FIELD OF THE INVENTION
[0001]The present invention relates to constructing a novel biosensor, which is a Laponite® RDCloisite-Na (MMT) cogel based non-enzymatic cholesterol sensor implanted with hydrogen ion and to study their sensing behavior under the influence of low energy hydrogen ion beam. It uses Laponite RD ® and Cloisite-Na (MMT) mixed clay hydrogels with electrodes prepared on indium tin oxide strips at room temperature (250C) irradiated with hydrogen ion beam. The electrode generates a measurable current directly proportional to the concentration of cholesterol sensor present in the sample.
BACKGROUND OF THE INVENTION
[0002]Ion beam irradiation has now become one of the interesting but routine techniques for processing as well as surface modification of systems comprising of bulk material, thin films and nanostructures. Ion beam irradiation is known to be a technique used in various research fields including bioengineering, radioactive breeding, tumour therapy etc.(Yu et al 2006;Forrest et al 1982; Schulz-Ertner et al 2006; Fujinami et al1998; Ruoff et al 1988; Bodo et al 1986).The use of radiation in polymer surface/thin films, where the ion beam interacts with the material and results in the modification of their physico-chemical properties, such as optical absorption, electrical conductivity, thermal stability etc. is increasing rapidly(Islam et al 2014; Loo et al 2005; Yoshihisa et al 2012).
[0003] Ion irradiation may result in severe damage, or enhancement of stability and/or conductivity of polymer thin films depending on the mass, energy and density of the interacting ions. Thus, its use can be tailored (used as tuning agent) to suit material processing. Lucca et al 2007 have studied the effect of ion irradiation on TEOS(tetraethylorthosilicate)Si(OC2H5)4/MTES(methyltriethoxysilane)CH3Si(OC2H5)3thin film resulting in the structural transformation of the films which led to increase in elastic modulus, and hardness of the film. A study of surface modification of Poly
3
(lactic-co-glycolic) acid due to irradiation was done using argon ion irradiation by Adhikari et al 2014 and structural changes were confirmed using X-ray photoelectron and atomic force spectroscopy. Ion bombardment promotes the formation of unsaturated bonds or the breakage of polymeric chains or emission of fragments that may be atomic or/and molecular (Lee et al 1996.).
[0004] The studies of nano-biosensors for the determination of concentration of analytes (glucose, cholesterol, ascorbic acid and oxalic acid etc.) which are of clinical interest have received considerable attention recently (White et al 1978).Cholesterol is known to be one of the main metabolites of human body being an active component of nerve and brain cells (Myant et al 1990).Cholesterol is one of the analytes which is determined in clinical pathology with its concentration being indeed indicatively related to human health. The determination of cholesterol is important for diagnosis and prevention of a number of clinical disorders like hypertension, coronary heart disease etc. Thus, it is of interest to both the biological science and food industries. The content of cholesterol in normal human blood serum is less than 200 mg/dl of which 70% is esterified with fatty acids and 30% is present as sterol (Li et al 2005).In literature, there have been a lot of attempts to develop cholesterol biosensors using enzyme Cholesterol Oxidase as well as without enzymes which is listed in Table 1.
Table 1: Comparison of different Cholesterol (ChOx)-based sensors. S.No. Electrode Enzyme used Linear dynamic range Sensitivity Limit of detection References 1 SiO2/CHIT/ MWCNT /ITO ChEt/ ChOx 10-500 mg/dl 3.84 μA/ mM - Solanki et al., 2009 2 (p(HEMA))/(polypyrrole) ChOx 5x10-4-1.5x10-2 M 6.63 nA/mM 120 μM Brahim et al 2001 3 Polypyrrole (PPY) ChEt/ ChOx 1 – 8 mM 0.15 μA/mM - Singh et al 2004
4
[0005] Nanoclays such as Na Montmorillonite (Cloisite Na) MMT are materials naturally occurring in the clay fraction of soil. In contrast, Laponite® RD is synthetic clay developed by Laporte Industries. These clays have found wide agricultural, industrial and mechanical applications. Thus, Laponite with a face diameter of ~30 nm and MMT with diameter of~ 300nm, and rim width of 1 nm each are known to be rheology modifier and have various applications in agriculture, building materials, surface coatings, household and personal care items (Hunter 1989). Nanoclay has the advantageous properties of high chemical stability, good absorption and penetrability due to its large surface area, and thus, its use in sensing can be promising. M. Seleci et al.2012 have developed a clay-based biosensor where they used MMT modified with methyl and dimethylamine to analyse glucose in wine.
[0006] In the present invention, the objective was to study the influence of low energy (20 keV) H2+ion beam of different fluence on L-MMT/ITO electrode resulting in the alteration in the properties (both morphological and electrochemical) of electrodes for detection of cholesterol. The effect of ion induced modification in the sensitivity of the thin films was also explored. In the present invention, the inventors have shown that the variation in fluence of molecular hydrogen ions can provide conditioning of the sensitivity of nanoclay-based electrodes towards the enhanced sensing of cholesterol at room temperature on an enzyme-free platform.
4 Ag nano-particle /GCE None 2.8x10-4- 3.3x10-2 M - 1.8x10-4 M Li et al 2010 5 CeO2-graphene None 12 μM - 7.2 mM - 4 μM Zhang et al 2013 6 PtNP/(CNT) None 0.005 – 10 mM 8.7μA/mM/cm2 - Yang et al2012 7 CSNF-AUNPs ChOx 1 – 45 μM 1.02 μA/mM - Gomathi et al 2011 8 ZnO nanoparticle ChOx 1 - 500 nM 2.3 μA/mM/cm2 0.37 nM Umar et al 2009 9 Irradiated L-MMT None 1 – 20 mM 8.08 μA/mM/cm2 1mM This invention
5
SUMMARY OF THE INVENTION
[0007] The present invention relates to constructing a novel biosensor, which is a Laponite® RD Cloisite-Na (MMT) cogel based non-enzymatic cholesterol sensor implanted with hydrogen ion and to study their sensing behavior under the influence of low energy hydrogen ion beam. It uses Laponite RD ® and Cloisite-Na (MMT) mixed clay hydrogels with electrodes prepared on indium tin oxide strips at room temperature (25 0C) irradiated with hydrogen ion beam. The electrochemical characterization of the L-MMT/ITO electrode is done using cyclic voltammetry (CV), scanning electron microscope (SEM), UV-Vis spectroscopy and Fourier Transform Infrared Spectroscopy (FTIR).The electrode generates a measurable current directly proportional to the concentration of cholesterol sensor present in the sample.
[0008] The present invention also includes the exponent n lies between 0 and 2, with n=0 corresponding to Hookean solid, and n=2 representing a Maxwellian viscoelastic matter. The hydrogen molecular ion (H2+) irradiation on the successfully fabricated L-MMT/ITO electrode, which was used for sensing of cholesterol. The present invention describes a follow-up study of the inventors previous work where it was found electrocatalytic behaviour of L-MMT/ITO electrode towards the detection of Cholesterol (Joshi et al 2015).
[0009] The objective of the present invention is to study the influence of low energy (20 keV) H2+ion beam of different fluence on L-MMT/ITO electrode resulting in the alteration in the properties (both morphological and electrochemical) of electrodes for detection of cholesterol. The effect of ion induced modification in the sensitivity of the thin films was also explored. In the present invention, the inventors have shown that the variation in fluence of molecular hydrogen ions can provide conditioning of the sensitivity of nanoclay-based electrodes towards the enhanced sensing of cholesterol at room temperature on an enzyme-free platform.
6
[0010] The present invention also relates to the sensing mechanism used for L-MMT toward cholesterol detection was based on ferro-ferri cyanide redox electrochemical reaction. Irradiation of ion beam produced surface modification of these electrodes and enhancement of sensitivity was noticed for low fluence (upto 1013 ions/cm2) towards cholesterol due to the breakage of hydrogen bonds. Irradiation by high fluence ions (above 1013 ions/cm2) caused the degradation of the electrode surface and resulted in offering lesser sensitivity due to the recombination of hydrogen atom. A mechanism is hypothesized for the electrode that low fluence irradiation induced certain active sites by breaking hydrogen bonds and these liberated hydrogen atoms were responsible for controlling (enhancing) the electron transfer kinetics. From this, it was further concluded that the fluence provides the tunability of the implantation induced sensitivity of the L-MMT/ITO electrodes.
[0011] In addition to this, low fluence irradiations from hydrogen molecular ion (H2+) beam on L-MMT electrode were found to be successful for enhancing electrochemical properties of the electrode. In general, such electrodes could be used for detection of cholesterol under room temperature conditions. This sensing platform can be developed to design cheap enzyme-free strip sensor for mass production and day to day use.
BRIEF DESCRIPTION OF THE FIGURES
[0012] Figure1.(a) Cyclic voltammogram of irradiated (1014 ions/cm2) L-MMT/ITO for different scan rates varying from 10 to 100 mV/s, and (b) anodic (Ia) and cathodic (Ic) current as a function of scan rate for as deposited L-MMT without and with irradiated (1014ions/cm2) L-MMT/ITO electrode. Note the better linearity of the irradiated sample.
7
[0013] Figure 2. Anodic and cathodic peak current as a function of ion fluences of H2+ ion beam in the absence and presence of cholesterol.
[0014] Figure 3.Variation of (a) anodic (Ia) and (b) cathodic (Ic) peak current with the concentration of cholesterol in the range of 1–20 mM shown for different ion beam fluence.
[0015] Figure 4. Sensitivity of L-MMT/ITO electrode for different irradiated electrode was shown, where the maximum sensitivity was noted for the H2+ion beam at the fluence of 1013 ions/cm2.
[0016] Figure 5. Nyquist plot of Electrochemical Impedance Spectroscopy for (a) irradiated L-MMT (1014 ions/cm2) with different concentration of cholesterol and (b) irradiated L-MMT electrode for different fluence.
[0017] Figure 6.SEM images for L-MMT/Chox/ITO irradiated with different fluence of hydrogen molecular ion beam. Figure depicts the (a) irradiated L-MMT/ITO thin film of ion fluence 5x1012 ions/cm2without cholesterol (b) irradiated with H2+ ion beam of fluence 1x1012(c) 5x1012 (d) 5x1013 (e)5x1014 and (f) 5x1015 ions/cm2for L-MMT/Chox/ITO electrode.
[0018] Figure 7. X-ray diffraction (XRD) spectra of the non-irradiated and irradiated samples.
[0019] Figure 8. FTIR spectroscopic data for L-MMT/Cholesterol/ITO irradiated with (a)low fluence and (b) high fluence of ion beam.
[0020] Figure 9. Schematic representation for fabrication method of L-MMT irradiated with H2+ ion beam for sensing cholesterol.
8
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention relates to constructing a novel biosensor, which is a Laponite® RD Cloisite-Na (MMT) cogel based non-enzymatic cholesterol sensor implanted with hydrogen ionand to study their sensing behavior under the influence of low energy hydrogen ion beam. It uses Laponite RD ® and Cloisite-Na (MMT) mixed clay hydrogels with electrodes prepared on indium tin oxide strips at room temperature (25 0C) irradiated with hydrogen ion beam.
[0022] The present invention includes constructing of cholesterol sensor using cogel of mixed nanoclay Laponite® RD and Cloisite-Na Montmorillonite(MMT), film deposited on indium tin oxide (ITO) glass electrode at room temperature (25 °C) irradiated with hydrogen ion beam.The biosensor electrode fabricated through this method was used for the determination of cholesterol present in a solution. The electrochemical characterization of the L-MMT/ITO electrode is done using cyclic voltammetry (CV), scanning electron microscope (SEM), UV-Vis spectroscopy and Fourier Transform Infrared Spectroscopy (FTIR).The electrode generates a measurable current directly proportional to the concentration of cholesterol sensor present in the sample.
[0023] Another embodiment of the present invention is to study the influence of low energy (20 keV) H2+ion beam of different fluence on L-MMT/ITO electrode resulting in the alteration in the properties (both morphological and electrochemical) of electrodes for detection of cholesterol.
[0024] In another embodiment of the present invention the effect of ion induced modification in the sensitivity of the thin films was also explored.
[0025] In one of the embodiment of the present invention, the inventors have shown that the variation in fluence of molecular hydrogen ions can provide conditioning of
9
the sensitivity of nanoclay-based electrodes towards the enhanced sensing of cholesterol at room temperature on an enzyme-free platform.
[0026] In another embodiment of the present invention the sensing mechanism used for L-MMT toward cholesterol detection was based on ferro-ferri cyanide redox electrochemical reaction. Irradiation of ion beam produced surface modification of these electrodes and enhancement of sensitivity was noticed for low fluence (upto 1013 ions/cm2) towards cholesterol due to the breakage of hydrogen bonds. Irradiation by high fluence ions (above 1013 ions/cm2) caused the degradation of the electrode surface and resulted in offering lesser sensitivity due to the recombination of hydrogen atom. A mechanism is hypothesized for the electrode that low fluence irradiation induced certain active sites by breaking hydrogen bonds and these liberated hydrogen atoms were responsible for controlling (enhancing) the electron transfer kinetics. From this, it was further concluded that the fluence provides the tunability of the implantation induced sensitivity of the L-MMT/ITO electrodes.
[0027] In addition to this, low fluence irradiations from hydrogen molecular ion (H2+) beam on L-MMT electrode were found to be successful for enhancing electrochemical properties of the electrode.
[0028] In general, such electrodes could be used for detection of cholesterol under room temperature conditions. This sensing platform can be developed to design cheap enzyme-free strip sensor for mass production and day to day use.
Construction of Cholesterol Biosensor
[0029] Laponite RD ® and Cloisite-Na (MMT) were purchased through Southern Clay Products, U.S. The individual dispersion of Laponite and MMT were prepared by dissolving 1.25% (w/v) in deionized water for 2 h and 12h, respectively. After their preparation, both the dispersions were mixed in 1:1 ratio and stirred vigorously for half
10
an hour. As the homogeneous dispersions were prepared, 40μl of the dispersion were drop casted onto ITO electrode and kept for drying for≈ 2 days. The final thickness of the drop-cast nanoclay L-MMT on ITO was of approximately 200-300 nm. After the preparation of electrode, these were irradiated with 20 keV hydrogen molecular ion (H2+) beam having different fluences (1012 – 1016 ions/cm2), and then used for further analysis. The pH of the prepared L-MMT solution was found to be 9.00.5.
[0030] The electrochemical studies (cyclic voltammetry and electrochemical impedance spectroscopy) were done on Auto lab Potentiostat/Galvanostat (Eco Chemie, Netherlands) in a three-electrode cell dipped in 3.3mM ferric and ferrous solution containing KCl. The three-electrode cell consisted of working electrode that was L-MMT/ITO, auxiliary electrode which was a platinum wire, and reference electrode that was Ag/AgCl.
[0031] Fourier transform infrared (FTIR) spectroscopic studies were done on PerkinElmer, Spectrum BX II instrument.
[0032] X-Ray diffraction (XRD) measurements were carried out on XRD, RigakuD/Max 2200 diffractometer with Cu- Kαradiation with wavelength of 1.54 Å in the 2θ range of 3° - 30°.
[0033] Scanning electron microscope (SEM) imaging was carried out using SEM, Zeiss, EVO-40instrument.
[0034] Ion beam irradiations on L-MMT samples were done using Table Top 50 kV Ion accelerator which is one of the low energy ion beam facility (LEIBF) available at IUAC (Kumar et al 2013).
11
[0035] The most optimized methodology for the present invention is explained in the form of examples below. The present invention is, however, not limited to these examples in any manner. The following examples are intended to illustrate the working of disclosure and not intended to take restrictively to apply any limitations on the scope of the present invention. Those persons skilled in the art will understand that the equivalent substitutes to the specific substances described herein, or the corresponding improvements in the process are considered to be within the scope of this invention.
Detailed Methodology and Experimental Data and Results
Example 1
[0036] Electrochemical Response: Cyclic Voltammetry (CV) is known to be a common technique which provides information about redox potentials of the sample. The cyclic voltammetry studies of the electrodes before and after irradiation with hydrogen molecular ion (H2+) beam using 1012,5x1012, 1013, 5x1013, 1014,5x1014, 1015,5x1015and 1016ions per cm2fluenceswere carried out using Zobell’s solution {3.3mM K4Fe(CN)6 (Potassium Ferrocyanide), 3.3mM K3Fe(CN)6 (Potassium Ferricyanide) containing 0.1 M KCl (Potassium Chloride)}at pH 7. The cyclic voltammogram were recorded in the potential range of -0.1 to 0.4 V. The various tuning parameters such as scan rate, pH, concentration etc. were optimized in an earlier work (Joshi et al 2015).Figure 1(a) shows the cyclic voltammogram of L-MMT electrode with, and without irradiation (1014ion/cm2) at different scan rates varying from 10 to100 mVs-1.The electrochemical profiles of all the L-MMT thin films (with and without irradiation) showed qualitatively same behavior with the oxidation and reduction process occurring at same potential of 0.22 V and 0.12V.Figure 1 (b) shows the scan rate dependence of peak current where both the anodic and cathodic current showed increasing behaviour with square root of scan rate. Thus, for both with and without irradiated samples, the anodic (Ia) and cathodic (Ic) current increased linearly with square root of scan rate with regression coefficient value (R2) of 0.98 and 0.96 for irradiated (1014 ions/cm2) and non-irradiated electrodes indicating that the electron
12
transfer process was a diffusion controlled process (Mabbott 1983).The same result
was found for all the other fluences.
[0037] Further electrochemical studies on all the samples were done in ferri/ferro
solution at pH 7, and at the scan rate of50 mVs-1. The effect of irradiation is shown in
Figure 2 where the electrochemical response of electrode irradiated with variant
fluences was compared. For low fluence H2
+ ion beam (≤ 1013ion/cm2), the increase in
magnitude of anodic and cathodic peak current were observed compared to L-MMT
electrode. However, the decrease in anodic peak current for high fluence H2
+ ion beam
(>1013ions/cm2) compared to L-MMT/ITO electrode was clearly noticed. The
hydrogen molecular (H2
+)ion beam irradiation to L-MMT/ITO electrode resulted in the
incorporation of these ions from the beam and thus, resulted in enhancement of the
anodic and cathodic currents. Both the current showed increasing behavior upto 1013
ions/cm2fluences while for higher fluences, it showed decreasing trend. This figure also
shows that in the absence of cholesterol the fluence of hydrogen molecular ion beam
had very negligible effect on the current value while noticeable changes were observed
in the presence of cholesterol. Thus, the irradiated electrode, with low fluence, provided
favourable environment for the electron transfer process in the electrode/electrolyte
interface for the cholesterol, but at higher fluence, the samples provided hindrance to
the electron transfer process due to excessive damage to the electrode surface.
[0038] At low fluence of irradiation, sufficient accessibility to electrons between
electrolyte and electrodes is facilitated. Further, we proceed to calculate diffusion
coefficient value from ferri/ferro electrolyte to different electrodes using the Randles-
Sevcik (Bard et al 1980) equation given by
3 1 1
(2.69 105 ) 2 2 2 p I   n AD CV (1)
where A is surface area of electrode which is 0.25 cm2, D is the diffusion coefficient,
n is the number of electron involved or electron stoichiometry which is 1, Ip is the peak
13
current, V is the scan rate (50 mVs-1) and C is the concentration of ferri/ferro solution
which is 3.3 mM.
[0039] The electro-active surface area of electrode was determined from the calculated
diffusion coefficient (D) and the Randles-Sevcik(Bard et al 1980) equation given by
3 1
(2.99 105 ) 2 2
c
S
A
n CD


(2)
where S is calculated through slope of peak current (Ip) versus square root of scan rate.
[0027] Also, the value of the electron transfer rate constant (Ks) for different electrodes
were determined using eqn (2) which was based on model of Laviron1979 given by
s
mnFV
K
RT

(3)
where m is the peak to peak separation, F is the Faraday constant which is 96485 Cmol-
1 and R is the universal gas constant.
[0040] The surface concentration (I*) of ionic species of the corresponding electrode
were calculated through Brown-Anson model (Bard et al 1980)
2 2 *
4 p
n F I AV
I
RT
 (4)
[0041] The diffusion coefficient value of redox species of bare (not irradiated) L-MMT
electrode was found to be 0.306×10-12cm2s-1 while irradiated L-MMT electrode has
diffusion coefficient as 0.890×10-12 cm2s-1.Therefore, irradiation led to increase in
electron diffusion coefficient of the electrode. The charge transfer rate constant and
average surface concentration for non-irradiated L-MMT electrode was found to be
0.194 s-1and 0.07×10-8 mol cm-2.
14
[0042] An increase in anodic current was observed with cholesterol concentration in the range of 1–20 mM (Figure 3). The magnitude of the oxidation peak (Ia) increased due to the increasing concentration of cholesterol. The maximum increase in magnitude of anodic and cathodic peak current with cholesterol was found for lower fluences of ion beam which revealed faster electron transfer process between electrode and electrolyte.
[0043] Increase in current, with increase in cholesterol concentration, showed the presence of interaction of cholesterol with L-MMT electrode (Zong et al 2007). Thus, addition of cholesterol in the electrolyte provided an easy conducting path for the electron to transfer from electrode to electrolyte or vice-versa for low fluence irradiated electrode.
[0044] The sensitivity which is defined as output of the sensor per unit input, of the L-MMT electrode toward cholesterol was calculated from the slope of the current versus cholesterol concentration plot. The sensitivity for electrode irradiated with different fluence is shown in Figure 4 where a clear enhancement of sensitivity for electrode which was irradiated with low fluence (≤ 1013 ions/cm2) was noticed. In addition to this, loss of sensitivity was found for higher fluence (>1013 ions/cm2) irradiated electrodes.
[0045] A well-known phenomenon which was observed in ion irradiation for various Si based polymer thin films is the loss of hydrogen which was due to breakage of hydrogen bonds (Cui et al 2001; Gelamo et al 2007; Rangel et al 2000; Baptista et al 2004).Cui et al 2001 studied the changes in protein secondary structure by low energy Nitrogen ion beam irradiation due to the scission of H-bonds and charge exchange. Polymer films synthesized from plasmas of a tetra methylsilane-Ar mixture using Helium ion beam irradiation showed loss of hydrogen was reported by Gelamo et al 2007. Rangel et al 2000 investigate the effect of argon ion irradiation on polymer film
15
where significant loss of hydrogen besides the formation of unsaturated bonds, and cross-links with decrease in electrical resistivity was found. Also the loss of hydrocarbon and the associated release of hydrogen is one of the consequences of the ion irradiation of polymer and polymer-like structures studied by Baptista et al 2004. Here, such a mechanism is hypothesized for the L-MMT colloidal thin film that the enhancement in sensitivity of the irradiated electrode could be due to the breakage of hydrogen bond induced by the as deposited L-MMT/ITO electrode. These liberated hydrogen atoms can diffuse out of the matrix resulting in the formation of active sites or defects which may result in increase in electron transfer (redox) process. The high fluence of ion beam result in the recombination of these liberated hydrogen atoms, and may block the active sites which were earlier responsible for the electron transfer kinetics in low fluence irradiated electrode. Thus, high fluence provided the hindrance (blockage) to the transfer process, and resulted in reduction of the sensitivity of the L-MMT electrode towards cholesterol due to physical degradation or damage to the current flow. The observation of enhancement of sensitivity by irradiation of hydrogen (H2+) molecular ion to L-MMT electrode clearly exhibited dependence on the fluence. From the CV analysis, low fluence irradiated electrode showed enhancement in the electron transfer process, and thus, helped in increasing the sensitivity towards this specific analyte (cholesterol). This is clearly depicted in Figure 4.
Example 2
[0046] Electrochemical Impedance Spectroscopy (EIS): Now, ElectrochemicalImpedance spectroscopy (EIS) study was done on the electrode in the frequency range of 0.01-106Hz so as to probe the surface of modified electrode. The Nyquist plot for electrochemical impedance (Z” vs Z’) of L-MMT electrode is shown in the Figure 5 and it provided information about the material conductivity, and dielectric constant.
16
[0047] The impedance data were analysed using Randle’s equivalent circuit results from the parallel combination of electron transfer resistance (RCT) and double layer capacitance (Cdl) from electrode impedance. The semicircle in the Nyquist plot corresponds to the electron transfer limited process. The electron transfer resistance (RCT) value was calculated from the diameter of the semicircle which defined the electron transfer process of the redox probe. The decrease in diameter of semicircle with the addition of cholesterol showed the low resistivity value and thus the addition of cholesterol enhanced the electron transfer process (conducting) and provided better environment for cholesterol to interact with L-MMT (Figure 5(a)).
[0048] A depressed semicircle for irradiated L-MMT in the presence of cholesterol having small diameter reflects the faster electron transfer process from electrode to electrolyte and vice versa as compared to the large semicircle which reflects the greater hindrance to the transfer process. The diameter of the semicircle of impedance spectra in Figure 5(b) provides the value of electron transfer resistance (RCT) known to be an index of the catalytic property of the electrode surface which varies from 319 KΩ to 38 KΩ for low fluences (≤1013 ions/cm2) while increases from 38 KΩ to 426 KΩ for high fluences (>1013 ions/cm2). This decrease in RCT value shows the enhanced electron transfer pathway from electrode to electrolyte and vice-versa. The ion beam irradiation of L-MMT/ITO electrode led to dielectric behaviour for electron transfer process. The high fluence ion beam irradiation resulted in the creation of a barrier (blockage due to recombination) so that electron transfer process couldn’t occur easily as observed through the Nyquist plot (Figure 5(b)). The decrease in RCT value upon addition of cholesterol to the electrolyte solution can be related to surface effects and it’s binding with the irradiated electrode. Thus, irradiation with low fluence results in improved charge transfer due to breakage of hydrogen bonds resulting in formation of active sites which enhanced diffusion of Ferri/Ferrocyanide ions towards the electrode surface and vice-versa. Hence, consistent results were obtained when the electrode was studied
17
using electrochemical techniques (Cyclic Voltammety and Electrochemical Impedance Spectroscopy).
Example 3
[0049] Surface Morphology: Ion irradiation can lead to the alteration in the surface morphology, which in turn may contribute to the changes in the electrochemical properties of the L-MMT electrode. The surface morphology of irradiated L-MMT electrode was studied by Scanning electron microscope (SEM), which shows huge irregularly flakes of cholesterol distributed on the surface of irradiated L-MMT(Figure 6b). These images show that with increase in ion fluence of ion beam, there is an increment of coverage area through flakes of cholesterol on the surface. For low fluence (≤ 1013 ions/cm2) the ion implantation result in breakage of hydrogen bond and formed the sites which were responsible for interaction of cholesterol. While for higher ion fluence (>1013 ions/cm2) the further recombination of hydrogen atoms takes place and thus blocked the sites for cholesterol. This result in hindrance for cholesterol to interact with high fluence irradiated L-MMT electrode and thus self-assembled irregular flakes of cholesterol were found on the whole surface. The images of irradiated electrode for high fluence in the presence of cholesterol showed rod like structures (huge irregular flakes) which were formed due to the linear self-assembly of the analyte (cholesterol). Therefore, the best change in surface morphology was found for low ion fluence which implied that incoming cholesterol was successfully aligned on the surface of pre-irradiated L-MMT electrode.
[0050] It was concluded that irradiation of the L-MMT electrode results in the alteration in surface morphology as observed from the SEM images and this provided the enhanced sensitivity for detection of cholesterol in case of low fluence of ion beam.
Example 4
[0051] X-Ray Diffraction: X-ray diffraction techniques (XRD) were used to investigate structural properties. Figure 7 shows the XRD patterns of the samples
18
without and with irradiation of 20 KeV H2
+ ion beam at the fluence of 1012 ions/cm2
and 1014 ions/cm2. It was found that the broadness of the irradiated electrodes increases
compared to non-irradiated one due to the destruction of crystallinity. The size of the
crystallite (L) can be obtained from equation used in Scherrer1918which is given as
cos
K
L
b



(5)
Where b is full width at half maxima (FWHM), λ is the wavelength of X-ray beam
(1.54 Å) and K is a Scherrer constant which depends upon the crystallise shape (=0.93).
The values obtained through these are given in Table 2.
Table 2: XRD parameters for the samples
Fluence
(ions/cm2)
2θ (°) FWHM (°) Crystallite size
(Å)
0 7.46 0.0156 92.30
1x1012 7.37 0.0189 76.44
1x1014 7.36 0.0206 70.09
[0052] The crystallite size was found to decrease with increase in ion fluence of H2
+
irradiated beam. Thus, Irradiation of L-MMT thin film with hydrogen molecular ion
beam causes 20% and 31% of decrease in crystallite size for low and high fluence of
1012ions/cm2 and 1014 ions/cm2. XRD measurements thus provide the information of
loss of crystallinity due to irradiation.
Example 5
[0053] FTIR: FTIR spectroscopy studies were used to quantify the chemical and
structural transformation induced by ion irradiation. The FTIR spectrum for irradiated
L-MMT thin films in the spectral range of 500-4000 cm-1 is shown in Figures 8(a) and
8(b). The bombardment of hydrogen molecular (H2
+) ion beam on the L-MMT
electrode may result in the alteration of their structure.
19
[0054] The cholesterol consists of C-H bond, O-H bond, C-O bond, C=C bond, and C-C bond. The obtained FTIR spectra for pure cholesterol shows peak at 680 and 790,1050 and 1236,1380, 1471, and, 859, 949 and 1037 cm-1 corresponding to the bonds C-H, O-H (of hydroxyl group), C-O (between carbon and hydroxyl), C=C (unsaturated carbon carbon)and C-C respectively (Silverstein et al 1991). Besides this peak at 2869 and 2994 cm-1corresponds to C-H bonds having asymmetric mode of stretching.
[0055] For L-MMT nanoclay, the spectrum shows a broad band at 3200-3460 cm-1 attributed to OH stretching due to presence of water. The broad bending band at 1042 cm-1 and the stretching band at 837 cm-1 attributes to the presence of -Si-O-Si- known to be siloxane groups. The characteristic of an asymmetric stretching vibration of Si-O-Si was found from the occurrence of these peaks. The bands which are located at 582cm-1 and 730 cm-1was associated with the stretching vibration of Si-OH or Si-O-bonds. Bands at around 3200-3460 cm-1 and 1537 cm-1 were associated with the stretching and deformation vibrations of the interlayer H2O of nanoclay. The small band at 2827cm-1was due to the asymmetrical stretching vibration of C-H in CH2 and CH3 and this presence of alkyl chain in the surface of electrode was due to the cholesterol which was drop-cast over the L-MMT electrode for observing the interactions.
[0056] The effect of irradiation on the L-MMT thin films can be noted by observing the gradual disappearance or reduction or shifting of various vibration bands. As it can be seen in Figure 8(a), after the L-MMT electrodes were irradiated with H2+ ion beam of low fluencies the band around 1537 cm-1 was shifted to 1637 cm-1 which suggest that the irradiation result in increment in interlayer H2O deformation due to the breakage of hydrogen bond.
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[0057] The broadness of the band at 1637 cm-1 was found to increase for low fluencies due to the increase in interaction and bonding with the hydroxyl group of cholesterol while it decreased for higher fluences. In addition to this, there were occurrence of sharp peaks correspond to cholesterol (1240, 1360, 1446 cm-1) in Figure 8(a) which occurs for low fluence in region 1200–1600 cm-1confirming the interaction of L-MMT with the cholesterol due to irradiating ions which lead to breakage of hydrogen bonds and this enhanced the electrochemical properties of the electrode. However, high fluence results in disappearance (collapse) of these peaks due to recombination of hydrogen atom resulting in less interaction or sensing of electrode towards cholesterol detection (Figure 8(b)). These peaks arises due to interaction of O-H bending and C-O stretching and C=C vibrations. The disappearance of various peaks in region 1600–2800 cm-1 shows the less binding (interaction) of cholesterol with L-MMT for high fluence as compared to the low fluence case. For high fluence, the diminished of peak 1240cm-1 and shifting of 1360 cm-1 and 1445 cm-1 shows the different behaviour of irradiated electrode with cholesterol. Thus, these results in the peak variation support the better interaction of irradiated electrode of low fluence with cholesterol.
[0058] The decrease in conductivity for irradiated samples (higher fluence) was due to loss of hydrogen due to recombination process or formation of (any) unsaturated bonds. For high fluence of ion beam the samples are sensitive to ion radiation and were physically damaged to certain extent resulting in the decrease in sensitivity for detection of cholesterol. The fabrication and sensing mechanism for irradiated L-MMT/ITO for sensing cholesterol is depicted in Figure 9 which shows that the electrode were fabricated through drop casting the L-MMT solution onto ITO and kept it drying (~ 2days). The prepared L-MMT electrodes were irradiated through 20 keV hydrogen molecular ion beam using Table Top Ion accelerator available at IUAC. The hydrogen ions through ion beam were incorporated into the L-MMT network formed and depending on the ion beam fluence the irradiated electrodes provides suitable electroactive surface area for electron conduction between the electrons through
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Ferri/Ferro electrolyte and electrode. The prepared irradiated electrode was then submerged in the Zobell’s solution with other electrodes present in the electrochemical cell whose instrumentation has been discussed earlier and the effect of ion beam fluence was observed for sensing cholesterol. The addition of cholesterol generates the electron during its oxidation and passes it to irradiated L-MMT electrode through the pathway provided by Zobell’s solution electrolyte present in the electrochemical cell. Due to the active sites provided by the hydrogen ion beam for electron transfer, the addition of cholesterol (Chox) enhances the diffusion of Ferri/Ferrocyanide ions towards the electrode surface from the electrolyte or vice-versa. These irradiated L-MMT electrode provides a good platform for electron transfer kinetics between cholesterol and electrode depending on the fluence of ion beam. Figure9represents the graphic representation of irradiated L-MMT/ITO electrode for sensing cholesterol.
INDUSTRIAL APPLICABILITY OF THE INVENTION
[0059] Cholesterol is known to be one of the main metabolites of human body being an active component of nerve and brain cells. Cholesterol is one of the analytes which is determined in clinical pathology with its concentration being indeed indicatively related to human health. The determination of cholesterol is important for diagnosis and prevention of a number of clinical disorders like hypertension, coronary heart disease etc. Thus, it is of interest to both the biological science and food industries.Therefore, the present invention provides the biosensor showing maximum sensitivity for cholesteroland also towards different analytes. This electrochemical strip-based analyte sensor that does not use any enzyme presented good characteristics in terms of stability, and reproducibility offering promise of applicability of this green sensor platform.
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We Claim:
1. A novel hydrogen ion implanted enzyme-free electrochemical biosensor, which is a mixed clay hydrogel based non-enzymatic cholesterol sensor used for studying sensing behavior towards different bio-analytes.
2. The biosensor as claimed in claim 1, wherein the said cholestrol sensor is constructed using cogel of mixed nanoclayLaponite® RD and Cloisite-Na Montmorillonite(MMT), film deposited on indium tin oxide (ITO) glass electrode at room temperature (25 °C) irradiated with hydrogen ion beam.
3. The biosensor as claimed in claim 1, wherein the electrode was fabricated through drop-casting the L-MMT solution onto ITO and kept it drying overnight (~ 2 days) and the prepared electrode was then subjected to hydrogen ion irradiation, and was then submerged in the Zobell’s solution with other electrodes present in the electrochemical cell and used for sensing cholesterol.
4. The biosensor as claimed in claim 1, wherein it is used particularly for determination of cholesterol present in a solution.
5. The biosensor as claimed in claim 1, wherein the electrode generates a measurable current directly proportional to the concentration of cholesterol present in the sample.
6. The biosensor as claimed in claims 1 and 3, wherein the hydrogen molecular ion (H2+) irradiation is done on fabricated L-MMT/ITO electrode, which is used for sensing of cholesterol.
7. The biosensor as claimed in claim 1, wherein irradiation of low energy (20 keV) H2+ion beam of different fluence on L-MMT/ITO electrode resulted in the alteration in the properties (both morphological and electrochemical) of electrodes for detection of cholesterol.
8. The biosensor claimed in claim 1, wherein electrochemical characterization of the L-MMT/ITO electrode is done using cyclic voltammetry (CV), scanning electron microscope (SEM), UV-Vis spectroscopy and Fourier Transform Infrared Spectroscopy (FTIR).
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9. The biosensor as claimed in claim 1, wherein hydrogen ion induced modification effected the sensitivity of the thin films.
10. The biosensor as claimed in claim 1, wherein the variation in fluence of molecular hydrogen ions provides conditioning of the sensitivity of nanoclay-based electrodes towards the enhanced sensing of cholesterol at room temperature on an enzyme-free platform.
11. The biosensor as claimed in claim 1, wherein the sensing mechanism used for L-MMT toward cholesterol detection was based on ferro-ferri cyanide redox electrochemical reaction.
12. The biosensor as claimed in any of the above claims, wherein irradiation of hydrogen ion beam produced surface modification of these electrodes and there was enhancement of sensitivity towards cholesterol at low fluence (upto 1013 ions/cm2) due to the breakage of hydrogen bonds.
13. The biosensor as claimed in any of the above claims, wherein irradiation by high fluence ions (above 1013 ions/cm2) caused degradation of the electrode surface resulting in lesser sensitivity towards cholesterol due to the recombination of hydrogen atom.
14. The biosensor as claimed in claim 1, wherein fluence from hydrogen ion beam provides the tunability of the implantation induced sensitivity of the L-MMT/ITO electrodes.
15. The biosensor as claimed in claim 7, wherein low fluence irradiations from hydrogen molecular ion (H2+) beam on L-MMT electrode enhanced electrochemical properties of the electrode.
16. The biosensor claimed in claim 1, has a maximum sensitivity for cholesterol in the range of 1-20 mM, with a variable sensitivity depending upon the hydrogen ion irradiation fluence.
17. The biosensor as claimed in any of the above claims, wherein such electrodes could be used for detection of cholesterol under room temperature conditions.
18. The biosensor as claimed any of the above claims, wherein the said sensing platform is developed to design cheap enzyme-free strip cholesterol sensor for mass production and day to day use.