Catalytic Pathway of Nanozyme "Artificial Peroxidase" with 100-Fold Greater Bimolecular Rate Constants Compared to Those of the Enzyme.

We report on the kinetic mechanism of the catalytically synthesized Prussian Blue nanoparticles denoted as "artificial peroxidase". In contrast to the enzyme, whose active site first interacts with hydrogen peroxide forming the so-called Compound I, in the case of the nanozymes, H2O2 oxidizes their complex with reducing substrate. Slow release of the product (oxidized form of the latter) from the nanozymes has been registered. The interaction of substrates with the nanozymes is 100 times faster than with enzyme peroxidases, and the rate-limiting constant for the nanozymes is also 2 orders of magnitude greater: for pyrogallol k2 = 1.3 ± 0.1 × 108 M-1 s-1 and for ferrocyanide k2 = 1.9 ± 0.1 × 108 M-1 s-1. Thus, the discovered novel advantage of nanozymes over the corresponding enzymes is the 100-fold greater bimolecular rate constants, resulting, most probably, from their uniformly accessible surface, avoiding the effect of rotation on the diffusion-controlled rate.

Wearable non-invasive monitors of diabetes and hypoxia through continuous analysis of sweat.

We present here wearable devices for continuous monitoring of diabetes and hypoxia based on continuous analysis of sweat. To induce sweating the clinically relevant procedure (pilocarpine electrophoresis) is used. Being a sufficient requirement for diagnostics, positive correlations in variation rates between glucose and lactate concentrations in sweat and the corresponding values in blood are shown. Continuous monitoring of human condition is possible only with the use of flow-through wearable devices providing a delivery of sweat to the biosensor almost immediately after secretion. Evaluating blood glucose through continuous sweat analysis upon glucose tolerance test, we clearly show that diabetics can actually be monitored reliably via non-invasive approach.

'Artificial peroxidase' nanozyme - enzyme based lactate biosensor.

In contrast to bienzyme biosensors, we propose the nanozyme-enzyme based ones substituting the enzyme peroxidase with the more active and stable nanoparticles "artificial peroxidase". The use of catalytically synthesized Prussian Blue based nanozymes simplifies assembling of hydrogen peroxide transducer providing its higher sensitivity. For immobilization of lactate oxidase the composite alkoxysilane - perfluorosulfonated ionomer (PFSI) membranes are proposed achieving the significantly improved operating stability. The resulting nanozyme-enzyme lactate biosensor displays twice higher sensitivity (>0.2 A M-1 cm-2) compared to the Prussian Blue film based one. Nanozymes "artificial peroxidase" are expected to find wide use in elaboration of oxidase based biosensors.

Constant Potential Amperometric Flow-Injection Analysis of Ions and Neutral Molecules Transduced by Electroactive (Conductive) Polymers.

We first report on constant potential (dc) amperometric flow-injection analysis (FIA) transduced by electroactive (conductive) polymers. Amperometric response is caused by the polymer recharging in order to maintain the electrode potential at a constant level when (i) ions are crossing the film|solution interface and polarizing electrode|film interface or (ii) ions or neutral molecules are specifically interacting with the polymer recharging it. The response under constant solution flow is a current peak and in flow-injection mode is a couple of current peaks directed opposite of the first sharp, analytically valuable peak. In both constant flow and flow-injection regimes, the peak current is dependent on analyte concentrations; obviously, the FIA mode provides more advantageous analytical characteristics. Constant potential amperometric flow-injection analysis is shown for boronate- and sulfate-functionalized polyanilines as well as for Prussian Blue, a member of the inorganic polymer family. As a proof of concept, the successful dc amperometric detection of lactate in human sweat with boronate-functionalized polyaniline has been shown. The proposed approach would revolutionize the field of conductive/electroactive polymer-supported ion sensing with the introduction of reliable and robust amperometry as a valuable alternative to existing potentiometry.

Noninvasive Diabetes Monitoring through Continuous Analysis of Sweat Using Flow-Through Glucose Biosensor.

We propose monitoring of diabetes through continuous analysis of undiluted sweat immediately after its excretion using a flow-through glucose biosensor. The used biosensors are based on Prussian Blue and glucose oxidase immobilized in perfluorosulfonated ionomer or gel of alkoxysilane; the resulting sensitivity with the latter reaches in batch mode 0.23 A M-1 cm-2, and the calibration range is from 1 µM to 1 mM (flow-through mode). On the basis of the glucose tolerance test known to be a clinically relevant procedure to mimic hyperglycemia, a positive correlation between the rates of glucose concentration increase in blood and in noninvasively collected sweat has been observed ( r = 0.75). The observed correlation between sweat and blood considering low-molecular weight metabolites is even better than that observed previously between capillary and vein blood, confirming diagnostic value of sweat for diabetes monitoring. The dynamics of sweat glucose concentration, recorded by means of the proposed biosensor, is in a good accordance with the dynamics of blood glucose content without any time delay, thus offering a prospect for noninvasive monitoring of diabetes.

Catalytically Synthesized Prussian Blue Nanoparticles Defeating Natural Enzyme Peroxidase.

We synthesized Prussian Blue (PB) nanoparticles through catalytic reaction involving hydrogen peroxide (H2O2) activation. The resulting nanoparticles display the size-dependent catalytic rate constants in H2O2 reduction, which are significantly improved compared to natural enzyme peroxidase: for PB nanoparticles 200 nm in diameter, the turnover number is 300 times higher; for 570 nm diameter nanoparticles, it is 4 orders of magnitude higher. Comparing to the known peroxidase-like nanozymes, the advantages of the reported PB nanoparticles are their true enzymatic properties: (1) enzymatic specificity (an absence of oxidase-like activity) and (2) an ability to operate in physiological solutions. The ultrahigh activity and enzymatic specificity of the catalytically synthesized PB nanoparticles together with high stability and low cost, obviously peculiar to noble metal free inorganic materials, would allow the substitution of natural and recombinant peroxidases in biotechnology and analytical sciences.

Nonenzymatic Sensor for Lactate Detection in Human Sweat.

For noninvasive diagnostics of hypoxia, we propose the nonenzymatic sensor based on screen-printed structures with the working surface modified in course of electropolymerization of 3-aminophenylboronic acid (3-APBA) with imprinting of lactate. Impedimetric sensor allows lactate detection in the range from 3 mM to 100 mM with the detection limit of 1.5 mM; response time is 2-3 min. Sensor sensitivity remains unchanged within 6 months of storage unpacked in dry state at a room temperature, which is unachievable for enzyme based devices. Analysis of human sweat with poly(3-APBA) based sensor is possible due to (i) much higher lactate content compared to other polyols and (ii) high sensor selectivity (Klactateglucose < 3 × 10-2). Successful detection of lactate in human sweat by means of the poly(3-APBA) based sensor has been confirmed using the highly specific reference method based on lactate oxidase enzyme (correlation coefficient r > 0.9). The attractive performance characteristics of poly(3-APBA) based enzyme-free sensors justify their future use for noninvasive clinical analysis and sports medicine.

Noiseless Performance of Prussian Blue Based (Bio)sensors through Power Generation.

In contrast to "self-powered" (bio)sensors aiming to generate maximum energy output, we propose the systems with the lowest potential difference between the working and the counter electrodes, which in galvanic mode would provide achievement of the best analytical performance characteristics. Prussian Blue based (bio)sensors known to operate at 0.00 V versus Ag|AgCl reference, in the short-circuit regime generate the current proportional to analyte concentration. Sensitivity and dynamic range of Prussian Blue based (bio)sensors in power generation mode are, respectively, even slightly higher and wider compared to the same (bio)sensors operated in the conventional three-electrode regime powered by a potentiostat. Selectivity of the (bio)sensors in power generation mode is similarly high relative to both oxygen, allowing H2O2 detection by its reduction, and reductants. Among the most important advantages of the proposed power generation mode is an order of magnitude decreased noise compared to performance in a conventional three-electrode setup powered by a potentiostat. Noiseless performances of Prussian Blue based (bio)sensors would open new horizons for electrochemical analysis.

Reagentless polyol detection by conductivity increase in the course of self-doping of boronate-substituted polyaniline.

We report on the novel reagentless and label-free detection principle based on electroactive (conducting) polymers considering sensors for polyols, particularly, saccharides and hydroxy acids. Unlike the majority of impedimetric and conductometric (bio)sensors, which specific and unspecific signals are directed in the same way (resistance increase), making doubtful their real applications, the response of the reported system results in resistance decrease, which is directed oppositely to the background. The mechanism of the resistance decrease is the polyaniline self-doping, i.e., as an alternative to proton doping, an appearance of the negatively charged aromatic ring substituents in polymer chain. Negative charge "freezing" at the boron atom is indeed a result of complex formation with di- and polyols, specific binding. Changes in Raman spectra of boronate-substituted polyaniline after addition of glucose are similar to those caused by proton doping of the polymer. Thermodynamic data on interaction of the electropolymerized 3-aminophenylboronic acid with saccharides and hydroxy acids also confirm that the observed resistance decrease is due to polymer interaction with polyols. The first reported conductivity increase as a specific signal opens new horizons for reagentless affinity sensors, allowing the discrimination of specific affinity bindings from nonspecific interactions.

Noninvasive hypoxia monitor based on gene-free engineering of lactate oxidase for analysis of undiluted sweat.

We report on the Prussian Blue based lactate biosensor with the remarkably increased upper detection limit suitable for analysis of undiluted sweat. Engineering of the enzyme lactate oxidase has been carried out upon its immobilization from water-isopropanol mixtures with the high (90%) content of organic solvent. To decrease the enzyme binding constant, we propose to shield the substrate binding sites in its active center with negatively charged polyelectrolyte. The biosensor made from the optimal mixture (3% γ- aminopropyltriethoxysilane and 5% perfluorosulfonated ionomer) is characterized by the calibration graph, which even in batch mode is shifted for 2 orders of magnitude toward high analyte concentrations as compared to it of lactate sensitive electrode made without Nafion analogue. In flow-injection mode, the biosensor allows lactate detection up to 0.5 M. The biosensor displays stable response for 4 h of continuous operation. The achieved analytical performance characteristics allow the monitoring of lactate content in undiluted sweat. A successful validation of the elaborated flow-through monitor with the integrated biosensor opens new horizons for noninvasive diagnostics of hypoxia-related conditions.

Transition metal hexacyanoferrates in electrocatalysis of H2O2 reduction: an exclusive property of Prussian Blue.

The ability of Prussian Blue, ferric hexacyanoferrate (FeHCF), to sensitively and selectively detect hydrogen peroxide by its reduction in the presence of oxygen is of high importance for analytical chemistry. Success with Prussian Blue (PB) provided an appearance of contradictory reports concerning electrocatalysis of the other transition metal hexacyanoferrates (HCFs) in H2O2 reduction. Investigating thermodynamics of the catalyzed reactions as well as electrochemical properties of the hexacyanoferrates, we are able to conclude that the noniron hexacyanoferrates themselves are completely electrocatalytically inactive, except for a minor electrocatalysis in the opposite reaction, hydrogen peroxide oxidation, registered for NiHCF. Concerning the most important reaction, H2O2 reduction, the observed electrocatalytic activity (by the way, 100 times decreased compared to PB) is due to the presence of FeHCF (Prussian Blue) as defects in the structure of noniron hexacyanoferrates. This finding, considering other unique properties of transition metal HCFs, will provide a systematic search for sensing materials with improved analytical performance characteristics.

Hydrogen peroxide detection in wet air with a Prussian Blue based solid salt bridged three electrode system.

We report on a novel electroanalytical system for hydrogen peroxide (H2O2) detection in humidity or droplets of aerosol, formed by air bubbling through a washing chamber; the resulting flow mimics the exhaled human breath. The system is based on a planar three-electrode structure (with a Prussian Blue based H2O2 transducer modified working electrode) bridged by a solid salt-saturated filament material (filter paper, cotton textile). Respective to the hydrogen peroxide content in the washing valve, the response of the aerosol-sensing system is linear in the concentration range of 0.1-10 µM, which overlaps the generally accepted H2O2 content in exhaled breath condensate (EBC), with the sensitivity of 8 A M(-1) cm(-2). The response to the upper limit of the calibration range is stable for more than 50 injection cycles recorded within 3 days. Both the stability and the suitable calibration range allow one to consider the reported aerosol-sensing system as a prototype for a simple (avoiding intermediate EBC collection) noninvasive diagnostic tool for pulmonary patients.

Reagentless biosensor based on glucose oxidase wired by the mediator freely diffusing in enzyme containing membrane.

Wiring glucose oxidase in the membrane with an immobilized mediator is possible due to the diffusion ability of the latter, if the enzyme containing membrane is formed according to the proposed protocol, including exposing proteins to water-organic mixtures with the high content of organic solvent. In the course of the study, the new glucose oxidase mediator, unsubstituted phenothiazine, was discovered. The diffusion coefficient of the mediator in the resulting membrane is independent of the presence of enzyme. The cyclic voltammograms of the enzyme electrode after appearance of the only glucose in solution obtain a well-defined catalytic shape, which is normally observed for both the enzyme and the mediator in solution. Analytical performances of the resulting biosensor are comparable to the advanced second generation ones, which, however, require covalent linking of the mediator either to the membrane forming polymer or to the enzyme. Even without such covalent linking, the reported biosensor is characterized by an appropriate long-term operational stability allowing reagentless sensing.

Sol-gel immobilization of lactate oxidase from organic solvent: toward the advanced lactate biosensor.

We report on the novel protocol for enzyme immobilization into gel of siloxanes using water-organic mixtures with the high content of organic solvent as a reaction medium. Hydrolysis of alkoxysilanes carried out without excessive dilution with water resulted in more active and stable enzyme containing membranes. Immobilization of an inherently labile lactate oxidase according to the proposed sol-gel protocol over Prussian Blue modified electrode resulted in an advanced lactate biosensor characterized with a sensitivity of 0.18 A M(-1) cm(-2) in the flow injection analysis (FIA) mode over a wide dynamic range. A comparison with the known sensors has shown that analytical performances of the elaborated lactate biosensor are advantageous over both published systems and commercialized devices. The biosensor shows an appropriate stability and is suitable for clinical analysis (including noninvasive diagnostics) and food quality control.

Kinetic approach for evaluation of total antioxidant activity.

We propose a novel approach for assessment of total antioxidant activity by monitoring kinetics of hydrogen peroxide (H(2)O(2)) scavenging after its injection into liquid sample under study. H(2)O(2) is known to be the strongest oxidant, really presented in human body in contrast to the majority of the model oxidative systems used for evaluation of antioxidant activity. In addition, kinetic approach, being more informative than the commonly used determination of the final product, obviously provides better discrimination of potential antioxidants. Prussian Blue based sensor due to its high sensitivity and operational stability allowed to monitor kinetics of hydrogen peroxide consumption in turbid and colored samples. The pseudo-first order kinetic constants of hydrogen peroxide scavenging in the presence of different food additives correlated with total antioxidant activity of these samples evaluated via standard procedure based on lipid peroxidation. However, in contrast to the standard method, the proposed kinetic approach is expressed and does not require fresh biological tissues.

Electropolymerized flavin adenine dinucleotide as an advanced NADH transducer.

Electropolymerizing the prosthetic group (flavin adenine dinucleotide, FAD) responsible in the active sites of dehydrogenases for NAD(+)|NADH regeneration, we succeeded in mimicking enzyme activity. Poly(FAD) characterized by an additional polymer-type redox reaction has been discovered as a highly effective electrocatalyst for NADH oxidation: operating at the lowest potentials reported for NADH transducers (0.00 V, pH 7.4), poly(FAD) is characterized by the electrochemical rate constant of 1.8 +/- 0.6 x 10(-3) cm s(-1), which is at the level of the NADH mass-transfer constant. Flow injection analysis of NADH with the poly(FAD)-modified wall-jet electrode as a detector has been characterized by a linear calibration range prolonged down to 5 x 10(-7) M and a sensitivity of 0.08 A M(-1) cm(-2), which taking into account the dispersion coefficient ( approximately 3), is at the diffusion-limiting value. In contrast to the low molecular weight mediators able to exhibit similar electrocatalytic properties, poly(FAD)-modified electrodes are characterized by the dramatically improved stability and, thus, can be considered as the most advantageous NADH transducers for analytical chemistry.

Prussian blue based nanoelectrode arrays for H(2)O(2) detection.

We propose to form nanoelectrode arrays by deposition of the electrocatalyst through lyotropic liquid crystalline templates onto inert electrode support. Whereas Prussian Blue is known to be a superior electrocatalyst in hydrogen peroxide reduction, carbon materials used as electrode support demonstrate only a minor activity. We report on the possibility for nanostructuring of Prussian Blue by its electrochemical deposition through lyotropic liquid crystalline templates, which is noticed from atomic force microscopy images of the resulting surfaces. The resulting Prussian Blue based nanoelectrode arrays in flow injection analysis mode demonstrate a sub-part-per-billion detection limit (1 x 10(-)(8) M) and a linear calibration range starting exactly from the detection limit and extending over 6 orders of magnitude of H(2)O(2) concentrations (1 x 10(-)(8) to 1 x 10(-)(2) M), which are the most advantageous analytical performances in hydrogen peroxide electroanalysis.

Electrochemical transducers based on surfactant bilayers for the direct detection of affinity interactions.

The simple methods for the preparing of direct affinity sensors are proposed. The proposed method consists of the immobilizations of either oligonucleotide or antibodies as recognizing elements onto the surfactant bilayer. For DNA-sensor we propose to immobilize oligonucleotide by spontaneous infiltration of hydrocarbon chain bound to oligonucleotide pentadecathymidylate (dT(15)) into the hydrophobic region of surfactant bilayer. The adsorption of antibodies on bilayer surface has resulted in immunosensor development. The direct detection of affinity interactions in both cases has been investigated by impedance spectroscopy. At both studies the significant changes in impedance spectra have observed. The dynamics of response manifestation have been followed the specific DNA-coupling causing the decrease of real part of impedance, whereas the antibody-antigen interaction caused the increase of real part. The obtained results are promising for the development of impedimetric affinity sensors for clinical or environmental applications.