chapter 7shodhganga.inflibnet.ac.in/bitstream/10603/51072/12... · these materials; constructed...

Chapter 7

True greatness is when your name is like ampere, watt, and fourier—when it's spelled with a lower case letter.

— Richard Hamming

Chapter 7

Center for Interdisciplinary Research, DYPU Kolhapur. 169

7.1. Introduction Biosensors based on oxidase enzymes have been described for over

eighty analytes; catalyzes oxidation of many substrates which are critical in

biological pathways. Among them, Flavin adenine dinucleotide (FAD)

dependent glucose oxidase and cholesterol oxidase are suitable for recognition

of important physiological metabolites like glucose and cholesterol,

respectively [1]. However, direct electrochemistry is rather hard for these

enzymes due to the bulky conformation. Improved electron transfer of FAD

dependent oxidase enzymes can be attained by proper orientation of enzymes

on electrode surface with retaining its native conformation.

Aimed for that, many immobilization matrices are employed such as

nanomaterials, polymers, and sol-gel matrices have been reported for enzyme

nanobiosensor. Among them, silica nanocomposite is a favorable candidate for

immobilization of enzyme offer many advantages due to its unique chemical,

electrochemical, and physical characteristics. The sol-gel method provides a

unique matrix in which various enzymes can be immobilized without loss of

enzyme functionality.

7.2. Background of Bienzymatic Cholesterol Nanobiosensor The clinical disorders such as arteriosclerosis, coronary artery disease,

cerebral thrombosis, hypothyroidism, and hypertension; owing to abnormal

levels of cholesterol in the blood has stimulated public concern about the

detection of cholesterol level. Development of a reliable cholesterol

nanobiosensor is vital in clinical diagnosis because the concentration of

cholesterol is a fundamental parameter for prevention and diagnosis of a

number of diseases. Furthermore, it is significant for patient suffering from

high blood pressure, nerve disease and other diseases that involve continuous

monitoring of cholesterol. The inherent specificity of cholesterol oxidase

provides the most exact means for detection of true blood cholesterol.

Chapter 7


Many enzymatic biosensors based on immobilized cholesterol oxidase

have been reported in the literature. Cholesterol can be analyzed indirectly by

monitoring hydrogen peroxide generated in enzymatic reactions using

voltammetry and amperometry. A kind of ferric redox enzyme, horseradish

peroxidase was also used as a cholesterol nanobiosensor. More recently,

cholesterol nanobiosensor based on physically adsorbed cholesterol oxidase

onto metal oxide nanoparticle have been applied to determine the cholesterol

concentration. The enzyme activity was well preserved upon binding onto the

nanoparticle when subjected to thermal and various pH conditions. Kinetic

studies indicated a substantial advance in magnetic nanoparticles bound

cholesterol oxidase owing to the large surface area and high chemical and

thermal stability of magnetic nanoparticles enhances the electrocatalytic

activity of the nanocomposite.

It has been described that the conductance of nanofibers, nanotubes,

nanoribbons, nanorods and nanowires were superior in regard to other

morphological nanomaterials. Referable to the high electrical conductivity of

these materials; constructed nanobiosensors amplify the signal-to-interference

ratio and the high sensitivity compared to that observed at bulk materials

electrodes. This improved analytic performance was due to both factors: the

high enzyme loading and better electrical communication ability [2]. This is

recognized as the direct electron transfer ability of nanotubes, nanorods,

nanofibers, and nanowires demonstrated that both FAD was found to

spontaneously adsorb (physical adsorption) to metal oxides nanoparticles and

to display the quasi-reversible one electron transfer reaction.

Using the rapid advances of nanotechnology, there has been bang of

interest in the role of biomolecules having sophisticated structures and

prominent physical and chemical properties as building blocks for the

development of multifunctional nanomaterial [3]. Due to the conformational

Chapter 7


polymorphism, sequence specific recognition and robust physicochemical

nature, DNA has been extensively investigated in nanotechnology and material

science [4].

The purpose of DNA as a template is promising avenues offered for

making up a variety of metallic nanomaterials with potential applications [5].

DNA is abundant in phosphate groups, amino groups and heterocyclic nitrogen

atoms; it offers nucleation sites for metallic nanoparticles and provides control

over the nanomaterial growth and stability [6]. DNA is inherently self-

assembly material due to its predictable base-pairing, high chemical stability.

DNA biomolecules are negatively charged polyelectrolytes; create the polyion

complex with positively charged polyelectrolytes [7]. The conductive DNA

stacked base pairs are considered as a system of connected π electrons to

transfer electrons. The efficient electron migration within the DNA duplex is

possible over the distance up to 40 A° [8].

Electrochemical study on nucleic acid hybridization indicated that

electron transfer could also take place by percolation or the physical

displacement of associated ions along a negatively charged phosphate

backbone [9]. In this regard, the DNA-based polyion complex can be used as a

host matrix of electrochemically active species (e.g., redox active intercalators)

and improve electron transfer characteristics between redox active, species and

the electrode surface [10]. DNA modified materials had been proposed for

immobilization of enzymes such as horseradish peroxidase [11], glucose

oxidase [12] for the fabrication of biosensors. Consequently, DNA utilized as

an economical, well-characterized, manageable, and easily adaptable material

to construct defined hybrid nanocomposites for immobilization of enzymes.

DNA-inorganic nanoparticles based biocompatible nanocomposite

possess biocompatible microenvironment around the enzyme, a host matrix of

Chapter 7


electrochemically active species and metal ions which specifically bind to

double stranded DNA, and unique electron transfer property improving

electron transfer characteristics between redox active species and the electrode

surface. Therefore, DNA is extensively used as biorecognition elements in

biosensors as well as unique building blocks in nanodevices [13]. The stacked

base pairs are considered as a system of ᴨ electrons; thus effective electron

transfer inside the DNA duplex is possible over a distance up to 40 Å. Besides

that, DNA has been used as easily flexible nano-biomaterial to construct

defined hybrid nanocomposite [14].

In recent times, core-shell nanoparticles is promising nanomaterial for

the biomedical applications in many arenas owing to its multifunctional

properties, which can be tailored by changing the core to shell ratio of

constituting materials. Core-shell nanoparticles composed of bare Fe3O4 NPs as

core and some other material as a shell have numerous benefits like to avoid

aggregation and oxidized in the air. Silver coated magnetic nanoparticles are

one of the most attractive core-shell nanoparticles for its chemical activity, and

biocompatibility utilized in antibacterial activity [15] and immunoassay [16].

Porous inorganic nanomaterials with high specific surface area have

emerged as appealing material for adsorption of various molecules into the

pores [17]. Meanwhile, due to the unique electrical and magnetic behavior,

DNA assembled nanomaterials have been extensively employed for biomedical

applications. Synthesis of DNA assembled Fe3O4@Ag NPs and its

incorporation in silica sol to produce silica nanocomposite is demonstrated in

Figure 7.1. This chapter illustrates the development of DNA-Fe3O4@Ag

nanorod embedded porous silica nanocomposite which is used to encapsulate

HRP and ChOx for development of bienzymatic cholesterol nanobiosensor.

Chapter 7


Figure 7.1: Synthesis of Silica/DNA-Fe3O4@Ag Nanocomposite

7.3. Experimental

7.3.1. Materials

Double stranded DNA was isolated from fresh ATCC 25922 E coli

culture. Cholesterol, Cholesterol Oxidase (ChOx, E.C. 1.1.3.6 25 U/mg of

protein), Horseradish peroxidase (HRP, E.C 1.11.1.7, 100 U/mg, from

horseradish), and Tetraethyl orthosilicate (TEOS) were procured from Sigma

Aldrich. Cholesterol, Silver Nitrate (AgNO3) Ferrous sulfate heptahydrate

(FeSO4.7H2O) and Ferric chloride hexahydrate (FeCl3.6H2O) were acquired

from Hi Media, India. All other reagents were of analytical grade purchased

from SD Fine Chemical Pvt. Ltd. India and used without further purification.

All solutions were prepared with deionized double distilled water.

7.3.2. Characterizations

All electrochemical experiments were performed at a CHI 650 D

electrochemical workstation (CH Instruments Inc., USA) using a conventional

three-electrode system with Silica/DNA-Ag@Fe3O4 nanocomposite modified

ITO electrode, Ag/AgCl and a platinum wire as the working, reference and

Chapter 7


counter electrodes, respectively. Cyclic voltammograms were collected in a

solution of potassium phosphate buffer containing 2-propanol 10% (v/v) and

Triton X-100 0.7% (v/v). Transmission electron microscopy (TEM) images

were recorded with a Model: JEM-100 CX II opened at an accelerating

potential difference of 100 KV. UV Vis spectroscopy experiments were

performed with a UV-2100S spectrophotometer (Shimadzu, Japan).

7.3.3. Synthesis of Silica/DNA-Ag@Fe3O4 Nanocomposite

i) Preparation of Silver coated iron oxide nanoparticles

The preparation of silver coated Fe3O4 NPs was carried out by the two-

step method. Initially, a core of 7 ± 2 nm Fe3O4 NPs using the co-precipitation

method was obtained as described in chapter 6. In a second step silver shell of

5 ± 1 nm using glucose as reducing agent was achieved. The Fe3O4 NPs were

synthesized by chemical co-precipitation method as per report [18]. Briefly,

Iron (III) chloride hexahydrate and iron (II) sulfate heptahydrate (1:1 Molar

ratio) were mixed with strongly stirred NaOH (3.0 M) solution in de-ionized

water at 88 °C for 15 min. The black precipitate product was magnetically

decanted, washed with ethanol and water. The prepared nanoparticles were

dried at 60 °C for 6 h in a vacuum oven.

Subsequently, Fe3O4 NPs suspension (1.0 mg mL-1

) was dispersed in

Ag(NH3)2+ solution (0.1 M) and stirred for sufficient adsorption Of Ag

+ ions

on Fe3O4 NPs. After addition of 5.0 mg of glucose as reducing agent in the

above solution, and heated in a water bath at 50 °C for 30 min. The slightly

brown product was obtained which is then magnetically decanted, washed and

dried at 60 °C for 6 h in a vacuum oven.

ii) Preparation of DNA-Fe3O4@Ag Nanorods

The fresh DNA sample was isolated from fresh E. Coli culure by using

Magnetic adsorption technology as per previous report [19]. The isolated DNA

Chapter 7


was kept at 80 °C in water bath for 10 min and then rapidly cooled in ice bath

for 10 min for relaxing the supercoils. The double stranded DNA was used for

the preparation of Fe3O4@Ag-DNA nanorods. Freshly synthesized Fe3O4@Ag

NPs were dispersed in DNA suspension in Tris HCl buffer for overnight.

The resulting material was magnetically decanted, i.e. by using magnet

Fe3O4@Ag-DNA nanorods are separated and washed three times to ensure the

removal of any unbound DNA. The sample was freeze dried for further

characterization. DNA assembled Fe3O4@Ag nanorods then inserted into silica

matrix to synthesize Silica/Fe3O4@Ag-DNA nanocomposite.

7.3.5. Fabrication of cholesterol bienzymatic nanobiosensor

Prior to use, ITO plates were sonicated with acetone, ethanol solution

and washed with distilled water and dried at room temperature. Pre-cleaned

ITO plates were immersed in a hydrolyzing solution of 1:1:5 (v/v)

H2O2:NH4OH:H2O for about 30 min at 80 °C. After hydrolysis plates were

rinsed with distilled water and dried at room temperature.

The enzyme solution of HRP (2.0 mg mL-1

) and ChOx (1.0 mg mL-1

) in

potassium phosphate buffer (pH 7.0) were added in the Silica/Fe3O4@Ag-DNA

nanocomposite solution for encapsulation of enzymes. Then resulting solution

coated onto a 1.0 cm2 area of ITO electrode by drop casting method and kept at

4 °C for 12 h in the humid chamber.

7.4. Results and discussion

7.4.1. Phase Confirmation

XRD patterns of Fe3O4 NPs (a), and Fe3O4@Ag NPs (b) were elucidated

in Figure 7.2. As compared to the standard XRD pattern of Fe3O4 NPs (JCPDS

Card no. 79-0419, magnetite) and Ag (JCPDS Card no. 89-3722, face

centered); the XRD pattern of Fe3O4@Ag NPs (c) displays diffraction peak at

35.56°, 38.098°, 44.419°, 62.970°, 64.540°, and 77.362° which are assigned to

Chapter 7


the (311), (111), (200), (440), (220), and, (311). The results indicate that

Fe3O4@Ag NPs composed of crystalline Fe3O4 NPs and Ag. The crystallite

sizes of nanomaterials were calculated from FWHM of the most intense peaks

using the Scherrer formula. The crystallite sizes are found to be 7.20 nm and

12.48 nm for bare Fe3O4 NP and Fe3O4@Ag NPs, respectively. These results

are consistent with the TEM results.

Figure 7.2: XRD pattern of (a) Fe3O4 NPs, and (b) Fe3O4@AgNPs

7.4.2. Spectroscopic Analysis

A. UV Visible spectroscopy

Silica/DNA-Fe3O4@Ag nanocomposite was characterized by UV Vis

spectroscopy. The two strong characteristic peaks appeared at 420 and 260 nm,

which are assigned to Fe3O4@Ag NPs and DNA, respectively. Appearance of

Chapter 7


peak at 260 nm in Figure 7.3 (c) confirms that Fe3O4@Ag NPs had been

successfully assembled on the DNA.

The shape and position of the Surface Plasmon absorption band of silver

nanomaterials at 420 nm are known to be intensely dependent on the particle

size and dielectric medium. Conferring to Mie’s theory [20], only a single SPR

band is anticipated in the absorption spectra of spherical metal nanoparticles,

whereas anisotropic particles could give rise to two or more SPR bands

depending on the configuration of the molecules. In Fe3O4@Ag NPs, a single

SPR band is observed, which suggests that nanoparticles are spherical in shape

and consistent with the TEM observations.

Figure 7.3: UV Visible spectra of (a) Fe3O4@Ag NPs, (b) DNA solution in

buffer, and (c) DNA- Fe3O4@Ag nanorods

The apparent enzyme activity (U cm−2

) was calculated using UV Vis

spectroscopy as per the reported method [21] based on the absorbance observed

at 500 nm before and after the incubation of ChOx modified electrode. The

Chapter 7


apparent enzyme activity 15.2×10 −3

Abs mg−1

dl −1

was evaluated using

equation,

( )

( )

Where A is the difference in absorbance before and after incubation, V

is the total volume (3.08 cm3), ε is the millimolar extinction coefficient (7.5 for

o-dianisidine at 500 nm), t is the reaction time (min), and s is the surface area

(1.0 cm2) of the electrode. For measurement, a solution of 20 µl HRP, 10 µl of

o-dianisidine solution and 50 µl of 100 mg dl−1

cholesterol was diluted by

adding 3 mL potassium phosphate buffer (pH 7.0) and was kept in a thermostat

at 25 °C. Silica/Fe3O4@Ag-DNA/HRP/ChOx/ITO electrode was immersed and

was incubated for approximately 3 min.

7.4.3. Morphological Characterizations

A. Transmission Electron Microscopy

The shape and size of nanoparticles and nanorods were confirmed using

TEM micrographs. The uniform spherical shape observed for Fe3O4 NPs (a),

and Ag@Fe3O4 NPs (b); with average particle size estimated 7 ± 2 nm and 15 ±

2 nm, respectively. TEM micrograph (Figure 7.4 c) illustrates that positively

charged Fe3O4@Ag NPs electrostatically attracted by the negatively charged

phosphate backbone of DNA forming nanorod structure.

Figure 7.4 (d) shows a TEM image at high magnification recorded for

single DNA-Fe3O4@Ag nanorods, demonstrating that the Fe3O4@Ag NPs grew

along the DNA direction. The SAED patterns of Fe3O4 NPs and Fe3O4@Ag NPs

[inset Figure 7.4 (a), and (b)] indicate the crystalline nature of materials while in

Fe3O4@Ag-DNA nanorods [inset Figure 7.4 (c)]; the intensity was decreased due to

the amorphous nature of DNA.

Chapter 7


Figure 7.4: TEM micrographs of (a) Fe3O4 NPs, (b) Fe3O4@Ag NPs, and

(c) DNA-Fe3O4@Ag nanorods [inset SAED pattern of (a) Fe3O4 NPs, (b)

Fe3O4@Ag NPs, and DNA-Fe3O4@Ag nanorods]; High magnification TEM

image of single DNA-Fe3O4@Ag nanorod

7.4.4. Cyclic Voltammetry

I) Electrochemical behavior of Silica/DNA-Fe3O4@Ag/HRP/ChOx

Cyclic voltammograms of differently modified electrodes in N2 gas

saturated potassium phosphate buffer of 0.1 M, pH 7.0 at scan rate of 0.1 V s-1

displayed in Figure 7.5. A small unsymmetrical reduction peak was observed

at the Fe3O4@Ag NPs modified electrode (a), indicates that these nanoparticles

are electroactive in the potential range from -0.2 to -0.8 V. Silica/DNA-

Chapter 7


Fe3O4@Ag modified electrode (b) had a very small single reduction peak; it

shows that the electrode was not reversible in this potential range.

Silica/DNA-Fe3O4@Ag/HRP/ChOx modified electrode (c) exhibited a

pair of stable and well-defined redox peaks at -0.40 and -0.43 V (vs. Ag/AgCl),

which could be attributed to the direct electron transfer of HRP encapsulated in

Silica/DNA-Fe3O4@Ag nanocomposite. Its formal potential is (defined as the

average Epa and Epc ), E0’ is -0.415. Cyclic voltammetric responses validate that

nanocomposite provides a favorable microenvironment which preserves the

biofunctionality of enzymes. Hence, the nanocomposite could improve direct

electron transfer between enzymes and the underlying electrode surface.

Figure 7.5: Cyclic voltammograms at different modified electrodes in

potassium phosphate buffer (0.1 M, pH 7.0) with scan rate of 0.1 V s-1

(a)

Fe3O4@Ag NPs/ITO, (b) Silica/DNA-Fe3O4@Ag/ITO, and (c) Silica/DNA-

Fe3O4@Ag/ChOx/HRP/ITO

Figure 7.6 shows cyclic voltammograms of Silica/DNA-

Fe3O4@Ag/ChOx/HRP/ITO in potassium phosphate buffer (0.1 M, pH 7.0)

Chapter 7


with varying scan rate in the range of 0.05 to 0.3 V s-1

(from a to i). The

cathodic and anodic peak current increases linearly with increase in scan rate

indicating a typical surface-controlled electrode process.

Figure 7.6: Cyclic voltammograms of Silica/DNA-Fe3O4@Ag/ChOx/HRP/

ITO in potassium phosphate buffer (0.1 M, pH 7.0) with varying scan rate

of 0.05 to 0.3 V s-1

(a-i)

The surface concentration of Silica/DNA-Fe3O4@Ag/HRP/ChOx/ITO

estimated from the plot of current versus potential using Brown-Anson model

based on the following equation [22],

( )

where n is the number of electrons transferred, F is the Faraday constant

(96,584 C mol−1

), I* is the surface concentration (mol cm2) obtained for the

Silica/DNA-Fe3O4@Ag/HRP/ChOx/ITO electrode film, A is the surface area

of the electrode (1.0 cm2), V is the scan rate (100 mV s

−1), R is the gas constant

(8.314 J−1

mol K), and T is the absolute temperature (298 K). The value of the

Chapter 7


surface concentration of the modified electrode has been found to be

10.709×10−6

mol cm2.

The electro-catalytic properties towards cholesterol were explored on

the Silica/DNA-Fe3O4@Ag/HRP/ChOx/ITO electrode. Figure 7.7 displays

cyclic voltammograms recorded for different concentration cholesterol. The

successive addition of cholesterol resulted in an increase in peak currents

corresponds to cholesterol oxidation. The increase of reduction peak current

was observed, accompanied by the decrease of the oxidation peak current with

the increasing cholesterol concentration. Thus, significant enhancement of the

peak current provides a clear evidence of excellent electro-catalytic activity of

Silica/DNA-Fe3O4@Ag/HRP/ChOx/ITO electrode towards cholesterol.

Figure 7.7: Cyclic voltammograms of Silica/DNA-Fe3O4@Ag/ChOx/HRP/

ITO modified electrode in potassium phosphate buffer (0.1 M, pH 7.0)

containing different concentrations of cholesterol from a to f at 0.1 V s-1

Chapter 7


II) Analytical performance of cholesterol nanobiosensor

A. Sensitivity

After the successive addition of cholesterol, rapid and prominent

increase in the reduction peak current was observed, the corresponding

calibration curve of cholesterol nanobiosensor is shown in Figure 7.8. The

catalytic reduction peak current increases with the linear calibration equation y

= 0.009 x + 0.7346 [mg dL-1

] (R2 = 0.9904) in the wide concentration range

from 5.0 to 195 mg dL-1

of cholesterol. The sensitivity of cholesterol

nanobiosensor is 0.009 µA [mg dL-1

] -1

with detection limits 5.0 mg dL-1

.

Figure 7.8: Calibration curve of cholesterol nanobiosensor (catalytic peak

current vs. cholesterol concentration in mg mL-1

).

B. Reproducibility, selectivity and stability

RSD of Silica/DNA-Fe3O4@Ag/HRP/ChOx/ITO modified electrode

response to 15 mg dL-1

cholesterol was within 3.3 % for six consecutive

measurements, indicating that the nanobiosensor had good reproducibility. The

Chapter 7


selectivity of proposed cholesterol nanobiosensor was evaluated by the

interference study using 1:1 solution of cholesterol and interference substances.

It was found that interfering substances glucose and ascorbic acid and

acetaminophen did not interfere significantly with the resulting biosensor,

indicating that biosensor has sufficient selectivity.

Silica/DNA-Fe3O4@Ag nanocomposite had remarkable compatibility

with enzymes as well as good conductivity, so it exhibited excellent stability.

The stability of the nanobiosensor was examined stored for 5 weeks at 4 °C.

The current response decreased by about 14 %. It revealed that the developed

biosensor possessed good stability.

Chapter 7


7.5. Conclusion A new Silica-DNA/Fe3O4@Ag has been successfully synthesized for the

immobilization of redox enzyme to detect free cholesterol. Noteworthy, the

method for the preparation of Silica-DNA/Ag@Fe3O4 deals with some

promising potential applications in nano-catalysis and nano-electronics. The

high sensitivity of nanobiosensor is attributed to the large surface area of

Fe3O4@Ag NPs for effective loading of enzymes as well its high electron

communication capability with the aid of enhanced selectivity and anti-

interference ability due to the Silica sol.

Chapter 7


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