Design and Fabrication of a Fully-Integrated, Miniaturised Fluidic System for the Analysis of Enzyme Kinetics.

The lab-on-a-chip concept, enabled by microfluidic technology, promises the integration of multiple discrete laboratory techniques into a miniaturised system. Research into microfluidics has generally focused on the development of individual elements of the total system (often with relatively limited functionality), without full consideration for integration into a complete fully optimised and miniaturised system. Typically, the operation of many of the reported lab-on-a-chip devices is dependent on the support of a laboratory framework. In this paper, a demonstrator platform for routine laboratory analysis is designed and built, which fully integrates a number of technologies into a single device with multiple domains such as fluidics, electronics, pneumatics, hydraulics, and photonics. This facilitates the delivery of breakthroughs in research, by incorporating all physical requirements into a single device. To highlight this proposed approach, this demonstrator microsystem acts as a fully integrated biochemical assay reaction system. The resulting design determines enzyme kinetics in an automated process and combines reservoirs, three-dimensional fluidic channels, optical sensing, and electronics in a low-cost, low-power and portable package.

In vivo application of an implantable tri-anchored methylene blue-based electrochemical pH sensor.

The development of robust implantable sensors is important in the successful advancement of personalised medicine as they have the potential to provide in situ real-time data regarding the status of health and disease and the effectiveness of treatment. Tissue pH is a key physiological parameter and herein, we report the design, fabrication, functionalisation, encapsulation and protection of a miniaturised, self-contained, electrochemical pH sensor system and characterisation of sensor performance. Notably for the first time in this environment the pH sensor was based on a methylene blue redox reporter which showed remarkable robustness, accuracy and sensitivity. This was achieved by encapsulation of a self-assembled monolayer containing methylene blue entrapped within a Nafion layer. Another powerful feature was the incorporation, within the same implanted device, of a fabricated on-chip Ag/AgCl reference electrode - vital in any electrochemical sensor, but often ignored. When utilised in vivo, the sensor allowed accurate tracking of externally induced pH changes within a naturally occurring ovine lung cancer model, and correlated well with single point laboratory measurements made on extracted arterial blood, whilst enabling in vivo time-dependent measurements. The sensors functioned robustly whilst implanted, and maintained in vitro function once extracted and together, these results demonstrate proof-of-concept of the ability to sense real-time intratumoral tissue pH changes in vivo.

One-Year Safety and Clinical Outcomes of a Transcatheter Interatrial Shunt Device for the Treatment of Heart Failure With Preserved Ejection Fraction in the Reduce Elevated Left Atrial Pressure in Patients With Heart Failure (REDUCE LAP-HF I) Trial: A Randomized Clinical Trial.

Importance: In patients with heart failure (HF) and left ventricular ejection fraction (LVEF) equal to or greater than 40%, a transcatheter interatrial shunt device (IASD; Corvia Medical) reduces exercise pulmonary capillary wedge pressure (PCWP) and is safe compared with sham control treatment at 1 month of follow-up. The longer-term safety and patency of the IASD has not yet been demonstrated in the setting of a randomized clinical trial (RCT). Objective: To evaluate the 1-year safety and clinical outcomes of the IASD compared with a sham control treatment. Design, Setting, and Participants: This phase 2, double-blind, 1-to-1 sham-controlled multicenter RCT of IASD implantation vs a sham procedure (femoral venous access and imaging of the interatrial septum without IASD) was conducted in 22 centers in the United States, Europe, and Australia on patients with New York Heart Association (NYHA) class III or ambulatory class IV HF, LVEF equal to or greater than 40%, exercise PCWP equal to or greater than 25 mm Hg, and PCWP-right atrial pressure gradient equal to or greater than 5 mm Hg. Main Outcomes and Measures: Safety was assessed by major adverse cardiac, cerebrovascular, or renal events (MACCRE). Exploratory outcomes evaluated at 1 year were hospitalizations for HF, NYHA class, quality of life, a 6-minute walk test, and device patency. Results: After 1 year, shunts were patent in all IASD-treated patients; MACCRE did not differ significantly in the IASD arm (2 of 21 [9.5%]) vs the control arm (5 of 22 [22.7%]; P = .41), and no strokes occurred. The yearly rate of hospitalizations for HF was 0.22 in the IASD arm and 0.63 in the control arm (P = .06). Median improvement in NYHA class was 1 class in the IASD arm (IQR, -1 to 0) vs 0 in the control arm (IQR, -1 to 0; P = .08). Quality of life and 6-minute walk test distance were similar in both groups. At 6 months, there was an increase in right ventricular size in the IASD arm (mean [SD], 7.9 [8.0] mL/m2) vs the control arm (-1.8 [9.6] mL/m2; P = .002), consistent with left-to-right shunting through the device; no further increase occurred in the IASD arm at 12 months. Conclusions and Relevance: The REDUCE LAP-HF I phase 2, sham-controlled RCT confirms the longer-term patency of the IASD. Through 1 year of follow-up, IASD treatment appears safe, with no significant differences in MACCRE in patients receiving IASD compared with those who received sham control treatment. Trial Registration: ClinicalTrials.gov identifier: NCT02600234.

A Microelectrode Array with Reproducible Performance Shows Loss of Consistency Following Functionalization with a Self-Assembled 6-Mercapto-1-hexanol Layer.

For analytical applications involving label-free biosensors and multiple measurements, i.e., across an electrode array, it is essential to develop complete sensor systems capable of functionalization and of producing highly consistent responses. To achieve this, a multi-microelectrode device bearing twenty-four equivalent 50 µm diameter Pt disc microelectrodes was designed in an integrated 3-electrode system configuration and then fabricated. Cyclic voltammetry and electrochemical impedance spectroscopy were used for initial electrochemical characterization of the individual working electrodes. These confirmed the expected consistency of performance with a high degree of measurement reproducibility for each microelectrode across the array. With the aim of assessing the potential for production of an enhanced multi-electrode sensor for biomedical use, the working electrodes were then functionalized with 6-mercapto-1-hexanol (MCH). This is a well-known and commonly employed surface modification process, which involves the same principles of thiol attachment chemistry and self-assembled monolayer (SAM) formation commonly employed in the functionalization of electrodes and the formation of biosensors. Following this SAM formation, the reproducibility of the observed electrochemical signal between electrodes was seen to decrease markedly, compromising the ability to achieve consistent analytical measurements from the sensor array following this relatively simple and well-established surface modification. To successfully and consistently functionalize the sensors, it was necessary to dilute the constituent molecules by a factor of ten thousand to support adequate SAM formation on microelectrodes. The use of this multi-electrode device therefore demonstrates in a high throughput manner irreproducibility in the SAM formation process at the higher concentration, even though these electrodes are apparently functionalized simultaneously in the same film formation environment, confirming that the often seen significant electrode-to-electrode variation in label-free SAM biosensing films formed under such conditions is not likely to be due to variation in film deposition conditions, but rather kinetically controlled variation in the SAM layer formation process at these microelectrodes.

Catheter-based renal denervation in patients with uncontrolled hypertension in the absence of antihypertensive medications (SPYRAL HTN-OFF MED): a randomised, sham-controlled, proof-of-concept trial.

BACKGROUND: Previous randomised renal denervation studies did not show consistent efficacy in reducing blood pressure. The objective of our study was to evaluate the effect of renal denervation on blood pressure in the absence of antihypertensive medications. METHODS: SPYRAL HTN-OFF MED was a multicentre, international, single-blind, randomised, sham-controlled, proof-of-concept trial. Patients were enrolled at 21 centres in the USA, Europe, Japan, and Australia. Eligible patients were drug-naive or discontinued their antihypertensive medications. Patients with an office systolic blood pressure (SBP) of 150 mm Hg or greater and less than 180 mm Hg, office diastolic blood pressure (DBP) of 90 mm Hg or greater, and a mean 24-h ambulatory SBP of 140 mm Hg or greater and less than 170 mm Hg at second screening underwent renal angiography and were randomly assigned to renal denervation or sham control. Patients, caregivers, and those assessing blood pressure were blinded to randomisation assignments. The primary endpoint, change in 24-h blood pressure at 3 months, was compared between groups. Drug surveillance was done to ensure patient compliance with absence of antihypertensive medication. The primary analysis was done in the intention-to-treat population. Safety events were assessed at 3 months. This study is registered with ClinicalTrials.gov, number NCT02439749. FINDINGS: Between June 25, 2015, and Jan 30, 2017, 353 patients were screened. 80 patients were randomly assigned to renal denervation (n=38) or sham control (n=42) and followed up for 3 months. Office and 24-h ambulatory blood pressure decreased significantly from baseline to 3 months in the renal denervation group: 24-h SBP -5·5 mm Hg (95% CI -9·1 to -2·0; p=0·0031), 24-h DBP -4·8 mm Hg (-7·0 to -2·6; p<0·0001), office SBP -10·0 mm Hg (-15·1 to -4·9; p=0·0004), and office DBP -5·3 mm Hg (-7·8 to -2·7; p=0·0002). No significant changes were seen in the sham-control group: 24-h SBP -0·5 mm Hg (95% CI -3·9 to 2·9; p=0·7644), 24-h DBP -0·4 mm Hg (-2·2 to 1·4; p=0·6448), office SBP -2·3 mm Hg (-6·1 to 1·6; p=0·2381), and office DBP -0·3 mm Hg (-2·9 to 2·2; p=0·8052). The mean difference between the groups favoured renal denervation for 3-month change in both office and 24-h blood pressure from baseline: 24-h SBP -5·0 mm Hg (95% CI -9·9 to -0·2; p=0·0414), 24-h DBP -4·4 mm Hg (-7·2 to -1·6; p=0·0024), office SBP -7·7 mm Hg (-14·0 to -1·5; p=0·0155), and office DBP -4·9 mm Hg (-8·5 to -1·4; p=0·0077). Baseline-adjusted analyses showed similar findings. There were no major adverse events in either group. INTERPRETATION: Results from SPYRAL HTN-OFF MED provide biological proof of principle for the blood-pressure-lowering efficacy of renal denervation. FUNDING: Medtronic.

Integration of Electrodeposited Ni-Fe in MEMS with Low-Temperature Deposition and Etch Processes.

This article presents a set of low-temperature deposition and etching processes for the integration of electrochemically deposited Ni-Fe alloys in complex magnetic microelectromechanical systems, as Ni-Fe is known to suffer from detrimental stress development when subjected to excessive thermal loads. A selective etch process is reported which enables the copper seed layer used for electrodeposition to be removed while preserving the integrity of Ni-Fe. In addition, a low temperature deposition and surface micromachining process is presented in which silicon dioxide and silicon nitride are used, respectively, as sacrificial material and structural dielectric. The sacrificial layer can be patterned and removed by wet buffered oxide etch or vapour HF etching. The reported methods limit the thermal budget and minimise the stress development in Ni-Fe. This combination of techniques represents an advance towards the reliable integration of Ni-Fe components in complex surface micromachined magnetic MEMS.

Advances in electroanalysis, sensing and monitoring in molten salts.

Microelectrodes have a number of advantages over macroelectrodes for quantitative electroanalysis and monitoring, including reduced iR drop, a high signal-to-noise ratio and reduced sensitivity to convection. Their use in molten salts has been generally precluded by the combined materials challenges of stresses associated with thermal cycling and physical and corrosive chemical degradation at the relatively high temperatures involved. We have shown that microfabrication, employing high precision photolithographic patterning in combination with the controlled deposition of materials, can be used to successfully address these challenges. The resulting molten salt compatible microelectrodes (MSMs) enable prolonged quantitative microelectrode measurements in molten salts (MSs). This paper reports the fabrication of novel MSM disc electrodes, chosen because they have an established ambient analytical response. It includes a detailed set of electrochemical characterisation studies which demonstrate both their enhanced capability over macroelectrodes and over commercial glass pulled microelectrodes, and their ability to extract quantitative electroanalytical information from MS systems. MSM measurements are then used to demonstrate their potential for shedding new light on the fundamental properties of, and processes in, MSs, such as mass transport, charge transfer reaction rates and the selective plating/stripping and alloying reactions of liquid Bi and other metals; this will underpin the development of enhanced MS industrial processes, including pyrochemical spent nuclear fuel reprocessing.

Label- and amplification-free electrochemical detection of bacterial ribosomal RNA.

Current approaches to molecular diagnostics rely heavily on PCR amplification and optical detection methods which have restrictions when applied to point of care (POC) applications. Herein we describe the development of a label-free and amplification-free method of pathogen detection applied to Escherichia coli which overcomes the bottleneck of complex sample preparation and has the potential to be implemented as a rapid, cost effective test suitable for point of care use. Ribosomal RNA is naturally amplified in bacterial cells, which makes it a promising target for sensitive detection without the necessity for prior in vitro amplification. Using fluorescent microarray methods with rRNA targets from a range of pathogens, an optimal probe was selected from a pool of probe candidates identified in silico. The specificity of probes was investigated on DNA microarray using fluorescently labeled 16S rRNA target. The probe yielding highest specificity performance was evaluated in terms of sensitivity and a LOD of 20 pM was achieved on fluorescent glass microarray. This probe was transferred to an EIS end point format and specificity which correlated to microarray data was demonstrated. Excellent sensitivity was facilitated by the use of uncharged PNA probes and large 16S rRNA target and investigations resulted in an LOD of 50 pM. An alternative kinetic EIS assay format was demonstrated with which rRNA could be detected in a species specific manner within 10-40min at room temperature without wash steps.

Utility of rotational atherectomy and outcomes over an eight-year period.

OBJECTIVES: To evaluate outcomes of patients undergoing rotational atherectomy (RA) in a multicenter percutaneous coronary intervention (PCI) registry. BACKGROUND: RA remains an important technique for plaque modification in PCI, particularly with complex calcification. METHODS: The study population consisted of consecutive patients undergoing PCI in nine major Australian hospitals, who were treated over an 8-year period (June 2004 to June 2012). RESULTS: Of 16,577 PCI's, 1.0% of patients n = 167 (214 lesions) underwent RA. Patients undergoing RA were more likely to be older (71.0 ± 9.7 vs. 64.4 ±11.9 years, P < 0.01), with greater incidence of diabetes (37.7% vs. 23.8%, P < 0.01) and renal impairment. There was no significant difference in procedural success (94.6% vs. 95.5%, P = 0.57), dissection (6.1% vs. 4.8%, P = 0.39), transient no reflow (4.4% vs. 2.8%, P = 0.23), or persistent no reflow (0% RA vs. 0.7% non-RA, P = 0.23). Those undergoing RA had a low but increased risk of death at 12 months (6.6 vs. 3.6%, P = 0.04). There was no significant difference in 12 month major adverse cardiovascular outcomes (MACE) between groups following adjustment for univariate predictors (OR 1.00, 95%CI; 0.93-1.08). Additionally, there was no significant difference in 30-day MACE (6.0% vs. 5.1%, P = 0.62) or 30-day mortality (2.4% vs. 1.8%, P = 0.54) between groups. CONCLUSIONS: In this large multicenter registry, RA continues to be used to treat complex lesions with low procedural complications and MACE rates. It is essential for interventional cardiologists to maintain skills in RA to enable effective percutaneous treatment of certain complex lesions.

Enhanced electroanalysis in lithium potassium eutectic (LKE) using microfabricated square microelectrodes.

Molten salts (MSs) are an attractive medium for chemical and electrochemical processing and as a result there is demand for MS-compatible analysis technologies. However, MSs containing redox species present a challenging environment in which to perform analytical measurements because of their corrosive nature, significant thermal convection and the high temperatures involved. This paper outlines the fabrication and characterization of microfabricated square microelectrodes (MSMs) designed for electrochemical analysis in MS systems. Their design enables precise control over electrode dimension, the minimization of stress because of differential thermal expansion through design for high temperature operation, and the minimization of corrosive attack through effective insulation. The exemplar MS system used for characterization was lithium chloride/potassium chloride eutectic (LKE), which has potential applications in pyrochemical nuclear fuel reprocessing, metal refining, molten salt batteries and electric power cells. The observed responses for a range of redox ions between 400 and 500 °C (673 and 773 K) were quantitative and typical of microelectrodes. MSMs also showed the reduced iR drop, steady-state diffusion-limited response, and reduced sensitivity to convection seen for microelectrodes under ambient conditions and expected for these electrodes in comparison to macroelectrodes. Diffusion coefficients were obtained in close agreement with literature values, more readily and at greater precision and accuracy than both macroelectrode and previous microelectrode measurements. The feasibility of extracting individual physical parameters from mixtures of redox species (as required in reprocessing) and of the prolonged measurement required for online monitoring was also demonstrated. Together, this demonstrates that MSMs provide enhanced electrode devices widely applicable to the characterization of redox species in a range of MS systems.

Nanoscale electrode arrays produced with microscale lithographic techniques for use in biomedical sensing applications.

A novel technique for the production of nanoscale electrode arrays that uses standard microfabrication processes and micron-scale photolithography is reported here in detail. These microsquare nanoband edge electrode (MNEE) arrays have been fabricated with highly reproducible control of the key array dimensions, including the size and pitch of the individual elements and, most importantly, the width of the nanoband electrodes. The definition of lateral features to nanoscale dimensions typically requires expensive patterning techniques that are complex and low-throughput. However, the fabrication methodology used here relies on the fact that vertical dimensions (i.e. layer thicknesses) have long been manufacturable at the nanoscale using thin film deposition techniques that are well established in mainstream microelectronics. The authors report for the first time two aspects that highlight the particular suitability of these MNEE array systems for probe monolayer biosensing. The first is simulation, which shows the enhanced sensitivity to the redox reaction of the solution redox couple. The second is the enhancement of probe film functionalisation observed for the probe film model molecule, 6-mercapto-1-hexanol compared with microsquare electrodes. Such surface modification for specific probe layer biosensing and detection is of significance for a wide range of biomedical and other sensing and analytical applications.

Comparison of the performance of an array of nanoband electrodes with a macro electrode with similar overall area.

The performance of two electrode architectures with broadly similar overall active electrode areas are examined. The first is an electrode comprising a single contiguous area (a disc) and the second is an electrode in which the cumulative electrode area is dispersed over a wide area as a 50 nm thickness platinum nanoband. A direct comparison of the electrochemical performance of these two electrodes has been made. The relatively simple nanoband electrode architecture is shown to have benefits, including two orders of magnitude greater mass transport limited currents, the ability to measure faster electrode kinetics (by a similar factor), a three orders of magnitude lowering of the Limit of Detection and a significantly reduced susceptibility to hydrodynamic perturbations. The consequences and implications of these performance characteristics on the uses of such a nanoband electrode have been considered.

Feasibility of catheter-based renal nerve ablation and effects on sympathetic nerve activity and blood pressure in patients with end-stage renal disease.

BACKGROUND AND OBJECTIVES: Sympathetic activation is a hallmark of ESRD and adversely affects cardiovascular prognosis. Efferent sympathetic outflow and afferent neural signalling from the failing native kidneys are key mediators and can be targeted by renal denervation (RDN). Whether this is feasible and effective in ESRD is not known. DESIGN, SETTING, PARTICIPANTS AND MEASUREMENTS: In an initial safety and proof-of-concept study we attempted to perform RDN in 12 patients with ESRD and uncontrolled blood pressure (BP). Standardized BP measurements were obtained in all patients on dialysis free days at baseline and follow up. Measures of renal noradrenaline spillover and muscle sympathetic nerve activity were available from 5 patients at baseline and from 2 patients at 12 month follow up and beyond. RESULTS: Average office BP was 170.8 ± 16.9/89.2 ± 12.1 mmHg despite the use of 3.8 ± 1.4 antihypertensive drugs. All 5 patients in whom muscle sympathetic nerve activity and noradrenaline spillover was assessed at baseline displayed substantially elevated levels. Three out of 12 patients could not undergo RDN due to atrophic renal arteries. Compared to baseline, office systolic BP was significantly reduced at 3, 6, and 12 months after RDN (from 166 ± 16.0 to 148 ± 11, 150 ± 14, and 138 ± 17 mmHg, respectively), whereas no change was evident in the 3 non-treated patients. Sympathetic nerve activity was substantially reduced in 2 patients who underwent repeat assessment. CONCLUSIONS: RDN is feasible in patients with ESRD and associated with a sustained reduction in systolic office BP. Atrophic renal arteries may pose a problem for application of this technology in some patients with ESRD.

A systematic study of the influence of nanoelectrode dimensions on electrode performance and the implications for electroanalysis and sensing.

Micron resolution photolithography has been employed to make microsquare nanoband edge electrode (MNEE) arrays with reproducible and systematic control of the crucial dimensional parameters, including array element size and spacing and nanoelectrode thickness. The response of these arrays, which can be reproducibly fabricated on a commercial scale, is first established. The resulting characteristics (including high signal and signal-to-noise, low limit of detection, insensitivity to external convection and fast, steady-state, reproducible and quantitative response) make such nanoband electrode arrays of real interest as enhanced electroanalytical devices. In particular, the nanoelectrode response is presented and analysed as a function of nanometre scale electrode dimension, to assess the impact and relative contributions of previously postulated nanodimensional effects on the resulting response. This work suggests a significant contribution of migration at the band edges to mass transfer, which affects the resulting electroanalytical response even at ionic strengths as large as 0.7 mol dm(-3) and for electrodes as wide as 50 nm. For 5 nm nanobands, additional nanoeffects, which are thought to arise from the fact that the size of the redox species is comparable to the band width, are also observed to attenuate the observed current. The fundamental insight this gives into electrode performance is discussed along with the consequent impact on using such electrodes of nanometre dimension.

Impedimetric detection of single-stranded PCR products derived from methicillin resistant Staphylococcus aureus (MRSA) isolates.

Using electrochemical impedance spectroscopy (EIS) the sensitive and specific detection of the antibiotic resistance gene mecA has been demonstrated. The gene sequence was obtained from clinical Staphylococcus aureus isolates. Initially a mecA specific probe was selected from hybridisation tests with a 3' and 5' version of a previously published probe sequence. When immobilised on a gold electrode in PNA form it was possible to detect hybridisation of mecA PCR product electrochemically at concentrations as low as 10nM. By incorporating an undecane-thiol and 1.8 nm glycol spacer into the PNA probe it was possible to extend the limit of detection for mecA to 10 pM. Most published studies on EIS and nucleic acid detection report the use of short artificial DNA sequences or novel signal amplification schemes which improve sensitivity whereas this study reports the successful detection of long DNA fragments produced by PCR following extraction from clinical isolates. Finally, using screen printed electrodes the paper demonstrates hybridisation monitoring of mecA in an "on-line" assay format under ambient conditions which paves the way for rapid mecA detection in point of care scenarios.

Development of immunosensors for direct detection of three wound infection biomarkers at point of care using electrochemical impedance spectroscopy.

A method for label-free, electrochemical impedance immunosensing for the detection and quantification of three infection biomarkers in both buffer and directly in the defined model matrix of mock wound fluid is demonstrated. Triggering Receptor-1 Expressed on Myeloid cells (TREM-1) and Matrix MetalloPeptidase 9 (MMP-9) are detected via direct assay and N-3-oxo-dodecanoyl-l-HomoSerineLactone (HSL), relevant in bacterial quorum sensing, is detected using a competition assay. Detection is performed with gold screen-printed electrodes modified with a specific thiolated antibody. Detection is achieved in less than 1h straight from mock wound fluid without any extensive sample preparation steps. The limits of detection of 3.3 pM for TREM-1, 1.1 nM for MMP-9 and 1.4 nM for HSL are either near or below the threshold required to indicate infection. A relatively large dynamic range for sensor response is also found, consistent with interaction between neighbouring antibody-antigen complexes in the close-packed surface layer. Together, these three novel electrochemical immunosensors demonstrate viable multi-parameter sensing with the required sensitivity for rapid wound infection detection directly from a clinically relevant specimen.

Fast DNA and protein microarray tests for the diagnosis of hepatitis C virus infection on a single platform.

Hepatitis C virus (HCV) is a major cause of chronic liver disease and liver cancer, and remains a large health care burden to the world. In this study we developed a DNA microarray test to detect HCV RNA and a protein microarray to detect human anti-HCV antibodies on a single platform. A main focus of this study was to evaluate possibilities to reduce the assay time, as a short time-to-result (TTR) is a prerequisite for a point-of-care test. Significantly reducing hybridisation and washing times did not impair the assay performance. This was confirmed first using artificial targets and subsequently using clinical samples from an HCV seroconversion panel derived from a HCV-infected patient. We were able to reduce the time required for the detection of human anti-HCV antibodies to only 14 min, achieving nanomolar sensitivity. The protein microarray exhibited an analytical sensitivity comparable to that of commercial systems. Similar results were obtained with the DNA microarray using a universal probe which covered all different HCV genotypes. It was possible to reduce the assay time after PCR from 150 min to 16 min without any loss of sensitivity. Taken together, these results constitute a significant step forward in the design of rapid, microarray-based diagnostics for human infectious disease, and show that the protein microarray is currently the most favourable candidate to fill this role.

Dielectrophoretic manipulation of ribosomal RNA.

The manipulation of ribosomal RNA (rRNA) extracted from E. coli cells by dielectrophoresis (DEP) has been demonstrated over the range of 3 kHz-50 MHz using interdigitated microelectrodes. Quantitative measurement using total internal reflection fluorescence microscopy of the time dependent collection indicated a positive DEP response characterized by a plateau between 3 kHz and 1 MHz followed by a decrease in response at higher frequencies. Negative DEP was observed above 9 MHz. The positive DEP response below 1 MHz is described by the Clausius-Mossotti model and corresponds to an induced dipole moment of 3300 D with a polarizability of 7.8×10(-32) F m(2). The negative DEP response above 9 MHz indicates that the rRNA molecules exhibit a net moment of -250 D, to give an effective permittivity value of 78.5 ε(0), close to that of the aqueous suspending medium, and a relatively small surface conductance value of ∼0.1 nS. This suggests that our rRNA samples have a fairly open structure accessible to the surrounding water molecules, with counterions strongly bound to the charged phosphate groups in the rRNA backbone. These results are the first demonstration of DEP for fast capture and release of rRNA units, opening new opportunities for rRNA-based biosensing devices.

Peptide-tags for enhanced DNA microarray performance.

DNA microarrays are powerful tools for gene expression analysis and genotyping studies in research and diagnostic applications. A high sensitivity and short time-to-result are prerequisites for their practical application in the clinic. The hybridization efficiency of DNA microarrays depends on the probe density and the probe orientation and thus their accessibility for target molecules. In order to find an optimal probe immobilization procedure a set of different oligonucleotide modifications was tested on epoxy silane functionalized glass slides. It was found that histidine-tagged oligonucleotides resulted in the highest amount of bound probe and by far the best hybridization efficiencies. The detection limit obtained with histidine-tagged probes was up to two orders of magnitude lower compared to commonly used probe modifications. In order to further investigate the binding mechanism of histidine-tags towards functionalized glass substrates a set of different peptide-tags with and without free terminal amino-groups and with different amino acid compositions was tested. The results indicate an impact of the terminal amino group on the covalent surface binding and of aromatic amino acid residues on the enhanced hybridisation efficiency.