Aptamer Lateral Flow Assays for Ultrasensitive Detection of ß-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers.

In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which ß-conglutin immobilized on the test line of a nitrocellulose membrane and ß-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the ß-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized ß-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay.

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10-11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need.

One-Port Electronic Detection Strategies for Improving Sensitivity in Piezoelectric Resonant Sensor Measurements.

This paper describes a one-port mechanical resonance detection scheme utilized on a piezoelectric thin film driven silicon circular diaphragm resonator and discusses the limitations to such an approach in degenerate mode mass detection sensors. The sensor utilizes degenerated vibration modes of a radial symmetrical microstructure thereby providing both a sense and reference mode allowing for minimization of environmental effects on performance. The circular diaphragm resonator was fabricated with thickness of 4.5 µm and diameter of 140 µm. A PZT thin film of 0.75 µm was patterned on the top surface for the purposes of excitation and vibration sensing. The device showed a resonant frequency of 5.8 MHz for the (1, 1) mode. An electronic interface circuit was designed to cancel out the large static and parasitic capacitance allowing for electrical detection of the mechanical vibration thereby enabling the frequency split between the sense and reference mode to be measured accurately. The extracted motional current, proportional to the vibration velocity, was fed back to the drive to effectively increase the Q factor, and therefore device sensitivity, by more than a factor of 8. A software phase-locked loop was implemented to automatically track the resonant frequencies to allow for faster and accurate resonance detection. Results showed that by utilizing the absolute mode frequencies as an indication of sensor temperature, the variation in sensor temperature due to the heating from the drive electronics was accounted for and led to an ultimate measurement sensitivity of 2.3 Hz.

Site-directed introduction of disulfide groups on antibodies for highly sensitive immunosensors.

The interface between the sample and the transducer surface is critical to the performance of a biosensor. In this work, we compared different strategies for covalent self-assembly of antibodies onto bare gold substrates by introducing disulfide groups into the immunoglobulin structure, which acted as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The disulfide moieties were chemically introduced to the antibody via the primary amines, carboxylic acids, and carbohydrates present in its structure. The site-directed modification via the carbohydrate chains exhibited the best performance in terms of analyte response using a model system for the detection of the stroke marker neuron-specific enolase. SPR measurements clearly showed the potential for creating biologically active densely packed self-assembled monolayers (SAMs) in a one-step protocol compared to both mixed SAMs of alkanethiol compounds and commercial immobilization layers. The ability of the carbohydrate strategy to construct an electrochemical immunosensor was investigated using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) transduction. Graphical Abstract Left: Functionalization strategies of bare gold substrates via direct bio-SAM using disulfide-containing antibody chemically modified via their primary amines (A), carbohydrates (B) and carboxylic acids (C). Right: Dependence of the peak height with NSE concentration at NSE21-CHO modified electrochemical immunosensor. Inset: Logarithmic calibration plot.

How much incisor decompensation is achieved prior to orthognathic surgery?

OBJECTIVES: To quantify incisor decompensation in preparation for orthognathic surgery. STUDY DESIGN: Pre-treatment and pre-surgery lateral cephalograms for 86 patients who had combined orthodontic and orthognathic treatment were digitised using OPAL 2.1 [http://www.opalimage.co.uk]. To assess intra-observer reproducibility, 25 images were re-digitised one month later. Random and systematic error were assessed using the Dahlberg formula and a two-sample t-test, respectively. Differences in the proportions of cases where the maxillary (1100 +/- 60) or mandibular (900 +/- 60) incisors were fully decomensated were assessed using a Chi-square test (p<0.05). Mann-Whitney U tests were used to identify if there were any differences in the amount of net decompensation for maxillary and mandibular incisors between the Class II combined and Class III groups (p<0.05). RESULTS: Random and systematic error were less than 0.5 degrees and p<0.05, respectively. A greater proportion of cases had decompensated mandibular incisors (80%) than maxillary incisors (62%) and this difference was statistically significant (p=0.029). The amount of maxillary incisor decompensation in the Class II and Class III groups did not statistically differ (p=0.45) whereas the mandibular incisors in the Class III group underwent statistically significantly greater decompensation (p=0.02). CONCLUSIONS: Mandibular incisors were decompensated for a greater proportion of cases than maxillary incisors in preparation for orthognathic surgery. There was no difference in the amount of maxillary incisor decompensation between Class II and Class III cases. There was a greater net decompensation for mandibular incisors in Class III cases when compared to Class II cases. Key words:Decompensation, orthognathic, pre-surgical orthodontics, surgical-orthodontic.

Health related quality of life in people with advanced chronic liver disease.

Cirrhosis has a long natural history with considerable symptomatic impacts, particularly in advancing disease. Measuring health related quality of life (HRQOL) in liver disease provides detail about the nature and extent of its effects on individuals. Understanding the drivers of impaired HRQOL can help identify targets for improvement through new treatments or health systems service delivery. Evaluation of novel therapies which target symptomatic improvement, should be done with suitable outcome measures, including HRQOL assessment. In this article, we provide an overview of HRQOL in advanced liver disease for the clinician. A clear description of the important HRQOL tools is given alongside a discussion of the factors, which are known to contribute to impaired HRQOL in advanced liver disease.

Real-time monitoring of superoxide generation and cytotoxicity in neuroblastoma mitochondria induced by 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline.

The potential neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) has recently been suggested to be a causative factor in the clinical development of parkinsonian symptoms after long-term exposure to precursor compounds such as the hypnotic chloral hydrate. TaClo is known to cause cell death in dopaminergic neuronal cells, however, the pathway and mechanisms remain undefined. This study reports for the first time that TaClo promotes cytotoxicity in SH-SY5Y neuroblastoma cells within 2 hours of initial exposure. TaClo also caused superoxide production from isolated mitochondria, which was comparable in response time and magnitude to production elicited by more established respiratory inhibitors such as rotenone and antimycin A. These findings present new evidence in support of TaClo-induced neuronal death via superoxide signalling and oxidative stress.

Electrochemical and optical sensing of reactive oxygen species: pathway to an integrated intracellular and extracellular measurement platform.

A comprehensive understanding of ROS (reactive oxygen species)-dependent cellular interaction requires the previously unmet ability to simultaneously monitor the intra- and extra-cellular environments. The present review assesses the potential of novel electrochemical and fluorescent-based nanosensor approaches to address the limitations of existing techniques for ROS analysis. Data generated by these new approaches have already contributed significantly to current understanding of the roles that these species play in various in vitro scenarios. However, integration of these novel approaches has the potential to offer, for the first time, the unparalleled ability to measure simultaneously and in real-time ROS flux in both the intra- and extra-cellular environments.

Biomolecule patterning on analytical devices: a microfabrication-compatible approach.

The present work describes a methodology for patterning biomolecules on silicon-based analytical devices that reconciles 3-D biological functionalization with standard resist lift-off techniques. Unlike classic sol-gel approaches in which the biomolecule of interest is introduced within the sol mixture, a two-stage scenario has been developed. It consists first of patterning micrometer/submicrometer polycondensate scaffold structures, using classic microfabrication tools, that are then loaded with native biomolecules via a second simple incubation step under biologically friendly environmental conditions. The common compatibility issue between the biological and microfabrication worlds has been circumvented because native recognition biomolecules can be introduced into the host scaffold downstream from all compatibility issues. The scaffold can be generated on any silicon substrate via the polycondensation of aminosilane, namely, aminopropyltriethoxy silane (APTES), under conditions that are fully compatible with resist mask lithography. The scaffold porosity and high primary amine content allow proteins and nucleic acid sequences to penetrate the polycondensate and to interact strongly, thus giving rise to micrometer/submicrometer 3-D structures exhibiting high biological activity. The integration of such a biopatterning approach in the microfabrication process of silicon analytical devices has been demonstrated via the successful completion of immunoassays and nucleic acid assays.

Direct, real-time monitoring of superoxide generation in isolated mitochondria.

Mitochondria are one of the major sources of reactive oxygen species (ROS) in mammalian cells. The generation of ROS underlies many physiological and pathophysiological processes that occur within cellular systems. Superoxide ([image omitted] ) is the proximal ROS generated during electron 'leakage' from the mitochondrial electron transport chain (mETC) and is known to be released at mitochondrial complex I and complex III. Monitoring mitochondrial [image omitted] production directly and in real-time offers the potential to improve understanding of the complex mechanisms involved during mitochondrial [image omitted] generation. This study reports the novel application of a cytochrome c functionalized amperometric sensor for monitoring [image omitted] generation in isolated mitochondrial fractions. The non-invasive sensor system described allowed a comparison of [image omitted] production following specific inhibition of complex I and complex III of the mETC to be made directly and in real-time.

The development and in vitro characterisation of an intracellular nanosensor responsive to reactive oxygen species.

Advances in sensor technologies have enhanced our understanding of the roles played by reactive oxygen species (ROS) in a number of physiological and pathological processes. However, high inter-reactivity and short life spans has made real-time monitoring of ROS in cellular systems challenging. Fluorescent dyes capable of intracellular ROS measurements have been reported. However, these dyes are known to be intrinsically cytotoxic and thus can potentially significantly alter cellular metabolism and adversely influence in vitro data. Reported here is the development and in vitro application of a novel ROS responsive nanosensor, based on PEBBLE (Probes Encapsulated By Biologically Localised Embedding) technology. The ROS sensitive fluorescent probe dihydrorhodamine 123 (DHR 123) was employed as the sensing element of the PEBBLE through entrapment within a porous, bio-inert polyacrylamide nanostructure enabling passive monitoring of free radical flux within the intracellular environment. Successful delivery of the nanosensors into NR8383 rat alveolar macrophage cells via phagocytosis was achieved. Stimulation of PEBBLE loaded NR8383 cells with phorbol-12-myristate-13-acetate (PMA) enabled real time monitoring of ROS generation within the cell without affecting cellular viability. These data suggest that PEBBLE nanosensors could offer significant advantages over existing technologies used in monitoring the intracellular environment.

Chemical introduction of disulfide groups on glycoproteins: a direct protein anchoring scenario.

The present work reports a direct glycoprotein immobilization protocol where the protein is chemically modified with disulfide groups which act as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The specificity of the chemical reaction, for disulfide introduction, toward carbohydrate moieties prevents any cross-reaction with other functional groups present in the protein structure. Horseradish peroxidase (HRP) was chosen as a model glycoprotein, and a biologically active densely packed SAM was obtained on gold, as demonstrated by spectrophotometry and surface plasmon resonance (SPR) spectroscopy. A hydrogen peroxide amperometric biosensor was designed using a freely diffusing mediator which exhibited high sensitivity (196 mA M-1 cm-2) and low apparent Michaelis-Menten constant (67 microM). By extension, a mixed bienzymatic monolayer, obtained by simultaneous cochemisorption of modified HRP and glucose oxidase (GOD), on a clean gold electrode displayed a high sensitivity toward glucose (13 mA M-1 cm-2). Far from competing with the versatility of the classic SAM scenario or the precision of genetic engineering, this work presents a rational and particularly rapid approach where the selectivity of chemical reactions takes advantage of the specific location of carbohydrates on glycosylated protein and antibody structures for creating highly active biological interfaces directly chemisorbed onto bare gold detection devices.

The biosensor based on the pyruvate oxidase modified conducting polymer for phosphate ions determinations.

An enzymatic biosensor was fabricated by the covalent immobilization of pyruvate oxidase (PyO) onto the nano-particle comprised poly-5,2':5',2''-terthiophene-3'-carboxylic acid, poly-TTCA (nano-CP) layers on a glassy carbon electrode (GCE) for the amperometric detection of the phosphate ions. The direct electron transfer reaction of the immobilized PyO onto the nano-CP layers was investigated and the electron transfer rate constant was determined to be 0.65 s(-1). The electrochemically prepared nano-CP lowered the oxidation potential (+0.40 V versus Ag/AgCl) of an enzymatically generated H(2)O(2) by PyO in a phosphate solution. Experimental parameters affecting the sensitivity of the biosensors, such as amounts of the cofactors, the pH, the applied potential, and the temperature were optimized. A linear response for the detection of the phosphate ion was observed between 1.0 microM and 100 microM and the detection limit was determined to be about 0.3 microM. The response time of the biosensors was about 6s. The biosensor showed good selectivity towards other interfering anions. The long-term storage stability of the phosphate biosensor was studied and the sensor was applied in a human serum sample for the phosphate ions detection.

One-step immunostrip test for the simultaneous detection of free and total prostate specific antigen in serum.

The development of a one-step lateral flow immunoassay on a strip format for the rapid and reliable simultaneous detection of serum levels of free and total prostate specific antigen (f-PSA and t-PSA) and estimation of f-PSA to t-PSA ratio (f/t-PSA) is reported. The f/t-PSA ratio has shown to be more specific for the correct diagnosis of prostate cancer than t-PSA alone, especially in the so-called diagnostic grey zone of 4-10 microg/l t-PSA. The performance of the system described relied on non-competitive immunoassay protocols. Herein, f-PSA and t-PSA were sandwiched between anti-f-PSA and anti-t-PSA monoclonal antibodies immobilised on the strip and a colloidal gold anti-PSA antibody tracer. In the presence of PSA in the sample, the tracer accumulated on the strip results in the appearance of specific pink colour lines. The colour intensity of these lines was found to be directly proportional to the PSA concentration and a semi-quantitative estimation could be carried out visually. Quantitative analysis was also possible by densitometry. Using PSA standards prepared in female serum, the strip could be calibrated up to a concentration of 60 microg/l for both PSA species, with an assay time of less than 20 min. The estimated detection limit was 1 microg/l in all cases. The immunostrip showed good storage stability for at least 2 months and the reproducibility was always between 12% and 17%. Fifty-one male serum samples were analysed with the strip and results compared with values obtained by two different commercial immunoassays taken as reference methods. The study yielded acceptable correlation and agreement. An estimation of the sensitivity and specificity demonstrated the strip validity as a potential front-line device for the early detection of prostate cancer and differentiation of benign prostatic anomalies. Small plastic cartridges incorporating the immunostrip and a small blister containing washing solution that helps remove unbound species from the strip were envisaged in order to avoid false positive readings and decrease background signals, thereby leading to better sensitivity and detection limits.

An electrochemical sensor array system for the direct, simultaneous in vitro monitoring of nitric oxide and superoxide production by cultured cells.

A new approach for an amperometric array sensor platform employing arrays of sensors in a 24-well cell culture plate format has been developed for simultaneous in vitro determination of nitric oxide (NO) and superoxide free radicals (O(2)(-)) produced by stimulated cells. The work reported focuses on the direct, real-time monitoring of extracellular production of these two analytes, as well as the effects of their interaction. The sensor platform was manufactured by a combination of sputtering gold electrodes and screen-printing carbon electrodes. The O(2)(-) sensor uses covalent immobilization of cytochrome c via a binder, DTSSP (3,3'-dithio-bis(sulphosuccinimidylpropionate) onto the surface of the Au electrodes, whereas the NO sensor system involves an NiTSPc (nickel tetrasulfonated phthalocyanine) film electrodeposited onto the surface of the carbon electrodes and subsequently covered with an external layer of Nafion. For in vitro demonstration of the platforms as a potential drug-screening system, A172 glioblastoma cells were cultured and transferred into the 24-well arrays. Simultaneous and direct monitoring of NO and O(2)(-) production as a response to chemicals of biomedical relevance was carried out. The results obtained demonstrated that it would be possible to envisage a drug screening platform for compounds designed to be inhibitors of nitric oxide synthase or to have an inhibitory effect on superoxide free radical production. By suitable modification of the electrodes employed it would also be possible to extend the platform to measure alternative species.

Combined system for the simultaneous optical and electrochemical monitoring of intra- and extracellular NO produced by glioblastoma cells.

A combined, optospectroscopic and electrochemical assay system for the simultaneous monitoring of intra- and extracellular production of biologically important species has been developed and assessed. The present model system evaluates intra- and extracellular nitric oxide produced by stimulated glioblastoma multiform cell line (A172). The production of endogenous NO was induced by phorbol-12-myristate-13-acetate and inhibited by N(omega)-nitro-l-arginine methyl ester. Intracellular production of NO was monitored via fluorescence image analysis using a 4,5-diaminofluorescein probe, while extracellular NO release was monitored via a chemically modified electrode, which was incorporated into an optically transparent cell chip. The results indicated that there was no mutual interference between the optical and electrochemical measurement systems. The response time of the combined optical/electrochemical system was found to be in the range of a few tens of seconds.

Simultaneous intra- and extracellular superoxide monitoring using an integrated optical and electrochemical sensor system.

A new integrated optical and electrochemical sensor system for simultaneous monitoring of intra- and extracellular superoxide (O(2)(-)) was developed using an array-based cell chip. For in vitro assays, A172 human glioblastoma cells were transferred into the cell chip and stimulated by phorbol 12-myristate 13-acetate (PMA). Intracellular O(2)(-) generation was detected via fluorescence image analysis with a dye probe, dihydrorhodamine 123 (DHR 123). Extracellular O(2)(-) was detected using an amperometric sensor constructed by immobilisation of cytochrome c using a binder, 3,3'-dithiobis(sulphosuccinimidylpropionate), to attach the redox protein onto the surface of electrodeposited Au electrodes incorporated into the optically transparent cell chip. The simultaneous intra- and extracellular production of O(2)(-) was successfully observed from PMA-stimulated A172 cells and inhibited by superoxide dismutase (SOD). The quantification of O(2)(-) concentration based on a mathematical model study and possible applications using the sensor system developed were discussed. The results confirm that there was no detectable interference or crosstalk between the optical and electrochemical assays. Feasibility of the integration of the two methods, optical and electrochemical, and the neutralisation of the intra- and extracellular O(2)(-) levels by SOD have been demonstrated.

Disposable noncompetitive immunosensor for free and total prostate-specific antigen based on capacitance measurement.

This work reports on the successful integration of a one-step lateral flow immunoassay format and impedance detection of the specific affinity event using an electrochemical transducer coated with a pH-sensitive polymer layer. This approach was particularly applied to the development of a rapid single-use immunosensor for the sensitive detection of free and total prostate-specific antigen (f-PSA, t-PSA) tumor marker. Strips of nitrocellulose membrane were coated with appropriate antibodies to f-PSA and t-PSA and used as solid supports for the performance of noncompetitive immunoassays where PSA was allowed to react with both immobilized anti-PSA antibody and anti-PSA urease enzyme conjugate for less than 1 min. An additional piece within the device consisting of a storage blister filled with a urea solution allowed the rapid washing of unbound species from the membrane strips and simultaneous urea hydrolysis catalyzed by the bound urease conjugate in an automatic fashion. The hydrolysis of urea increased the pH of the reaction media, which in turn induced a breakdown of the polymer layer on the transducer and a consequent measurable change in capacitance of the system. This was easily recorded at a given frequency over a 30-min period. Overall, we describe a one-step immunosensor prototype that exhibits enough sensitivity to detect both forms of PSA at concentration levels down to 3 ng/mL. With the possibility of being portable and considering its ease of use, robustness, and simplicity, this device has great potential as a tool for the screening and early detection of prostate cancer.

Comparisons of platinum, gold, palladium and glassy carbon as electrode materials in the design of biosensors for glutamate.

Four electrode materials: Pt, Au, Pd and glassy carbon (GC), were studied to investigate their suitability as substrates in the development of two different classes of glutamate biosensor. Glutamate oxidase cross-linked onto poly(o-phenylenediamine) was chosen as the type 1 biosensor (PPD/GluOx), incorporating PPD as the permselective element to detect H(2)O(2) directly on the electrode surface at relatively high applied potentials. GluOx and horseradish peroxidase/redox polymer modified electrodes (Os(2+)PVP/HRP/GluOx) that relied on enzyme-catalysed H(2)O(2) detection at lower applied potentials were used as type 2 biosensors. The voltammetric and amperometric responses to the enzyme signal transduction molecule, H(2)O(2), and the archetypal interference species in biological applications, ascorbic acid, were determined on the bare and PPD/GluOx-modified surfaces. The amperometric responses of these electrodes were stable over several days of continuous recording in phosphate buffered saline (pH 7.4). The sensitivity of the type 1 biosensors to H(2)O(2) and glutamate showed parallel trends with low limits of detection and good linearity at low concentrations: Pt>Au approximately Pd>>GC. Type 2 biosensors out-performed the type 1 design for all electrode substrates, except Pt. However, the presence of the permselective PPD membrane in the type 1 biosensors, not feasible in the type 2 design, suggests that Pt/PPD/GluOx might have the best all-round characteristics for glutamate detection in biological media containing interference species such as ascorbic acid. Other points affecting a final choice of substrate should include factors such as mass production issues.

Disposable tyrosinase-peroxidase bi-enzyme sensor for amperometric detection of phenols.

A new disposable amperometric bi-enzyme sensor system for detecting phenols has been developed. The phenol sensor developed uses horseradish peroxidase modified screen-printed carbon electrodes (HRP-SPCEs) coupled with immobilized tyrosinase prepared using poly(carbamoylsulfonate) (PCS) hydrogels or a poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) matrix. Optimization of the experimental parameters has been performed with regard to buffer composition, pH, operating potential and storage stability. A co-operative reaction involving tyrosinase and HRP occurs at a potential of -50 mV versus Ag/AgCl without the requirement for addition of extraneous H(2)O(2), thus, resulting in a very simple and efficient system. Comparison of the electrode responses with the 4-aminoantipyrine standard method for phenol sample analysis indicated the feasibility of the disposable sensor system for sensitive "in-field" determination of phenols. The most sensitive system was the tyrosinase immobilized HRP-SPCE using PCS, which displayed detection limits for phenolic compounds in the lower nanomolar range e.g. 2.5 nM phenol, 10 nM catechol and 5 nM p-cresol.