Encyclopedia
Please note this is a comparison between Version 2 by Jason Zhu and Version 1 by Yang LI.
Glycated hemoglobin (HbA1c) is the gold standard for measuring glucose levels in the diagnosis of diabetes due to the excellent stability and reliability of this biomarker. HbA1c is a stable glycated protein formed by the reaction of glucose with hemoglobin (Hb) in red blood cells, which reflects average glucose levels over a period of two to three months without suffering from the disturbance of the outside environment. A number of simple, high-efficiency, and sensitive electrochemical sensors have been developed for the detection of HbA1c. Indirect type electrochemical HbA1c sensors work based on the measurement of FV or FVH, which is a form of enzymatic determination. According to the type of enzymes, indirect sensors are divided into fructosyl amino acid oxidase (FAO) type, fructosyl peptide oxidase (FPOX) type, and molecularly imprinted catalyst (MIC) type sensors. These enzymes usually need to be processed with nanotechnology or imprinting technology to immobilize them on the electrode surface.
- electrochemical sensor
- HbA1c sensor
- Indirect Type
1. Introduction
Diabetes mellitus (DM) is one of the three most harmful non-communicable diseases to humans [1], with an estimated global prevalence of 9.3% (463 million people) in 2019, which is projected to increase to 10.2% (578 million) by 2030 [2]. DM is a group of metabolic diseases associated with high blood glucose [3]. Conventionally, diabetes detection is based on glucose sensing, which is a continuous process, such as impaired fasting glucose (IFG) and impaired glucose tolerance (IGT), and which can easily cause diagnosis errors [4,5]. However, more recently, glycated hemoglobin (HbA1c) has been shown as an index of blood glucose levels in patients in the past 60 to 90 days, and therefore, it could be an excellent biomarker for continuous glucose monitoring. This protein is a stable product of a non-enzymatic reaction of glucose and human hemoglobin (Hb) β-chain N-terminal valine in serum, and its concentration is insensitive to short-term fluctuations in glucose [6,7,8]. Therefore, HbA1c levels reflect the long-term glucose levels of a patient, which can improve diabetes diagnostic accuracy [9] and is crucial for the diagnosis of diabetes [10]. HbA1c levels are defined as the ratio of HbA1c concentration to total hemoglobin concentration and are ~4–6.5% for a normal person, while the clinical reference range for its concentration is 5–20% [4], and the physiological levels range from 3 to 13 mg/mL in human blood samples [5]. In addition, current diagnostic criteria for diabetes include the requirement of monitoring fasting blood glucose or plasma glucose measured 2 h after an oral glucose tolerance test (OGTT). By contrast, HbA1c is more convenient, requiring no preparation, and has the lowest intra-individual variation [11].
Diabetes mellitus (DM) is one of the three most harmful non-communicable diseases to humans [1], with an estimated global prevalence of 9.3% (463 million people) in 2019, which is projected to increase to 10.2% (578 million) by 2030 [2]. DM is a group of metabolic diseases associated with high blood glucose [3]. Conventionally, diabetes detection is based on glucose sensing, which is a continuous process, such as impaired fasting glucose (IFG) and impaired glucose tolerance (IGT), and which can easily cause diagnosis errors [4][5]. However, more recently, glycated hemoglobin (HbA1c) has been shown as an index of blood glucose levels in patients in the past 60 to 90 days, and therefore, it could be an excellent biomarker for continuous glucose monitoring. This protein is a stable product of a non-enzymatic reaction of glucose and human hemoglobin (Hb) β-chain N-terminal valine in serum, and its concentration is insensitive to short-term fluctuations in glucose [6][7][8]. Therefore, HbA1c levels reflect the long-term glucose levels of a patient, which can improve diabetes diagnostic accuracy [9] and is crucial for the diagnosis of diabetes [10]. HbA1c levels are defined as the ratio of HbA1c concentration to total hemoglobin concentration and are ~4–6.5% for a normal person, while the clinical reference range for its concentration is 5–20% [4], and the physiological levels range from 3 to 13 mg/mL in human blood samples [5]. In addition, current diagnostic criteria for diabetes include the requirement of monitoring fasting blood glucose or plasma glucose measured 2 h after an oral glucose tolerance test (OGTT). By contrast, HbA1c is more convenient, requiring no preparation, and has the lowest intra-individual variation [11].
Several clinical methods are currently available for determining the level of HbA1c in bodies, including liquid chromatography [12], electrophoresis [13], affinity chromatography [14], ion exchange chromatography [15], and immunoassays [16]. Although the effectiveness of these methods has been demonstrated in clinical practice, they require expensive and professional equipment, operation by experienced professionals, and complicated testing processes [17]. In contrast, electrochemical methods require no professional equipment or well-trained operators, and the testing processes are simple and quick [18]. Furthermore, using captured biomolecules, proteins, or antibodies to activate the surface of the electrodes and enable repeatable electrical output for HbA1c detection has significant applications in point-of-care testing (POCT) [19,20].
Several clinical methods are currently available for determining the level of HbA1c in bodies, including liquid chromatography [12], electrophoresis [13], affinity chromatography [14], ion exchange chromatography [15], and immunoassays [16]. Although the effectiveness of these methods has been demonstrated in clinical practice, they require expensive and professional equipment, operation by experienced professionals, and complicated testing processes [17]. In contrast, electrochemical methods require no professional equipment or well-trained operators, and the testing processes are simple and quick [18]. Furthermore, using captured biomolecules, proteins, or antibodies to activate the surface of the electrodes and enable repeatable electrical output for HbA1c detection has significant applications in point-of-care testing (POCT) [19][20].
2. Fructosyl Amino Acid Oxidase (FAO) Type
Currently, fructosyl amino acid oxidase (FAO) has been proven to be reproducible [83,84,85] and suitable for simple, convenient, and economical real-time HbA1c detection [85]. The emergence of nanotechnology has led to a new generation of electrochemical biosensors with nanostructured interfaces that enable faster detection with smaller volumes [86]. Currently, a wide range of nanomaterials, such as gold, silver, carbon nanotubes, graphene, and metal oxides, have been successfully applied in biosensors [87]. These materials have excellent properties, such as high biocompatibility, good water dispersibility, and large surface areas [88,89,90]. Doping nanoparticles into normal materials or substrates can improve the resulting electrochemical properties [91].
Utkars et al. [92] formed a stable scaffold with AuNPs and point-like tubular TiO2; 12-phosphotungstic acid was used as a reductant after depositing well-dispersed AuNPs on TiO2 nanotubes, which accelerated electron transfer between proteins and conductors. The response time was only three seconds, and the linear range for HbA1c detection was 0.5–2000 µM. Furthermore, Utkarsh et al. [93] fabricated a fixed nitrogen-doped graphene/AuNP/fluorine-doped tin oxide (FTO) glass electrode based on fructosyl amino acid oxidase, with a half-life of up to four months. This biosensor could detect HbA1c in human whole blood, exhibiting a low detection limit of 0.2 μM.
To further improve the reaction efficiency, Utkarsk et al. [94] synthesized 3D-structured reduced graphene oxide, multi-walled carbon nanotubes, and platinum nanoparticles (PtNPs) to coat an Au electrode. Additional reaction sites were exposed, the response time of the sensor was reduced to less than three seconds, and the linear range was 0.05–1000 µM. This sensor had good repeatability and was successfully used to determine the concentration of HbA1c in human blood samples. Moreover, Utkarsh et al. mixed AuNPs-PtNPs and polyindole-5-carboxylic acid (PIN5COOH) and modified them onto the surface of a gold electrode [95], which showed good storage stability and retained 50% of the initial activity after 12 weeks. The unique characteristics of the two different metal nanoparticles helped improve the sensitivity and specificity of the sensor; its linear detection range for FV was 0.1–1000 µM, and its detection limit was 0.2 µM. Sheetal et al. prepared a film of a ZnO nanoparticle/polypyrrole hybrid [96] and fixed it onto a gold electrode surface with FAO. The sensor had a low detection limit of 0.05 mM, and its linear detection range for FV was 0.1–3 mM. The modified electrodes could be stored for 160 days and be used to analyze human whole blood samples.
Magnetic nanoparticles have drawn considerable research attention as special biomolecular immobilized carriers [97,98]. Sheetal et al. introduced amino groups onto the surface of core-shell magnetic nanoparticles [99]. The sensor was built by immobilizing FAO on the modified nanoparticle surface to achieve high sensitivity. The linear detection range for FV was 0–2 mM, and the detection limit was as low as 0.1 mM. Moreover, this enzyme electrode could be stored for three months and used up to 250 times.
Recently, some semiconductor nanomaterials have been applied in the research of nanometer sensors due to their good stability [100]. Chauhan et al. [101] established a direct, rapid, and sensitive HbA1c sensor, which was constructed by immobilizing FAO onto a ZnO nanorod-modified indium tin oxide (ITO)-coated glass plate electrode. The sensor presented significant sensitivity and detection limit advantages (0.1 µM), a fast response time (4 s), and a wide linear range (0.1–2000 µM). The working electrode was stable for approximately 4 months at 4 °C. This sensor could be used to distinguish HbA1c in blood samples from healthy and diabetic patients.
In addition, Leng et al. fabricated an amperometric biosensor by drop-coating an FAO enzyme onto an SPE surface [102]. The biosensor showed high current output, high linearity, and effectiveness for FV (0–8000 µM), as well as human blood samples. The drawback of the Prussian blue (PB) electrode for indirect HbA1c sensors is that Fe
2. Fructosyl Amino Acid Oxidase (FAO) Type
Currently, fructosyl amino acid oxidase (FAO) has been proven to be reproducible [21][22][23] and suitable for simple, convenient, and economical real-time HbA1c detection [23]. The emergence of nanotechnology has led to a new generation of electrochemical biosensors with nanostructured interfaces that enable faster detection with smaller volumes [24]. Currently, a wide range of nanomaterials, such as gold, silver, carbon nanotubes, graphene, and metal oxides, have been successfully applied in biosensors [25]. These materials have excellent properties, such as high biocompatibility, good water dispersibility, and large surface areas [26][27][28]. Doping nanoparticles into normal materials or substrates can improve the resulting electrochemical properties [29]. Utkars et al. [30] formed a stable scaffold with AuNPs and point-like tubular TiO2; 12-phosphotungstic acid was used as a reductant after depositing well-dispersed AuNPs on TiO2 nanotubes, which accelerated electron transfer between proteins and conductors. The response time was only three seconds, and the linear range for HbA1c detection was 0.5–2000 µM. Furthermore, Utkarsh et al. [31] fabricated a fixed nitrogen-doped graphene/AuNP/fluorine-doped tin oxide (FTO) glass electrode based on fructosyl amino acid oxidase, with a half-life of up to four months. This biosensor could detect HbA1c in human whole blood, exhibiting a low detection limit of 0.2 μM. To further improve the reaction efficiency, Utkarsk et al. [32] synthesized 3D-structured reduced graphene oxide, multi-walled carbon nanotubes, and platinum nanoparticles (PtNPs) to coat an Au electrode. Additional reaction sites were exposed, the response time of the sensor was reduced to less than three seconds, and the linear range was 0.05–1000 µM. This sensor had good repeatability and was successfully used to determine the concentration of HbA1c in human blood samples. Moreover, Utkarsh et al. mixed AuNPs-PtNPs and polyindole-5-carboxylic acid (PIN5COOH) and modified them onto the surface of a gold electrode [33], which showed good storage stability and retained 50% of the initial activity after 12 weeks. The unique characteristics of the two different metal nanoparticles helped improve the sensitivity and specificity of the sensor; its linear detection range for FV was 0.1–1000 µM, and its detection limit was 0.2 µM. Sheetal et al. prepared a film of a ZnO nanoparticle/polypyrrole hybrid [34] and fixed it onto a gold electrode surface with FAO. The sensor had a low detection limit of 0.05 mM, and its linear detection range for FV was 0.1–3 mM. The modified electrodes could be stored for 160 days and be used to analyze human whole blood samples. Magnetic nanoparticles have drawn considerable research attention as special biomolecular immobilized carriers [35][36]. Sheetal et al. introduced amino groups onto the surface of core-shell magnetic nanoparticles [37]. The sensor was built by immobilizing FAO on the modified nanoparticle surface to achieve high sensitivity. The linear detection range for FV was 0–2 mM, and the detection limit was as low as 0.1 mM. Moreover, this enzyme electrode could be stored for three months and used up to 250 times. Recently, some semiconductor nanomaterials have been applied in the research of nanometer sensors due to their good stability [38]. Chauhan et al. [39] established a direct, rapid, and sensitive HbA1c sensor, which was constructed by immobilizing FAO onto a ZnO nanorod-modified indium tin oxide (ITO)-coated glass plate electrode. The sensor presented significant sensitivity and detection limit advantages (0.1 µM), a fast response time (4 s), and a wide linear range (0.1–2000 µM). The working electrode was stable for approximately 4 months at 4 °C. This sensor could be used to distinguish HbA1c in blood samples from healthy and diabetic patients. In addition, Leng et al. fabricated an amperometric biosensor by drop-coating an FAO enzyme onto an SPE surface [40]. The biosensor showed high current output, high linearity, and effectiveness for FV (0–8000 µM), as well as human blood samples. The drawback of the Prussian blue (PB) electrode for indirect HbA1c sensors is that Fe3+
in PB easily reacts with OH
3+
in PB to avoid reactions of Fe
3+
and OH
− in solution. The modified Tris-PB/SPE was applied in the detection of H2O2, presenting a linear range of 0–2000 µM FV.
3. FPOX Type
Compared with other enzymatic HbA1c sensors, FPOX type HbA1c sensors have high specificity because substantial specific measurements of HbA1c can be realized through mutagenesis and modification [104,105]. Shahbazmohammadi et al. immobilized a recombinant engineered FPOX enzyme to specifically hydrolyze FVH on an electrode surface modified by CHIT, graphene oxide (GO), and gold nanoparticles (AuNPs). The biosensor showed a linear response within the range of 0.1–2 mM [104]. In addition, to prove that the FPOX-modified electrode can specifically measure FVH, significant changes in electron transfer resistance were observed after incubation of the FPOX-modified electrode with FVH, but there was no response in the control group, indicating the specific measurement of FVH. Hatada et al. replaced Arg414 with Lys to form the PnFPOX (FPOX from Phaeosphaeria nodorum) N56A/R414K mutant and modified PnFPOX near FAD with amine-reactive phenazine ethosulfate (arPES), which showed quasi-direct electron transferability [106]. This electrode was combined with an enzyme flow injection analysis (FIA) system. The linear range of the system for both FV and FVH was 20–500 μM, and the sensitivities and detection limits of the system were 0.49 nA·μM
in solution. The modified Tris-PB/SPE was applied in the detection of H2O2, presenting a linear range of 0–2000 µM FV.
3. FPOX Type
Compared with other enzymatic HbA1c sensors, FPOX type HbA1c sensors have high specificity because substantial specific measurements of HbA1c can be realized through mutagenesis and modification [42][43]. Shahbazmohammadi et al. immobilized a recombinant engineered FPOX enzyme to specifically hydrolyze FVH on an electrode surface modified by CHIT, graphene oxide (GO), and gold nanoparticles (AuNPs). The biosensor showed a linear response within the range of 0.1–2 mM [42]. In addition, to prove that the FPOX-modified electrode can specifically measure FVH, significant changes in electron transfer resistance were observed after incubation of the FPOX-modified electrode with FVH, but there was no response in the control group, indicating the specific measurement of FVH. Hatada et al. replaced Arg414 with Lys to form the PnFPOX (FPOX from Phaeosphaeria nodorum) N56A/R414K mutant and modified PnFPOX near FAD with amine-reactive phenazine ethosulfate (arPES), which showed quasi-direct electron transferability [44]. This electrode was combined with an enzyme flow injection analysis (FIA) system. The linear range of the system for both FV and FVH was 20–500 μM, and the sensitivities and detection limits of the system were 0.49 nA·μM−1
and 1.3 μM for FV, respectively, and 0.13 nA μM
−1 and 2.0 μM for FVH. The oxidative activity and specificity of PnFPOX for this method are commendable, but the detection range needs to be expanded if this method is to be adopted for clinical measurements of HbA1c. In addition, the PnFPOX electrode was engineered for continuous operation of the FIA system. This case shows that an electrochemical sensor can be combined with an FIA system to develop an integrated measurement system for sample pretreatment and sample electrochemical measurement.
There are also some sensors that modify both FPOX and FAO, which can selectively determine only glycated N-terminal peptides from the β chain without any interference in the presence of glycated peptides from the α chain [107]. Nanjo et al. adopted an FIA system with a flow-type spectrophotometer and electrochemical detector (with FPOX immobilized on amino-alkyl-bonded Uniport C and FAO immobilized on dialdehyde-activated Uniport C) [107]. The total hemoglobin in the sample is determined by spectrophotometry, and then, the FVH released from HbA1c is determined by an electrochemical sensor with FPOX or FAO. As a result, at pH 8.0–8.5, FAO showed high activity against FV but no activity against FVH, and at nearly pH 7.0, FPOX showed the maximum activity of FV and FVH. The FIA system with the FAO reactor showed a linear detection range for FV of 2–200 µM, and the FPOX reactor showed linear detection ranges for FV and FVH of 2–100 µM and 7–110 µM, respectively. However, when blood cell samples were digested by protease, only FVH was released, not FV. Therefore, according to the definition of HbA1c by the International Federation of Clinical Chemistry (IFCC), the combination of protease and FPOX reactor systems can determine only FVH.
4. MIC Type
The molecular imprinting catalyst is the formation of molecular recognition sites in a polymer by performing synthesis in the presence of a target template. Therefore, MIPs can selectively identify target analytes. The use of MIP technology is the most common, stable, scalable, and economical method at present [108,109]. In this technology, a functional monomer is combined with imprinted molecules and fixed by a cross-linking agent, and then, the imprinted molecules are washed away. The goal is to develop a polymer material with high affinity and selectivity to bind the target substance[82,108]. The content of the template monomer used in the preparation process is usually less than 5%, while that of the cross-linking agent is as high as 95% [110].
MIC is an artificial enzyme catalyst. Molecular imprinting technology can be used to construct molecular recognition elements, and the premise of detecting FV by sensor elements based on the enzyme method is to develop a catalytic center, which is used to mimic fructosamine dehydrogenase [81]. Koji et al. found that polyvinyl imidazole (PVI) could be used as an oxidant in the oxidation reaction of FV in the presence of electron receptors under alkaline conditions. Similarly, the same team used a carbon paste electrode to fix PVI and built an amperometric sensor to detect FV, with a linear response range of 0.02–0.7 mM [81]. These results indicated that PVI can be used as a catalyst for oxidizing fructosamine compounds and HbA1c. This team also prepared an artificial fructosyl amine dehydrogenase, a polymer catalyst prepared by copolymerizing allylamine, 1-vinylimidazole, and 4-vinylphenylboronate to improve the selectivity of MIC to FV, showing a typical response curve of amperometric enzyme sensors for FV in the range of 0.2–0.8 mM [111]. Subsequently, the team used a higher concentration of buffer solution to improve the selectivity and sensitivity of MIC to FV by changing the operating conditions [112]. When 100 mM of potassium phosphate buffer were used instead of 10 mM, the sensitivity ratio of FV and Fru-ε-Lys with the sensor increased significantly from 1.9 to 5.7. However, the synthesized MIC was characterized by low flexibility, and most of the active catalyst sites were located inside the polymer. Therefore, the FV oxidation activity of the MIC needs to be improved [113]. Tomohiko proved that changing the flexibility of the polymer chain can improve the FV oxidation activity of the MIC [113]. A more water-soluble polyacrylamide gel with a relatively low cross-linking degree and macroporous structure was used to improve the conformational flexibility of the polymer. This material was fixed on a gold electrode, and an amperometric sensor was prepared that could detect 0.05 to 0.6 mM FV. Therefore, this soluble MIC sensor achieves the detection range required for HbA1c measurement.
In brief, existing indirect type HbA1c detection sensors that work by detecting FV/FVH have several performance advantages. Specifically, the detection time is generally only 2–120 s; the pH value of the measurement system is near that of human physiology (7.0–7.5). The linear detection range of FV by indirect sensors based on nanotechnology can reach 3000 µM at most, and the detection limit can reach at least 0.05 µM. The linear detection range of FV by indirect sensors based on imprinting technology can reach 800 µM, and the minimum detection limit is 50 µM. Due to the unique properties of nanomaterials, including adjustable chemical properties, large surface-to-volume area, strong biocompatibility, good stability, excellent conductivity, high sensitivity, and non-participation in redox reaction, electrochemical HbA1c sensors based on nanotechnology have attracted extensive attention (e.g., see [87]). For the FAO- and FPOX-based sensors, graphene, AuNPs, core-shell magnetic nanoparticles, metal oxide nanolayers, and nanotubes are the common materials used as electrodes. Using novel nanomaterials as sensing and conduction materials, electrochemical sensors were developed and are expected to provide more convenient, accurate and reliable platforms for the diagnosis of diabetes in the future [88,89,90,114,115,116]. In the MIC type indirect sensors based on imprinting technology, the specific binding sites of the target molecules can be easily customized in the polymer network. At present, these sensors can cover the scope of clinical detection, but most of them are highly cross-linked rigid polymers with low flexibility, so their activity is far lower than that of natural enzymes. Molecularly imprinted microgels and other flexible sensors combined with artificial enzymes may promote the application of imprinted sensors in the clinic. In addition, electrochemical measurements can be combined with the FIA system, which has broad prospects in the field of HbA1c detection.
and 2.0 μM for FVH. The oxidative activity and specificity of PnFPOX for this method are commendable, but the detection range needs to be expanded if this method is to be adopted for clinical measurements of HbA1c. In addition, the PnFPOX electrode was engineered for continuous operation of the FIA system. This case shows that an electrochemical sensor can be combined with an FIA system to develop an integrated measurement system for sample pretreatment and sample electrochemical measurement.
There are also some sensors that modify both FPOX and FAO, which can selectively determine only glycated N-terminal peptides from the β chain without any interference in the presence of glycated peptides from the α chain [45]. Nanjo et al. adopted an FIA system with a flow-type spectrophotometer and electrochemical detector (with FPOX immobilized on amino-alkyl-bonded Uniport C and FAO immobilized on dialdehyde-activated Uniport C) [45]. The total hemoglobin in the sample is determined by spectrophotometry, and then, the FVH released from HbA1c is determined by an electrochemical sensor with FPOX or FAO. As a result, at pH 8.0–8.5, FAO showed high activity against FV but no activity against FVH, and at nearly pH 7.0, FPOX showed the maximum activity of FV and FVH. The FIA system with the FAO reactor showed a linear detection range for FV of 2–200 µM, and the FPOX reactor showed linear detection ranges for FV and FVH of 2–100 µM and 7–110 µM, respectively. However, when blood cell samples were digested by protease, only FVH was released, not FV. Therefore, according to the definition of HbA1c by the International Federation of Clinical Chemistry (IFCC), the combination of protease and FPOX reactor systems can determine only FVH.
