Colorimetric cholesterol sensor based on peroxidase like activity of zinc oxide nanoparticles incorporated carbon nanotubes

Author’s Accepted Manuscript Colorimetric cholesterol sensor based on peroxidase like activity of zinc oxide nanoparticles incorporated carbon nanotubes Akhtar Hayat, Waqar Haider, Yousuf Raza, Jean Louis Marty www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(15)30008-4 http://dx.doi.org/10.1016/j.talanta.2015.05.051 TAL15643

To appear in: Talanta Received date: 9 April 2015 Revised date: 19 May 2015 Accepted date: 22 May 2015 Cite this article as: Akhtar Hayat, Waqar Haider, Yousuf Raza and Jean Louis Marty, Colorimetric cholesterol sensor based on peroxidase like activity of zinc oxide nanoparticles incorporated carbon nanotubes, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.05.051 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Colorimetric cholesterol sensor based on peroxidase like activity of zinc oxide nanoparticles incorporated carbon nanotubes Akhtar Hayata*, Waqar Haiderb, Yousuf Razab, Jean Louis Martyc a

Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS Institute of Information Technology (CIIT), Lahore, Pakistan b

Institute of Chemical Sciences, Bahauddin Zakariya University, Multan, Pakistan

c

BAE: Biocapteurs-Analyses-Environnement, Universite de Perpignan Via Domitia, 52 Avenue Paul Alduy, Perpignan Cedex 66860, France

*Corresponding author; [email protected]

Abstract A sensitive and selective colorimetric method based on the incorporation of zinc oxide nanoparticles (ZnO NPs) on the surface of carbon nanotubes (CNTs) was shown to posses synergistic peroxidase like activity for the detection of cholesterol. The proposed nanocomposite catalyzed the oxidation of 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) in the presence of hydrogen peroxide (H2O2) to produce a green colored product which can be monitored at 405 nm. H2O2 is the oxidative product of cholesterol in the presence of cholesterol oxidase. Therefore, the oxidation of cholesterol can be quantitatively related to the colorimetric response by combining these two reactions. Under the optimal experimental conditions, the colorimetric response was proportional to the concentration of cholesterol in the range of 0.5-500 nmol/L, with a detection limit of 0.2 nmol/L. The applicability of the proposed assays was

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demonstrated for the determination of cholesterol in milk powder samples with good recovery results. Keywords: ZnO incorporated carbon nanotubes; synergic effect; enzyme mimic; colorimetric assays; Cholesterol detection

1. Introduction Cholesterol is a construction unit of hormonal system of mammals and an important component of cell membrane. It plays a vital role in the synthesis of several vitamins, steroid hormones and bile acids. Cholesterol is also related to the immune system, brain synapses and protection from cancer. The abnormal concentration of cholesterol can result in certain diseases such as brain thrombosis, anemia, hypolipoproteinemia, malnutrition hypertension, septicemia and arteriosclerosis [1, 2]. Therefore, the concentration of cholesterol is monitored most often in food and clinical samples. Several method are reported for the determination of cholesterol such as high performance liquid chromatography [3], electrochemical methods [4, 5] and electrogenerated chemiluminescence [6]. However, most of these methods undergo the problems of low sensitivity, selectivity and expensive instrumentation. The alternative approach for the determination of cholesterol level can be a simple colorimetric method with added advantages such as rapid analysis, good sensitivity and low background singles. Recently, artificial enzymes are gaining vital importance over the natural enzymes for catalysis of colorimetric reactions due to ease of their preparation, low cost and stability under harsh reaction conditions. Although natural enzymes have impressive catalytic

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activity and substrate specificity, but their expensive and long preparation time, purification and storage conditions critically limit their real time applications. Therefore, the recent research has focused on the exploration of new materials that can be used as artificial enzymes mimics. These materials included hematin[7], hemin [8, 9], cyclodextrin [10], DNAzyme [11], porphyrin [12]. In this context, nanomaterials have been emerged as the most attractive artificial enzymes due to their low cost, easy preparation and large surface area. The synergistic catalytic effect of different nanocomposites has been investigated in biochemical reactions by combing the catalytic activities of two different materials of known properties and structures. The synergistic catalytic phenomena is based on the integration of two or more catalytic materials into a single nanocompsite in such a way that all the integrated materials retain their catalytic properties, and finally obtained nanocomposite is characterized with enhanced/added catalytic activity. Various novel combinations of inorganic nano hybrids such as PtIr/CNT [13] and Fe3O4/graphene oxide[14] have been employed for divers applications. Thus, there is immense scope to explore new nanocomposites having enzymatic activities. Carbon nanotubes have fascinating feature of good catalytic activity even without catalytic factors [15]. The peroxides like activity of CNTs have been explored in the field of biosensors and biofuel cells. Similarly, ZnO NPs are known to have high catalytic activity due to their large surface area and high adsorption ability. The tremendous electrical and optical properties, high chemical stability and the reduced nontoxicity make them the most suitable candidate for sensing applications [16, 17]. Keeping in view the above mentioned exciting features of CNTs and ZnO NPs, the catalytic activity of ZnO incorporated CNTs was explored for the detection of cholesterol. Herein, we have reported a novel, simple and sensitive assay for the colorimetric detection of cholesterol via H2O2, during which 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) was oxidized by H2O2 to

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produce a green colored product using a new combination of ZnO and CNTs to replace the commonly used natural enzyme. 2. Experimental 2.1 Reagents and instruments Cholesterol, Cholesterol oxidase (100 UN), hydrogen peroxide (H2O2), 2,2’-azino-bis(3ethylbenzthiazoline-6-sulfonic acid (ABTS), zinc acetate, Sodium hydroxide (NaOH) and sodium borohydride (NaBH4) were purchased from Sigma-Aldrich. The possible interfering compounds of the assay including phenol, histidine, uric acid, glucose, and absorbic acid were also purchased from Sigma. All purchased chemicals were of analytical grade, and used without further purification. The serial dilutions of stock solutions were prepared in order to obtain working solutions. Ultra-pure water was used to prepare stock solutions, as well as for all the dilutions. 96 Well Microplates were obtained from Greiner bio-one. The colorimetric measurements were carried out with Multiskan EX micro titer plate reader. A Perkin-Elmer Lambda UV/Vis spectrophotometer was used for the characterization of proposed assay. 2.2 Incorporation of ZnO on carbon nanotubes To prepare the hybrid material, 5 mg of CNTs were dispersed in the neutral medium and subsequently sonicated for 2 h. Afterwards, the CNTs were mixed with zinc acetate (5mM) and stirred for 3 h. NaOH was used to adjust the pH at 10. The obtained mixture was again stirred for 30 min. Subsequently, 20 mg of NaBH4 were added with vigorous stirring for 30 min and then heated at 130 °C for 6 h. After completion of the reduction process, the final product was filtered and washed to remove the impurities[18]. The obtained nanocomposite was dispersed at a

4

concentration of 0.5 mg/mL through sonication process. The obtained ZnO incorporated CNTs were integrated in the construction of H2O2 and cholesterol colorimetric assays. 2.3 Procedure for the colorimetric detection of H2O2 The peroxidase like efficiency of ZnO incorporated CNTs determined by using ABTS solution. In the proposed assay, the reaction mixture of H2O2 and ABTS was incubated with 10 µL of nanocomposite. The intensity of green color of oxidized ABTS was determined by monitoring the absorbance at 405 nm. In reaction mixture, H2O2 was incubated in a concentration range between 0.1 ˗ 37.5 µM in order to establish concentration dependence response and sensitivity of the nanocomposite. The calibration curve was obtained by plotting the values of absorbance against concentration. The kinetic parameters of the catalytic reaction were also performed by following the above described methodology. 2.4 Quantitative analysis of cholesterol For the measurement of cholesterol, 65 µL of Cholesterol (the concentration range from 0.5-500 nmol), 25 µL of cholesterol oxidase (25 U/mL) and 162.5 µL of Phosphate buffer saline (PBS, PH 6.5) were incubated in the well of 96 microplate at room temperature for 10 min to obtain the testing sample solution . Afterwards, 20 µL of ABTS and 10 µL of ZnO incorporated CNTs were added to the reaction mixture. The reaction mixture was mixed well and the absorbance was measured after 20 min of incubation period at room temperature. The proposed assay was also applied to milk sample for the determination of cholesterol.

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2.5 Selectivity of the proposed assay The selectivity of the method was demonstrated by applying the same assay for the determination of glycine, uric acid, glucose, and absorbic acid which are the possible interfering compounds. 2.6 Preparation of milk powder sample 2.0 g of milk powder sample were dissolved in 10 mL of KOH/ethanol solution with subsequent sponification in a water bath for 1 h. Then, 10 mL of water and 20 mL of n-hexane were added to the solution and the reaction mixture was centrifuged for 5 min. The n-hexane was separated and solvent was evaporated under a steam of nitrogen. The residue was dissolved in isopropanol and triton. 3. Results and Discussion 3.1 Principle for the colorimetric detection of Cholesterol The cholesterol sensor was developed by a colorimetric method during which cholesterol was oxidized in the presence of cholesterol oxidase to produce H2O2. The H2O2 formed during first step was systematically quantified by the oxidation of ABTS to give a green colored product that can be monitored at 405 nm by colorimetric analysis(see eq. 1 & 2).The second step was catalyzed by the peroxidase like activity of ZnO incorporated CNTs. The overall depiction is provided in the scheme 1 (Scheme 1)

(1)

(2)

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3.2 Colorimetric analysis under different experimental condition The peroxidase like catalytic activity of ZnO nanoparticles incorporated CNTs was demonstrated by the oxidation of ABTS in the presence and absence of H2O2. The oxidation rate of chromogenic substrate (ABTS) by H2O2 was significantly increased by the addition of catalyst, thus a green color was observed due to absorption of the radiations by the oxidized product in visible range(405 nm) (Fig S1). A negligible green color was found in the presence of only ABTS and nanocomposite. Similarly the reaction did not proceed in the absence of ABTS. These results show that ZnO nanoparticles incorporated CNTs have excellent peroxidase like characteristics that can be used to mimic HRP enzyme for the colorimetric detection of H2O2. Furthermore, ZnO nanoparticles incorporated CNTs were characterized by enzyme kinetic methodology for catalytic oxidation of ABTS by H2O2, which was employed to determine the kinetic parameters. To conduct the kinetic experiments, the concentration of one substrate was varied while the other was kept constant. The Lineweaver-Burk plots were used for the calculation of kinetic parameters including Michaelis-Menton (Km) and maximum initial velocity (Vm) (table 1). The Km value for ZnO nanoparticles incorporated CNTs towards H2O2 was found lower than the values obtained with previously reported nanomaterials peroxidase mimics[19-21]. The lower Km value indicates that the lower concentration of oxidizing agent (H2O2) is required to achieve maximum catalytic response with ZnO nanoparticles incorporated CNTs. The catalytic efficiency of an enzyme is directly related to Km value towards the substrate. A lower Km value of ZnO nanoparticles incorporated CNTs reveals that the synergistic catalytic effect of combined ZnO and CNTs has much profound effect, thus increasing the affinity of nanocomposite towards H2O2. The hybrid material can be applied in a variety of fields due to enhanced features. To have further insight on the nanocomposite catalysis

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mechanism, the double reciprocal of velocity against one of the component concentrations were achieved while the concentrations for other substrates were fixed. All the experiments were under the above optimized conditions. The absorbance of reaction mixture was observed to increase with the increasing concentration of H2O2 (Fig S2). A similar trend was observed with the increasing concentration of ABTS (Fig S3). The slopes of the lines were parallel, revealing a ping pong mechanism and demonstrating that the designed enzyme mimic material binds and reacts with the first substrate and then releases the first product prior to its reaction with other substrate (Fig S2, S3). 3.3 Optimization of experimental conditions The catalytic activity of artificial enzyme mimics was investigated under varying concentrations of ZnO nanoparticles incorporated CNTs, H2O2, ABTS, and reaction mixture’s pH. The maximum catalytic activity was observed at pH 7.4 with an optimized concentration of nanocomposite (10 µL) (Supporting information, Fig S4 A, B). Similarly, the observed optimal concentrations for H2O2 and ABTS were 35 mM and 750 µM respectively (Fig S5 A, B). The obtained optimal values of ZnO nanoparticles incorporated CNTs were quite similar with those reported for HRP and other artificial peroxidase enzymes[22]. The effect of nanocomposite incubation time in the reaction was investigated and an optimal incubation time of 20 min was selected based on the product color intensity. In the same context, the impact of cholesterol oxidase quantity was studied over a range of 0- 25 U/mL. The colorimetric response was found to increase with increasing concentration of enzyme. However, when enzyme concentration was 25 U/mM, the colorimetric response was strong enough for qualitative analysis (Fig S6). The optimal experimental conditions were employed to perform further experiments. 8

3.4 Detection of hydrogen peroxide The colorimetric method combining the synergisitic peroxides like activity of ZnO nanoparticles incorporated CNTs was employed for the determination of H2O2 and cholesterol. As the concentration of H2O2 is directly related to the oxidation of ABTS, therefore, H2O2 level was monitored by the absorbance of ABTS at 405 nm. The calibration curve for varying concentrations of hydrogen peroxide (0.1 – 37.5 µM) is presented in the Fig. 1. The gradual increase in color intensity with increasing concentration of H2O2 was observed with naked eye and a visual limit of detection was found to be 1.2 µM. Analytical parameters such as linearity, limits of detection and precisions were determined at optimal reaction conditions (table 1). The proposed method showed good linearity (R2≥0. 0.997) for the quantification. Figure 1 3.5 Analytical application for cholesterol detection Cholesterol level is most frequently monitored in clinical laboratories due to its important role in human health. Generally, cholesterol is oxidized by cholesterol oxidase to produce hydrogen peroxide and Cholest-4-en-3-one in the presence of oxygen. In our proposed method, the produced H2O2 is reacted with ABTS in the presence of ZnO nanoparticles incorporated CNTs to produce a green colored product. The concentration of H2O2 is monitored by the color intensity of the product which is indirectly related to the concentration of cholesterol. The method showed a linear response for the determination of cholesterol in the range of 0.5 – 500 nM, with a better limit of detection (0.2 nM) as compared to those obtained with the previously reported methods [23-30]. The calibration curve and visual inset is presented in Fig 2. Analytical characteristics

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including linearity, limits of detection and precisions are provided in the table 1. The color changes can also be observed with naked eye in order to monitor the level of cholesterol. Figure 2

Table 2: Analytical performance comparison of the proposed method with literature reported cholesterol biosensors Sr No

Method Principle

Limit of detection

Linear range

Ref

1

Based on peroxidase-like activity of cupric oxide nanoparticles Amperometric biosensor based on a conducting polymer

170 nmol/L

0.625-12.5 µmol/L

[24]

406 nmol/L

-

[25]

Electrochemical biosensor based on 2-(4-fluorophenyl)4,7-di(thiophene-2-yl)-1Hbenzo[d]imidazole (BIPF) Based on Cholesterol Oxidase Co-Immobilized with α-Fe2O3 Micro-Pine Shaped Hierarchical Structures Based on Bi-pseudoenzyme synergetic catalysis

4 nmol/L

-

[26]

7 nmol/L

100-8000 nmol/L

[27]

1 nmol/L

3.3-1500 nmol/L

[28]

6

Paper-based cholesterol

1µmol/L

50 µM-10 mM

[29]

7

Based on a bi-enzyme immobilized on conducting poly(thionine) film Based on the peroxidase like activity of zinc oxide nanoparticles incorporated carbon nanotubes

6.3µmol/L)

25-125 µM

[30]

0.2 (nmol/L)

0.5-500 (nmol/L)

Present work

2 3

4

5

8

10

In order to demonstrate the applicability of the purpose methods, the peroxidase nanocomposite catalyzed assays was employed to monitor cholesterol level in milk powder samples. The results including precision and recovery of the method are presented in the Table S1. The recovery of the spiked samples was found between 96.5% and 97.5%. Similarly, low values for relative standard deviations (3.5-4.1%) were observed for milk samples spiked with different concentrations of cholesterol (5 nM, 150 nM, 400 nM). Thus, good precision and recovery values of ZnO nanoparticles incorporated CNTs catalyzed colorimetric detection indicate that the method is not affected by the matrix of milk samples for the determination of cholesterol. Taking in account the effectiveness, the proposed method may find spread widespread application in the domain of oxidase based colorimetric assays. 3.6 Selectivity of the method The method specificity for cholesterol was also checked by investigating the developed method for possible interfering compounds including glucose, ascorbic acid, uric acid and glycine. As shown in Fig. 3, the common interfering compounds have no evident absorbance even at much higher concentrations than cholesterol. It can be concluded from these results that the proposed colorimetric method involving ZnO nanoparticles incorporated CNTs has good selectivity for determination of cholesterol. In broad spectrum, the catalytic efficiency of the nanocompiste is not only limited to the use of ABTS dye, but can be easily extended to other dyes of HRP enzyme. The stability of the assay is related to the nature of the dye and is independent of the nanocomposite. Similarly, cholesterol was selected as model analyte, however, the oxidase like properties of the proposed nanocomposite can be integrated to any type of biosensors or sensors where H2O2 is the product of the enzymatic reaction. The nanocomposite can also be employed for direct monitoring of H2O2 based on its peroxidase like properties. It is expected that the

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proposed biomimic material may find widespread applications in the field of sensors and biosensor. Figure 3 4. Conclusion In the present work, we have explored a new peroxidase like catalyst, ZnO nanoparticles incorporated CNTs which was used for colorimetric monitoring of cholesterol. The nanocomposite has shown impressive synergistic catalytic activity which permitted to monitor cholesterol level at very lower concentration. The catalytic oxidation of ABTS in the presence of H2O2 was dependent on different factors such as pH, time, concentrations of nanocomposite and substrates. The assay presented good selectivity, sensitivity and linearity in a range between 0.5 to 500 nM for the detection of cholesterol. Furthermore, the method is quick, easy and economical. Thus, the nanocomposite with peroxidase like activity can be a good replacement of natural enzyme because of easy preparation and robustness under varying reaction conditions. References [1] S. Aravamudhan, N.S. Ramgir, S. Bhansali, Electrochemical biosensor for targeted detection in blood using aligned Au nanowires, Sensors and Actuators B: Chemical, 127 (2007) 29-35. [2] L. Hong, A.L. Liu, G.W. Li, W. Chen, X.H. Lin, Chemiluminescent cholesterol sensor based on peroxidase-like activity of cupric oxide nanoparticles, Biosens Bioelectron, 43 (2013) 1-5. [3] Y.-T. Lin, S.-S. Wu, H.-L. Wu, Highly sensitive analysis of cholesterol and sitosterol in foods and human biosamples by liquid chromatography with fluorescence detection, Journal of Chromatography A, 1156 (2007) 280-287. [4] Z. Matharu, P.R. Solanki, V. Gupta, B.D. Malhotra, Mediator free cholesterol biosensor based on self-assembled monolayer platform, Analyst, 137 (2012) 747-753. [5] C. Wang, X. Tan, S. Chen, R. Yuan, F. Hu, D. Yuan, Y. Xiang, Highly-sensitive cholesterol biosensor based on platinum–gold hybrid functionalized ZnO nanorods, Talanta, 94 (2012) 263270.

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[6] M. Zhang, R. Yuan, Y. Chai, S. Chen, X. Zhong, H. Zhong, C. Wang, A cathodic electrogenerated chemiluminescence biosensor based on luminol and hemin-graphene nanosheets for cholesterol detection, RSC Advances, 2 (2012) 4639-4641. [7] G. Zhang, P.K. Dasgupta, Hematin as a peroxidase substitute in hydrogen peroxide determinations, Analytical Chemistry, 64 (1992) 517-522. [8] Q. Wang, Z. Yang, X. Zhang, X. Xiao, C.K. Chang, B. Xu, A Supramolecular-HydrogelEncapsulated Hemin as an Artificial Enzyme to Mimic Peroxidase, Angewandte Chemie International Edition, 46 (2007) 4285-4289. [9] L. Fruk, C.M. Niemeyer, Covalent Hemin–DNA Adducts for Generating a Novel Class of Artificial Heme Enzymes, Angewandte Chemie International Edition, 44 (2005) 2603-2606. [10] Z. Liu, R. Cai, L. Mao, H. Huang, W. Ma, Highly sensitive spectrofluorimetric determination of hydrogen peroxide with [small beta]-cyclodextrin-hemin as catalyst, Analyst, 124 (1999) 173-176. [11] G. Pelossof, R. Tel-Vered, J. Elbaz, I. Willner, Amplified biosensing using the horseradish peroxidase-mimicking DNAzyme as an electrocatalyst, Anal Chem, 82 (2010) 4396-4402. [12] M. Sono, M.P. Roach, E.D. Coulter, J.H. Dawson, Heme-Containing Oxygenases, Chemical Reviews, 96 (1996) 2841-2888. [13] V. Georgakilas, D. Gournis, V. Tzitzios, L. Pasquato, D.M. Guldi, M. Prato, Decorating carbon nanotubes with metal or semiconductor nanoparticles, Journal of Materials Chemistry, 17 (2007) 2679-2694. [14] Y.-l. Dong, H.-g. Zhang, Z.U. Rahman, L. Su, X.-j. Chen, J. Hu, X.-g. Chen, Graphene oxide-Fe3O4 magnetic nanocomposites with peroxidase-like activity for colorimetric detection of glucose, Nanoscale, 4 (2012) 3969-3976. [15] Y. Ma, P.L. Chiu, A. Serrano, S.R. Ali, A.M. Chen, H. He, The Electronic Role of DNAFunctionalized Carbon Nanotubes: Efficacy for in Situ Polymerization of Conducting Polymer Nanocomposites, Journal of the American Chemical Society, 130 (2008) 7921-7928. [16] A. Ali, A.A. Ansari, A. Kaushik, P.R. Solanki, A. Barik, M.K. Pandey, B.D. Malhotra, Nanostructured zinc oxide film for urea sensor, Materials Letters, 63 (2009) 2473-2475. [17] S.P. Singh, S.K. Arya, P. Pandey, B.D. Malhotra, S. Saha, K. Sreenivas, V. Gupta, Cholesterol biosensor based on rf sputtered zinc oxide nanoporous thin film, Applied Physics Letters, 91 (2007) 063901. [18] K.-Y. Hwa, B. Subramani, Synthesis of zinc oxide nanoparticles on graphene–carbon nanotube hybrid for glucose biosensor applications, Biosensors and Bioelectronics, 62 (2014) 127-133. [19] R. Cui, Z. Han, J.-J. Zhu, Helical Carbon Nanotubes: Intrinsic Peroxidase Catalytic Activity and Its Application for Biocatalysis and Biosensing, Chemistry – A European Journal, 17 (2011) 9377-9384. [20] X.-X. Wang, Q. Wu, Z. Shan, Q.-M. Huang, BSA-stabilized Au clusters as peroxidase mimetics for use in xanthine detection, Biosensors and Bioelectronics, 26 (2011) 3614-3619.

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[21] W. Haider, A. Hayat, Y. Raza, A. Anwar Chaudhry, I.-U. Rehman, J.L. Marty, Gold nanoparticle decorated single walled carbon nanotube nanocomposite with synergistic peroxidase like activity for d-alanine detection, RSC Advances, 5 (2015) 24853-24858. [22] L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, T. Wang, J. Feng, D. Yang, S. Perrett, X. Yan, Intrinsic peroxidase-like activity of ferromagnetic nanoparticles, Nat Nano, 2 (2007) 577-583. [23] S. Lata, B. Batra, P. Kumar, C.S. Pundir, Construction of an amperometric d-amino acid biosensor based on d-amino acid oxidase/carboxylated mutliwalled carbon nanotube/copper nanoparticles/polyalinine modified gold electrode, Analytical Biochemistry, 437 (2013) 1-9. [24] L. Hong, A.-L. Liu, G.-W. Li, W. Chen, X.-H. Lin, Chemiluminescent cholesterol sensor based on peroxidase-like activity of cupric oxide nanoparticles, Biosensors and Bioelectronics, 43 (2013) 1-5. [25] S. Soylemez, Y.A. Udum, M. Kesik, C. Gündoğdu Hızlıateş, Y. Ergun, L. Toppare, Electrochemical and optical properties of a conducting polymer and its use in a novel biosensor for the detection of cholesterol, Sensors and Actuators B: Chemical, 212 (2015) 425-433. [26] S. Soylemez, F. Ekiz Kanik, M. Ileri, S.O. Hacioglu, L. Toppare, Development of a novel biosensor based on a conducting polymer, Talanta, 118 (2014) 84-89. [27] A. Umar, R. Ahmad, S.W. Hwang, S.H. Kim, A. Al-Hajry, Y.B. Hahn, Development of Highly Sensitive and Selective Cholesterol Biosensor Based on Cholesterol Oxidase CoImmobilized with α-Fe2O3 Micro-Pine Shaped Hierarchical Structures, Electrochimica Acta, 135 (2014) 396-403. [28] J. Zhang, W. Wang, S. Chen, Y. Ruo, X. Zhong, X. Wu, Bi-pseudoenzyme synergetic catalysis to generate a coreactant of peroxydisulfate for an ultrasensitive electrochemiluminescence-based cholesterol biosensor, Biosensors and Bioelectronics, 57 (2014) 71-76. [29] N. Ruecha, R. Rangkupan, N. Rodthongkum, O. Chailapakul, Novel paper-based cholesterol biosensor using graphene/polyvinylpyrrolidone/polyaniline nanocomposite, Biosensors and Bioelectronics, 52 (2014) 13-19. [30] M.M. Rahman, X.-b. Li, J. Kim, B.O. Lim, A.J.S. Ahammad, J.-J. Lee, A cholesterol biosensor based on a bi-enzyme immobilized on conducting poly(thionine) film, Sensors and Actuators B: Chemical, 202 (2014) 536-542.

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Figure Captions

Scheme 1. The proposed mechanism for the detection of cholesterol Figure 1. The calibration plots for H2O2 detection: Inset; images of end colored product under varying concentration of analyte Figure 2. The calibration plots for cholesterol detection: Inset; images of end colored product under varying concentration of two analyte Figure 3. Selectivity analysis for cholesterol detection

Table 1. Analytical performance of the proposed colorimetric method for H2O2 and cholesterol detection Analyte H2O2

Km [mM] 0.6

Vm [10-8 MS-1] 5

Cholesterol

-

-

ABTS

0.5

15

Linear range 0.1-37.5 (µmol/L) 0.5-500 (nmol/L)

Slope 0.28834

Intercept Correlation coefficient 0.31865 0.997

0.18615

-

-

0.14632 -

0.997 -

LOD (µmol/L) 0.05 (µmol/L) 0.2 (nmol/L) -

Highlights    

A new peroxidase like ZnO nanoparticles incorporated CNTs catalyst was explored The nanocomposite has shown impressive synergistic catalytic activity for cholesterol detection The assay presented good selectivity, sensitivity and linearity The nanocomposite can be a good replacement of natural enzyme

15

Figure 1

Increasing concentration of Cholesterol

[Cholesterol] Figure 2

16

Figure 3

Scheme 1

17

Relative activity (%)

Graphical abstract

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