Elaine Spain

High sensitivity DNA detection using gold nanoparticle functionalised polyaniline nanofibres

Polyaniline (PANI) nanofibres (PANI-NF) have been modified with chemically grown gold nanoparticles to give a nanocomposite material (PANI-NF-AuNP) and deposited on gold electrodes. Single stranded capture DNA was then bound to the gold nanoparticles and the underlying gold electrode and allowed to hybridise with a complementary target strand that is uniquely associated with the pathogen, Staphylococcus aureus (S. aureus), that causes mastitis. Significantly, cyclic voltammetry demonstrates that deposition of the gold nanoparticles increases the area available for DNA immobilisation by a factor of approximately 4. EPR reveals that the addition of the Au nanoparticles efficiently decreases the interactions between adjacent PANI chains and/or motional broadening. Finally, a second horseradish peroxidase (HRP) labelled DNA strand hybridises with the target allowing the concentration of the target DNA to be detected by monitoring the reduction of a hydroquinone mediator in solution. The sensors have a wide dynamic range, excellent ability to discriminate DNA mismatches and a high sensitivity. Semi-log plots of the pathogen DNA concentration vs. faradaic current were linear from 150×10(-12) to 1×10(-6) mol L(-1) and pM concentrations could be detected without the need for molecular, e.g., PCR or NASBA, amplification.

DNA sensor based on vapour polymerised pedot films functionalised with gold nanoparticles.

Poly-3,4-ethylenedioxythiophene, PEDOT, films have been deposited on gold electrodes using polymerization from the vapor-phase in which the surface is first covered with a Fe (III) tosylate oxidant and then exposed to 3,4-ethylenedioxythiophene, EDOT, vapor. Gold nanoparticles were then electrodeposited to give a nanocomposite material, PEDOT-AuNP. Thiolated capture strand DNA, that is complementary to the sequence associated with the pathogen S. aureus that causes mammary gland inflammation, was then immobilized onto the gold nanoparticles and the underlying gold electrode. The target oligo was then hybridized to the capture strand DNA. A probe strand, labeled with horse radish peroxidase, was then hybridized to the target. The concentration of the target was determined by measuring the current required to reduce hydroquinone oxidized during the regeneration of the HRP label. Semi-log plots of the pathogen DNA concentration vs. faradaic current are linear from 150 pM to 1 μM and pM concentrations can be detected without the need for molecular, e.g., PCR or NASBA, amplification.

High sensitivity DNA detection based on regioselectively decorated electrocatalytic nanoparticles.

Self-assembled monolayers (SAMs) of dodecanethiol have been formed on gold electrodes to produce nanoscale defects. These defects define nucleation sites for the electrodeposition of mushroom shaped platinum nanoparticles (PtNPs). The top surfaces of these PtNPs have been selectively functionalized with single stranded probe DNA. These regioselectively modified particles were desorbed by applying a current jump to yield nanoparticles capable of biorecognition on the top curved side and efficient electrocatalysis on the nonfunctionalized lower surface. A second electrode was functionalized with single stranded capture DNA that has a sequence that is complementary to the pathogen, Staphylococcus aureus but leaves a section of the target available to bind the probe strand immobilized on the PtNPs. Following hybridization of the target and capture strands, the surface was exposed to the probe DNA labeled electrocatalytic PtNPs. Target binding was detected by monitoring the current associated with the reduction of hydrogen peroxide in a solution of 0.01 M H(2)SO(4). Calibration plots of the log[DNA] versus faradaic current were linear from 10 pM to 1 μM and picomolar concentrations could be detected without the need for amplification of the target, for example, using PCR or NASBA. As well as a wide dynamic range, this detection strategy has an excellent ability to discriminate DNA mismatches and a high analytical sensitivity.

Direct, non-amplified detection of microRNA-134 in plasma from epilepsy patients

Biofluid-based molecular biomarkers support clinical decision-making around diagnosis, prognosis and treatment for various diseases. MicroRNAs (miRNAs) have emerged as important regulators of gene expression in the brain and levels of miR-134 have been found to be elevated in experimental and human epilepsy. Here we show direct detection of miR-134 in human plasma without the need for PCR amplification using platinum nanoparticles (PtNPs) that are region-selectively decorated with probe strand nucleic acids. The nanoparticles were produced by electrodeposition using nanoscale defects within self-assembled monolayers of dodecanethiol. The template breaks the symmetry of the particle allowing one side to be selectively modified with the probe and leaving the other clean and capable of catalytically reducing hydrogen peroxide. The detection electrode was functionalised with single-stranded capture miRNA, miRNA that is complementary to miR-134 but leaves a section of the target available to bind the nucleic acid sequence bound to the PtNPs. Thus, electrocatalytic nanoparticles become confined on the electrode surface only when the target is present. Significantly, the relatively large current associated with the binding of small numbers of nanoparticles and their small area of occupation leads to attomolar limits of detection and a wide dynamic range without the need for molecular, e.g., PCR amplification. Using plasma samples from healthy volunteers and epilepsy patients we show highly linear correlation in miR-134 measurement to results with Taqman-based PCR. The present study demonstrates rapid and simple detection of miRNA that if developed further could provide simple point-of-care devices for detection of epilepsy biomarkers.

“TORNADO” – Theranostic One-Step RNA Detector; microfluidic disc for the direct detection of microRNA-134 in plasma and cerebrospinal fluid

Diagnosis of seizure disorders such as epilepsy currently relies on clinical examination and electroencephalogram recordings and is associated with substantial mis-diagnosis. The miRNA, miR-134 (MIR134 in humans), has been found to be elevated in brain tissue after experimental status epilepticus and in human epilepsy cells and their detection in biofluids may serve as unique biomarkers. miRNAs from unprocessed human plasma and human cerebrospinal fluid samples were used in a novel electrochemical detection based on electrocatalytic platinum nanoparticles inside a centrifugal microfluidic device where the sandwich assay is formed using an event triggered release system, suitable for the rapid point-of-care detection of low abundance biomarkers of disease. The device has the advantage of controlling the rotation speed of the centrifugal device to pump nanoliter volumes of fluid at a set time and manipulate the transfer of liquids within the device. The centrifugal platform improves reaction rates and yields by proposing efficient mixing strategies to overcome diffusion-limited processes and improve mass transport rates, resulting in reduced hybridization times with a limit of detection of 1 pM target concentration. Plasma and cerebrospinal fluid samples (unprocessed) from patients with epilepsy or who experienced status epilepticus were tested and the catalytic response obtained was in range of the calibration plot. This study demonstrates a rapid and simple detection for epilepsy biomarkers in biofluid.

Ligand capture and activation of human platelets at monolayer modified gold surfaces

Blood platelet adhesion is crucial in dictating haemocompatibility of medical implants and in platelet capture in diagnostics. Understanding the role of platelet activation in dictating platelet adhesion at chemically modified interfaces is important but relatively unexplored. Using scanning electron microscopy and confocal fluorescence microscopy a quantitative assessment of capture of blood platelets at self-assembled monolayers and mixed monolayers (SAMs) on gold as a function of the activation status of the platelets was conducted. Single and mixed monolayers were prepared using thiol-functionalized arginine-glycine-aspartic acid (RGD), C-Ahx-GRGDS (Ahx = aminohexanoic acid linker), thiolated poly(ethylene)glycol (PEG-COOH) and 1-octanethiol. When incubated with suspensions of resting platelets, RGD promoted platelet adhesion compared to bare or alkanethiol modified gold. Increasing the alkanethiol ratio in the deposition solution decreased the extent of platelet adhesion. Platelet adhesion increased approximately 3 fold at PEG-COO- modified surfaces compared to RGD-alone. Platelets adhered to RGD or mixed RGD : alkane SAM surfaces were found to be captured in their resting state. In contrast, platelets captured at PEG-COO- SAM surfaces were activated by these substrates. The effect of treating platelets with the chemical activators, Mn2+ or DTT or the physiological activator, thrombin, on the capture efficiency and activation at RGD modified surfaces was also investigated. Mn2+ treated platelets presented similar adhesion to untreated platelets, while surprisingly DTT yielded a very significant decrease in platelet adhesion. And, any platelets that were captured, were in a resting state. Thrombin activated platelets were captured with similar efficiencies as untreated platelets. However, the platelets captured were fully activated. The distinction between capture of chemically and physiologically activated platelet is interesting and likely to originate from differences in the conformation of the integrin induced by each process. Finally, platelet adhesion to each surface could be reversed by incubation with a solution of linear or cyclical RGD or PEG-COO- for the RGD and PEGCOO- surfaces respectively. The specificity of platelet removal confirmed that platelet adhesion at RGD surfaces is occurring through integrin-RGD interactions.

Fibrinogen Motif Discriminates Platelet and Cell Capture in Peptide-Modified Gold Micropore Arrays

Human blood platelets and SK-N-AS neuroblastoma cancer-cell capture at spontaneously adsorbed monolayers of fibrinogen-binding motifs, GRGDS (generic integrin adhesion), HHLGGAKQAGDV (exclusive to platelet integrin αIIbβ3), or octanethiol (adhesion inhibitor) at planar gold and ordered 1.6 μm diameter spherical cap gold cavity arrays were compared. In all cases, arginine/glycine/aspartic acid (RGD) promoted capture, whereas alkanethiol monolayers inhibited adhesion. Conversely only platelets adhered to alanine/glycine/aspartic acid (AGD)-modified surfaces, indicating that the AGD motif is recognized preferentially by the platelet-specific integrin, αIIbβ3. Microstructuring of the surface effectively eliminated nonspecific platelet/cell adsorption and dramatically enhanced capture compared to RGD/AGD-modified planar surfaces. In all cases, adhesion was reversible. Platelets and cells underwent morphological change on capture, the extent of which depended on the topography of the underlying substrate. This work demonstrates that both the nature of the modified interface and its underlying topography influence the capture of cancer cells and platelets. These insights may be useful in developing cell-based cancer diagnostics as well as in identifying strategies for the disruption of platelet cloaks around circulating tumor cells.