A micro-potentiometric hemoglobin immunosensor based on electropolymerized polypyrrole–gold nanoparticles composite

We report a novel micro-potentiometric hemoglobin (Hb) immunosensor based on electrochemically synthesized polypyrrole (PPy)–gold nanoparticles (AuNPs) composite. PPy–AuNPs film with AuNPs uniformly distributed in it was deposited on gold electrode surface by a simple and direct procedure, without the addition of any nanoparticles or reducing agent. And this generic method makes it possible to deposite different polymers on miniaturized electrodes. With the existence of AuNPs, the antibody immobilization onto the electrode surface was facilitated. Morphology study by field emission scanning electron microscope (FE-SEM) confirms the presence of AuNPs in PPy. Based on an ion-sensitive field-effect transistors (ISFETs) integrated chip, a micro-potentiometric immunosensor for Hb and hemoglobin-A1c (HbA1c) has been constructed. The sensor response was linear over the concentration range 60–180 μg/ml Hb and 4–18 μg/ml HbA1c. The Hb concentration in whole blood samples has also been analysed, with a linear dose–response behavior between 125 and 197 μg/ml and a sensitivity of 0.20 mV μg⁻¹ ml. The measuring ranges of the developed Hb and HbA1c immunosensors meet the clinical demand for measuring the HbA1c/Hb ratio of 5–20%. This sensor results in simple and rapid differential measurement of Hb and HbA1c, and has great potential to become an inexpensive and portable device for monitoring of diabetes.     

Study of mixed Langmuir-Blodgett films of immunoglobulin G/amphiphile and their application for immunosensor engineering

Langmuir-Blodgett (LB) technique appears to be quite suitable for generating biospecific surfaces and it has potential application for fabricating biosensors. In this work, mixed Langmuir-Blodgett films of immunoglobulin G/amphiphile have been transferred onto hydrophobic silver surface previously modified by 1-octadecanethiol (ODT) SAMs. In order to obtain stable LB films, the influences of different parameters - type of amphiphile, surface pressure and pH - on the properties of mixed IgG/amphiphile monolayer, were investigated. Electrochemical properties of the engineered immunosensor have been measured by impedimetric spectroscopy. The immunosensor obtained exhibits a high sensitivity and a good specificity in a linear dynamic range from 200 to 1000 ng ml(-1).     

Gold nanoparticles-coated magnetic microspheres as affinity matrix for detection of hemoglobin A1c in blood by microfluidic immunoassay

A novel microfluidic immunoassay system for specific detection of hemoglobin A1c (HbA1c) was developed based on a three-component shell/shell/core structured magnetic nanocomposite Au/chitosan/Fe(3)O(4), which was synthesized with easy handling feature of Fe(3)O(4) by magnet, high affinity for gold nanoparticles of chitosan and good immobilization ability for anti-human hemoglobin-A1c antibody (HbA1c mAb) of assembled colloidal gold nanoparticles. The resulting HbA1c mAb/Au/chitosan/Fe(3)O(4) magnetic nanoparticles were then introduced into microfluidic devices coupled with a gold nanoband microelectrode as electrochemical detector. After that, three-step rapid immunoreactions were carried out in the sequence of HbA1c, anti-human hemoglobin antibodies (Hb mAb) and the secondary alkaline phosphatase (AP)-conjugated antibody within 20 min. The current response of 1-naphtol obtained from the reaction between the secondary AP-conjugated antibody and 1-naphthyl phosphate (1-NP) increased proportionally to the HbA1c concentration. Under optimized electrophoresis and detection conditions, HbA1c responded linearly in the concentration of 0.05-1.5 μg mL(-1), with the detection limit of 0.025 μg mL(-1). This system was successfully employed for detection of HbA1c in blood with good accuracy and renewable ability. The proposed method proved its potential use in clinical immunoassay of HbA1c.     

The investigation of recognition interaction between phenylboronate monolayer and glycated hemoglobin using surface plasmon resonance

Glycated hemoglobin (HbA1c) is formed by a nonenzymatic reaction of glucose with the N-terminal valine of adult hemoglobin's beta-chain. The amount of HbA1c reflects the average concentration of glucose variation level over the preceding 2 to 3 months. Because the boronate has antibody mimicking for HbA1c, often it is used to detect HbA1c. However, factors such as the ratio of the phenylboronic acid derivatives and diol composition, the pH of the solution, and the stereostructure of phenylboronic acid derivatives could influence the interactions between phenylboronic acid derivatives and diol composition. In this study, the factors were evaluated using surface plasmon resonance (SPR). The results show that pH value is an important factor affecting HbA1c and phenylboronic acid to form the complex and Lewis bases. This could change the stereostructure of phenylboronic acid to form B(OH)(3) for binding with saccharine easily. In addition, linear response appeared in HbA1c in the range of 0.43 to 3.49 mug/ml, and the detection limit was 0.01 microg/ml. The results also demonstrated that an SPR biosensor can be used as a sensitive technique for improving the accuracy and correctness of HbA1c measurement.     

Bioelectrocatalytic detection of glycated hemoglobin (HbA1c) based on the competitive binding of target and signaling glycoproteins to a boronate-modified surface

We developed an electrochemical glycated hemoglobin (HbA(1c)) biosensor for diagnosing diabetes in whole human blood based on the competitive binding reaction of glycated proteins. Until now, no studies have reported a simple and accurate electrochemical biosensor for the quantification of HbA(1c) in whole blood. This is because it is very difficult to correctly distinguish HbA(1c) from large amounts of hemoglobin and other components in whole blood. To detect glycated hemoglobin, we used electrodes modified with boronic acid, which forms a covalent bond between its diol group and the cis-diol group of the carbohydrate moiety of glycated proteins. For accurate HbA(1c) biosensing, we first removed blood components (except for hemoglobin) such as glycated proteins and blood glucose as they interfere with the boronate-based HbA(1c) competition analysis by reacting with the boronate-modified surface via a cis-diol interaction. After hemoglobin separation, target HbA(1c) and GOx at a predetermined concentration were reacted through a competition onto the boronate-modified electrode, allowing HbA(1c) to be detected linearly within a range of 4.5-15% of the separated hemoglobin sample (HbA(1c)/total hemoglobin). This range covers the required clinical reference range of diabetes mellitus. Hence, the proposed method can be used for measuring %HbA(1c) in whole human blood, and can also be applied to measuring the concentration of various glycated proteins that contain peripheral sugar groups.     

Novel potentiometric immunosensor for hepatitis B surface antigen using a gold nanoparticle-based biomolecular immobilization method

A novel potentiometric immunosensor for the detection of hepatitis B surface antigen has been developed by means of self-assembly to immobilize hepatitis B surface antibody on a platinum disk electrode based on gold nanoparticles, Nafion, and gelatin as matrices in this study. The modification procedure of the immunosensor was further characterized by using cyclic voltammetry and the enzyme-linked immunosorbent assay (ELISA) method. The detection is based on the change in the electric potential before and after the antigen-antibody reaction. In contrast to the commonly applied methods (e.g., the glutaraldehyde crosslinking procedure), this strategy could allow for antibodies immobilized with a higher loading amount and better retained immunoactivity, as demonstrated by the potentiometric measurements. A dynamic concentration range of 4-800 ng ml(-1) and a detection limit of 1.3 ng ml(-1) were observed. Analytical results of several human serum samples obtained using the developing technique are in satisfactory agreement with those given by ELISA. In addition, the technique presents some distinct advantages over the traditional sandwich format in that the analyzing performances are direct, rapid, and simple without multiple separation and labeling steps.     

Effect of nonenzymatic glycation on functional and structural properties of hemoglobin

HbA(1c), the major glycated hemoglobin increases proportionately with blood glucose concentration in diabetes mellitus. H(2)O(2) promotes more iron release from HbA(1c) than that from nonglycated hemoglobin, HbA(0). This free iron, acting as a Fenton reagent, might produce free radicals and degrade cell constituents. Here we demonstrate that in the presence of H(2)O(2), HbA(1c) degrades DNA and protein more efficiently than HbA(0). Formation of carbonyl content, an index of oxidative stress, is higher by HbA(1c). Compared to HbA(0), HbA(1c) is more rapidly autooxidized. Besides these functional changes, glycation also causes structural modifications of hemoglobin. This is demonstrated by reduced alpha-helix content, more surface accessible hydrophobic tryptophan residues, increased thermolability and weaker heme-globin linkage in HbA(1c) than in its nonglycated analog. The glycation-induced structural modification of hemoglobin may be associated with its functional modification leading to oxidative stress in diabetic patients.     

Electrochemical detection of HbA1c, a marker [correction of maker] for diabetes, using a flow immunoassay system

An on-chip electrochemical flow immunoassay system for the detection of hemoglobin A1c (HbA1c) was developed using anti-human hemoglobin (Hb) IgG labeled with ferrocene monocarboxylic acid (Fc-COOH) and boronate-affinity chromatography. An on-chip column packed with boronate-activated agarose beads was used for the separation of HbA1c from both non-glycated Hb and free antibody. Anti-human Hb IgG conjugated to Fc-COOH (Fc-IgG) was used for the electrochemical detection of HbA1c. The assay procedure included immunoreactions with Fc-IgG and HbA1c, separation of immunocomplexes by boronate affinity, and electrochemical detection of Fc-IgG-HbA1c immunocomplexes. The immunoreaction mixtures were injected onto a boronate-affinity column. HbA1c-antibody complexes were then trapped onto the column by the affinity of HbA1c to boronic acid. Subsequently, elution buffer containing sorbitol was applied to elute HbA1c-antibody complexes and a current was detected by applying 600 mV versus Ag/AgCl. The elution signal was an estimation of the HbA1c amount. A linear correlation between the increase of current and HbA1c concentration was obtained up to an HbA1c concentration of 500 microg/ml. The HbA1c flow immunoassay was successfully achieved using hemolysates. This electrochemical flow immunoassay system enabled us to construct a novel point-of-care testing device for the monitoring of glycated proteins including HbA1c.     

Covalently coupling the antibody on an amine-self-assembled gold surface to probe hyaluronan-binding protein with capacitance measurement

Hyaluronan-binding proteins (HABPs), the important structural components of extracellular matrices, served important structural and regulatory functions during development and in maintaining adult tissue homestats. A sensitive, specific and rapid-responsing immunosensor to probe hyaluronan-binding cartilage protein was presented in this work. The novel immunosensor supplied a label-free detection method for HABP, which was based on measuring the capacitance change in-between the unlabeled HABP (antigen) and rabbit-anti-HABP (Ra-HABP, antibody). The HABP immunosensor was prepared by covalently coupling Ra-HABP on an amine-self-assembled gold surface with glutaraldehyde. The capacitance change corresponding to the concentration of HABP, the target antigen, was evaluated by an electrochemical approach called potentiostatic-step in microseconds. The immunosensor showed a specific response to HABP in the range 10-1000 ng/ml. The presented work supplied a promising clinical screening method.     

An impedimetric immunosensor for the label-free detection of bisphenol A

Label-free detection of bisphenol A based on the impedance measurement was achieved with an impedimetric immunosensor. The immunosensor was fabricated by the covalent bond formation between a polyclonal antibody and a carboxylic acid group functionalized onto a nano-particle comprised conducting polymer. By using a commercial reagent 4,4-bis(4-hydroxyphenyl) valeric acid (BHPVA), which has an analogous structure of BPA, we have prepared the antigen through the conjugation of BHPVA with bovine serum albumin (BSA) and then produced a specific polyclonal antibody. The immobilization of antibody and the interaction between antibody and antigen were studied using quartz crystal microbalance (QCM) and electrochemical impedance spectroscopic (EIS) techniques. The impedance and mass changes due to the specific immuno-interaction at the sensor surface were utilized to detect antigen and bisphenol A (BPA). The immunosensor showed specific recognition of BPA with less interference than 4.5% from other common phenolic compounds. Under an optimized condition, the linear dynamic range of BPA detection was between 1 and 100 ng/ml. The detection limit of bisphenol A was determined to be 0.3+/-0.07 ng/ml. The proposed immunosensor was applied to a human serum sample and the BPA concentration was determined by the standard addition method.     

Novel approach for the analysis of glycated hemoglobin using capillary isoelectric focusing with chemical mobilization

In this work, a novel CIEF methodology for the analysis of the glycated hemoglobin, HbA(1c), in dimethylpolysiloxane coated fused-silica capillaries (DB-1, 50 microm I.D., 27 cm, 0.20 microm coating thickness), using a narrow pH ampholyte mixture (4% pH 6-8:pH 3-10, 10:1, v/v) in 0.30% methylcellulose, was developed. In the focusing procedure, a 0.100-mol l(-1) phosphoric acid solution was used as anolyte and a 0.040-mol l(-1) NaOH solution was used as catholyte. During method development, two types of mobilization of the focused hemoglobins were tested: pressure and chemical mobilization. Chemical mobilization performed better, allowing the complete baseline resolution of the hemoglobin of interest, HbA(1c), from its adjacent peak, HbA, in less than 8 min. In the chemical mobilization procedure, the catholyte was replaced by a 0.040-mol l(-1) NaOH solution containing 0.080 mol l(-1) NaCl. The proposed methodology was applied to the analysis of 31 hemolysate samples and validated with respect to the selectivity, inter-assay and intra-assay precision (both migration time and hemoglobin percentage concentration). In addition, HbA(1c) determinations were compared for the CIEF method and a chromatographic standardized procedure using cation-exchanger columns (Variant, Bio-Rad), adopted in a local clinical laboratory, showing excellent correlation (r(2)=0.872, n=31). The slope was found to be statistically equal to one but the intercept differed from zero. Also the Bland-Altman plot indicates bias, implying that the CIEF method yields HbA(1c) concentration higher than the reference method. The separation of the hemoglobins HbA, HbA(2), HbF and HbA(1c) and the variants HbS and HbC was also demonstrated (8 min run). The resolving power of the proposed CIEF method allowed baseline resolution of hemoglobins with a pI difference as small as ca. 0.03, as it is the case for the pairs HbC/HbA(2) and HbA/HbA(1c).     

Continuous-flow fluoro-immunosensor for paclitaxel measurement

A fluorescence-based continuous-flow immunosensor for sensitive, precise, accurate and fast determination of paclitaxel was developed. The sensor utilizes anti-paclitaxel antibody immobilized through its Fc region and crosslinked by dimethylpimelimidate to protein A attached covalently onto the silanized inner walls of a glass capillary column followed by saturation of the paclitaxel-binding sites with rhodamine-labeled paclitaxel. The assay is based on the displacement and detection downstream of the rhodamine-labeled paclitaxel, by a flow-through spectrofluorometer, as a result of the competition with paclitaxel introduced as a pulse into the stream of carrier buffer flowing through the system. The peak height of the fluorescence intensity profile of the displaced rhodamine-labeled paclitaxel was directly proportional to the concentration of paclitaxel applied and was a function of the carrier buffer flow rate. The sensitivity of the immunosensor response ranged from 0.31 relative fluorescence units (RFU)/ng/ml at a flow rate 0.1 ml/min to 0.52 RFU/ng/ml at 1 ml/min, while the lower detection limit ranged from 1 ng/ml at 0.1 ml/min to 4 ng/ml at 1 ml/min. The immunosensor response was very reproducible (RSD=4.8%; n=10) and linear up to 100 ng/ml. The assay time ranged from 2 min at 1 ml/min to 8 min at 0.1 ml/min. A technique developed to resaturate the antigen binding sites of the immobilized antibody with rhodamine-labeled paclitaxel was successful in regenerating the capillary column without affecting its performance, thus enhancing the economic viability of the immunosensor. The immunosensor was successfully applied for the determination of paclitaxel in human plasma.     

Amperometric immunosensor for the determination of IgA deficiency in human serum samples

An electrochemical immunosensor for the detection of human IgA deficiency in real human blood serum has been developed. The performance of the immunosensor presents a large but sensitive dynamic range that allows the determination of non-deficient IgA levels (>70 μg/mL) as well as of severe IgA deficiencies (0.5-5.0 μg/mL). The assay architecture involves the immobilisation of a coating antibody on an electrode surface using carboxylic-ended bipodal alkane-thiol self-assembled monolayers (SAMs). The long chain bipodal SAM presents intercalated poly(ethylenglycol) groups that confer the immunosensor the ability to retain its optimum performance in very complex matrices and serum with negligible non-specific adsorption phenomena. Amperometric optimisation of the assay resulted in limits of detection of 142 ng/mL in just 30 min total assay time. Real patients' serum samples were analysed using the developed electrochemical immunosensor demonstrating an excellent correlation in terms of sensitivity and reproducibility compared with standard enzyme linked immunosorbent assays (ELISA).     

A label-free immunosensor by controlled fabrication of monoclonal antibodies and gold nanoparticles inside the mesopores

A label-free immunosensor for the detection of α-fetoprotein (AFP) is proposed based on controlled fabrication of monoclonal antibodies of AFP (anti-AFP) and gold nanoparticles (GNPs) inside the pores of mesoporous silica (MPS). The silanol groups on the internal pore walls were grafted by aminopropyltriethoxyl silane, whereas the silanol groups on the external surface of MPS were blocked by trimethylchlorosilane (TMCS). Thus, anti-AFP and GNPs could be confined inside the mesopores of TMCS-MPS by the covalent linking with the amino groups. The prepared anti-AFP/GNPs/TMCS-MPS particles were used to modify glassy carbon electrode (GCE) to construct a label-free immunosensor. After incubating the sample AFP with the anti-AFP/GNPs/TMCS-MPS/GCE, the immunoconjugates were formed on the surface of GCE and the spatial block increased. Thus, the peak current decreased with increasing concentrations of AFP. GNPs inside the mesopores could promote the electron transportation through the pore channel. Under the optimal experimental conditions, the fabricated immunosensor could detect AFP in a linear range from 1.0 to 90 ng ml(-1) with a detection limit of 0.2 ng ml(-1) (3σ). It provided a novel alternative method for the label-free determination of other antigens.     

Detection of sulfamethoxazole by a piezoquarz immunosensor]

A mass susceptible immunosensor for FIA of sulfamethoxazole residues in liquid products was designed. The immunosensor is based on piezoelectric transducer. Hapten-protein conjugate (SMX-Diazo-BSA) immobilized on the preliminarily silanized electrode surface of piezoelectric quartz crystal was used as the bioreceptor coating. Optimization of the FIA conditions permitted to develop a simple and express procedure for one-step detection of sulfamethoxazole in a sample and further regeneration of the bioreceptor layer. The measuring ranges are 1 to 50 ng/ml and the detection limit is 0.15 ng/ml. The detection results were compared with the HPLC data. The advantages of the new procedure are its simplicity and rapid provision of the analysis results, possible direct detection of the analyte without additional label and repeated use of the bioreceptor layer. The new immunosensor was applied to testing of various milk specimens. It was shown that the quantity of sulfamethoxazole in all the specimens was lower than the recommended Euroresidue standards (100 ng/ml).     

A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice

A sensitive and simple electrochemical immunosensor based on enzymatic silver deposition amplification was constructed for the detection of aflatoxin B₁ (AFB₁) in rice. The immunosensor was based on an indirect competitive format between free AFB₁ and aflatoxin B₁-bovine serum albumin (AFB₁-BSA) conjugate immobilized on the electrode surface for binding to a fixed amount of anti-AFB₁ antibody. Then the alkaline phosphatase (ALP)-labeled anti-mouse immunoglobulin G (IgG) secondary antibody was bound to the electrode surface through reaction with primary antibody. Finally, ALP catalyzed the substrate, ascorbic acid 2-phosphate, into ascorbic acid that reduced silver ions in solution to metal silver deposited onto the electrode surface. Linear sweep voltammetry was carried out to quantify the metal silver, which indirectly reflected the amount of the analyte. The experimental parameters, such as the dilution ratio of antibody and the concentration of AFB₁-BSA conjugate, have been evaluated and optimized. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. So, the proposed method in this article could find a good use for screening AFB₁ in real samples.     

Disposable amperometric immunosensor for the detection of polycyclic aromatic hydrocarbons (PAHs) using screen-printed electrodes

An amperometric immunosensor for polycyclic aromatic hydrocarbons (PAHs) was developed. The immunosensor was based on disposable screen-printed carbon electrodes. The coating antigen used was phenanthrene-9-carboxaldehyde coupled to bovine serum albumin (BSA) via adipic acid dihydrazide. Antibodies were monoclonal mouse anti-phenanthrene. The enzyme alkaline phosphatase (AP) was used in combination with the substrate p-aminophenyl phosphate (pAPP) for detection at +300 mV (vs. Ag/AgCl). Various assay types were compared. Good results were achieved with an indirect co-exposure competition assay with a LOD of 0.8 ng/ml (800 ppt) and an IC(50) of 7.1 ng/ml (7.1 ppb) for phenanthrene. An indirect competition assay could detect phenanthrene with a LOD of 2 ng/ml (IC(50): 15 ng/ml) and an indirect displacement assay with a LOD of 2 ng/ml (IC(50): 11 ng/ml) at a 5 microl surface coating of 8.8 microg/ml phenanthrene-BSA conjugate. A coating concentration of 2.2 microg/ml allowed detection with a LOD of 0.25 ng/ml (250 ppt) with the indirect competition assay. The influence of the coating concentration on the sensor performance was investigated. Cross-reactivities were tested for 16 important PAHs. Anthracene and chrysene showed strong cross-reactivity, whereas benzo[g,h,i]perylene and dibenzo[a,h]anthracene showed no cross-reactivity.     

Enhanced performance of a surface plasmon resonance immunosensor for detecting Ab-GAD antibody based on the modified self-assembled monolayers

To enhance the feasibility of surface plasmon resonance (SPR) immunosensor as a tool for diagnosing type I diabetes, we enhanced the sensitivity of immunoresponse for detecting the monoclonal anti-glutamic acid decarboxylase (GAD) antibody by modification of mixed self-assembled monolayers (SAMs). The effects of the different mixed SAMs were evaluated with respect to the degree of streptavidin immobilization, the degree of biotin-GAD immobilization, and the immunoresponse sensitivity. Consequently, the sensitivity of the immunoresponse for the detection of anti-GAD antibody was enhanced as a result of the reduction in steric hindrance brought about by using SAMs of heterogeneous lengths. The immunoresponse for detecting the monoclonal anti-GAD antibody was also enhanced with the reduction of the excess immobilization of biotin-GAD and the minimization of non-specific binding that resulted from the simple substitution of the spacer from a carboxylic-terminated SAM for the hydroxyl-terminated SAM.     

Serum thyroglobulin concentration in patients with diabetes mellitus

The serum thyroglobulin (Tg) concentration was measured in 97 patients with diabetes mellitus (39 males, 58 females). Hyper Tg-nemia which exceeds the normal range (1.0-26.6 ng/ml) was observed in 10 patients (3 out of 21 cases treated with diet alone, 3 out of 50 cases treated with oral hypoglycemic agents, 4 out of 26 cases treated with insulin). There was no significant correlation between concentrations of serum Tg and triiodothyronine (T3), thyroxine (T4), fasting plasma glucose (FPG), and hemoglobin A1c (HbA1c). However, a positive correlation was observed between serum concentrations of Tg and thyroid stimulating hormone (TSH). Patients with diabetes were divided into three groups according to the mode of treatment (Group I; diet alone, n = 21, Group II; oral hypoglycemic agents, n = 50, Group III; insulin, n = 26). No significant difference in the serum Tg concentration was observed among the three groups. They were also divided into two groups; normal Tg-nemia (Group A, n = 87) and hyper Tg-nemia (Group B, n = 10). There was no difference between levels of T3, T4, FPG, and HbA1c in the two groups. The serum TSH concentration measured by double antibody RIA and two site immunoradiometric assay in Group B was significantly higher than that in Group A. These results suggest that hyper Tg-nemia in patients with diabetes could be due to the increased TSH concentration which reflects latent subclinical primary hypothyroidism in them.     

Effect of inositol hexakisphosphate on the spectroscopic properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA1c

The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA1c (HbA1cNO) has been investigated quantitatively. The results obtained show that 1) both in the absence and presence of IHP, the EPR and absorbance spectra of HbA1cNO show the same basic characteristics described for the nitrosyl derivative of ferrous HbA0, the nonglycated major component of human hemoglobin (HbA0NO); and 2) HbA1cNO binds IHP with an apparent dissociation equilibrium constant (upsilon = 1.8 x 10(-2) M), which is at least four orders of magnitude higher than that estimated for the polyphosphate interaction with HbA0NO (less than or equal to 3 x 10(-6) M). These data provide further independent evidence that interaction(s) of polyphosphates at the specific cleft between beta-chains along the dyad-axis is sterically hindered in HbA1c by the presence of the two glucose residues covalently bound to the N-termini of beta-chains, this finding being in agreement with the reduced effect of polyanions on HbA1c spectral and ligand-binding properties.