FORM-2
THE PATENTS ACT, 1970 (39 OF 1970) & The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10 and rule 13)
NON-ENZYMATIC ELECTROCHEMICAL METHOD FOR
SIMULTANEOUS DETERMINATION OF TOTAL HEMOGLOBIN AND GLYCATED HEMOGLOBIN


PIRAMAL HEALTHCARE LIMITED, a company incorporated under the Companies Act, 1956, of Piramal Tower, Ganpatrao Kadam Marg, Lower Parel, Mumbai - 400 013, State of Maharashtra, India, an Indian company and
COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH, of Anusandhan Bhavan, 2, Rafi Marg, New Delhi 110 001, India, an Indian registered body incorporated under the Registration of Societies Act (Act XXI of 1860).
The following specification particularly describes the invention and the manner in which it is to be performed.



FIELD OF THE INVENTION
The present invention relates to a method which uses a non-enzymatic, disposable screen-printed electrode strip (SPE strip) for simultaneous measurement of total hemoglobin (Hb) and percentage of glycated hemoglobin (%HbAlc) in a blood sample wherein the total Hb is estimated by amperometry or differential pulse voltammetry, and the amount of HbAlc is estimated by potentiometry. Modification of a SPE strip for potentiometric measurement of HbAlc is also disclosed.
BACKGROUND OF THE INVENTION
The importance of diagnosis and monitoring of diabetes is emphasized by a recent report in which it was stated that 20% of the total world population is affected by this chronic disease. One of the proactive measures needed to control diabetes mellitus is periodic monitoring and control of blood glucose levels either with the help of clinicians or using "do-it-yourself kits. HbA1c is a stable minor variant of Hb, formed in vivo by non-enzymatic post-translational modification of N-terminal valine of the [3-chains of Hb. Estimation of HbAlc is extremely valuable for long-term control of diabetes mellitus unlike direct estimation of glucose wherein one obtains information of blood sugar at the time of measurement. Hence, in addition to the monitoring of blood glucose levels, it is extremely important that one monitors the overall level of glucose by monitoring HbAlc. This is a better way to manage diabetes, and may result in the prevention or reduction of long-term complications. In recent years, various types of kits for monitoring HbA1c levels in blood have been described or developed.
U.S. Patent No. 7,005,273 describes enzyme catalyzed electrochemical methods to measure Hb and HbAlc, and a spectrophotometric method to measure HbAlc. The method is based on an indirect electrochemical estimation of Hb using a measurement of dissolved oxygen and enzyme-catalyzed reactions. Disadvantages of this method relate to the stability of the enzyme and the shelf

life of the system. It is well known that the dissolved oxygen levels are temperature dependent and hence a constant temperature environment needs to be maintained for the reliability of the analysis. Further, oxygen solubility in an aqueous environment is not sufficient to provide the required current signals for the indirect determination of Hb.
U.S. Patent No. 6,677,158 describes a colorimetric method for HbAlc estimation that can be performed outside of the medical laboratory and includes several steps involving chemical addition and colour read-out devices for Hb measurement which require high dilution of the sample. This technique is rather complex and requires several manual operations. Moreover, in colorimetric measurements, sensitivity is relatively less compared to other methods. U.S. Patent No. 4,876,205 describes a method for assaying Hb in blood in which the blood is contacted with a sufficient amount of a ferricyanide (redox mediator) so that hemoglobin in the blood is reacted therewith and the hemoglobin is electrochemically assayed by monitoring the change in current, produced on reduction of ferricyanide by hemoglobin. The assay method incorporates a dry strip sensor with a dry mixture containing finely divided ferricyanide and a non-ionic surfactant, clerol (a mix of polyethylene oxide and polypropylene oxide and emulsifiers). However, this is a method useful only for total hemoglobin in whole blood. It is an indirect estimation of Hb and it has certain limitations, such as the dependence of the current signal on the kinetics of the redox transformations of the mediator. The use of redox mediators is not cost-effective for commercialization of the process.
EP 1,225,449 Al describes the use of a non-enzymatic disposable electrode strip for detection of uric acid and Hb. The strip contains non-ionic or neutral surfactants such as Triton X-100 for Hb and a cationic surfactant for uric acid. The strip is used subsequently as an amperometric sensor. Neither anionic nor cationic surfactants are used in this method for sensing Hb. There are known methods for analysis of HbAlc. For example, the DCA2000 analyzer from Siemens Diagnostics is an automated enzyme immunoassay

method for determination of HbA1c. Most of the commercially available analyzers employ HPLC as a tool for the assay of HbAlc [Clinical Biochemistry, 2005, 38, 88-91]. There has been a report of use of a quartz crystal biosensor for detection of HbAlc using complexation reactions of diol groups with 3-aminophenylboronic acid [Analytica Chimica Acta, 2005, 530, 75-84].
There are reports about exploring electrochemical methods such as amperometry and variants to develop disposable sensors for determination of HbAlc. [Biosensors and Bioelectronics, 2006, 21, 1952-1959; Biosensors and Bioelectronics, 2007, 22, 2051-2056; Sensors and Actuators B, 2006, 113, 623-629; Sensors and Actuators A, 2006, 130-131, 267-272; Clinical Biochemistry, 2008, CLB6720, doi: 10.1016/j.clinbiochem.208.01.113]. The clinical estimation of HbAlc based on enzymatic conversion is rather complicated and requires the use of analytical methods such as cation exchange chromatography, affinity chromatography, gel electrophoresis, immunochemical and other spectroscopic methods. These techniques are complex, reagent-intensive and time-consuming. The cost per analysis is also relatively high. Though several methods for estimation of HbAlc are commercially available, there is a need for quick, robust and cost effective diagnostic tool for the analysis of HbAlc so that decisions can be made for better management of diabetes mellitus and complications thereof. Therefore, an aspect of the present invention is to provide a rapid, non-enzymatic and direct method for simultaneous determination of HbAlc by potentiometry and total Hb by amperometry or differential pulse voltammetry in blood in a single analysis.
SUMMARY OF THE INVENTION
The present invention relates to a screen-printed electrode (SPE) strip for simultaneous measurement of total Hb and %HbAlc in a blood sample. The strip includes four electrodes.

In one aspect of the invention, the SPE strip is non-enzymatic. In another aspect of the invention, the SPE strip is disposable. The invention also relates to a non-enzymatic, disposable screen-printed electrode (SPE) strip for simultaneous measurement of total Hb by amperometry or differential pulse voltammetry, and %HbAlc by potentiometry in a blood sample. The strip facilitates the analysis in a single step operation. Still another aspect of the invention is that the strip is used in a method for simultaneous measurement of total Hb and % HbAlc in a blood sample. The present invention also relates to a kit for simultaneous measurement of total Hb and %HbA1c in blood sample comprising a SPE strip (as described above), a lysis solution, and a surfactant solution. The kit may also include a lancet, a blotting paper strip, an empty vial and an instruction insert.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a block diagram of the SPE strip and its connection to a meter.
Figure 2 is a block diagram of the hardware and the functional details of the
meter.
Figure 3 is a diagram of a screen-printed electrode (SPE) strip.
Figure 4A shows a typical calibration plot for Hb by amperometry.
Figure 4B shows the electrode response for Hb by amperometry.
Figure 5A shows a typical calibration plot for Hb by differential pulse
voltammetry (DPV).
Figure 5B shows the electrode response for Hb by differential pulse
voltammetry (DPV).
Figure 6 shows the DPV response of Hb in 1.5 mM of Sodium dodecylsulphate
(SDS) in acetate buffer of pH 5.0 [Hb cone. 0.7-1.7 g/dl].
Figure 7 shows the potentiometric estimation of HbAlc (the graph line having
square symbols ■) using aminophenylboronic acid polymer film on the
electrode surface and estimation of Hb (the graph line with triangle symbols
A).

Figure 8 shows the potentiometric estimation of HbA 1 c using aminophenylboronic acid in solution.
Figure 9 shows the potentiometric estimation of HbAlc by using an electrode that has been modified with carbon ink using water-insoluble 4-phenyl-vinyl boronic acid (the graph line having square symbols ■ )and an electrode that has been modified with carbon ink using 3-thiophene boronic acid (the graph line with triangle symbols A).
DETAILED DESCRIPTION OF THE INVENTION Definitions
Before describing the present invention in detail, it has to be understood that
this invention is not limited to particular embodiments. It is also to be
understood that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to be limiting.
As used in the specification and claims, the singular forms "a", "an" and "the"
include plural references unless the context clearly indicates otherwise.
The term "screen printed electrode (SPE) strip" refers to an electrode strip
described below. It is to be understood that although the electrodes can be
formed by using screen-printing, the invention is not limited to the use of
screen-printing to form the electrodes. Other printing methods or other methods
to form the electrodes can be used.
The term "Nernstian response range" refers to a range of concentration in which
the slope (defined by mV/decade of concentration) is less than the "ideal"
Nernstian slope of 59 mV/decade.
The term "modified electrode" refers to an electrode whose surface is coated
with layers of the desired functional materials specific to the application. In an
embodiment of this invention, a screen-printed carbon or graphite electrode is
modified by a water insoluble boronic acid compound.
The term "differential pulse voltammetry" refers to an electro-analytical
technique in which a square wave pulse superimposed on a potential dc ramp

(linear increase of potential with time) is applied on the sensing electrode and
the differential current output is plotted against the applied dc potential.
The term "reaction area" refers to the area on the electrode, which is exposed to
the blood sample.
The term "Glassy carbon" also called vitreous carbon refers to a non-
graphitizing carbon, which combines glassy and ceramic properties with those
of non-graphitizing carbon. The most important properties are high temperature
resistance, extreme resistance to chemical attack and impermeability to gases
and liquids. Glassy carbon is widely used as an electrode material in
electrochemistry.
The term "meter" refers to an instrument, which measures potential difference
and current signal generated at the electrode surface when the electrode comes
in contact with the blood sample. The concentration of HbAlc is converted into
potential difference, the concentration of Hb is converted into current signal,
and both Hb and %HbAlc values are displayed on the screen of the meter.
Measurement of Total Hb
Hb consists of four protein chains with four heme portions (Fe2+/Fe3+) and is
located in the erythrocytes. The approach of this invention involves analyzing
Hb by exploiting the redox behaviour of the heme portions (Fe2+/Fe3+) in Hb
molecule using a disposable, screen printed electrode surface coated with a
material such as carbon, graphite, gold, platinum, palladium, or a printing ink as
described below.
The method according to this invention includes determining the total amount
of Hb in a sample by electrochemical ly measuring the voitammetric current due
to iron (II) and iron (III) redox centers in Hb using surfactant-enhanced current
signal amplification methodologies. The electrode potential is fixed at a level
where the heme molecule interacts with the electrode surface to undergo
electron transfer reaction. Thus the current observed is directly proportional to
the amount of heme present, which in turn is related to the concentration of total
Hb present in the given test solution. As the heme centers in Hb are buried deep

into the bulky protein molecules, it is difficult to get an appreciable current signal. To overcome this, heme portions should be released or made available before performing amperometric measurement. For this purpose, the Hb is treated as described below with a current-enhancing surfactant. The released heme group shows significant redox characteristics at the electrode without a redox mediator. The heme group can also be released from the Hb molecule using sonication followed by centrifugation or by providing the Hb molecule a chemical link to redox mediators (such as ferrocene, methylviologen, etc.). The current signal can thus be amplified by using the ionic surfactant and is converted to g/dL of Hb and displayed on the screen of the meter.
Measurement of HbAlc
HbAlc is the glycated form of Hb resulting from the condensation reaction between hexose sugars and Hb. In the present invention, HbAlc has been analyzed using a potentiometric approach unlike optical, red ox-mediated amperometry, immunoassay and other methods such as quartz crystal mass balance methods.
The water-insoluble boronic acid compound added as described below results in a complex being formed between boronate and the cis-diol groups of sugars present in the HbAlc. During these chemical changes, an equilibrium potential of the electrode surface is developed depending on the HbAlc concentration. The potential difference arises due to change in pKa value of boronic acid compound at the electrode surface. This results in a linear relationship between HbAlc concentration and the potential difference measured by making use of the sub-Nernstian response range of the potentiometric technique. This potential difference is measured with respect to the reference electrode and is converted into %HbAlc and displayed on the screen of a meter.
After the SPE strip (which is connected directly or indirectly to a meter) is wetted by the solution containing red blood corpuscles (RBCs), the presence of Hb and HbAlc is detected by amperometry or differential pulse voltammetry,

and potentiometry respectively. The current signal can be amplified by using an ionic surfactant, which is converted to g/dL of Hb and displayed on the screen of the meter.
Description of the SPE strip
The SPE strip includes contact pads that are the upper portion of the electrodes and are illustrated by 12 in Figure 3; insulating material, which is the substrate, and insulating non-porous film, which is the electrical insulating film. The strip, which may be disposable, is used for non-enzymatic detection of Hb and %HbAlc. It comprises:
(i) A substrate, which is an electrical insulator. Types of electrical insulators
that can be used include but are not limited to glass epoxy board;
electrically non-conducting polymer material such as polystyrene; or
fiber-reinforced epoxy (FRE) substrates of thickness varying from 0.3 mm
to 1.0 mm. In an aspect of the invention, the substrate is FRE.
(ii) A conducting film, which is coated on one side of the substrate to form
four independent electrodes, namely, (a) counter electrode, (b) working
electrode- (c) reference electrode and (d) modified electrode.
(iii) An electrical insulating film. The electrical insulating film is coated on a
part of the conducting film such that one end of all the electrodes are
uncovered for connecting with the measuring device and the opposite end
is uncovered and is intended to be in contact with the solution containing
the sample to be tested. The electrical insulating film may be a
commercially available material, having properties of electrical insulation
with very high impedance of greater than 1012 ohms. This material is used
to coat the conducting film to provide the electrical insulation. An
example of an electrical insulating film is XV1300U.V. -NOTATION
WHITE", INK NO. CFSN6022, supplied by Sun Chemical, UK.
In an aspect of the present invention, the SPE strip comprises four electrodes
wherein electrode 1 (counter electrode), electrode 2 (working electrode) and
electrode 3 (reference electrode) are used for estimation of Hb by amperometry

or differential pulse voltammetry; and electrode 3 and electrode 4 (modified electrode) are used for estimation of HbA1c by potentiometry. Electrode 3 is a common reference electrode for both amperometry and potentiometry. The electrodes are independent of each other and do not touch each other. Electrodes 1, 2, 3 and 4 are shown in Figure 3.
According to an aspect of the invention, the locations for the electrodes are marked and one side of the substrate is coated with a conducting film using screen-printing or a similar printing method to form the electrodes. Other methods can also be used to form the electrodes. In this process only the electrodes are coated and not the entire substrate. The conducting film is selected from gold, platinum, palladium, silver, carbon or graphite or a printing ink which has the property of adhering to the surface of the substrate without any smearing so that the electrodes remain independent of each other. The conducting film accepts or donates electrons and can be used as the mediator to transfer electrons between the analyte and the electrode in the redox reaction. In an embodiment of the invention, printing ink is used as the conducting film and the printing ink typically used is a carbon or graphite ink or a mixture of a carbon and silver ink. In an aspect of the present invention, the material for coating the electrodes is a carbon conducting film or carbon printable ink. Any commercially available conductive carbon ink which gives an electrochemical response for standard cyclic voltammetry experiments can be used. A material that can be used for coating the substrate using screen-printing is a conductive carbon paste procured from Coates, Inc. (USA). This conductive carbon paste can be used as an ink to print on predetermined areas of the substrate to form the electrodes.
In one aspect of the invention the thickness of the conducting film on the substrate is between 20 to 60 microns. In another aspect of the invention, the thickness of the conducting film is about 30 microns.
The range of the dimensions of each electrode of the SPE strip, which is exposed to the solution containing the red blood corpuscles (RBCs) (region 15

in Figure 3), may be:
Electrode 1 (counter electrode):
Length - 3.0 mm to 10.0 mm, preferably 5.0 mm
Width - 0.3mm to 2.0 mm, preferably 0.5 mm
Thickness - 20 microns to 150 microns, preferably 60 microns.
Electrode 2 (working electrode):
Length - 2.0 mm to 9.0 mm, preferably 4.0 mm
Width - 0.3mm to 2.0 mm, preferably 1 -0 mm
Thickness - 20 microns to 150 microns, preferably 60 microns.
Electrode 3 (reference electrode):
Length - 3.0 mm to 10.0 mm, preferably 5.0 mm
Width - 0.3mm to 2.0 mm, preferably 0.5 mm
Thickness - 20 microns to 150 microns, preferably 60 microns.
Electrode 4 (modified electrode):
Length - 3,0 mm to 10.0 mm, preferably 5.0 mm
Width - 0.3mm to 2.0 mm, preferably 0.5 mm
Thickness - 20 microns to 150 microns, preferably 60 microns.
After the substrate is coated with the conducting film, it is dried at a
temperature from 90°C to 150°C, preferably at about 120°C, for 30 minutes to
60 minutes, preferably for about 45 minutes. After drying, the substrate may be
dipped in 10% chromic acid, 10% sulfuric acid, 5-10% nitric acid or 10%
hydrochloric acid solution for 10.0 minutes. In an aspect of the invention, the
coated substrate is dipped in 10% chromic acid solution. The substrate is
removed from the chromic acid solution and washed with water three times for
2 to 15 minutes per wash, preferably, about 10 minutes per wash. The substrate
is again dried, preferably at about 70°C for about 20 minutes.
An electrical insulating film is applied to the strip by screen printing or another
method except on the contact pads and the section of the strip identified as
region 15 in Figure 3.
The conducting film of the fourth electrode (modified electrode), is modified by


a water-insoluble boronic acid compound using screen printing or the like at the portion of the electrode that will be immersed in the sample of RBCs shown as 16 in Figure 3. This modified coating enables changes such as potential, resistance by electrochemical reaction between the modified electrode and reference electrode to be used to determine the % HbAlc. Electrode modification is not possible with the soluble form of boronic acid compounds because the electrode will lose its sensing ability due to the leaching of HbA1c-selective boronic acid and the associated functional groups. Thus, in the present invention, water-insoluble boronic acid compounds have been used to modify the fourth electrode (electrode 4). The water-insoluble boronic acid compound may be selected from 4-phenyl-vinyl boronic acid, aminophenyl boronic acid and thiophene boronic acid. In one aspect of the invention 4-phenyl vinyl boronic acid is used. The fourth electrode can be modified according to the following procedures:
(a) A water-insoluble boronic acid compound is dissolved in a suitable low volatile solvent that can dissolve the water insoluble boronic acid compound. The solvent may be selected from isopropyl alcohol, ethanol, propanol and acetone. The solution obtained can be blended with the conductive carbon paste in a weight ratio of 1:0.5 to 1:4, preferably in a ratio of about 1:1 and used for printing on the substrate for potentiometric estimation of HbA 1c.
(b) In an alternative configuration, (for potentiometry) the printed carbon electrode is modified with a film (thickness: approx.5-10 μm) of a water-insoluble boronic acid compound, by electro-deposition on the carbon electrode using electro-polymerization procedure/conditions. The water-insoluble boronic acid compound and sodium fluoride are dissolved in hydrochloric acid solution. Polymerization is effected by dipping the screen-printed fourth carbon electrode in this solution without stirring. The fourth electrode potential is scanned between 0.0 and 1.1 V until the charge in the cathodic scan reaches 10 mC cm-2. A deep bluish-green film is obtained and it is washed with water. The electrode is thus modified and then rinsed with water, followed by rinsing in phosphate


buffered saline (PBS) solution.
Other processes can be used to prepare the modified electrode. Only the portion of the fourth electrode that will be immersed in the sample of RBCs is modified.
Figure 3 describes a screen-printed electrode (SPE) strip. It consists of four electrodes, namely, counter electrode 1, working electrode 2, reference electrode 3 and modified electrode 4. Basically, the electrodes are screen printed on the substrate 13 using a conducting film. Preferably, the conductive carbon ink of resistance in the range 15 ohms to 25 ohms is used to screen print the electrodes 1, 2, 3 and 4 on substrate 13. Contact pads 12 are at the top end of the electrodes and are used to provide the electrical connection with the connector 8 in Figure 1. Preferably, the width of the contact pads is the same for all four electrodes. An electrically insulating film 14 is screen printed on all the electrode surfaces except for the contact pads and the section of the electrodes identified as region 15. Region 15 is the portion of the electrodes that come in contact with the sample containing the RBCs (5 in Figure 1) for determination of concentration of hemoglobin and glycated hemoglobin. Only the portion of electrode 4 that is to be immersed in the sample is modified using a water insoluble boronic acid compound and is shown as 16 in Figure 3. Additionally, the invention also relates to a non-enzymatic, electrochemical method for simultaneous measurement of total Hb and % HbAlc in blood sample using the SPE strip (as described above) comprising the steps of:
(a) treating a blood sample with a lysis solution;
(b) removing the plasma to obtain red blood corpuscles (RBCs) either by decanting the plasma or by dipping a blotting paper strip in sample obtained in step (a);
(c) treating the sample containing the RBCs obtained in step (b) with a surfactant solution;
(d) contacting the sample obtained in step (c) with the SPE strip;
(e) measurement of total Hb by amperometry or differential pulse


voltammetry; and measurement of HbAlc by potentiometry; and
(f) calculating the %HbA1c relative to the total Hb in blood sample.
The blood sample collected from the patient is subjected to pre-treatment to
separate red blood corpuscles (RBCs) from plasma by adding a lysis solution.
Plasma is removed either by decanting or by dipping a blotting paper in the
blood sample with lysis solution and the RBCs obtained are treated with the
surfactant solution.
The lysis solution may be selected from 50% ethanol; 1M acetic acid (in water)
0.2M acetic acid (in water) 0.2M citric acid (in water); ethyl alcohol/water (1:1)
and NaCl (in water).
The ratio of the lysis solution to the sample is I: I to 1:20 (v/v), preferably, 1:10
(v/v).
The surfactant may be selected from all types of cationic, anionic, e.g. ionic
surfactants and preferably is selected from gemini surfactants,
didodecyldimethylammonium bromide, cetyltrimethyl ammonium bromide,
benzyltrimethylammonium bromide, phenacylthiazolium bromide,
aminoguanidine hydrochloride, thiourea, phenacyl-thiazoliunV-pyridinium
bromide, sodium dodecylsulfate, sodium polystyrenesulfonate, and sodium salts
of benzene-/naphthalene-mono-/di-/tri-sulfonic acids.
The ratio of the surfactant to the sample is of RBCs 1:1 to 1:20 (v/v) and
preferably 1:10 (v/v).
The SPE strip is introduced into the sample containing treated RBCs. A
potential difference is generated due to reaction of HbA lc on the surface of the
boronic acid modified electrode. This potential difference is measured with
respect to the reference electrode and is converted into %HbAlc and displayed
on the screen of a meter. Similarly, a current signal is generated between
electrodes 1, 2 and 3 proportional to the concentration of hemoglobin wherein
the Fe2+/Fe3+ reaction takes place on the electrode surface. The current signal is
converted to g/dL of Hb and displayed on the screen of the meter. The
functional details of the meter are shown in Figure 2. The dotted line separates


the components of the Printed Circuit Board (PCB) comprising a preamplifier and Microcontroller Unit (MCU) modules. The Hb electrodes (electrode 1, 2 and 3) generate the current signal, which is subsequently converted into equivalent voltage signal through a current to voltage converter. The modified electrode directly generates a potential difference, which in turn is measured as a voltage signal. Both the voltage signals corresponding to Hb and HbAlc respectively are amplified through Instrumentation Amplifier. The Analog to Digital Converter (ADC) converts the amplified analog voltage signals to equivalent digital signals. The MCU processes the digital data and directly displays the Hb value in terms of g/dL and HbAlc as a percentage value on Alphanumeric Display.
In an aspect of the present invention, both the values of total Hb and HbA 1c are required to calculate the value of %HbAlc. The percentage of HbAlc is calculated as follows:
%HbAlc = [(HbAlc/total Hb) x 100]. The entire analysis may be completed within five to ten minutes after collection of the blood. As shown, for example, in figure 7, according to the invention, Hb and HbAlc can each be measured and quantified and there is no interference between the measurements and quantification of each as it pertains to the other. Figure 1 shows block diagram of how a typical analysis is carried out by connecting the SPE strip with the meter. The sample in vial 6 contains red blood corpuscles (RBCs) 5, which have been isolated from plasma. The surfactant solution, preferably an ionic surfactant solution is added to vial 6 to preferentially release Heme proteins. The RBCs are mixed with the surfactant solution and can be analyzed. The SPE strip 7 is connected to the connector end 8 of the meter 10, through the cable 9. The sensor measures the concentration of Hb and HbAlc in the vial, the MCU calculates both Hb and HbA1c in g/dL and % unit respectively. The meter 10 indicates these values on the display 11. The present invention also relates to a kit for simultaneous measurement of total Hb and %HbAlc in blood sample comprising a SPE strip (as described above),


a lysis solution, and a surfactant solution. The kit may also include a lancet, a
blotting paper strip, an empty vial and an instruction insert.
In one embodiment of the present invention, the lancet is used for pricking the
skin so the blood can be collected in the empty vial.
The instruction insert provides instructions for use of the kit. The insert may
include instructions describing the steps needed to measure Hb and %HbA 1c in
the sample including describing how the blood is drawn, and mixed with the
lysis and surfactant solutions.
The invention thus provides a method for the estimation of %HbAlc and total
Hb in a single step using a disposable, non-enzymatic screen-printed electrode
strip, which incorporates electrodes for amperometry or differential pulse
voltammetry and potentiometry.
An example of an apparatus that can be used is a tabletop device that can be
used in a medical practitioner's office. In some embodiments, an apparatus that
can be used may be operated by non-technically trained people.
The above disclosure generally describes the present invention. More details of
the above invention can be understood from the following specific examples.
These examples are herein provided for the purpose of illustration only and are
not intended to limit the scope of the invention.
Examples Example 1
Preparation of electrodes
Starting material used for the preparation of electrodes of the screen-printed
sensor strip was conductive carbon paste procured from Coates, Inc. (USA).
This conductive carbon paste was used as an ink to print on the predetermined
areas of the fibre-reinforced epoxy (FRE) substrates using a screen-printing
process.


Example 2
Modification of electrode 4
As shown in Figure 3, region 16 of electrode 4 of the SPE strip prepared in Example 1 was modified by dissolving 4-vinylphenyl boronic acid in iso-propyl alcohol (~ 10 ml) and blended with the conductive carbon paste in 1:1 ratio (by weight) and was used for screen printing for potentiometric estimation of HbAlc from blood sample.
Example 3
Process for modification of electrode 4
In this process, the screen-printed carbon electrode of Example 1 was modified with a conducting polymer film (thickness: approx.5-10 μm) of amino phenyl boronic acid (PABA). It was electro-deposited on the carbon electrode using the electro-polymerization procedure/conditions, which are briefly described as follows: 3-amino phenyl boronic acid (0.04 M) of quantity 87.0 mg and sodium fluoride (0.2 M) of quantity 105.0 mg were dissolved in 12.5 ml of 0.2 M HC1 solution. Polymerization was effected by dipping one of the screen-printed carbon electrodes in the above solution under unstirred conditions and the electrode potential was scanned between 0.0 and 1.1 V until the charge in the cathodic scan reached 10 mC cm-2. A deep bluish-green film was obtained and it was washed with water. The electrode was thus modified and then rinsed with water, followed by PBS solution and it was ready for use.
Example 4
Calibration curve for estimation of Hb by amperometry using didodecyldimethyl ammonium bromide (DDDMAB) as a surfactant. The standard hemoglobin sample (Catalog No. 400294022, Nicholas Piramal India Limited) (15 g/dl) was diluted ranging from concentration of 0.5g/dl to 1.9g/dl using the surfactant solution containing DDDMAB dissolved in 0.IM potassium chloride solution. The SPE strip, prepared in Example 1, was


introduced into the above sample solution. Then the electrodes were connected to the potentiostat using appropriate connectors and the potential was swept between 0.1 to 0.8 volt at a scan rate of 100 mV/s. The peak current was measured in the peak potential range of 0.25 to 0.30 V. This was repeated with five standard samples and a calibration plot of "peak current vs. Hb concentration" was plotted. From the calibration plot, the slope of the graph was calculated and the latter was used for determination of total Hb in the test sample. A typical calibration plot and the electrode response for Hb in 5mM DDDMAB-1M KC1 solution is shown in Figures 4A and 4B. The experimental calibration graph for Hb, carried by amperometry, is linearly fitted by a straight line. The equation y = 0.3759x gives the best fit with regression coefficient R2 = 0.9857. This equation is used to determine the concentration of Hb present in the sample.
Example 5
Calibration curve for estimation of Hb by differential pulse voltammetry using DDDMAB as surfactant
The standard hemoglobin sample (catalog no. 400294022, Nicholas Piramal India Limited) (15 g/dl) was diluted ranging from concentration of 0.5g/dl to 1.9g/dl using the surfactant solution containing didodecyldimethyl ammonium bromide (DDDMAB) dissolved in 0.1M potassium chloride solution. The SPE strip, prepared in Example 1 was introduced into the above sample solution. Then the electrodes were connected to the potentiostat using appropriate connectors in the differential pulse voltammetry (DPV) mode. The potential was swept between -0.2 and 0.4 V at a scan rate of 5 mV/s using the parameters: step potential: 2 mV; pulse width: 50 mV; pulse period: 200 ms. The DPV peak current was measured in the above potential range. This was repeated with five standard samples and a calibration plot of "peak current vs. Hb concentration" was plotted. From the calibration plot, the slope of the graph was calculated and the latter was used for determination of total Hb in the test sample. A typical


calibration plot and the electrode response for Hb in 5mM DDDMAB-1M KG solution is shown in Figure 5A and 5B.
The experimental calibration graph for Hb, carried by differential pulse voltammetry, is linearly fitted by a straight line. The equation y = 3.2409x gives the best fit with regression coefficient R2 = 0.9952. This equation is used to determine the concentration of Hb present in the sample.
Example 6
Calibration curve for estimation of Hb by differential pulse voltammetry using sodium dodecyl sulphate as the surfactant.
The standard hemoglobin sample (Catalog No. 400294022, Nicholas Piramal India Limited) (15 g/dl) was diluted ranging from concentration of 0.5g/dl to l.9g/dl using the surfactant solution containing sodium dodecyl sulphate (SDS) dissolved in 0.1M potassium chloride solution. The SPE strip, prepared in Example 1, was introduced into the above sample solution. Then the electrodes were connected to the potentiostat using appropriate connectors and the potential was swept between 0.1 to 0.8 volt at a scan rate of 100 mV/s. The peak current was measured in the peak potential range of 0.25 to 0.30 V. This was repeated with five standard samples and a calibration plot of "peak current vs. Hb concentration" was plotted. From the calibration plot, the slope of the graph was calculated and the latter was used for determination of total Hb in the test sample. A typical calibration plot and the electrode response for Hb in 5mM SDS - 1M KCI solution is shown in the Figure 6.
The experimental calibration graph for Hb, carried by differential pulse voltammetry, is linearly fitted by a straight line. The equation y = 0.475x+0.4199 gives the best fit with regression coefficient R2 = 0.9802.
Example 7
Calibration curve for estimation of %HbAlc by potentiometry using SPE strip modified by aminophenylboronic acid.


A film of aminophenylboronic acid (PABA) was deposited on the glassy carbon electrode. 3-amino phenyl boronic acid (0.04 M) and sodium fluoride (0.2 M) were dissolved in hydrochloric acid (0.2M) solution. Polymerization was effected by keeping the working electrode in this solution along with platinum foil as counter electrode and saturated calomel as reference electrode. The electrode potential was scanned between 0.0 and 1.1 V for 3-5 scans. The modified electrode was then rinsed with water followed with PBS solution and used for further experiments.
Based on these results, a linear relationship was established between concentration of HbAlc and the potential difference, enabling potentiometric estimation of the HbAlc as shown in the Figure 7.
The graph line in figure 7 having square symbols (■) indicates the change in the potential difference of HbAlc as a function of concentration of HbAlc. The graph line in the figure with triangle symbols (A) indicate the change in the concentration of Hb alone. The separation of these two graph lines show that that there is no interference from Hb in the detection and quantification of HbAlc when both Hb and HbAlc are measured simultaneously.
Example 8
Calibration curve for estimation of %HbAlc by potentiometry using SPE strip modified by water-soluble aminophenylboronic acid.
SPE strip, prepared in Example 1, was used for the experiment. Water-soluble aminophenylboronic acid (APBA) was dissolved in an electrolyte solution containing the sample and the consequent shift in electrode potential due to addition of HbAlc was measured. Aminophenylboronic acid in solution interacts with HbAlc, yielding a relationship between the concentration of HbAlc and the measured potential difference. This potential difference arises due to change in pKa value at the electrode surface. Based on these results, a linear relationship (Figure 8) was established between concentration of HbAlc and the potential difference, enabling potentiometric estimation of HbAlc.


Example 9
Calibration curve for estimation of %HbA1c by potentiometry using SPE strip modified by vinylphenylboronic acid.
This method estimates the potential of an electrode modified by a carbon ink of (water-insoluble) vinylphenylboronic acid, which was immersed in an electrolyte solution containing the sample (TruLab HbAlc liquid level 1 to level 4; Diagnostic System GmbH, Germany) and the consequent shift in electrode potential due to addition of HbAlc. This modified electrode interacts with HbAlc, yielding a relationship between the concentration of HbAlc and the measured potential difference. This potential difference arises due to change in pKa value at the electrode surface. Based on these results, a linear relationship was established between concentration of HbAlc and the potential difference, enabling potentiometric estimation of HbA1c as shown in the Figure 9. This experiment demonstrates the linear relationship between HbAlc and potential difference.
Figure 9 shows the potentiometric estimation of HbAlc by using an electrode that has been modified with carbon ink using water-insoluble 4-phenyl-vinyl boronic acid (the graph line having square symbols ■ )and an electrode that has been modified with carbon ink using 3-thiophene boronic acid (the graph line with triangle symbols ▲).
Example 10
Calibration curve for estimation of %HbA lc by potentiometry using SPE strip modified by thiopheneboronic acid.
An electrode modified by a carbon ink of (water-in soluble) thiopheneboronic acid was immersed in an electrolyte solution containing the sample (TruLab HbAlc liquid level lto level 4 Diagnostic System GmbH, Germany) and the consequent shift in electrode potential due to addition of standard HbAlc was measured. This modified electrode interacts with HbAlc, yielding a relationship between the concentration of HbAlc and the measured potential difference.


This potential difference arises due to changes in pKa value at the electrode surface. Based on these results, a linear relationship was established between concentration of HbAIc and the potential difference, enabling potentiometric estimation of HbAIc as shown in Figure 9.
Example 11
Measurement of total Hb and %HbAlc from a patient's blood sample. A blood sample from a diabetic patient was collected at a clinical laboratory. 20 uL of blood sample was taken in a test vial and 200μL of lysis solution consisting of 50% ethanol was added. The vial was kept for two minutes without shaking so that plasma was separated from RBCs. The separated plasma was decanted by tilting the vial. RBC, being a thick fluid did not flow out of the vial while decanting the plasma. Then, 200 uL of surfactant 5mM ionic surfactant, cetyl trimethyl ammonium bromide (CTAB) was added and the solution was manually shaken approximately for a minute for mixing of RBC with the surfactant solution. The solution was ready for analysis. The SPE strip modified by 4-phenyl-vinyl-boronic acid was inserted in the vial. A potential difference was generated due to reaction of HbAIc on the surface of boronic acid modified electrode which was measured with respect to the reference electrode and was converted into %HbAlc and displayed on the screen of the meter. Similarly, a current signal generated between electrodes 1, 2 and 3 proportional to the concentration of hemoglobin was converted to g/dL of Hb and displayed on the screen of the meter. The Hb concentration was 10.52 g/dl and the %HbAlc value was 9.3%. Sample from the same patient was analysed using Chloestech GDX A1C testing system and HbAIc was estimated to be 9.2%.


CLAIMS
What is claimed is:
1. A screen printed electrode (SPE) strip for simultaneous measurement of total
hemoglobin and percentage of glycated hemoglobin in a blood sample
comprising four electrodes comprising a counter electrode, working electrode,
reference electrode and an electrode modified by a water - insoluble boronic
acid compound;
wherein the counter, working and reference electrodes are used for estimation of Hb by amperometry or differential pulse voltammetry; the reference and modified electrodes are used for estimation of HbA lc by potentiometry; and the reference electrode is used for both amperometry and potentiometry.
2. The screen-printed electrode strip according to claim 1, wherein the electrodes are coated with a material selected from the group consisting of carbon, graphite, gold, platinum, palladium and silver.
3. The screen-printed electrode strip according to claim 2, wherein the electrodes are coated with carbon or graphite.
4. The screen-printed electrode strip according to claim 2, wherein the electrodes are coated with carbon.
5. The screen-printed electrode strip according to claim 4, wherein carbon is in
the form of printable ink.
6. The screen-printed electrode strip according to claim 1, wherein the
electrodes are formed by using printing.
7. The screen-printed electrode strip according to claim 1, wherein the strip is
non-enzymatic.

8. The screen-printed electrode strip according to claim 1, wherein the strip is disposable.
9. The screen-printed electrode strip according to claim 1, wherein the boronic acid compound is selected from a group consisting of 4-phenyl-vinyl boronic acid, aminophenyl boronic acid and thiophene boronic acid.
10. The screen-printed electrode strip according to claim 1, wherein the strip
is prepared by a process comprising the steps of:
a) coating with a conducting film on one side of the substrate to form the electrodes comprising of a counter electrode, working electrode, reference electrode and a modified electrode wherein the electrodes are isolated and disconnected;
b) washing the substrate in an acid solution and drying;
c) coating an insulating film on a part of the electrodes wherein one end of the electrodes is uncovered to make contact with the meter and the other end of the electrodes, which is opposite to the end which can be connected to the meter, is also uncovered;
d) modifying the portion of the electrode 4, which is opposite to
the end which can be connected to the meter, using a water-
insoluble boronic acid compound.
11. A kit for simultaneous measurement of total Hb and %HbAlc in blood sample comprising a SPE strip of claim 1.
12. The kit according to claim 11, further comprising a lysis solution and a surfactant solution.
13. The kit according to claim 12, further comprising a lancet, a blotting paper strip, an empty vial and an instruction insert.


14. The kit according to claim 11, wherein the lysis solution is selected from 50% ethanol; IM acetic acid (in water) 0.2M acetic acid (in water) 0.2M citric acid (in water); ethyl alcohol/water (1:1) and NaCl (in water).
15. The kit according to claim 12, wherein the lysis solution is a premeasured amount of 50% ethanol.
16. The kit according to claim 12, wherein the surfactant solution is a
premeasured amount of ionic surfactant selected from the group consisting of
gemini surfactants, didodecyldimethylammonium bromide,
cetyltrimethylammonium bromide, benzyltrimethylammonium bromide,
phenacylthiazolium bromide, aminoguanidine hydrochloride, thiourea,
phenacyl-thiazolium/-pyridinium bromide, sodium dodecylsulfate, sodium
polystyrenesulfonate, and sodium salts of benzene-/naphthalene-mono-/di-/tri-
sulfonic acids.
17. A method for simultaneous measurement of total Hb and % HbAlc in blood
sample comprising:
(a) treating a blood sample with a lysis solution;
(b) removing the plasma to obtain red blood corpuscles (RBCs) either by decanting the plasma or by dipping a blotting paper strip in sample obtained in step (a);
(c) treating the solution obtained in step (b) with a surfactant solution;
(d) contacting the solution obtained in step (c) with the screen printed electrode strip of claim 1;
(e) measuring total Hb by amperometry or differential pulse voltammetry; and measuring of HbAlc by potent iometry; and
(f) calculating the %HbAlc relative to the total Hb in the


solution of red blood corpuscles.
18. The method according to claim 17, wherein the lysis solution is selected from 50% ethanol; IM acetic acid (in water) 0.2M acetic acid (in water) 0.2M citric acid (in water); ethyl alcohol/water (1:1) and NaCl (in water).
19. The method of claim 17, wherein the surfactant solution is an ionic surfactant selected from the group consisting of Gemini surfactants, phenacyl-thiazolium/-pyridinium bromide, aminoguanidine hydrochloride, thiourea, dodecyldimethylammontum bromide, cetyltrimethylammonium bromide, benzyltrimethylammonium bromide, phenacylthiazolium bromide, sodium dodecylsulfate and sodium polystyrenesulfonate.
Dated this14th day of October 2008
Council of Scientific and Industrial Research
Dr.Swati Bal-Tembe Piramal Healthcare Limited