Alexander Butlerov Institute of Chemistry, Kazan Federal University, 18 Kremlevskaya Street, Kazan 420008, Russia.
Analytical Chemistry Department, Chemical Technology Institute, Ural Federal University, 19 Mira Street, Ekaterinburg 620002, Russia.
Biosensors (Basel). 2024 Feb 23;14(3):120. doi: 10.3390/bios14030120.
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 -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, 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.
一种用于测定尿酸的流通式生物传感器系统是在由聚乳酸（PLA）通过 3D 打印制造的流通式电化学池平台上开发的，该平台配备了经过修饰的丝网印刷石墨电极（SPE）。尿酸酶被固定在可更换的反应室的内表面上。与之前报道的类似电池相比，其工作体积减少到 10μL。SPE 是在不与酶反应器接触的情况下用碳黑、[5]轮烷、基于叔丁基硫杂杯[4]芳烃（PAMAM-杯芳烃树状大分子）平台的聚（酰胺-胺）树状大分子和电聚合的 3,7-双（4-氨基苯基氨基）吩噻嗪-5-氯化物进行修饰的。将 PAMAM-杯芳烃树状大分子引入电极涂层中，导致电活性聚合物的氧化还原电流增加了五倍。结果表明，较高代数的 PAMAM-杯芳烃树状大分子导致测量电流的增加更大。涂层由与实施的[5]轮烷和 PAMAM-杯芳烃树状大分子的吩噻嗪电聚合产物组成，在尿酸酶氧化生成的过氧化氢的电化学还原中表现出高效。涂层中存在 PAMAM-杯芳烃树状大分子 G2 使与尿酸测定相关的氧化还原信号增加了 1.5 倍以上。该生物传感器系统成功地应用于酶法在计时安培模式下测定尿酸。在流通条件下建立了用于尿酸酶的酶促测定的最佳参数：pH 8.0，流速 0.2 mL·min，每个反应器 5 U 的尿酸酶。在这些条件下，该生物传感器系统能够在 10 nM 至 20 μM 的尿酸范围内进行测定，检测限（LOD）为 4 nM。葡萄糖（高达 1 mM）、多巴胺（高达 0.5 mM）和抗坏血酸（高达 50 μM）对生物传感器向尿酸的信号没有影响。该生物传感器在加标人工尿液样品上进行了测试，对十倍稀释的样品回收率为 101%。流通池的组装简单且更换部件成本低廉，为该生物传感器系统在常规临床分析中的未来应用提供了广阔的前景。
