Fructosyl Valine Research Articles

Fructosyl Amino Oxidase as a Therapeutic Enzyme in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is an age-related disorder that is a global public health problem. The non-enzymatic Maillard reaction results in the formation of advanced glycation end products (AGEs). Accumulation of AGEs in drusen plays a key role in AMD. AGE-reducing drugs may contribute to the prevention and treatment of AGE-related disease. Fructosamine oxidase (FAOD) acts on fructosyl lysine and fructosyl valine. Based upon the published results of fructosamine 3-kinase (FN3K) and FAOD obtained in cataract and presbyopia, we studied ex vivo FAOD treatment as a non-invasive AMD therapy. On glycolaldehyde-treated porcine retinas, FAOD significantly reduced AGE autofluorescence (p = 0.001). FAOD treatment results in a breakdown of AGEs, as evidenced using UV fluorescence, near-infrared microspectroscopy on stained tissue sections of human retina, and gel permeation chromatography. Drusen are accumulations of AGEs that build up between Bruch's membrane and the retinal pigment epithelium. On microscopy slides of human retina affected by AMD, a significant reduction in drusen surface to 45 ± 21% was observed following FAOD treatment. Enzymatic digestion followed by mass spectrometry of fructose- and glucose-based AGEs (produced in vitro) revealed a broader spectrum of substrates for FAOD, as compared to FN3K, including the following: fructosyllysine, carboxymethyllysine, carboxyethyllysine, and imidazolone. In contrast to FN3K digestion, agmatine (4-aminobutyl-guanidine) was formed following FAOD treatment in vitro. The present study highlights the therapeutic potential of FAOD in AMD by repairing glycation-induced damage.

Bacterial diversity and metabolites: Exploring correlations with preservative properties in soybean pastes.

Soybean paste, a traditional fermented condiment, exhibits distinct quality attributes by its microbial communities. This study employed Illumina sequencing and LC-MS to scrutinize the bacterial biota and metabolome of highly preserved (HP) and easily spoiled (ES) soybean pastes. Firmicutes were prevalent in both pastes, with HP showcasing greater microbial α-diversity compared to ES pastes. Bacillus predominated in HP pastes, whereas Lactobacillus was most abundant in ES pastes. Significant metabolic differences were observed between HP and ES samples in lipids, peptides, nucleic acids, secondary metabolite biosynthesis, protein digestion, amino acid metabolism, inflammatory mediator regulation, and neomycin, kanamycin, and gentamicin biosynthesis. Lactobacillus exhibited positive associations with daidzein and 3,4,5-trihydroxypentanoylcarnitine, whereas Bacillus showed negative correlations with 1,n6-ethenoadenosine, 2-deoxy-2,3-dehydro-n-acetyl-neuraminic acid, 3,4,5-trihydroxypentanoyl carnitine, and fructosyl valine. These findings highlight the collaborative impact of bacterial communities and metabolites on soybean paste quality attributes. This research enhances our comprehension of preservation mechanisms in fermented foods, particularly soybean pastes. PRACTICAL APPLICATION: The investigation would provide insights into the soybean pastes fermentation, safe and quality control methods, bio-preservative development strategies, and so on of soybean pastes for related studies and the consumers. Bacteria and their metabolites could be used to optimize the fermentation processes for the preservative and safe regulations.

Molecular two-point recognition of fructosyl valine and fructosyl glycyl histidine in water by fluorescent Zn(II)-terpyridine complexes bearing boronic acids.

Selective recognition of fructosyl amino acids in water by arylboronic acid-based receptors is a central field of modern supramolecular chemistry that impacts biological and medicinal chemistry. Fructosyl valine (FV) and fructosyl glycyl histidine (FGH) occur as N-terminal moieties of human glycated hemoglobin; therefore, the molecular design of biomimetic receptors is an attractive, but very challenging goal. Herein, we report three novel cationic Zn-terpyridine complexes bearing a fluorescent N-quinolinium nucleus covalently linked to three different isomers of strongly acidified phenylboronic acids (ortho-, 2Zn; meta-, 3Zn and para-, 4Zn) for the optical recognition of FV, FGH and comparative analytes (D-fructose, Gly, Val and His) in pure water at physiological pH. The complexes were designed to act as fluorescent receptors using a cooperative action of boric acid and a metal chelate. Complex 3Zn was found to display the most acidic -B(OH)2 group (pKa = 6.98) and exceptionally tight affinity for FV (K = 1.43 × 105 M-1) with a strong quenching analytical response in the micromolar concentration range. The addition of fructose and the other amino acids only induced moderate optical changes. On the basis of several spectroscopic tools (1H, 11B NMR, UV-Vis, and fluorescence titrations), ESI mass spectrometry, X-ray crystal structure, and DFT calculations, the interaction mode between 3Zn and FV is proposed in a 1 : 1 model through a cooperative two-point recognition involving a sp3 boronate-diol esterification with simultaneous coordination bonding of the carboxylate group of Val to the Zn atom. Fluorescence quenching is attributed to a static complexation photoinduced electron transfer mechanism as evidenced by lifetime experiments. The addition of FGH to 3Zn notably enhanced its emission intensity with micromolar affinity, but with a lower apparent binding constant than that observed for FV. FGH interacts with 3Zn through boronate-diol complexation and coordination of the imidazole ring of His. DFT-optimized structures of complexes 3Zn-FV and 3Zn-FGH show a picture of binding which shows that the Zn-complex has a suitable (B⋯Zn) distance to the two-point recognition with these analytes. Molecular recognition of fructosyl amino acids by transition-metal-based receptors has not been explored until now.

Point-of-care detection of glycated hemoglobin using a novel dry chemistry-based electrochemiluminescence device

As a good biomarker to reflect the average level of blood glucose, glycated hemoglobin (HbA1c) is mainly used for long-term glycemic monitoring and risk assessment of complications in diabetic patients. Previous analysis methods for HbA1c usually require complex pretreatment processes and large-scale biochemical analyzers, which makes it difficult to realize the point-of-care testing (POCT) of HbA1c. In this work, we have proposed a three-electrode dry chemistry-based electrochemiluminescence (ECL) biosensor and its self-contained automatic ECL analyzer. In this enzymatic biosensor, fructosyl amino-caid oxidase (FAOD) reacts with the hydrolysis product of HbA1c, and the produced hydrogen peroxide further reacts with luminol under the appropriate driving voltage, generating photons to realize the quantitative detection of HbA1c. Under optimized conditions, the biosensors have a good linear response to different concentrations of fructosyl valine (FV) ranging from 0.05 to 2 mM, with a limit of detection of 2 μM. The within-batch variation is less than 15%, and the biosensors still have 78% of the initial response after the accelerated aging test of 36 h at 37 °C. Furthermore, the recoveries for different concentrations of samples in whole blood were within 92.3–99.7%. These results illustrate that the proposed method has the potential for use in POCT of HbA1c.

Topical Application of Deglycating Enzymes as an Alternative Non-Invasive Treatment for Presbyopia.

Presbyopia is an age-related vision disorder that is a global public health problem. Up to 85% of people aged ≥40 years develop presbyopia. In 2015, 1.8 billion people globally had presbyopia. Of those with significant near vision disabilities due to uncorrected presbyopia, 94% live in developing countries. Presbyopia is undercorrected in many countries, with reading glasses available for only 6-45% of patients living in developing countries. The high prevalence of uncorrected presbyopia in these parts of the world is due to the lack of adequate diagnosis and affordable treatment. The formation of advanced glycation end products (AGEs) is a non-enzymatic process known as the Maillard reaction. The accumulation of AGEs in the lens contributes to lens aging (leading to presbyopia and cataract formation). Non-enzymatic lens protein glycation induces the gradual accumulation of AGEs in aging lenses. AGE-reducing compounds may be effective at preventing and treating AGE-related processes. Fructosyl-amino acid oxidase (FAOD) is active on both fructosyl lysine and fructosyl valine. As the crosslinks encountered in presbyopia are mainly non-disulfide bridges, and based on the positive results of deglycating enzymes in cataracts (another disease caused by glycation of lens proteins), we studied the ex vivo effects of topical FAOD treatment on the power of human lenses as a new potential non-invasive treatment for presbyopia. This study demonstrated that topical FAOD treatment resulted in an increase in lens power, which is approximately equivalent to the correction obtained by most reading glasses. The best results were obtained for the newer lenses. Simultaneously, a decrease in lens opacity was observed, which improved lens quality. We also demonstrated that topical FAOD treatment results in a breakdown of AGEs, as evidenced by gel permeation chromatography and a marked reduction in autofluorescence. This study demonstrated the therapeutic potential of topical FAOD treatment in presbyopia.

Cloning, expression and purification of fructosyl amino acid oxidase (FAOX) in Escherichia Coli

Introduction: The level of serum HbA1c is an indicator of the average blood sugar level in the last three months. HbA1c can be quantified using assays involving the enzyme fructosyl amino acid oxidase (FAOX). This study aims to produce GST-tagged FAOX-TE (GST/FAOX-TE), a thermal stable and specific variant of FAOX, for future application studies.
 Materials and methods: The E. coli strains DH5α and BL21 (DE3) were used as cloning and expression hosts, respectively. The FAOX-TE sequence was synthesized at IDT (US) and clonned into pGEX-4T3 vector, which was confirmed by Colony PCR. The expression was induced at 16°C, 0.5 mM IPTG in LB media containing 50 µg/ml ampicilin. The protein expression profile was analyzed by SDS-PAGE. The cell pellet was sonicated and purified by Glutathione Sepharose 4 Fast Flow (Cytiva, US). The catalytic activity of GST/FAOX-TE with fructosyl valine was determined using high performance anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD).
 Results: The fusion protein was successfully expressed in Escherichia coli using the plasmid pGEX-4T3 and purified to high purity 93%. Recombinant GST/FAOX-TE was shown to be active on fructosyl valine.
 Conclusions: Active GST/FAOX-TE was successfully expressed in E. coli BL21 (DE3) and purified, which will be used for future development of biosensors for fructosyl valine quantification.

Development of glycated peptide enzyme sensor based flow injection analysis system for haemoglobin A1c monitoring using quasi-direct electron transfer type engineered fructosyl peptide oxidase

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.

Gold Nanostar Colorimetric Detection of Fructosyl Valine as a Potential Future Point of Care Biosensor Candidate for Glycated Haemoglobin Detection.

Diabetes Mellitus is a growing global concern. The current methods used to detect glycated haemoglobin are precise, however, utilise expensive equipment, reagents and consumables. These are luxuries which rural communities cannot access. The nanotechnology methods which have been developed for glycated haemoglobin detection are predominantly electrochemically based, have complicated lengthy fabrication processes and utilise toxic chemicals. Here a fructosyl amino acid oxidase gold nanostar biosensor has been developed as a potential future point of care biosensor candidate for glycated haemoglobin detection. The workup done on this biosensor showed that it was able to give a spectrophotometric readout and colorimetric result with naked eye detection in blank serum spiked with fructosyl valine.

Development of the 2.5th Generation Biosensor for HbA1c Using Engineered Fructosyl Peptide Oxidase

Introduction Haemoglobin A1c (HbA1c) is an important glycaemic control marker for diabetes mellitus. The level of HbA1c reflects the average blood glucose level over the prior 2–3 months. Recently an enzymatic assay has been introduced, which employs fructosyl peptide oxidase (FPOx). These methods have high accuracy, however, they are time-consuming, expensive, and require complicated analytical procedures. There is an increasing need for simple, rapid and miniaturized point-of-care testing (POCT). Our research group has been engaged in the development of novel electrochemical methods for glycated proteins, including HbA1c. We previously reported on the isolation and molecular engineering of FPOx derived from Phaeosphaeria nodorum (PnFPOx), which catalyzes the oxidative deglycosylation of fructosyl peptide and yield glucosone and free peptide, and it is specific to α-fructosyl amino acid (Kim et al., 2010). We reported the engineering of this enzyme to yield the mutant FPOx, Asn56Ala (N56A) with minimized oxidase activity whereas its dye-mediated dehydrogenase activity is higher than the wild-type, and its application for the electrochemical measurement of HbA1c (Kim et al., 2012). The X-ray structure of both of wild-type and mutant PnFPOxs were elucidated, which revealed the existence of the channel where oxygen may access to FAD (Shimasaki et al., 2017). Considering that the protease digestion pretreatment to release the fructoseyl peptide is necessary for the current FPOx based HbA1c assay, flow injection analysis (FIA) would be ideal platform for HbA1c electrochemical sensing system. To avoid the impact of oxygen (1st generation) and the use of electron mediator (2nd generation) in the carrier solution, direct electron transfer principle, the 3rd generation principle, is ideal for FIA based enzyme electrochemical sensing. However, FPOx is not able to transfer electron directly to electrode. In this study, we report our challenge in the development of enzyme electrochemical measurement of HbA1c by FIA system using engineered FPOx based on 2.5th generation principle. We recently reported the electrochemical sensor using mediator-modified enzyme, which doesn’t need external electron acceptor (Hatada et al., 2018), designated as 2.5th generation principle. We first engineered PnFPOx mutant to construct mediator-modified PnFPOx and evaluated the electrochemical behavior of mediator modified-PnFPOx for further application for the FIA system. Method The recombinantly prepared PnFPOx wild type and mutants were modified with amine reactive phenazine ethosulfate (arPES), according to our previous study. The modification by PES was confirmed by dye mediated dehydrogenase activity using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as electron acceptors. Fructosyl valine (FV) was used as the substrate. Result and discussion Most of FAD dependent oxidoreductases, including FPOx, cannot utilize MTT as the primary external electron acceptor, therefore, successful modification of PES at the residues proximate to FAD, can be confirmed by the dye-mediate dehydrogenase activity using MTT as the color indicating dye, which will be occurred by the intra-molecular electron transfer between FAD and covalently bound PES, and by the electron transfer from reduced PES to external MTT. Regarding the PES modification of wild type and N56A mutant according to our previous protocol, no dye-mediated dehydrogenase activity was observed when MTT was used as the electron acceptor. However, cyclic voltammetry (CV) measurements of PES modified wild type and N56A mutant showed typical redox peaks corresponding to PES, revealing that PES molecules were modified on the surface of wild type and N56A mutant FPOxs, but not at the residue(s) proximate to FAD. Therefore, Lys residues were introduced by substituting adequate residue(s) at the proximate of FAD, where no significant impact on structural nor activity would be predicted. As the results, the PES modified lysine introduced mutant showed the dye-mediated dehydrogenase activity with MTT without losing catalytic activity. Accordingly, the double mutant with N56A and lysine, was constructed, which showed the highest dye-mediated dehydrogenase activity with MTT with negligible oxidase activity. This constructed PES-modified wild type and engineered FPOx were then immobilized on the gold electrode using self-assembled monolayer (SAM), and their electrochemical properties were investigated. CV analysis revealed that electrodes with PES-modified lysine introduced mutant enzymes showed catalytic current in the presence of enzyme substrate, FV, but the electrodes with wild type nor N56A did not. The characterization of FIA system will be also reported. References Kim et al., Biotechnol. Bioeng. 106 (3), 358-366 (2010)Kim et al., Biotechnol. Lett. 34, 491-497 (2012)Shimasaki et al., Sci. Rep. 7, 2790 (2017)Hatada et al., Bioelectrochemistry 121, 185-190 (2018)

Development of Disposable Single-Use Biosensor for Fructosyl Valine and Glycated Hemoglobin A1c

A novel amperometric biosensor prototype was fabricated using screen printing technique. The disposable single-use strips were made from conductive carbon ink and modified with fructosyl amino acid oxidase. The electrodes and conducting paths were made solely with carbon ink and characterized by conductivity and cyclic voltammetry. The biosensor showed high current output, large linearity, and effectiveness for fructosyl valine as well as human blood samples. Amperometric studies were carried out using both fructosyl valine and human blood samples. With 5 uL sample volume, the biosensor showed strong amperometric response with good linearity for a wide range (0 to 8 mM). Diabetic and healthy blood samples showed sufficient difference in their amperometric responses that correlate well with their different hemoglobin A1c levels. These results demonstrate the feasibility of using this type of inexpensive single-use biosensor strips as the basis for determining hemoglobin A1c levels for diabetic patients.

Affinity sensor for haemoglobin A1c based on single-walled carbon nanotube field-effect transistor and fructosyl amino acid binding protein

Haemoglobin A1c (HbA1c) is a significant glycaemic marker for diabetes mellitus. The level of HbA1c reflects the mean blood glucose level over the prior 2–3 months and it is useful for the assessment of therapeutic effectiveness and for diagnosis. In this study, we report the label-free affinity sensor for HbA1c based on the chemiresistor-type field-effect transistor, which has a simple sensor configuration. Single-walled carbon nanotubes (SWNTs) were used as the transducing element. The fructosyl amino acid binding protein from Rhizobium radiobacter (SocA), which binds to α-fructosyl amino acid specifically, was used as the biorecognition element for fructosyl valine (FV), the product of the proteolytic hydrolysis of HbA1c. The developed sensor shows the ability to measure as low as 1.2 nM FV, which is 14-fold more sensitive compared to the previously reported fluorescence-based sensor using SocA. This sensor also exhibits high specificity where no significant response is observed from either fructosyl lysine (FK) or glucose, which are potential interferents. FK is the ε-fructosyl amino acid from glycated albumin, another glycated protein, whereas glucose is naturally present at very high concentration in the blood. We propose that the modulation of the surface charges on the SWNTs caused by the conformational change in SocA upon ligand binding leads to the proportionate changes in the number of carriers in the SWNT channel.

Recombinant fructosyl peptide oxidase preparation and its immobilization on polydopamine coating for colorimetric determination of HbA1c

Recombinant fructosyl peptide oxidase (FPO) from Phaeosphaeria nodorum SN15 was functionally expressed by Escherichia coli cells and one-step purified from crude cells extract using immobilized metal affinity chromatography (IMAC) to achieve a specific activity of 26 U/mg. A ready-use colorimetric detection of HbA1c level in blood sample was developed based on FPO immobilized membrane. Facile bio-inspired polydopamine coating on the surface of a microporous membrane was employed for effective FPO immobilization. Glutaraldehyde activation of the polydopamine coating significantly enhanced FPO immobilization yield that at least 5-fold higher activity could be achieved. The stability of FPO membrane was also enhanced by glutaraldehyde activation that 85% activity could be maintained after 7 repeated uses. Highly correlated optical densities at 727 nm (OD727) against fructosylvaline (FV) in the range of 0.02 mM to 0.7 mM (R2 = 0.988) could be achieved using FPO membrane. At least 80% of the initial activity was maintained for FPO membrane stored at 4 °C for 7 days. Rather low OD727 but good correlation still could be obtained by using FPO membrane for the detection HbA1c levels (6–14%) in blood samples.

Gold Nanoparticle‐based Novel Biosensors for Detecting Glycated Hemoglobin

We report here on novel biosensors for the highly sensitive and specific enzymatic analysis of glycated hemoglobin (HbA1c), which we measured primarily to identify three‐month average plasma glucose concentrations. Hydrogen peroxide (H2O2) was generated in this enzymatic measuring of glycated hemoglobin from oxidative cleavage of fructosyl valine by fructosyl amino acid oxidase, and we then used the generated hydrogen peroxide as a reducing agent of gold (III) ion to synthesize gold (0) as a nanoparticle. We were able to measure the gold nanoparticles obtained from this novel approach using colorimetry under a UV–VIS spectrophotometer. For this colorimetric detection, we analyzed 96‐well plates for glycated hemoglobin, and this system worked very well, with a detection limit of 0.142% and coefficient of variation below 10%. Finally, this method can measure glycated hemoglobin in patients with diabetes.

Assay of hemoglobin A1c using lectin from Aleuria aurantia.

Hemoglobin A1c (HbA1c) has an N-terminal fructosyl valine on the β-chain, and this modification is caused by the non-enzymatic glycosylation of hemoglobin (Hb). The relative concentration ratio of HbA1c to total Hb is an important biomarker for the diagnosis of diabetes. HbA1c-binding lectins were screened from 29 sources of lectin, and the lectin from Aleuria aurantia (AAL) was revealed to have higher affinity to HbA1c than to Hb. The concentration of HbA1c was determined by lectin-based enzyme-linked immunosorbent assay (ELISA) using the AAL lectin. Higher reproducibility of the assay was observed at 4 °C than at 25 and 37 °C. This observation is consistent with the known temperature-dependent behavior of lectins. Preincubation of HbA1c with an anti-HbA1c antibody inhibited the binding, suggesting that AAL binds to the N-terminal fructosyl valine epitope of HbA1c. Higher inhibitory effect was observed for 10 mM d-fructose than for the same concentrations of l-fucose, d-fucose, or d-glucose.

Titania nanotubes decorated with gold nanoparticles for electrochemiluminescent biosensing of glycosylated hemoglobin

A glycated hemoglobin (HbA1c) biosensor with high performance has been constructed in this work. Here the fructosyl amino acid oxidase was immobilized onto a pre-functionalized indium tin oxide glass with titania nanotubes decorated with gold nanoparticles. The property of nanocomposite was characterized by transmission electromicroscopy, scanning electron microscopy, electrochemistry and spectroscopy. Under the optimum conditions, fructosyl valine was detected by this biosensor. It exhibited a linear detection range from 4.0 × 10−9 M to 7.2 × 10−7 M, and a limit of detection for 3.8 × 10−9 M at the signal-to-noise ratio of 3. Thus the HbA1c level in whole blood samples of healthy individuals or diabetic patients were evaluated with designed biosensor after pre-treatment of hydrolysis. The results of our detection were closely consistent with that of the standard method. At the same time, our biosensor has some advantages including high sensitivity, disposable usage and low cost, which implies its great promising application in point-of-care testing of HbA1c.

Glycated hemoglobin detection with electrochemical sensing amplified by gold nanoparticles embedded N-doped graphene nanosheet

In the diabetic patients the level of glucose must be determined without any short term fluctuations. The level of Glycated hemoglobin (HbA1c) is accordingly examined for checking diabetes mellitus. HbA1c is considered one of the primarily factor to discern the concentration of average plasma glucose over a long-drawn-out period. In our work, we describe a construction of biosensor which is based on fructosyl amino-acid oxidase (FAO) immobilized nitrogen-doped graphene/gold nanoparticles (AuNPs)/fluorine doped tin oxide (FTO) glass electrode. This constructed biosensor exhibits a wide linear range of 0.3 to 2000μM in response to HbA1c at +0.2V. Consequently, the detection limit of 0.2μM and good stability (4 months) were achieved. The electrocatalytic activity of this sensor was good as a result of synergistic effect of graphene and AuNPs (2D and 0D nanomaterials). The charge transfer resistance was decreased which was observed by electrochemical impedance spectroscopy (EIS) study. The graphene/AuNPs composites film reveals a distinguished electrochemical response to fructosyl valine (FV) which demonstrates a promising application for electrochemical detection of HbA1c in human blood samples.

Recent Progress in Electrochemical HbA1c Sensors: A Review.

This article reviews recent progress made in the development of electrochemical glycated hemoglobin (HbA1c) sensors for the diagnosis and management of diabetes mellitus. Electrochemical HbA1c sensors are divided into two categories based on the detection protocol of the sensors. The first type of sensor directly detects HbA1c by binding HbA1c on the surface of an electrode through bio-affinity of antibody and boronic acids, followed by an appropriate mode of signal transduction. In the second type of sensor, HbA1c is indirectly determined by detecting a digestion product of HbA1c, fructosyl valine (FV). Thus, the former sensors rely on the selective binding of HbA1c to the surface of the electrodes followed by electrochemical signaling in amperometric, voltammetric, impedometric, or potentiometric mode. Redox active markers, such as ferrocene derivatives and ferricyanide/ferrocyanide ions, are often used for electrochemical signaling. For the latter sensors, HbA1c must be digested in advance by proteolytic enzymes to produce the FV fragment. FV is electrochemically detected through catalytic oxidation by fructosyl amine oxidase or by selective binding to imprinted polymers. The performance characteristics of HbA1c sensors are discussed in relation to their use in the diagnosis and control of diabetic mellitus.

An amperometric hemoglobin A1c biosensor based on immobilization of fructosyl amino acid oxidase onto zinc oxide nanoparticles–polypyrrole film

Measurement of hemoglobin A1c (HbA1c, glycated hemoglobin) level in blood provides the long-term glucose level in diabetic patients without the influence of short-term fluctuations. The existing methods for HbA1c determination, including biosensors, suffer from insufficient sensitivity, detection limit, response time, and storage stability. These problems were overcome in the current biosensor. A method is described for construction of an amperometric HbA1c biosensor by immobilizing a fructosyl amino acid oxidase (FAO) onto zinc oxide nanoparticles/polypyrrole (ZnONPs/PPy) hybrid film deposited onto gold (Au) electrode and using it as working electrode, Ag/AgCl as reference electrode, and platinum (Pt) as auxiliary electrode. The whole blood samples were hemolyzed and digested by protease before measuring their HbA1c level by the biosensor. The enzyme electrode detected fructosyl valine (FV) as low as 50μM at a signal-to-noise ratio of 3 within 2s at +0.27V versus Ag/AgCl, pH7.0, and 35°C with a linear working range of 0.1 to 3.0mM for FV and sensitivity of 38.42μAmM−1. The electrode showed only a 30% loss of its initial response over a period of 160days when stored at 4°C. The biosensor measured HbA1c in whole blood of apparently healthy individuals and diabetic patients and found it to be in the ranges of 4.0% to 5.6% and 5.7% to 12.0%, respectively.

An electrochemical biosensor for fructosyl valine for glycosylated hemoglobin detection based on core–shell magnetic bionanoparticles modified gold electrode

A high-performance amperometric fructosyl valine (FV) biosensor was developed, based on immobilization of fructosyl amino-acid oxidase (FAO) on core–shell magnetic bionanoparticles modified gold electrode. Chitosan was used to introduce amino groups onto the surface of core–shell magnetic bionanoparticles (MNPs). With FAO as an enzyme model, a new fructosyl valine biosensor was fabricated. The biosensor showed optimum response, when operated at 50 mV s −1 in 0.1 M potassium phosphate buffer, pH 7.5 and 35 °C. The biosensor exhibited excellent sensitivity [the detection limit is down to 0.1 mM for FV], fast response time (less than 4 s), wide linear range (from 0 to 2 mM). Analytical recovery of added FV was 95.00–98.50%. Within batch and between batch coefficients of variation were <2.58% and <5.63%, respectively. The enzyme electrode was used 250 times over 3 months, when stored at 4 °C.

Glassy Carbon Paste Electrodes for the Determination of Fructosyl Valine

AbstractNearly 200 million people worldwide have type‐2 diabetes. Glucose sensors are routinely used for diagnosis; however, the relative amount of glycosylated hemoglobin (HbA1c) may be a better marker. A working electrode made from bare glassy carbon paste was used for sensing fructosyl valine (Fru‐Val), a component of HbA1c. Amperometric measurements revealed a linear relationship between Fru‐Val concentration and the sensing current. The square correlation coefficient and the sensitivity were 0.999 and 5.26 μA mM−1, respectively. The minimum detection limit was less than 0.05 mM.