Development of an Electrochemical Immunoassay For Determination of Glycated Albumin Using Nanozymes

Abstract

We developed a new nanozyme-based electrochemical immunoassay method for the monitoring of glycated albumin (GA) known to reflect short-term glycaemic levels. For this study, we synthesized urchin-like Pt nanozymes (uPtNZs) and applied them to colorimetric and electrochemical assays for sensitive determination of GA in total human serum albumin (tHSA) using 3,3’,5,5’-tetramethylbenzidine (TMB) and thionine as substrates, respectively. The uPtNZs showed peroxidase-mimic activity in the presence of hydrogen peroxide. Boronic acid (BA)-agarose bead was used to capture GA through specific cis-diol interactions. uPtNZs were modified with GA antibody (GA-Ab) to form sandwich complexes with GA/BA-agarose bead. The amount of Ab-uPtNZ/GA/BA-agarose bead complex increased with increasing percentage of GA in 50 mg/mL tHSA. The colorimetric assay exhibited linearity from 0.02 to 10 % (10 µg/mL – 5 mg/mL) GA with an LOD of 0.02 % (9.2 µg/mL). For electrochemical assay, GA was detected from 0.01 to 20 % (5 µg/mL – 10 mg/mL) with an LOD of 0.008 % (3.8 µg/mL). The recovery values of measured GA in human plasma samples were from 106 to 107 %. These results indicate that electrochemical assay using uPtNZs is a promising method for determining GA. |우리는 단기 혈당 수준을 반영하는 것으로 알려진 GA의 모니터링을 위한 나노자임 기반의 전기화학 면역측정법을 개발했다. 본 연구를 위해 성게 유사 모양인 백금 기반의 나노자임 (uPtNZs)을 합성하여, 총 인간 혈청 알부민 (tHSA)에 있는 GA의 민감한 검출을 위한 3,3,5,5,-tetramethylbenzidine (TMB)와 thionine을 기질로 하는 색도 측정 및 전기화학적 분석법에 적용하였다. uPtNZs는 과산화수소 존재 하에 페록시다아제와 같은 성질을 보였다. 보론산 (BA)-아가로스 비드는 특정 시스-다이올 작용을 통해 GA를 잡는 것에 사용되었으며, uPtNZs는 GA 항체 (GA-Ab)를 수식하여 GA/BA-아가로스 비드와 샌드위치 형태의 복합체 (Ab-uPtNZ/GA/BA-아가로스 비드)를 형성하였다. Ab-uPtNZ/GA/BA-아가로스 비드 복합체의 양은 50 mg/mL tHSA에서 GA 비율이 증가할수록 증가하였다. 색도측정 분석법은 0.02 - 10 % (10 µg/mL – 5 mg/mL) GA 구간에서 선형성을 보였고, 0.0184 % (9.2 µg/mL) 검출 한계를 가졌다. 전기화학적 분석법에서 GA는 0.01 - 20 % (5 µg/mL – 10 mg/mL)에서 선형구간을 보였고, 0.0076 % (3.8 µg/mL) 검출한계를 가졌다. 또한, 인간 혈장 샘플의 GA 회수 값은 106 - 107 % 이었다. 이러한 결과들은 uPtNZs를 이용한 전기화학적 분석법이 GA를 검출하는 유망한 방법임을 보여줬다.; We developed a new nanozyme-based electrochemical immunoassay method for the monitoring of glycated albumin (GA) known to reflect short-term glycaemic levels. For this study, we synthesized urchin-like Pt nanozymes (uPtNZs) and applied them to colorimetric and electrochemical assays for sensitive determination of GA in total human serum albumin (tHSA) using 3,3’,5,5’-tetramethylbenzidine (TMB) and thionine as substrates, respectively. The uPtNZs showed peroxidase-mimic activity in the presence of hydrogen peroxide. Boronic acid (BA)-agarose bead was used to capture GA through specific cis-diol interactions. uPtNZs were modified with GA antibody (GA-Ab) to form sandwich complexes with GA/BA-agarose bead. The amount of Ab-uPtNZ/GA/BA-agarose bead complex increased with increasing percentage of GA in 50 mg/mL tHSA. The colorimetric assay exhibited linearity from 0.02 to 10 % (10 µg/mL – 5 mg/mL) GA with an LOD of 0.02 % (9.2 µg/mL). For electrochemical assay, GA was detected from 0.01 to 20 % (5 µg/mL – 10 mg/mL) with an LOD of 0.008 % (3.8 µg/mL). The recovery values of measured GA in human plasma samples were from 106 to 107 %. These results indicate that electrochemical assay using uPtNZs is a promising method for determining GA.