DESC:FIELD OF THE INVENTION
The present invention relates to a sensor for homocysteine detection and method thereof. More particularly, present invention relates to an electrochemical sensor for the detection of homocysteine and a method for using said electrochemical sensor.

BACKGROUND OF THE INVENTION
Homocysteine is an important molecule within the human body. It's derived from dietary methionine and plays a role in building proteins and other essential functions. Maintaining a healthy balance of homocysteine is crucial because both high and low levels are linked to various health issues. On the one hand, homocysteine acts as a precursor for cysteine, an amino acid that contributes to the structure and function of proteins throughout the body. It's also involved in a process called methylation, which is critical for DNA regulation, gene expression, and cellular maintenance. Additionally, homocysteine helps maintain a cellular balance.

However, if homocysteine levels become elevated (hyperhomocysteinemia), it can increase the risk of various diseases. These include cardiovascular disease, neurological disorders like Alzheimer's and Parkinson's, and even birth defects. Conversely, severe deficiency of homocysteine can also lead to developmental problems in children. Several factors influence homocysteine levels, including dietary intake of methionine and B vitamins, as well as genetics. Maintaining a balanced diet rich in fruits, vegetables, and whole grains, combined with adequate B vitamin intake, can help promote healthy homocysteine levels.

Understanding the role of homocysteine in human health is important for developing strategies to prevent and manage diseases associated with its imbalance.

There are number of patent and non-patent documents published to cater for this problem. Reference is made to CN114577875A titled “Electrochemical immunodetection method for total homocysteine” which discloses an electrochemical immunosensor successfully assessing homocysteine. While the reported immunosensor demonstrates success in homocysteine detection, its production process presents significant challenges. The intricate nature of electrode fabrication, particularly the use of delicate and expensive materials like graphene and gold, raises concerns about cost-effectiveness. These fragile components necessitate meticulous handling throughout the process, increasing production time and the risk of errors that could compromise sensor function.

Another reference is made to CN116448842A titled “Homocysteine electrochemical sensor and preparation method and application thereof”. The reported sensor employs a two-step electrode modification process involving carbon nanomaterial baking followed by gold electrodeposition. While this approach seems promising, it suffers from inherent limitations. The primary concern lies in the inconsistency of surface modification. Baking alone cannot guarantee uniform distribution of the carbon nanomaterial, potentially leading to variations in sensor performance and unreliable results. Additionally, the use of gold nanoparticles and carbon nanotubes significantly increases the sensor's overall production cost.

Despite the significance of homocysteine monitoring for health assessment, readily available and efficient technologies for its detection remain scarce. Existing methods often lack sensitivity or necessitate the use of complex equipment, hindering their widespread adoption.

In order to obviate the drawbacks in the existing state of the art, there is pressing need for a sensing device for homocysteine detection, which has potential to significantly contribute to this field by enabling more efficient and accessible monitoring of homocysteine levels.

OBJECT OF THE INVENTION
In order to overcome the shortcomings in the existing state of the art, the objective of the present invention is to provide a medical sensing device for homocysteine detection and quantification.

Yet another objective of the invention is to provide an electrochemical sensor with a superior linear range for homocysteine detection.

Yet another objective of the invention is to modify the working electrode i.e. glassy carbon electrode using novel synthesized nanocomposite for better sensitivity for homocysteine detection.

Yet another objective of the invention is to provide a miniaturize / portable device by incorporating screen-printed electrodes, suitable for convenient homocysteine monitoring at home or in point-of-care settings.

Yet another objective of the invention is to develop a cost-effective solution for homocysteine detection and quantification using in-house synthesized material.

Yet another object of the present invention is to provide a medical device which is consistent, efficient and provides reliable results.

Yet another objective of the invention is to provide a method for detection of homocysteine using electrochemical sensor.

Yet another object of the present invention is to provide a method for detection of homocysteine which is rapid yet efficient.
SUMMARY OF THE INVENTION:
The present invention discloses a medical device for detection of homocysteine and a method thereof. More particularly, the present invention discloses an electrochemical sensor for the detection of homocysteine, a crucial amino acid metabolite within the human body and method thereof.

The invention is described below by way of non-limiting example:

The present sensor is an electrode based electrochemical sensor, which incorporates a glassy carbon electrode (GCE) as working electrode, as the heart of the detection process. A key innovation lies in the modification of this working electrode with a unique in-house synthesized nanocomposite. Said nanocomposite is prepared using a simple one-step solvothermal method. The modification process significantly enhances the sensitivity of the electrode towards homocysteine detection compared to unmodified electrodes.

Said sensor also includes a platinum counter electrode and a calomel reference electrode to complete the electrochemical circuit. Standard techniques like differential pulse voltammetry (DPV) and cyclic voltammetry (CV) are employed to measure the current response generated by the oxidation of homocysteine at the modified working electrode surface.

Said nanocomposite including but not limited to Fe2(MoO4)3/FeS (FMO/FeS)., modified working electrode plays a critical role in the sensing process. When a biological sample, containing homocysteine, is introduced, the homocysteine molecules interact with the electrode surface. This interaction leads to the electrochemical oxidation of homocysteine, generating a current response. The measured current is then correlated with the homocysteine concentration in the sample using a pre-established calibration curve.

The FMO/FeS nanocomposite offers a significant advantage by enabling the sensor to detect homocysteine across a wider linear range compared to existing methods. This translates to more accurate detection across a broader spectrum of homocysteine concentrations.

Accordingly, present invention provides a novel electrochemical sensor for homocysteine detection with enhanced sensitivity due to the unique nanocomposite modification. The sensor offers the potential for a portable device due to its planned miniaturization and holds promise for improved accessibility and early disease diagnosis through efficient homocysteine monitoring.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1 displays detailed diagram of the medical device with all constructional features.
Figure 2 displays electrochemical cell set up with three electrode configuration.
Figure 3 displays surface modified glassy carbon electrode.
Figure 4 displays schematic representation of electrochemical detection of homocysteine.
Figure 5 displays method of preparation/fabrication of Fe2(MoO4)3/FeS (FMO/FeS).
Figure 6 displays cyclic voltammogram of bare GCE, FeS/GCE and FMO/FeS/GCE with 1500 µM homocysteine.
Figure 7 displays plot between peak potential Vs pH strength Vs oxidation current.
Figure 8(a) displays DPV of FMO/FeS/GCE in the concentration range of 13 µM to 9061 µM and 8(b) displays linear calibration plot framed between current and concentration of homocysteine.
Figure 9(a) displays reproducibility analysis of sensor, 9(b) displays effect of interfering agents in the presence of 300.0 µM Hcy, 9(c) displays long term stability of the senor and 9(d) at rest stability of the sensor.
Figure 10 displays real sample analysis of Hcy in (a) human blood serum and (b) human urine sample.

DETAILED DESCRIPTION OF THE INVENTION WITH ILLUSTRATIONS AND EXAMPLES
While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of “a”, “an”, and “the” include plural references. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The system, methods, and examples provided herein are only illustrative and not intended to be limiting.

The abbreviations used in the invention are represented in table 1 as below:
Table 1: Legend of abbreviations
S.no. Particulars Legend
1 Iron molybdate FMO
2 Differential pulse voltammetry DPV
3 Cyclic voltammetry CV
4 Deionised water DI
5 Dimethylformamide DMF
6 Glassy carbon electrode GCE
7 Limit of detection LOD
8 Homocysteine Hcy
9 Square-wave voltammetry SWV
10 Carbon nanotubes CNT
11 Phosphate buffer solution PBS
12 Relative standard deviation RSD
13 Acetate buffer ABS

Some of the technical terms used in the specification are elaborated as below:
- Electrochemical sensor: Sensors that convert the information associated with electrochemical reactions (the reaction between an electrode and analyte) into an applicable qualitative or quantitative signal.
- Linear range: Linear range or linear dynamic range refers to the range of concentrations where the signals are directly proportional to the concentration of the analyte in the sample.
- Limit of detection (LOD): The 'Limit of Detection' (LOD) is the lowest concentration of a substance in a sample that can be consistently detected with a certain level of certainty, typically 95%.
- Differential pulse voltammetry (DPV): It is a highly sensitive and low detection limit electrochemical technique and is more appropriate for irreversible systems, since the electron transfer kinetics in the electrode surface is slow.
- Cyclic Voltammetry: Cyclic voltammetry (CV) is an electrochemical technique used to study the redox properties of a chemical species. It provides information about the electron transfer processes that occur during oxidation and reduction reactions.
- Subject: A patient, person, or organism that is the object of research, treatment, experimentation, or dissection. Here in the description the term subjects include humans or animals who have been researched upon for detection of homocysteine. The term includes the patients or persons who undergo tests for detection and quantification of homocysteine.

The reference numerals used in the present invention are tabulated below in table 2.
Table 2: Legend of Reference numerals
Ser No. Item description Reference signs/ numerals
1 Medical device D
- Electrochemical sensor E
• Working electrode WE
• Counter electrode CE
• Reference electrode RE
• Electrochemical cell EC
• Supporting electrolyte EE
- Potentiostat EP
- Computer monitor EM
- Power source P
2 Subject/ Patient S

Homocysteine (Hcy) or 2-amino-4-mercapatobutyric acid is a non-proteinogenic a-amino acid consisting prominent thiol group and play significant role in many biological courses like metabolism. They are highly useful for clinical applications as they can act as biomarker for number of diseases like Alzheimer's, cardiovascular risks and loss of cognitive utility. Homocysteine is therefore closely associated with wellbeing of humans as it is involved in various biological reactions taking place in human body. The appropriate level of homocysteine in a healthy person is in the range of 5 µM- 15 µM. The elevated values reveal the unhealthiness of human body. The abnormal homocysteine ranges are identified as potential reason behind Parkinson’s disease, stroke, cancer and they can even act as an efficient biomarker for cardiovascular diseases. The utility of homocysteine to forecast risks due to COVID 19 has also been reported in recent studies. All these facts emphasize the need for regular monitoring of homocysteine in human body.

STATEMENT OF INVENTION
The present invention discloses a medical device (D) for detection and quantification of homocysteine that comprises of one electrochemical sensor (E) of three electrode configuration. The electrochemical sensor (E) in turn comprises of one working electrode (WE) where the primary electrochemical reaction due to homocysteine interacting with the electrode surface occurs to reveal its presence and quantification. The sensor (E) also comprises of one counter electrode (CE) to pass all the current needed to balance the current observed at the working electrode (WE). The sensor also comprises of one reference electrode (RE) to act as a reference in measuring and controlling the working electrode potential, without passing any current. There is one electrochemical cell (EC) for accommodating the analyte and various components of the electrochemical sensor (E). A supporting electrolyte (EE) to maintain charge neutrality within the electrochemical cell (EC) by allowing the movement of ions forms part of the electrochemical sensor (E).

The medical device (D) also comprises of a potentiostat or electrochemical workstation (EP), connected to the electrodes of the electrochemical sensor (E) for measurements by means of copper plated wires. The potentiostat (EP) is an analytical instrument to control the working electrode's potential and process the signals produced as a result of the electrochemical reactions occurring at the working electrode interface and convert it into readable signals. The device (D) includes one computer monitor (EM), connected to the potentiostat (EP), to display the readable signals indicating detection of homocysteine and its quantity, produced by the potentiostat (EP). There is a power source (P) that facilitates the connection of said potentiostat (EP) to the computer monitor (EM). The medical device (D) of the invention is differentiated in that the surface of the working electrode (WE) is modified using a nanocomposite material such as but not limited to Fe2(MoO4)3/FeS (FMO/FeS). The nanocomposite FMO/FeS utilized for modification of surface of working electrode (WE) is uniquely synthesised through a single step facile solvothermal method. The invention thereby provides an efficient, low cost lab scale medical device (D) that is accurate and sensitive providing a broad linear range of detection and quantification of homocysteine.

According to an embodiment of the invention the working electrode (WE) comprises of glassy carbon electrode. The counter electrode (CE ) comprises of platinum electrode and the reference electrode (RE) comprises of calomel electrode. The electrochemical analysis was done with techniques such as differential pulse voltammetry (DPV) and cyclic voltammetry (CV) using an electrochemical workstation. The medical device (D) of the present invention used for analyzing human blood serum is highly practicable and yields good results in detection. Figure 1 illustrates the detailed diagram of the medical device of the present invention with all constructional features.

The synthesis of nanocomposite FMO/FeS is carried out through a facile solvothermal method comprising of steps of adding specific quantities of precursors including Ferric Chloride Hexahydrate, Ammonium Molybdate Heptahydrate and Thiourea to a solution mixture containing specific amount of Deionized water (DI) and Dimethylformamide (DMF) in the range of 15.0 -50.0 ml. The solution mixture containing all the precursors is stirred thoroughly for about 10.0 min to 25 mins at room temperature. The solution mixture is then moved to a Teflon vessel in an autoclave and kept in oven for time ranging from 18 to 30 hours at a reaction temperature ranging from 150 - 250 °C. The reaction is allowed to complete and subsequently black coloured precipitate as obtained is collected. The precipitate is washed twice in DI water and three times in DMF. It is then dried for a time in the range of 18 hrs to 30 hrs at a temperature in the range of 40 to 80 °C to obtain a powder sample of nanocomposite FMO/FeS. This powder obtained is collected to be used to modify surface of working electrode (WE) of the medical device (D) for detection of homocysteine.

The method of fabrication of the medical device (D) for detection and quantification of homocysteine comprises of following steps. The surface of working electrode (WE) of the electrochemical sensor (E) with three electrode configuration is modified using a nanocomposite material such as Fe2(MoO4)3/FeS that is synthesized using a single step facile solvothermal method, to obtain a surface modified working electrode (WE). This surface modified working electrode (WE) is immersed in specific amount of supporting electrolyte (EE) such as phosphate buffer with pH value 9.0 taken in the electrochemical cell (EC) of the electrochemical sensor (E) of the medical device (D) along with reference and counter electrodes (CE). The analyte that has to be analyzed for detection and quantification of homocysteine is added to the electrolyte. The electrochemical reaction is allowed to take place at the interface between the modified working electrode (WE) and the electrolyte at a temperature ranging between 15? to 35 ? with the voltage window applied ranging between 0.0 to +1.6 V, to produce electrochemical output. The output is analyzed and processed at a potentiostat/ electrochemical workstation (EP), connected to the electrochemical sensor (E), using analytical techniques such as DPV and CV. The electrochemical output is then converted by the potentiostat (EP) into readable signals by correlating the electrochemical output with the homocysteine concentration in the analyte using a pre-established calibration curve. The readable signals indicating detection of homocysteine detected and its quantity are then displayed at a computer monitor (EM).

The specifications of the various materials and components of the medical device (D) are described in detail in the following paragraphs.

The medical device of the present invention comprises mainly of an electrochemical sensor (E) that comprises of a typical three electrode system to include glassy carbon electrode used as working electrode (WE), Calomel electrode (SCE, Hg/Hg2Cl2 in saturated KCl) used as reference electrode (RE), and platinum electrode used as a counter electrode (CE). The working electrode (WE) surface is modified with nanocomposite material such as but not limited to Fe2(MoO4)3/FeS (FMO/FeS). Phosphate buffer with pH value 9.0. in the range of 5- 15.0 ml is used as supporting electrolyte (EE). The working electrode (WE) is immersed into the electrolyte solution along with reference and counter electrode (CE). Upon addition of analyte into the electrolyte solution, the thiol groups get attached to electrode surface and Fe2+ favours the reactions occurring. When the homocysteine gets oxidised the Fe from FMO gets reduced and produces Fe2+. During the reaction, FeS can get oxidised to hydroxides and thereby providing more and more actives sites for facilitating the reaction. Hence the electrons generated due to the redox reactions constitute the current in here and as the concentration of homocysteine in the system increases peak current also increases. A typical scheme of an electrochemical cell (EC) set up with three electrode configuration is illustrated in figure 2.

As per an embodiment of the present invention, CH instrument is utilized to function as potentiostat and is connected to the three electrodes. CHI 610 E, CH instruments, USA, was specifically used in the invention for measurements by means of copper plated wires. CH instrument processes the signals produced as a result of electrochemical reactions occurring at the working electrode (WE) interface and converts it into readable signals in the computer monitor.

As described earlier, the surface of working electrode (WE) is modified with an innovative nanocomposite material and immersed into the electrochemical cell (EC) along with counter and reference electrodes (RE). The interaction between the surface coating material and homocysteine that is added to the system takes place wherein homocysteine analyte undergoes electrochemical oxidation at the surface of working electrode (WE). This enables the electrochemical detection of analyte viz. homocysteine. The specific interaction occurring produces the superior linear range of detection for the sensor.

Method of preparation/fabrication of the device as a whole including the method of modification of the glassy carbon electrode is described in detail as follows. Prior to surface modification, the electrode surface is carefully cleaned with alumina powder of different sizes, including 0.05 micron, 0.3 micron, and 1 micron from electrode cleaning kit. The homogenous suspension of FMO/FeS is prepared by dispersing 5.0 mg hybrid material in 150.0 µl of DI water using ultrasonication for approximately 15 min without adding any binder. From the dispersion prepared, about 5.0 µl is drop casted gently on electrode surface and allowed to dry at room temperature. This results in the fabrication of FMO/FeS/GCE that is employed for homocysteine quantification through procedures such as Differential Pulse Voltammetry (DPV) and Cyclic Voltammetry (CV). Figure 3 depicts an image of surface modified glassy carbon electrode.

The surface modified glassy carbon electrode is immersed in supporting electrolyte (EE) with PB pH 9.0. preferably about 10.0 ml taken in an electrochemical cell (EC) along with reference and counter electrodes (CE). The analyte is added in the said electrolyte. The electrochemical reaction taking place at the interface between the electrode and electrolyte is responsible for the electrochemical output obtained through the analytical techniques such as DPV and CV. DPV is preferably used for linear range study as it is more sensitive than CV.
The electrochemical oxidation of homocysteine happening can be expressed as follows. Homocysteine is oxidised to form disulfide bond and releases electrons as well as protons.
FMO (Fe3+)/FeS + 2Hcy (SH) ? FMO (Fe2+)/FeS + HcySScyH+ 2H+ + 2e-
All the electrochemical experimentations are preferably executed at 25 ?. The voltage window applied for the detection of homocysteine is 0.0 to +1.6 V. The pulse width of DPV were in the range of 0.01 V to 0.1 V, preferably 0.05 V and pulse amplitude of DPV in the range of 0.01s to 0.1s, preferably 0.05 s respectively. For the CV study scan rate is to be fixed in the range of 50 mV/s to 150 mV/s preferably as 100 mV/s. Figure 4 illustrates a schematic representation of electrochemical detection of homocysteine.

Fe2(MoO4)3/FeS (FMO/FeS) nanocomposite is an innovative material utilized in the device of the present invention and has been synthesized uniquely following a one-step solvothermal method in lab. The material has not been reported before for the construction of an electrochemical sensor (E). The created composite material is observed to balance out limitations of its original constituents and improves the electrochemical properties as a whole performing efficiently as part of an electrochemical sensor (E). The Fe2(MoO4)3/FeS nanocomposite is expected to possess an open 3D network, and the ratio between the constituents is maintained as 1:1.

The FMO/FeS nanoparticles are synthesized effectively through a one-step solvothermal method. As per the method about 0.374 g of Ferric Chloride Hexahydrate, 0.8 g of Ammonium Molybdate Heptahydrate and 0.7 g of Thiourea are added in to solution mixture containing 25.0 ml Deionized water and 25.0 ml DMF.

The quantities of precursors to be added are summarized as below.
Precursor Range of quantity (g) Quantity (g)
Ferric Chloride Hexahydrate 0.2- 0.5 0.374
Ammonium Molybdate Heptahydrate 0.5- 1.2 0.8
Thiourea 0.2- 1.2 0.7

The solution mixture containing all these precursors is stirred thoroughly for 15.0 min at room temperature as depicted in figure 5. The solution mixture is then moved to a Teflon vessel in an autoclave and kept in oven for 24.0 h at a temperature of 200 °C. After the completion of reaction, the generated black coloured precipitate is collected, washed twice in DI water and three times in DMF and then dried for 24.0 h at 60 °C. The powder sample is then collected and used to modify electrode. It is essential to maintain the reaction temperature is at 200.0 ? for 24.0 h for the synthesis of FMO/FeS. Figure 5 illustrates the method of preparation/fabrication of Fe2(MoO4)3/FeS (FMO/FeS).

The medical device (D) has applications in the field of clinical pathology finding relevance in various medical specialties such as but not limited to cardiology, neurology, genetics and metabolic medicine, internal medicine, endocrinology etc.

EXAMPLES
The present invention shall now be explained with accompanying examples. These examples are non-limiting in nature and are provided only by way of representation. While certain language has been used to describe the disclosure, any limitations arising on account of the same are not intended. As would be seeming to a person skilled in the art, various working alterations may be made to the method in order to implement the inventive concept as taught herein. The figures and the preceding description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, order of steps of method or processes of data flow described herein may be changed and is not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts need to be necessarily performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples.

The performance of the device of the present invention is compared to existing literature considering the electrochemical detection of homocysteine in terms of linear range and limit of detection (LOD) precisely. The electrochemical sensor (E) of the present invention shows broadest linear range starting from the normal Hcy level in blood as compared to existing literature. The sensitivity of the FMO/FeS/GCE is significant as well. Therefore, it can be established that electrochemical sensor (E) od the medical device (D) introduced in the present invention successfully delivers noteworthy electrochemical output in terms of linear range, sensitivity, reproducibility and repeatability. The table 3 illustrating the comparison of performance of the device of present invention with devices available in the prior art is presented below.

Table 3: Comparison study of important works discussing homocysteine analysis
Electrode Analyte Method Linear range LOD
N-GNs/Pt NPsa Homocysteine DPV
10-70 µM 0.20 nM
AuNP/rGO/GCEb
Homocysteine CV 2-14 mM 6.9 µM
BFCNPEc Homocysteine SWV 0.1-80 µM 50 nM
CNTPd
Homocysteine Amperometry 5-200 µM 4.6 µM
CNT/Nafion/GCE Homocysteine Amperometry 0.10-60 µM 0.06 µM
Hcyaptamer/Au NPs/GSe Homocysteine DPV 1-100 µM 1 µM
BSA-AuCNs/MXene/GCEf Homocysteine Amperometry 5.29 nM-103 µM 1.76 nM
AuNPs/AB-DHP/GCEg
Homocysteine Amperometry 3 µM – 1 mM 0.6 µM
FMO/FeS/GCE Homocysteine DPV 13µM-9061 µM 0.05 µM

Legend of suffixes used in the above table:
- aN doped graphene supported Pt nanoparticles
- bAu particle incorporated reduced graphene oxide
- cBenzoylferrocene and CNTs
- dCarbon nanotube paste electrode
- eHcy aptamer/ Au Nanoparticles/ Graphene sponge
- fMXene matrix supported with gold nanoclusters
- g Gold and acetylene black dihexadecyl phosphate film on GCE

The figure 6 successfully illustrates the voltammogram of bare GCE, FeS/GCE and FMO/FeS loaded GCE individually after the addition of 1500 µM homocysteine. The figure well establishes the significance of surface modification with FMO/FeS in promoting the electrochemical activity of GCE electrode towards homocysteine analyte. The oxidation current produced with modified electrode is two times larger than that obtained with unmodified electrode here. Moreover, the potential at which peak appeared was 1.2557 V for bare GCE but when it comes to modified electrode the peak potential shifted to 1.175 V, which is sign of electrocatalytic effect exhibited by the modified electrode of the present invention.

The optimization of experimental settings was performed by selecting the best supporting electrolyte (EE) medium for the sensor. And for this, various electrolytes like 0.1 M H2SO4, 0.1 M HCl, 0.1 M KCl, 0.1 M NaOH, 0.1 M acetate buffer (ABS) and 0.1 M phosphate buffer (PBS) were tested with the device of the present invention. Based on the result recorded, 0.1 M PBS (pH=7) gave adequate analytical response and thus is chosen for further investigations. In the next step, the pH value of PBS electrolyte had to be finalised to maximise the analytical result. To do that, the sensor's performance was examined using a 0.1 M phosphate buffer, whose pH ranges were 2 to 10. Figure 7 illustrates the graph obtained between pH and potential values at which peak appeared. Figure 7 also highlights the gradual change in amount of current produced as the pH strength varied. The peak current recorded in the acidic media was considerably lower as compared to that with alkaline surroundings. It is observed that electrochemical oxidation of Hcy at the FMO/FeS/GCE surface is more favourable under alkaline conditions and exhibited more peak current than even neutral condition. Additionally, the Fe3+ ions present catalyses the oxidation process and contribute to increment in peak current. However, anodic current reached maximum value when the pH was 9 and fell down after further increase. Therefore, PBS 9 was chosen as optimum pH here for subsequent studies.

The linear range of Hcy concentration, which can be detected with the device of the present invention is found to be 13 µM to 9061 µM and it is an exceptional value higher than reported till date as given figure 8(a). The linear regression equation framed with concentration of Hcy added against current produced is expressed as, Ipa (µA) = 15.95 + 0.0176 * C (µM) and the regression coefficient, R2= 0.986 as given in figure 8 (b). The medical device of the invention fabricated effectively unveiled significant linearity even with a broadest concentration range here. The limit of detection (LOD) of the sensor is calculated using the equation 3 s/b and it is gotten as 0.05 µM.

The reproducibility of FMO/FeS/GCE for the detection of Hcy was evaluated by executing trials with six electrodes. The oxidative current produced after the addition of 300 µM homocysteine recorded each time for the six electrodes and analysed with each other. The DPV graph of reproducibility analysis is given in Figure 9 (a). The sensor demonstrated impressive reproducibility with relative standard deviation (RSD) value as 3.682%. Six successive anodic peak current measurements in the presence of 300 µM Hcy are taken into consideration in order to assess the repeatability of the developed FMO/FeS/ GCE sensor. The medical device shows RSD value of 4.32% exposing the noteworthy repeatability of the device. The sensitivity of the electrode also has to be analysed to determine the efficiency of the sensor. Here in the sensitivity of the device sensor is acquired from the ratio of slope to area of electrode surface. The sensitivity of FMO/FeS/GCE is determined as 0.0235 µA/µM/cm2. It is imperative to verify the device sensor's selective analysis towards homocysteine to ensure that unique sensor remains free from the intervention of other electrochemically active species. The device of the present invention tested in the presence of other biomolecules like glucose, ascorbic acid, uric acid, sucrose, dopamine, serotonin and urea and amino acids such as cysteine and cysteamine and metal holding species like KCl, CaCl2, and NaCl. The selectivity of FMO/FeS/GCE towards Hcy substantiated in the presence of 300.0 µM Hcy and in the presence of excess interfering agents in PBS 9. The figure 9 (b) shows that no species succeeded in producing any serious impact to the electrochemical response of the sensor towards Hcy. In addition to that, the current variance procured lies within the negligible margin of 12%. Thus, the device sensor presented efficacious elimination of potential interfering candidates and proven its selectivity for the selected analyte and thereby illustrated anti-interference capability of sensor. The at rest stability of developed electrode was also evaluated by determining the current after 15 days in room temperature and yielded satisfactory results. The RSD value obtained was 2.56 %. In order to analyse the long-term stability, the current response of the one electrode was observed in the 1st day, 5th day, 7th day,10th day and at 15th day. This analysis obtained notable results with RSD value of 3.28 %. The DPV graphs are given in Figure 9 (c) and (d).

To validate the feasibility of thus developed FMO/FeS/GCE device sensor, Hcy analysis was performed in human serum with this innovative sensor by following typical standard spiking method.

Biological samples such as human blood serum and urine samples were used for the real sample study of the sensor for the electrochemical detection of homocysteine following traditional spiking approach.

For the purpose of real sample testing, about 50.0 µl of blood serum from a healthy subject (S) was added to the electrochemical cell (EC) possessing 9.95 ml PBS 9.0 solution followed by performing DPV analysis. Subsequently about 16.0 µM, 26.0 µM and 36.0 µM Hcy were added to the same cell and the peak current produced measured. This was repeated for three times to reassure the responses recorded. Similarly, about 1.0 ml of urine was collected from a healthy person to execute the real sample testing. Next about 50.0 µl urine was added to cell having 9.95 ml PBS 9.0 solution and DPV analysis carried out. Prefixed amount of Hcy such as 26.0 µM, 36.0 µM and 46.0 µM were added in to the above cell and the oxidation current produced logged. This step was repeated for three times. It is to be noted that the blood serum and urine samples used here are without any preprocessing.

The data entered in the table 4 confirmed the reasonable results obtained in the form of remarkable recovery rates. The real sample spiked with comparatively higher concentrations of Hcy since the linear range determined for the sensor is extended over a wide range. The recovery rates and RSD values recorded were within the margins of acceptable rates and this clearly expresses the efficiency of thus fabricated medical device sensor for the examination of homocysteine in human serum samples. The DPV graph of real sample analysis executed in blood serum and urine sample is displayed in Figure 10.

Table 4: Demonstration of FMO/FeS/GCE in human serum samples (n=3)
Sample Electrode Added (µM) Found (µM) Recovery (%) RSD (%)
Blood serum FMO/FeS/GCE 16 15.73 98.31 0.188
26 25.43 97.8 2.72
36 35.62 98.94 2.7
Urine

FMO/FeS/GCE
26 25.73 98.96 1.33
36 35.54 98.72 1.6
46 45.82 99.6 3.367

The primary advantage of the medical device(D) or the sensor of the present invention is that it possesses superior linear range of 13 µM- 9061 µM, which means that device has the efficiency to detect homocysteine if its concentration is in this range and it is higher than reported by any other system till now.
,CLAIMS:We claim:
1. A medical device (D) for detection and quantification of homocysteine, said medical device (D) comprising of
- one electrochemical sensor (E) of three electrode configuration for detecting and quantifying an analyte homocysteine, said electrochemical sensor (E), comprising of
• one working electrode (WE) where the primary electrochemical reaction due to homocysteine interacting with the electrode surface occurs to reveal its presence and quantification,
• one counter electrode (CE), to pass all the current needed to balance the current observed at the working electrode (WE),
• one reference electrode (RE) to act as a reference in measuring and controlling the working electrode potential, without passing any current,
• one electrochemical cell (EC) for accommodating the analyte and various components of the electrochemical sensor (E) and
• one supporting electrolyte (EE) to maintain charge neutrality within the electrochemical cell (EC) by allowing the movement of ions,
- one potentiostat (EP), connected to the electrodes of the electrochemical sensor (E) for measurements by means of copper plated wires, that is an analytical instrument to control the working electrode's potential and process the signals produced as a result of the electrochemical reactions occurring at the working electrode interface and convert it into readable signals,
- one computer monitor (EM), connected to said potentiostat (EP), to display the readable signals indicating detection of homocysteine and its quantity, produced by the potentiostat (EP) and
- power source (P) that facilitates the connection of said potentiostat (EP) to the computer monitor,
wherein
- said surface of working electrode (WE) is modified using a nanocomposite material such as but not limited to Fe2(MoO4)3/FeS (FMO/FeS) and
- said FMO/FeS utilized for modification of surface of working electrode (WE) is synthesised through a single step facile solvothermal method,
thereby providing an efficient, low cost lab scale medical device (D) that is accurate, sensitive and provides a broad linear range of detection and quantification of homocysteine.
2. The medical device (D) as claimed in claim 1, wherein said working electrode (WE) of electrochemical sensor (E) comprises of glassy carbon electrode.
3. The medical device (D) as claimed in claim 1, wherein said counter electrode (CE) of electrochemical sensor (E) comprises of platinum electrode including in the form of platinum wire.
4. The medical device (D) as claimed in claim 1, wherein said reference electrode (RE) of electrochemical sensor (E) comprises of calomel electrode.
5. The medical device (D) as claimed in claim 1, wherein said potentiostat (EP) facilitates electrochemical analysis of the analyte with techniques such as differential pulse voltammetry (DPV) and cyclic voltammetry (CV).
6. The medical device (D) as claimed in claim 1, wherein said medical device (D) possesses linear range of detection of homocysteine in the range of 13 µM- 9061 µM.
7. The medical device (D) as claimed in claim 1, wherein said synthesis of nanocomposite FMO/FeS is carried out through a facile solvothermal method comprising of steps of
- adding specific quantities of precursors including Ferric Chloride Hexahydrate, Ammonium Molybdate Heptahydrate and Thiourea to a solution mixture containing specific amount of Deionized water (DI) and Dimethylformamide (DMF),
- stirring the solution mixture containing all the precursors thoroughly for about 10.0 min to 25 mins at room temperature,
- moving the solution mixture to a Teflon vessel in an autoclave and keeping it in oven for time ranging from 18 to 30 hours at a reaction temperature ranging from 150 - 250 °C,
- allowing the reaction to complete and subsequently collecting black coloured precipitate as obtained,
- washing said black coloured precipitate twice in DI water and three times in DMF,
- drying the washed precipitate for a time in the range of 18 hrs to 30 hrs at a temperature in the range of 40 to 80 °C to obtain a powder sample of nanocomposite FMO/FeS and
- collecting the powder sample obtained in the previous step to be used to modify surface of working electrode (WE) of the medical device (D) for detection of homocysteine.
8. The medical device (D) as claimed in claim 7, wherein said quantities of precursors to be added are as below.
Precursor Range of quantity (g) Quantity (g)
Ferric Chloride Hexahydrate 0.2- 0.5 0.374
Ammonium Molybdate Heptahydrate 0.5- 1.2 0.8
Thiourea 0.2- 1.2 0.7

9. The medical device (D) as claimed in claim 7, wherein said amount of DI water and DMF to which the precursors are added are each in the range of 15.0 -50.0 ml preferably 25.0 ml.
10. The medical device (D) as claimed in claim 7, wherein said stirring of the solution mixture containing all the precursors thoroughly is preferably carried out for about 15.0 mins at room temperature.
11. The medical device (D) as claimed in claim 7, wherein said reaction temperature to be maintained in autoclave oven is preferably 200.0 ? and the time is preferably 24.0 h.
12. The medical device (D) as claimed in claim 7, wherein said drying of the washed precipitate is carried out for a time of about 24 hrs at a temperature of preferably 60°C to obtain the powder sample of nanocomposite FMO/FeS.
13. The medical device (D) as claimed in claim 1, wherein said supporting electrolyte (EE) utilized of the electrochemical sensor (E) comprises of phosphate buffer preferably of pH value 9.0.
14. The medical device (D) as claimed in claim 1, wherein said medical device (D) has applications in the field of clinical pathology finding relevance in various medical specialties such as but not limited to cardiology, neurology, genetics and metabolic medicine, internal medicine, endocrinology etc.
15. A method of fabrication of a medical device (D) for detection and quantification of homocysteine, steps of said method comprising of
- modifying surface of working electrode (WE) of an electrochemical sensor (E) of three electrode configuration that is part of said medical device (D) using a nanocomposite material such as Fe2(MoO4)3/FeS, synthesized using a single step facile solvothermal method, to obtain a surface modified working electrode (WE),
- immersing said surface modified working electrode (WE) in specific amount of supporting electrolyte (EE) such as phosphate buffer with pH value 9.0 taken in the electrochemical cell (EC) of the electrochemical sensor (E) of the medical device (D) along with reference and counter electrodes (CE),
- adding analyte for detection and quantification of homocysteine, to said electrolyte,
- allowing and executing an electrochemical reaction to take place at the interface between the modified working electrode (WE) and the electrolyte at a temperature ranging between 15? to 35 ? with the voltage window applied ranging between 0.0 to +1.6 V, to produce electrochemical output,
- analyzing and processing said obtained electrochemical output at a potentiostat (EP), connected to the electrochemical sensor (E), using analytical techniques such as DPV and CV,
- converting said electrochemical output from previous step by the potentiostat (EP) into readable signals by correlating the electrochemical output with the homocysteine concentration in the analyte using a pre-established calibration curve and
- displaying the readable signals produced by the potentiostat (EP) at a computer monitor (EM), indicating detection of homocysteine detected and its quantity.
16. The method as claimed in claim 15, wherein said step of modifying of surface of working electrode (WE) of the electrochemical sensor (E) of three electrode configuration comprises of steps of
- cleaning of said electrode surface with alumina powder of a plurality of sizes selected from group of 0.05 micron, 0.3 micron, and 1 micron from an electrode cleaning kit,
- preparing a dispersion of homogenous suspension of FMO and FeS by dispersing 5.0 mg of said hybrid material in 150.0 µl of DI water using ultrasonication for approximately 15 min without adding any binder and
- drop casting from the dispersion prepared about 5.0 µl on electrode surface and allowing it to dry at room temperature to obtain a surface modified working electrode (WE).
17. The method as claimed in claim 15, wherein said DPV is executed with the pulse width in the range of 0.01 V to 0.1 V, preferably 0.05 V and pulse amplitude in the range of 0.01s to 0.1s, preferably 0.05 s.
18. The method as claimed in claim 15, wherein said CV is executed with scan rate fixed in the range of 50 mV/s to 150 mV/s preferably as 100 mV/s.
19. The method as claimed in claim 15, wherein said amount of supporting electrolyte (EE) is in the range of 5- 15.0 ml preferably 10.0 ml.