Advancing the Development of Glycated Protein Biosensing Technology

Abstract

Research advances in biochemical molecules have led to the development of convenient and reproducible biosensing molecules for glycated proteins, such as those based on the enzymes fructosyl amino acid oxidase (FAOX) or fructosyl peptide oxidase (FPOX). Recently, more attractive biosensing molecules with potential applications in next-generation biosensing of glycated proteins have been aggressively reported. We review 2 such molecules, fructosamine 6-kinase (FN6K) and fructosyl amino acid-binding protein, as well as their recent applications in the development of glycated protein biosensing systems. Research on FN6K and fructosyl amino acid-binding protein has been opening up new possibilities for the development of highly sensitive and proteolytic-digestion-free biosensing systems for glycated proteins.

Electrochemical sensing system employing fructosamine 6‐kinase enables glycated albumin measurement requiring no proteolytic digestion

“…To determine the reactivity of FN6K against intact GA, the colorimetric measurement system coupling pyruvate kinase, pyruvate oxidase and peroxidase (Fig. A) which was previously reported as novel enzymatic sensing for ε‐FK was applied to GA. In the colorimetric measurement system, the concentration of each enzyme was determined based on a preliminary investigation using ε‐FK as the substrate: the increase of the FN6K, pyruvate kinase and pyruvate oxidase concentration in the reaction mixture resulted in the increase of absorbance change.…”

“…Enzymatic activity of purified FN6K was measured colorimetrically by coupling with pyruvate kinase, pyruvate oxidase, and peroxidase as previously described . One unit is defined as the enzyme quantity able to phosphorylate 1 μmol of substrate per minute under the defined reaction conditions.…”

“…To develop novel diagnostic kits for monitoring HbA1c or GA in patients with diabetes, our group has been engaged in the development of a variety of molecules and principles for glycated protein biosensing. These include fructosyl amino acid/peptide oxidase (FAOX/FPOX) , engineered fructosyl amino acid/peptide oxidases , molecularly imprinted polymers , and fructosyl amino acid binding proteins . Sensing systems based on FAOXs/FPOXs are commercially available and have been gradually penetrating into clinical practice because they are rapid, reproducible, and suitable for automated analyzers.…”

“…Fructosamine kinases, which recognize glycated proteins and glycated amino acids have been reported . The kinases are divided into two types – fructosamine 3‐kinase and fructosamine 6‐kinase (FN6K) – according to the catalytic mechanism . Fructosamine 3‐kinases, found only in mammals, cleave fructosamines via two consecutive reactions .…”

“…We previously reported that the enzymatic activity of FN6Ks for ε‐FK can be measured colorimetrically by coupling with pyruvate kinase, pyruvate oxidase, and peroxidase reactions and detecting quinoneimine dye, as shown in Fig. . The application of this sensing scheme for the detection of GA is expected to yield a novel sensing system which is rapid, reproducible, and suitable for automated analyzers.…”