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Plastic Antibodies for the detection of Bacterial Proteins and Microorganisms

  • Autores: Azizur Rahman Khan
  • Directores de la Tesis: Jordi Riu Rusell (dir. tes.)
  • Lectura: En la Universitat Rovira i Virgili ( España ) en 2016
  • Idioma: inglés
  • Tribunal Calificador de la Tesis: Francesc Xavier Rius Ferrus (presid.), Alicia Maroto Sánchez (secret.), Ana M. Benito (voc.)
  • Materias:
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  • Resumen
    • The diagnosis of most illnesses is of vital importance for providing the appropriate cure and hence controlling public health concerns. The standard methods that are used to confirm the presence of microorganisms typically consist of specific enrichment media to multiply, separate, identify and count bacterial cells. The duration of the process depends on the microorganism, but in most cases a confirmatory result can take from a few days to even weeks. One of the major objectives in this area is to detect microorganisms quickly, accurately and cheaply. Molecularly imprinted polymers (MIPs) offer in principle a robust, cost-efficient alternative to natural antibodies, but it is still a challenge to develop such materials for large molecule recognition. In this thesis we present a variety of molecular imprinting approaches for detecting bacterial proteins and/or microorganisms using techniques such as impedimetry, square wave voltammetry and potentiometry.

      The field of molecular imprinting in synthetic polymers is one of the most exciting areas in chemistry and materials science today. Molecular imprinting may be described as the formation of sites or cavities within the polymer that are receptive to a specific molecule or group of related molecules. These cavities are formed by the synthesis of the polymer in the presence of a specific molecule. The molecule around which the polymer is synthesized is known as the template, and the subsequent removal of the template after the polymer has formed creates the selective sites. These sites are then available for rebinding the template on the basis of shape, size and, perhaps most importantly, functionality, all of which combine to promote a high degree of molecular recognition. The polymer thus formed is known as a molecularly imprinted polymer (MIP) or plastic antibody or man-made antibody which may replace natural antibodies and have advantages of cost-effectiveness, reusability and long term stability.

      This doctoral thesis has been structured in chapters, each one of which contains the following information:

      Chapter 1 gives a brief overview of general methods for detecting proteins and microorganisms, and an introduction to molecular imprinting approaches for detecting proteins and/or microorganisms. This chapter also includes a brief discussion about the detection techniques (impedimetry, square wave voltammetry and potentiometry) used in this thesis, and states its specific objectives at the end of this chapter.The main focus and objective of this thesis is to develop a new approach for detecting bacterial surface proteins and microorganisms based on artificial antibodies for the construction of label-free and cost-effective portable devices. These general objectives are achieved by implementing a series of specific objectives: i. development of an easy pathway to make artificial antibodies by molecular imprinting process, ii. application of impedimetry, square wave voltammetry and potentiometry as detection techniques using molecularly imprinting polymers as the sensing layer, iii. use of homemade and commercially available screen-printed electrodes for the electrochemical detection of targets in the search for disposable and portable devices iv. electrochemical imprinting and detection of bacterial surface proteins and/or microorganisms.

      Chapter 2 is the experimental part and describes the reagents, materials, protocols, instruments and techniques.

      Chapter 3 demonstrates the detection of bacterial surface proteins from Staphylococus aureus. As first approach for the detection of bacterial surface proteins (protein A from Staphylococcus aureus) we proposed to make MIPs to be used as the sensing part in the construction of potentiometric sensors based on conductive materials (oxidized multi-walled carbon nanotubes (MWCNTs-COOH), graphene oxide (GO), polypyrrole). But since the obtained results were not good, we decided to change the detection method from potentiometry to impedimetry. In this chapter (section 3.1), electrochemical bulk imprinting and electrochemical impedance spectroscopy have been used to capture and detect protein A from Staphylococcus aureus, and the results were published in ‘Sensors and Actuators B: Chemical’, year 2011, volume 233, pages 697-704, entitle with “Plastic antibody for the electrochemical detection of bacterial surface proteins”. This work presents a novel molecularly imprinted polymer (MIP) for the indirect detection of bacteria, by targeting an outer membrane protein on a disposable device. Protein A (PA) was selected for this purpose, as a representative protein of the outer surface of Staphylococcus aureus. The imprinted polymer was assembled directly on a film of single walled carbon nanotubes (SWCNTs), placed on screen-printed electrodes (SPEs). The MIP material was produced by electropolymerizing 3-aminophenol in the presence of the protein template (PA) using cyclic voltammetry (CV). The proteins entrapped at the polymeric backbone were digested by the action of proteolytic activity of proteinase K and then washed away to create vacant sites.The performance of the corresponding imprinted and non-imprinted electrodes was evaluated by EIS and the effect of several variables, such as monomer and template concentrations, thickness of imprinting surface, was controlled and optimized by the number of CV cycles. The detection limit of the MIP-based sensors was 0.60 nM in MES buffer. High repeatability and good selectivity was observed in the presence of a model protein BSA. The sensor performance was also tested to check the effect of inorganic ions in tap water. The detection limit observed was 16.83 nM, with a recovery factor of 91.1±6.6%. The sensor described in this work is a potential tool for screening PA on-site, due to the simplicity of fabrication, disposability, short response time, low cost, good sensitivity and selectivity.

      Chapter 4 demonstrates the fabrication of electrodes in inexpensive paper-based substrates (homemade paper-based carbon-printed electrode, HP C-PEs). Standard filter paper was modified with wax to make it hydrophobic and the electrodes were manually printed over the hydrophobic paper at closer distance (≈ 1 mm). Two different configurations of devices are presented: two electrodes (to be used for instance in potentiometric measurements) or three electrodes (for instance to be used in voltammetry experiments). The three electrodes system shows similar electrochemical characteristics as the commercial ones, but a reduced cost. Electropolymerization performance with various electroactive monomers has been thoroughly studied on the HP C-PEs for the first time when three electrodes are assembled together. Moreover, the ability of these electrodes to be used as a reference electrode has been tested (in the two electrodes configuration) by adding a model protein (bovine serum albumin, BSA) in the potentiometric cell and observing stable potential upon BSA additions. Paper based electrode fabrication is a promising technology and limitations still exist regarding the rigidity, flexibility and designing of the electrode including the simplicity of fabrication with affordable cost. Herein, we have shown that manually homemade carbon-printed electrodes on paper-based substrate have been successfully fabricated with a simple, easy and cost-effective procedure. The fabricated electrodes are portable and can be used as disposable devices. We also show that the fabricated paper-based electrodes have special mechanical properties such as rigidity to make easy and quick connection with portable connector boxes and flexibility to bend. Furthermore, the fabricated three-electrode system has been used in chapter 5 section 5.1 for the construction of biosensors to detect flagella from Proteus mirabilis.

      Chapter 5 describes the detection of bacterial flagella and Proteus mirabilis. The chapter is divided into three subsections: in the first section (5.1), we successfully detect for the first time bacterial flagella from Proteus mirabils using molecularly imprinted-based artificial receptors. These receptors acted as a sensing layer, assembled by imprinting flagella proteins on a polymeric backbone of electropolymerized phenol. In brief, flagella were absorbed onto a carbon support, phenol was electroplolymerized around it through the carbon conductive matrix to create the protein molecular molds and lastly flagella were removed by enzymatic and electrochemical action. Each removed flagella gave rise to an imprinted site with eventual rebinding ability.

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