Haemoglobin A1c (hemoglobin A1c, HbA1c) is an important long-term glycemic control marker for diabetes. The aim of this study was to develop an enzyme flow injection analysis (FIA) system using engineered fructosyl peptide oxidase (FPOx) based on 2.5th generation principle for an HbA1c automated analytical system. FPOx from Phaeosphaeria nodorum (PnFPOx) was engineered by introducing a Lys residue at the R414 position, to be modified with amine reactive phenazine ethosulfate (arPES) in proximity of FAD. The engineered PnFPOx mutant with minimized oxidase activity, N56A/R414K, showed quasi-direct electron transfer (quasi-DET) ability after PES-modification. The FIA system was constructed by employing a PES-modified PnFPOx N56A/R414K and operated at 0 V against Ag/AgCl. The system showed reproducible responses with a linear range of 20–500 μM for both fructosyl valine (FV) and fructosyl valylhistidine (FVH), with sensitivities of 0.49 nA μM⁻¹ and 0.13 nA μM⁻¹, and the detection limits of 1.3 μM and 2.0 μM for FV and FVH, respectively. These results indicate that the enzyme electrochemical FIA system covers the clinical range of HbA1c detection for more 200 consecutive measurements. Protease digested three different levels of HbA1c samples including healthy and diabetic range subjects were also measured with the FIA system. Thus, it will be possible to develop an integrated system consisting of sample pretreatment and sample electrochemical measurement based on an FIA system possessing quasi-DET type PnFPOx.

血红蛋白A1c（血红蛋白A1c，HbA1c）是糖尿病的重要长期血糖控制指标。这项研究的目的是基于HbA1c自动化分析系统的第2.5代原理，使用工程果糖基肽氧化酶（FPOx）开发酶流动注射分析（FIA）系统。Nophaum夜蛾的FPOx（PnFPOx）通过在R414位置引入Lys残基进行工程改造，并在FAD附近用胺反应性吩嗪乙硫酸盐（arPES）进行修饰。经过改造的具有最小氧化酶活性的PnFPOx突变体N56A / R414K，在PES修饰后显示出准直接电子转移（quasi-DET）的能力。通过使用PES改性的PnFPOx N56A / R414K构建FIA系统，并在0 V电压下对Ag / AgCl进行操作。该系统显示出对果糖基缬氨酸（FV）和果糖基缬氨酸组氨酸（FVH）的线性范围为20–500μM的可再现响应，灵敏度分别为0.49 nAμM ^-1和0.13 nAμM ^-1，对于FV和FVH的检测限分别为1.3μM和2.0μM。这些结果表明，酶电化学FIA系统涵盖了200多个连续测量的HbA1c检测的临床范围。还使用FIA系统测量了蛋白酶消化的三种不同水平的HbA1c样品，包括健康和糖尿病范围的受试者。因此，有可能基于具有准DET型PnFPOx的FIA系统开发一个包括样品预处理和样品电化学测量的集成系统。