Selen Ayaz, Teslime Erşan, Yusuf Dilgin, Reşat Apak
{"title":"使用二元酶(乳酸-丙酮酸氧化酶)固定化硅烷化磁铁矿纳米粒子,基于 CUPRAC 试剂的新型比色乳酸生物传感器。","authors":"Selen Ayaz, Teslime Erşan, Yusuf Dilgin, Reşat Apak","doi":"10.1007/s00604-024-06531-w","DOIUrl":null,"url":null,"abstract":"<p><p>A novel optical lactate biosensor is presented that utilizes a colorimetric interaction between H<sub>2</sub>O<sub>2</sub> liberated by a binary enzymatic reaction and bis(neocuproine)copper(II) complex ([Cu(Nc)<sub>2</sub>]<sup>2+</sup>) known as CUPRAC (cupric reducing antioxidant capacity) reagent. In the first step, lactate oxidase (LOx) and pyruvate oxidase (POx) were separately immobilized on silanized magnetite nanoparticles (SiO<sub>2</sub>@Fe<sub>3</sub>O<sub>4</sub> NPs), and thus, 2 mol of H<sub>2</sub>O<sub>2</sub> was released per 1 mol of the substrate due to a sequential enzymatic reaction of the mixture of LOx-SiO<sub>2</sub>@Fe<sub>3</sub>O<sub>4</sub> and POx-SiO<sub>2</sub>@Fe<sub>3</sub>O<sub>4</sub> NPs with lactate and pyruvate, respectively. In the second step, the absorbance at 450 nm of the yellow-orange [Cu(Nc)<sub>2</sub>]<sup>+</sup> complex formed through the color reaction of enzymatically produced H<sub>2</sub>O<sub>2</sub> with [Cu(Nc)<sub>2</sub>]<sup>2+</sup> was recorded. The results indicate that the developed colorimetric binary enzymatic biosensor exhibits a broad linear range of response between 0.5 and 50.0 µM for lactate under optimal conditions with a detection limit of 0.17 µM. The fabricated biosensor did not respond to other saccharides, while the positive interferences of certain reducing compounds such as dopamine, ascorbic acid, and uric acid were minimized through their oxidative removal with a pre-oxidant (NaBiO<sub>3</sub>) before enzymatic and colorimetric reactions. The fabricated optical biosensor was applied to various samples such as artificial blood, artificial/real sweat, and cow milk. The high recovery values (close to 100%) achieved for lactate-spiked samples indicate an acceptable accuracy of this colorimetric biosensor in the determination of lactate in real samples. Due to the increase in H<sub>2</sub>O<sub>2</sub> production with the bienzymatic lactate sensor, the proposed method displays double-fold sensitivity relative to monoenzymatic biosensors and involves a neat color reaction with cupric-neocuproine having a clear stoichiometry as opposed to the rather indefinite stoichiometry of analogous redox dye methods.</p>","PeriodicalId":705,"journal":{"name":"Microchimica Acta","volume":null,"pages":null},"PeriodicalIF":5.3000,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11233342/pdf/","citationCount":"0","resultStr":"{\"title\":\"A new colorimetric lactate biosensor based on CUPRAC reagent using binary enzyme (lactate-pyruvate oxidases)-immobilized silanized magnetite nanoparticles.\",\"authors\":\"Selen Ayaz, Teslime Erşan, Yusuf Dilgin, Reşat Apak\",\"doi\":\"10.1007/s00604-024-06531-w\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>A novel optical lactate biosensor is presented that utilizes a colorimetric interaction between H<sub>2</sub>O<sub>2</sub> liberated by a binary enzymatic reaction and bis(neocuproine)copper(II) complex ([Cu(Nc)<sub>2</sub>]<sup>2+</sup>) known as CUPRAC (cupric reducing antioxidant capacity) reagent. In the first step, lactate oxidase (LOx) and pyruvate oxidase (POx) were separately immobilized on silanized magnetite nanoparticles (SiO<sub>2</sub>@Fe<sub>3</sub>O<sub>4</sub> NPs), and thus, 2 mol of H<sub>2</sub>O<sub>2</sub> was released per 1 mol of the substrate due to a sequential enzymatic reaction of the mixture of LOx-SiO<sub>2</sub>@Fe<sub>3</sub>O<sub>4</sub> and POx-SiO<sub>2</sub>@Fe<sub>3</sub>O<sub>4</sub> NPs with lactate and pyruvate, respectively. In the second step, the absorbance at 450 nm of the yellow-orange [Cu(Nc)<sub>2</sub>]<sup>+</sup> complex formed through the color reaction of enzymatically produced H<sub>2</sub>O<sub>2</sub> with [Cu(Nc)<sub>2</sub>]<sup>2+</sup> was recorded. The results indicate that the developed colorimetric binary enzymatic biosensor exhibits a broad linear range of response between 0.5 and 50.0 µM for lactate under optimal conditions with a detection limit of 0.17 µM. The fabricated biosensor did not respond to other saccharides, while the positive interferences of certain reducing compounds such as dopamine, ascorbic acid, and uric acid were minimized through their oxidative removal with a pre-oxidant (NaBiO<sub>3</sub>) before enzymatic and colorimetric reactions. The fabricated optical biosensor was applied to various samples such as artificial blood, artificial/real sweat, and cow milk. The high recovery values (close to 100%) achieved for lactate-spiked samples indicate an acceptable accuracy of this colorimetric biosensor in the determination of lactate in real samples. Due to the increase in H<sub>2</sub>O<sub>2</sub> production with the bienzymatic lactate sensor, the proposed method displays double-fold sensitivity relative to monoenzymatic biosensors and involves a neat color reaction with cupric-neocuproine having a clear stoichiometry as opposed to the rather indefinite stoichiometry of analogous redox dye methods.</p>\",\"PeriodicalId\":705,\"journal\":{\"name\":\"Microchimica Acta\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":5.3000,\"publicationDate\":\"2024-07-09\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11233342/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Microchimica Acta\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1007/s00604-024-06531-w\",\"RegionNum\":2,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"CHEMISTRY, ANALYTICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Microchimica Acta","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1007/s00604-024-06531-w","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
A new colorimetric lactate biosensor based on CUPRAC reagent using binary enzyme (lactate-pyruvate oxidases)-immobilized silanized magnetite nanoparticles.
A novel optical lactate biosensor is presented that utilizes a colorimetric interaction between H2O2 liberated by a binary enzymatic reaction and bis(neocuproine)copper(II) complex ([Cu(Nc)2]2+) known as CUPRAC (cupric reducing antioxidant capacity) reagent. In the first step, lactate oxidase (LOx) and pyruvate oxidase (POx) were separately immobilized on silanized magnetite nanoparticles (SiO2@Fe3O4 NPs), and thus, 2 mol of H2O2 was released per 1 mol of the substrate due to a sequential enzymatic reaction of the mixture of LOx-SiO2@Fe3O4 and POx-SiO2@Fe3O4 NPs with lactate and pyruvate, respectively. In the second step, the absorbance at 450 nm of the yellow-orange [Cu(Nc)2]+ complex formed through the color reaction of enzymatically produced H2O2 with [Cu(Nc)2]2+ was recorded. The results indicate that the developed colorimetric binary enzymatic biosensor exhibits a broad linear range of response between 0.5 and 50.0 µM for lactate under optimal conditions with a detection limit of 0.17 µM. The fabricated biosensor did not respond to other saccharides, while the positive interferences of certain reducing compounds such as dopamine, ascorbic acid, and uric acid were minimized through their oxidative removal with a pre-oxidant (NaBiO3) before enzymatic and colorimetric reactions. The fabricated optical biosensor was applied to various samples such as artificial blood, artificial/real sweat, and cow milk. The high recovery values (close to 100%) achieved for lactate-spiked samples indicate an acceptable accuracy of this colorimetric biosensor in the determination of lactate in real samples. Due to the increase in H2O2 production with the bienzymatic lactate sensor, the proposed method displays double-fold sensitivity relative to monoenzymatic biosensors and involves a neat color reaction with cupric-neocuproine having a clear stoichiometry as opposed to the rather indefinite stoichiometry of analogous redox dye methods.
期刊介绍:
As a peer-reviewed journal for analytical sciences and technologies on the micro- and nanoscale, Microchimica Acta has established itself as a premier forum for truly novel approaches in chemical and biochemical analysis. Coverage includes methods and devices that provide expedient solutions to the most contemporary demands in this area. Examples are point-of-care technologies, wearable (bio)sensors, in-vivo-monitoring, micro/nanomotors and materials based on synthetic biology as well as biomedical imaging and targeting.