The sixth workshop of the European Community Concerted Action on Chemical sensors for in vivo monitoring was held at Snogeholm, Sweden, in October 1991. The meeting reviewed recent in vivo and ex vivo results, and also included a shorter session on ethical and safety problems.
The latest workshop of the European Community (EC) Concerted Action on Chemical sensors for in vivo monitoring was held in Nauplion, Greece, in April this year. This fifth workshop focused on The design and development of new sensors for in vivo monitoring, and was organized into five sessions: design and development of new sensors; operational considerations; performance of analytical systems; novel sensors/tissue heterogeneity; and infra-red spectroscopy.
Melanophores are pigmented cells in lower vertebrates capable of quick color changes and thereby suitable as whole cell biosensors. In the frog dermis skin layer, the large and dark pigmented melanophore surrounds a core of other pigmented cells. Upon hormonal stimulation the black-brown pigment organelles will redistribute within the melanophore, and thereby cover or uncover the core, making complex color changes possible in the dermis. Previously, melanophores have only been cultured on flat surfaces. Here we mimic the three dimensional biological geometry in the frog dermis by culturing melanophores on fluorescent plastic microbeads. To demonstrate biosensing we use the hormones melatonin and α-melanocyte stimulating hormone (α-MSH) as lightening or darkening stimuli, respectively. Cellular responses were successfully demonstrated on single cell level by fluorescence microscopy, and in cell suspension by a fluorescence microplate reader and a previously demonstrated computer screen photo-assisted technique. The demonstrated principle is the first step towards "single well/multiple read-out" biosensor arrays based on suspensions of different selective-responding melanophores, each cultured on microbeads with distinctive spectral characteristics. By applying small amount of a clinical sample, or a candidate substance in early drug screening, to a single well containing combinations of melanophores on beads, multiple parameter read-outs will be possible. © 2004 Elsevier B.V. All rights reserved.
Regeneration is a key goal in the design of immunosensors. In this study, we report the temperature-regulated interaction of N-isopropylacrylamide (PNIPAAm) functionalised cardiac troponin T (cTnT) with anti-cTnT. Covalently bonded PNIPAAm on an anti-cTnT bioelectrode showed on/off-switchability, regeneration capacity and temperature triggered sensitivity for cTnT. Above the lower critical solution temperature (LCST), PNIPAAm provides a liphophilic microenvironment with specific volume reduction at the bioelectrode surface, making available binding space for cTnT, and facilitating analyte recognition. Computational studies provide details about the structural changes occurring at the electrode above and below the LCST. Furthermore, free energies associated with the binding of cTnT with PNIPAAm at 25 (Delta G(coil)=-6.0 Kcal/mole) and 37 degrees C (Delta G(globular)=-41.0 kcal/mole) were calculated to elucidate the interaction and stability of the antigen-antibody complex. The responsiveness of such assemblies opens the way for miniaturised, smart immuno-technologies with built-in programmable interactions of antigen-antibody upon receiving stimuli.
In this article, we report a functionalised ZnO-nanorod-based selective electrochemical sensor for intracellular glucose. To adjust the sensor for intracellular glucose measurements, we grew hexagonal ZnO nanorods on the tip of a silver-covered borosilicate glass capillary (0.7 mu m diameter) and coated them with the enzyme glucose oxidase. The enzyme-coated ZnO nanorods exhibited a glucose-dependent electrochemical potential difference versus an Ag/AgCl reference microelectrode. The potential difference was linear over the concentration range of interest (0.5-1000 mu M). The measured glucose concentration in human adipocytes or frog oocytes using our ZnO-nanorod sensor was consistent with values of glucose concentration reported in the literature; furthermore, the sensor was able to show that insulin increased the intracellular glucose concentration. This nanoelectrode device demonstrates a simple technique to measure intracellular glucose concentration.
Zinc oxide nanorod-extended gate field effect transistor (MOSFET) is demonstrated for the detection of calcium (Ca2+) ions. ZnO nanorods were grown on the surface of a silver wire to produce an electrochemical nanosensor for selectively detecting Ca2+. The electrochemical response from the interaction between the ZnO nanorods and Ca2+ in an aqueous solution is coupled directly to the gate of a field effect transistor (MOSFET). The induced voltage change on the gate results in a measureable current response. In order to adapt the sensors for Ca2+ ions measurements in biological fluids with sufficient selectivity and stability, a plastic membrane coating containing ionophores was applied on the nanorods. The sensor exhibited a linear response within the range of interest from 1 μM to 1 mM. This work demonstrates a simple technique for sensitive detection of Ca2+ ions by efficient transfer of the chemical response directly to a standard electronic component producing a low impedance signal.
ZnO nanorods were grown on a silver-coated tip of a borosilicate glass capillary (0.7 mu m in tip diameter) and used as selective potentiometric sensor of intracellular free Mg2+. To functionalize the ZnO nanorods for selectivity of Mg2+, a polymeric membrane with Mg2+-selective ionophores were coated on the surface of the ZnO nanorods. These functionalized ZnO nanorods exhibited a Mg2+-dependent electrochemical potential difference versus an Ag/AgCl reference microelectrode within the concentration range from 500 nM to 100 mM. Two types of cells, human adipocytes and frog oocytes, were used for the intracellular Mg2+ measurements. The intracellular concentration of free Mg2+ in human adipocytes and frog oocytes were 0.4-0.5 and 0.8-0.9 mM, respectively. Such type of nanoelectrode device paves the way to enable analytical measurements in single living cells and to sense other bio-chemical species at the intracellular level.
Alteration in expression of miRNAs has been correlated with different cancer types, tumour stage and response to treatments. In this context, a structurally responsive oligonucleotide-based electrochemical impedimetric biosensor has been developed for the simple and sensitive detection of miRNA-21. A highly specific biotinylated DNA/LNA molecular beacon (MB) probe was conjugated with gold nanoparticles (AuNPs) to create an integrated, dual function bio-label (biotin-MB-AuNPs) for both biorecognition and signal generation. In the presence of target miRNA-21, hybridisation takes place resulting in the "activation" of the biotin-MB; this event makes the biotin group, which was previously "protected" by the steric hindrance of the MB stem-loop structure, accessible. The activated biotin-MB-AuNPs/miRNA complexes become available for capture, via supramolecular interaction, onto a nentravidin-modified electrode for electrochemical transduction. The binding event results in a decrease of the charge transfer resistance at the working electrode/electrolyte interface. The biosensor responded linearly in the range 1-1000 pM of miRNA-21, with a limit of detection of 0.3 pM, good reproducibility (Relative Standard deviation (RSD) =3.3%) and high selectivity over other miRNAs (i.e. miRNA221 and miRNA-205) sequences. Detection of miRNA-21 in spiked serum samples at clinically relevant levels (low pM range) was also demonstrated, thus illustrating the potential of the biosensor for point-of-care clinical applications. The proposed biosensor design, based on the combination of a neutravidin transducing surface and the dual-function biotin-MB-AuNPs bio-label, provides a simple and robust approach for detection of short-length nucleic acid targets, such as miRNAs.
The detection of bacterial infections in a mammalian cell culture process is realised using a gas sensor array. In production-scale and laboratory-scale cultivations of a perfused recombinant CHO-cell culture producing human blood coagulation Factor VIII, we show that the gas sensor array identifies bacterial contamination earlier than conventional methods. The sensitivity of the instrument is verified by inoculation of a blank cell culture medium with defined bacterial cell counts. © 2002 Elsevier Science B.V. All rights reserved.
Fast and accurate detection of microorganisms is of key importance in clinical analysis and in food and water quality monitoring. Salmonella typhimurium is responsible for about a third of all cases of food borne diseases and consequently, its fast detection is of great importance for ensuring the safety of foodstuffs. We report the development of a label-free impedimetric aptamer-based biosensor for S. typhimurium detection. The aptamer biosensor was fabricated by grafting a diazonium-supporting layer onto screen printed carbon electrodes (SPEs), via electrochemical or chemical approaches, followed by chemical immobilisation of aminated-aptamer. FTIR-ATR, contact angle and electrochemical measurements were used to monitor the fabrication process. Results showed that electrochemical immobilisation of the diazonium-grafting layer allowed the formation of a denser aptamer layer, which resulted in higher sensitivity. The developed aptamer-biosensor responded linearly, on a logarithm scale, over the concentration range 1 x 10(1) to 1 x 10(8) CFU mL(-1), with a limit of quantification (LOQ) of 1 x 10(1) CFU mL(-1) and a limit of detection (LOD) of 6 CFU mL(-1). Selectivity studies showed that the aptamer biosensor could discriminate S. typhimurium from 6 other model bacteria strains. Finally, recovery studies demonstrated its suitability for the detection of S. typhimurium in spiked (1 x 10(2), 1 x 10(4) and 1 x 10(6) CFU mL(-1)) apple juice samples. (C) 2016 Elsevier B.V. All rights reserved.
The detection of dopamine by differential pulse voltammetry (DPV) and square wave voltammetry (SWV) at the interface between twoimmiscible electrolyte solutions (ITIES) has been studied. Voltammetry at the liquid/liquid (water/1,2-dichloroethane) interface provides asimple method for overcoming the major problem associated with dopamine detection by voltammetry at solid electrodes: the co-existenceof ascorbate at higher concentrations. Selectivity for dopamine was achieved by the use of dibenzo-18-crown-6 as an ionophore for thefacilitated transfer voltammetry of protonated dopamine across the ITIES. Under these conditions, ascorbate is not transferred and hence doesnot interfere in the ion transfer current for dopamine. By use of DPV and SWV, the lowest concentration detectable can be lowered from ca.0.1mM (obtained with cyclic voltammetry) to 2 M. Evaluation of the effect of some other physiologically important species (acetylcholine,sodium, potassium and ammonium ions) on the dopamine transfer voltammetry has been studied, indicating the need for improved ionophoredesigns in order to achieve practically useful selectivity.
Cystic fibrosis is one of the most common genetically inherited diseases in Northern Europe, consistingof an inherited defect of chloride transport in the epithelium. Of the several mutations related to CF, theF508 mutation occurs in ca. 70% of the cases. In this work the use of a gold nano-particle supportedfluorescence molecular beacon was investigated as an optical sensing platform for the detection of theF508 cystic fibrosis associated mutation. Different parameters such as molecular beacon design, Aunano-particle size, molecular beacon–nano-particle conjugation protocol, molecular beacon loading aswell as experimental conditions were evaluated. A 31-base long molecular beacon, containing a 15-baserecognition sequence specific for the mutant target, was linked via a thiol modified poly thymine linker(10 bases long) to a 13 nm gold nano-particle and was exposed to mutant and wild type targets, and aclear differentiation was achieved at target concentrations as low as 1 nM.
Recent advances in nanotechnology have allowed significant progress in utilising cutting-edge techniques associated with nanomaterials and nano-fabrication to expand the scope and capability of biosensors to a new level of novelty and functionality. The aim of this work was the development and characterisation of conductive polyaniline (PANI) nanostructures for applications in electrochemical biosensing. We explore a simple, inexpensive and fast route to grow PANI nanotubes, arranged in an ordered structure directly on an electrode surface, by electrochemical polymerisation using alumina nanoporous membranes as a nano-mould. The deposited nanostructures have been characterised electrochemically and morphologically prior to grafting with a molecularly imprinted polymer (MIP) receptor in order to create a model sensor for catechol detection. In this way, PANI nanostructures resulted in a conductive nanowire system which allowed direct electrical connection between the electrode and the synthetic receptor (MIP). To our knowledge, this is the first example of integration between molecularly imprinted polymers and PANI nanostructured electrodes. The advantages of using nanostructures in this particular biosensing application have been evaluated by comparing the analytical performance of the sensor with an analogous non-nanostructured MIP-sensor for catechol detection that was previously developed. A significantly lower limit of detection for catechol has been obtained (29 nM, one order of magnitude), thus demonstrating that the nanostructures are capable of improving the analytical performance of the sensor. (C) 2010 Elsevier B.V. All rights reserved.
The possibility of introducing a computationally assisted method to study aptamer-protein interaction was evaluated with the aim of streamlining the screening and selection of new aptamers. Starting from information on the 15-mer (5-GGTTGGTGTGGTTGG-3 thrombin binding aptamer (TBA), a library of mutated DNA sequences (994 elements) was generated and screened using shapegauss a shape-based scoring function from openeye software to generate computationally derived binding scores. The TBA and three other mutated oligonucleotides, selected on the basis of their binding score (best, medium, worst), were incorporated into surface plasmon resonance (SPR) biosensors. By reducing the ionic strength (binding buffer, 50 mMTrisHC1pH 7.4, 140 mM NaCl, 1 mM MgCl(2), diluted 1:50) in order to match the simulated condition, the analytical performances of the four oligonucleotide sequences were compared using signal amplitude, sensitivity (slope), linearity (R(2)) and reproducibility (CVav %). The experimental results were in agreement with the simulation findings.
The synthesis of poly-aminophenylboronic acid (ABPA) imprinted beads for the recognition of the protein human serum albumin (HSA) is reported. In order to create homogeneous recognition sites, covalent immobilisation of the template HSA was exploited. The resulting imprinted beads were selective for HSA. The indirect imprinting factor (IF) calculated from supernatant was 1.6 and the direct IF, evaluated from the protein recovered from the beads, was 1.9. The binding capacity was 1.4 mg/g, which is comparable to commercially available affinity materials. The specificity of the HSA recognition was evaluated with competitive experiments, indicating a molar ratio 4.5/1 of competitor was necessary to displace half of the bound HSA. The recognition and binding of the imprinted beads was also tested with a complex sample, human serum and targeted removal of HSA without a loss of the other protein components was demonstrated. The easy preparation protocol of derivatised beads and a good protein recognition properties make the approach an attractive solution to analytical and bio-analytical problems in the field of biotechnology. (c) 2007 Elsevier B.V. All rights reserved.
A fast, specific and sensitive homogeneous assay for Staphylococcus aureus detection was developed by measuring the activity of secreted nuclease from the bacteria via a modified DNA oligonucleotide. As biosensor format, an effective system, Nanokeepers as previously reported, were used for triggered release of confined fluorophores, and hence specific detection of S. aureus on nuclease activity was obtained. The interference from blood components for fluorescent quantification was eliminated by a pre-purification by aptamer-functionalized silica magnetic nanoparticles. The reported assay system was exclusively formed by nucleic acid oligos and magnetic or mesoporous silica nanoparticles, that can be used on blood samples in a stepwise manner. The assay was successfully used as a sensing platform for the specific detection of S. aureus cells as low as 682 CFU in whole blood.
Molecular imprinting has proved to be an effective technique for the creation of recognition sites on a polymer scaffold. Protein imprinting has been a focus for many chemists working in the area of molecular recognition, since the creation of synthetic polymers that can specifically recognise proteins is a very challenging but potentially extremely rewarding objective. It is expected that molecularly imprinted polymers (MIPs) with specificity for proteins will find application in medicine, diagnostics, proteomics, environmental analysis, sensors and drug delivery. In this review, the authors provide an overview of the progress achieved in the decade between 1994 and 2005, with respect to the challenging area of MIPs for protein recognition. The discussion furnishes a comparative analysis of different approaches developed, underlining their relative advantages and disadvantages and highlighting trends and possible future directions. (c) 2006 Elsevier B.V. All rights reserved.
An entirely new means of printing molecular information on a planar film, involving casting nanoscale impressions of the template protein molecules in molten gallium, is presented here for the first time. The metallic imprints not only replicate the shape and size of the proteins used as template. They also show specific binding for the template species. Such a simple approach to the creation of antibody-like properties in metallic mirrors can lead to applications in separations, microfluidic devices, and the development of new optical and electronic sensors, and will be of interest to chemists, materials scientists, analytical specialists, and electronic engineers.
This paper reports the synthesis and screening of a combinatorial peptide library for new affinity ligands for glycosylated haemoglobin (HbA(1c)), which is an important indicator of diabetes control. The new ligands are suitable for large-scale synthesis and overcome the disadvantages of antibodies (unstable and expensive to produce etc.), while remaining as efficient as antibodies in binding to the analyte. The library consisted of 262 144 hexapeptides synthesised using the one-bead-one-compound technique. The hexapeptides attached onto beads were screened with glycosylated haemoglobin HbA(1c). The structures of the peptides exhibiting high affinity were characterised by Edman microsequencing. Computer modelling simulation of one of the lead sequences has shown that this class of ligand has a high affinity and specificity for glycosylated haemoglobin. (C) 1998 Elsevier Science S.A. All rights reserved.
Microsystin-LR is one of the most widespread and dangerous toxins produced by the freshwater Cyanobacteria. The contamination of water supplies with microcystin-LR has been reported in several areas around the world and the development of an easy-to-use, rapid, robust and inexpensive sensor for this toxin is urgently required. In this work an artificial receptor for microcystin-LR was synthesised using the technique of molecular imprinting. The composition of the molecularly imprinted polymer (MIP) was optimised using computer modelling. The synthesised polymer was used both as a material for solid-phase extraction (SPE) and as a sensing element in a piezoelectric sensor. Using the combination of SPE followed by detection with a piezoelectric sensor the minimum detectable amount of toxin was 0.35 nM. The use of MIP-SPE provided up to 1000 fold preconcentration, which was more than sufficient for achieving the required detection limit for microcystin-LR in drinking water (I nM). This work is the first example where the same MIP receptor has been used successfully for both SPE and the corresponding sensor. (C) 2002 Elsevier Science B.V. All rights reserved.
Modern cell phones are a ubiquitous resource with a residual capacity to accommodate chemical sensing and biosensing capabilities. From the different approaches explored to capitalize on such resource, the use of autonomous disposable lab-on-a-chip (LOC) devices conceived as only accessories to complement cell phones underscores the possibility to entirely retain cell phones ubiquity for distributed biosensing. The technology and principles exploited for autonomous LOC devices are here selected and reviewed focusing on their potential to serve cell phone readout configurations. Together with this requirement, the central aspects of cell phones resources that determine their potential for analytical detection are examined. The conversion of these LOC concepts into universal architectures that are readable on unaccessorized phones is discussed within this context. (C) 2015 Elsevier B.V. All rights reserved.
Melanophores, dark pigment cells from the frog Xenopus laevis, have the ability to change light absorbance upon stimulation by different biological agents. Hormone exposure (e.g. melatonin or α-melanocyte stimulating hormone) has been used here as a reversible stimulus to test a new compact microplate reading platform. As an application, the detection of the asthma drug formoterol in blood plasma samples is demonstrated. The present system utilizes a computer screen as a (programmable) large area light source, and a standard web camera as recording media enabling even kinetic microplate reading with a versatile and broadly available platform, which suffices to evaluate numerous bioassays. Especially in the context of point of care testing or self testing applications these possibilities become advantageous compared with highly dedicated comparatively expensive commercial systems.
The computer screen photo-assisted technique (CSPT), a method for substance classification based on spectral fingerprinting, which involves just a computer screen and a web camera as measuring platform is used here for the evaluation of a prospective enzyme-linked immunosorbent assay (ELISA). A anti-neutrophil cytoplasm antibodies (ANCA-ELISA) test, typically used for diagnosing patients suffering from chronic inflammatory disorders in the skin, joints, blood vessels and other tissues is comparatively tested with a standard microplate reader and CSPT, yielding equivalent results at a fraction of the instrumental costs. The CSPT approach is discussed as a distributed measuring platform allowing decentralized measurements in routine applications, whereas keeping centralized information management due to its natural network embedded operation. © 2004 Elsevier B.V. All rights reserved.
New thiophene-carbazole functional and cross-linking monomers electropolymerizing at potentials sufficiently low for molecular imprinting of an electroactive aripiprazole antipsychotic drug were herein designed and synthesized. Numerous conducting molecularly imprinted polymer (MIP) films are deposited by electro-polymerization at relatively low potentials by electm-oxidation of pyrmle, aniline, phenol, or 3,4-ethylenediox-ythiophene (EDOT). However, their interactions with templates are not sufficiently strong. Hence, it is necessary to introduce additional recognizing sites in these cavities to increase their affinity to the target molecules. For that, functional monomers derivatized with substituents forming stable complexes with the templates are used. However, oxidation potentials of these derivatives are often, disadvantageously, higher than that of parent monomers. Therefore, we designed and synthesized new functional and cross-linking monomers, which are oxidized at sufficiently low potentials. The deposited MIP and non-imprinted polymer (NIP) films were characterized by PM-IRRAS and UV-vis spectroscopy and imaged with AFM. The structure of the aripiprazole prepolymerization complex with functional monomers was optimized with density functional theory (DFT), and aripiprazole interactions with imprinted cavities were simulated with molecular mechanics (MM) and molecular dynamics (MD). MIP-aripiprazole film-coated electrodes were used as extended gates for selective determination of aripiprazole with the extended-gate field-effect transistor (EG-FET) chemosensor. The linear dynamic concentration range was 30-300 pM, and the limit of detection was 22 fM. An apparent imprinting factor of the MIP-1 was IF = 4.95. The devised chemosensor was highly selective to glucose, urea, and creatinine interferences. The chemosensor was successfully applied for aripiprazole determination in human plasma. The results obtained were compared to those of the validated HPLC-MS method.
Biosensors can deliver the rapid bacterial detection that is needed in many fields including food safety, clinical diagnostics, biosafety and biosecurity. Whole-cell imprinted polymers have the potential to be applied as recognition elements in biosensors for selective bacterial detection. In this paper, we report on the use of 3-aminophenylboronic acid (3-APBA) for the electrochemical fabrication of a cell-imprinted polymer (CIP). The use of a monomer bearing a boronic acid group, with its ability to specifically interact with cis-diol, allowed the formation of a polymeric network presenting both morphological and chemical recognition abilities. A particularly beneficial feature of the proposed approach is the reversibility of the cis-diol-boronic group complex, which facilitates easy release of the captured bacterial cells and subsequent regeneration of the CIP. Staphylococcus epidermidis was used as the model target bacteria for the CIP and electrochemical impedance spectroscopy (EIS) was explored for the label-free detection of the target bacteria. The modified electrodes showed a linear response over the range of 103–107 cfu/mL. A selectivity study also showed that the CIP could discriminate its target from non-target bacteria having similar shape. The CIPs had high affinity and specificity for bacterial detection and provided a switchable interface for easy removal of bacterial cell.
A surface plasmon resonance (SPR) based flow chamber device was designed for real time detection of blood coagulation and platelet adhesion in platelet rich plasma (PRP) and whole blood. The system allowed the detection of surface interactions throughout the 6 mm length of the flow chamber. After deposition of thromboplastin onto a section of the sensor surface near the inlet of the flow chamber, coagulation was detected downstream of this position corresponding to a SPR signal of 7 to 8 mRIU (7 to 8 ng/mm2). A nonmodified control surface induced coagulation 3.5 times slower. Platelet adhesion to gold and fibrinogen coated surfaces in the magnitude of 1.25 and 1.66 mRIU was also shown with platelets in buffer, respectively. SPR responses obtained with PRP and whole blood on surfaces that were methylated or coated with von Willebrand factor (vWF), fibrinogen, or collagen, coincided well with platelet adhesion as observed with fluorescence microscopy in parallel experiments. The present SPR detection equipped flow chamber system is a promising tool for studies on coagulation events and blood cell adhesion under physiological flow conditions, and allows monitoring of short-range surface processes in whole blood. © 2007 Elsevier B.V. All rights reserved.
In the haemostatic system a multitude of processes are intertwined in fine-tuned interactions that arrest bleeding, keep the circulatory system open, and the blood flowing. The occurrence of both surface and bulk interactions adds an additional dimension of complexity. These insights have led to the belief that global overall procedures can inform on the likely behaviour of the system in health and disease. Two sensing procedures: surface plasmon resonance (SPR), which senses surface interactions, and free oscillation rheometry (FOR), which senses interactions within the bulk, have been combined and evaluated. The contribution of blood cells, mainly platelets, to the SPR and FOR signals was explored by simultaneous SPR and FOR measurement during native whole blood coagulation, accelerated via the platelets through addition of SFLLRN peptide and inhibition of platelet aggregation with abciximab (ReoPro®) and of shape change with cytochalasin E. The SPR technique was found to be sensitive to inhibition of blood cell functions such as adhesion to and spreading on surfaces, as well as platelet aggregation. SPR seemed not to be directly sensitive to fibrin polymerisation in coagulating whole blood. The FOR technique detected the coagulation as a bulk phenomenon, i.e. the gelation of the blood due to fibrin formation was detected. The combination of SPR and FOR may therefore be suitable for studies on blood cell functions during coagulation.
In haemostatic and biomaterial research biological processes at surfaces and in the bulk phase of the surface-contacting medium are important. The present work demonstrates the usefulness of the combination of surface plasmon resonance (SPR), sensitive to changes in refractive index at surfaces, and free oscillation rheometry (FOR), sensitive to rheological properties of the bulk, for simultaneous real-time measurements on coagulation and fibrinolysis of blood plasma and coagulation of whole blood. SFLLRN stimulated coagulation of native whole blood presented a higher SPR signal with different appearance than plasma coagulation, while the FOR signals corresponding to plasma and whole blood coagulation were similar. This indicated that the SPR technique was more sensitive to cell-surface interactions than to fibrin formation in whole blood during coagulation, while the FOR technique were equally sensitive to coagulation in whole blood and plasma. Spontaneous coagulation of native whole blood in contact with methyl- and hydroxyl-terminated self-assembled monolayers (SAM) on gold and gold surfaces regenerated after coagulation were also studied. The regenerated gold surfaces displayed the shortest coagulation times, although the contact-activation of blood coagulation for these surfaces was low. The methylated and hydroxylated surfaces were comparable in terms of coagulation activation, while the hydroxylated surfaces presented FOR signals that indicated detaching of the coagulum from the surface. The combination of SPR and FOR is well suited for studies of cell– and protein–surface interactions and simultaneous bulk processes. Possible applications are investigations of blood cell defects in patients and monitoring of native whole blood interactions with artificial surfaces.
It is previously shown that surface plasmon resonance (SPR) can be used to study blood plasma coagulation. This work explores the use of this technique for the analysis of tissue factor induced coagulation, i.e. prothrombin time (PT) analysis, of whole blood and plasma. The reference method was nephelometry. The prothrombin time analysis by SPR was performed by mixing two volumes of blood/plasma, one volume of thromboplastin, and one volume of CaCl2 solution directly on a sensor surface. The measurements show good agreement between nephelometry and SPR plasma analysis and also between SPR plasma and whole blood analysis. The effect of anticoagulant treatment on the clotting times was significant both quantitatively and qualitatively. The impact on the SPR signal of different physiological events in the coagulation process is discussed, and tentative interpretations of the sensorgram features are given. The major advantage of the SPR method compared to nephelometry is the possibility to perform analysis on whole blood instead of plasma. In conclusion, SPR is a promising method for whole blood coagulation analysis.
Prototype electrochemical lactate electrodes based on lactate oxidase were produced by screen-printing and application of membranes by dip-coating or ink-jet printing. The link between enzyme and electrode was made by electro-deposition of platinum onto graphite pads. The linear range of the sensors was small; up to 2 mM lactate. Electrodes retained their activity when stored dry; activity remained high for 244 days. The properties of the electrodes indicate that screen- and ink-jet printing are feasible techniques for the machine production of lactate sensors.
There is a widespread need for commercial instrumentation for the rapid and inexpensive detection of microbial contamination of food, industrial waste water and clinical samples. A large number of detection methods have been developed utilizing the optical, electrochemical, biochemical and physical properties of microorganisms. The need for a device which can produce a rapid, accurate, sensitive, real-time analysis for clinical, industrial and environmental applications has led to considerable progress being achieved in recent years in the development of biosensors for microbial detection. This intense research has resulted in the commercialization of several instruments. Techniques used for the quantification of microorganisms are reviewed under the general categories of non-bioelectrochemical and bioelectrochemical methods.
We report a novel procedure for the electrochemical modification of graphite disc electrodes with potassium hexacyanoferrate(III) using cyclic voltammetry. Cyclic voltammograms of the modified electrodes in 0.1 M potassium chloride showed two redox couples, at 0.22 and 0.148V and at 0.923 and 0.798 V. These redox potentials correspond to those cited for Prussian blue type films. These modified electrodes exhibited excellent stability during repeated potential cycling in 0.1 M potassium chloride with no difference in peak heights between the second and thirtieth scans. We used glucose oxidase as the model enzyme to investigate application of these novel modified graphite disc electrodes in the development of biosensors. The resulting glucose sensors exhibited a response to glucose at potentials as low as 450 mV vs. saturated calomel electrode with a linear range up to 1.5 mM glucose and sensitivity of 50 +/- 10 nA mM(-1) cm(-2) (geometric area). The modified graphite disc electrodes displayed an electrocatalytic effect on hydrogen peroxide with sensitivity of 0.70 mu A mM(-1) cm(-2) (geometric area). The effect of ascorbic acid, 4-acetamidophenol and uric acid, common interferents found in blood, is also reported. (C) 1996 Published by Elsevier Science Limited
A DNA-based surface plasmon resonance (SPR) biosensor has been developed for the detection of TP53 mutation using the inexpensive and commercially available instrument, SPREETA (TM) SPR-EVM-BT, from Texas Instruments. A direct immobilisation procedure, based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces, was optimized using synthetic oligonucleotides. Hybridisation reactions between the immobilised probe and a short sequence (26 mer) complementary, non-complementary and one-point mutation DNA were then investigated. The main analytical parameters of the sensor system were studied in detail including selectivity, sensitivity, reproducibility and analysis time. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples, DNA extracted from both normal, wild-type, (Jurkat) and mutated (Molt 4), carrying the mutation at codon 248 of the TP53 cell lines. The results obtained demonstrate that the DNA-based SPR biosensor was able to distinguish sequences present in the various samples that differ only by one base; and hence, it appears to be a strong candidate technique for the detection of gene mutation. (c) 2004 Elsevier B.V. All rights reserved.
Many scientific instruments utilise multiple element detectors, e.g. CCD's or photodiode arrays, to monitor the change in a position of an optical pattern. For example, instruments for affinity biosensing based on surface plasmon resonance (SPR) or resonant mirror are equipped with such detectors. An important and desired property of these bioanalytical instruments is that the calculation of the movement or change in shape follows the true change. This is often not the case and it may lead to linearity errors, and to sensitivity errors. The sensitivity is normally defined as the slope of the calibration curve. A new parameter is introduced to account for the linearity errors, the sensitivity deviation, defined as the deviation from the undistorted slope of the calibration curve. The linearity error and the sensitivity deviation are intimately related and the sensitivity deviation may lead to misinterpretation of kinetic data, mass transport limitations and concentration analyses. Because the linearity errors are small (e.g. 10 pg/mm2 of biomolecules on the sensor surface) with regard to the dynamic range (e.g. 30 000 pg/mm2), they can be difficult to discover. However, the linearity errors are often not negligible with regard to a typical response (e.g. 0-100 pg/mm2), and may therefore cause serious problems. A method for detecting linearity errors is outlined. Further on, this paper demonstrates how integral linearity errors of less than 1% can result in a sensitivity deviation of 10%, a value that in our opinion cannot be ignored in biospecific interaction analysis (BIA). It should also be stressed out that this phenomenon also occurs in other instruments using array detectors. (C) 2000 Elsevier Science S.A.Many scientific instruments utilize multiple element detectors, e.g. CCD's or photodiode arrays, to monitor the change in a position of an optical pattern. For example, instruments for affinity biosensing based on surface plasmon resonance (SPR) or resonant mirror are equipped with such detectors. An important and desired property of these bioanalytical instruments is that the calculation of the movement or change in shape follows the true change. This is often not the case and it may lead to linearity errors, and to sensitivity errors. The sensitivity is normally defined as the slope of the calibration curve. A new parameter is introduced to account for the linearity errors, the sensitivity deviation, defined as the deviation from the undistorted slope of the calibration curve. The linearity error and the sensitivity deviation are intimately related and the sensitivity deviation may lead to misinterpretation of kinetic data, mass transport limitations and concentration analyses. Because the linearity errors are small (e.g. 10 pg/mm2 of biomolecules on the sensor surface) with regard to the dynamic range (e.g. 30 000 pg/mm2), they can be difficult to discover. However, the linearity errors are often not negligible with regard to a typical response (e.g. 0-100 pg/mm2), and may therefore cause serious problems. A method for detecting linearity errors is outlined. Further on, this paper demonstrates how integral linearity errors of less than 1% can result in a sensitivity deviation of 10%, a value that in our opinion cannot be ignored in biospecific interaction analysis (BIA). It should also be stressed out that this phenomenon also occurs in other instruments using array detectors.
Ligands for identifying protein aggregates are of great interest as such deposits are the pathological hallmark of a wide range of severe diseases including Alzheimers and Parkinsons disease. Here we report the synthesis of an azide functionalized fluorescent pentameric oligothiophene that can be utilized as a ligand for multimodal detection of disease-associated protein aggregates. The azide functionalization allows for attachment of the ligand to a surface by conventional click chemistry without disturbing selective interaction with protein aggregates and the oligothiophene-aggregate interaction can be detected by fluorescence or surface plasmon resonance. In addition, a methodology where the oligothiophene ligand is employed as a capturing molecule selective for aggregated proteins in combination with an antibody detecting a distinct peptide/protein is also presented. We foresee that this methodology will offer the possibility to create a variety of multiplex sensing systems for sensitive and selective detection of protein aggregates, the pathological hallmarks of several neurodegenerative diseases.
The selective detection and quantification of macromolecular targets is a fundamental biological mechanism in nature. Molecularly imprinted polymers (MIPs) have been identified as one of the most promising synthetic alternatives to bioreceptors. However, expanding this methodology towards selective recognition of bulky templates such as proteins appears to be extremely challenging due to problems associated with removal of the template from the polymeric network. In this study, polymer imprinted with troponin T (TnT) was assessed using electrochemical methods and the influence of various extraction methods, including conventional immersion extraction, thermal annealing and ultrasonic-assisted extraction, on the binding characteristics of the troponin-to-imprinted polymer receptor was elucidated. Cyclic voltammetric deposition of o-phenylenediamine (o-PD) film in the presence of TnT as a template was performed in acetate buffer (0.5 M, pH 5.2) on a gold substrate. Solvent extraction of the target molecule was optimised and followed by subsequent washing with water. The electrochemistry of a ferro/ferricyanide probe was used to characterise the TnT MIP receptor film. The incubation of the TnT MIP receptor-modified electrode with respect to TnT concentration resulted in a suppression of the ferro/ferricyanide redox current. The dissociation constant (KD) was calculated using a two-site model of template affinity for the TnT MIP receptor. The synthetic TnT MIP receptor had high affinity for TnT with a KD of 2.3×10−13 M.
Cardiac troponin T (TnT) is a highly sensitive cardiac biomarker for myocardial infarction. In this study, the fabrication and characterisation of a novel sensor for human TnT based on a molecularly-imprinted electrosynthesised polymer is reported. A TnT sensitive layer was prepared by electropolymerisation of o-phenylenediamine (o-PD) on a gold electrode in the presence of TnT as a template. To develop the molecularly imprinted polymer (MIP), the template molecules were removed from the modified electrode surface by washing with alkaline ethanol. Electrochemical methods were used to monitor the processes of electropolymerisation, template removal and binding. The imprinted layer was characterised by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). The incubation of the MIP-modified electrode with respect to TnT concentration resulted in a suppression of the ferro/ferricyanide redox process. Experimental conditions were optimised and a linear relationship was observed between the peak current of [Fe(CN)(6)](3-)/[Fe (CN)(6)](4-) and the concentration of TnT in buffer over the range 0.009-0.8 ng/mL, with a detection limit of 9 pg/mL. The TnT MIP sensor was shown to have a high affinity to TnT in comparison with nonimprinted polymer (NIP) electrodes in both buffer and blood serum.
Advanced multifunctional protein particles encapsulated enzymes and antibodies were developed for enzymatic bioassays and immunoassays with colorimetric and fluorescent channels. A colorimetric channel based on color-substrate precipitation was assigned for enzymatic bioassays for the measurement of hydrogen peroxide with the lowest detectable concentration of 10 μM. A fluorescent channel based on fluorescent labeled antibodies was assigned for immunoassays for the measurement of mouse immunoglobulin G (M IgG) with the lowest detectable concentration of 1.25 μg L−1. The protein microparticles were fabricated with a template-assisted self-assembly technique termed “Protein Activation Spontaneous Self-assemble” (PASS). The multifunctional protein particles prepared with the PASS method have the advantages of high loading of analytical biomolecules, integrated biological functions, porous structure, and more importantly, they are optically transparent and fluorescence inactive. These unique features make our protein particles a new generation of bead-based platforms to perform enzyme bioassays and immunoassays.
Immobilisation of biorecognition elements on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. A novel protocol for the covalent immobilisation of biomolecules containing primary amines using an inexpensive and simple polymer is presented. This tridimensional (3D) network leads to a random immobilisation of antibodies on the polymer and ensures the availability of a high percentage of antibody binding sites. The reactivity of the polymer is based on the reaction between primary amines and thioacetal groups included in the polymer network. These functional groups (thioacetal) do not need any further activation in order to react with proteins, making it attractive for sensor fabrication. The novel polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on a metal transducer surface. For demonstration purposes the polymer was immobilised on Au Biacore chips. The resulting polymer layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. A general protocol suitable for the immobilisation of bovine serum albumin (BSA), enzymes and antibodies such as polyclonal anti-microcystin-LR antibody and monoclonal anti-prostate specific antigen (anti-PSA) antibody was then optimised. The affinity characteristics of developed immunosensors were investigated in reaction with microcystin-LR, and PSA. The calculated detection limit for analytes depended on the properties of antibodies. The detection limit for microcystin-LR was 10 ng mL(-1) and for PSA 0.01 ng mL(-1). The non-specific binding of analytes to synthesised polymers was very low. The polymer-coated chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. These findings make this new polymer very promising for the development of low-cost, easy to prepare and sensitive biosensors. (C) 2008 Elsevier B.V. All rights reserved.
The immobilisation of bio-receptors on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. The development of a protocol for biomolecule immobilisation onto a surface plasmon resonance (SPR) sensor surface using inexpensive polythiol compounds is presented here. The method used here is based on the reaction between primary amines and thioacetal groups, formed upon reaction of o-phthaldialdehyde (OPA) and thiol compounds. The self-assembled thiol monolayers were characterised using contact angle and XPS. The possibility to immobilise proteins on monolayers was assessed by employing BSA as a model protein. For the polythiol layers exhibiting the best performance, a general protocol was optimised suitable for the immobilisation of enzymes and antibodies such as anti-prostate specific antigen (anti-PSA) and anti Salmonella typhimurium. The kinetic data was obtained for PSA binding to anti-PSA and for S. typhimurium cells with a detection limit of 5 x 10(6) cells mL(-1) with minimal non-specific binding of other biomolecules. These findings make this technique a very promising alternative for amine coupling compared to peptide bond formation. Additionally, it offers opportunity for immobilising proteins (even those with low isoelectric point) on neutral polythiol layers without any activation step. (C) 2009 Elsevier B.V. All rights reserved.
Highly sensitive and label-free detection of the biomarker carbohydrate antigen 15-3 (CA 15-3) remains a challenge in the diagnosis of breast cancer. Here, a novel electrochemical immunosensor capable of sensitive and label-free detection of CA 15-3 is reported. This unique immunosensor, equipped with a highly conductive graphene (i.e., N-doped graphene sheets)-modified electrode, exhibited significantly increased electron transfer and high sensitivity toward CA 15-3. This novel immunosensor, with a low detection limit of 0.012U/mL, worked well over a broad linear range of 0.1-20U/mL. Unlike conventional immunosensors, which usually involve complicated label processing and time-consuming separations, the use of highly conductive graphene avoids the need for labels and is simple in nature. The strategy developed for this immunosensor provides a promising approach for clinical research and diagnostic applications.
A method based on a surface plasmon resonance technique for detection of changes in concentration and glycosylation of proteins in cell culture supernatant is described. The method was used to analyze α1-acid glycoprotein (AGP) produced by a human hepatoma cell line (HepG2). Cell culture supernatant was injected to a BIACORE 2000 instrument and AGP was captured on the sensor chip by immobilized antibodies. The captured glycoprotein was then analyzed for content of carbohydrate epitopes using three different lectins, Aleuria aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), and Triticum vulgaris agglutinin (wheat germ agglutinin, WGA). The method was used to analyze changes in concentration and glycosylation of AGP produced by HepG2 cells grown with or without three different cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6), and transforming growth factor β-1 (TGFβ1). Using the described method it was shown that when HepG2 cells were grown in the presence of IL-6 both AGP concentration and fucosylation increased. When HepG2 cells instead were grown in the presence of TGFβ1 AGP fucosylation increased whereas AGP concentration decreased.
A new approach for the facile fabrication of electrochemical biosensors using a biohybrid conducting polymer was demonstrated using glucose oxidase (GOx) and poly (3, 4-ethylenedioxythiophene) (PEDOT) as a model. The biohybrid conducting polymer was prepared based on a template-assisted chemical polymerisation leading to the formation of PEDOT microspheres (PEDOT-MSs), followed by in-situ deposition of platinum nanoparticles (PtNPs) and electrostatic immobilisation of glucose oxidase (GOx) to form water processable GOx-PtNPs-PEDOT-MSs. The morphology, chemical composition and electrochemical performance of the GOx-PtNPs-PEDOT-MS-based glucose biosensor were characterised using scanning electron microscopy (SEM), energy-dispersive X-ray spectrometry (EDS), Fourier transform infrared (FTIR) spectroscopy, zeta potential and electrochemical measurements, respectively. The biosensor delivered a linear response for glucose over the range 0.1-10 mM (R-2 = 0.9855) with a sensitivity of 116.25 mu A mM(-1) cm(-2), and limit of detection of 1.55 mu M (3 x SD/sensitivity). The sensitivity of the developed PEDOT-MS based biosensor is significantly higher (2.7 times) than the best reported PEDOT-based glucose biosensor in the literature. The apparent Michaelis Menten constant (K-m(app)) of the GOx-PtNPs-PEDOT-MS-based biosensors was calculated as 7.3 mM. Moreover, the biosensor exhibited good storage stability, retaining 97% of its sensitivity after 12 days storage. This new bio-hybrid conducting polymer combines the advantages of micro-structured morphology, compatibility with large-scale manufacturing processes, and intrinsic biocatalytic activity and conductivity, thus demonstrating its potential as a convenient material for printed bioelectronics and sensors.
A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BlAcore 3000(TM). The gold surface was first functionalised with a self-assembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethyl amino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BlAcore 3000(TM). Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BlAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip did not affect its recognition properties and continuous measurement was possible over a period of at least 2 months. (C) 2004 Elsevier B.V. All rights reserved.
Methylamine dehydrogenase, a tryptophan tryptophyl quinone (TTQ) containing quinoprotein, catalyzes the oxidation of a variety of primary aliphatic monoamines and diamines to their respective aldehydes and ammonia. This paper reports the construction and characterization of an enzyme electrode capable of detecting histamine and methylamine at +200 mV versus a saturated calomel reference electrode. The methylamine dehydrogenase isolated from Paracoccus denitrificans was used in conjunction with the insoluble mediator tetracyanoquinodimethane (TCNQ) to construct enzyme electrodes which will potentially provide simple rapid analysis of histamine without the need for the extensive sample pretreatments currently required in HPLC and GLC analysis. The linear response of this amperometric sensor, between 0 and 200 mu M, correlates well with elevated histamine levels predominant in patients with chronic myelogenous leukaemia, whilst the observed limit of detection, 4.8 mu M, compares favourably with the lower limits of detection reported for a potentiometric histamine sensitive enzyme electrode.
Genosensor technology relying on the use of carbon and gold electrodes is reviewed. The key steps of each analytical procedure, namely DNA-probe immobilisation, hybridisation, labelling and electrochemical investigation of the surface, are discussed in detail with separate sections devoted to label-free and newly emerging magnetic assays. Special emphasis has been given to protocols that have been used with real DNA samples. (C) 2003 Elsevier B.V. All fights reserved.