Dynamic pH mapping in microfluidic devices by integrating adaptive coatings based on polyaniline with colorimetric imaging techniques

In this paper we present a microfluidic device that has integrated pH optical sensing capabilities based on polyaniline. The optical properties of polyaniline coatings change in response to the pH of the solution that is flushed inside the microchannel offering the possibility of monitoring pH in continuous flow over a 10 wide pH range throughout the entire channel length. This work also features an innovative detection system for spatial localisation of chemical pH gradients along microfluidic channels through the use of a low cost optical device. Specifically, the use of a microfluidic channel coated with polyaniline is shown to respond colorimetrically to pH and that effect is detected by the detection system, even when pH gradients are induced within the channel. This study explores the capability of detecting this gradient by means of imaging techniques and the mapping of the camera’s response to its corresponding pH after a successful calibration process. The provision of an inherently responsive channel means that changes in the pH of a sample moving through the system can be detected dynamically using digital imaging along the entire channel length in real time, without the need to add reagents to the sample. This approach is generic and can be applied to other chemically responsive coatings immobilised on microchannels

NDM-3: A Study of the Expression, Purification, and Characterisation of a B1-type metallo β-lactamase

Antibiotic resistance is a prevalent and global problem. It is one of the biggest, if not the biggest, health care challenge we face this century. Metallo-beta-lactamases (MBLs) are a group of enzymes that play a pivotal role in the spread of resistance. There are many contributing factors for the success of MBLs at overcoming current antibiotics, and why they evade and evolve to overcome potential new antibiotics before said antibiotics would make it to market. One of these reasons is the sheer number of MBLs and more specifically, the number of mutants of these MBLs. There are as many as four subgroups in the MBL family, labelled B1 to B4. B1 is the best-studied subgroup, and the one that is most prevalent in pathogenic microorganisms. Members of this subgroup employ two metal ions (generally Zn2+) in their active sites for hydrolysis of antibiotics. NDM (New Delhi MBL) is a prominent example that has spread quickly and poses a real threat to society’s use of antibiotics. Known as a “superbug,” NDM-1 has made use of various tools to spread rapidly (horizontal gene transfer), evade inhibition, and evolve catalytic efficiency. The introduction of specific mutations has led to the emergence of at least 15 known variants of this enzyme that vary in their substrate preference and catalytic properties. NDM-3 is one such variant and is the topic of this research project, which aims to determine and characterise its catalytic properties and compare those to corresponding parameters reported for the original NDM variant discovered, NDM-1. What was discovered during this thesis was the optimised process to isolate, express, and purify NDM-3. This was completed with two constructs of NDM-3: NDM-3+Ub, and NDM-3(NC) – NDM-3(NC) did not have the ubiquitin tag attached. Purification of both of these constructs were completed using hexahistidine IMAC chromatography. NDM-3+Ub then underwent cleavage using USP2cc protease, however no activity was ever witnessed on this cleaved enzyme. Activity assays were performed on both the uncleaved NDM-3+Ub, and NDM-3(NC). These results were compared with the NDM-3(NC) showing stronger activity than the construct with the ubiquitin tag. Overall, NDM-3(NC) has lower activity than NDM-1. From these works it is shown that like NDM-1, NDM-3(NC) was shown to have activity across a wide range of substrates but NDM-1 has superior hydrolytic activity against most β-lactam antibiotics tested. As an example, the kcat values of NDM-1 for the hydrolysis of ampicillin and penicillin are 182 s-1 and 142 s-1, respectively. The corresponding parameters for NDM-3(NC) are only 47 s-1 and 38 s-1, respectively, effectively showing only ~40% of NDM-1 activity. Similarly, NDM-3(NC) underwent pH profile assays to determine the pH range it has activity against various beta lactam antibiotic substrates, and to determine the pH point that activity is the strongest. As would be expected for a virulent and pathogen that infects humans, NDM-3(NC) like NDM-1, has strongest activity at physiological pH points (i.e. around pH 7.5) and showing the strongest activity overall against ampicillin and meropenem across the pH ranges

NDM-3: A Study of the Expression, Purification, and Characterisation of a B1-type metallo β-lactamase

Antibiotic resistance is a prevalent and global problem. It is one of the biggest, if not the biggest, health care challenge we face this century. Metallo-beta-lactamases (MBLs) are a group of enzymes that play a pivotal role in the spread of resistance. There are many contributing factors for the success of MBLs at overcoming current antibiotics, and why they evade and evolve to overcome potential new antibiotics before said antibiotics would make it to market. One of these reasons is the sheer number of MBLs and more specifically, the number of mutants of these MBLs. There are as many as four subgroups in the MBL family, labelled B1 to B4. B1 is the best-studied subgroup, and the one that is most prevalent in pathogenic microorganisms. Members of this subgroup employ two metal ions (generally Zn2+) in their active sites for hydrolysis of antibiotics. NDM (New Delhi MBL) is a prominent example that has spread quickly and poses a real threat to society’s use of antibiotics. Known as a “superbug,” NDM-1 has made use of various tools to spread rapidly (horizontal gene transfer), evade inhibition, and evolve catalytic efficiency. The introduction of specific mutations has led to the emergence of at least 15 known variants of this enzyme that vary in their substrate preference and catalytic properties. NDM-3 is one such variant and is the topic of this research project, which aims to determine and characterise its catalytic properties and compare those to corresponding parameters reported for the original NDM variant discovered, NDM-1. What was discovered during this thesis was the optimised process to isolate, express, and purify NDM-3. This was completed with two constructs of NDM-3: NDM-3+Ub, and NDM-3(NC) – NDM-3(NC) did not have the ubiquitin tag attached. Purification of both of these constructs were completed using hexahistidine IMAC chromatography. NDM-3+Ub then underwent cleavage using USP2cc protease, however no activity was ever witnessed on this cleaved enzyme. Activity assays were performed on both the uncleaved NDM-3+Ub, and NDM-3(NC). These results were compared with the NDM-3(NC) showing stronger activity than the construct with the ubiquitin tag. Overall, NDM-3(NC) has lower activity than NDM-1. From these works it is shown that like NDM-1, NDM-3(NC) was shown to have activity across a wide range of substrates but NDM-1 has superior hydrolytic activity against most β-lactam antibiotics tested. As an example, the kcat values of NDM-1 for the hydrolysis of ampicillin and penicillin are 182 s-1 and 142 s-1, respectively. The corresponding parameters for NDM-3(NC) are only 47 s-1 and 38 s-1, respectively, effectively showing only ~40% of NDM-1 activity. Similarly, NDM-3(NC) underwent pH profile assays to determine the pH range it has activity against various beta lactam antibiotic substrates, and to determine the pH point that activity is the strongest. As would be expected for a virulent and pathogen that infects humans, NDM-3(NC) like NDM-1, has strongest activity at physiological pH points (i.e. around pH 7.5) and showing the strongest activity overall against ampicillin and meropenem across the pH ranges

Identification and characterization of an unusual metallo-β-lactamase from Serratia proteamaculans

Metallo-β-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing β-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequenc

Identification and characterization of an unusual metallo-beta-lactamase from Serratia proteamaculans

Metallo-beta-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing beta-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding. Steady-state kinetic measurements further indicate functional differences and identify two relevant pK (a) values for catalysis (3.8 for the enzyme-substrate complex and 7.8 for the free enzyme) and a preference for penams with modest reactivity towards some cephalosporins. An analysis of the metal ion content indicates the presence of only one zinc ion per active site in the resting enzyme. In contrast, kinetic data suggest that the enzyme may operate as a binuclear enzyme, and it is thus proposed that a catalytically active di-Zn2+ center is formed only once the substrate is present