Quantification of tRNA fragments by electrochemical direct detection in small volume biofluid samples.

Elevated levels of transfer RNA (tRNA) fragments were recently identified in plasma samples from people with epilepsy in advance of a seizure, indicting a potential novel class of circulating biomarker. Current methods for detection and quantitation of tRNA fragments (tRFs) include northern blotting, RNA sequencing or custom Taqman-based PCR assays. The development of a simple, at home or clinic-based test, would benefit from a simple and reliable method to detect the tRFs using small volumes of biofluids. Here we describe an electrochemical direct detection method based on electrocatalytic platinum nanoparticles to detect 3 specific tRFs: 5'AlaTGC, 5'GlyGCC, and 5'GluCTC. Using synthetic tRF mimics we showed this system was linear over 9 orders of magnitude with sub-attomolar limits of detection. Specificity was tested using naturally occurring mismatched tRF mimics. Finally, we quantified tRF levels in patient plasma and showed that our detection system recapitulates results obtained by qPCR. We have designed a tRF detection system with high sensitivity and specificity capable of quantifying tRFs in low volumes of plasma using benchtop apparatus. This is an important step in the development of a point-of-care device for quantifying tRFs in whole blood.

Elevated Plasma microRNA-206 Levels Predict Cognitive Decline and Progression to Dementia from Mild Cognitive Impairment.

The need for practical biomarkers for early diagnosis of Alzheimer's disease (AD) remains largely unmet. Here we investigated the use of blood-based microRNAs as prognostic biomarkers for AD and their application in a novel electrochemical microfluidic device for microRNA detection. MicroRNA transcriptome was profiled in plasma from patients with mild cognitive impairment (MCI) and AD. MicroRNAs Let-7b and microRNA-206 were validated at elevated levels in MCI and AD, respectively. MicroRNA-206 displayed a strong correlation with cognitive decline and memory deficits. Longitudinal follow-ups over five years identified microRNA-206 increases preceding the onset of dementia. MicroRNA-206 was increased in unprocessed plasma of AD and MCI subjects, detected by our microfluidic device. While increased Let-7b levels in plasma may be used to identify patients with MCI, changes in plasma levels of microRNA-206 may be used to predict cognitive decline and progression towards dementia at an MCI stage. MicroRNA quantification via a microfluidic device could provide a practical cost-effective tool for the stratification of patients with MCI according to risk of developing AD.

Cardiac Troponin I: Ultrasensitive Detection Using Faradaic Electrochemical Impedance.

An electrochemical biosensor for the detection of cardiac troponin I, cTnI, an important cardiac biomarker, is described. A combination of a novel monoclonal antibody, mAb20B3, and a novel Ir(III)-based metal complex was used for detection using faradaic electrochemical impedance spectroscopy. A limit of detection of 10 ag/mL was achieved, which is significantly lower than established assays. The ability to detect these ultralow concentrations enables rapid and early stage detection of cardiac events and opens up the possibility of developing a point-of-care device.

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 αIIbß3), 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, αIIbß3. 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.

Hemispherical platinum : silver core : shell nanoparticles for miRNA detection.

Defects within a self-assembled monolayer (SAM) of dodecanethiol on gold have been used as nucleation sites for the electrodeposition of mushroom shaped platinum nanoparticles (PtNPs). The top surfaces of these PtNPs were then decorated with a layer of silver creating a hemispherical - platinum : silver core : shell nanoparticle (Pt-AgNP). Thiolated probe strand miRNA was then immobilised onto the upper silver surface. These regioselectively modified particles were desorbed by applying a current jump to yield nanoparticles capable of hybridising to a complementary miRNA target with electrocatalysis occurring on the non-functionalized lower surface. A second electrode was functionalized with single stranded capture miRNA that has a sequence that is complementary to an miRNA, miR-132, associated with the childhood cancer, Neuroblastoma but leaves a section of the target available to bind the nucleic acid sequence on the core : shell Pt-AgNPs. Following hybridization of the target and capture strands the surface was exposed to the miRNA labelled electrocatalytic Pt-AgNPs. The concentration of the target was then determined by monitoring the current associated with the reduction of hydrogen peroxide in a solution of H2SO4. Calibration plots of the log[miRNA] vs. faradaic current were linear from 1 aM to 1 µM and aM concentrations could be detected without the need for chemical amplification of the target, e.g., using PCR or NASBA. The regioselectively modified particles were also immobilised within the interior of gold microcavity arrays via miRNA hybridisation and their Raman properties investigated.

Peptide-Mediated Platelet Capture at Gold Micropore Arrays.

Ordered spherical cap gold cavity arrays with 5.4, 1.6, and 0.98 µm diameter apertures were explored as capture surfaces for human blood platelets to investigate the impact of surface geometry and chemical modification on platelet capture efficiency and their potential as platforms for surface enhanced Raman spectroscopy of single platelets. The substrates were chemically modified with single-constituent self-assembled monolayers (SAM) or mixed SAMs comprised of thiol-functionalized arginine-glycine-aspartic acid (RGD, a platelet integrin target) with or without 1-octanethiol (adhesion inhibitor). As expected, platelet adhesion was promoted and inhibited at RGD and alkanethiol modified surfaces, respectively. Platelet adhesion was reversible, and binding efficiency at the peptide modified substrates correlated inversely with pore diameter. Captured platelets underwent morphological change on capture, the extent of which depended on the topology of the underlying substrate. Regioselective capture of the platelets enabled study for the first time of the surface enhanced Raman spectroscopy of single blood platelets, yielding high quality Raman spectroscopy of individual platelets at 1.6 µm diameter pore arrays. Given the medical importance of blood platelets across a range of diseases from cancer to psychiatric illness, such approaches to platelet capture may provide a useful route to Raman spectroscopy for platelet related diagnostics.

Detection of prostate specific antigen based on electrocatalytic platinum nanoparticles conjugated to a recombinant scFv antibody.

Highly sensitive and label free detection of prostate specific antigen (PSA) still remains a challenge in prostate cancer diagnosis. In this paper, we propose a sensitive electrochemical immunosensor based on electrocatalytic platinum nanoparticles conjugated to a recombinant scFv antibody. Gold disc electrodes functionalised with a l-Cysteine (Cys) self-assembled monolayer (SAM) were used to covalently bind PSA specific monoclonal antibody (anti-PSA) using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry. Immunosensing was completed using sandwich-type immunoreaction of the PSA-antigen (1-30 ng/mL) between anti-PSA immobilized on the l-Cys modified electrode using label free electrochemical impedance (EIS) technique. Furthermore, highly specific in-house generated scFv fragments as receptor proteins were utilised for one step site-directed immobilisation on the surface of platinum nanoparticles (PtNPs). To improve the sensitivity of the immunoassay, these scFV labelled electrocatalytic PtNPs were then used for covalent hybridisation to the PSA modified electrode and then applied in a hybridisation assay to determine the concentration of the PSA by measuring the faradaic current associated with reduction of peroxide in solution. Semi-log plots of the PSA concentration vs. faradaic current are linear from 1 to 30 ng/mL and pM concentrations can be detected without the need for molecular, e.g., PCR or NASBA, amplification.

RGD labeled Ru(II) polypyridyl conjugates for platelet integrin αIIbß3 recognition and as reporters of integrin conformation.

The ability of two novel ruthenium(II) polypyridyl-Arg-Gly-Asp (RGD) peptide conjugates to act as molecular probes for reporting on the presence and conformation of integrin αIIbß3 in solution and in live cells was described. The compounds are [Ru(bpy)2PIC-RGD](2+), bpy-RGD, and [Ru(dpp)2PIC-RGD](2+), dpp-RGD, where dpp is 4,7-diphenyl-1,10-phenanthroline, bpy is 2,2'-bipyridine, and PIC is 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline. Bpy-RGD is hydrophilic, whereas dpp-RGD is comparatively hydrophobic. Both probes exhibited good affinity and high specificity for purified αIIbß3 in solution. Binding of either complex to the resting integrin resulted in an approximately 8-fold increase of emission intensity from the metal center with dissociation constants (Kd) in the micromolar range for each complex. The Kd for each conjugate/αIIbß3 assembly were compared following treatment of the integrin with the activating agents, Mn(2+) and dithiothreitol (DTT), which are commonly used to induce active-like conformational changes in the integrin. For bpy-RGD/αIIbß3 Kd showed relatively little variation with integrin activation, presenting the following trend: denatured αIIbß3 > resting αIIbß3 = pretreated DTT = pretreated Mn(2+). Kd for dpp-RGD/ αIIbß3 showed greater variation with integrin activation and the following trend was followed: denatured αIIbß3 > resting αIIbß3 > pretreated Mn(2+) = pretreated DTT. Time resolved luminescence anisotropy was carried out to obtain the rotational correlation time of bpy-RGD and dpp-RGD bound to resting or nominally activated integrin. The rotational correlation times of bpy-RGD and dpp-RGD, too fast to measure unbound, decreased to 1.50 ± 0.03 µs and 2.58 ± 0.04 µs, respectively, when the complexes were bound to resting integrin. Addition of Mn(2+) to bpy-RGD/αIIbß3 or dpp-RGD/αIIbß3 reduced the rotational correlation time of the ruthenium center to 1.29 ± 0.03 µs and to 1.72 ± 0.03 µs, respectively. Following treatment, the rotational correlation time decreased to 1.04 ± 0.01 µs and 1.29 ± 0.03 µs for bpy-RGD/αIIbß3, and dpp-RGD/αIIbß3, respectively. The large relative changes in rotational correlation times observed for Mn(2+) or DTT activated integrin indicates significant change in protein conformation compared with the resting integrin. The results also indicated that the metal complex itself affects the final conformational and/or aggregation status of the protein obtained. Furthermore, the extent of conformational change was influenced by whether the probe was bound to the integrin before or after activator treatment. Finally, in vitro studies indicated that both probes selectively bind to CHO cells expressing the resting form of αIIbß3. In each case the probe colocalized with αIIb specific SZ22 antibody. Overall, this work indicates that bpy-RGD and dpp-RGD may be useful peptide-probes for rapid assessment of integrin structural status and localization in solution and cells.

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.

Peptide directed transmembrane transport and nuclear localization of Ru(II) polypyridyl complexes in mammalian cells.

The targeted delivery of luminescent Ru(II) polypyridyl complexes to the nucleus of live mammalian cells by a Nuclear Localisation Signal (NLS) peptide; NF-κB is demonstrated.