TECHNICAL FIELD OF INVENTION:
The present invention relates to anelectrode fabricated with a novel biointerface
for electrochemical detection and quantification of creatine in a
biological fluid. The present invention also relates to method of preparation
5 of said electrode and use of said electrode for electrochemical detection and
quantification of creatine in a biological fluid.
BACKGROUND OF INVENTION:
Creatine (2-amino-1-methyl-2-imidazoline-4-one) is a bio-molecule found in
vertebrates performing several vital metabolic functions in the body.
10 However, excessive uptake of creatine inthe body is associated with health
disorders including thyroid malfunction, muscular disorders and kidney
related disorders. Normal concentration of creatine has been reported to be
within 40 - 150 μM in serum. The concentration of creatine increases
significantly in serum and other body fluids during these health-related
15 disorders. In muscular disorder the concentration of creatine in serum may
raise up to 1000 μM. Similarity during thyroid malfunction and kidney
related disorders, the creatine concentration in the blood rise significantly.
Therefore, detection and quantification of creatine content in plasma, serum
or other body fluid act an effective tool for detection ofsuchhealth-related
20 disorders.
As per the existing art, there are several methods for determination of
creatine concentration in body fluids. Majority of the methods for quantifying
creatine concentration in the body fluids involvespectrophotometric,
fluorimetric and colorimetric techniques which depend on a colour change of
25 a chromophore with respect to creatine concentration. Limitations associated
3
with these methods are, these detection methods have been affected by
interferences of other compounds present in serum or body fluids such as
glucose, ascorbic acid, proteins, amino acids, ketones, etc. and likewise
analysis in general takes longer time and the sensitivity used to be poor. To
5 overcome these limitations, modern methods use electrochemical biosensors.
A biosensor is an analytical device, used for the detection of a chemical
substance that combines a biological component with
a physicochemical detector. The Electrochemical biosensors are normally
based on enzymatic catalysis of a reaction that produces or consumes
10 electrons, where the production and the consumption of electrons in the fluid
sample is detected in form of electric signals through an electrode.
The region of contact between a biomolecule, biological cell, tissue or living
organism or organic material considered living, with another biomolecule,
inorganic or organic material is known as a bio-interface.
15 In the context of the biosensors using one or more enzyme for catalysis of any
reaction, the contact region between the enzymes and the substrates or
intermediate of a reaction is a bio-interface.
Typically, electrochemical creatine biosensors are based on dual enzyme
system of creatine amidinohydrolase (CAH) and sarcosine oxidase (SOx). The
20 enzyme creatine amidinohydrolase (CAH) hydrolyses creatine present in
sample for analysis to sarcosine and urea. Whereas, the enzyme sarcosine
oxidase (SOx) catalyzes hydrolysis of sarcosine to glycine, hydrogen
peroxideand formaldehyde. The formaldehydegetscatalytically reduced and
produced two electrons (2e-).The electrons thus released is detected in form of
4
electrical signal through the electrodes and directly correlated to the
concentration of the creatine present in the biological fluid sample.
The limitations associated with this type of creatine biosensors are their poor
stability due to low functional ability of interfaces for creatine
5 amidinohydrolase (CAH) and sarcosine oxidase (SOx). To overcome
limitations associated with electrochemical biosenors and improve their
functional ability and sensitivity there has been reports of use of
nanomaterials-enzyme interfaces.
OBJECT OF INVENTION:
10 The object of present invention is to provide a bio-interface for detection of
and quantification of creatine in biological fluids, with substantially
improved functional ability and sensitivity. The object of present invention is
also to provide an electrode fabricated with said bio-interface for detection
and quantification of creatine in biological fluid sample.The object of present
15 invention is also to provide a method of fabrication of said bio-interface on
the electrode.
SUMMARY OF INVENTION:
The present invention discloses a dual enzymatic, highly sensitive
nanomaterial-based bio-interface for the electrochemical detection of creatine,
20 wherein the nanomaterialcomprises of both graphene oxide and chitosan, and
the dual enzyme comprises of both creatine amidinohydrolase (CAH) and
sarcosine oxidase (SOx),
In the present invention, the nanomaterial is coated on surface ofan electrode
responsible for sensing the electrochemical signals. The enzymes creatine
5
amidinohydrolase (CAH) and sarcosine oxidase (SOx) are sequentially
immobilized on said coated surface of the electrode, wherein the
immobilization is achieved through covalent bonding between the nanomaterial
and the enzymes.
5 BRIEF DESCRIPTION OF DRAWINGS:
Fig 1represents the mechanism associated with the present invention,
wherein creatine in the fluid sample is hydrolyzed to sarcosine and urea,
followed by hydrolysis of sarcosine by sarcosine oxidase to formaldehyde,
glycine and hydrogen peroxide, which is further followed by catalytic
10 oxidation of hydrogen peroxide and release of electrons.
In Fig 2, (a)(I) represents Atomic Force Microscopy image of surface of
electrode when coated with nano-material only; (a)(II)represents Atomic
Force Microscopy image of surface of the electrode coated with nano-material
followed by sequential immobilization of enzymes creatine amidinohydrolase
15 (CAH) and sarcosine oxidase (SOx) as disclosed in the present invention;
(b)(I) represents water contact angle of nano-material coated on the surface of
the electrode; (b)(II) represents water contact angle of the surface of the
electrode when coated with nano-material followed by sequential
immobilization of enzymes creatine amidinohydrolase (CAH) and sarcosine
20 oxidase (SOx) as disclosed in the present invention;
Fig 3represents amperometric response of the electrode fabricated with the
nanomaterial composite along with immobilized enzymes creatine
amidinohydrolase (CAH) and sarcosine oxidase (SOx) (hereinafter “the biointerface”),
when the creatine concentration ranges from 0.01 to 1.25M at
25 working potential of +0.4 V vs Ag/AgCl using 50 mM phosphate buffer
6
solution containing 1% NaCl. Inset of figure 3 shows linear calibration plot
corresponding to current responses, for different creatinine concentration by
electrode fabricate with the bio-interface.
Fig. 4, (a) represent effect of pH and (b) represents effect of temperature on
5 the electrode fabricated with the bio-interfaceat fixed value of creatine 250
M in the phosphate buffer solution (50 mM, pH 7) with scan rate of 50
mV/s.
DESCRIPTION OF INVENTION:
Graphene is an atom thick 2D semi-conducting carbon nanomaterial
10 possessing honeycomb lattice arrangement of sp2 hybridized carbon atoms
with very high aspect ratio. Few layered graphene is more suitable for device
fabrication especially where on/off switchable property is desired. Graphene
is an attractive electrode material for various electrochemical processes
including energy storage and conversions, electrochemical sensors, fuel cells,
15 bioreactors and many other devices. Consequently, graphene has become one
of the most utilized material in the design and fabrication of electrode
interfaces in the recent times, this is because it (graphene) is additionally
cheap, non-toxic and compatible with all biomolecules. The presence of
graphene improves the stability and activity of immobilized biomolecules,
20 consequently facilitating electron transfer.
Chitosan is a linear polysaccharide composed of randomly distributed -
(14)-linked D-glucosamine (deacetylated unit) and N-acetyl-Dglucosamine
(acetylated unit).
7
The present invention discloses an electrode fabricated with a novel biointerface,
wherein the bio-interface comprises of a layer of nanomaterial made
from graphene oxide and chitosan; enzymes creatine amidinohydrolase
(CAH) and sarcosine oxidase (SOx) are immobilized on said layer of
5 nanomaterial through covalent linkages. Wherein the ratio of the creatine
amidinohydrolase (CAH) to sarcosine oxidase (SOx) is 3:2
In the present invention the enzyme creatine amidinohydrolase (CAH)
hydrolyses creatine present in sample for analysis to sarcosine and urea.
Whereas, the enzyme sarcosine oxidase (SOx) catalyzes hydrolysis of
10 sarcosine to glycine, hydrogen peroxideand formaldehyde. The
hydrogenperoxide gets catalytically oxidized and produceselectrons.
The electron thus released is detected in form electrical signal through the
electrodes and directly correlated to the concentration of the creatine present
in the biological fluid sample.
15 A schematic representation of the fabricated bienzyme electrode is presented
in Fig. 1(a) while the electrobiocatalytic sensing mechanism process is
presented in Fig. 1(b) showing the electrobiocatalytic hydrolysis of creatine to
sarcosine and urea by CAH and a simultaneous secondary electrobiocatalytic
reaction involving the conversion of sarcosine to formaldehyde, glycine and
20 hydrogen peroxide by SOx and at the same time, the transducer signals the
sensing of electrons released.
The method of preparation of the electrode fabricated with biointerfacecomprises
following steps:
8
1. Preparation of CHIT/GrO nanocomposite using physical agitation
method;
2. Coating said nanocomposite onto the surface of electrode responsible for
sensing the electro-chemical signal;
5 3. Sequential immobilization of enzyme creatine amidinohydrolase (CAH)
and sarcosine oxidase (SOx) by glutaraldehyde coupling; wherein the
ratio of conc. (in units) of creatine amidinohydrolase (CAH) to sarcosine
oxidase (SOx)is 3:2.
10 The detailed step by step procedure of preparation of electrodes fabricated
with bio-interface as claimed in the present invention is disclosed under
Example 1, and different physicochemical characteristics of said electrode is
disclosed under Example 2.
Examples 1:
15 Materials required
• Chitosan (CHIT, Mw = 50,000 - 190,000 Da, DDA = 75–85%, 20 - 300 cP,
1 wt. % in 1% acetic acid at 25 °C),
• Graphene oxide (GrO aqueous solution, 500 mg/L),
• Creatine monohydrate (98%),
20 • Creatine amidinohydrolase (CAH) (Pseudomonas sp, 100-300
units/mg),
• Sarcosine oxidase (SOx) (Bacillus sp. 25-50 units/mg),
• Glutaraldehyde solution (25% w/v in H2O),
• Potassium dihydrogen phosphate (KH2PO4, 99.0%),
25 • Dipotassium hydrogen phosphate (K2HPO4, 99.0%),
9
• Potassium chloride (KCl, 99.0%),
• Phosphate buffer saline (1X PBS),
• Ferri/ferro cyanide solutions (were used as supporting electrolyte for
some amperometric measurements).
5
All the solution used in the invention were prepared using double-distilled
water
The method of fabrication of the electrode with bio-interface is represented as
follows:
10 a) Preparation of CHIT solution: CHIT solution (0.5%, w/v) was prepared
by mixing 10 mL of chitosan solution (1 wt. %) with 10 mL of 1% acetic
acid solution at 25 °C, the mixture was sonicated for 60 sec. to ensure
complete dispersion of the solution.
15 b) Preparation of GrO Solution:

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20 "%&%

c) Preparation of CHIT/GrO nanocomposite:The CHIT/GrO
nanocomposite was prepared by physical agitation method in which
250μL of the prepared CHIT solution was magnetically stirred in 250
25 μLof GrO aqueous solution at 500 rpm for 48 h, at RT. Subsequently, the
10
resulting nanocomposite solution was dialyzed with a dialysis tube
(MW cut-off 12 kDa) against the double distilled water.
d) Coating of CHIT/GrOnanocomposite
5 10 μl of CHIT/GrO nanocomposite solution was mixed with 5 μL of
glutaraldehyde (50% v/v), then gently shaken for 30 min to ensure
proper mixing. The mixture was then drop cast on Glassy carbon (GC)
electrodes which had been pre-cleaned in 1.0 μm, 0.3 μm and 0.05 μm
alumina slurries respectively. The modified electrode was left to dry for
10 4 hrs.
e) Immobilization of Enzymes:
The enzymes creatine amidinohydrolase (CAH) (350 units/mL) and
sarcosine oxidase (SOx) (350 units/mL) were added to the surface of
CHIT/GrO nanocomposite coated electrode sequentially so that the
15 optimal ratio of creatine amidinohydrolase (CAH) to sarcosine oxidase
(SOx) (350 units/mL) on the electrode is 3: 2. This is followed by drying
of the electrode for 4hrs at room temperature (RT). The unbound
enzyme was washed out with phosphate buffer saline (PBS, pH 7.4) and
resulting material was dried under vacuum at 25 °C. All experiments
20 were carried out at RT.
Example 2:
The electrode coated with nanocomposite only and electrode fabricated
with nanocompositealong with immobilized enzymes (bio-interface) were
thoroughly characterized using the electrochemical measurements.Their
25 surface morphology was characterized by Atomic force microscopy
11
(AFM). Contact angle measurement was used to analyze their hydrophilic
interactions and hydrophilicity.
Water contact angle measurements were performed using an optical
contact angle meter. All voltammetric measurements were carried out
with an 5 Ivium Stat. XR electrochemical analyser. A three-electrode cell
with glassy carbon working electrode, having 0.07 cm2 surface area,
platinum wire auxiliary and Ag/AgCl (3M KCl) reference electrode was
used for the voltammetric measurements. All measurements were carried
out at 20 ± 2 °C.
10 a) Characterization of electrode with nanocomposite only and electrode
fabricated with bio-interface
i) Surfacecharacterization of electrodes through atomic force
microscopy (AFM)
15 The surface morphology of the electrodes was characterized using atomic
force microscopy (AFM) as presented in Fig. 2(a). It is observed that the
electrodes with nanocomposite only (Fig. 2(a-i) possesses a relatively
rougher surface as compared to that of electrode with bio-interface.
Rougher surface of the electrode with nanocomposite only, facilitated the
20 entrapment and consequent covalent interaction of graphene withCAH
and SOx thereby facilitating the superior stability of the enzymes
modified electrode structure. It is observed that the surface of the
electrode with bio-interface becomes smoother after the immobilization of
the enzymes as presented in Fig. 2(a-ii), thus providing a favorable
25 platform for the interaction of the enzymes with the analytes.
12
ii) Surface characterization of electrodes through water contact angle
measurement
Water contact angle measurement is a common and very useful way of
measuring the hydrophilic or hydrophobic character of a substrate and
consequently, the 5 affinity of that substrate to aqueous media. The water
contact angle measurements for both the electrodes were performed and
the result is presented respectively in Fig. 2b (i) and (ii). The water contact
angle measurement results, clearly indicate that the immobilization of
CAH and SOx on the modified electrodes causes an increase in the
10 wettability and hydrophilic character of the modified electrodes, thus
signifying that the electrode with bio-interface with water contact angle of
48.7±2° possesses a higher affinity for the aqueous analytes than the
electrode with nanocomposite only, with water contact angle of 78.4±2°.
The surface of electrode with bio-interface showed more hydrophilic
15 character that of electrode with nanocomposite only.
b) Electrochemical Measurements (Amperometric Response) of electrode
fabricated with bio-interface:
Fig. 3 showed the cyclic voltammograms of the electrochemical cells using
20 electrode with bio-interface at a constant 50 mVs-1 scan rate in 50 mM
phosphate buffer saline (PBS) solution (pH 7, 1% NaCl).
Chronoamperometry was performed for determining of Amperometric
Response of the electrode with bio-interface with respect to different
concentration of creatine in the aqueous medium. The observed current
25 response represents the cathodic oxidation of H2O2 at a constant potential
of +0.4 V against Ag/AgCl reference electrode. It was also observed that
13
the cathodic current peak increases linearly with respect to the increasing
concentration of creatine. The linear regression was calculated to be
r2=0.941. The electrode responded within 3s to change in creatine
concentration. The current sensitivity of the electrode with bio-interface
5 toward creatine concentration was approximately 2 A/mM/cm2. This
new type of creatine sensor has demonstrated a shorter response time and
a broader detection range compared to those previously reported (Table
1).
The detection limit of the nanocomposite creatine biosensor is 10 M, with
10 a linear response from 0.01 to 1.25 mM.
c) Electrochemical Measurements of the electrodes with biointerface,
effect of pH, temperature, interference and reproducibility:
The effect of pH on the electrodes with bio-interface was determined by
15 measuring the current response for a constant creatine concentration of
250 μM within the pH range of 5.0-9.0 and the optimum current obtained
at pH 7.0 (Fig. 4 (a)) that the result illustrating that optimum CAH and
SOx enzyme activity for the electrodes with bio-interface was at pH 7.0.
The thermal stability of the electrodes with bio-interface was also studied
20 by measuring the current response at different temperatures (ranging
from 20 to 50 °C) in the presence of creatine at concentration of 250 Min
PBS (50 mM, pH 7.0). The result showedthat catalytic property of the
enzymeswere increased till 30 °C, while catalytic response begins to be
decreased after 30 °C (Fig. 4 (b)).
25 The effect of interferents was measured on the amperometric responses of
the electrodes with bio-interface in presence of 250 M creatine in
14
phosphate buffer (50 mM, pH 7). The interference effects of L-serine, Lthreonine,
-ketoglutaric acid, L-alanine, L-phenylalanine, uric acid, Lcystine,
L (+)-glutamic acid, sodium pyruvate, L-glutamine, and Lascorbic
acid. These substances were added into the reaction mixture at
5 their normal physical concentration, i.e., 0.2 mM. It was found that the
presence of interferents had a relative error of less than 10% in the current
measured by the amperometric method; therefore, this electrodes with
bio-interface can detect creatine with negligible interference. Also,
biosensor showed a relatively high reusability with less than 10%
10 standard deviation for 12 measurements in a row.
we provide herein below a detailed comparison of electrochemical
characteristics of the bio-interfaces available in art, with those of electrode
with bio-interfaceas disclosed in this specification
Table (Comparison of electrochemical characteristics of the bio-interfaces
15 available in art, with those of electrode with bio-interface as disclosed in this
specification)
For the table as indicated above, reference 1 is a publication identified as E.Karaku, . Pekyardmci, E. Kilic, Process Biochem., 2006, 41 (6), 1371-1377. DOI:

20 https://doi.org/10.1016/j.procbio.2006.01.017
15
For the table as indicated above, reference 2 is a publication identified as A.
Ramanavicius, Anal. Bioanal. Chemistry, 387, 2007, 1899-1906.
DOI: 10.1007/s00216-006-1065-2
5
In this invention, we havedisclosed an electrode with bio-interface for
electrobiocatalytic detection and quantification of creatine in a biological
fluid, which shows higher sensitivity by detecting conc. of creatine in the
fluid sample as less as 10 μM, as well as providing very shorter response time
10 of 3s. Thus, the electrode with bio-interface as disclosed in this specification
can be used for fast and efficient detection of creatine in the fluid samples,
particularly the biological sample.

We claim,
1. An electrode for detection and quantification of creatine in a fluid
sample, wherein the electrode is fabricated with a bio-interface,
wherein the bio-interface comprises of nanomaterial and enzymes, wherein the nanomaterial comprises of both graphene oxide and
chitosan, wherein the enzymes comprise of both creatine
amidinohydrolase (CAH) and sarcosine oxidase (SOx), wherein the
nanomaterial is coated on surface of the electrode and the enzyme
creatine amidinohydrolase (CAH) and sarcosine oxidase (SOx) are immobilized on said nanomaterial, wherein the ratio of creatine
amidinohydrolase (CAH) to sarcosine oxidase (SOx) is 3:2.
2. The electrode as claimed in claim 1, wherein the electrode can detect
and quantify creatine concentration as less as 10μM in a fluid sample.
3. The electrode as claimed in claim 1, wherein the electrode showsresponse time as less as 3 seconds for detection and
determination of creatine in a fluid sample.
4. A method for fabrication of the electrode as claimed in claim 1,
wherein the method comprises of
a. Creation of nanomaterial composite from graphene oxide and chitosan;
b. Coating said nano-composite on surface of the electrode
responsible for sensing electrochemical signals;
c. Sequential Immobilization of creatine amidinohydrolase (CAH)
and sarcosine oxidase (SOx) on the nanomaterial coated surface of
the electrode by covalent bond wherein the ratio of creatine
amidinohydrolase (CAH) to sarcosine oxidase (SOx) is 3:2.
5. A method of detection and quantification of creatine in a fluid sample
using the electrode as claimed in claim 1.
6. The electrode as claimed in claim 1 to 3 and 5, wherein the fluid
sample is a biological fluid sample.
7. The electrode as claimed in claim 6, wherein the biological fluid
sample is a serum or plasma.
8. The electrode as claimed in claim 6, wherein the biological fluid
sample is a serum.