Membrane-immobilized haptoglobin as affinity matrix for a hemoglobin-A1c immunosensor

An amperometric immunosensor for hemoglobin-A1c (HbA1c) determination has been developed utilizing membrane-immobilized haptoglobin as affinity matrix fixed in front of a Pt-working electrode. The HbA1c assay was carried out in a two-step procedure including the selective hemoglobin enrichment on the sensor surface and the specific HbA1c detection by a glucose oxidase (GOx) labeled anti-HbA1c antibody. Hydrogen peroxide generated by the enzyme label was oxidized at +600 mV versus Ag/AgCl. A standard curve for HbA1c was obtained with a linear range between 0 and 25% HbA1c of total hemoglobin which correspond to 7.8–39 nM. ELISA studies confirmed the advantage of a sandwich-type format with haptoglobin as capture molecule for selective hemoglobin binding over the direct adsorption method. Results by the sandwich immunoassay showed a linear correlation within the clinically relevant range 5–20% (CV < 3). For sensor application the immobilization procedure of haptoglobin onto CDI-activated cellulose membranes was optimized.     

FET immunosensor for hemoglobin A1c using a gold nanofilm grown by a seed-mediated technique and covered with mixed self-assembled monolayers

A micro FET-based immunosensor was developed for the determination of hemoglobin-A1c (HbA1c). The HbA1c/hemoglobin ratio is an important index in diabetes control. The sensor was fabricated by Complementary Metal-Oxide-Semiconductor Transistor (CMOS) and Micro Electronic Mechanical System (MEMS) techniques. The antibodies were immobilized via mixed self-assembled monolayers (SAMs) on a gold nanofilm. The nanofilm was deposited on a gold electrode by seed-mediated growth and gave a uniform and well distributed coverage. Nonspecific sites and interferences by noise were eliminated by covering the AuNPs with mixed SAMs. Compared to the immunosensor fabricated via the mixed SAMs method without gold nanofilm, the immunosensor displays a more than 2-fold sensitivity. The immunosensor is capable of detecting HbA1c and hemoglobin in hemolyzed and diluted whole blood, and results showed good agreement with the established clinical method.

Development of an electrophoretic method based on nanostructured materials for HbA1c determination

Glycosylated hemoglobin (HbA1c) detection is performed routinely in hospitals as it is the most widespread confirmatory diagnosis of diabetes mellitus. Here we present a novel CE method for measuring HbA1c by introducing silica nanoparticles (NPs) modified with a boronic acid derivative (sugar loadings of 51 ± 2 μg/mg) as pseudo‐stationary phase. Before the sample injection, SiO₂NP─B(OH)₂ were introduced via pressure. Electrophoretic separation was explored through variation of the buffer pH and separation voltage, being the best separation, resolution and shorter separation time achieved with a 25 mM phosphate buffer pH 6.5. The calibration curve obtained was expressed as Area = 182.05%⁻¹ × HbA1c − 377.02; R² = 0.9826, using a UV/VIS absorbance detector at 415 nm (diode array). No interferences were observed from carbamylated or acetylated hemoglobin and the method shows a noteworthy stability. A paired t‐test was applied to compare the developed CE method with a commercial HbA1c test and no significant variations have been observed at a 90% significance level.     

Point‐of‐Care Amperometric Testing of Diabetic Marker (HbA1c) Using Specific Electroactive Antibodies

Specific immune detection of glycated hemoglobin is still a great challenge owing to the small epitopic difference between Hemoglobin (Hb) and HbA1c. We report a new electrochemical immunoassay format for point of care testing of HbA1c. A conducting self‐assembled monolayer of mercaptophenyl boronic acid (MPBA) was used as a capture layer for binding of glycated proteins and ferrocene tagged anti‐HbA1c antibody (FcAb) as a tracer molecule on a gold screen printed electrode. Validation of the new HbA1c assay was carried out using 6 clinical samples with known HPLC values and a correlation coefficient of 98 % was observed.     

Glassy Carbon Paste Electrodes for the Determination of Fructosyl Valine

Nearly 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.     

In-Depth Comparative Characterization of Hemoglobin Glycation in Normal and Diabetic Bloods by LC-MSMS

The glycation level at β-Val-1 of the hemoglobin β chain in human blood (HbA1c%) is used to diagnose diabetes and other diseases. However, hemoglobin glycation occurs on multiple sites on different isoforms with different kinetics, but its differential profile has not been clearly demonstrated. In this study, hemoglobin was extracted from the blood of normal and diabetic individuals by protein precipitation. Triplicate solutions prepared from each sample were directly analyzed or digested with multiple enzymes and then analyzed by nano-LC/MS via bottom-up approach for side-by-side characterization. Intact hemoglobin analysis indicated a single glucose-dominant glycation, which showed good correlation with the HbA1c% values. Moreover, full sequence (100 %) of α/β globin was mapped and seven glycation sites were unambiguously assigned. In addition to β-Val-1, two other major sites at α-Lys-61 and β-Lys-66, which contain the common sequence HGKK, and four minor sites (<1 %) on α-Val-1, β-Lys-132, α-Lys-127, and α-Lys-40 were identified. All sites were shown to exhibit similar patterns of site distribution despite different glucose levels. Both the intact mass measurement and bottom-up data consistently indicated that the total glycation percentage of the β-globin was twice higher than the α-globin. Using molecular modeling, the 3D structure of the consensus sequence (HGKK) was shown to contain a phosphate triangle cavity, which helps to catalyze the glycation reaction. For the first time, hemoglobin glycation in normal and diabetic bloods was comparatively characterized in-depth with 100 % sequence coverage. The results provide insight about the HbA1c parameter and help define the new and old markers.

Assessment of microwave-assisted enzymatic digestion by measuring glycated hemoglobin A1c by mass spectrometry

Enzymatic digestion of proteins and analysis of the resulting peptides by mass spectrometry is an established approach in proteomics and in clinical and environmental chemistry. The long digestion times of several hours prevent the fast turnover of samples and results. Qualitative applications showed that microwave radiation profoundly shortens enzymatic digestion. However, its usefulness for quantitative applications had not been assessed. In this study, the microwave-assisted enzymatic digestion of hemoglobin at different temperatures, buffer concentrations, and digestion times was assessed and compared with conventional digestion for the proteolytic enzymes trypsin and Glu-C. A microwave-assisted enzymatic digestion method optimized for digestion time and temperature was applied for the analysis of glycated hemoglobin HbA1c and compared with a reference method. Using trypsin, complete digestion was obtained at 50 degrees C within 20 min. Under these conditions, the digestion efficiency was 20% higher than with conventional trypsin digestion. These effects were not observed with Glu-C as enzyme, probably because of the decreased stability of Glu-C at elevated temperatures in comparison with the trypsin used. The comparison of the optimized microwave-assisted digestion method using trypsin with the reference method for HbA1c using Glu-C gave a close correlation in the results (R2: 0.996). A significant bias of 0.33% HbA1c was observed, with higher values obtained with the microwave-assisted tryptic digest; this finding might have resulted from the use of a different enzyme. This study showed that microwave-assisted enzymatic digestion can substantially reduce digestion times to minutes and can be used in qualitative as well as quantitative applications.     

Spectroelectrochemical study of hemoglobin A, alpha- and beta-fumarate crosslinked hemoglobins; implications to autoxidation reaction

The thermodynamics and kinetics of the reaction DeoxyHb-Fe(2+)<-->MetHb-Fe(3+) for human hemoglobin A (HbA), alpha- and beta-fumarate crosslinked hemoglobins were investigated by spectroelectrochemistry. Information from this study is used to determine what structural features and experimental conditions stabilize ferrous vs. ferric form of hemoglobin, and what implications this stabilization may have on the autoxidation reaction. Alpha- and beta-fumarate crosslinked hemoglobins, alphaXL-HbA and betaXL-HbA, were obtained by crosslinking deoxyhemoglobin and oxyhemoglobin, respectively, with bis(3,5-dibromosalicyl) fumarate (DBSF). Formal redox potentials, E(0), and reduction/oxidation rates were measured in the presence of mediator, hexammineruthenium(III) chloride. It was found that E(0) shifted positive for the alpha-, and negative for the beta-fumarate crosslinked hemoglobin compared to HbA for all experimental conditions investigated. This shift was consistent with stabilization of the tense (positive shift) or relaxed conformation (negative shift) conferred by crosslinking. Formal redox potentials shifted positive with addition of nitrate and chloride ions for alphaXL-HbA, indicating additional stabilization of the T quaternary. The slopes of the Nernst plots showed evidence of cooperativity as expressed by n(max). The data points (E(0), n(max)) were fitted by the MWC model which states that the electron transfer and the addition/removal of water are concerted. The set of K(R) and c values, where the parameter c is the ratio K(R)/K(T) and K(R) and K(T) are the ligand (water molecule and an electron-hole) dissociation constants for the R and T states, for the beta-crosslinked hemoglobin compared to that of HbA and alpha-crosslinked hemoglobin indicated that crosslinking of oxyhemoglobin affected differently the inner-coordination sphere at the heme site. By modulating the electrolyte concentration the reduction rates were measured as a function of DeltaE(0), the difference in E(0) between hemoglobin molecules and mediator. Linearization of the Marcus cross-relationship (based on the concerted water and electron transfer) was good for HbA, and poor for alphaXL-HbA and betaXL-HbA, consistent with results obtained by the MWC analysis. This may imply that the reduction of HbA is controlled by the driving force, DeltaE(0), whereas the reduction of alphaXL-HbA and betaXL-HbA occurs by a non-concerted mechanism controlled by structural features brought about by crosslinking. The autoxidation reaction, conversion of oxygen-bound ferrous hemoglobin to ferric hemoglobin, was found independent of E(0). Alpha-fumarate crosslinked hemoglobin showed the highest autoxidation rate despite its positive shift in formal redox potential as compared to HbA, followed by beta-fumarate crosslinked hemoglobin, and by native hemoglobin. These data suggest that the chemical mechanism of oxygen dissociation and accessibility of water and oxygen radicals to heme site control autoxidation.     

电位无标型糖化血红蛋白免疫微传感器

研制了基于标准CMOS工艺和微加工技术的电位无标型免疫微传感器,可实现糖化血红蛋白浓度与血红蛋白浓度的简便检测。该微传感器包括含有信号读出电路的场效应型微传感集成芯片和一次性测试试条。微传感集成芯片由本实验室设计并经新加坡Chartered半导体公司流片制备。一次性测试试条采用微加工技术制备于柔性塑料片上,包括敏感电极阵列和三维微结构测量池。基于自组装单层膜并引入纳米金颗粒的方法,在测试试条电极表面固定抗体。采用循环伏安法和交流阻抗法对电极表面的修饰过程进行了测试和分析。该传感器对糖化血红蛋白和血红蛋白检测的线性范围分别为4～24mg/L和60～180mg/L。     

Glycated hemoglobin (HbA1c) measurement in frozen whole blood depends on baseline values of fresh samples

Glycated hemoglobin (HbA1c) has been recently adopted as a diagnostic marker of type 2 diabetes. However, its usage is currently limited to fresh blood samples. To allow retrospective HbA1c measurement in blood banks developed in large epidemic studies, here, we contribute to validate HbA1c assessment in frozen versus fresh blood samples from a cohort of diabetic/nondiabetic adult subjects. HbA1c was measured by HPLC in 237 fresh whole blood samples and on the same samples after a 12-month storage and a further 6-month-refrozen storage. Mean HbA1c?±?SD in fresh, frozen, and refrozen samples was 6.9?±?1.2, 6.6?±?1.1, and 6.4?±?1.0 % for the Diabetes Control and Complications Trial and 52?±?13, 49?±?12, and 46?±?11 mmol/mol for the International Federation of Clinical Chemistry and Laboratory Medicine reference, respectively. A significant correlation was found between fresh/frozen and fresh/refrozen (R?=?0.994 and 0.993, P?<?0.001) samples. HbA1c relative error ratio (%RER) between frozen/refrozen and fresh samples significantly correlated with HbA1c and depended on fresh value range, increasing in the five HbA1c classes (<6.0, 6.0–6.5, 6.5–7, 7–8, ≥8 %, corresponding to <42, 42–48, 48–53, 53–64, ≥64 mmol/mol, P?<?0.001). In particular, the 6.5 % (48 mmol/mol) HbA1c diagnostic cutoff of fresh samples identified two classes reflecting significant differences in %RER (2.8?±?2.0 and 3.3?±?1.7; P?<?0.05) between frozen and fresh samples. In conclusion, our results demonstrate a high correlation between data from fresh and frozen samples, with a very limited %RER between the two measurements, which increases with baseline HbA1c levels. Accordingly, when analyzing biobank frozen specimens for diagnostic purpose, the effect of the HbA1c range should be taken into account.

Direct monitoring of glycohemoglobin A1c in the blood samples of diabetic patients by capillary electrophoresis. Comparison with an immunoassay method

The capillary electrophoresis (CE) study was focused on quantifying glycohemoglobin A1c, HbA1c, in whole blood samples of patients suffering diabetes mellitus. The results showed that dynamic polyionic coating of the capillary made the method very reproducible. The precision evaluation, method comparison and bias estimation in CE were performed during 20 days with patient blood samples and with four control samples. The influence of the storage time and temperature on the glycohemoglobin levels were also tested. High resolution in CE could be used to show evidences of the ageing of the samples stored at -70 degrees C. The results showed that the ageing peak was not originated from HbA1c, because it did not affect the HbA1c level which was in balance with the results of fresh samples measured with immunoassay. The HbA1c % values of blood samples of 105 patients measured with the CE technique varied between 3.6 and 11.8 and they were approximately 2-3% lower than measured with immunoassay. The correlation (R2) of CE results with immunoassay and HPLC results were 0.962 and 0.781, respectively.     

Momordica charantia Extract Protects against Diabetes-Related Spermatogenic Dysfunction in Male Rats: Molecular and Biochemical Study

More than 90% of diabetic patients suffer from sexual dysfunction, including diminished sperm count, sperm motility, and sperm viability, and low testosterone levels. The effects of Momordica charantia (MC) were studied by estimating the blood levels of insulin, glucose, glycosylated hemoglobin (HbA1c), testosterone (TST), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in diabetic rats treated with 250 and 500 mg/kg b.w. of the total extract. Testicular antioxidants, epididymal sperm characteristics, testicular histopathology, and lesion scoring were also investigated. Testicular mRNA expression of apoptosis-related markers such as antiapoptotic B-cell lymphoma-2 (Bcl-2) and proapoptotic Bcl-2-associated X protein (Bax) were evaluated by real-time PCR. Furthermore, caspase-3 protein expression was evaluated by immunohistochemistry. MC administration resulted in a significant reduction in blood glucose and HbA1c and marked elevation of serum levels of insulin, TST, and gonadotropins in diabetic rats. It induced a significant recovery of testicular antioxidant enzymes, improved histopathological changes of the testes, and decreased spermatogenic and Sertoli cell apoptosis. MC effectively inhibited testicular apoptosis, as evidenced by upregulation of Bcl-2 and downregulation of Bax and caspase-3. Moreover, reduction in apoptotic potential in MC-treated groups was confirmed by reduction in the Bax/Bcl-2 mRNA expression ratio.     

Autofluorescence characterization of advanced glycation end products of hemoglobin

This article describes the analysis of autofluorescence of advanced glycation end products of hemoglobin (Hb-AGE). Formed as a result of slow, spontaneous and non-enzymatic glycation reactions, Hb-AGE possesses a characteristic autofluorescence at 308/345 nm (lambda(ex)/lambda(em)). Even in the presence of heme as a quenching molecule, the surface presence of the glycated adduct gave rise to autofluorescence with the quantum yield of 0.19. The specificity of monoclonal antibody developed against common AGE structure with Hb-AGE was demonstrated using reduction in fluorescence polarization value due to increased molecular volume while binding. The formation of fluorescent adduct in hemoglobin in the advanced stage of glycation and the non-fluorescent HbA(1c) will be of major use in distinguishing and to know the past status of diabetes mellitus. While autofluorescence correlated highly with HbA(1c) value under in vivo condition (r = 0.85), it was moderate in the clinical samples (r = 0.55). The results suggest a non-linear relation between glycemia and glycation, indicating the application of Hb-AGE as a measure of susceptibility to glycation rather than glycation itself.     

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.     

Development of a flow-injection analysis (FIA) enzyme sensor for fructosyl amine monitoring

An enzyme-sensor system with flow-injection analysis (FIA) has been developed for the detection of fructosyl amine compounds; the sensor utilizes fructosyl amine oxidase isolated from the marine yeast Pichia sp. N1-1 strain. With this FIA system 0.2 to 10 mmol L(-1) fructosyl valine can be determined. The sensor is approximately five times more sensitive to fructosyl valine, a model compound for glycated hemoglobin HbA1c, than to N(epsilon)-fructosyl lysine, a model compound for glycated albumin. This FIA system can also be used to detect fructosyl dipeptides. The operational stability of the sensor enabled more than 120 consecutive sample injections over a period of approximately 20 h.     

Glycated Albumin Measurement Using an Electrochemical Aptasensor for Screening and Monitoring of Diabetes Mellitus

Glycated albumin (GHSA) is a medium‐term glycaemic control marker of diabetes, which can be used as an alternative to or together with glycated haemoglobin (HbA1c). Currently available methods for the measurement of GHSA are limited in clinical practice because they involve slow and cumbersome processes of sample preparation, proteolytic digestion, and thermal incubation, and they suffer from limited analytical performance, and/or a lack of normalization to total albumin (HSA) levels. In this paper, we developed a simple electrochemical biosensor to measure GHSA values based on two DNA aptamers that specifically bind to GHSA and HSA. We used square wave voltammetry (SWV) to measure binding of the target proteins to their specific biotinylated aptamers, which had been immobilised on separate streptavidin (STR)‐modified screen‐printed carbon electrodes (SPCEs), in the presence of the redox mediator ferricyanide (Fe(CN)₆^3?). This electrochemical aptasensing system had a detection limit of 3 ng/ml for GHSA and 0.2 μg/ml for HSA. The results exhibited high selectivity for GHSA over other molecules present in the blood. The developed sensor was able to measure the amount of GHSA in plasma samples. A statistically significant difference was observed in the elevated plasma GHSA levels in diabetic versus non‐diabetic patients. Moreover, the trends in these GHSA levels were consistent with those obtained using the HbA1c test. The sensing system reported herein could be applied as a point‐of‐care‐testing (POCT) device for measurements of clinically relevant GHSA values.     

A new quality control method for lateral flow assay

We proposed a lateral flow assay (LFA) based on internal quality control microspheres to realize the accurate diagnosis of HbA1c in human body. This method can improve the precision and accuracy of HbA1c detection.     

A multiplexed three-dimensional paper-based electrochemical impedance device for simultaneous label-free affinity sensing of total and glycated haemoglobin: The potential of using a specific single-frequency value for analysis

A novel three-dimensional paper-based electrochemical impedance device (3D-PEID) is first introduced for measuring multiple diabetes markers. Herein, a simple 3D-PEID composed of a dual screen-printed electrode on wax-patterned paper coupled with a multilayer of magnetic paper was fabricated for label-free electrochemical detection. The results clearly demonstrated in a step-wise manner that the haptoglobin (Hp)-modified and 3-aminophenylboronic acid (APBA)-modified eggshell membranes (ESMs) were highly responsive to a clinically relevant range of total (0.5–20 g dL⁻¹; r² = 0.989) and glycated haemoglobin (HbA1c) (2.3%–14%; r² = 0.997) levels with detection limits (S/N = 3) of 0.08 g dL⁻¹ and 0.21%, respectively. The optimal binding frequencies of total haemoglobin and HbA1c to their specific recognition elements were 5.18 Hz and 9.99 Hz, respectively. The within-run coefficients of variation (CV) were 1.84%, 2.18%, 1.72%, and 2.01%, whereas the run-to-run CVs were 2.11%, 2.41%, 2.08%, and 2.21%, when assaying two levels of haemoglobin and HbA1c, respectively. The CVs for the haemoglobin and HbA1c levels measured on ten independently fabricated paper-based sheets were 1.96% and 2.10%, respectively. These results demonstrated that our proposed system achieved excellent precision for the simultaneous detection of total haemoglobin and HbA1c, with an acceptable reproducibility of fabrication. The long-term stability of the Hp-modified eggshell membrane (ESM) was 98.84% over a shelf-life of 4 weeks, enabling the possibility of storage or long-distance transport to remote regions, particularly in resource-limited settings; however, for the APBA-modified ESM, the stability was 92.35% over a one-week period. Compared with the commercial automated method, the results demonstrated excellent agreement between the techniques (p-value < 0.05), thus permitting the potential application of 3D-PEID for the monitoring of the glycaemic status in diabetic patients.     

富含微量元素中药煎剂对2型糖尿病疗效观察

为了观察富含微量元素中药煎剂对2型糖尿病（T2DM）的疗效,将门诊64名血糖控制不满意的T2DM患者分为增加富含微量元素中药煎剂组（观察组）和不增加组（对照组）,服药3个月后比较。结果表明,观察组的空腹血糖（FPG）、空腹血清胰岛素（Fins）和糖化血红蛋白（HbA1c）3月后明显下降（P〈0．01）;3月后观察组的FPG、Fins和HbA1c较对照组明显下降（P〈0．01）。可见增加富含微量元素中药煎剂对血糖难以控制的T2DM的FPG、Fins和HbA1c能够起到有效控制作用。     

冠心病患者糖化血红蛋白水平检测对评估患者冠状动脉病变程度的价值探究

目的通过观察了解糖化血红蛋白HbA1c水平与冠状动脉病变严重程度的相关关系。方法随机选取2013年1月—2015年1月来遵义医学院第五附属(珠海)医院心血管内科住院并进行选择性冠脉造影(CAG)的96例患者,同时对这96例患者进行糖化血红蛋白测定,回顾性分析冠心病患者的临床症状、体征、诊疗等相关指标,以了解HbA1c水平与冠状动脉病变程度的关系。结果对冠心病患者进行临床调查,并对结果进行记录,冠心病组和对照组HbA1c水平比较可知,冠心病组HbA1c水平高于对照组(P0.05)。结论对冠心病患者进行糖化血红蛋白水平检测,严格控制高血糖,降低HbA1c水平,通过改善血管内皮细胞功能,对延缓或阻止动脉粥样硬化,减少心血管并发症和后遗症的发生有着重要意义。