David R. Klug

Correlating long-lived photogenerated hole populations with photocurrent densities in hematite water oxidation photoanodes

Photogenerated charge carrier dynamics are investigated as a function of applied bias in a variety of different hematite photoanodes for solar water oxidation. Transient absorption spectroscopy is used to probe the photogenerated holes, while transient photocurrent measures electron extraction. We report a general quantitative correlation between the population of long-lived holes and the photocurrent amplitude. The yield of long-lived holes is shown to be determined by the kinetics of electron-hole recombination. These recombination kinetics are shown to be dependent upon applied bias, exhibiting decay lifetimes ranging from ca 5 μs to 3 ms (at −0.4 and +0.4 V versus Ag/AgCl, respectively). For Si-doped nanostructured hematite photoanodes, electron extraction and electron-hole recombination are complete within ∼20 ms, while water oxidation is observed to occur on a timescale of hundreds of milliseconds to seconds. The competition between electron extraction and electron-hole recombination is electron-density-dependent: the effect on recombination of applied bias and excitation intensity is discussed. The timescale of water oxidation is independent of the concentration of photogenerated holes, indicating that the mechanism of water oxidation on hematite is via a sequence of single-hole oxidation steps.

Efficient Suppression of Electron–Hole Recombination in Oxygen-Deficient Hydrogen-Treated TiO2 Nanowires for Photoelectrochemical Water Splitting

There is an increasing level of interest in the use of black TiO2 prepared by thermal hydrogen treatments (H:TiO2) due to the potential to enhance both the photocatalytic and the light-harvesting properties of TiO2. Here, we examine oxygen-deficient H:TiO2 nanotube arrays that have previously achieved very high solar-to-hydrogen (STH) efficiencies due to incident photon-to-current efficiency (IPCE) values of >90% for photoelectrochemical water splitting at only 0.4 V vs RHE under UV illumination. Our transient absorption (TA) mechanistic study provides strong evidence that the improved electrical properties of oxygen-deficient TiO2 enables remarkably efficient spatial separation of electron–hole pairs on the submicrosecond time scale at moderate applied bias, and this coupled to effective suppression of microsecond to seconds charge carrier recombination is the primary factor behind the dramatically improved photoelectrochemical activity.

Charge carrier separation in nanostructured TiO2 photoelectrodes for water splitting

There is intense interest in developing new novel nanostructured photoanodes for water splitting. It is therefore important that methods to analyze the effect of nanostructuring on water splitting yields are developed in order to rationalize the relative merits of this approach for different materials. In this study the dependence of charge separation efficiency (η(sep)) on potential during photoelectrochemical water splitting at pH 2 has been quantified in a model electrode system (nanocrystalline, mesoporous TiO2) using two independent methods. These are (i) analysis of incident photon conversion efficiency (IPCE) measurements and (ii) transient absorption (TA) spectroscopy measurements. The techniques provide good agreement with each other and show that a low maximum value of η(sep) (~0.18) is the primary cause of the low IPCE for water oxidation on these nc-TiO2 electrodes.

Quantitative single cell and single molecule proteomics for clinical studies

A central aspect of cellular systems biology is the study of cell-to-cell variability driven by network control of molecular noise. Proteins are produced in stochastic bursts and, although time averaging smoothes their accumulated levels, variation in their copy number is substantial in members of environmental sensing and signalling networks. We have developed a label-free, microfluidic antibody capture chip platform called the MAC chip, to quantify precisely the copy numbers of many proteins from a single cell in a multiplexed single assay format. We intend to investigate protein noise in circulating tumour cells (CTCs) isolated from biopsies of cancer patients through the identification of biomolecular signatures, such as p53 tumour suppressor protein, which correlate with biological properties and clinical outcomes during treatment.

Chemical-free lysis and fractionation of cells by use of surface acoustic waves for sensitive protein assays

We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 μm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.

Dynamical hologram generation for high speed optical trapping of smart droplet microtools

This paper demonstrates spatially selective sampling of the plasma membrane by the implementation of time-multiplexed holographic optical tweezers for Smart Droplet Microtools (SDMs). High speed (>1000fps) dynamical hologram generation was computed on the graphics processing unit of a standard display card and controlled by a user friendly LabView interface. Time multiplexed binary holograms were displayed in real time and mirrored to a ferroelectric Spatial Light Modulator. SDMs were manufactured with both liquid cores (as previously described) and solid cores, which confer significant advantages in terms of stability, polydispersity and ease of use. These were coated with a number of detergents, the most successful based upon lipids doped with transfection reagents. In order to validate these, trapped SDMs were maneuvered up to the plasma membrane of giant vesicles containing Nile Red and human biliary epithelial (BE) colon cancer cells with green fluorescent labeled protein (GFP)-labeled CAAX (a motif belonging to the Ras protein). Bright field and fluorescence images showed that successful trapping and manipulation of multiple SDMs in x, y, z was achieved with success rates of 30-50% and that subsequent membrane-SDM interactions led to the uptake of Nile Red or GFP-CAAX into the SDM.

Acoustic suppression of the coffee-ring effect

We study the influence of acoustic fields on the evaporative self-assembly of solute particles suspended inside sessile droplets of complex fluids. The self-assembly process often results in an undesirable ring-like heterogeneous residue, a phenomenon known as the coffee-ring effect. Here we show that this ring-like self-assembly can be controlled acoustically to form homogeneous disc-like or concentrated spot-like residues. The principle of our method lies in the formation of dynamic patterns of particles in acoustically excited droplets, which inhibits the evaporation-driven convective transport of particles towards the contact line. We elucidate the mechanisms of this pattern formation and also obtain conditions for the suppression of the coffee-ring effect. Our results provide a more general solution to suppress the coffee-ring effect without any physiochemical modification of the fluids, the particles or the surface, thus potentially useful in a broad range of industrial and analytical applications that require homogenous solute depositions.

Absolute quantification of protein copy number using a single-molecule-sensitive microarray

We report the use of a microfluidic microarray incorporating single molecule detection for the absolute quantification of protein copy number in solution. In this paper we demonstrate protocols which enable calibration free detection for two protein detection assays. An EGFP protein assay has a limit of detection of <30 EGFP proteins in a microfluidic analysis chamber (limited by non-specific background binding), with a measured limit of linearity of approximately 6 × 10(6) molecules of analyte in the analysis chamber and a dynamic range of >5 orders of magnitude in protein concentration. An antibody sandwich assay was used to detect unlabelled human tumour suppressor protein p53 with a limit of detection of approximately 21 p53 proteins and a dynamic range of >3 orders of magnitude. We show that these protocols can be used to calibrate data retrospectively to determine the absolute protein copy number at the single cell level in two human cancer cell lines.

Identification and relative quantification of tyrosine nitration in a model peptide using two-dimensional infrared spectroscopy

Nitration of tyrosine in proteins and peptides is a post-translational modification that occurs under conditions of oxidative stress. It is implicated in a variety of medical conditions, including neurodegenerative and cardiovascular diseases. However, monitoring tyrosine nitration and understanding its role in modifying biological function remains a major challenge. In this work, we investigate the use of electron-vibration-vibration (EVV) two-dimensional infrared (2DIR) spectroscopy for the study of tyrosine nitration in model peptides. We demonstrate the ability of EVV 2DIR spectroscopy to differentiate between the neutral and deprotonated states of 3-nitrotyrosine, and we characterize their spectral signatures using information obtained from quantum chemistry calculations and simulated EVV 2DIR spectra. To test the sensitivity of the technique, we use mixed-peptide samples containing various levels of tyrosine nitration, and we use mass spectrometry to independently verify the level of nitration. We conclude that EVV 2DIR spectroscopy is able to provide detailed spectroscopic information on peptide side-chain modifications and to detect nitration levels down to 1%. We further propose that lower nitration levels could be detected by introducing a resonant Raman probe step to increase the detection sensitivity of EVV 2DIR spectroscopy.

Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line

We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.