Myoglobin biofunctionalized electrochemical biosensor platform for the dual determination of nitrite and H2O2 as oxidative stress markers

Myoglobin biofunctionalized electrochemical biosensor platform for the dual determination of nitrite and H2O2 as oxidative stress markers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Myoglobin biofunctionalized electrochemical biosensor platform for the dual determination of nitrite and H2O2 as oxidative stress markers Santharaman Paulraj, Karunakaran Chandran, Venkatesan Raju, Arun Thesingu Rajan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8131544/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract The myoglobin (Mb) biofunctionalized carboxylic acid functionalized multi walled carbon nanotubes (FMWCNT) modified polypyrrole (PPy) electrodeposited screen printed carbon electrode (SPCE) showed an excellent biocatalytic activity towards the one electron redox reaction of nitrite and the reduction of H 2 O 2 . The direct electron transfer of the Mb immobilized into the FMWCNT was greatly facilitated. The influence of various experimental conditions was examined for the characterization of fabricated biosensor for optimum analytical performance. The each and every step of surface morphological changes were studied by scanning electron microscope (SEM) while electrochemical changes were monitored via cyclic voltammetry (CV). The voltammetric response of the fabricated biosensor platform varied linearly with varying NO 2 – concentration of 0.1 to 800 µM by a detection limit 0.1 µM. Further, the electrochemical response of the constructed biosensor changed linearly into the H 2 O 2 concentration ranges between 1 to 600 µM by the detection limit of 0.5 µM. Moreover, a low cost electrochemical virtual cyclic voltammetric analyzer was developed based on home-made potentiostat and graphical user-interface software LabVIEW 10.0. The performance of the developed virtual analyzer has been further evaluated by applying it for the measurement of NO 2 – and H 2 O 2 in L-NAME treated embryonic rat heart-derived H9c2 cardiomyoblasts and the results are validated with commercial electrochemical analyzer. Myoglobin biofunctionalized cardiomyoblasts biosensor LabVIEW L-NAME Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Hypoxia, a condition of low oxygen can cause oxidative stress (OS), which has been associated with several diseases such as atherosclerosis, cardiomyopathy, ischemia reperfusion injury, congestive heart failure, rheumatoid arthritis, chronic fatigue syndrome, and also affect the central nervous system can lead to Alzheimer’s disease, Amyotrophic lateral sclerosis (ALS), Huntington’s disease and Parkinson’s disease [ 1 – 9 ]. The mechanism through OS forced to damage heart function includes oxidative devastation to cellular membranes and proteins, thus prompting cellular loss or dysfunction. It arises while the formation of free radicals and active intermediates in the biological system exceeds its ability to neutralize and exclude them [ 10 – 12 ]. Reactive oxygen species (ROS) viz. hydroxyl radical (HO·), hydrogen peroxide (H 2 O 2 ), superoxide radical (O 2 •- ), and singlet oxygen ( 1 O 2 ) and reactive nitrogen species (RNS) viz . peroxynitrite (ONOO ། ) and nitric oxide (NO) are frequently produced in biological conditions [ 13 , 14 ], is the critical occurrence in living systems. Thus, ROS and RNS are commonly known as reactive species (RS). They react with biological components such as carbohydrates, proteins and lipids with ensuing modifications in the intracellular and intercellular homeostasis, leads to possible cell death and regeneration [ 15 – 17 ]. Indirect evidence via monitoring RS indicates, oxidative damage may be caused in the pathogenesis of these sicknesses [ 18 , 19 ]. Particularly, the accurate determination of RS of H 2 O 2 and NO 2 – are of great importance because they are involved in the etiology of ageing, progressive neurodegenerative and cardiovascular diseases [ 20 , 21 ]. Further, ROS is also able to generate other forms of RS. Particularly, H 2 O 2 have the capacity to produce additional destructive ·OH, via a blend of the Fenton and Haber-Weiss reactions. Furthermore, H 2 O 2 is an essential mediator in various significant field analyses [ 22 – 24 ]. On the other hand there has been growing interest about the role of nitrite anion as an important precursor in the formation of N-nitrosamines, many of these have been shown as significant carcinogens in human physiques [ 25 , 26 ]. However, various methods have been established to determine both nitrite and H 2 O 2 , such as spectrophotometry [ 27 – 29 ], chromatography [ 30 , 31 ] capillary electrophoresis [ 32 – 34 ], chemiluminescence [ 35 , 36 ] and electrochemistry [ 37 – 39 ]. Especially, the biosensors based on the electrochemical techniques such as voltammetry, coulometry, amperometry and impedance are promising with potential sensitivity, selectivity, low cost, fast response, easy to fabricate, portability and real-time monitoring ability [ 40 , 41 ]. However, if cofactors are shielded by the apoprotein, it is often durable to attain an effective electronic contact among the enzyme with the signal transducer then the controlled contact of the redox active sites inhibits facile electron transfer (ET) concerning the sensing area [ 42 ]. Since, the approach of direct electron transfer (DET) between the active center of the protein and sensor surface to resolve this issues [ 43 ]. The critically important step in the biosensor design is the consideration of the recognition surface [ 44 ]. Screen-printed electrode provides advantages such as easy miniaturization, low cost, disposable nature due to their large-scale making competency and portability, making potential on-site detection of various target molecules [ 45 , 46 ]. Further, modification of sensor surfaces can improve protein interaction with transducers, thus assisting DET and/or electrocatalysis [ 47 ]. Moreover, nanoparticles play variety of roles in different biosensing systems based on their unique physical, chemical and electrocatalytic properties. The binding of nanoparticles onto the sensor platforms drastically improves the conductivity then ET from the redox moieties to create them electroanalytical sensors [ 48 ]. Iijima’s discovery of carbon nanotubes (CNTs) in 1991 marked the beginning of a new era in the field of nanotechnology [ 49 ]. Owing to their exceptional characteristics such as size, hollow geometry, high surface area (> 1500m 2 /g) and high aspect ratio, CNTs have been the subject of considerable research aimed at electrocatalytic with sensing purposes including construction of electrochemical sensors also biosensors platforms [ 50 , 51 ]. The enriched electrochemical reactivity of H 2 O 2 and NADH at CNT adapted platforms creates these nanomaterials exceptionally attractive for abundant oxidase and dehydrogenase centered biosensors [ 52 ]. Moreover, CNT functionalization can be used to bind nearly any chosen substance to them, which permit us-for instance-to boost the biocompatibility and solubility of the nanotubes [ 53 ]. Recent years, the application of virtual instrumentation (VI) for the electroanalytical measurement have substantial interest since it affords practical benefits, such as low expense of fabrication, operation simplicity and suitability for real-time monitoring [ 54 , 55 ]. Earlier, we developed and reported the virtual instrumentation performing cyclic voltammetry using home-made potentiostat coupled with data acquisition system processed by LabVIEW software for nitric oxide [ 56 ]. The potential applications of this VI are used here for the determination of nitrite (NO 2 – ) and H 2 O 2 using the developed electrochemical biosensor. In this work, the biosensor systems were designed and constructed based on the redox activities of Myoglobin (Mb), a kind of heme protein which is an perfect substance for the characterization of electron transfer responses of sensing and redox proteins [ 57 , 58 ], covalently bonded on FMWCNT-PPy coated SPCE by EDC and NHS for NO 2 – and H 2 O 2 sensing. Cyclic voltammetry (CV) measurements are used to study the electrochemical behavior of the developed biosensor while SEM characterize the electrode surface morphologies. Moreover, the biosensors were developed to detect NO 2 – and H 2 O 2 in cultured biological samples using LabVIEW. 2. Experimental 2.1. Chemicals and reagents Myoglobin from equine heart (M1882), pyrrole, carboxylic acid functionalized multi-walled carbon nanotube (755125), chitosan (Chit), sodium nitrite, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium chloride, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), diethylenetriaminepentaacetic acid (DTPA), were purchased from Sigma Aldrich (USA). Acetic acid was purchased from Alfa Aesar. All the solutions were made through deionized water. 2.2. Instruments The morphological variations of the functionalised electrodes surface were studied using FEI Quanta FEG High Resolution scanning electron microscope (SEM) (FEI Co., Netherlands). CV characterization were carried out by CHI 1200B electrochemical workstation (CHI, USA). SPCEs were used as the sensor podia for designing the sensor (Zensor R&D, Taiwan). The SPCE strip consisting of carbon working, carbon counter and an Ag/AgCl reference electrode. The exterior area of the working electrode is 0.0071 cm –2 . National Instruments supply MyDAQ instrument having digital input, output (DIO), analog input (AI), analog output (AO) functions through USB connectivity. LabVIEW 10.0 software offered by National Instruments was helped to create the electrochemical regulator programs. PC running windows 2007 with XP was monitored for observing the results. 2.3. Fabrication of myoglobin biofunctionalized FMWCNT-PPy-SPCE 2.3.1. Electropolymerization of pyrrole on SPCE Electropolymerization of pyrrole on SPCE was followed our earlier procedure. Prior to deposition of PPy, the sensing surface of SPCE was electrochemically cleaned by using 40 µL 1 M H 2 SO 4 and cycling the voltage value at -0.5 V to 1.0 V for 10 cycles at 100 mV s − 1 and then cleaned with double distilled water. The major role of pretreatment process was to eliminate the excess organic ink ingredients, impurities and to rise the electrochemical behavior of the working area of the electrode surface [ 59 ]. After pretreatment, 10 complete cycles of pyrrole was deposited on to SPCE by the irreversible process of 0.4 M pyrrole in a auxiliary electrolyte of 0.1 M KCl, through giving a voltage of 0 to 0.9 V vs. Ag/AgCl with a scan rate of 50 mV/s [ 60 , 61 ]. 2.3.2. Myoglobin biofunctionalization on FMWCNT-PPy-SPCE After the preparation of PPy-SPCE, FMWCNT nanoparticles were immobilized onto the PPy-SPCE. For this first, 2 h sonicating 2 mg of FMWCNTs in 200 µL (1.0% m/m) Chitosan (Chit) in 1.0% (v/v) acetic acid to form FMWCNTs in 1.0% (m/m) chitosan dispersion. Then, on the surface of PPy-SPCE, drop 10 µL of this dispersion and kept to dry. After the initial drying, additional aliquot of 10 µL of the same dispersion was dropped and the sensor surface was further left for drying at room temperature for 1 h in air. By rinsing the electrode surface with PBS (pH 7.0) to activate the modified electrode and this step was repetitive twice so as to deprotonate the NH 2 – groups of Chit by altering the pH at the constructed sensor area [ 51 ]. In addition 5 µL of a blend comprising EDC (200 mM) and NHS (50 mM) in water was placed onto the electrode for nearly 10 min. Then the excess EDC–NHS mixture was detached by washing the surface of the working sensor platform by using 0.1 M PBS. The Mb enzyme biofunctionalized sensor was made by placing 5 µL of Mb (4 mg/mL) onto the exterior of FMWCNT-PPy-SPCE for one day [ 62 ]. The electrode was then washed with 0.1 M PBS to eliminate any electrostatically occupied Mb and washed by water earlier use. Figure 1 represents the construction of Mb biofunctionalized FMWCNT-PPy nanocomposite modified screen printed carbon electrode surface. 2.4. H9c2 cardiomyoblasts cell culture H9c2 embryonic rat heart cardio myoblasts were cultured in Dulbecco's adapted Eagle's medium (DMEM) through excess glucose. The supplementary media was supplied through Penicillin- Streptomycin with fetal bovine serum (FBS-10%) in a humidified 5% CO 2 maintained CO 2 incubator at 37°C [ 63 , 64 ]. Cells were consistently passaged when they reached 70–80% confluency by detaching them with Trypsin-EDTA. For this, cells were initiated for the experiment by seeding them in 60 mm dishes to achieve an initial density of 30%. They were maintained in culture until they reached approximately 60–70% confluency. Following this growth period, the media was changed, and the cells were stimulated as described in the subsequent steps. To prepare for analysis, the cells were detached using Trypsin-EDTA, centrifuged 10 minutes with a speed of 1500 rpm at 4°C, and the resulting pellet was re-suspended in 150 µl of 1X PBS. This suspension was then used for analysis with various biosensors. 3. Results and discussion Nowadays, there is an emerging interest for the invention of cost effective instruments for the measurement of RS. So, we have developed a virtual electrochemical analyzer for RS viz. H 2 O 2 and NO 2 – by using home-made potentiostat coupled with LabVIEW 10.0 (graphical user interface software). In order to test the performance of the developed biosensor, linearity, detection limit, sensitivity, selectivity and effect of scan rate & pH were investigated using standard physical voltammetric instruments. Further, the linearity and sensitivity of the biosensor were also checked by developed virtual electrochemical analyzer. 3.1. SEM characterization of functionalized biosensor platforms The surface morphology of bare SPCE, PPy-SPCE and FMWCNT-PPy-SPCE platforms were examined by SEM. Fig. 2. (a), (b) and (c) illustrate the morphological pictures of bare SPCE, PPy-SPCE, and FMWCNT-PPy-SPCE sensor platforms correspondingly. Image 2.b exhibits the typical vastly porous morphology of PPy matrix onto the SPCE electrode platform. This great porous nature of PPy probably offers considerable larger surface zone to bind further FMWCNTs, and Mb at the biosensor surface. Further, image 2.c displays the FMWCNT were uniformly deposited on to the PPy-SPCE matrix. Further, it shows the highly microporous structure, favorable for high loading of Mb. 3.2. Conformation of Mb immobilization on the modified electrode surface The direct electrochemistry of Mb modified SPCE was investigated by cyclic voltammetry. Fig. 3. shows the CVs of SPCE, PPy-SPCE, FMWCNT-PPy-SPCE and Mb-FMWCNT-PPy-SPCE (curve a, b, c and d correspondingly) electrodes in 0.1 M PBS of pH 7.0 with 0.1 M KCl as a supportive electrolyte and a scan rate of 50 mVs -1 . In the specified voltage range, there were no signifying Faradaic ET process because there were no significant redox peaks showed for bare SPCE, PPy-SPCE and FMWCNT-PPy-SPCE. Additionally, there were noticeable broad peaks detected on the CV ranges of -0.8 V to +0.2 V for PPy-SPCE and FMWCNT-PPy-SPCE exteriors probably because of the complex redox process of PPy and high capacitive currents [65]. On the other hand, the coverage of FMWCNT matrix on PPy-SPCE surface ensued in an observable increase in anodic and cathodic currents (Fig. 3, curve c) as compared with the previous PPy-SPCE and bare SPCE surfaces. This rise in peak current response is presumably owing to the rising ET rate of the FMWCNT. Moreover, when the binding of Mb on the FMWCNT-PPy-SPCE, a duo of typical reversible redox peaks at -0.2 and -0.38 V vs . reference electrode. This electrochemical response related to the reversible transformation among Mb–Fe (III) and Mb–Fe (II) redox pair and the nanocomposite altered electrode surface [66]. Based on the cyclic voltammetry, the formal potential (E 0 ) of Mb, determined by the midpoint of redox peak potentials, was −260 mV ( vs . Ag/AgCl), and the peak-to-peak potential change (ΔE p ) was 165 mV. The effect of pH and the scan rate of the biosensor was also studied [67]. 3.3. Electrochemical response to NO 2 – The conversion of nitrite ion to nitric oxide (NO·) is triggered by low oxygen strain. However this process modifies mitochondrial respiration and hypoxic NO· signaling and confines myocardial infarction in mammalian health, the pathways to nitrite bioactivation remain uncertain [68]. Research recommend that hemoglobin and myoglobin may subserve a crucial physiological role as hypoxia reliant enzyme nitrite reductases [69]. The research conducted by Ulrike and his group show that myoglobin is the cause for nitrite-dependent NO· production and cardiomyocyte protein iron-nitrosylation [68]. The mechanism of this reaction is Mb-Fe 2+ + NO 2 – + e – → Mb-Fe 3+ + NO· The electrochemical study of the Mb bonded electrode in several concentrations of NO 2 – by the same scan rate are shown in Fig. 4. The anodic current response increases with corresponding with increasing concentration linearly at the potential of 0.8 V. The electrochemical response obtained for NO 2 – is 0.8 V as previously reported by Madasamy et al and Balamurugan et al [65,70]. It is attributed to the response of electrochemical conversion of NO 2 – to NO by a cyclic redox reaction of Mb active site Fe(II/III) moiety. The concentrations of NO 2 – was plotted with the observed anodic peak currents as shown in Fig. 5. The linear calibration curve was obtained for NO 2 – exhibited linear range from 100 nM to 800 µM but for visual clarity in the accompanying fig. the range from 100 µM to 800 µM (r 2 = 0.965 and n = 3) is existing in a detection limit of 100 nM and the sensitivity of 22.763 nA µM – 1 . In addition, the linear calibration measurements of nitrite was done in developed LabVIEW program. Fig. 6. represents the front panel view of virtual instrumentation for the electrochemical responses of the Mb-FMWCNT-PPy-SPCE electrode in (a) 100 µM (b) 200 µM, (c) 400 µM, (d) 600 µM and (e) 800 µM of NO 2 – solution in 0.1 M PBS by the scan rate of 50 mVs -1 vs. Ag/AgCl. 3.4. Electrochemical response to H 2 O 2 The electrochemical oxidation of H₂O₂ to H₂O is facilitated by a cyclic redox reaction involving the iron (Fe(II/III)) centre at the Mb active site. Figure 7 shows the electrochemical behaviors of the Mb-FMWCNT-PPy-SPCE electrode to different concentrations of H₂O₂ in 0.1 M PBS, measured in a scan rate of 50 mV s⁻¹ against the reference electrode of Ag/AgCl. For instance the concentration increases, the response of the anodic current value is too linearly rises at -0.4 V. The reaction mechanism of the electrochemical response by myoglobin was given as follows Mb-Fe 3+ + e - → Mb-Fe 2+ Mb-Fe 2+ + H 2 O 2 + 2H + + e - → Mb-Fe 3+ + 2H 2 O which gives the overall reaction: H 2 O 2 + 2H + + 2e - → 2H 2 O The obtained anodic peak currents against H 2 O 2 concentrations were graphed as given in Fig. 8. The plotted curve thus obtained demonstrates a linear range of response over the concentration of H 2 O 2 from 1 µM to 600 µM but for simplicity here we have revealed from 100 µM to 600 µM (r 2 = 0.973 and n = 3) with the detection limit of 1 µM and the sensitivity 74.26 nA µM – 1 . Further, Fig. 9. represents the front panel view of LabVIEW for the electrochemical responses of (a) 100 µM (b) 200 µM, (c) 300 µM, (d) 400 µM, and (e) 500 µM of H 2 O 2 solution in 0.1 M PBS at the scan rate of 50 mVs -1 with Ag/AgCl by using the fabricated Mb-FMWCNT-PPy-SPCE biosensor. 3.5. Stability and reproducibility Stability and reproducibility are two significant factors for the assessment of any biosensors to be functional for therapeutic diagnosis. The stability of the biosensing platform was monitored periodically using CV and the results confirmed that no noticeable decrease in current level was observed after storage in 4 weeks (data not shown). The reproducibility of Mb based biosensor was separately examined in 0.1 M PBS containing 100 µM NO 2 – and H 2 O 2 by repetitive determination in triplicate respectively. Due to its robust reproducibility, demonstrated by low relative standard deviations (3.6% for NO 2 – and 5.2% for H₂O₂) across three distinct Mb-based biosensors, this electrochemical method could be a viable candidate for clinical trials. 3.7. Application of Mb biofunctionalized FMWCNT-PPy-SPCE biosensor The developed biosensor was used for the detection of NO 2 – and H 2 O 2 in cultured H9c2 cells under stress induced condition by different concentrations of L-NAME using standard cyclic voltammetry. In order to further investigate NO 2 – and H 2 O 2 measurements in real samples was performed in LabVIEW instrument by using the fabricated biosensor. The nitrite and H 2 O 2 levels of cardiac H9c2 cells treated with different concentration of L-NAME for 1 hour to induce stress were estimated using both standard CV instrument and LabVIEW. For this experiments, cells treated with 0.1 mM L-NAME for 1 hr, cells kept at 1 mM L-NAME for 1 hr and cells kept without L-NAME treatment. H9c2 cardiomyocytes cell lines were treated with L-NAME to induce the stress and make the variation in both nitrite and H 2 O 2 . Before that, 0.1 M CA membrane was coated onto the Mb-FMWCNT-PPy-SPCE electrode surface and eliminate possible interferences viz. NO, ascorbic acid present in the sample [65,71]. After that, the amount of NO 2 – was determined by interposing the current level into the standardization plot prepared by the standard NO 2 – solutions and the obtained results were given in table 1. The NO 2 – levels in H9c2 cells were in control (0.61 ± 0.18), 0.1 M L-NAME (1.02 ± 0.15), and 1.0 M L-NAME (1.58 ± 0.20) by using LabVIEW program. As expected, these results were comparable with the NO 2 – determination done by standard CHI instrument. These results thus confirm the suitability of the LabVIEW for the quantification of cellular NO 2 – levels by using constructed Mb-FMWCNT-PPy-SPCE biosensor. Similarly, Nafion coated Mb-FMWCNT-PPy-SPCE biosensor was used to eliminate the possible interference substances viz. AsA, UA and NO 2 – during the measurement of H 2 O 2. Afterward, the amount of H 2 O 2 was meausred by interposing the acquired current values into the calibration plot able by the standard H 2 O 2 solutions and the obtained results were displayed in table 1. The H 2 O 2 levels present in H9c2 cells were in control (0.39 ± 0.20), 0.1 M L-NAME (0.68 ± 0.30), and 1.0 M L-NAME (0.98 ± 0.25) by using LabVIEW. S. No Samples treated with different conc. of L-NAME Nitrite conc. by standard CV using Mb biosensor (µM) Nitrite conc. by LabVIEW using Mb biosensor (µM) H 2 O 2 conc. by standard CV using Mb biosensor (µM) H 2 O 2 conc. by LabVIEW using Mb biosensor (µM) 1 2 3 Control 0.1 M 1 M 0.58±0.12 0.98±0.16 1.46±0.08 0.61±0.18 1.02±0.15 1.58±0.20 0.42±0.25 0.75±0.20 0.98±0.30 0.39±0.20 0.68±0.30 0.98±0.25 3.8. Conclusion The electrochemical biosensor for NO 2 – and H 2 O 2 was developed by biofunctionalizing Mb as a biorecognizing element to FMWCNT on PPy nanocomposite based on SPCE. The FMWCNT nanoparticles assist the ET among Mb and the sensing electrode surface and afford additional binding spots for the immobilization of Mb, hence significantly increasing the sensitivity of the biosensor. The biosensor revealed good reproducibility, stability, low detection limit and a wide linear range. Moreover, a cost effective graphical user-interface software LabVIEW 10.0 based electrochemical analyzer for the concurrent measurement of NO 2 – and H 2 O 2 was developed by using home-made potentiostat. The electroanalytical performance of the biosensor responses obtained by using LabVIEW were also related with the standard CV results and found in correlation with each other. Declarations Author Contribution P.S and C.K design the work and writing the manuscript.R.V helps to study the morphalogical characterization.T.A helps to measure the calibration in labview. A.M, T.R.K, C.M, S.D helps to edit and correct the manuscript. Acknowledgement This work was supported by the Managing Board of Virudhunagar Hindu Nadar’s Senthikumara Nadar College (Autonomous), Virudhunagar, Tamil Nadu, India and G.Venkataswamy Naidu College (Autonomous), Kovilpatti - 628502, Tamil Nadu, India. References Erica N.C., Vincent, P., 2012. Life, 64 (1), 72-80. Venket, R., Balachandran, B., 2002. Nutritional Neuroscience 5 (5), 291-309. Taibur, R., Ismail, H., Towhidul Islam, M.M., Hossain, U.S., 2012, Adv Biosci. Biotech. 3, 997-1019. Saber, H., Syed, F. Ali., 1998. Antioxidant enzymes developmental profiles and their role in metal-induced oxidative stress. William Slikker, Jr., Louis W. Chang (Eds). Handbook of Developmental Neurotoxicology, Academic Press, Medical. Sian, C.B., Pamela, J.S., 2010. Free Radic. Biol. Med. 48, 629-641. Derek, A.D., Manisha, P., 2008. Free Radic. Biol. Med. 44, 1873-1886. Rodrigo, A.Q., Juan, A.O., Rommy, V.B., 2012. Arch. Med. Res. 43, 632-644. Shigenaga, M.K., Hagen, T.M., Ames, B.N., 1994. Proc. Natl. Acad. Sci. USA, 91, 10771- 10778. Shyamal, K.G., Nilanjana, M., Dipak, K.D., 2007. Annals of Medicine 39, 275-289. Khalid R., 2007. Clin Interv Aging 2 (2), 219-236. Gino, A.K., Rashmi, R., Srinivasan, V., Mohanraj, R., 2016. Oxidative Medicine and Cellular Longevity 1-14. Simone, F., Adrienne, M.G., Osamu, H., Afshin, S., 2010. Int J Cell Biol. 1-23. Ayla, O., Metin, O., 2015. Biochemistry of Reactive Oxygen and Nitrogen Species, Sivakumar Joghi Thatha Gowder (Ed.), Basic Principles and Clinical Significance of Oxidative Stress. InTech. 37-58. Edwin, H., Keyvan, K.G., Chia-Chi, L., Ravi, B., Gemma, A.F., 2013. Red. Biol. 1, 483-491. Alessandro, F., Floriana, M., Concetta, T., Carmela, L., 2007. International Journal of Cancer 121 (11), 2381-2386. Min, Z., Alessia, P., Alessandra, G., Emilio, H., Ajay, M.S., 2013. Antioxid Redox Signal. 18 (9), 1024-1041. Santosh, K., Sahdeo, P., Sandhya, L.S., 2013. PLOS ONE 8 (7), 1-12. Mrakic-Sposta, S., Maristella, G., Michela, M, Simone P., Alessandra, V., 2014. Oxidative Medicine and Cellular Longevity 1-10. Ramón, R., Matías, L., Felipe, F., Daniel, H., 2013. Disease Markers 35 (6), 773-790. Jian-Ming, L. Peter, H. L., Qizhi, Y., Changyi, C., 2010. J Cell Mol Med. 14 (4), 840- 860. Barry, H., Matthew W., 2004. Br J Pharmacol. 142 (2):, 231-255. Xueping, C., Chunyan, G., Jiming, K.,2012. Neural Regen Res. 7 (5), 376-385. Poljsak, B., Milisav, I., 2012. Oxid Med Cell Longev. 1-12. James P.K., 2000. Toxicology 149, 43-50. Rosana, A.M.,Tomás E.B. , Ana M.B. , Velia M.S. , Silvia A. L.B., 2011. American Journal of Analytical Chemistry 2, 683-688. Stuart, W.K., William, A.M., Daniel, L.M., David, H., Paul, W., 2013. Water Research 47, 4433-4450. Flamerz S., Bashir, W.A., 1981. Analyst , 106 , 243-247. Raquel, F.P.N., Mirela, C.O., Willian, C.P., 2005. Talanta 66 (1), 86-91. Philip, A.C., Dennis, F.E., Tippu, S.S.S., 1989. Anal Chem. Acta 218, 331-334. Kissner, R., Koppenol, W.H., 2005. Methods Enzymol. 396, 61-8. Steinberg, S.M., 2013. Environ Monit Assess. 185 (5), 3749-57. Asopuru, A.O., Herbert, H.H., Steven, G.M., Michael, A.B., 1999. Journal of Chromatography A, 844, 387-394. David, S.H., Anuja, C., Carrie, A.C.W., Julie, G., Paul, K., 1998. J. Chem. Educ. , 75 (12), 1588. Samuele, M., Elisa, R., Marco, B., Orfeo, Z.,2009. J. Environ. Eng. Manage., 19 (5), 307-314. Enika, N., Joseph, M.R., 2010. Methods Mol Biol. 610, 41-49. Eremin, S.A., Vlasenko, S.B., Osipov, A.P., Eremina, I.D., Egerov., A.M., 1989. Analytical Letters 22 (9), 2037-2050. Ningning, Z., Qin, X., Sini, L., Huan, G., 2009. Electrochem Comm. 11 (12), 2308- 2311. Danton, D.N., 1981. Anal Chim Acta 130 (2) 391-394. Ali, A.E., Mokhtari A.M., Rezaei, B., 2014. Sens Actuators B Chem. 196, 398-405. Danielle, W.K., Gabriel, L., Mika, E.M, David, E.C., 2012. Anal Chem. 84 (2), 685- 707. Chengzhou, Z., Guohai, Y.,He, L., Dan, D., Yuehe, L., 2015. Anal. Chem. 87, 230- 249. Célia, M.S., Sofia, P.G., Alberto, N.A., M. Conceicão B.S.M.M., Smilja, T., Ana, S.V., Rui, J.C.S., José, J.G.M., Gabriela, A.M., 2010. Biosens Bioelectron. 25, 2026- 2032. Shengshui, H., Qing, L., Yanxia, X., Biosensors based on direct electron transfer of protein, 2011. Electrochemical Sensors, Biosensors and their Biomedical Applications. Xueji, Z., Huangxian, J., Joseph, W., (Eds). Academic Press. Erik, R., Fredrik, H., 2015. Sensors (Basel). 15 (1), 1635-1675. Tatianny, A.F., Alessandra, B.M., Bárbara, V.M.S., Rosa, F.D., 2014. Biomed Res Int. 1-9. Lukasz, T., Stanisław, G., Robert, K., 2006. Sensors 6, 390-396. William, P., Niina, J.R., 2013. Sensors (Basel). 13 (4), 4811-4840. Farough, S., Masoud, N., Gholamreza, M., Hajar, A., Yousoof, G., Sholeh, J., Seyed hosein, I., Maryam, M., Mohammadhani, S., Aida S,. 2012. Int. J. Electrochem. Sci., 7, 7225-7234. Iijima, S., 1991. Nature 354, 56-58. Sayago, I., Santos, H., Horrillo, M.C., Aleixandre, M., Fernandez, M.J., Terrado, E., Tacchini, I., Aroz, R., Maser, W.K., Benito, A.M., Martinez, M.T., Gutierrez, J., Munoz, E., 2008. Talanta 77, 758-764. Mariana, E.G., Rasa, P., Orlando, F.F., Christopher, M.A.B., 2009. Sens Actuators B Chem. 142, 308-315. Joseph, W., 2005. Electroanalysis 17 (1), 7-14. Kannan, B., Marko, B., 2006. Anal Bioanal Chem. 385, 452-468. Economou, A.S., Volikakis, G.J., Efstathiou, C.E., 1999. Journal of Automated Methods & Management in Chemistry 21 (2) 33-38. Shankar, C.B., 2015. IJESTA 1 (3), 27-46. Madasamy, T., Pandiaraj, M., Balamurugan, M., Santosh K., Benjamin, A.R., Arun Venkatesh, K., Vairamani, K., Kotamraju, S., Karunakaran, C., 2012. Talanta 100 168-174. Miao-Qing, X., Jian-Feng, W., Guang-Chao, Z., 2013. Int. J. Electrochem. Sci., 8, 6502-6512. Yancai, L., Yuanjun, L., Yiyun, Y., 2011. Bioelectrochemistry 82, 112-116. Nassef, H.M., Civit, L., Fragoso, A., O’Sullivan, C.K., 2008. Analyst 133, 1736- 1741. Rajesh, S., Koteswararao, A.K., Kalpana, B., Ilavazhagan, G., Kotamraju, S., Karunakaran, C., 2010. Biosens. Bioelectron. 26, 689-695. Pandiaraj, M., Niroj K. S., Kalpana, B., Vepa K., Karunakaran, C., 2014. Biosens. Bioelectron. 54, 115-121. Ricardo, M.S., Marcelo, S.R., Joao, L., Rui, M.B., 2013. Biosens. Bioelectron. 18 (44), 152-159. Andrey, V.K., Sabzali, Javadov, S.S., Sandra, F., Michael, G., 2015. Biochim Biophys Acta. 1853 (2), 276-284. Kai, L., Yong-Chun, C., Hong, Z., Xiao-Peng, L., Dong, Z., Ai-Li, W., Jian J.L., Yue, T., 2015. PLOS ONE 10 (7), e0132402. Madasamy, T., Pandiaraj, M., Balamurugan, M., Kalpana, B., Niroj K. Sethy, Karunakaran, C., 2014. Biosens. Bioelectron. 52, 209-215. Ajay Kumar, Y., Taek, L., Junhong, M., Jeong-Woo, C., 2013. Biosens. Bioelectron. 47, 385-390. Ruiyi, L., Qianfang, X., Zaijun, L., Xiulan, S., Junkang, L., 2013. Biosens. Bioelectron. 44, 235-240. Ulrike, B.H.C., Malte, K., Tienush, R, . 2010. Nitric Oxide 22 (2), 75-82. Chad, K.N., Jonathan, P.L., Chi-Wing, C., David, J.L., John, W.C., 2013. J Mol Cell Cardiol. 64, 1-10. Balamurugan, M., Madasamy, T., Pandiaraj, M., Kalpana, B., Niroj K. Sethy, Karunakaran, C., 2015. Anal Biochem. 478, 121-127. Badea, M., Amine, A., Palleschi, G., Moscone, D., Volpe, G., Curulli, A., 2001. J Electroanal Chem. 509, 66-72. Additional Declarations No competing interests reported. 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16:17:59","extension":"png","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52426,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/2fbf1445562b8deaacd8992b.png"},{"id":97256039,"identity":"8bcc3dbb-41b4-4ca6-95b2-aaf5e82bf515","added_by":"auto","created_at":"2025-12-02 13:28:36","extension":"xml","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120809,"visible":true,"origin":"","legend":"","description":"","filename":"0dcb6999454742abab7a3854fe312e511structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/c70980a37c0f883f403418d9.xml"},{"id":97367147,"identity":"7ff399dd-44cb-4adf-a7d8-48d52cdd20e7","added_by":"auto","created_at":"2025-12-03 16:17:08","extension":"html","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133246,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/47a6209f41f755ca524ed351.html"},{"id":97255990,"identity":"d1876f0e-52f8-42c8-a4a2-39997d0a57e3","added_by":"auto","created_at":"2025-12-02 13:28:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":334570,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the construction of myoglobin biofunctionalized FMWCNT-PPy nanocomposite modified SPCE surface.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/eee7705afd9004e753511bd2.png"},{"id":97256032,"identity":"75f9ff28-9ef8-4217-801f-769dbf118f00","added_by":"auto","created_at":"2025-12-02 13:28:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":358585,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of the bare SPCE (a), PPy functionalized SPCE (b) and FMWCNT-PPy integrated SPCE (c).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/c3d0db7e70e51b2bb5325a63.png"},{"id":97367442,"identity":"99fbb65d-8ee5-486e-916f-07849db61094","added_by":"auto","created_at":"2025-12-03 16:18:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":234267,"visible":true,"origin":"","legend":"\u003cp\u003eTypical cyclic voltammetric responses of (a) bare SPCE, (b) PPy-SPCE, (c) FMWCNT-PPy-SPCE and (d) Mb-FMWCNT-PPy-SPCE electrodes in 0.1 M PBS (pH 7.0) containing 100 µM DTPA in 0.1 M KCl at a scan rate of 50 mVs\u003csup\u003e-1\u003c/sup\u003e \u003cem\u003evs\u003c/em\u003e. Ag/AgCl.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/dc4b328d82ae09dca01575e3.png"},{"id":97256012,"identity":"bf433c2b-de33-4479-b335-e74730a19377","added_by":"auto","created_at":"2025-12-02 13:28:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":234333,"visible":true,"origin":"","legend":"\u003cp\u003eThe electrochemical responses of the Mb-FMWCNT-PPy-SPCE electrode in (a) 100 µM (b) 200 µM, (c) 300 µM, (d) 400 µM, (e) 600 µM and (f) 800 µM of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e–\u0026nbsp; \u003c/sup\u003esolution\u003csup\u003e\u0026nbsp; \u003c/sup\u003ein 0.1 M PBS using the scan rate of 50 mVs\u003csup\u003e-1\u003c/sup\u003e \u003cem\u003evs.\u003c/em\u003e Ag/AgCl.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/8f1d0b630d1afed126278f43.png"},{"id":97367740,"identity":"4faab72d-eec2-4cce-a641-c0e7c77fdab3","added_by":"auto","created_at":"2025-12-03 16:20:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74310,"visible":true,"origin":"","legend":"\u003cp\u003eA linear calibration plot of anodic peak currents against NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e– \u003c/sup\u003econcentrations (y = -0.141x + 23.01, r\u003csup\u003e2 \u003c/sup\u003e= 0.965). Each point represents the average of three measurements.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/681f72c9902065810dd8403c.png"},{"id":97256033,"identity":"d74d405a-9f6a-496f-91d8-9f21ac7b0cca","added_by":"auto","created_at":"2025-12-02 13:28:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":441262,"visible":true,"origin":"","legend":"\u003cp\u003eThe front panel view of the electrochemical responses of the Mb-FMWCNT-PPy-SPCE electrode in (a) 100 µM (b) 200 µM, (c) 400 µM, (d) 600 µM and (e) 800 µM of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e–\u0026nbsp; \u003c/sup\u003esolution\u003csup\u003e\u0026nbsp; \u003c/sup\u003ein 0.1 M PBS using the scan rate of 50 mVs\u003csup\u003e-1\u003c/sup\u003e \u003cem\u003evs.\u003c/em\u003e Ag/AgCl by LabVIEW.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/564d5af43f3c4756b97943a0.png"},{"id":97256003,"identity":"74df071d-6305-4e9b-b4de-8050a11507b8","added_by":"auto","created_at":"2025-12-02 13:28:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":279790,"visible":true,"origin":"","legend":"\u003cp\u003eThe electrochemical responses of the Mb-FMWCNT-PPy-SPCE electrode in (a) 100 µM, (b) 200 µM, (c) 300 µM, (d) 400 µM, (e) 500 µM and (f) 600 µM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026nbsp; \u003c/sup\u003ein 0.1 M PBS solution\u003csup\u003e\u0026nbsp; \u003c/sup\u003eusing the scan rate of 50 mVs\u003csup\u003e-1\u003c/sup\u003e \u003cem\u003evs.\u003c/em\u003e Ag/AgCl.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/e0131f553d2ef980c26a3d12.png"},{"id":97256025,"identity":"bea03f41-778d-4ed4-bddf-becb37ad7ac3","added_by":"auto","created_at":"2025-12-02 13:28:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":81524,"visible":true,"origin":"","legend":"\u003cp\u003eThe electrochemical responses of the Mb-FMWCNT-PPy-SPCE electrode in (a) 100 µM, (b) 200 µM, (c) 300 µM, (d) 400 µM, (e) 500 µM and (f) 600 µM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026nbsp; \u003c/sup\u003ein 0.1 M PBS solution\u003csup\u003e\u0026nbsp; \u003c/sup\u003eusing the scan rate of 50 mVs\u003csup\u003e-1\u003c/sup\u003e \u003cem\u003evs.\u003c/em\u003e Ag/AgCl.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/86abad9c527e6417a2122a5c.png"},{"id":97367121,"identity":"8e3d4ceb-9e96-4055-9362-8dc4f4b5aba6","added_by":"auto","created_at":"2025-12-03 16:16:33","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":455450,"visible":true,"origin":"","legend":"\u003cp\u003eThe front panel view of the electrochemical responses of the Mb-FMWCNT-PPy-SPCE electrode in (a) 100 µM (b) 200 µM, (c) 300 µM, (d) 400 µM, and (e) 500 µM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026nbsp; \u003c/sup\u003esolution\u003csup\u003e\u0026nbsp; \u003c/sup\u003ein 0.1 M PBS using the scan rate of 50 mVs\u003csup\u003e-1\u003c/sup\u003e \u003cem\u003evs.\u003c/em\u003e Ag/AgCl by using LabVIEW.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/f1a6f8908869a6182cbf7c04.png"},{"id":97665478,"identity":"d47ac5dc-1341-49bb-8504-2d0654774d57","added_by":"auto","created_at":"2025-12-08 09:18:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3362045,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8131544/v1/e0161b53-b9f8-4f09-9ea9-515887f87ca9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Myoglobin biofunctionalized electrochemical biosensor platform for the dual determination of nitrite and H2O2 as oxidative stress markers","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHypoxia, a condition of low oxygen can cause oxidative stress (OS), which has been associated with several diseases such as atherosclerosis, cardiomyopathy, ischemia reperfusion injury, congestive heart failure, rheumatoid arthritis, chronic fatigue syndrome, and also affect the central nervous system can lead to Alzheimer\u0026rsquo;s disease, Amyotrophic lateral sclerosis (ALS), Huntington\u0026rsquo;s disease and Parkinson\u0026rsquo;s disease [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The mechanism through OS forced to damage heart function includes oxidative devastation to cellular membranes and proteins, thus prompting cellular loss or dysfunction. It arises while the formation of free radicals and active intermediates in the biological system exceeds its ability to neutralize and exclude them [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eReactive oxygen species (ROS) viz. hydroxyl radical (HO\u0026middot;), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), superoxide radical (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;-\u003c/sup\u003e), and singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) and reactive nitrogen species (RNS) \u003cem\u003eviz\u003c/em\u003e. peroxynitrite (ONOO\u003csup\u003e།\u003c/sup\u003e) and nitric oxide (NO) are frequently produced in biological conditions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], is the critical occurrence in living systems. Thus, ROS and RNS are commonly known as reactive species (RS). They react with biological components such as carbohydrates, proteins and lipids with ensuing modifications in the intracellular and intercellular homeostasis, leads to possible cell death and regeneration [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Indirect evidence \u003cem\u003evia\u003c/em\u003e monitoring RS indicates, oxidative damage may be caused in the pathogenesis of these sicknesses [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Particularly, the accurate determination of RS of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e are of great importance because they are involved in the etiology of ageing, progressive neurodegenerative and cardiovascular diseases [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Further, ROS is also able to generate other forms of RS. Particularly, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e have the capacity to produce additional destructive \u0026middot;OH, via a blend of the Fenton and Haber-Weiss reactions. Furthermore, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is an essential mediator in various significant field analyses [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOn the other hand there has been growing interest about the role of nitrite anion as an important precursor in the formation of N-nitrosamines, many of these have been shown as significant carcinogens in human physiques [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, various methods have been established to determine both nitrite and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, such as spectrophotometry [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], chromatography [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] capillary electrophoresis [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], chemiluminescence [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and electrochemistry [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Especially, the biosensors based on the electrochemical techniques such as voltammetry, coulometry, amperometry and impedance are promising with potential sensitivity, selectivity, low cost, fast response, easy to fabricate, portability and real-time monitoring ability [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, if cofactors are shielded by the apoprotein, it is often durable to attain an effective electronic contact among the enzyme with the signal transducer then the controlled contact of the redox active sites inhibits facile electron transfer (ET) concerning the sensing area [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Since, the approach of direct electron transfer (DET) between the active center of the protein and sensor surface to resolve this issues [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe critically important step in the biosensor design is the consideration of the recognition surface [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Screen-printed electrode provides advantages such as easy miniaturization, low cost, disposable nature due to their large-scale making competency and portability, making potential on-site detection of various target molecules [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Further, modification of sensor surfaces can improve protein interaction with transducers, thus assisting DET and/or electrocatalysis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Moreover, nanoparticles play variety of roles in different biosensing systems based on their unique physical, chemical and electrocatalytic properties. The binding of nanoparticles onto the sensor platforms drastically improves the conductivity then ET from the redox moieties to create them electroanalytical sensors [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Iijima\u0026rsquo;s discovery of carbon nanotubes (CNTs) in 1991 marked the beginning of a new era in the field of nanotechnology [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Owing to their exceptional characteristics such as size, hollow geometry, high surface area (\u0026gt;\u0026thinsp;1500m\u003csup\u003e2\u003c/sup\u003e/g) and high aspect ratio, CNTs have been the subject of considerable research aimed at electrocatalytic with sensing purposes including construction of electrochemical sensors also biosensors platforms [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The enriched electrochemical reactivity of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NADH at CNT adapted platforms creates these nanomaterials exceptionally attractive for abundant oxidase and dehydrogenase centered biosensors [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Moreover, CNT functionalization can be used to bind nearly any chosen substance to them, which permit us-for instance-to boost the biocompatibility and solubility of the nanotubes [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent years, the application of virtual instrumentation (VI) for the electroanalytical measurement have substantial interest since it affords practical benefits, such as low expense of fabrication, operation simplicity and suitability for real-time monitoring [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Earlier, we developed and reported the virtual instrumentation performing cyclic voltammetry using home-made potentiostat coupled with data acquisition system processed by LabVIEW software for nitric oxide [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The potential applications of this VI are used here for the determination of nitrite (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e using the developed electrochemical biosensor.\u003c/p\u003e\u003cp\u003eIn this work, the biosensor systems were designed and constructed based on the redox activities of Myoglobin (Mb), a kind of heme protein which is an perfect substance for the characterization of electron transfer responses of sensing and redox proteins [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], covalently bonded on FMWCNT-PPy coated SPCE by EDC and NHS for NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sensing. Cyclic voltammetry (CV) measurements are used to study the electrochemical behavior of the developed biosensor while SEM characterize the electrode surface morphologies. Moreover, the biosensors were developed to detect NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in cultured biological samples using LabVIEW.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chemicals and reagents\u003c/h2\u003e\u003cp\u003eMyoglobin from equine heart (M1882), pyrrole, carboxylic acid functionalized multi-walled carbon nanotube (755125), chitosan (Chit), sodium nitrite, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium chloride, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N\u0026prime;-ethylcarbodiimide hydrochloride (EDC), diethylenetriaminepentaacetic acid (DTPA), were purchased from Sigma Aldrich (USA). Acetic acid was purchased from Alfa Aesar. All the solutions were made through deionized water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Instruments\u003c/h2\u003e\u003cp\u003eThe morphological variations of the functionalised electrodes surface were studied using FEI Quanta FEG High Resolution scanning electron microscope (SEM) (FEI Co., Netherlands). CV characterization were carried out by CHI 1200B electrochemical workstation (CHI, USA). SPCEs were used as the sensor podia for designing the sensor (Zensor R\u0026amp;D, Taiwan). The SPCE strip consisting of carbon working, carbon counter and an Ag/AgCl reference electrode. The exterior area of the working electrode is 0.0071 cm\u003csup\u003e\u0026ndash;2\u003c/sup\u003e. National Instruments supply MyDAQ instrument having digital input, output (DIO), analog input (AI), analog output (AO) functions through USB connectivity. LabVIEW 10.0 software offered by National Instruments was helped to create the electrochemical regulator programs. PC running windows 2007 with XP was monitored for observing the results.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Fabrication of myoglobin biofunctionalized FMWCNT-PPy-SPCE\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Electropolymerization of pyrrole on SPCE\u003c/h2\u003e\u003cp\u003eElectropolymerization of pyrrole on SPCE was followed our earlier procedure. Prior to deposition of PPy, the sensing surface of SPCE was electrochemically cleaned by using 40 \u0026micro;L 1 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and cycling the voltage value at -0.5 V to 1.0 V for 10 cycles at 100 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and then cleaned with double distilled water. The major role of pretreatment process was to eliminate the excess organic ink ingredients, impurities and to rise the electrochemical behavior of the working area of the electrode surface [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAfter pretreatment, 10 complete cycles of pyrrole was deposited on to SPCE by the irreversible process of 0.4 M pyrrole in a auxiliary electrolyte of 0.1 M KCl, through giving a voltage of 0 to 0.9 V \u003cem\u003evs.\u003c/em\u003e Ag/AgCl with a scan rate of 50 mV/s [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Myoglobin biofunctionalization on FMWCNT-PPy-SPCE\u003c/h2\u003e\u003cp\u003eAfter the preparation of PPy-SPCE, FMWCNT nanoparticles were immobilized onto the PPy-SPCE. For this first, 2 h sonicating 2 mg of FMWCNTs in 200 \u0026micro;L (1.0% m/m) Chitosan (Chit) in 1.0% (v/v) acetic acid to form FMWCNTs in 1.0% (m/m) chitosan dispersion. Then, on the surface of PPy-SPCE, drop 10 \u0026micro;L of this dispersion and kept to dry.\u003c/p\u003e\u003cp\u003eAfter the initial drying, additional aliquot of 10 \u0026micro;L of the same dispersion was dropped and the sensor surface was further left for drying at room temperature for 1 h in air. By rinsing the electrode surface with PBS (pH 7.0) to activate the modified electrode and this step was repetitive twice so as to deprotonate the NH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e groups of Chit by altering the pH at the constructed sensor area [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In addition 5 \u0026micro;L of a blend comprising EDC (200 mM) and NHS (50 mM) in water was placed onto the electrode for nearly 10 min. Then the excess EDC\u0026ndash;NHS mixture was detached by washing the surface of the working sensor platform by using 0.1 M PBS.\u003c/p\u003e\u003cp\u003eThe Mb enzyme biofunctionalized sensor was made by placing 5 \u0026micro;L of Mb (4 mg/mL) onto the exterior of FMWCNT-PPy-SPCE for one day [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. The electrode was then washed with 0.1 M PBS to eliminate any electrostatically occupied Mb and washed by water earlier use. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e represents the construction of Mb biofunctionalized FMWCNT-PPy nanocomposite modified screen printed carbon electrode surface.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.4. H9c2 cardiomyoblasts cell culture\u003c/h2\u003e\u003cp\u003eH9c2 embryonic rat heart cardio myoblasts were cultured in Dulbecco's adapted Eagle's medium (DMEM) through excess glucose. The supplementary media was supplied through Penicillin- Streptomycin with fetal bovine serum (FBS-10%) in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e maintained CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Cells were consistently passaged when they reached 70\u0026ndash;80% confluency by detaching them with Trypsin-EDTA.\u003c/p\u003e\u003cp\u003eFor this, cells were initiated for the experiment by seeding them in 60 mm dishes to achieve an initial density of 30%. They were maintained in culture until they reached approximately 60\u0026ndash;70% confluency. Following this growth period, the media was changed, and the cells were stimulated as described in the subsequent steps. To prepare for analysis, the cells were detached using Trypsin-EDTA, centrifuged 10 minutes with a speed of 1500 rpm at 4\u0026deg;C, and the resulting pellet was re-suspended in 150 \u0026micro;l of 1X PBS. This suspension was then used for analysis with various biosensors.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eNowadays, there is an emerging interest for the invention of cost effective instruments for the measurement of RS. So, we have developed a virtual electrochemical analyzer for RS \u003cem\u003eviz.\u003c/em\u003e H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e by using home-made potentiostat coupled with LabVIEW 10.0 (graphical user interface software). In order to test the performance of the developed biosensor, linearity, detection limit, sensitivity, selectivity and effect of scan rate \u0026amp; pH were investigated using standard physical voltammetric instruments. Further, the linearity and sensitivity of the biosensor were also checked by developed virtual electrochemical analyzer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1. SEM characterization of functionalized biosensor platforms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe surface morphology of bare SPCE, PPy-SPCE and FMWCNT-PPy-SPCE platforms were examined by SEM. Fig. 2. (a), (b) and (c) illustrate the morphological pictures of bare SPCE, PPy-SPCE, and FMWCNT-PPy-SPCE sensor platforms correspondingly. Image 2.b exhibits the typical vastly porous morphology of PPy matrix onto the SPCE electrode platform. This great porous nature of PPy probably offers considerable larger surface zone to bind further FMWCNTs, and Mb at the biosensor surface. Further, image 2.c displays the FMWCNT were uniformly deposited on to the PPy-SPCE matrix. Further, it shows the highly microporous structure, favorable for high loading of Mb.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Conformation of Mb immobilization on the modified electrode surface\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe direct electrochemistry of Mb modified SPCE was investigated by cyclic voltammetry. Fig. 3. shows the CVs of SPCE, PPy-SPCE, FMWCNT-PPy-SPCE and Mb-FMWCNT-PPy-SPCE (curve a, b, c and d correspondingly) electrodes in 0.1 M PBS of pH 7.0 with 0.1 M KCl as a supportive electrolyte and a scan rate of 50 mVs\u003csup\u003e-1\u003c/sup\u003e. In the specified voltage range, there were no signifying Faradaic ET process because there were no significant redox peaks showed for bare SPCE, PPy-SPCE and FMWCNT-PPy-SPCE. Additionally, there were noticeable broad peaks detected on the CV ranges of -0.8 V to +0.2 V for PPy-SPCE and FMWCNT-PPy-SPCE exteriors probably because of the complex redox process of PPy and high capacitive currents [65]. On the other hand, the coverage of FMWCNT matrix on PPy-SPCE surface ensued in an observable increase in anodic and cathodic currents (Fig. 3, curve c) as compared with the previous PPy-SPCE and bare SPCE surfaces. This rise in peak current response is presumably owing to the rising ET rate of the FMWCNT. Moreover, when the binding of Mb on the FMWCNT-PPy-SPCE, a duo of typical reversible redox peaks at -0.2 and -0.38 V \u003cem\u003evs\u003c/em\u003e. reference electrode.\u0026nbsp;This electrochemical response related to the reversible transformation among Mb\u0026ndash;Fe (III) and Mb\u0026ndash;Fe (II)\u0026nbsp;redox pair and the nanocomposite altered electrode surface [66]. Based on the cyclic voltammetry,\u0026nbsp;the formal potential (E\u003csup\u003e0\u003c/sup\u003e) of Mb, determined by the midpoint of redox peak potentials, was \u0026minus;260 mV (\u003cem\u003evs\u003c/em\u003e. Ag/AgCl), and the peak-to-peak potential change (\u0026Delta;E\u003csub\u003ep\u003c/sub\u003e) was 165 mV. The effect of pH and the scan rate of the biosensor was also studied [67].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Electrochemical response to\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe conversion of nitrite ion to nitric oxide (NO·) is triggered by low oxygen strain. However this process modifies mitochondrial respiration and hypoxic NO· signaling and confines myocardial infarction in mammalian health, the pathways to nitrite bioactivation remain uncertain [68]. Research recommend that hemoglobin and myoglobin may subserve a crucial physiological role as hypoxia reliant enzyme nitrite reductases [69]. The research conducted by Ulrike and his group show that myoglobin is the cause for nitrite-dependent NO· production and cardiomyocyte protein iron-nitrosylation [68]. The mechanism of this reaction is\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMb-Fe\u003csup\u003e2+\u003c/sup\u003e + NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e + e\u003csup\u003e\u0026ndash;\u003c/sup\u003e \u0026rarr; Mb-Fe\u003csup\u003e3+\u003c/sup\u003e + NO·\u003c/p\u003e\n\u003cp\u003eThe electrochemical study of the Mb bonded electrode in several\u0026nbsp;concentrations of\u0026nbsp;NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003eby the same scan rate are shown in Fig. 4. The anodic current response increases with corresponding with increasing concentration linearly at the potential of 0.8 V. The electrochemical response obtained for\u0026nbsp;NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e is\u003csup\u003e\u0026nbsp;\u003c/sup\u003e0.8 V as previously reported by Madasamy et al and Balamurugan et al [65,70]. It is attributed to the response of electrochemical conversion of\u0026nbsp;NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003eto\u0026nbsp;NO\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003cem\u003eby\u003c/em\u003e a cyclic redox reaction of Mb active site Fe(II/III) moiety.\u003c/p\u003e\n\u003cp\u003eThe concentrations of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003ewas plotted with the observed anodic peak currents as shown in Fig. 5.\u0026nbsp;The linear calibration curve was obtained for\u0026nbsp;NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e exhibited linear range from 100 nM to 800 \u0026micro;M but for visual clarity in the accompanying fig. the range from 100 \u0026micro;M to 800 \u0026micro;M (r\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 0.965 and n = 3) is existing in a detection limit of 100 nM and the sensitivity of 22.763 nA \u0026micro;M\u003csup\u003e\u0026ndash;\u003c/sup\u003e\u003csup\u003e1\u003c/sup\u003e. In addition, the linear calibration measurements of nitrite was done in developed LabVIEW program.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFig. 6. represents the front panel view of virtual instrumentation for the electrochemical responses of the Mb-FMWCNT-PPy-SPCE electrode in (a) 100 \u0026micro;M (b) 200 \u0026micro;M, (c) 400 \u0026micro;M, (d) 600 \u0026micro;M and (e) 800 \u0026micro;M of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003esolution\u003csup\u003e\u0026nbsp;\u003c/sup\u003ein 0.1 M PBS by the scan rate of 50 mVs\u003csup\u003e-1\u003c/sup\u003e \u003cem\u003evs.\u003c/em\u003e Ag/AgCl.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Electrochemical response to\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe electrochemical oxidation of H₂O₂ to H₂O is facilitated by a cyclic redox reaction involving the iron (Fe(II/III)) centre at the Mb active site.\u0026nbsp;Figure 7 shows the electrochemical behaviors of the Mb-FMWCNT-PPy-SPCE electrode to different concentrations of H₂O₂ in 0.1 M PBS, measured in a scan rate of 50 mV s⁻\u0026sup1; against the reference electrode of Ag/AgCl.\u0026nbsp;For instance the concentration increases, the response of the anodic current value is too linearly rises at -0.4 V. The reaction mechanism of the electrochemical response by myoglobin was given as follows\u003c/p\u003e\n\u003cp\u003eMb-Fe\u003csup\u003e3+\u003c/sup\u003e + e\u003csup\u003e-\u003c/sup\u003e \u0026rarr; Mb-Fe\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eMb-Fe\u003csup\u003e2+\u003c/sup\u003e + H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e + 2H\u003csup\u003e+\u003c/sup\u003e + e\u003csup\u003e-\u003c/sup\u003e \u0026rarr; Mb-Fe\u003csup\u003e3+\u003c/sup\u003e + 2H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n\u003cp\u003ewhich gives the overall reaction:\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e + 2H\u003csup\u003e+\u003c/sup\u003e + 2e\u003csup\u003e-\u003c/sup\u003e \u0026rarr; 2H\u003csub\u003e2\u003c/sub\u003eO\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe obtained anodic peak currents against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003econcentrations were graphed as given in Fig. 8.\u0026nbsp;The plotted curve thus obtained demonstrates a linear range of response over the concentration of\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003efrom 1 \u0026micro;M to 600 \u0026micro;M but for simplicity here we have revealed from 100 \u0026micro;M to 600 \u0026micro;M (r\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e= 0.973 and n = 3) with the detection limit of 1 \u0026micro;M and the sensitivity 74.26 nA \u0026micro;M\u003csup\u003e\u0026ndash;\u003c/sup\u003e\u003csup\u003e1\u003c/sup\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFurther, Fig. 9. represents the front panel view of LabVIEW for the electrochemical responses of (a) 100 \u0026micro;M (b) 200 \u0026micro;M, (c) 300 \u0026micro;M, (d) 400 \u0026micro;M, and (e) 500 \u0026micro;M of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026nbsp;\u0026nbsp;\u003c/sup\u003esolution\u003csup\u003e\u0026nbsp;\u0026nbsp;\u003c/sup\u003ein 0.1 M PBS\u0026nbsp;at the scan rate of 50 mVs\u003csup\u003e-1\u003c/sup\u003e with Ag/AgCl by using the fabricated Mb-FMWCNT-PPy-SPCE biosensor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Stability and reproducibility\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStability and reproducibility are two significant factors for the assessment of any biosensors to be functional for therapeutic diagnosis. The stability of the biosensing platform was monitored periodically using CV and the results confirmed that no noticeable decrease in current level was observed after storage in 4 weeks (data not shown). The reproducibility of Mb based biosensor was\u0026nbsp;separately\u0026nbsp;examined in 0.1 M PBS containing 100 \u0026micro;M\u0026nbsp;NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003eand\u003csub\u003e\u0026nbsp;\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eby repetitive determination in triplicate respectively. Due to its robust reproducibility, demonstrated by low relative standard deviations (3.6% for NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash; \u0026nbsp;\u003c/sup\u003eand 5.2% for H₂O₂) across three distinct Mb-based biosensors, this electrochemical method could be a viable candidate for clinical trials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7. Application of Mb biofunctionalized FMWCNT-PPy-SPCE biosensor\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe developed biosensor was used for the detection of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003eand\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ein\u003csub\u003e\u0026nbsp;\u003c/sub\u003ecultured H9c2 cells under stress induced condition by different concentrations of L-NAME using standard cyclic voltammetry. In order to further investigate NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003eand H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003emeasurements in real samples was performed in LabVIEW instrument by using the fabricated biosensor. The nitrite and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels of cardiac H9c2 cells treated with different concentration of L-NAME for 1 hour to induce stress were estimated using both standard CV instrument and LabVIEW. For this experiments, cells treated with 0.1 mM L-NAME for 1 hr, cells kept at 1 mM L-NAME for 1 hr and cells kept without L-NAME\u003csub\u003e\u0026nbsp;\u003c/sub\u003etreatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH9c2 cardiomyocytes cell lines were treated with L-NAME to induce the stress and make the variation in both nitrite and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Before that, 0.1 M CA membrane was coated onto the Mb-FMWCNT-PPy-SPCE electrode surface and eliminate possible interferences \u003cem\u003eviz.\u003c/em\u003e NO, ascorbic acid present in the sample [65,71]. After that, the amount of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003ewas determined by interposing the current level into the standardization plot prepared by the standard NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003esolutions and the obtained results were given in table 1. The NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003elevels in H9c2 cells were in control (0.61 \u0026plusmn; 0.18), 0.1 M L-NAME (1.02 \u0026plusmn; 0.15), and 1.0 M L-NAME (1.58 \u0026plusmn; 0.20) by using LabVIEW program. As expected, these results were comparable with the NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e determination done by standard CHI instrument. These results thus confirm the suitability of the LabVIEW for the quantification of cellular NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003elevels by using constructed Mb-FMWCNT-PPy-SPCE biosensor. Similarly, Nafion coated Mb-FMWCNT-PPy-SPCE biosensor was used to eliminate the possible interference substances \u003cem\u003eviz.\u003c/em\u003e AsA, UA and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003eduring the measurement of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2.\u0026nbsp;\u003c/sub\u003eAfterward, the amount of\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewas meausred by interposing the acquired current values into the calibration plot able by the standard\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003esolutions and the obtained results were displayed in table 1. The\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003elevels present in H9c2 cells were in control (0.39 \u0026plusmn; 0.20), 0.1 M L-NAME\u0026nbsp;(0.68 \u0026plusmn; 0.30), and 1.0\u0026nbsp;M L-NAME\u0026nbsp;(0.98 \u0026plusmn; 0.25) by using LabVIEW.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"587\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.01361%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; S. No\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.1973%;\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;treated\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;with different \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;conc. of\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;L-NAME\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.1973%;\"\u003e\n \u003cp\u003eNitrite conc.\u003c/p\u003e\n \u003cp\u003eby standard\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCV using Mb\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ebiosensor\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.2177%;\"\u003e\n \u003cp\u003eNitrite conc.\u003c/p\u003e\n \u003cp\u003eby LabVIEW\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;using Mb\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ebiosensor\u003c/p\u003e\n \u003cp\u003e(\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.1973%;\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e conc.\u003c/p\u003e\n \u003cp\u003eby standard\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCV using Mb\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;biosensor\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1769%;\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e conc.\u003c/p\u003e\n \u003cp\u003eby LabVIEW\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eusing Mb\u003c/p\u003e\n \u003cp\u003ebiosensor\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 9.01361%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.1973%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Control\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; 0.1 M\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1 M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.1973%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; 0.58\u0026plusmn;0.12\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; 0.98\u0026plusmn;0.16\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1.46\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.2177%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; 0.61\u0026plusmn;0.18\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; 1.02\u0026plusmn;0.15\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;1.58\u0026plusmn;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.1973%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; 0.42\u0026plusmn;0.25\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; 0.75\u0026plusmn;0.20\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;0.98\u0026plusmn;0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.1769%;\"\u003e\n \u003cp\u003e0.39\u0026plusmn;0.20\u003c/p\u003e\n \u003cp\u003e0.68\u0026plusmn;0.30\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; 0.98\u0026plusmn;0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.8. Conclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe electrochemical biosensor for NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003eand H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewas developed by biofunctionalizing Mb as a biorecognizing element to FMWCNT on PPy nanocomposite based on SPCE. The FMWCNT nanoparticles assist the ET among Mb and the sensing electrode surface and afford additional binding spots for the immobilization of Mb, hence significantly increasing the sensitivity of the biosensor. The biosensor revealed good reproducibility, stability, low detection limit and a wide linear range. Moreover, a cost effective graphical user-interface software LabVIEW 10.0 based electrochemical analyzer for the concurrent measurement of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u0026nbsp;\u003c/sup\u003eand H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewas developed by using home-made potentiostat. The electroanalytical performance of the biosensor responses obtained by using LabVIEW were also related with the standard CV results and found in correlation with each other.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eP.S and C.K design the work and writing the manuscript.R.V helps to study the morphalogical characterization.T.A helps to measure the calibration in labview. A.M, T.R.K, C.M, S.D helps to edit and correct the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Managing Board of Virudhunagar Hindu Nadar\u0026rsquo;s Senthikumara Nadar College (Autonomous), Virudhunagar, Tamil Nadu, India and G.Venkataswamy Naidu College (Autonomous), Kovilpatti - 628502, Tamil Nadu, India.\u003c/p\u003e"},{"header":"References","content":"\u003col class=\"decimal_type\"\u003e\n\u003cli\u003eErica N.C., Vincent, P., 2012. Life, 64 (1), 72-80.\u003c/li\u003e\n\u003cli\u003eVenket, R., Balachandran, B., 2002. Nutritional Neuroscience 5 (5), 291-309.\u003c/li\u003e\n\u003cli\u003eTaibur, R., Ismail, H., Towhidul Islam, M.M., Hossain, U.S., 2012, Adv Biosci. Biotech. 3, 997-1019.\u003c/li\u003e\n\u003cli\u003eSaber, H., Syed, F. Ali., 1998. Antioxidant enzymes developmental profiles and their role in metal-induced oxidative stress. William Slikker, Jr., Louis W. Chang (Eds). Handbook of Developmental Neurotoxicology, Academic Press, Medical.\u003c/li\u003e\n\u003cli\u003eSian, C.B., Pamela, J.S., 2010. Free Radic. Biol. Med. 48, 629-641.\u003c/li\u003e\n\u003cli\u003eDerek, A.D., Manisha, P., 2008. Free Radic. Biol. Med. 44, 1873-1886.\u003c/li\u003e\n\u003cli\u003eRodrigo, A.Q., Juan, A.O., Rommy, V.B., 2012. Arch. Med. Res. 43, 632-644.\u003c/li\u003e\n\u003cli\u003eShigenaga, M.K., Hagen, T.M., Ames, B.N., 1994. Proc. Natl. Acad. Sci. USA, 91, 10771- 10778.\u003c/li\u003e\n\u003cli\u003eShyamal, K.G., Nilanjana, M., Dipak, K.D., 2007. Annals of Medicine 39, 275-289.\u003c/li\u003e\n\u003cli\u003eKhalid R., 2007. Clin Interv Aging 2 (2), 219-236.\u003c/li\u003e\n\u003cli\u003eGino, A.K., Rashmi, R., Srinivasan, V., Mohanraj, R., 2016. Oxidative Medicine and Cellular Longevity 1-14.\u003c/li\u003e\n\u003cli\u003eSimone, F., Adrienne, M.G., Osamu, H., Afshin, S., 2010. Int J Cell Biol. 1-23. \u003c/li\u003e\n\u003cli\u003eAyla, O., Metin, O., 2015. Biochemistry of Reactive Oxygen and Nitrogen Species, Sivakumar Joghi Thatha Gowder (Ed.), Basic Principles and Clinical Significance of Oxidative Stress. InTech. 37-58.\u003c/li\u003e\n\u003cli\u003eEdwin, H., Keyvan, K.G., Chia-Chi, L., Ravi, B., Gemma, A.F., 2013. Red. Biol. 1, 483-491.\u003c/li\u003e\n\u003cli\u003eAlessandro, F., Floriana, M., Concetta, T., Carmela, L., 2007. International Journal of Cancer 121 (11), 2381-2386.\u003c/li\u003e\n\u003cli\u003eMin, Z., Alessia, P., Alessandra, G.,\u003csup\u003e \u003c/sup\u003eEmilio, H., Ajay, M.S., 2013. Antioxid Redox Signal. 18 (9), 1024-1041.\u003c/li\u003e\n\u003cli\u003eSantosh, K., Sahdeo, P., Sandhya, L.S., 2013. PLOS ONE 8 (7), 1-12.\u003c/li\u003e\n\u003cli\u003eMrakic-Sposta, S., Maristella, G., Michela, M, Simone P., Alessandra, V., 2014. Oxidative Medicine and Cellular Longevity 1-10.\u003c/li\u003e\n\u003cli\u003eRam\u0026oacute;n, R., Mat\u0026iacute;as, L., Felipe, F., Daniel, H., 2013. Disease Markers 35 (6), 773-790.\u003c/li\u003e\n\u003cli\u003eJian-Ming, L. Peter, H. L., Qizhi, Y., Changyi, C., 2010. J Cell Mol Med. 14 (4), 840- 860.\u003c/li\u003e\n\u003cli\u003eBarry, H., Matthew W., 2004. Br J Pharmacol. 142 (2):, 231-255.\u003c/li\u003e\n\u003cli\u003eXueping, C., Chunyan, G., Jiming, K.,2012. Neural Regen Res. 7 (5), 376-385.\u003c/li\u003e\n\u003cli\u003ePoljsak,\u003csup\u003e \u003c/sup\u003e B., Milisav, I., 2012. Oxid Med Cell Longev. 1-12.\u003c/li\u003e\n\u003cli\u003eJames P.K., 2000. Toxicology 149, 43-50.\u003c/li\u003e\n\u003cli\u003eRosana, A.M.,Tom\u0026aacute;s E.B. , Ana M.B. , Velia M.S. , Silvia A. L.B., 2011. American Journal of Analytical Chemistry 2, 683-688.\u003c/li\u003e\n\u003cli\u003eStuart, W.K., William, A.M., Daniel, L.M., David, H., Paul, W., 2013. Water Research 47, 4433-4450.\u003c/li\u003e\n\u003cli\u003eFlamerz S., Bashir, W.A., 1981. \u003cstrong\u003eAnalyst\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e \u003cstrong\u003e106\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 243-247.\u003c/li\u003e\n\u003cli\u003eRaquel, F.P.N., Mirela, C.O., Willian, C.P., 2005. Talanta 66 (1), 86-91. \u003c/li\u003e\n\u003cli\u003ePhilip, A.C., Dennis, F.E., Tippu, S.S.S., 1989. Anal Chem. Acta 218, 331-334.\u003c/li\u003e\n\u003cli\u003eKissner, R., Koppenol, W.H., 2005. Methods Enzymol. 396, 61-8.\u003c/li\u003e\n\u003cli\u003eSteinberg, S.M., 2013. Environ Monit Assess. 185 (5), 3749-57.\u003c/li\u003e\n\u003cli\u003eAsopuru, A.O., Herbert, H.H., Steven, G.M., Michael, A.B., 1999. Journal of Chromatography A, 844, 387-394.\u003c/li\u003e\n\u003cli\u003eDavid, S.H., Anuja, C., Carrie, A.C.W., Julie, G., Paul, K., 1998. \u003ccite\u003eJ. Chem. Educ.\u003c/cite\u003e\u003cem\u003e,\u003c/em\u003e 75 (12), 1588.\u003c/li\u003e\n\u003cli\u003eSamuele, M., Elisa, R., Marco, B., Orfeo, Z.,2009. J. Environ. Eng. Manage., 19 (5), 307-314.\u003c/li\u003e\n\u003cli\u003eEnika, N.,\u003csup\u003e \u003c/sup\u003e Joseph, M.R., 2010. Methods Mol Biol. 610, 41-49.\u003c/li\u003e\n\u003cli\u003eEremin, S.A., Vlasenko, S.B., Osipov, A.P., Eremina, I.D., Egerov., A.M., 1989. Analytical Letters 22 (9), 2037-2050.\u003c/li\u003e\n\u003cli\u003eNingning, Z., Qin, X., Sini, L., Huan, G., 2009. Electrochem Comm. 11 (12), 2308- 2311.\u003c/li\u003e\n\u003cli\u003eDanton, D.N., 1981. Anal Chim Acta 130 (2) 391-394.\u003c/li\u003e\n\u003cli\u003eAli, A.E., Mokhtari A.M., Rezaei, B., 2014. Sens Actuators B Chem. 196, 398-405.\u003c/li\u003e\n\u003cli\u003eDanielle, W.K., Gabriel, L., Mika, E.M, David, E.C., 2012. Anal Chem. 84 (2), 685- 707.\u003c/li\u003e\n\u003cli\u003eChengzhou, Z., Guohai, Y.,He, L., Dan, D., Yuehe, L., 2015. Anal. Chem. 87, 230- 249.\u003c/li\u003e\n\u003cli\u003eC\u0026eacute;lia, M.S., Sofia, P.G., Alberto, N.A., M. Conceic\u0026atilde;o B.S.M.M., Smilja, T., Ana, S.V., Rui, J.C.S., Jos\u0026eacute;, J.G.M., Gabriela, A.M., 2010. Biosens Bioelectron. 25, 2026- 2032. \u003c/li\u003e\n\u003cli\u003eShengshui, H., Qing, L., Yanxia, X., Biosensors based on direct electron transfer of protein, 2011. Electrochemical Sensors, Biosensors and their Biomedical Applications. Xueji, Z., Huangxian, J., Joseph, W., (Eds). Academic Press. \u003c/li\u003e\n\u003cli\u003eErik, R., Fredrik, H., 2015. Sensors (Basel). 15 (1), 1635-1675.\u003c/li\u003e\n\u003cli\u003eTatianny, A.F., Alessandra, B.M., B\u0026aacute;rbara, V.M.S., Rosa, F.D., 2014. Biomed Res Int. 1-9.\u003c/li\u003e\n\u003cli\u003eLukasz, T., Stanisław, G., Robert, K., 2006. Sensors 6, 390-396. \u003c/li\u003e\n\u003cli\u003eWilliam, P., Niina, J.R., 2013. Sensors (Basel). 13 (4), 4811-4840.\u003c/li\u003e\n\u003cli\u003eFarough, S., Masoud, N., Gholamreza, M., Hajar, A., Yousoof, G., Sholeh, J., Seyed hosein, I., Maryam, M., Mohammadhani, S., Aida S,. 2012. Int. J. Electrochem. Sci., 7, 7225-7234. \u003c/li\u003e\n\u003cli\u003eIijima, S., 1991. Nature 354, 56-58.\u003c/li\u003e\n\u003cli\u003eSayago, I., Santos, H., Horrillo, M.C., Aleixandre, M., Fernandez, M.J., Terrado, E., Tacchini, I., Aroz, R., Maser, W.K., Benito, A.M., Martinez, M.T., Gutierrez, J., Munoz, E., 2008. Talanta 77, 758-764.\u003c/li\u003e\n\u003cli\u003eMariana, E.G., Rasa, P., Orlando, F.F., Christopher, M.A.B., 2009. Sens Actuators B Chem. 142, 308-315.\u003c/li\u003e\n\u003cli\u003eJoseph, W., 2005. Electroanalysis 17 (1), 7-14.\u003c/li\u003e\n\u003cli\u003eKannan, B., Marko, B., 2006. Anal Bioanal Chem. 385, 452-468.\u003c/li\u003e\n\u003cli\u003eEconomou, A.S., Volikakis, G.J., Efstathiou, C.E., 1999. Journal of Automated Methods \u0026amp; Management in Chemistry 21 (2) 33-38.\u003c/li\u003e\n\u003cli\u003eShankar, C.B., 2015. IJESTA 1 (3), 27-46.\u003c/li\u003e\n\u003cli\u003eMadasamy, T., Pandiaraj, M., Balamurugan, M., Santosh K., Benjamin, A.R., Arun Venkatesh, K., Vairamani, K., Kotamraju, S., Karunakaran, C., 2012. Talanta 100 168-174.\u003c/li\u003e\n\u003cli\u003eMiao-Qing, X., Jian-Feng, W., Guang-Chao, Z., 2013. Int. J. Electrochem. Sci., 8, 6502-6512.\u003c/li\u003e\n\u003cli\u003eYancai, L., Yuanjun, L., Yiyun, Y., 2011. Bioelectrochemistry 82, 112-116. \u003c/li\u003e\n\u003cli\u003eNassef, H.M., Civit, L., Fragoso, A., O\u0026rsquo;Sullivan, C.K., 2008. Analyst 133, 1736- 1741.\u003c/li\u003e\n\u003cli\u003eRajesh, S., Koteswararao, A.K., Kalpana, B., Ilavazhagan, G., Kotamraju, S., Karunakaran, C., 2010. Biosens. Bioelectron. 26, 689-695.\u003c/li\u003e\n\u003cli\u003ePandiaraj, M., Niroj K. S., Kalpana, B., Vepa K., Karunakaran, C., 2014. Biosens. Bioelectron. 54, 115-121.\u003c/li\u003e\n\u003cli\u003eRicardo, M.S., Marcelo, S.R., Joao, L., Rui, M.B., 2013. Biosens. Bioelectron. 18 (44), 152-159.\u003c/li\u003e\n\u003cli\u003eAndrey, V.K., Sabzali, Javadov, S.S., Sandra, F., Michael, G., 2015. Biochim Biophys Acta. 1853 (2), 276-284.\u003c/li\u003e\n\u003cli\u003eKai, L., Yong-Chun, C., Hong, Z., Xiao-Peng, L., Dong, Z., Ai-Li, W., Jian J.L., Yue, T., 2015. PLOS ONE 10 (7), e0132402.\u003c/li\u003e\n\u003cli\u003eMadasamy, T., Pandiaraj, M., Balamurugan, M., Kalpana, B., Niroj K. Sethy, Karunakaran, C., 2014. Biosens. Bioelectron. 52, 209-215.\u003c/li\u003e\n\u003cli\u003eAjay Kumar, Y., Taek, L., Junhong, M., Jeong-Woo, C., 2013. Biosens. Bioelectron. 47, 385-390. \u003c/li\u003e\n\u003cli\u003eRuiyi, L., Qianfang, X., Zaijun, L., Xiulan, S., Junkang, L., 2013. Biosens. Bioelectron. 44, 235-240.\u003c/li\u003e\n\u003cli\u003eUlrike, B.H.C., Malte, K., Tienush, R,\u003cem\u003e.\u003cem\u003e 2010.\u003c/em\u003e \u003cem\u003eNitric Oxide 22 (2), 75-82.\u003c/em\u003e\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eChad, K.N., Jonathan, P.L., Chi-Wing, C., David, J.L., John, W.C., 2013. \u003cem\u003eJ Mol Cell Cardiol. 64, 1-10.\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eBalamurugan, M., Madasamy, T., Pandiaraj, M., Kalpana, B., Niroj K. Sethy, Karunakaran, C., 2015. Anal Biochem. 478, 121-127.\u003c/li\u003e\n\u003cli\u003eBadea, M., Amine, A., Palleschi, G., Moscone, D., Volpe, G., Curulli, A., 2001. J Electroanal Chem. 509, 66-72.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ionics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":" Learn more about [Ionics](https://www.springer.com/journal/11581) ","snPcode":"11581","submissionUrl":"https://mc.manuscriptcentral.com/ionics","title":"Ionics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Myoglobin, biofunctionalized, cardiomyoblasts, biosensor, LabVIEW, L-NAME","lastPublishedDoi":"10.21203/rs.3.rs-8131544/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8131544/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe myoglobin (Mb) biofunctionalized carboxylic acid functionalized multi walled carbon nanotubes (FMWCNT) modified polypyrrole (PPy) electrodeposited screen printed carbon electrode (SPCE) showed an excellent biocatalytic activity towards the one electron redox reaction of nitrite and the reduction of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The direct electron transfer of the Mb immobilized into the FMWCNT was greatly facilitated. The influence of various experimental conditions was examined for the characterization of fabricated biosensor for optimum analytical performance. The each and every step of surface morphological changes were studied by scanning electron microscope (SEM) while electrochemical changes were monitored \u003cem\u003evia\u003c/em\u003e cyclic voltammetry (CV). The voltammetric response of the fabricated biosensor platform varied linearly with varying NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e concentration of 0.1 to 800 \u0026micro;M by a detection limit 0.1 \u0026micro;M. Further, the electrochemical response of the constructed biosensor changed linearly into the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration ranges between 1 to 600 \u0026micro;M by the detection limit of 0.5 \u0026micro;M. Moreover, a low cost electrochemical virtual cyclic voltammetric analyzer was developed based on home-made potentiostat and graphical user-interface software LabVIEW 10.0. The performance of the developed virtual analyzer has been further evaluated by applying it for the measurement of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in L-NAME treated embryonic rat heart-derived H9c2 cardiomyoblasts and the results are validated with commercial electrochemical analyzer.\u003c/p\u003e","manuscriptTitle":"Myoglobin biofunctionalized electrochemical biosensor platform for the dual determination of nitrite and H2O2 as oxidative stress markers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 13:28:24","doi":"10.21203/rs.3.rs-8131544/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-06T16:16:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-26T11:33:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"306425809605686810547563415912887584833","date":"2025-12-20T15:52:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199602497564176622938596618694648724783","date":"2025-12-19T14:23:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-10T05:01:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76535399139927932243422016857238357763","date":"2025-12-06T07:10:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212878264717068579002186293201527344594","date":"2025-12-02T02:08:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-01T01:31:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-19T09:20:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-19T09:16:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Ionics","date":"2025-11-17T05:51:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ionics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":" Learn more about [Ionics](https://www.springer.com/journal/11581) ","snPcode":"11581","submissionUrl":"https://mc.manuscriptcentral.com/ionics","title":"Ionics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f8c7f24c-b879-4774-acb4-16fccab73e59","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T22:38:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 13:28:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8131544","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8131544","identity":"rs-8131544","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}