A method for the determination of amitriptyline, a tricyclic antidepressant, in human urine by immunosensors has been developed using supramolecular systems based on hexamolybdenum cluster complexes. These complexes have electrochemical activity and give a stable analytical signal, which was used in the development of amperometric immunosensors. Luminescence and dynamic light scattering methods were used to demonstrate the formation of a supramolecular system of self-organized hexamolybdenum nanoparticles and chitosan molecules. A composite material based on hexamolybdenum cluster complexes in combination with reduced graphene oxide has been developed. The working range of amitriptyline concentrations to be determined by an amperometric immunosensor was 1 × 10–9–1 × 10–4 M, the limit of determination was at a level of 5 × 10–10 M, and the amitriptyline content of urine samples was at a level of (n – 7) × 10–8 M. A comparison of the results of analysis performed using an amperometric immunosensor and a fluorescence polarization immunoassay showed the absence of significant systematic errors. The ability to determine amitriptyline in biological fluids makes it possible to select an optimal therapeutic dose of the drug, that is, to develop approaches to creating personalized medicine.

Biosensor devices that include hybrid nanostructures as transducer surface modifiers meet current requirements for methods of research and determination of drugs, including antidepressants. Here, we consider the features of amperometric monoamine oxidase biosensors based on screen-printed graphite electrodes modified with nanocomposite consisting of C60/cobalt nanoparticles/amino derivative of a second-generation polyether polyol/chitosan in the determination of the tricyclic antidepressant amitriptyline. The best modifier was selected using transmission electron microscopy, scanning electron microscopy, electrochemical impedance spectroscopy, and differential pulse voltammetry. In the biosensor development, the conditions for applying the composite based on cobalt nanoparticles/amino derivative of polyether polyol to the electrode surface were varied: electrochemical deposition, sequential deposition by the layer-on-layer method, and deposition of a mixture. As an analytical signal of the biosensor, we used the peak of the electrochemical oxidation of hydrogen peroxide, which is formed during the enzymatic oxidation of serotonin under the action of monoamine oxidase. The operating principle of the biosensor is based on the inhibitory effect of amitriptyline on the catalytic activity of immobilized monoamine oxidase. For the selected modifier, the determined concentration range of amitriptyline is 1 × 10–4–1 × 10–8 mol/L and the lower limit of the determined contents is 5 × 10–9 mol/L under optimal operating conditions. Comparison of the results of the amitriptyline determination in a pharmaceutical preparation and urine that were obtained using a monoamine oxidase biosensor and the method of fluorescence polarization immunoassay (dilution of the tracer of 1 : 32, dilution of antibodies of 1 : 128, range of working concentrations from 5 × 10–8 to 5 × 10–9 mol/L), which has proven itself in the determination of medicinal substances, confirmed the correctness of the developed method.

Methods using fluorescence polarization analysis and an amperometric immunosensor for the immunochemical determination of the non-steroidal anti-inflammatory drug diclofenac in drugs, artificial urine, and surface water are proposed. High analytical sensitivity was achieved by using promising nanomaterials, in particular, silicon nanoparticles doped with highly luminescent ruthenium complexes and electrochemically active hexarhenium cluster complexes. The relative standard deviation was (0.070. It was shown that the developed procedures for immunochemical determination of diclofenac had a wider range of determined concentrations than other methods.

Предложены способы иммунохимического определения нестероидного противовоспалительного средства диклофенака в лекарственном препарате (таблетках), искусственной моче и поверхностных водах с помощью поляризационного флуоресцентного анализа и амперометрического иммуносенсора. Высокая чувствительность анализа достигается за счет использования перспективных наноматериалов, в частности силикатных наночастиц, допированных высоколюминесцентными комплексами рутения, и гексарениевых кластерных комплексов, обладающих электрохимическими свойствами. Относительное стандартное отклонение не превышает 0,070. Показано, что разработанные варианты иммунохимического определения диклофенака имеют более широкий диапазон определяемых концентраций по сравнению с другими способами.

The present work introduces nanocomposites based on carbon materials of various nature and hexarhenium chalcogenide clusters as surface modifiers of screen-printed graphite electrodes for amperometric immunosensing of the tricyclic antidepressant desipramine. The surface modification of the electrodes by chronoamperometric reduced graphene oxide in combination with chalcogenide hexarhenium clusters or nanodiamonds was developed for the determination of desipramine. The properties of the nanocomposites studied by voltammetry and electrochemical impedance spectroscopy (EIS) were correlated with the roughness parameters of their modified surface associated with the height properties of the irregularities estimated by atomic force microscopy. The composition of the hybrid nanostructures was optimized to provide the following analytical figures of merit for the immunosensors: the range of working concentrations from 1 x 10(-4) to 1 x 10(-9) \M and the lower limit of the determined concentrations of (5-8) x 10(-10) \M. The optimal parameters for sequential injection analysis were obtained at a flow rate of 25 mu l s(-1) and the injected volume of 3125 mu l of the supporting buffer at pH 7.5. It is possible to determine lower concentrations of desipramine equal to (7-9) x 10(-11) M using the proposed immunosensors in the flow mode. The testing of the developed immunosensors was characterized by the determination of the analyte in a pharmaceutical preparation and in urine. The relative standard deviation was less than 0.072.

We developed a procedure for determining tricyclic antidepressant amitriptyline using fluorescence polarization immunoassay against the background of a buffer solution, urine, and breast milk with a relative standard deviation of no more than 5%. Tracers were synthesized by the noncovalent adsorption of amitriptyline on the surface of silicate nanoparticles doped with ruthenium(II) complexes. For the practical application of the proposed method, we selected the optimal concentrations of the tracer and antibodies by dilution. The amounts of reagents were selected based on fluorescence intensity and fluorescence polarization. According to the sensitivity requirements of the tracer, a dilution of 1 : 2 was selected, the fluorescence intensity of which was ten times higher than the background value, while the optimal fluorescence polarization value was fixed at 32. The optimal dilution of antibodies, corresponding to 70% of the fluorescence polarization value from the maximum signal, was 1 : 200. The range of working concentrations of amitriptyline was 1 × 10 –10 –1 × 10 –5 M; the lower limits of determination were at a level of (7–8) × 10 –11 M. The results are promising for monitoring the concentration of amitriptyline both in the pharmaceutical preparation and biological fluids to determine the effectiveness of ongoing therapeutic treatments and for medical and forensic purposes.

A method of sequential injection determination of tricyclic antidepressants (amitriptyline, desipramine, imipramine) using amperometric immunosensors based on screen-printed graphite electrodes modified by carbon nanomaterials (carbon nanotubes, graphene oxide or fullerene) in combination with hexarhenium chalcogenide clusters is proposed. Sequential injection analysis was performed at a sweep rate of 10 mV/s; the flow rate (supporting electrolyte – phosphate buffer solution with pH 7.0) was also varied from 20 to 40 µl/s. Composition of the modifier affects the analytical capability of immunosensors. Screening of various types of modifiers showed that the immunosensor based on graphene oxide and cyanide complexes of rhenium chalcogenide clusters exhibited the highest sensitivity of 23.9±0.9 with a range of working concentrations of 1·10–10–1·10–5 M, and even wider range of detectable concentrations, 1·10–10–1·10–4 M was observed for the immunosensor based on fullerene and cyanide complexes of rhenium chalcogenide clusters. The lowest limit of the determined contents was (4–7)·10–11 M. Relative standard deviation of repeatability (Sr) did not exceed 0.046. The results obtained show that the proposed immunosensors can be used in sequential injection determination of trace amounts of medicinal compounds (antidepressants) in biomedical objects.

The properties of hexarhenium chalcogenide nanoclusters (K4[{Re6S8}(OH)6]·8H2O and K4[{Re6S8}(CN)6]·8H2O) in combination with carbon nanomaterials (carbon nanotubes and graphene oxide) are studied by voltammetry, electrochemical impedance spectroscopy, atomic force microscopy, and spectrophotometry and their screening is performed for use as hybrid modifiers of screen-printed graphite electrodes in immunosensors in order to improve analytical characteristics. The high negative charge of nanoclusters can be considered the driving force of the adsorption of clusters in the formation of electrodes modified by hybrid nanomaterials. It was found that hexarhenium chalcogenide nanoclusters possess electrochemical activity, which was first used to register immunochemical interactions. The change in the resistance of electron transfer made it possible to choose the best hybrid nanomaterials. The parameters of the surface roughness of the modified electrodes associated with the height properties of the irregularities were estimated. The use of hexarhenium chalcogenide nanoclusters in combination with carbon nanomaterials as hybrid nanomodifiers has made it possible to develop highly sensitive and selective amperometric and impedimetric immunosensors for the determination of tricyclic antidepressants (amitriptyline, desipramine, and imipramine) in pharmaceuticals and urine. The limit of quantification (LOQ) is at the level (4–7) × 10–11 M. The relative standard deviation does not exceed 5%.

Various nanostructured materials (graphene, fullerene C60, carbon nanotubes, and cobalt nanoparticles as a label) are used as nanocomposites to modify the surface of primary signal converters (screen-printed graphite electrode) in the development of amperometric immunosensors for the determination of tricyclic antidepressant amitriptyline. The use of nanomaterials improved the analytical characteristics of the corresponding immunosensors. The range of working concentrations of the immunosensor is 1 × 10–9–1 × 10–4 M, the lower limit of the analytical range is at a level of 5 × 10–10 M. The binding constants of antigen–antibody immune complexes are determined. Immunosensors are tested in the control of contents of medicinal preparations in urine.

Amperometric monoamine oxidase biosensors based on screen-printed graphite electrodes modified with nanostructured reduced graphene oxide (RGO) composites and cobalt nanoparticles (CoNPs) were developed to determine antidepressant drug substances: tianeptine, thioridazine, and fluoxetine. Combinations of carbon nanomaterials with metal nanoparticles (nanocomposites) along with retaining the properties of individual components, also provide a new quality of the developed devices due to their joint contribution. The nanomaterial-modifier was applied to the surface of screen-printed graphite electrodes using dropwise evaporation. Fixing of RGO on the surface of the screen-printed graphite electrodes occurs due to electrostatic interaction between RGO carboxyl groups and amine groups of the amine derivative on the platform of polyester polyol (H20–NH2). The CoNPs were obtained electrochemically by the method of chronoamperometry at a potentialE= – 1.0 V and different time of their accumulation (about 50 – 60 sec) on the electrode surface. According to the data of atomic force microscopy, the predominant size of CoNPs is (40 ± 2) and (78 ± 8) nm, depending on the time of electrochemical deposition of NPs. Data of electrochemical impedance spectroscopy show that nanocomposites RGO-chitosan/CoNPs and RGO-amine derivative on the polyester polyol (H20–NH2)/CoNPs platform are characterized by the lowest values of the charge transfer resistance. The use of those nanocomposites modifying the electrode surface significantly improved the analytical characteristics of the developed biosensors providing a wider range of operating concentrations from 1 × 10–4to 5 × 10–9mol/liter, greater sensitivity coefficient, better correlation coefficient, and lower limit of the detectable concentrations. A possibility of using biosensors to control the quality of antidepressants upon determination of the main active substance in medicinal drugs and biological fluids is shown. The lower limit of detectable concentrations (7 – 9) × 10–10mol/liter is attained when using tyramine as a substrate for determination of fluoxetine, thioridazine and tianeptine, respectively.

Surface modification of screen-printed graphite electrodes with nanostructured materials (multiwall carbon nanotubes, gold and silver nanoparticles) allow their application as supports of amperometric monoaminoxidase biosensors for the determination of antidepressant drugs (moclobemide, tianeptine, and amitriptyline). This approach improves analytical characteristics of the corresponding biosensors because of the inhibitory effect of antidepressants (two-parameter concerted inhibition) on the catalytic activity of an immobilized enzyme. The analytical capabilities of the developed biosensor types were compared. The range of working concentrations was from 5 × 10 –9 to 1 × 10 –4 M and the lower limit of the analytical range was of about 8 × 10 –10 M. Biosensors based on electrodes modified with nanostructured materials were tested in the control of the concentration of drugs in body fluids (urine) and dosage forms.

The use of hyperbranched polyesterpolyols of different generations favors firmer fixation of carbon nanotubes and silver nanoparticles as components of composite materials on the electrode surface (0.028 mg cm –2 ), which improves the operation characteristics of monoamine oxidase biosensors. The size of silver nanoparticles (18–52 nm) depends on the conditions for preparing hyperbranched polyesterpolyols, and their use as electrode modifiers influences the analytical possibilities of amperometric biosensors. Silver nanoparticles (18 nm, data of atomic force microscopy) in polyesterpolyols of third generation (pH 10.0) as components of the developed biosensors extend the interval of determinable concentrations to 1 × 10 –4 –1 × 10 –8 M and decrease the lower limit of determination to 3 × 10 –9 M, compared to the unmodified sensors, owing to enhancement of the analytical signal. The developed biosensors were tested in monitoring of drugs (antidepressants) in Coaxil and Auroriks drug forms with the relative standard deviation on the level of 0.052.

Modification of a printed graphite electrode surface by carbon nanomaterials (carbon nanotubes and graphene oxide) was used to improve the analytical capabilities of amperometric monoamine oxidase biosensors in the determination of medicinal substances with antidepressant activity (moclobemide and amitriptyline). It was found that the range of determined concentrations of pharmaceutical agents varied from 1 × 10–4 to 1 × 10–8 mol/L. The developed monoamine oxidase biosensors were used for determination of medicinal agents in their dosage forms.

Effect of multi-walled carbon nanotubes and silver nanoparticles as surface modifiers of screen-printed graphite electrodes, the basis of amperometric monoamine oxidase biosensors, on their analytical characteristics was studied. The presence of carbon nanotubes and silver nanoparticles was confirmed by UV spectroscopy and scanning electron microscopy. The use of the oxidation current of hydrogen peroxide, the product of oxidative deamination of biogenic amines in the presence of immobilized monoamine oxidase as a representative of the class of oxidoreductases, as the analytical signal made it possible to choose the synthesis method and to suggest ways to deposit the modifiers under study on the electrode surface. The curve of the inhibiting effect of tricyclic antidepressants on the catalytic activity of the immobilized enzyme on the solution pH and amount of modifiers was considered. It was found that the lower limit of the determinable concentrations decreases to a level of 4 × 10−9 M, the sensitivity coefficient is improved, and the range of determinable concentrations of amitryptiline and imipramine is extended to 1 × 10−4–1 × 10−8 M. It is shown that the suggested biosensors can be used to monitor the residual amounts of medical substances in urine.

Amperometric biosensors based on planar screen-printed graphite electrodes modified with multiwall carbon nanotubes (MWCNTs) and immobilized monoamine oxidase enzyme (MAO) have been proposed for the determination of antidepressants (imipramine, afobazole, and phenazepam). The operation of the proposed biosensors is based on the inhibiting ability of antidepressants. The analytical capabilities of the proposed devices have been compared to those of biosensors based on the electrodes unmodified with MWCNTs. The proposed biosensors can be used for the control of both residual amounts of drug substances in biological fluids (urine) and the active ingredient in dosage forms.

Novel monoamine oxidase amperometric biosensors based on screen-printed graphite electrodes modified with nanostructured material graphene oxide ( GO ) was developed for the determination of antidepressants («Melipraminum», «Coaxil» and «Fenazepam»). Response of the biosensor created is based on a combination of monoamine oxidase biochemical action towards biogenic amines (dopamine, serotonin) and electrochemical oxidation of hydrogen peroxide (a product of the enzymatic reaction) as well as the inhibition effect of the studied antidepressants of the immobilized enzyme. Тhe analytic signal is hydrogen peroxide oxidation current at potentials 0.7-0.75 V. The usage of chitosan acetate solution as a dispersant for carbon nanotubes and graphene oxide provides a more pronounced analytical biosensor signals compared to suspension in DMF. The antidepressants under investigation were determined within the wide range concentration of 1·10 -4 - 1·10 -8 M. The analytical capabilities of the GO-based biosensor with biosensor modified with carbon nanotubes in chitosan have been compared. GO-modified biosensor had certain advantages over biosensors modified with carbon nanotubes, in particular the higher sensitivity coefficient and lower detection limit. The application of acid-base titration as a reference method for determination on example of imipramine has shown the absence of systematic error in measurements using the developed biosensors. The biosensors developed can be applied for the control for residual amounts of drugs in biological fluids (urine) at 9·10 -9 M as well as contents of active substance in pharmaceutical dosage forms. Keywords: biosensor, graphene oxide, carbon nanotubes, chitosan, antidepressants, monoamine oxidase, biological fluid (Russian) DOI: http://dx.doi.org/10.15826/analitika.2014.18.4.011 E. P. Medyantseva, D. V. Brusnitsyn, R. M. Varlamova, R. R. Sitdikova, A.N. Galiavina, G.K. 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