A flow-through biosensor system for the determination of uric acid was developed on the platform of flow-through electrochemical cell manufactured by 3D printing from poly(lactic acid) and equipped with a modified screen-printed graphite electrode (SPE). Uricase was immobilized to the inner surface of a replaceable reactor chamber. Its working volume was reduced to 10 μL against a previously reported similar cell. SPE was modified independently of the enzyme reactor with carbon black, pillar[5]arene, poly(amidoamine) dendrimers based on the p-tert-butylthiacalix[4]arene (PAMAM-calix-dendrimers) platform and electropolymerized 3,7-bis(4-aminophenylamino) phenothiazin-5-ium chloride. Introduction of the PAMAM-calix-dendrimers into the electrode coating led to a fivefold increase in the redox currents of the electroactive polymer. It was found that higher generations of the PAMAM-calix-dendrimers led to a greater increase in the currents measured. Coatings consisted of products of the electropolymerization of the phenothiazine with implemented pillar[5]arene and PAMAM-calix-dendrimers showing high efficiency in the electrochemical reduction of hydrogen peroxide that was formed in the enzymatic oxidation of uric acid. The presence of PAMAM-calix-dendrimer G2 in the coating increased the redox signal related to the uric acid assay by more than 1.5 times. The biosensor system was successfully applied for the enzymatic determination of uric acid in chronoamperometric mode. The following optimal parameters for the chronoamperometric determination of uric acid in flow-through conditions were established: pH 8.0, flow rate 0.2 mL·min⁻¹, 5 U of uricase per reactor. Under these conditions, the biosensor system made it possible to determine from 10 nM to 20 μM of uric acid with the limit of detection (LOD) of 4 nM. Glucose (up to 1 mM), dopamine (up to 0.5 mM), and ascorbic acid (up to 50 μM) did not affect the signal of the biosensor toward uric acid. The biosensor was tested on spiked artificial urine samples, and showed 101% recovery for tenfold diluted samples. The ease of assembly of the flow cell and the low cost of the replacement parts make for a promising future application of the biosensor system in routine clinical analyses.

Simple and reliable electrochemical sensors are highly demanded in medicine and pharmacy for the fast determination of metabolites and biomarkers of diseases. In this work, a flow-through biosensor system was developed on the base of a screen-printed carbon electrode modified with pillar[3]arene[2]quinone and ferrocene implemented in carbon black. The modification was performed in a single step and resulted in the formation of a stable layer with good operation characteristics. Uricase was immobilized on the inner walls of a replaceable reactor by carbodiimide binding. A flow-through cell was manufactured by 3D printing from poly(lactic acid). The flow-through system was first optimized on the hydrogen peroxide assay and then used for the determination of 1 nM–0.1 mM uric acid (limit of detection 0.3 nM, 20 measurements per hour). Implementation of ferrocene resulted in a synergetic increase in the cathodic current of H₂O₂ reduction measured by flow switching in chronoamperometric mode. The developed system was tested on the determination of uric acid in artificial urine and Ringer–Locke solution and showed a recovery rate of 96–112%. In addition, the possibility of determination of H₂O₂ in commercial disinfectants was shown. Easy assembly, fast and reliable signal and low consumption of the reagents make the system developed attractive for routine clinical analysis of metabolites.

Electrochemical enzyme biosensors are extensively utilized in clinical analysis and environmental monitoring, yet achieving effective enzyme immobilization while maintaining high activity remains a challenge. In this work, we developed a flow-through enzyme biosensor system using a 3D-printed flow-through electrochemical cell fabricated from commercially available poly (lactic acid). After modification with thiacalixarene-functionalized oligo (lactic acids) (OLAs), the material enabled efficient immobilization of uricase on the inner surface of a replaceable reactor of the cell. Swelling and hydrolytic stability of OLAs in cone, partial cone, and 1,3-alternate conformations were studied, with 1,3-alernate conformation demonstrating superior stability and enzyme immobilization performance. The use of OLAs enhanced immobilization efficiency by over 30% and protected the reactor from swelling, hydrolytic degradation, and enzyme loss. The biosensor was validated for amperometric uric acid determination, with a screen-printed carbon electrode modified with carbon black and Prussian Blue. This modification reduced the cathodic potential for uric acid detection to –0.05 V. The biosensor exhibited a linear detection range of 10 nM to 30 μM with a detection limit of 7 nM, and it performed effectively in artificial urine and synthetic blood plasma. The novel cell design, featuring easy assembly and low-cost replaceable parts, makes this biosensor a promising candidate for routine clinical analysis and other practical applications.

The approaches to the synthesis of newly obtained pillar[3]arene[2]hydroquinone and its derivative with functional ammonium substituents (P[3]A[2]HQae) are suggested. The structures of the macrocycles synthesized were confirmed and characterized by a complex of physical methods. P[3]A[2]HQae has found its application as an electron transfer mediator for the determination of dopamine and ascorbic acid with the voltammetric sensor developed. For a successful sensor assembly the glassy carbon electrode (GCE) was modified with carbon black (CB), P[3]A[2]HQae and electropolymerized form of Neutral red (polyNR). P[3]A[2]HQae demonstrated better mediator properties compared to unsubstituted pillar[5]arene due to the 5–6-fold increase in the redox currents of Neutral red (NR) during its electropolymerization. Under optimal conditions the sensor allowed performing the determination of dopamine and ascorbic acid in the ranges from 10 nM to 1.0 mM and from 1 nM to 1 mM, respectively. The limits of detection were of 10 nM for dopamine and and 1 nM for ascorbic acid. The polyNR peaks positions after its interaction with the analytes provided the opportunity to discriminate the sensor response towards dopamine and ascorbic acid. The possibility of dopamine and ascorbic acid determination in real drug samples was demonstrated.