in sol gel derived matrices, but detection limit was in micromolar

range (Shen et al., 2007). Conjugation of HRP mimicking DNAzyme with aptamers

has also been used for amplified Pb (II) detection, achieving a detection limit of 10

nM (Elbaz et al., 2008). Recent trend is the immobilization of DNAzyme on gold

surface to lower down the detection limit to 1 nM (Swearingen et al., 2005; Shen et

al., 2008). The present work has been focused to develop a micro-array based optical

DNAzyme lead biosensor for multiple sample analysis, in which the enzyme and

substrate strands has been labeled with fluorescent dyes and the FRET phenomenon in

the presence and absence of Pb (II) ions has been studied. Two Immobilization

strategies have been optimized for DNAzyme based studies. In first method

DNAzyme has been immobilized in TMOS based hydrosol gel in a microarray plate

and the microarray analysis of lead in hydrosol gel system has been successfully

miniaturized up to 10ul volume, with a detection limit of 0.24 nanomoles (24 μM).

This method has the advantage of multiple samples testing at the same time in a cost

effective way.


134

Secondly, Immobilization of fluorophore bearing DNAzyme through

SAM on gold solid phase surface has been achieved. In this case, 6 x 10-3

nanomoles

(1.2 μM) biosensing of lead at 5ul volume has been achieved in synthetic samples and

milk samples. This method has an advantage of fast analysis along with testing of

several samples at the same time. The present biosensor has a novelty of sample

analysis directly on the gold chip as compared to the already developed samples in

which first the reaction is done on the gold surface and then transferred to other

system for analysis.

4.7.4 Mechanistic conformation of DNAzyme based lead analysis

The validation of Substrate strand cleavage was obtained by running the samples of

DNAzyme hybrid in the absence and presence of lead along with the individual 17 E

and 17 DS strands. As visible in the fig 4.30 the 33 bp 17 E strand (lane 2) was

obtained slightly above the 20 bp 17 DS strand (lane 1). Then the hybridized

DNAzyme incubated with deionized water (lane 4 & 8) showed a single band close to

17 E position. The samples incubated with lead (lane 5-7) showed the clear cleavage

of the substrate strand in which two distinct bands one corresponding to 17 E and the

lower one of cleaved 10 bp 17 DS were obtained. The cleaved 10 bp substrate strand

was obtained lower to the 20 bp 17DS strand confirming its cleavage in to short

fragments of 10 bp.


135

Figure 4.30: PAGE analysis of 17 E and 17DS. Lane 1: 17S, Lane 2: 17E, Lane 4

& 8: Hybrid + water (control), lane 5-7: Hybrid + Pb2+

ions

4.8 Microarray based optical lead biosensor

To develop an optical microarray based biosensor for multiple sample analysis,

Rhodamine G (Ex = 526, Em = 555) was used as an indicator dye and read against

HEX (Ex = 535, Em = 556) in the plate reader. The dye was optimized for linear

concentration response on the microarray plate. A stock of 2mg/ 10 ml was diluted

340 times and then it’s different volume in a total of 680 µl was prepared and

emission read through plate reader. A volume of 80 µl dye in total of 680 µl was

optimized according to the linear curve (4.31) which showed the maximum emission

after which the intensity of fluorescence reached the pseudozero order stage.


136

Figure 4.31: Calibration curve for Rhodamine-G dye

4.8.1 Analysis of lead in microarray plate

The lead was analyzed by setting the hydrosol- gel directly into the microarray plate

and then adding different lead concentrations in the wells with same initial

fluorescence. Whole cells of B. sphaericus was used as the sensing component with

enzyme activity 14.97 ± 2.01 IU (250 ml broth) and the fluorescent dye used was

Rhodamine – 6G (Ex = 526, Em = 555) which was read against HEX (Ex = 535, Em =

556) in the plate reader. The lead inhibition effect was found to be logarithmic in this

case also and the linear range of detection was found from 4.83 X 10-5

nanomoles to

4.8 X 10-2

nanomoles (4.83 nM – 4.83 μM ) as shown in figure 4.32.

The lower limit of detection in this method was 4.83 X 10-5

nanomoles (4.83 nM).


137

Figure 4.32: Linear range of lead detection by direct hydrosol- gel

immobilization method

Although this method was sensitive but had the disadvantage of the selection of wells

with same fluorescence and results in many unutilised wells, hence wastage of

biomass and reagents. To overcome these limitations a modified method was

developed in which the hydrosol- gel was prepared in air tight tubes and lead was

added to them after all the required incubations. The analysis was done after pouring

20 µl from each reaction mixture in different wells of the microarray plate. This

method gave repeatability of the results and linear range of lead detection was

achieved upto to 4.8 X 10-4

to 9.7 X 10-3

nanomoles (0.48 – 9.66 nM), with lowest

detection limit of 4.8 X 10-4

nanomoles (0.48 nM) lead (figure 4.33).

Figure 4.33: Linear range detection of lead in modified microarray method


138

Owing to the repeatibility and lower limit of detection of lead, this method was

further applied for lead analysis on milk samples (10 µl and 1000 µl ). In case of 1000

µl milk samples the linear range of detection was from 4.8 X 10-4

to 4.8 X 10-2

nanomoles (0.48 – 48.3 nM) in an logarithmic pattern as shown in figure 4.34 with

lowest limit of detection as 4.8 X 10-4


Figure 4.34: Linear range of detection of lead in spiked milk (1000 µl )

The linear range in 10 µl spiked milk was from 2.4 X 10-3

to 4.8 X 10-2

nanomoles

(2.4 – 48.3 nM) as shown in figure 4.35 and the lowest limit of detection obtained was

4.8 X 10-4


Figure 4.35: Linear range of detection of lead in spiked milk (10 µl)


139

From the above shown data it is very evident that the developed method is suitable for

lead analysis in milk samples with a very low detection limit. The obtained limit of

detection in milk has not been reported by any other worker yet and the developed

method surpasses the need of any pretreatment to the milk samples. It was observed

that the complex matrix of milk imparts some increase in fluorescence of the dye as

compared to the synthetic system. So, to detect that interferrance due to milk and to

apply the developed method on unknown milk smples taking synthetic systen as

reference, the analysis of lead in synthetic and spiked milk samples was carried out

and the fluorescence read at different lead concentrations and in control samples was

compared. As shown in table 4.22, it was observed that the difference in fluoresence

of spiked milk and synthetic sample was following a regular pattern. So it could be

suggested that the effect of milk on fluorescence is corresponding to 0.12 which

should be considered at the time of lead determination in unknown milk samples.

Table 4.22: Comparison of fluorescence in synthetic and spiked milk samples

Lead

Concentration

(nanomoles)

Fluorescence in

Synthetic system

Fluorescence in

spiked milk

sample (10 μl)

Fluorescence

difference

Control 0.502 0.641 0.139

4.8 X 10-4

0.525 0.654 0.129

2.4 X 10-3

0.534 0.653 0.119

4.8 X 10-3

0.547 0.667 0.120

9.7 X 10-3

0.553 0.671 0.119

2.4 X 10-2

0.581 0.683 0.102

Av=0.12


140

The detection limit achieved in the developed biosensor is far below the limits

obtained till now and is under the permissible limit of lead in milk (96.6 nM or 20 ng

ml-1

). No optical based study has been done so far for lead contamination in milk. So

the comparison has been done with studies on water samples. Lee and Lee, 2002 had

developed a conductometric biosensor based on sol- gel immobilized urease and

obtained a detection limit of 0.9 mM for lead. Later Tsai et al. (2003) used FITC-

dextran as fluorescent dye to develop urease based optical biosensor for heavy metals

and achieved a detection limit of 0.1 mM. Kuswandi (2003) obtained a detection limit

of 1 X 10-2

mM lead in 6 min response time using immobilized urease on a optical

fiber. Then a sol- gel immobilized urease conductometric biosensor was developed by

Illangovan et al. (2006) and a percentage inhibition of 35% was observed with 1 mM

lead. A very low detection limit of 4.83 nM (4.83 X 10-3

nanomoles) was achieved

using cyclotetrachronotropylene (CTCT) as an indicator by Haron and Ray (2006).

They developed an optical biosensor based on total reflection at interface between

SiN4 core and composite polyelectrolyte self assembled (PESA) membrane caused by

the catalytic activities of urease and acetylcholine esterase.

4.8.2 Detection of lead in the presence of cadmium

The detection of lead and cadmium simultaneously was possible by using two urease

producing strains, B. sphaericus and B. badius respectively. Two different fluorescent

dyes Rhodamine 6G (Ex = 526, Em = 555) and Acridine orange (Ex = 493, Em = 535)

with different emission wavelengths were selected for this purpose and were read

against HEX (Ex = 535, Em = 556) and Sybergreen (Ex = 497, Em = 520 nm)

respectively. Also there was an advantage that B. badius urease is insensitive to lead

at nanomolar levels and only shows inhibition at micromolar range, wheres as B.

sphaericus is sensitive to lead at nanomolar levels. So lead does not interfere in


141

cadmium detection by B. badius and the fluorescence read against sybergreen in the

system corresponds to cadmium effect only, but the fluorescence read against HEX is

the cumulative effect of lead and cadmium on B. sphaericus. The standard curve of

cadmium was prepared for synthetic and spiked milk samples (figure 4.36 and figure

4.37).

Figure 4.36: Cadmium standard curve in simultaneous study with Pb (II) in

synthetic system

Figure 4.37: Cadmium standard curve in simultaneous study with Pb (II) in

spiked milk samples (10 µl)


142

The lead concentration in an unknown sample was detected by carrying out

simultaneous detection of lead and cadmium in the synthetic and spiked milk samples.

According to the standard curve of cadmium effect on urease, its concentration could

be deduced from the change in fluorescence pattern against sybergreen dye

(corresponding to Cd2+

). Taking that concentration as reference the respective

fluorescence fraction of Cd2+

was subtracted against the HEX fluorescence to get the

lead contamination value. The present method is the first of its kind for simultaneous

detection of Pb (II) and Cd (II) in milk in the same well. Haron and Ray, (2006) had

developed an optical biosensor for monitoring Pb (II) and Cd (II) in water samples

based on inhibition of urease and acetylcholinesterase and achieved a detection limit

of 1ng ml-1

whereas the present work could detect upto 0.1 ng ml-1

in milk samples.

4.8.3 Application of developed sample on different areas milk samples

Twenty milk samples each from rural, urban and industrial area were collected and

lead analysis was done on them according to the developed method. As per the

standards of spiked lead samples and cadmium interference values, two formulas for

quantitative analysis of lead was devised which could be directly applied on unknown

samples.

X nanomoles of Pb2+

= [ (A – M1) – F1] X B1 , when only Rhodamine dye used

Where, A = Fluorescence read for the sample (corresponding to Pb2+

)

M1 = Milk factor with Rhodamine dye,

F1 = Fluorescence read of Rhodamine G

B1 = Pb2+

Standard / Fluorescence of that standard sample

To detect lead in the presence of cadmium a formula was devised for cadmium

detection and then the fluorescence factor (interference factor) according to that


143

cadmium concentration was subtracted from Rhodamine G fluorescence to detect lead

contamination.

Y nanomoles of Cd2+

= [(C – M2) – F2] X B2

Where, C = Fluorescence read for the sample (corresponding to Cd2+

),

M2 = Milk factor with acridine orange dye,

F2 = Fluorescence read of acridine orange dye

B2 = Cd2+


Formula for detection of lead in the presence of cadmium

X nanomoles of Pb2+

= [ {[ A – M1) – F3] – C} X B1

Where, A = Fluorescence read for the sample (corresponding to Pb2+

),

M1 = Milk factor,

F3 = Florescence read of combined dyes rhodamine and acridine orange

C = Cadmium conc. calculated from standard curve using dye acridine orange,

B1 = Pb2+


The reliability of the developed formula was checked by carrying out analysis of lead

and cadmium in unknown samples and then again after spiking of the samples with

known concentration of lead and cadmium. The lead analysis was done using

Rhodamine G dye alone and in the presence of acridine orange to detect the presence

of cadmium simultaneously in the milk samples. All the samples were analyzed in

triplicates and the devised formula was applied to the fluorescence read to detect lead

and cadmium contamination.

All the rural and urban samples were found free of any lead or cadmium

contamination (table 4.23 and table 4.24). In the case of industrial samples lead and

cadmium was found in two samples at the levels shown in the table 4.25.


144

Table 4.23 Lead and cadmium contamination in rural samples

Sample Place Lead detected

(nanomoles)

Lead detected

(nanomoles) in

the presence of

Cd2+

Cadmium

detected

(nanomoles)

Sample R1 VPO- Banur,

Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND


Rajpura

ND ND ND

*ND = No heavy metal detected


145

Table 4.24 Lead and cadmium contamination in urban samples

Sample Place Lead

detecte

d

(micro

moles)

Lead detected

(micromoles)

in the

presence of

Cd2+

Cadmium

detected

(nanomoles)

Sample U1 Tripuri, Patiala ND ND ND










Sample U11 Urban State Ph- II,

Patiala

ND ND ND


Patiala

ND ND ND


Patiala

ND ND ND


Patiala

ND ND ND


Patiala

ND ND ND


Patiala

ND ND ND


Patiala

ND ND ND


Patiala

ND ND ND


Patiala

ND ND ND


Patiala

ND ND ND


146

Table 4.25 Lead and cadmium contamination in industrial samples

Sample Place Lead

detected

(nanomoles)

Lead detected

(nanomoles) in

the presence of

Cd2+

Cadmium

detected

(nanomoles)

Sample D1 Industrial Area,

Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

5.9 ± 0.32 7.1 ± 0.29 0.026 ± 0.022


Ludhiana

6.0 ± 0.33 6.2 ± 0.25 ND


Ludhiana

ND ND 0.162 ± 0.039


Ludhiana

ND ND ND


Ludhiana

ND ND ND

Sample D11 Tajpur Road,

Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

ND ND ND


Ludhiana

ND ND ND


147

Reliability of the developed method and formulas was checked by applying the same

on three unknown samples D4, D19, D20 (which were detected negative for both Pb2+

and Cd2+

earlier) after spiking with 5 ng lead and cadmium each. The results are

shown in table 4.26.

Table 4.26: Reliability studies of Optical Biosensor

Sample Pb2+

and

Cd2+

Added

(ng)

Lead found

(ng)

Cadmium found (ng)

Sample D4 5 4.64 ± 0.33 4.69 ± 0.38

Sample D19 5 4.88 ± 0.26 5.01 ± 0.17

Sample D20 5 5.3 ± 0.10 4.77 ± 0.19

Finally the present work has developed new technologies for the successful

construction of enzyme inhibition based colorimetric, potentiometric, amperometric

and optical biosensor along with DNAzyme based optical biosensor for monitoring

lead in milk.

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