TECHNICAL FIELD
The present invention relates to a field of detection of bio-molecules in a sample.
More specifically, the present invention relates to a gold-nanoparticles for
determination of glucose.
BACKGROUND/PRIOR 5 R ART
Diabetes mellitus is a serious life-time health issue which has been increasing
among the greater population, approximately 285 million people carrying this
disease worldwide. In present diagnosis, health professionals have reported
greater number of children being diagnosed with diabetes. Thus, diabetes is
10 characterized as a chronic metabolic disorder with hyperglycemia (high blood
sugar) and abnormal energy metabolism. It has number of complications and can
occasionally be life threatening. This has concerned researchers and has led them
to overcome the issue by early diagnosis of diabetes using the self-monitoring
glucose meters for monitoring glucose concentration in the blood. However,
15 precise detection of glucose is essential to monitor the biological level of glucose
in the body. Therefore, nanotechnology has essential application in medicine and
considers development for advanced treatments and diagnosis of diabetes.
Several attempts have been made in the nanomedicine using their application to
develop advanced techniques that can be applied to detect or control the disease
20 such as fabrication of glucose biosensor by covalent attachment of glucose
oxidase (GOx) to a gold nanoparticle monolayer modified Au electrode.
Researchers and scientists are paying much attention on developing effective and
accurate diagnostic devices based on nanotechnology and its application which is
an extremely interesting area to explore since my interest has always been
25 learning about dynamic developments in medicine.
Diabetes and current methodology for detection of glucose: Diabetes mellitus is
commonly referred to as diabetes, it is caused by blood glucose being excluded
by pancreas because pancreatic hormone insulin is not efficiently produced or not
3
used properly by the body. Insulin is an essential hormone which allows the body
to regulate amount of glucose in the blood and convert glucose into energy
needed for the body. There are two forms of diabetes mellitus, which are Type 1
and Type 2:
Type 5 1 diabetes generally affect 5-10% of all people due to destruction of insulin
producing beta-cells by immune system in the pancreas which prevent efficient
production of insulin. The most common cause of type 1 is genetics and
environmental factors.
Type 2 diabetes makes up about 90-95% people affected. It is predominant
10 compare to type 1. It is caused by body being resistance to insulin and unable to
respond to its action. It is associated with a person’s lifestyle and diet since
commonly obesity results the rise of this type diabetes.
To detect glucose concentration in the blood, strip glucometer is one of the
popular device. In general, it is an electrochemical device configurated on the
15 digital platform. The strip has enzyme electrode containing glucose oxidase. The
enzyme is reoxidized with an excess of a mediator reagent, by the reaction of
reoxidization on the electrode which generates an electric current. Thereby, the
total charge passing through the electrode is proportional to the amount of
glucose in the blood that has reacted with the enzyme.
20 Nanotechnology in medicine: Nanotechnology is a field of encompassing
nanostructures, nanomaterials and nanoparticles. Nanoscales are used to increase
the capability of observing changes and properties of materials and analyze in
depth involving atoms and molecules, describe their surface area, how they are
bonded, geometry of the material etc. The spherical AuNPs has the optical and
25 geometrical properties, size and shape-related exhibit a range of colours in
aqueous solution as the core size increases from 1 to 100 nm and generally show
a size relative absorption peak from 500 – 550 nm. This absorption band arises
from collective oscillation of the conduction electrons due to resonant excitation
by the incident photons which is called a surface Plasmon band. It is influenced
4
not only by size but also by shape, solvent, surface ligand, core charge and
temperature. Gold nanoparticles are widely used in the area of nanotechnology
based on their wide range surface functionality and bioconjuagtes coupled with
outstanding physical properties. Functionalized AuNPs provides a versatile
platform for nanobiological assemblies, 5 lies, the binding event between the analytes
and the AuNPs can alter the physicochemical properties of AuNPs such as
Plasmon resonance. There are wide varieties of applications of gold nanoparticles
such as lateral flow, optical sensing, light scattering applications, drug delivery
and cancer therapy. These applications of AuNps are essential for the
10 nanotechnology to develop advanced techniques such as sensors, probes,
diagnostics, catalysis etc.
Inventor of the present application have developed an innovative citrate capped
gold-nanoparticles with glucose oxidase (GOx) for determination of glucose.
This minimizes the risk of destabilizing during the binding process that is
15 aggregation forms a cluster of particles, which causes Plasmon shift and colour
change. There are several other factors which could prevent aggregation such as
store particles at the recommended temperature and maintain pH of the particles.
In another aspect inventor of the present application have developed a
spectrophotometric detection of glucose concentration, using ionically
20 immobilized glucose oxidase onto gold nanoparticles.
The enzyme-catalyzed reaction for the determination of glucose:
→ Photometric detection
SUMMARY
25 The present invention provides a photometric nano-transducer for detection of
glucose concentration.
5
In an embodiment, the invention provides a photometric nano-transducer for
detection of glucose concentration by functionalizing and modulating gold
nanoparticles with an glucose oxidase comprising: (i) a citrate-stabilized AuNPs;
and (ii) a glucose oxidase (GOx).
In another embodiment, 5 nt, the invention provides a photometric nano-transducer,
wherein the AuNPs are having a diameter of 3 to 7 nm.
In another embodiment, the invention provides a photometric nano-transducer,
wherein the GOx molecules are firmly attached on the surface of the AuNPs via
electrostatic/ionic interactions.
10 In another embodiment, the invention provides a photometric nano-transducer,
wherein the glucose concentration is detected on the basis of glucose biocatalysis
on the surface of AuNPs.
In still another embodiment, the invention provides a method for the preparation
of photometric nano-transducer comprising incubating AuNPs and GOx for 80 to
15 160 minutes at 30°C to 40°C.
In another embodiment, the invention provides a method for the preparation of
photometric nano-transducer, wherein the AuNPs and GOx are used in a ratio of
1:2 v/v.
In another embodiment, the invention provides a method for the preparation of
20 photometric nano-transducer, wherein the AuNPs are citrate stabilized.
Other aspects, advantages of certain embodiments, and salient features of the
invention will become apparent to those skilled in the art from the detailed
description, which, taken in conjunction with the annexed drawings and disclosed
exemplary embodiments of the invention.
6
DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1: Stepwise electrostatic self-assembly of GOx onto AuNPs.
Figure 2: Spectrophotometry of fabricated bioconjugate complex (in absence of
glucose).
Figure 3: Mechanism of GOx assembly 5 ly and AuNPs and oxidation reaction of
glucose in the presence of glucose oxidase and nano-photometric detection of
glucose.
Figure 4: Uv -visible absorption spectra changes of bioconjugate complex after
addition of various concentrations of glucose.
10 Figure 5: Uv-visible absorption spectra addition of glucose concentration in the
range of 2.5-10mg/100dl.
Figure 6: Uv-visible absorption spectra addition of glucose concentration in the
range of 15-25mg/100dl.
Figure 7: Calibration curve of the absorbance of GOx/AuNPs vs glucose
15 concentration
DETAILED DESCRIPTION
The embodiments herein and the various features and advantageous details
thereof are explained more fully with reference to the non-limiting embodiments
that are illustrated in the following description and drawings. Descriptions of
20 well-known components and process techniques are omitted so as to not
unnecessarily obscure the embodiments herein. The examples considered along
with drawings used herein are intended merely to facilitate an understanding of
ways in which the embodiments herein can be practiced and to further enable
those skilled in the art to practice the embodiments herein. Accordingly, the
7
examples should not be construed as limiting the scope of the invention described
herein.
Present application have developed a photometric nano-transducer for detection
of glucose concentration by functionalizing and modulating gold nanoparticles
with an enzyme (glucose oxidase). Molecular 5 ecular binding interaction between analyte
molecule and surface of gold nanoparticle was determined using surface
plasmonic resonance technique. Glucose biocatalysis was studied through the
nano-optical function of glucose on the surface of AuNPs. On the basis of visible
spectrum, a successful immobilization of glucose oxidase on gold nanoparticle
10 and biocatalysis of glucose were demonstrated. Thus, the resulting change in the
absorption peak exhibited the response of oxidation of different concentrations of
glucose on the surface of GOx/AuNPs.
Enzyme - Glucose oxidase: Glucose oxidase is secreted by fungi Aspergillus
Niger. It catalyses the oxidation of Beta D-glucose present in the plasma to D
15 glucono-1, 5 - lactone with the formation of hydrogen peroxide; the lactone is
then slowly hydrolysed to D-gluconic acid. Glucose oxidase enzyme produces
gluconic acid. Due to production of gluconic acid, absorption level is changed.
Therefore, glucose oxidase is used as an analytical reagent. The enzymatic assay
is specific for glucose allowing more accurate determination. The enzyme is to
20 create the desired functionalities for specific applications of the gold
nanoparticles.
Citrate-stabilized AuNPs was purchased from Sigma-Aldrich with diameter of 5
nm, OD 1 and absorption at 510 – 525 nm. Stored at 2-8°C. Glucose oxidase
(GOx) from the fungi Aspergillus Niger, 100,000 - 250,000 units/g in solid form
25 was also purchased from Sigma-Aldrich and stored at -20 °C.
Citrate buffer with concentration of 0.1 moldm−3 were prepared at pH 5.4. To
prepare the solution 0.1 M of citric acid with the volume of 16 ml and 0.1 M
sodium citrate of 34 ml was used.
8
This equation gave the mass of citric acid powder:
(Citric acid) mass(g) = Molar mass (g/mol) x concentration (M) x volume (l)
210.14 g/mol x 0.1 M x 0.016 l =0.336224g
Accordingly, 0.3362 g (± 0.0001, 0.03%) was added to a 25 ml of graduated
cylinder, distilled water was poured till the mark of 16 ml (± 0.4 ml, 5 , 2.5%) was
reached.
In similar way mass of sodium citrate powder was calculated:
(sodium citrate) mass(g) = Molar mass (g/mol) x concentration (M) x volume (l)
294.10 g/mol x 0.1 M x 0.034 l = 0.99994 g
10 0.9994 g (± 0.0001, 0.01%) was added to a 100ml of graduated cylinder, distilled
water was poured till the mark of 34 ml (±1, 2.94%), was reached.
Thereafter, a stir bar was placed in the beaker with the solution of 16 ml of citric
acid. The beaker was put on the magnetic stirrer and it was turned on to start
stirring the solution. Added approximately 34 ml of sodium citrate to make sure
15 the pH meter displayed the value 5.4 (±0.1, 1.85%) pH electrode was placed in
the solution and waited 20 to 30 seconds. Switched off the magnetic stirrer, took
off the pH electrode and poured the solution into a volumetric flask. Added
distilled water into the solution and made final volume of 100 ml (±0.1, 0.1%).
Repeated the procedure seven times to make 700 ml of citric buffer. In seven
20 volumetric flasks 100ml of citric buffer was poured.
The other solution, glucose oxidase was prepared by pouring 0.012 g (± 0.001,
0.83%) of GOx in 10 ml (±0.2, 2%) of citrate buffer solution, pH 5.4. Due to its
highly inflammable nature, gloves and safely glasses were worn throughout the
whole procedure.
9
Stock solution of glucose (100 mg/dl, 0.00106) was prepared in citrate buffer pH
5.4. It was prepared by adding 0.1044 g (±0.0001, 0.96%) of glucose in a
volumetric flask, then citrate buffer was poured till the 100 ml (±0.1, 0.1%) mark
had reached. Using the glucose stock and addition of citrate buffer, six different
5 concentrations of glucose solution was prepared shown in (Table 1). All the
apparatus and equipments were thoroughly cleaned to reduce random errors.
Table 1: Preparation of six different concentrations of glucose solution, converted
from mg/100cm3 to mol/ dm3, their absolute uncertainties are also stated.
TABLE 1
Volume of
glucose(±0.1cm3)
Volume
of citrate
buffer (±
0.1cm3)
Concentration
of glucose
(mg/100cm3)
Concentration
of glucose
(mol/dm3)
Absolute
uncertainty
(%)
25.0 75.0 25.0 1.39x10–5 1.59
20.0 80.0 20.0 1.11x10–5
1.69
15.0 85.0 15.0 8.33x10–6
1.85
10.0 90.0 10.0 5.55x10–6
2.17
5.0 95.0 5.0 2.77x10–6
3.17
2.5 97.5 2.5 1.39x10–6
5.16
10
Using the formula:
10
All the percentage uncertainties are calculated for all the values using the formula
below:
Absolute uncertainty 5 certainty of stock solution of glucose is calculated by adding
percentage uncertainty of the added solute and added volume of citrate buffer and
then multiplied by concentration it has reached.
(0.96%+0.1%)x0.001g/ml = 0.00106
Absolute uncertainty of different concentrations of glucose is calculated in this
10 certain way:
% uncertainty of mass of glucose + % uncertainty of volume of stock of glucose
+ % uncertainty of glucose volume added + % uncertainty of citric buffer added
0.96%+ 0.1%+ 0.4% + 0.13% = 1.59%
0.96%+ 0.1%+0.5% + 0.13% = 1.69%
15 0.96%+ 0.1%+ 0.67% +0.12% = 1.85%
11
0.96%+ 0.1%+ 1%+ 0.11% = 2.17%
0.96%+ 0.1%+ 2%+0.11% = 3.17%
0.96%+ 0.1%+4% +0.10% = 5.16%
Preparation of glucose oxidase functionalized AuNPs: In 7 eppendorfs, using a
micropipette with volume of 100 μl, 100 μl (±0.8 μl, 5 l, 0.8%) of AuNPs was added
with 200 μl (±0.8 μl, 0.4%) of GOx. Both reagents reacted for 2 hours at
approximately 37°C (±0.5, 1.35%) in the incubator. The reaction was stopped
with addition of 25 μl (±0.0175, 0.07%) of glucose in 6 eppendorfs with varied
concentration of glucose, the solutions stayed in the incubator for approximately
10 20 minutes at 37°C. AuNPs and GOx had a volumetric ratio of 1:2 v/v which is
an ideal ratio for glucose catalysis. This procedure was repeated three times to
obtain average absorption value.
Spectrophotometric detection of glucose: UV-vis absorption spectroscopy was
done with a Genesis 10 UV-vis spectrophotometer. Fingerprints from cuvettes
15 were removed with tissue to avoid false reading. Spectrophotometer was used to
determine the absorbance of the enzyme glucose oxidase (GOx). Thereby,
spectrum of AuNPs was carried out to make comparison in the absorbance. The
absorbance of the fabricated bioconjugate complex was monitored in the absence
of glucose and presence of various concentrations of glucose.
20 Fabrication process of the biocongugate complex: Figure 1 shows the stepwise
electrostatic self-assembly of GOx onto AuNPs which was followed by
incubation of citrate stabilized gold nanoparticles (100 μl) with glucose oxidase
(200 μl, pH 5.4) at 37°C for 2 hours. The incubation caused absorption of the
enzyme glucose oxidase onto the gold nanoparticles due to GOx have opposite
25 charges with respect to surface charge of AuNPs. Thus, electrostatic attractive
force between the GOx molecules and the AuNps surface caused strong
interaction of the molecules. GOx has been exploited as a stabilizing agent at pH
5.4 which minimized the risk of aggregation.
12
Binding event between the analyte and the AuNps can alter the physicochemical
properties of AuNps such as Plasmon resonance. Figure 2 shows the absorption
band exhibited by bare AuNPs (diameter of 5nm) at 520nm is the characteristic
surface Plasmon resonance band and also demonstrates the visible spectrum of
5 successful immobilization of glucose oxidase onto gold nanoparticles’ surface.
The attachment of the enzyme glucose oxidase was monitored by the
spectrophotometer, it showed the significant difference in the absorption peak at
520nm. The drastic change in absorption band was caused by attachment of
inactive GOx on the surface of the particles which slows down the light intensity
10 and less light penetrated through glucose oxidase. Low light permeability on
AuNPs and less light being absorbed compared to bare AuNPs therefore there is a
decrease in plasmonic band after the immobilization of GOx onto the AuNPs.
Additionally, the graph explained the assembly of gold nanoparticles and GOx
were consistent and GOx molecules are firmly attached on the surface of the gold
15 nanoparticles via electrostatic/ionic interactions. The surface of gold
nanoparticles was fully or incompletely covered by GOx function which
regulates the plasmonic peaks during UV-visible measurements in
presence/absence of glucose. It was observed that during the experiment when
eppendorfs of one of the trial were taken out after two hours’ incubation, the
20 solution still had some stances of yellow colour which indicates there were still
some glucose oxidase left in the solution and all were not completely
immobilized onto AuNPs.
Performance of the nano-photometric transducer: The coupled nanoparticle
system could be used in a potential nano-photometric transducer application.
25 Spectrophotometry detection of the immobilized enzyme was investigated to
monitor nano-photometric transducer’s performance. Therefore, various
concentration of substrate (glucose) were added to check the oxidization. The
surface of glucose oxidase onto gold nanoparticles reacted with glucose and
exhibited the catalytic activities for the oxidation of glucose. Figure 3 represents
30 the enzyme catalyzes the oxidation of D-glucose to D-glucono-lactone and it is
oxidized by molecular oxygen to produce hydrogen peroxide. D-gluconolactone
13
is then slowly hydrolyses spontaneously to produce gluconic acid. Production of
gluconic acid in the medium may provide the possible reattachment of GOx
present in the reacting solution on the surface of AuNPs. In the enzyme-catalysed
reaction there is increase in substrate concentration (glucose) and the amount of
GOx or covered area of GOx onto AuNPs decreases so 5 AuNP’s pority increases.
Coverage area on AuNPs decreases and light penetration increases. On the
addition of glucose concentration, the size of the particle has increased. Increase
in the lambda max which indicates the formation of a thicker monolayer around
the nanoparticles, resulting change in the absorption peak of nano-assembly.
10 Table 3 provide absorption spectrum of the nano-photometric detection of
different glucose concentrations in numerical form. Whereas, Figure 4 represents
the Uv-visible absorption spectra changes of bioconjugate complex after addition
of various concentrations of glucose. It demonstrates as there is increases in the
concentration of glucose there is increase in the plasmonic surface. However, the
15 Figure 5 shows that the pattern is followed only between the range of 2.5-
10mg/100dl. Glucose concentration in the range between 15-25 mg/100dl does
not show any changes in the lambda max even though there is increase in the
glucose concentration in the solution, this is presented by the Figure 6 the oddity
is due to reactivity of GOx because glucose seems to be saturated, this means that
20 all glucose particles fully covered the surface of AuNPs therefore, low light
permeability on AuNPs and less light being absorbed which could reflect in the
plasmonic peaks (there is no increase in the lamda max) of GOx functionalized
AuNPs. The graphs also represent that there is no shifting in the absorption peaks
which means aggregation has not formed during the whole procedure.
25 Table 3. Averaged absorption spectrum (wavelength 480-514nm) of the nanophotometric
detection of different glucose concentrations.
14
Standard Calibration Curve: By the use of Uv-vis absorption spectra quantitative
measurements have been obtained. Six dilutions of a standard of known
concentrations are prepared. Unit of the glucose concentration are converted from
mg/100cm3 to mol/dm3 since blood sugar level is measured 5 easured in mg/dl but in
chemistry more commonly measurement of concentration is mol/dm3. The
absorbance of glucose concentration is generated by reacting on immobilized
GOx onto AuNPs. Absorbance of various concentrations of glucose at Lambda
max was plotted on a graph to obtain calibration curve (Table 4). Figure 7
10 represents calibration plot of the absorbance vs glucose concentration, the
intensity absorption bands at 520nm gradually increases which indicates increase
15
in glucose concentration leading to higher rate of reaction and more gluconic acid
produced and causing increase in the absorption peak. The Beer-Lambert graph
shows recordings of the nano-photometric transducer on successive changes of
glucose concentration. The nano-photometric transducer exhibited a rapid
response to changes of glucose concentration 5 tration in the range between 1.39 x 10-5 –
8.33 x10-6 mol/dm3. Since Beer-Lambert law expresses the linear relationship
between the absorbance and concentration of a compound at a fixed wavelength.
However, Lambda max of GOx/AuNPs vs. concentration of glucose ranging 5.55
x10-6 – 1.39 x10-6 mol/dm3 does not follow the trend and there is a decrease in
10 absorption which is said to be because of saturation.
Table 4: Lambda max of GOx/AuNPs vs. concentration of glucose ranging 1.39
x 10-5 – 1.39 x 10-6 mol/dm3.
16
Table 5: Averaged absorption spectrum (wavelength 515-650nm) of the nanophotometric
detection of different glucose concentrations.
17
The inventors of present application have functionalized gold nanoparticles
(AuNPs) with an enzyme glucose oxidase for detection of glucose and
development of photometric nano-transducer. The photometric 5 ric nano-transducer
was examined through the nano-optical function of glucose on the surface of
18
AuNPs. The molecular binding interaction between analyte molecule and gold
nanoparticle surface was determined using surface plasmonic resonance
technique. Since binding event between the analyte and the AuNps has altered its
Plasmon resonance. The incubation of (100 μl) with glucose oxidase (200 μl, pH
5.4) at 37°C for 2 hours had caused absorption of the enzyme 5 glucose oxidase
onto the gold nanoparticles due to GOx carrying opposite charges with respect to
surface charge of AuNPs. Thus, electrostatic attractive force between the GOx
molecules and the AuNps surface caused strong interaction of the molecules.
Based on the visible spectrum successful immobilization of glucose oxidase onto
10 gold nanoparticle was demonstrated. The attachment of the inactive enzyme
glucose oxidase had caused drastic change in absorption band, it showed the
significant difference in the absorption peak at 520nm.
To monitor nano-photometric transducer’s performance, different concentrations
of glucose was incubated in the solution of coated AuNPs with GOx. Since the
15 GOx functionalized AuNP exhibits catalytic activities for the oxidation of
glucose and resulting change in the absorption peak of nanoassembly. It was
observed that the absorbance at 520 nm is proportional to the concentration of
glucose in the test samples. The Lambert-Beer law expresses the linear
relationship between the net absorbance and glucose concentration at a fixed
20 wavelength, i.e, λmax at 520 nm. The precise detection of glucose is essential to
monitor the biological level of glucose in the body. It can be concluded that the
nano-photometric transducer exhibits a rapid photometric response with changes
of glucose concentration in the test samples.
The foregoing description of the present invention will fully reveal the general
25 nature of the embodiment/aspect herein that others can, by applying current
knowledge, readily modify and/or adapt for various applications such specific
embodiments without departing from the generic concept, and, therefore, such
adaptations and modifications should and are intended to be comprehended
within the meaning and range of equivalents of the disclosed embodiment. It is to
19
be understood that the phraseology or terminology employed herein is for the
purpose of description and not of limitation.

We claim,
1. A photometric nano-transducer for detection of glucose concentration by
functionalizing and modulating gold nanoparticles with an glucose oxidase
comprising: (i) a citrate-stabilized AuNPs; and (ii) a glucose oxidase (GOx).
2. The photometric nano-transducer as claimed in claim 1, wherein the AuNPs
are having a diameter of 3 to 7 nm.
3. The photometric nano-transducer as claimed in claims 1 to 2, wherein the
GOx molecules are firmly attached on the surface of the AuNPs via
electrostatic/ionic interactions.
4. The photometric nano-transducer as claimed in claims 1 to 3, wherein the
glucose concentration is detected on the basis of glucose biocatalysis on the
surface of AuNPs.
5. A method for the preparation of photometric nano-transducer as claimed in
claims 1 to 4 comprising incubating AuNPs and GOx for 80 to 160 minutes at
30°C to 40°C.
6. The method for the preparation of photometric nano-transducer as claimed in
claim 5, wherein the AuNPs and GOx are used in a ratio of 1:2 v/v.
7. The method for the preparation of photometric nano-transducer as claimed in
claims 5 to 6, wherein the AuNPs are citrate stabilized.