A disposable biosensor for urea determination in blood based on an ammonium-sensitive transducer

A potentiometric urea-sensitive biosensor using a NH4(+)-sensitive disposable electrode in double matrix membrane (DMM) technology as transducer is described. The ion-sensitive polymer matrix membrane was formed in the presence of an additional electrochemical inert filter paper matrix to improve the reproducibility in sensor production. The electrodes were prepared from one-side silver-coated filter paper, which is encapsulated for insulation by a heat-sealing film. A defined volume of the NH4(+)-sensitive polymer matrix membrane cocktail was deposited on this filter paper. To obtain the urea-biosensor a layer of urease was cast onto the ion-sensitive membrane. Poly (carbamoylsulfonate) hydrogel, produced from a hydrophilic polyurethane prepolymer blocked with bisulfite, served as immobilisation material. The disposable urea sensitive electrode was combined with a disposable Ag/AgCl reference electrode to obtain the disposable urea biosensor. The sensor responded rapidly and in a stable manner to changes in urea concentrations between 7.2 x 10(-5) and 2.1 x 10(-2)mol/l. The detection limit was 2 x 10(-5) mol/l urea and the slope in the linear range 52 mV/decade. By taking into consideration the influence of the interfering K(+)- and Na(+)-ions the sensor can be used for the determination of urea in human blood and serum samples (diluted or undiluted). A good correlation was found with the data obtained by the spectrophotometric routine method.     

A novel ternary NiFe2O4/CuO/FeO-chitosan nanocomposite as a cholesterol biosensor

We report the results of studies relating to the in situ synthesis of a novel ternary NiFe₂O₄/CuO/FeO-chitosan nanocomposite, which could be utilized as a cholesterol biosensor. The phase identification, morphology and particle size of the NiFe₂O₄/CuO/FeO nanocomposite have been investigated via X-ray diffraction pattern (XRD), scanning electron microscopy (SEM), high resolution transmission electron microscope (HR-TEM) and Fourier transform infrared (FTIR) spectroscopy. The quantification of cholesterol was accomplished by immobilizing cholesterol oxidase (ChOx) onto a chitosan-NiFe₂O₄/CuO/FeO nanocomposite (NiFe₂O₄/CuO/FeO-CH NC) deposited onto an indium-tin-oxide (ITO) glass substrate via the sol–gel technique. The electrochemical study results of the biocompatible ChOx/NiFe₂O₄/CuO/FeO-CH/ITO electrode reveal good linearity (50–5000 mg/L), a low detection limit (313 mg/L), high sensitivity (0.043 μA/(mg/L cm^?2)), a fast response time (10 s) and a shelf-life of 3 months. The low Michaelis–Menten constant (K_m) of 80 mg/L (0.21 mM) indicates the high affinity of ChOx for the analytes. Further, this bioelectrode has been used in clinical applications to estimate cholesterol levels with negligible interference (2%) from analytes present in human serum samples.     

Nanostructured transducer surfaces for electrochemical biosensor construction—Interfacing the sensing component with the electrode

For fabrication of effective electrochemical biosensors, interfacing the biomolecular receptor with the underlying transducer represents a critical step. The actual approach taken depends on the tethering layer covering the transducer, which is typically either a conducting polymeric matrix, or a thin film, such as an alkanethiol monolayer. Non-specific immobilisation methods can be either covalent, or non-covalent affinity attachment, with multipoint electrostatic attachment of the sensing biomolecule to either a polyanionic or polycationic layer representing the most common approach. Many specific affinity immobilisation strategies exist, but the majority make use of one of two binding systems. The first relies on the specific and strong affinity between biotin and proteins of the avidin family, with both bioreceptor and transducer bearing pendant biotins and avidin used as the crosslinker. The second approach employs a metal chelating group on the transducer to which can be bound a polyhistidine tag present on the N- or C-terminus of the receptor protein and which can be introduced genetically, when the expression sequence for a recombinant proteins is designed.     

High-performance electrochemical biosensor for the detection of total cholesterol

We report on a highly sensitive electrochemical biosensor for the determination of total cholesterol. The novel biosensor was fabricated by co-immobilizing three enzymes, cholesterol oxidase (ChO(x)), cholesterol esterase (ChE) and horseradish peroxidase (HRP), on nanoporous gold networks directly grown on a titanium substrate (Ti/NPAu/ChO(x)-HRP-ChE). The morphology and composition of the fabricated nanoporous gold were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS) and X-ray diffraction spectroscopy (XRD). The electrochemical behaviour of the Ti/NPAu/ChO(x)-HRP-ChE biosensor was studied using cyclic voltammetry (CV), showing that the developed biosensor possessed high selectivity and high sensitivity (29.33 μA mM?1 cm?2). The apparent Michaelis-Menten constant, K(M)(app) of this biosensor was very low (0.64 mM), originating from the effective immobilization process and the nanoporous structure of the substrate. The biosensor exhibited a wide linear range up to 300 mg dL?1 in a physiological condition (pH 7.4), which makes it very promising for the clinical determination of cholesterol. The fabricated biosensor was further tested using real food samples margarine, butter and fish oil, showing that the biosensor has the potential to be used as a facile cholesterol detection tool in food and supplement quality control.     

Resonance phenomenon of the ATP motor as an ultrasensitive biosensor

We designed a rotary biosensor as a damping effector, with the rotation of the F₀F₁-ATPase driven by Adenosine Triphosphate (ATP) synthesis being indicated by the fluorescence intensity and a damping effect force being induced by the binding of an RNA molecule to its probe on the rotary biosensor. We found that the damping effect could contribute to the resonance phenomenon and energy transfer process of our rotary biosensor in the liquid phase. This result indicates that the ability of the rotary motor to operate in the vibration harmonic mode depends on the environmental conditions and mechanism in that a few molecules of the rotary biosensor could induce all of the sensor molecules to fluoresce together. These findings contribute to the theory study of the ATPase motor and future development of biosensors for ultrasensitive detection.     

Amperometric cholesterol biosensor based on in situ reconstituted cholesterol oxidase on an immobilized monolayer of flavin adenine dinucleotide cofactor

A new amperometric biosensor for determining cholesterol based on deflavination of the enzyme cholesterol oxidase (ChOx) and subsequent reconstitution of the apo-protein with a complexed flavin adenine dinucleotide (FAD) monolayer is described. The charge transfer mediator pyrroquinoline quinone (PQQ) was covalently bound to a cystamine self-assembled monolayer (SAM) on an Au electrode. Boronic acid (BA) was then bound to PQQ using the carbodiimide procedure, and the BA ligand was complexed to the FAD molecules on which the apo-ChOx was subsequently reconstituted. The effective release of the FAD from the enzyme and the successful reconstitution were verified using molecular fluorescence and cyclic voltammetry. The optimal orientation of FAD toward the PQQ mediator and the distances between FAD and PQQ and between PQQ and electrode enhance the charge transfer, very high sensitivity (about 2,500 nAmM(-1)cm(-2)) being obtained for cholesterol determination. The biosensor is selective toward electroactive interferents (ascorbic acid and uric acid) and was tested in reference serum samples, demonstrating excellent accuracy (relative errors below 3% in all cases). The biosensor activity can be successfully regenerated in a simple process by successive reconstitution with batches of recently prepared apo-ChOx on the same immobilized Au/SAM-PQQ-BA-FAD monolayer (it was tested five times); the lifetime of the biosensor is about 45-60 days.     

A piezoelectric biosensor as an olfactory receptor for odour detection: electronic nose

Humans can detect and differentiate the presence of different odours even at trace levels of these odorous compounds. The odour quantification of any particular samples is normally based on conventional panel decisions. Other analytical instruments could be used to detect trace levels of odorous molecules. This study presents the results of a biological sensor system subject to different odorants. The system consists of a sensor in which the isolated olfactory receptor proteins (ORPs) from bullfrogs (Rana spp.) were coated onto the surface of a piezoelectric (PZ) electrode, similar to the mechanism of human olfaction. The PZ crystal served as a signal transducer. The results indicate rapid (about 400 s), reversible, and longterm (up to 3 months) stable responses to different volatile compounds such as n-caproic acid, isoamyl acetate, n-decyl alcohol, beta-ionone, linalool, and ethyl caporate. The sensitivity of the sensor ranges from 10(-6)-10(-7) g, fully correlated with the olfactory threshold values of human noses. An array of six sensors consisting of five fractionated ORPs and one referenced phospholipid probe is able to respond to different odorants and form a typical fingerprint for each odorant.     

Bis-pyrene labeled DNA aptamer as an intelligent fluorescent biosensor

The site-directed incorporation of bis-pyrenyl fluorophore into anti-ATP DNA aptamer results in a creation of an intelligent fluorescent sensor with high signal intensity and specificity for detecting the target ligand in a homogeneous system.     

Development of disposable lipid biosensor for the determination of total cholesterol

A prototype chronoamperometric biosensor for the determination of total cholesterol was developed that consists of a homemade potentiostat and disposable strips immobilized with Fe(3)O(4), cholesterol oxidase (ChOx), and cholesterol esterase (ChE). The principle of sensing cholesterol is based on the detection of reduction signal of hydrogen peroxide generated in two enzymatic reactions. The co-immobilization of ChE and ChOx allows the sensor to detect both concentrations of esterified and free cholesterol. The effects of biosensor on catalyst, enzymes, applied potential, and buffer pH was investigated, and the operation conditions were optimized. The detection of cholesterol can be accomplished in one step, a 10 microL of sample was dropped onto the area of sensing strip and the reduction signal was obtained at an applied potential of -200 mV (vs. Ag/Ag(+)). The pre-reaction time was set at 15s before applying potential on the strip and the sampling time was 5s. The sensing device displays a linear response over the range of 100-400mg/dL (R(2)=0.999) for cholesteryl oleate. The coefficient variation was determined as 5.06% (N=20) for 100mg/dL cholesteryl oleate and the detection limit is 19.4 mg/dL (S/N=3). The probable interferences in bio-matrix were selected to test the selectivity and no significant response was observed in the biosensor.     

A fiber-optic microarray biosensor using aptamers as receptors

A fiber-optic biosensor using an aptamer receptor has been developed for the measurement of thrombin. An antithrombin DNA aptamer was immobilized on the surface of silica microspheres, and these aptamer beads were distributed in microwells on the distal tip of an imaging fiber. A different oligonucleotide bead type prepared using the same method as the aptamer beads was also included in the microwells to measure the degree of nonspecific binding. The imaging fiber was coupled to a modified epifluorescence microscope system, and the distal end of the fiber was incubated with a fluorescein-labeled thrombin (F-thrombin) solution. Nonlabeled thrombin could be detected using a competitive binding assay with F-thrombin. The aptamer beads selectively bound to the target and could be reused without any sensitivity change. The fiber-optic microarray system has a detection limit of 1 nM for nonlabeled thrombin, and each test can be performed in ca. 15 min including the regeneration time.     

A feasibility study for the use of a silastic gage as an in vivo muscle force transducer

This study investigates the feasibility of utilizing silastic gages for in vivo dynamic muscle force measurement. The gastrocnemius muscle of a fifty-one pound black short hair dog was selected for the test. The study shows that such measurements can be reliably performed in vivo for short durations without interfering with the natural movement of the animal. The durability of the gage appears to be primarily limited by the biological rejection process at the gage site.     

Influence of phosphatidylcholine/cholesterol molar ratios in liposomes on cholesterol reactivity with cholesterol:oxygen oxidoreductase]

The reactivity of sonicated phosphatidylcholine-cholesterol liposomes with cholesterol : oxygene oxydoreductase, an enzyme which catalyses the oxidation of the 3 beta hydroxyl group of cholesterol to a ketone group, is compared with that of ternary system phosphatidylcholine-cholesterol-Thesit. Regardless to the phosphatidylcholines nature and the phosphatidylcholine/cholesterol molar ratio (R), the enzymatic oxidation rate of liposomal cholesterol is slower than when the reaction is developed in the present of Thesit, a surfactif agent which destroyes the lamellar particles. This is true whether Thesit is added during preparation of dispersions or during incubation with cholesterol oxydase. The enzymatic oxydation rate of cholesterol of ternary systems phosphatidylcholine-cholesterol-Thesit is independent of the (R) value and the phosphatidylcholine fatty acid unsaturation, whereas that of phosphatidylcholine-cholesterol dispersions depends on these two parameters. The reaction rate increases in the order: dipalmitoylphosphatidylcholine to yolk egg phosphatidylcholines, and dioleylphosphatidylcholine. The optimal conditions for cholesterol oxidation were found to be R = 0.5. This result is not affected by the phosphatidylcholines nature. In order to explain these data, various hypotheses are considered. In particular, the weak liposomal cholesterol reactivity with cholesterol oxidase could result from an inhibitory effect on the enzyme-substrate combination due to the polar phosphorylcholine groups.     

On the oxygen reactivity of flavoprotein oxidases: an oxygen access tunnel and gate in brevibacterium sterolicum cholesterol oxidase

The flavoprotein cholesterol oxidase from Brevibacterium sterolicum (BCO) possesses a narrow channel that links the active center containing the flavin to the outside solvent. This channel has been proposed to serve for the access of dioxygen; it contains at its "bottom" a Glu-Arg pair (Glu-475-Arg-477) that was found by crystallographic studies to exist in two forms named "open" and "closed," which in turn was suggested to constitute a gate functioning in the control of oxygen access. Most mutations of residues that flank the channel have minor effects on the oxygen reactivity. Mutations of Glu-311, however, cause a switch in the basic kinetic mechanism of the reaction of reduced BCO with dioxygen; wild-type BCO and most mutants show a saturation behavior with increasing oxygen concentration, whereas for Glu-311 mutants a linear dependence is found that is assumed to reflect a "simple" second order process. This is taken as support for the assumption that residue Glu-311 finely tunes the Glu-475-Arg-477 pair, forming a gate that functions in modulating the access/reactivity of dioxygen.     

Bacteria-degraders as the base of an amperometric biosensor for detection of anionic surfactants

Several strains belonging to genera Pseudomonas and Achromobacter and characterized by the ability to degrade anionic surfactants were tested as potential bases of microbial biosensors for surfactant detection. For each strain the substrate specificity and stability of sensor signals were studied. The total amount of the substrates tested (including carbohydrates, alcohols, aromatics, organic acids, etc.) was equal to 60; the maximal signals were observed towards the anionic surfactants. The lower limit of detection for sodium dodecyl sulfate used as a model surfactant was in the field of 1 microM for all the strains. The created microbial biosensor model can extend the practical possibilities for rapid evaluation of surfactants in water media.     

Characterisation of an antibody coated microcantilever as a potential immuno-based biosensor

In this study, we investigated the activity, stability, lifetime and re-usability of monoclonal antibodies to myoglobin covalently immobilised onto microfabricated cantilever surfaces. These sensing surfaces are of interest to us in the development of novel cantilever-based immunosensors. For such sensors the antibody layer represents the sensing element while the microcantilever acts as a mechanical transducer. A procedure for producing re-usable biological coatings has been tested with different independent techniques. An Enzyme Linked Immunosorbent Assay (ELISA) was used to determine the presence of an active antibody coating, and to monitor the lifetime and stability of the immobilised antibody. Through this analysis, the activity of the immobilised antibody layer was found to be more stable with the introduction of sucrose, as a stabilising agent. Sucrose was applied to the immobilised antibody layer after each regeneration step. The immobilised antibody was found to have a stable active lifetime for up to 7 weeks. Fluorescence microscopy was used to give information on the distribution of the coating on the gold and silicon nitride sides of the cantilever. Atomic Force Microscopy was used to determine the presence of the biological coating on the cantilever and to obtain information on the surface morphology of the biological element of the sensor. The combined results provide valuable information on the development of an optimised sensing element and demonstrate a set of methods to use for future sensor-to-sensor characterisation. Preliminary experimental results showing the antibody activity against myoglobin, detected with a microcantilever based sensor prototype confirmed the motivations and potentialities of the proposed immunosensing technique.     

Long-period gratings in photonic crystal fiber as an optofluidic label-free biosensor

Using long-period gratings (LPG) inscribed in photonic crystal fiber (PCF) and coupling this structure with an optically aligned flow cell, we have developed an optofluidic refractive index transduction platform for label-free biosensing. The LPG-PCF scheme possesses extremely high sensitivity to the change in refractive index induced by localized binding event in different solution media. A model immunoassay experiment was carried out inside the air channels of PCF by a series of surface modification steps in sequence that include adsorption of poly(allylamine hydrochloride) monolayer, immobilization of anti-rat bone sialoprotein monoclonal primary antibody, and binding interactions with non-specific goat anti-rabbit IgG (H+L) and specific secondary goat anti-mouse IgG (H+L) antibodies. These adsorption and binding events were monitored in situ using the LPG-PCF by measuring the shift of the core-to-cladding mode coupling resonance wavelength. Steady and significant resonance changes, about 0.75 nm per nanometer-thick adsorbed/bound bio-molecules, have been observed following the sequence of the surface events with monolayer sensitivity, suggesting the promising potential of LPG-PCF for biological sensing and evaluation.     

Silica sol-gel composite film as an encapsulation matrix for the construction of an amperometric tyrosinase-based biosensor

An amperometric tyrosinase enzyme electrode for the determination of phenols was developed by a simple and effective immobilization method using sol-gel techniques. A grafting copolymer was introduced into sol-gel solution and the composition of the resultant organic-inorganic composite material was optimized, the tyrosinase retained its activity in the sol-gel thin film and its response to several phenol compounds was determined at 0 mV vs. Ag/AgCl (sat. KCl). The dependences of the current response on pH, oxygen level and temperature were studied, and the stability of the biosensor was also evaluated. The sensitivity of the biosensor for catechol, phenol and p-cresol was 59.6, 23.1 and 39.4 microA/mM, respectively. The enzyme electrode maintained 73% of its original activity after intermittent use for three weeks when storing in a dry state at 4 degrees C.