Chemical analysis of biomarkers and silver nanoparticles

This thesis is focused on the development of chemical sensors for the detection of molecular biomarkers and silver nanoparticles, and the application of the techniques to authentic saliva samples. As an important antioxidant in humans, the first aim of this thesis is to develop sensors for the detection of glutathione. Two different assays for glutathione detection based on electrochemical and spectrophotometric techniques are developed. The spectrophotometric assay is further applied to authentic human plasma and saliva samples, where the stability of glutathione is investigated in-vitro. The copper(II)-catalysed oxidation process which is the major cause of the loss of reduced glutathione from biological samples is next investigated and the mechanism is proposed. The spectrophotometric glutathione assay is then employed to study the potential of salivary glutathione as a biomarker for bipolar disorder. An electroanalytical assay is also developed for the detection of salivary uric acid, a potential biomarker for gout, oral cavity cancer and other several diseases. The significantly different electrochemical behaviour in authentic as compared to synthetic saliva of commonly used redox mediators such as ferrocenemethanol and ferrocyanide is further highlighted in this thesis. In addition to molecular biomarkers such as glutathione and uric acid, an electrochemical sensor is developed for the detection of ensembles and individual silver nanoparticles (AgNPs). The oxidative dissolution of AgNPs is first investigated by two electrochemical techniques: nano-impacts and anodic stripping voltammetry, where the results reveal the relative kinetics of the process. The electrochemical techniques are then directly applied to investigate the physicochemical properties of AgNPs in authentic human saliva.</p

Chemical analysis of biomarkers and silver nanoparticles

This thesis is focused on the development of chemical sensors for the detection of molecular biomarkers and silver nanoparticles, and the application of the techniques to authentic saliva samples. As an important antioxidant in humans, the first aim of this thesis is to develop sensors for the detection of glutathione. Two different assays for glutathione detection based on electrochemical and spectrophotometric techniques are developed. The spectrophotometric assay is further applied to authentic human plasma and saliva samples, where the stability of glutathione is investigated in-vitro. The copper(II)-catalysed oxidation process which is the major cause of the loss of reduced glutathione from biological samples is next investigated and the mechanism is proposed. The spectrophotometric glutathione assay is then employed to study the potential of salivary glutathione as a biomarker for bipolar disorder. An electroanalytical assay is also developed for the detection of salivary uric acid, a potential biomarker for gout, oral cavity cancer and other several diseases. The significantly different electrochemical behaviour in authentic as compared to synthetic saliva of commonly used redox mediators such as ferrocenemethanol and ferrocyanide is further highlighted in this thesis. In addition to molecular biomarkers such as glutathione and uric acid, an electrochemical sensor is developed for the detection of ensembles and individual silver nanoparticles (AgNPs). The oxidative dissolution of AgNPs is first investigated by two electrochemical techniques: nano-impacts and anodic stripping voltammetry, where the results reveal the relative kinetics of the process. The electrochemical techniques are then directly applied to investigate the physicochemical properties of AgNPs in authentic human saliva.</p

Magnetic control: Switchable ultrahigh magnetic gradients at Fe3O4 nanoparticles to enhance solution-phase mass transport

© 2015, Tsinghua University Press and Springer-Verlag Berlin Heidelberg. Enhancing mass transport to electrodes is desired in almost all types of electrochemical sensing, electrocatalysis, and energy storage or conversion. Here, a method of doing so by means of the magnetic gradient force generated at magnetic-nanoparticle-modified electrodes is presented. It is shown using Fe3O4-nanoparticle-modified electrodes that the ultrahigh magnetic gradients (&gt;108 T·m–1) established at the magnetized Fe3O4 nanoparticles speed up the transport of reactants and products at the electrode surface. Using the Fe(III)/Fe(II)-hexacyanoferrate redox couple, it is demonstrated that this mass transport enhancement can conveniently and repeatedly be switched on and off by applying and removing an external magnetic field, owing to the superparamagnetic properties of magnetite nanoparticles. Thus, it is shown for the first time that magnetic nanoparticles can be used to control mass transport in electrochemical systems. Importantly, this approach does not require any means of mechanical agitation and is therefore particularly interesting for application in micro- and nanofluidic systems and devices. [Figure not available: see fulltext.

The fate of silver nanoparticles in authentic human saliva

The physicochemical properties of silver nanoparticles (AgNPs) in human whole saliva are investigated herein. In authentic saliva samples, AgNPs exhibit a great stability with over 70% of the nanomaterial remaining intact after a 24-hour incubation in the presence of ~0.3 mM dissolved oxygen. The small loss of AgNPs from the saliva sample has been demonstrated to be a result of two processes: agglomeration/aggregation (not involving oxygen) and oxidative dissolution of AgNPs (assisted by oxygen). In authentic saliva, AgNPs are also shown to be more inert both chemically (silver oxidative dissolution) and electrochemically (electron transfer at an electrode) than in synthetic saliva or aqueous electrolytes. The results thus predict based on the chemical persistence (over a 24-hour study) of AgNPs in saliva and hence the minimal release of hazardous Ag+ and reactive oxygen species that the AgNPs are less likely to cause serious harm to the oral cavity but this persistence may enable their transport to other environments

Rapid method for the quantification of reduced and oxidized glutathione in human plasma and saliva

A new method is developed to determine the concentrations of reduced (GSH) and oxidized glutathione (GSSG), and hence the GSH:GSSG ratios in human plasma and saliva samples. The assay is based on the masking of GSH in a GSH and GSSG mixture via a 1,4-addition reaction with p-benzoquinone (BQ), followed by enzymatic kinetic measurement. The enzyme, glutathione reductase, is highly specific to glutathione. Excess BQ can thus be easily removed by the addition of non-GSH thiols. The assay takes less than 2 min, is suitable for short timescale study and minimizes the in-vitro underestimation of the GSH:GSSG ratio arising from the degradation of GSH and formation of GSSG. We further show in this paper that the stability of the total glutathione content (GSH + GSSG) and GSH in saliva is significantly greater than in plasma, encouraging the development of non-invasive saliva sensing

The Copper(II)-Catalyzed Oxidation of Glutathione.

The kinetics and mechanisms of the copper(II)-catalyzed GSH (glutathione) oxidation are examined in the light of its biological importance and in the use of blood and/or saliva samples for GSH monitoring. The rates of the free thiol consumption were measured spectrophotometrically by reaction with DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)), showing that GSH is not auto-oxidized by oxygen in the absence of a catalyst. In the presence of Cu(2+) , reactions with two timescales were observed. The first step (short timescale) involves the fast formation of a copper-glutathione complex by the cysteine thiol. The second step (longer timescale) is the overall oxidation of GSH to GSSG (glutathione disulfide) catalyzed by copper(II). When the initial concentrations of GSH are at least threefold in excess of Cu(2+) , the rate law is deduced to be -d[thiol]/dt=k[copper-glutathione complex][O2 ](0.5) [H2 O2 ](-0.5) . The 0.5(th) reaction order with respect to O2 reveals a pre-equilibrium prior to the rate-determining step of the GSSG formation. In contrast to [Cu(2+) ] and [O2 ], the rate of the reactions decreases with increasing concentrations of GSH. This inverse relationship is proposed to be a result of the competing formation of an inactive form of the copper-glutathione complex (binding to glutamic and/or glycine moieties)

Rapid method for the quantification of reduced and oxidized glutathione in human plasma and saliva

A new method is developed to determine the concentrations of reduced (GSH) and oxidized glutathione (GSSG), and hence the GSH:GSSG ratios in human plasma and saliva samples. The assay is based on the masking of GSH in a GSH and GSSG mixture via a 1,4-addition reaction with p-benzoquinone (BQ), followed by enzymatic kinetic measurement. The enzyme, glutathione reductase, is highly specific to glutathione. Excess BQ can thus be easily removed by the addition of non-GSH thiols. The assay takes less than 2 min, is suitable for short timescale study and minimizes the in-vitro underestimation of the GSH:GSSG ratio arising from the degradation of GSH and formation of GSSG. We further show in this paper that the stability of the total glutathione content (GSH + GSSG) and GSH in saliva is significantly greater than in plasma, encouraging the development of non-invasive saliva sensing

Chemical analysis in saliva and the search for salivary biomarkers – a tutorial review

Biomarkers refer to analytes that can be used in the diagnosis of diseases or disorders. In saliva, there are many components that are potential biomarkers, and an increasingly research has focussed on the development of saliva as a diagnostic fluid. This review summarizes existing uses of salivary biomarkers and highlights the importance of the choice of saliva collection as well as the storage procedures. A case study on the effect of collection tools on the concentrations of one of the potential biomarkers, glutathione, is highlighted. Moreover, molecular diagnosis requires reliable measurement assays. This review presents electroanalytical methods for the detection of salivary biomarkers. It further reviews approaches that can be taken to improve the selectivity of the electroanalytical assays without the use of biologically selective materials, notably without the use of enzymes or antibodie

The fate of silver nanoparticles in authentic human saliva

The physicochemical properties of silver nanoparticles (AgNPs) in human whole saliva are investigated herein. In authentic saliva samples, AgNPs exhibit a great stability with over 70% of the nanomaterial remaining intact after a 24-hour incubation in the presence of ~0.3 mM dissolved oxygen. The small loss of AgNPs from the saliva sample has been demonstrated to be a result of two processes: agglomeration/aggregation (not involving oxygen) and oxidative dissolution of AgNPs (assisted by oxygen). In authentic saliva, AgNPs are also shown to be more inert both chemically (silver oxidative dissolution) and electrochemically (electron transfer at an electrode) than in synthetic saliva or aqueous electrolytes. The results thus predict based on the chemical persistence (over a 24-hour study) of AgNPs in saliva and hence the minimal release of hazardous Ag+ and reactive oxygen species that the AgNPs are less likely to cause serious harm to the oral cavity but this persistence may enable their transport to other environments

Anodic stripping voltammetry of silver in the absence of electrolytes: Theory and experiment

This work demonstrates that anodic stripping voltammetry at a microdisc electrode allows the detection of silver in the absence of added supporting electrolyte. The voltammetry however becomes distorted under low-support conditions. A semi-analytical model is developed to investigate the effects of the lack of supporting electrolyte on the voltammetric responses. First, the alteration in the double-layer kinetics is studied. In particular, we show that the classical Frumkin correction cannot explain the distorted shape of the voltammetry and only accounts for no more than ~50% of the shift in the voltammetric peaks. Second, compared to the Frumkin correction, Ohmic drop contributes much more significantly to the shift in the peak potential and the distorted wave shape upon lowering electrolyte concentrations. The Ohmic drop determined on the basis of predicted constriction resistance as expressed by Newman gives a reasonable approximation to the solution resistances, especially at ionic strengths above 200 μM. In deionized water or ultra-low conductivity water with the ionic strength of sub-micromolar level, a discrepancy in the resistances of up to a factor of 25 was observed. This discrepancy likely arises due to the assumptions made in applying the Newman model to the experimental system. Finally, we show that a model which considers simultaneously the electrode kinetics and the ohmic drop effects give a consistent fit with experimental data, when the values of the resistances are appropriately adjusted to account for the inaccuracy in the approximation of constriction resistances