Zein as biodegradable material for effective delivery of alkaline phosphatase and substrates in biokits and biosensors.

A biodegradable material, zein, is proposed as a reagent delivery platform for biokits and biosensors based on alkaline phosphatase (ALP) activity/inhibition in the presence of phosphatase substrates. The immobilization and release of both the substrate and/or the active ALP, in a biodegradable and low-cost material such as zein, a prolamin from maize, and in combination with glycerol as plasticizer have been investigated. Three zein-based devices are proposed for several applications: (1) inorganic phosphorus estimation in water of different sources (river, lake, coastal water and tap water) with a detection limit of 0.2mg/L - compared to at least 1mg/L required by legislation, (2) estimation of ALP in saliva and (3) chlorpyrifos control in commercial preparations. The single-use kits developed are low cost, easy and fast to manufacture and are stable for at least 20 days at -20°C, so the zein film can preserve and deliver both the enzyme and substrates.

A step towards mobile arsenic measurement for surface waters.

Surface modified quantum dots (QDs) are studied using a bio-inspired cysteine rich ligand (glutathione, GSH) and their quenching response and selectivity to arsenic examined. As predicted from As(3+) binding with highly crosslinked phytochelatin-(PCn)-like molecules, better arsenic selectivity is obtained for a thicker more 3-dimensional GSH surface layer, with exposed sulfhydryl groups. A detection limit of at least 10 µM can be achieved using CdSe/ZnS core-shell QDs capped with this GSH structure. The system is also demonstrated using a mobile phone camera to record the measurement, producing a detection limit of 5 µM. However, copper remains the main interferent of concern. Water-soluble CdTe QDs show little sensitivity to As(3+) even with a GSH surface, but they remain sensitive to Cu(2+), allowing a copper baseline to be established from the CdTe measurement. Despite anticipating that spectrally non overlapping fluorescence would be required from the two types of QDs to achieve this, a method is demonstrated using RGB channels from a mobile phone and processing the raw data for CdTe QDs, with an emission wavelength of 600 nm, and CdSe/ZnS QDs, with emission maximum of 630 nm. It is shown that As(3+) measurement remains feasible at the WHO guideline value of 10 µg L(-1) up to a copper concentration of around 0.3 µM Cu(2+), which corresponds to the highest recorded level in a selection of large rivers world-wide.

Assessment of protein phosphatase in a re-usable rapid assay format in detecting microcystins and okadaic acid as a precursor to biosensor development.

The feasibility of developing an immobilised protein phosphatase (PP) biosensor was tested by immobilising PP onto CNBr-activated Sepharose beads placed in Millipore microfilter plate wells. Under optimised immobilised enzyme assay conditions, okadaic acid (OA) and microcystin LR (MC-LR) inhibited Upstate Biotechnology PP (PP-2A), with IC50 values of 12.5 and 11nM respectively. Similarly, immobilised recombinant PP type 1 (rec PP-1) was inhibited by MC-LR and OA, with IC50 values of 150 and >1000nM respectively. The IC50 values for free PP-2A against OA and MC-LR were 2.5 and 3.5nM, and 0.7nM and 200nM for rec PP-1 against the same substrates respectively. For free and immobilised Neptunea arthritic PP (PP-2Ana) against OA the IC50 values were 0.45 and >1000nM respectively. Of the three immobilised enzyme systems, PP-2A showed greatest sensitivity to OA and MC-LR followed by rec PP-1 and PP-2Ana. In assessments for re-usability (determined by removal of > or =70% OA or MC-LR inhibition of PP-2A by washing), <50% of the original activity remained after 20 washings. Including 1M NaCl in the wash buffer did not increase enzyme activity with wash frequency, but rather "salted in" the inhibitor. The LoD of immobilised PP-2A to MC-LR meets the WHO guideline of 1microgl(-1) for drinking water, and the sensitivity to OA (3.5microgl(-1)) would allow detection of DSP during the peak of some phytoplankton blooms.

Rapid detection of toxicity in wastewater: recent developments with manometric respirometry.

Manometric respirometry can be used for the direct toxicity assessment (DTA) of wastewater. A first prototype portable system for field use as well as the principle of operation has been reported previously [A. Tzoris, D. Cane, P. Maynard, E.A.H. Hall, Anal. Chim. Acta 460 (2002) 257; A. Tzoris, D. Fearnside, M. Lovell, E.A.H. Hall, Environ. Toxicol. 17 (2002) 284; A. Tzoris, V. Fernandez-Perez, E.A.H. Hall, Sens. Actuators B 105 (2005) 39]. In this report we examine the simple mathematical derivation of the principle and identify some design parameters that could be altered or improved. Hence by changing the geometry of the respiration chamber aiming at a larger surface area and smaller air headspace volume combined with sample stirring for improved aeration, the response of the instrument was improved by at least 50%. Data obtained from real samples as well as data from a comparative study with a lab-based respirometer routinely used in toxicity assessment is presented. Correlation was excellent with the advantage that a test could be completed in 6min or with five repeat measurements the data could be collected within a 10min measurement period. Using a 25% inhibition threshold, all samples found toxic by the standard method were also found toxic by the Baroxymeter method and dilution factors could be estimated for pretreatment of the toxic waste prior to release to a processing plant.

Detection of the steroidogenic acute regulatory protein, StAR, in human liver cells.

Overexpressing StAR (a mitochondrial cholesterol transporter) increases (>5-fold) the rate of 27-hydroxylation of cholesterol and the rates of bile acid synthesis in primary rat hepatocytes; suggesting that the transport of cholesterol into mitochondria is rate-limiting for bile acid biosynthesis via the CYP27A1 initiated 'acidic' pathway. Our objective was to determine the level of StAR expression in human liver and whether changes in StAR would correlate with changes in CYP27A1 activity/bile acid synthesis rates in human liver tissues. StAR mRNA and protein were detected in primary human hepatocytes and HepG2 cells by RT-PCR/Northern analysis and by Western analysis, respectively. In immunocompetition assays, liver StAR was competed away with the addition of purified human adrenal StAR. Overexpressing CYP27A1 in both cell types led to >2-fold increases in liver StAR concentration. StAR protein levels also increased approximately 2-fold with the addition of 27-hydroxycholesterol to HepG2 cell culture medium. Overexpressing StAR increased the rates of 27-hydroxylation of cholesterol/bile acid synthesis in both cell lines and increased intracellular levels of 27-hydroxycholesterol. In conclusion, human liver cells contain regulable StAR protein whose level of expression appears capable of regulating cellular cholesterol homeostasis, representing a potential therapeutic target in the management of hyperlipidemia.

Mitochondrial cholesterol transport: a possible target in the management of hyperlipidemia.

Sterol 27-hydroxylase (CYP27A1) may defend cells against accumulation of excess cholesterol, making this enzyme a possible target in the management of hyperlipidemia. The study objective was to analyze cholesterol homeostatic responses to increases in CYP27A1 activity in HepG2 cells and primary human hepatocytes. Increasing CYP27A1 activity by increasing enzyme expression led to significant increases in bile acid synthesis with compensatory increases in HMG-CoA reductase (HMGR) activity/protein, LDL receptor (LDLR) mRNA, and LDLR-mediated cholesterol uptake. Under these conditions, only a small increase in cellular 27-hydroxycholesterol (27OH-Chol) concentration was observed. No changes were detected in mature sterol regulatory element-binding proteins (SREBP) 1 or 2. Increasing CYP27A1 activity by increasing mitochondrial cholesterol transport (i.e., substrate availability) led to greater increases in bile acid synthesis with significant increases in cellular 27OH-Chol concentration. Mature SREBP 2 protein decreased significantly with compensatory decreases in HMGR protein. No change was detected in mature SREBP 1 protein. Despite increasing 27OH-Chol and lowering SREBP 2 protein concentrations, LDLR mRNA increased significantly, suggesting alternative mechanisms of LDLR transcriptional regulation. These findings suggest that regulation of liver mitochondrial cholesterol transport represents a potential therapeutic strategy in the treatment of hyperlipidemia and atherosclerosis.

Hybridization of DNA at the surface of phospholipid monolayers. Effect of orientation of oligonucleotide chains.

We studied the properties of lipid monolayers formed at the air-water interface composed of dioleoylphosphatidylcholine (DOPC) with incorporated short (19-mer) oligonucleotides. These oligonucleotides were modified by oleylamine at both (3' and 5') terminals or only at one (3') terminal. Interaction of single-stranded (19-mer) oligonucleotides without oleylamine with DOPC monolayers resulted only in slight increase of surface pressure and the area per phospholipid molecule, while more substantial and significant increase of these values were observed following incorporation of oligonucelotides modified by oleylamine. This influence is similar for both types of oligonucleotide modifications. However, considerable differences in changes of monolayer properties took place after hybridization with complementary oligonucleotides. The hybridization of oligonucleotides with the DNA modified by oleic acid at both 3' and 5' terminals at the surface of lipid monolayer resulted in further increase of surface pressure and in the increase of the area per phospholipid molecule, while decrease of both the surface pressure and the area per phospholipid molecules were observed for hybridization with DNA modified by oleic acid at 3' terminal. It is possible that in latter case, the hybridization caused the loss of hybridized molecules from monolayers. Interaction of noncomplementary chains with DOPC monolayers with incorporated oleyl acid-modified DNA also influenced the properties of monolayers, but the effect was weaker in comparison with that observed for complementary chains.

Assessment of the fifth ligand-binding repeat (LR5) of the LDL receptor as an analytical reagent for LDL binding.

The fifth ligand binding repeat (LR5) of the low density lipoprotein (LDL) receptor was assessed ex vivo as an 'analytical reagent' to distinguish LDL state, in atherosclerosis risk monitoring. LR5 was immobilized to mercaptoundecanoic acid modified gold surfaces via a glycine linker. Surface plasmon resonance (SPR) was used to monitor LDL binding. Unfolded LR5 was ineffectual as an affinity ligand for LDL but refolded LR5 showed a high affinity for native LDL but little affinity for oxidized LDL. LR5 refolded in the presence of calcium or EDTA gave the equivalent LDL binding capacity. However, EDTA-LR5 was less stable than Ca-LR5 at pH 5 and, from tryptophan fluorescence evidence, they appeared to involve different regions of LR5 and/or LDL in the binding. Involvement of amino acid residues of the calcium cage of LR5 was tested in LDL binding by monitoring calcium ion release with a calcium ionophore. The results were consistent with approximately 7-8 LR5 binding per LDL, of which only some induce calcium release (a maximum of approximately 25 mol% calcium, based on LR5, was released during LDL binding). For LDL binding to the LDL receptor in vivo more than one ligand-binding repeat is needed and this may be consistent with LR5 acting here also at binding sites which other LRs normally occupy in the LDL-LDL receptor complex. This initial study is encouraging for the use of a minimum peptide repeat array based on the conserved region of the LRs as an affinity surface for atherosclerosis risk monitoring.

Detection of PCR products using PNA strand invasion.

The unique ability of homopyrimidine peptide nucleic acid (PNA) to strand invade homopurine sites of duplex DNA offers a potential alternative to existing techniques for rapid detection of PCR products. From gel shift studies, PNA was found to specifically strand invade homopurine sites that had been incorporated into an amplicon during the PCR cycle. This was achieved by adding a homopyrimidine sequence to the 5'-terminus of a PCR primer. The position of the strand invasion sites at the termini of the DNA duplex offers kinetic advantages for PNA strand invasion, since the termini of DNA duplexes are known to be unwound. This unwound state was demonstrated using a novel assay that determined single-stranded regions within the amplicon. The presence of the PNA moiety in strand invasion complexes was confirmed by a novel electroblot, an Enzyme Linked Nucleic Acid assay and by an increase in stability as demonstrated by T(m)studies with the Idaho RapidCycler. Since the strand invasion sites can be controlled through selection of the homopurine sequence there is great flexibility for designing strand invasion motifs unique to a particular PCR amplicon, thus providing a huge potential for differentiating and detecting multiplex PCR products.

Producing "self-plasticizing" ion-selective membranes.

Polymer membranes have been explored for the analysis of ions that do not require plasticizers and with photocurable properties. This work was focused on investigating the viability of the methacrylic-acrylic copolymers as new self-plasticizing membrane matrixes for ion-selective electrodes or other ion-sensor applications. Copolymers with glass transition temperatures ranging from -20 to -44 degrees C could be prepared without added plasticizer and were found to be functional as ion-selective membranes. Both free-radical solution polymerization and photopolymerization could be used, and "self-plasticizing" behavior of copolymers was observed with a high alkylacrylate (R = C4) content. This was found to be compatible with most commercially available ionophores, and sensors for potassium, sodium, calcium and pH were fabricated entirely by photocure procedures; single-step procedures for the immobilization of benzo-15-crown-5 ionophore on these self-plasticizing copolymer matrixes were also developed. Even though the ionophore was immobilized, potentiometric studies revealed that the ionophore remained functional, and thus, these copolymers have the advantage of suffering neither leaching of ionophore nor plasticizers. All these sensors exhibited a Nernstian or near Nernstian response with selectivity comparable to plasticized PVC membranes or other plasticized and photocurable polymer membranes. The long-term response of the potassium sensor with immobilized ionophore and the sodium sensor showed little deterioration for as long as one month and three months, respectively, under continuous use.

Phasor transform to extract glucose and ascorbic acid data in an amperometric sensor.

A method for separating the signals from glucose and ascorbic acid on a single recognition surface using an ac immittance technique is presented. It is proposed that each oxidation process can be represented by a unique vector based on psi and YO, and that the concentration of each analyte can be determined by monitoring the change in the admittance magnitude in the direction of the characteristic angle for that particular species. The total Faradaic admittance (YF,total) for all electroactive species present is given by a linear combination of the independent vectors from the different species. In the system tested, the analytes are glucose and ascorbic acid, the former being estimated via the measurand, hydrogen peroxide. Thus, one of the electroactive species (hydrogen peroxide) is not a bulk solution species, but is 'generated' in the enzyme matrix. The admittance measurements from ascorbic acid and the enzyme-generated hydrogen peroxide showed the characteristic phase angles of each oxidation signal, allowing for good spatial resolution. The behaviour of each of these analytes is presented and calibration curves tested. Based on the calibration curves and the basis vectors, samples containing both glucose and ascorbic acid were measured by transforming the measured total admittance from the complex Cartesian space into 'analyte space', where the X-Y axes are given by the basis vectors yEGHP,GOD and yAA,GOD, respectively.

Dipicolinic acid (DPA) assay revisited and appraised for spore detection.

Delayed gate fluorescence detection of dipicolinic acid (DPA), a universal and specific component of bacterial spores, has been appraised for use in a rapid analytical method for the detection of low concentrations of bacterial spores. DPA was assayed by fluorimetric detection of its chelates with lanthanide metals. The influence of the choice and concentration of lanthanide and buffer ions on the fluorescence assay was studied as well as the effects of pH and temperature. The optimal system quantified the fluorescence of terbium monodipicolinate in a solution of 10 microM terbium chloride buffered with 1 M sodium acetate, pH 5.6 and had a detection limit of 2 nM DPA. This assay allowed the first real-time monitoring of the germination of bacterial spores by continuously quantifying exuded DPA. A detection limit of 10(4) Bacillus subtilis spores ml-1 was reached, representing a substantial improvement over previous rapid tests.

Detection of oxidized low-density lipoproteins using surface plasmon resonance.

Management of atherosclerosis is a high priority target. If this is to be achieved, the early detection of risk and risk factors are paramount and integrated with this is a need for the detection of the oxidation state of a patient's low density lipoprotein (LDL). Presently no readily usable technique exists for their rapid determination and in order to develop such a technique a monitoring system must be devised which distinguishes a parameter which changes on oxidation and distinguishes critical and noncritical oxidation products. The strategy which is investigated here is based on the use of a heparin-modified Au-surface plasmon resonance (SPR) device as a modulator of LDL binding, according to its oxidation state. Heparin is strongly negatively charged and seven binding sites for heparin have been identified on the LDL apoprotein consisting of arginine and lysine clusters; these are regarded as identical to the LDL receptor binding sites. The heparin-modified surface was calibrated for LDL and a calibration factor of 1.84 × 10(9) particles mm(-)(2) Δ(o)(-)(1) SPR and instrumental resolution of 9 × 10(6) particles mm(-)(2) obtained which gives sufficient scope to distinguish LDL dependent binding. LDL oxidation could involve the protein and/or lipoprotein, the latter being of interest for athersclerosis risk and the LDL binding to heparin was shown to decrease with degree of protein oxidation as determined by the free amino groups (fluorescamine assay), but was not influenced by lipid oxidation (determined by thiobarbituric acid reactive substances assay, TBARS). The SPR based assay was tested for LDL in plasma and the calibration found to follow that obtained in buffer, although the scatter was higher, probably due to interference from other plasma species. Nevertheless, in the context of the normal distribution of LDL in healthy patients, the assay would almost certainly be able to determine Ox-LDL in atherosclerotic patients.

A lactulose sensor based on coupled enzyme reactions with a ring electrode fabricated from tetrathiafulvalen-tetracyanoquinodimetane.

A dual enzyme electrode is explored for measuring lactulose in milk. A ring electrode (diameter = 3 mm; ring width = 10-20 microns) is proposed onto which tetrathiafulvalen-tetracyanoquinodimetane (TTF-TCNQ) salt was physically packed. The electrode is a band electrode with dimensions approaching those for micro electrodes, so that the improved faradaic current/charging current ratio lead to improved detection limits. Fructose dehydrogenase (FDH) and beta-galactosidase (beta-gal) were immobilized by covering the electrode surface with a dialysis membrane. Lactulose was hydrolyzed to D-fructose and D-galactose by beta-gal. The hydrolyzed D-fructose was oxidized by FDH which was simultaneously reduced to the reduced form (FDH-PQQH2). The FDH-PQQH2 was directly reoxidized by TTF-TCNQ on the ring electrode, whose current was monitored at 200 mV vs Ag/AgCl. The detection limit of the lactulose sensor was 1.0 microM and the selectivity for lactulose was at least 1000 times higher than that for lactose. Pasteurized, UHT and sterilized milks were applied to the lactulose sensor, showing good accuracy and precision and, furthermore, good correlation to a reference photometric method, even though no rigorous procedure for the electrode fabrication has presently been addressed.

Platinum-catalyzed enzyme electrodes immobilized on gold using self-assembled layers.

The bonding of enzymes to self-assembled monolayers (SAMs) of alkanethiols onto gold electrode surfaces is exploited to produce an enzyme biosensor. The attachment of glucose oxidase to a SAM of 3-mercaptopropionic acid was achieved using carbodiimide coupling. The resultant biosensor showed good sensitivity to glucose and a large dynamic range when measured amperometrically via the p-benzoquinone mediator. On the other hand, subsequent platinization of the enzyme-SAM electrode allowed hydrogen peroxide produced in the enzyme reaction to be detected directly, thus obviating the need for an artificial redox mediator. The performance of such sensors constructed on bulk gold electrodes was evaluated and finally compared to that of some preliminary thin-film gold electrodes. Biosensors constructed using the two alternative electrode surfaces have quite different sensitivities, thus reflecting the influence of the anchoring surface on the performance of the biosensor.

Surface plasmon resonance sensor for heparin measurements in blood plasma.

Surface plasmon resonance (SPR) was used as an affinity biosensor to determine absolute heparin concentrations in human blood plasma samples. Protamine and polyethylene imine (PEI) were evaluated as heparin affinity surfaces. Heparin adsorption onto protamine in blood plasma was specific with a lowest detection limit of 0.2 U/ml and a linear window of 0.2-2 U/ml. Although heparin adsorption onto PEI in buffer solution had indicated superior sensitivity to that on protamine, in blood plasma it was not specific for heparin and adsorbed plasma species to a steady-state equilibrium. By reducing the incubation time and diluting the plasma samples with buffer to 50%, the non-specific adsorption of plasma could be controlled and a PEI pre-treated with blood plasma could be used successfully for heparin determination. Heparin adsorption in 50% plasma was linear between 0.05 and 1 U/ml so that heparin plasma levels of 0.1-2 U/ml could be determined within a relative error of 11% and an accuracy of 0.05 U/ml.

A Fill-and-Flow Biosensor.

An alternative design for an amperometric biosensor for lactate is described in which the analyte flows through a rectangular duct without the aid of external pumping or a flow system. With this deviation from the traditional "stacked layers" of the biosensor geometry, the biorecognition matrix is located upstream of the detector electrode rather than directly over the electrode; this general design is reminiscent of the "dipstick" technology of the home test kits, but in the latter case the "flow" is a function of the nature of the wicking material rather than the dimensions of the channel. A droplet of analyte, l-lactate, is placed in the inlet well of the channel which flows through the cell and reacts with an immobilized lactate oxidase surface matrix zone, and the coproduct, hydrogen peroxide, is swept downstream to the monitoring electrode, where it is detected. The flow through the channel biosensor is shown to be laminar, and the biosensor response varies with the rate of flow of the analyte, which could be controlled by the outlet porosity; the sensitivity was observed to increase, but the dynamic range decreased with an increase in flow rate until a threshold loading, where the biosensor response became independent of the enzyme loading.

Clinical characteristics and management of acute stroke in patients with atrial fibrillation admitted to US university hospitals.

UNLABELLED: The optimal evaluation and management of patients with atrial fibrillation who suffer an acute ischemic stroke remains controversial. METHODS: Medical records of 171 consecutive patients with atrial fibrillation and acute stroke at six U.S. university hospitals were reviewed. Data collected included the use of antithrombotic therapy, brain and cardiac imaging, bleeding complications, stroke risk factors, and contraindications to anticoagulation. RESULTS: Mean age was 75.4 years. Cardiovascular risk factors associated with increased stroke risk were present in 87%; 35% had at least one contraindication to anticoagulation. Half of the patients with stroke risk factors and no contraindications to anticoagulation were not receiving any antithrombotic therapy at the time of admission. Of the 22 patients who were treated with warfarin, and had INR values on admission, 16 had levels of < 2.0; only six had INR values between 2.0 and 3.0. Transthoracic echocardiography was performed in 107 patients (63%); intracardiac thrombi were visualized in only 5%. Initial brain imaging revealed hemorrhagic transformation in nine. Heparin was used in 93 patients (54%), usually within 48 hours of stroke onset. Patients who received delayed heparin typically did not have repeat brain imaging prior to starting heparin. One patient had a delayed symptomatic cerebral hemorrhage. Of the survivors, 47% were discharged and treated with warfarin (or warfarin plus aspirin), 28% with ASA, 7% with other antithrombotic therapies, and 18% with no antithrombotic therapy. CONCLUSION: Antithrombotic therapy was underutilized and inadequately monitored in atrial fibrillation patients prior to stroke onset. After hospital admission, a wide range of diagnostic and management strategies, which often did not follow current recommendations, were employed.