On-chip type cation-exchange chromatography with ferrocene-labeled anti-hemoglobin antibody and electrochemical detector for determination of hemoglobin A1c level

An on-chip type cation-exchange chromatography system with electrochemical detection of HbA_1c, which is one of the most important diabetes marker protein, was developed using ferrocene-conjugated anti-human hemoglobin (Hb) monoclonal antibody (FcAb). The FcAb was used as an electrochemical probe for the detection of each Hb. The system contains syringe pump, on-chip type cation-exchange column consisted of PDMS and cation-exchange resin beads, and a three-electrode flow-cell system. The separation conditions of HbA_1c in blood calibrator samples from other Hbs, e.g. HbA₀, HbA_1a or HbA_1b, were optimized using the on-chip type system. The electrochemical oxidation current from FcAb reacting with each Hb was measured at 350 mV (vs. Ag/AgCl). Hbs including HbA_1a and HbA_1b, HbA_1c and HbA₀ fractions were eluted in this order. A linear relationship between HbA_1c levels and electrochemical oxidation currents was obtained in the range from 4.0% to 12.6% HbA_1c. All procedure including antigen-antibody reaction was completed in 15 min. Furthermore, a good correlation was obtained between KO500 method (HPLC) and our proposed method. These results indicate that the on-chip type system with electrochemical detection can be applied to a novel POCT device for rapid and precise detection of HbA_1c.     

Determination of fructosyl amino acids and fructosyl peptides in protease-digested blood sample by a flow-injection system with an enzyme reactor.

A flow-injection system with an enzyme reactor was proposed for the measurement of fructosyl amino acids and fructosyl peptides in protease-digested blood samples. A fructosyl-amino acid oxidase (FAOX-TE) and two fructosyl-peptide oxidases (FPOX-CE and FPOX-CET) were covalently immobilized onto an inert support. They were used as the enzyme reactor in a FIA system with a hydrogen peroxide electrode. In particular, the FPOX-CET reactor possessed high selectivity for the detection of fructosyl valine (FV) and fructosyl valyl histidine (FVH) and an excellent operational stability. The proposed FIA system with the FPOX-CET reactor responded linearly to the concentration of FV over the dynamic range of 7.8 x 10(-6) to 5.8 x 10(-4) M. The present method could be successfully applied to the assay of FV and FVH in the protease-digested blood samples.     

Glycated hemoglobin biosensing integration formed on Au nanoparticle-dotted tubular TiO2 nanoarray

Excessive glucose present in the blood of diabetic patients binds with the hemoglobin of red blood cells resulting in the formation of glycated hemoglobin (HbA_1c). Measurement of HbA_1c levels may help in identifying the efficacy of the ongoing treatment and hence provide a better control over the disease. In the present study, we have synthesized a sensitive and stable scaffold, which consists of Au nanoparticles (GNPs)-dotted tubular TiO₂, for the construction of an electrochemical HbA_1c biosensor. 12-phosphotungstic acid has been used as a reducer after depositing well-dispersed GNPs on TiO₂ nanotubes (TiO₂ NTs) and an electron mediator by accelerating the electron transfer between the conductor and protein. The fabricated electrode was characterized using scanning electron microscopy (SEM), cyclic voltammetry (CV), Fourier transform infrared spectroscopy (FTIR) and electrochemical impedance spectroscopic analysis (EIS). Biosensor exhibited low detection limit (0.5 μM), fast response time (3 s) and wide linearity (from 0.5 to 2000 μM). The working electrode was used 100 times over 4 months, when stored at 4 °C. The HbA1c biosensor was then effectively used to measure the % of HbA_1c in the blood of apparently healthy persons and diabetic patients.     

Development of a Laser Nephelometric Method for the Quantitation of Human Glycohemoglobins

Abstract

A nephelometric procedure for quantitative measurement of glycohemoglobin (Hb A₁) was developed, evaluated, and compared with the semi-quantitative mini-column chromatographic procedure. Hb A₁ was purified from human red cell hemolystate by Bio-Rex 70 ion-exchange liquid chromatography and was used for standards and immunization. The antisera raised in rabbits showed high cross-reactivities with normal human hemoglobin (Hb A). The latter was separated by mixing 25 μl of patients' hemolystate with 2 ml ion-exchange resin suspension for 15 minutes. One hundred microiters of the supernatant was incubated with 900 μl antiserum (dilution 1:50) for 45 minutes at room temperature. Samples were then read on a laser nephelometer. The sensitivity of the assay was found to be 0.1 mg Hb A₁. The intraassay relative standard deviation (RSD) was 5.6% and the interassay RSD was 6.5%.     

Ferrocene-Labelled Electroactive Aptamer-Based Sensors (Aptasensors) for Glycated Haemoglobin

Glycated haemoglobin (HbA_1c) is a diagnostic biomarker for type 2 diabetes. Traditional analytical methods for haemoglobin (Hb) detection rely on chromatography, which requires significant instrumentation and is labour-intensive; consequently, miniaturized devices that can rapidly sense HbA_1c are urgently required. With this research, we report on an aptamer-based sensor (aptasensor) for the rapid and selective electrochemical detection of HbA_1c. Aptamers that specifically bind HbA_1c and Hb were modified with a sulfhydryl and ferrocene group at the 3′ and 5′-end, respectively. The modified aptamers were coated through sulfhydryl-gold self-assembly onto screen printed electrodes, producing aptasensors with built in electroactivity. When haemoglobin was added to the electrodes, the current intensity of the ferrocene in the sensor system was reduced in a concentration-dependent manner as determined by differential pulse voltammetry. In addition, electrochemical impedance spectroscopy confirmed selective binding of the analytes to the aptamer-coated electrode. This research offers new insight into the development of portable electrochemical sensors for the detection of HbA_1c     

Development of hemoglobin A1c certified reference material by liquid chromatography isotope dilution mass spectrometry

We report the development of a National Institute of Metrology (NIM) hemoglobin A_1c (HbA_1c) certified reference material (CRM). Each CRM unit contains about 10 μL of hemoglobin. Both hemoglobin and glycated hemoglobin were quantitatively determined by high-performance liquid chromatography (HPLC)–isotope dilution mass spectrometry (IDMS) with synthesized VHLTPE and glycated VHLTPE as standards. The mass fraction of synthesized VHLTPE or glycated VHLTPE was also quantitatively determined by HPLC-IDMS with NIM amino acid CRMs as standards. The homogeneity and stability of the CRMs were examined with a commercial HbA_1c analyzer based on the HPLC principle. Fifteen units were randomly selected for homogeneity examination, and statistical analysis showed there was no inhomogeneity. Examination of the stability showed that the CRM was stable for at least 6 months at -80 °C. Uncertainty components of the balance, amino acid purity, hydrolysis and proteolysis efficiency, method reproducibility, homogeneity, and stability were taken into consideration for uncertainty evaluation. The certified value of NIM HbA_1c CRM was expressed as the ratio of HbA_1c to total hemoglobin in moles, and was (9.6 ± 1.9)% . The CRM can be used as a calibration or validation standard for clinical diagnostics. It is expected to improve the comparability for HbA_1c measurement in China.     

Electroanalytical Study of the Pesticide Asulam

The electrochemical behaviour of the herbicide Asulam was studied by cyclic and square wave voltammetry. Asulam may be irreversibly oxidised at a glassy carbon electrode. Maximum currents were obtained at pH = 1.9 in aqueous electrolyte solution. Based on the electrochemical behaviour of Asulam, two analytical methodologies were developed for its determination in water samples, using square wave voltammetry (SWV) and flow injection analysis (FIA) coupled with an amperometric detector. Limits of detection of 7.1 2 10 m 6 mol L m 1 and 1.2 2 10 m 8 mol L m 1 for SWV and FIA respectively, were achieved. Repeatability was calculated by assessing the relative standard deviation (%) for 10 consecutive determinations of one sample. The found values were 2.1% for SWV and 5.0% for FIA. Validation of the results provided by SWV and FIA methodologies was performed by comparison with results from an HPLC-DAD technique. Good relative deviations were found (< 5%). Recovery trials were performed to assess the accuracy of the results and the obtained values were between 84% and 107% for both methods.     

A Nonenzymatic Amperometric Method for Fructosyl‐Valine Sensing Using Ferroceneboronic Acid

Fructosyl valine (Fru‐Val) is a glycosylated component of hemoglobin (HbA_1c) that can serve as a diagnostic target for type 2 diabetes. While average blood glucose levels fluctuate significantly, the more stable levels of HbA_1c can serve as a better long‐term diagnostic marker. Here a diagnostic system, incorporating an amperometric method, for detecting Fru‐Val (at +0.1 V vs. Ag/AgCl), using ferrocene boronic acid (FcBA) is presented. FcBA can complex diols, and has easily detectable redox properties. The boronic acid group in FcBA mediates complexation, while the Fe(II)/Fe(III) couple serves as a transducer. The diagnostic system, based on a miniaturized bare glassy carbon paste electrode (GCPE), has a fast response time.     

Superoxide sensor based on cytochrome c immobilized on mixed-thiol SAM with a new calibration method

Cytochrome c was immobilized on a mixed-thiol (mercaptoundecanoic acid/mercaptoundecanol) modified gold electrode (MUA:MU/cyt c electrode). Characterization of the cyt c electrode showed a quasi-reversible, electrochemical redox behavior with a formal potential of −13±5 mV (versus Ag/AgCl) for the surface adsorbed protein and 3±5 mV for covalently immobilized cyt c. The heterogeneous electron transfer rate constants were determined to be about 70 and 40 s⁻¹ for both states of the protein, respectively. They were found to be significantly higher than those of pure MUA-modified cyt c electrodes (MUA/cyt c electrodes). The interaction of superoxide radicals (O₂⁻) with the (MUA:MU)/cyt c electrode was characterized and used for an amperometric O₂⁻ detection. The influence of H₂O₂ and uric acid on the sensor signal was investigated. The sensitivity of the (MUA:MU)/cyt c electrode to O₂⁻ was significantly improved compared with that of the MUA/cyt c electrode. Based on a kinetic model for the superoxide detection system, a new calibration method was established. This simple and fast method used the spontaneous dismutation of KO₂ and was compared with the enzymatic superoxide generation system using xanthine oxidase.     

Development of a long-life capillary enzyme bioreactor for the determination of blood glucose

A long-life capillary enzyme bioreactor was developed that determines glucose concentrations with high sensitivity and better stability than previous systems. The bioreactor was constructed by immobilizing glucose oxidase (GOx) onto the inner surface of a 0.53 mm i.d. fused-silica capillary that was part of a continuous-flow system. In the presence of oxygen, GOx converts glucose to gluconic acid and hydrogen peroxide (H₂O₂). Hydrogen peroxide detection was accomplished using an amperometric electrochemical detector. The integration of this capillary reactor into a flow-injection (FIA) system offered a larger surface-to-volume ratio, reduced band-broadening effects, and reduced reagent consumption compared to packed column in FIA or other settings. To obtain operational (at ambient temp) and storage (at 4 °C) stability for 20 weeks, the glucose biosensing system was prepared using an optimal GOx concentration (200 mg/mL). This exhibited an FIA peak response of 7 min and a detection limit of 10 μM (S/N = 3) with excellent reproducibility (coefficient of variation, CV < 0.75%). It also had a linear working range from 10¹ to 10⁴ μM. The enzyme activity in this proposed capillary enzyme reactor was well maintained for 20 weeks. Furthermore, 20 serum samples were analyzed using this system, and these correlated favorably (correlation coefficient, r² = 0.935) with results for the same samples obtained using a routine clinical method. The resulting biosensing system exhibited characteristics that make it suitable for in vivo application.     

直接竞争荧光免疫法测定中药材中的黄曲霉毒素B1

本文采用Mannich反应合成黄曲霉毒素B1(AFB1)人工抗原,免疫小鼠制备AFB1单克隆抗体。采用直接搅拌法将异硫氰酸荧光素(FITC)标记AFB1抗体,经Sephadex-50凝胶柱纯化,制得FITC-AFB1荧光标记抗体,分析其免疫学特性,从而建立了一种快速灵敏的直接竞争荧光免疫分析方法(FIA)以用于检测中药材中的AFB1含量。结果表明,AFB1-FITC标记抗体的结合比率为4.19。通过对检测体系多项影响因素的筛选优化,FIA检测方法的标准曲线方程为I=33.45 log C+25.55,R=0.9913,线性检测范围1~100 ng/m L,检测限0.69ng/m L,回收率90.4%~106.6%。该方法具有操作简单、快速灵敏、特异性高等特点,可用于中药材中AFB1的分析测定。     

Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test

To elucidate the protein–protein interactions of hemoglobin (Hb) variants A and A₂, HbA was first shown to bind with HbA₂ in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA₂ outside the RBC was shown by cross electrophoresis. The starch–agarose gel electrophoresis of hemolysate, RBCs, freeze‐thawed RBCs and the supernatant of freeze‐thawed RBCs showed that the interaction between HbA and HbA₂ was affected by membrane integrity. To identify the proteins involved in the interaction, protein components located between HbA and HbA₂ in RBCs (RBC HbA‐HbA₂) and hemolysate (hemolysate HbA‐HbA₂) were isolated from the starch–agarose gel and separated by 5–12% SDS‐PAGE. The results showed that there was a ≈22 kDa protein band located in the RBC HbA‐HbA₂ but not in hemolysate HbA‐HbA₂. Sequencing by LC/MS/MS showed that this band was a protein complex that included mainly thioredoxin peroxidase B, α‐globin, δ‐globin and β‐globin. Thus, using our unique in vivo whole blood cell electrophoresis release test, Hbs were proven for the first time to interact with other proteins in the live RBC.     

A new screening procedure for the estimation of oxidizable organic compounds in water samples

Summary Immobilized lead dioxide (supported on SiO₂) has been used as the packing material in a solid phase reactor for the oxidation of organic compounds in water samples by flow injection analysis (FIA). On-line oxidation takes place in a FIA-system; this allows the detection of mobilized Pb²⁺ either photometrically, after complex formation with 4-(2-pyridylazo)-recorcinol (PAR), or directly with flame-AAS. The oxidation yield is quite different (0–100%) for a variety of organic compounds; however, calibration was possible in all cases investigated. Thus the systems can be used for the screening of polluted waters and as a post-column chemical-reaction detector (e.g. after HPLC-separation of organic compounds). After modification the FIA determination of COD equivalent values should be possible.     

纳米CoHCF修饰电极的制备及其对血红蛋白的电化学测定研究

A cobalt hexacyanoferrate （CoHCF） nanoparticle （size ca. 60 nm） chemically modified electrode （CME） was fabricated and the electrochemical behavior of hemoglobin （Hb） at this nanosized CoHCF CME was studied. In comparison with a bare glassy carbon electrode （GCE） and a general CoHCF CME electrodeposited in a traditional manner, the present nanosized CoHCF CME performed efficiently electrocatalytic reduction for Hb with relatively high sensitivity, stability, and longlife, Combined with liquid chromatography （LC）, the nanosized CoHCF CME was used as the electrochemical detector of Hb in the established flow injection analysis-electrochemical determination （FIA-ECD） system. The peak current was a linear function of concentrations in the range from 2.5×10^-8 to 5.0×10^-6mol/L for Hb, with detection limit of 1.4×10^-8 mol/L. The FIA-ECD system has been successfully applied to assess the Hb content of clinic blood samples with advantages of sensitiveness, speediness, easy control and small sample-consumption.     

纳米ZnO修饰电极的制备及其对血红蛋白, 细胞色素c的直接电化学研究

A novel electrochemical method as a sensitive and convenient technique for the determination of heme proteins based on their interaction with ZnO nanorods was developed. A ZnO nanorod modified glassy carbon electrode (ZnO/GCE) was prepared and the electrochemical behaviors of heme proteins, such as hemoglobin (HB) and cytochrome c (Cyt-c), on this modified electrode have been studied. The results showed that both HB and Cyt-c could be oxidized on the modified electrode and the oxidation currents were linear to the concentrations of the analytes in aqueous solutions. In addition, the results of flow injection analysis (FIA) further suggested the high stability and reproducibility of the ZnO nanorod modified electrode. So this method can be applied to the determination of HB and Cyt-c in biological systems.     

低温合成超导体Ba1-xKxBiO3

先以BaCO₃和Bi₂O₃为原料由传统的固相反应制备纯BaBiO₃,然后在低温下将用KOH-KF熔盐处理这样的拓扑反应合成了Ba_1-xK_xBiO₃超导体. 所有样品均进行了粉末X射线衍射（XRD）和磁性表征. XRD结果表明,所得Ba_1-xK_xBiO₃样品均为纯相,且均可用赝立方晶胞指标化. 磁性测量表明所有样品具有超导电性,最高超导转变温度（T_c）为30.6 K. 讨论了反应时间、前驱体与熔盐质量比对超导转变温度的影响. 最佳的反应条件为：反应温度450 ℃,反应时间4 h,BaBiO₃：KOH：KF质量比1：5：2.5.     

Role of proficiency testing in monitoring of standardization of hemoglobin A1c methods

After Hemoglobin A_1c (HbA_1c), therapeutic targets for monitoring diabetes therapy were recommended, first, National Glycohemoglobin Standardization Program (NGSP), then, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) developed standardization initiatives. The aim of this article is to demonstrate the role of a proficiency testing (PT) programs in monitoring the long-term effect of these initiatives and the current status of HbA_1c measurement. Measurement precision as a coefficient of variation (CV), measurement bias (bias), and satisfactory HbA_1c result rates in proficiency testing (PT) surveys were evaluated using fresh single donor whole blood PT items and assigned values from a NGSP-certified secondary reference laboratory. Between 2000 and 2010, both CV and bias of the IC measurement method showed a decreasing trend. While the CV of the HPLC measurement method decreased, no significant change was observed in its bias. The rates of satisfactory HbA_1c results in PT surveys were higher in HPLC users than IC users. In 2010, the average CVs in HPLC and IC groups were 2.6 and 3.4?%, biases were 2.7 and 1.8?%, and corresponding total error (TE) estimates were 7.8 and 8.5?%, respectively. These TE values were higher than the maximum permissible measurement error of 7?%, developed based on clinical use of the test. The NGSP and the IFCC networks have promoted improvements in HbA_1c testing; however, tightening of NGSP method certification criteria seems to be necessary to achieve a maximum permissible measurement error of 7?%.     

Sodium and silver phosphate glasses doped with chlorides of Fe,Mn and Zn

A number of samples of sodium and silver phosphate glasses doped with various compositions of some transition metals viz. iron, manganese and zinc chlorides alongwith undoped samples of sodium and silver phosphate glasses were synthesized and characterized by X-ray diffraction, IR spectral, electrical conductivity and differential scanning calorimetry (DSC). The glass transition temperature (T _g) and crystallization temperature (T _c) values obtained from DSC curves were found to increase with increasing concentration of the dopant Fe/Mn/Zn chlorides in both sodium and silver phosphate glasses and the following sequence is observed: T _g(–FeCl₃)>T _g(–MnCl₂)>T _g(–ZnCl₂) T _c(–FeCl₃)>T _c(–MnCl₂)>T _c(–ZnCl₂) The increase in T _g and T _c values indicate enhanced chemical durability of the doped glasses. The electrical conductivity values and the results of FTIR spectral studies have been correlated with the structural changes in the glass matrix by the addition of different transition metal cations as dopants.     

The results of HbA1c measurement and its comparison with reference method values in an EQA programme

This article describes the effect of a change in reference method for HbA_1c measurement on results in our EQA programme. Important indicators of analytical quality, particularly reproducibility, bias and uncertainty, were assessed. Basic analytical quality indicators for the measurement of HbA_1c in an EQA survey during 2000–2008 were evaluated. Three EQA surveys per year were performed. Usually there were more than 200 participating laboratories in each survey. Reproducibility, systematic differences, bias, and methodology changes before and after IFCC reference method implementation were determined. In the EQA surveys, target values traceable to the IFCC reference method are used. Estimation of the combined uncertainty of the result was a voluntary part of the surveys and was reported by 50% of participating laboratories. Reproducibility of all participants’ measurements was close to the target value of CV = 5.0% (coefficient of variation). The group of participants using HPLC methodology had reproducibility lower than 5.0% in each survey, whilst the immunochemistry group usually had CV values more than 6.0%. The differences in individual results from target values ranged in the interval ±12% for HPLC methods but more (−8 to +30%) for immunochemistry methods. During the monitored time interval the number of participants using HPLC methods increased up to 70% whilst the number of participants using immunochemistry methods decreased approximately to 30%. The combined uncertainty (u _c ) reported by participants varies between 4.0 and 4.5% for HbA_1c values between 30 and 90 mmol/mol. No significant differences between precision and systematic errors (bias) after the implementation of IFCC method of measurement for HbA_1c to routine processes for HPLC were observed. Immunochemical methods do not fulfil requirements for analytical reproducibility.     

Direct spectroelectrochemistry of peroxidases immobilised on mesoporous metal oxide electrodes: Towards reagentless hydrogen peroxide sensing

In this paper, we employ two peroxidases (horseradish peroxidase, HRP and cytochrome c peroxidase, CcP) to demonstrate their ability to retain their redox and biological functions after their immobilisation on mesoporous TiO₂ and SnO₂ electrodes. We will also demonstrate the use of HRP immobilised on the metal oxide electrodes for the development of reagentless optical and electrochemical biosensors for the detection of hydrogen peroxide (H₂O₂) with low detection limit of 0.04 and 1 μM, respectively.