Farzana Darain

On-chip detection of myoglobin based on fluorescence

A disposable immunosensor cartridge was developed that allows antibodies to be immobilized on the surface for the detection of myoglobin, a marker for the early assessment of acute myocardial infarction (AMI) using fluorescence techniques. The anti-myoglobin antibody was immobilized on a polystyrene substrate based on covalent bonding via silanization. The immunosensor chip layers were fabricated from sheets by CO(2)-laser ablation. The functionalized polystyrene surfaces were characterized by contact angle measurement, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). After the antigen-antibody reaction as a sandwich enzyme-linked immunosorbent assay (ELISA) with a horseradish peroxidase-conjugated secondary antibody (HRP-anti-myoglobin), addition of fluorogenic substrate produced a fluorescent dye which was quantified on-chip using fluorescent technique. The immunosensor response was linear for myoglobin concentrations between 20 and 230 ng/ml (r=0.991, n=3). The detection limit was found to be 16 ng/ml, which is lower than the clinical cut-off value for myoglobin in healthy patients. This protocol could be extended to the detection of other important cardiac markers simultaneously in microchannels.

Performance of Surface-Modified Polycaprolactone on Growth Factor Binding, Release, and Proliferation of Smooth Muscle Cells

This study investigates the performance of surface modification of polycaprolactone (PCL) membrane on the binding and release behavior of basic fibroblast growth factor (bFGF) for in vitro proliferation of porcine eosophageal smooth muscle cells (PESMCs). The PCL membrane surfaces were treated using UV/ozone and the surface modified PCL was characterized using water contact angle measurement, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy. The immobilization of bFGFs on the treated and non-treated PCL surfaces was also investigated using atomic force microscopy (AFM). It was found that the growth factor uptake on the PCL membrane was increased about 2-fold after treatment, which was attributed to significant contribution of oxygen containing polar groups resulting from UV/ozone treatment. Compared to non-treated PCL the treated PCL showed a prolonged bFGF release indicated by a linear increase over the first 3 days followed by a moderate and slow release profile. Moreover, the proliferation assay of PESMCs revealed that bFGF released from treated PCL had significantly higher proliferation than that of untreated PCL film. Thus, the UV/ozone-treated PCL membranes immobilized with bFGF accelerate the proliferation of PESMCs and may play an important role in soft tissue engineering.

Nano-hard template synthesis of pure mesoporous NiO and its application for streptavidin protein immobilization

A simple and efficient immobilization of streptavidin protein (with hexa-histidine tag) onto the surface of mesoporous NiO is described. Before immobilization of streptavidin protein (with hexa-histidine tag) onto the surface of mesoporous NiO, we first synthesized well-organized mesoporous NiO by a nanocasting method using mesoporous silica SBA-15 as the hard template. Then, the well-organized mesoporous NiO particles were characterized by small angle x-ray diffraction (XRD), wide angle XRD, nitrogen adsorption/desorption, and transmission electron microscopy (TEM). TEM and small angle XRD suggested the formation of mesoporous NiO materials, whereas the wide angle XRD pattern of mesoporous NiO indicated that the nickel precursor had been transformed into crystalline NiO. The N2 sorption experiments demonstrated that the mesoporous NiO particles had a high surface area of 281 m2 g(-1), a pore volume of 0.51 cm3 g(-1) and a pore size of 4.8 nm. Next, the immobilization of streptavidin protein (with hexa-histidine tag) onto the surface of mesoporous NiO was studied. Detailed analysis using gel electrophoresis confirmed that this approach can efficiently bind his-tagged streptavidin onto the surface of mesoporous NiO material since the mesoporous NiO provides sufficient surface sites for the binding of streptavidin via non-covalent ligand binding with the histidine tag.

Surface activation of poly(methyl methacrylate) by plasma treatment : stable antibody immobilization for microfluidic enzyme-linked immunosorbent assay

With the aim of obtaining stable antibody immobilization on the poly(methyl methacrylate), PMMA channel surface, PMMA substrates were activated with O2 plasma treatment to introduce surface polar groups on it. The plasma-treated PMMA surfaces were characterized using water contact angle measurement, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). It was observed that plasma treatment significantly improved the surface wettability with changing surface chemistry and topography. The strategy of immobilization of a model antibody, anti-goat IgG on plasma-treated PMMA involved two steps. First the plasma-treated PMMA was functionalized with (3-aminopropyl)thriethoxy silane, APTES off-chip which facilitated covalent capturing of antibody via a crosslinking agent in the inner surface of PMMA channel in the second step. The antibody immobilization on plasma-treated PMMA was also confirmed using AFM, XPS, and fluorescence microscopy. The anti-IgG covalently captured on channel surface was evaluated with sandwich ELISA protocol on-chip using fluorescence microscopy. The observed results demonstrate that this technique could be extended to integrate the current diagnostic techniques into the plastic chip for important biomarker diagnosis.

Nano-confined synthesis of fullerene mesoporous carbon (C60-FMC) with bimodal pores: XRD, TEM, structural properties, NMR, and protein immobilization

Nanoconfined synthesized crystalline fullerene mesoporous carbon (C60-FMC) with bimodal pore architectures of 4.95 nm and 10-15 nm pore sizes characterized by XRD, TEM, nitrogen adsorption/ desorption isotherm and solid-state NMR, and the material was used for protein immobilization. The solid-state 13C NMR spectrum of C60-FMC along with XRD, BET and TEM confirms the formation of fullerene mesoporous carbon structure C60-FMC. The immobilization of albumin (from bovine serum, BSA) protein biomolecule in a buffer solution at pH 4.7 was used to determine the adsorption properties of the C60-FMC material and its structural changes investigated by FT-IR. We demonstrated that the C60-FMC with high surface area and pore volumes have excellent adsorption capacity towards BSA protein molecule. Protein adsorption experiments clearly showed that the C60-FMC with bimodal pore architectures (4.95 nm and 10-15 nm) are suitable material to be used for protein adsorption.

Nano/Mesoporous Carbon from Rice Starch for Voltammetric Detection of Ascorbic Acid

Rice starch (RS-)based nano/mesoporous carbon (RSNMC) was prepared via a hard-templating route using cheap rice starch as a carbon source. XRD and TEM characterization indicated the formation of organized nanoporous RSNMC. Nitrogen absorption–desorption studies revealed a high surface area of up to 488 m2∙g−1, uniform pore size of 3.92 nm, and pore volume of 1.14 cm3∙g−1. A RSNMC-modified glassy carbon (GC) electrode was employed for the determination of ascorbic acid (AA) and exhibited a linear response in the concentration range of 0.005–6.0 mM with a detection limit of 0.003 mM. These results demonstrate that RSNMC has potential as an advanced and cheap electrode material for electrochemical sensing and other electrocatalytic applications.

CHAPTER 13:Nanostructure-Modified Electrodes for Food Sensors

This chapter focuses on nano/mesoporous nanostructures as modified electrodes for the analysis of food. These are of interest due to their narrow and tunable pore size distributions, high surface area, and the ability to introduce various functions into the final framework. These materials can easily be prepared with a variety of porous structures with uniform pore sizes in the mesoporous range, making them more suitable and effective for targeted applications. Extensive research has been carried out recently, focusing on their synthesis and characterization. Various applications have been reported, but recent developments in bioadsorption ability and capacity, separation, color science, determination of contaminants and aqueous stability of these materials have particularly promoted the use of nano/mesoporous nanostructures as electrode materials for food analysis.