Immobilisation of Bio-molecules on Magnetisable Solid Supports for Applications in Bio-catalysts and Bio-sensors

Hodgson, Ben Joseph (2014) Immobilisation of Bio-molecules on Magnetisable Solid Supports for Applications in Bio-catalysts and Bio-sensors. Doctoral thesis, University of Central Lancashire.

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A series of core and core-shell nanoparticles with superparamagnetic properties were synthesised and surface functionalised using three different amino-silanes by a chemical conjugation method. The functionalised nanoparticles were characterised and further modified by chemical conjugation with two different classes of bio-molecules; (i) enzymes and (ii) single stranded DNA primers. The resultant nanoparticles (nano-bio conjugates) were used for applications in (i) enzyme catalysis and (ii) bio-separation / bio-sensing.

Magnetite and amorphous silica-coated core-shell nanoparticles were synthesised on both small (5 g) and large (20 g) scales and were characterised using transmission electron microscopy (TEM), X-ray diffraction (XRD), Brunauer-Emmett-Teller (BET) surface area measurement and vibrating sample magnetometry (VSM). Silica-coated core-shell nanoparticles were functionalised by silanisation with three different aminosilanes [3-aminopropyl tri-ethoxysilane (APTS), 3-aminopropyl di-ethoxymethylsilane (APDS) and 3-aminopropyl mono-ethoxydimethylsilane (APMS)] and two different methods: water (classical method) or a Tri-phasic Reverse Emulsion (TPRE) using toluene and a surfactant (Triton X-100). It was observed that the materials prepared using the TPRE method produced higher surface amine density values on average.

The first application involved bio-catalysis where lipases [Pseudomonas Fluorescens lipase (PFL) and Candida Rugosa lipase (CRL)] were chemically conjugated (covalently linked) via glutaraldehyde-modification onto the amino-functionalised nanoparticles for applications such as: (i) hydrolysis of p-nitrophenyl palmitate to produce palmitic acid and p-nitrophenol (model reaction), (ii) transesterification of ethyl butyrate with n-butanol to produce butyl butyrate and (iii) partial and selective hydrolysis of cis-3,5-diacetoxy-1-cyclopentene to produce pharmaceutically important and expensive chiral intermediate molecules. Various reaction parameters such as (a) water concentration in a bi-phasic solvent mixture and (b) temperature were investigated to determine the optimum conditions. All reactions were carried out using free lipases and the physically adsorbed lipases in order to compare the performance with chemically conjugated nano-biomaterials.

It was observed from the bio-catalytic reaction (i) that the conversion values given by lipase-immobilised materials were comparable to those given by free lipases with the added advantage of being re-usable for further catalytic cycles. PFL-immobilised nanoparticles were shown to be more effective catalysts than CRL-immobilised materials. In the bio- catalytic reaction (ii), Lipase-immobilised materials were shown to exhibit reasonable conversion values (maximum 53%) along with easy separability by one-step magnetic separation from the reaction mixture and re-usability. Finally, in the bio-catalytic reaction (iii), lipase-immobilised materials were shown to give lower total conversion values compared to free enzymes, but a higher proportion of desired products [(1S,4R)-cis-4-acetoxy-2-cyclopenten-1-ol and (1R,4S)-cis-4-acetoxy-2-cyclopenten-1-ol]. PFL (both free and immobilised) materials were shown to give higher conversion and enantioselectivity towards the desired (1S,4R)-enantiomer (93-100% ee) than CRL materials (30-40% ee).

The second application involved bio-separation and bio-sensing where 5ʹ-NH2-modified oligonucleotide sequences specific to either Listeria Monocytogenes (LM) or Escherichia Coli (EC) were immobilised onto the surface of glutaraldehyde modified nanoparticles to assess the specific capture and enhance the sensitivity of detection of pathogenic bacterial DNAs from food samples. Firstly, the oligonucleotide-grafted nanoparticles were used in a hybrid capture assay (model assay) at UCLan using specific single stranded DNA primers of our interest followed by the application in real food samples at Q-Bioanalytic GmbH, Germany. Capture of the complementary sequences was reasonably high (48-70% for LM-specific materials and 48-55% for EC-specific materials) when calculated as a molar ratio of conjugated oligonucleotides to complementary oligonucleotides captured. Specific capture was determined to be 33-52% for LM-specific oligonucleotide-grafted nano-materials and 59-60% for EC-specific oligonucleotide-grafted nano-materials. Dehybridisation of captured sequences was shown to be efficient for all oligonucleotide-grafted materials (72-97% for LM-specific materials and 86-87% for EC-specific materials), indicating that the materials were ready for real applications using food matrices at Q-Bioanalytic GmbH, Germany.

Nucleic acid DNA was extracted from a real food sample inoculated with either LM or EC and the extracted DNA was used for specific capture using the oligonucleotide-grafted materials tested at UCLan. Dehybridised oligonucleotides were amplified and analysed using quantitative real-time PCR (qPCR). The results showed that using a one-step hybrid capture assay, LM-specific oligonucleotide-grafted materials were successful at detecting LM from an undiluted solution of LM only and from a 1:1 mixture of LM and EC. Using a two-step assay where the forward and reverse oligonucleotide-grafted materials were applied for capture separately, only EC-specific materials were successful for the detection of EC from an undiluted solution, and also from a 1:1 mixture of LM and EC.

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