Synthesis of Redox-Active Probes for the Multiplex Detection of DNA

Abstract

The research presented within this thesis is concerned with the design and synthesis of redox-active derivatives for the use as labels for DNA probes to be used in a commercial DNA detection assay.Chapter 1: Introduces the area of electrochemical DNA sensing, the methods used and the transducer derivatives used. Also included within this chapter is the discussion on the use of ferrocene and its derivatives as redox-active transducer moieties and their use in a DNA sensing capacity.Chapter 2: Introduces the sponsoring company, Atlas genetics, and the DNA detection assay they are developing for use in a point of care device (POC). The chapter also details the design, synthesis and electrochemical analysis via differential pulse voltammetry of mono-ferrocenyl derivatives for the use as redox-active labels for DNA sensing. The chapter outlines the development of labels containing a variety of functionality as well as possessing a range of oxidation potentials from 95 to 610 mV (vs Ag/AgCl).Chapter 3: Introduces and details the sensitivity issues between mono-ferrocenyl and di-ferrocenyl labels and the effect this could have on the commercial DNA detection assay. Within this chapter there are the details of the design, synthesis and electrochemical analysis via differential pulse voltammetry of the di-ferrocenyl labels. The chapter shows the synthesis of di-ferrocenyl labels containing a wide range of functionality on both the ferrocene core as well as the linker unit, with the labels processing a range of oxidation potentials from 242 to 500 mV (vs. Ag/AgCl).Chapter 4: Discusses the use of the labels designed and synthesised in chapters 2 and 3 in the commercial DNA detection assay developed by Atlas genetics. The development of the labels used as DNA probes in the DNA detection assay and their ability to be used in multiplex DNA detection assays are also described. Demonstrated within this chapter is the use of the labels synthesised in this thesis that give both a duplex between two different probes and also the development of a triplex assay using three different labels to detect for two different target DNAs as well as provide an internal control.Chapter 5: Introduces the synthetic methods towards the synthesis of 2-oxazolines and discusses their use within ring-opening reactions. This chapter details the optimisation of the ring-opening reaction of phenyl-2-oxazoline with a range of carboxylic acid derivatives. The synthesis of a range of 2-oxazolines with various functionality aimed towards the analytical detection of carboxylic acids through the direct conjugation with 2-oxazolines. The ring-opening reaction was found to tolerate a wide range of carboxylic acid derivatives as well as a variety of functionality on the 2-oxazolines such as ferrocene-2-oxazolines and also 3,5 – bis(trifluoromethyl)phenyl-2-oxazoline. The use of ferrocene-2-oxazoline allowed for the electrochemical detection of carboxylic acids and the 3,5 – bis(trifluoromethyl)phenyl-2-oxazoline allows for thedetection of carboxylic acids through its ring-opened form via 19F NMR. These two functionalised 2-oxazolines were used to further analyse the reactions viability as a possible analytical tool for the detection of carboxylic acids by carring out the ring-opening reaction conditions for the detection of ibuprofen from an over the counter tablet.

Date of Award	17 Sept 2013
Original language	English
Awarding Institution	University of Bath
Supervisor	Christopher Frost (Supervisor)