Highly Sensitive Biofunctionalized Mesoporous Electrospun TiO 2 Nanofiber Based Interface for Biosensing Kunal Mondal, † Md. Azahar Ali, ‡ Ved V. Agrawal, ‡ Bansi D. Malhotra,* ,‡,§ and Ashutosh Sharma* ,† † Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur, 208016, India ‡ Department of Science and Technology Centre on Biomolecular Electronics, Biomedical Instrumentation Section, National Physical Laboratory, New Delhi, 110012, India § Department of Biotechnology, Delhi Technological University, Shahbad Daulatpur, Main Bawana Road, New Delhi, 110042, India * S Supporting Information ABSTRACT: The surface modified and aligned mesoporous anatase titania nanofiber mats (TiO 2 -NF) have been fabricated by electrospinning for esteri fied cholesterol detection by electrochemical technique. The electrospinning and porosity of mesoporous TiO 2 -NF were controlled by use of polyvinylpyrrolidone (PVP) as a sacrificial carrier polymer in the titanium isopropoxide precursor. The mesoporous TiO 2 - NF of diameters ranging from 30 to 60 nm were obtained by calcination at 470 °C and partially aligned on a rotating drum collector. The functional groups such as -COOH, -CHO etc. were introduced on TiO 2 -NF surface via oxygen plasma treatment making the surface hydrophilic. Cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) were covalently immobilized on the plasma treated surface of NF (cTiO 2 -NF) via N-ethyl-N0- (3-dimethylaminopropyl carbodiimide) and N-hydroxysuccinimide (EDC-NHS) chemistry. The high mesoporosity (∼61%) of the fibrous film allowed enhanced loading of the enzyme molecules in the TiO 2 -NF mat. The ChEt-ChOx/cTiO 2 -NF-based bioelectrode was used to detect esterified cholesterol using electrochemical technique. The high aspect ratio, surface area of aligned TiO 2 -NF showed excellent voltammetric and catalytic response resulting in improved detection limit (0.49 mM). The results of response studies of this biosensor show excellent sensitivity (181.6 μA/mg dL -1 /cm 2 ) and rapid detection (20 s). This proposed strategy of biomolecule detection is thus a promising platform for the development of miniaturized device for biosensing applications. KEYWORDS: mesoporous titania nanofibers, oxygen plasma, cholesterol detection, enzyme, electrochemical technique 1. INTRODUCTION There is intense interest in the application of one dimensional (1D) materials such as nanowires, nanofibers, nanotubes, etc., for the development of medical diagnostics devices. 1-6 This is because 1D nanomaterials can facilitate the fast mass transfer to and from a molecular recognition event along the entire wire resulting in significant gain in the sensing signal. 7-9 In particular, aligned 1D nanofibers may play important role in an electro- chemical sensor device because of its electrocatalytic activity, mechanical strength and excellent electron transfer properties resulting in higher diffusion of redox species. 10,11 It has been found that the 1D structure can provide faster electron transport as compared to a nanopartilcles-based film of the same material. 12 The nanostrutured titania (TiO 2 ) has found applications in tissue engineering and drug delivery, membranes, filters, solar cells, and biosensors because of its high specific surface area, structural uniformity, chemical stability, and excellent biocompatibility. 7,13-15 However, the use of TiO 2 nanofibers as biosensing platforms has been limited to a few analytes (glucose and urea) 13,16 with a rather moderate performance owing to the low isoelectric point (∼5.5) of TiO 2 , which prevents stable attachment of biomolecules for functionalization. In this work, we fabricate an ultrasensitive, rapid detection, stable biosensing platform based on electrospun mesoporous aligned anatase TiO 2 nanofiber (30-60 nm) mats with hydrophilic surfaces containing -COOH, -CO, etc., which allow covalent grafting of the sensing biomolecules. In particular, we demonstrate the efficacy of this platform for the sensing of esterified cholesterol, with the results that are comparable to or exceed some of the critical performance matrices achievable by the use of other nanomaterials based platforms. The 1D nanostrutured titania such as nanobelts and nanotubes have excellent electrochemical performances. 17,18 Interestingly, TiO 2 nanofibers have found several interesting applications requiring efficient charge transfer, large surface area, and chemical stability. 19,20 Kim et al. have reported an ultrasensitive chemiresistor using electrospun TiO 2 nanofibers (TiO 2 -NF). 10 Lee et al. used electrospun TiO 2 nanorod photoelectrodes to Received: November 5, 2013 Accepted: January 21, 2014 Published: January 21, 2014 Research Article www.acsami.org © 2014 American Chemical Society 2516 dx.doi.org/10.1021/am404931f | ACS Appl. Mater. Interfaces 2014, 6, 2516-2527