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.     

Testing the performance of pure spectrum resolution from Raman hyperspectral images of differently manufactured pharmaceutical tablets

Chemical imaging is a rapidly emerging analytical method in pharmaceutical technology. Due to the numerous chemometric solutions available, characterization of pharmaceutical samples with unknown components present has also become possible. This study compares the performance of current state-of-the-art curve resolution methods (multivariate curve resolution-alternating least squares, positive matrix factorization, simplex identification via split augmented Lagrangian and self-modelling mixture analysis) in the estimation of pure component spectra from Raman maps of differently manufactured pharmaceutical tablets. The batches of different technologies differ in the homogeneity level of the active ingredient, thus, the curve resolution methods are tested under different conditions. An empirical approach is shown to determine the number of components present in a sample. The chemometric algorithms are compared regarding the number of detected components, the quality of the resolved spectra and the accuracy of scores (spectral concentrations) compared to those calculated with classical least squares, using the true pure component (reference) spectra. It is demonstrated that using appropriate multivariate methods, Raman chemical imaging can be a useful tool in the non-invasive characterization of unknown (e.g. illegal or counterfeit) pharmaceutical products.     

HbA/H2O2: an efficient biomimetic catalytic system for the oxidation of sulfides to sulfoxides

Human hemoglobin (HbA) catalyzed chemo-selective oxidation of sulfides to sulfoxides has been reported using hydrogen peroxide as an oxidant in a phosphate buffer. This biomimetic catalytic procedure was found to be simple, environmentally friendly, selective, and high yielding. Purified human oxyhemoglobin (HbA₀), crude human oxyhemoglobin, and ferryl oxyhemoglobin were used for sulfoxidation. HbA₀ and ferryl oxyhemoglobin (Fe⁺⁴O) efficiently catalyze the sulfoxidation reaction without over oxidation to the sulfone.     

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.     

Analysis of the essential oils of Coriandrum sativum Using GC-MS coupled with chemometric resolution methods

The essential oils extracted from Coriandrum sativum L. were analyzed by GC-MS coupled with chemometric resolution methods. Through the chemometric resolution methods, peak clusters were uniquely resolved into the pure chromatographic profiles and mass spectra of each component. Qualitative analysis was performed by comparing the pure mass spectra with those in the NIST 05 mass spectral library. Quantitative analysis was performed using the total volume integration method. A total of 118 constituents were detected, of which 104 were identified, accounting for 97.27% of the total content. The results indicate that GC-MS combined with chemometric resolution methods can greatly enhance the capability of separation and the reliability of qualitative and quantitative results. The combined method is an economical and accurate approach for the rapid analysis of the complex essential oil samples in Coriandrum sativum L.     

Characterization of essential oil components of Iranian geranium oil using gas chromatography-mass spectrometry combined with chemometric resolution techniques

The essential oil components of geranium oil cultivated in center of Iran were identified and determined using gas chromatography-mass spectrometry data combined with the chemometric resolution techniques. A total of 61 components accounting for 91.51% were identified using similarity searches between the mass spectra and MS database. This number was extended to 85 components using chemometric techniques. Various chemometric methods such as morphological scores, simplified Borgen method (SBM) and fixed size moving window evolving factor analysis (FSMWEFA) were used for determining the number of components, pure variables, zero concentration and selective regions. Then the overlapping peak clusters were resolved into pure chromatograms and pure mass spectra using heuristic evolving latent projections (HELP) method. A characteristic feature of the Iranian geranium oil is the absence of 10-epi-gamma-eudesmol in its constituents compared with the oil from northern and southern parts of India. The results of this work show that combination of hyphenated chromatographic methods and resolution techniques provide a complementary method for accurate analysis of essential oils.     

Resolution of chemical species in flow injection analysis by using self-modeling curve resolution

Mixtures can be resolved into their components by separation, spectrometric and kinetic (chemical) procedures, which have varying degrees of efficiency. Chemometric methods of data processing have proved useful for increasing resolution, especially in spectral and chromatographic procedures. This study shows that chemometric methods are also useful for kinetic flow-injection methods based on chemical reactions combined with multichannel spectral detection, where neither the reaction nor the detector provides adequate resolution. The problem of background absorbance of a sample in a flow-injection system with an ultraviolet/visible photodiode array detector is solved by applying a self-modeling curve resolution method. The example used is a mixture of methyl violet and phenolphthalein.     

Chemometric resolution of ATR-IR spectra data for polycondensation reaction of bis(hydroxyethylterephthalate) with a combination of self-modeling curve resolution (SMCR) and local rank analysis

Self-modeling curve resolution (SMCR) methods, simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) and alternating least squares (ALS) were used to calculate pure concentration profiles and pure spectra for the two-way spectral data collected during the on-line polycondensation reaction of bis(hydroxyethylterephthalate) with an ATR-FT-IR spectrometer. In order to improve the resolution results, SIMPLISMA was combined with local rank analysis method, fixed size moving window evolving factor analysis (FSMWEFA) to search for selective regions of various components and then look for the purest wavenumber variables in the selective regions. Such combination allows more accurate determination of the number of chemical components in the reaction system and the calculations of more accurate concentration profiles and spectra.     

An automatic method for determination of glycosylated hemoglobins using low-pressure liquid chromatography

Several studies have revealed a correlation between blood levels of glucose and hemoglobin A1c (HbA1c), a minor form of hemoglobin (Hb) present at elevated concentrations in patients with diabetes mellitus. To facilitate a clinical study of the level of circulating HbA1c we have developed an automatic chromatographic system. An efficient separation of HbA1c from HbA0 and other rapid hemoglobins (HbA1a, HbA1b) was achieved on Bio-Rex-70 columns using three buffers. This system allows the daily analysis of 40 samples. The mean level of HbA1c in normal subjects was 5.4 +/- 0.4%. The method also detects the presence of elevated levels of HbF and the most frequent forms of abnormal hemoglobin (HbS, HbC).     

3-己炔-2, 5-二醇在镍电极上电还原反应研究

采用化学计量学中新近发展的二维数据分辨方法,直观推导式演进特征投影(HELP)算法,对3-己炔-2,5-二醇在镍电极上的电还原反应得到的混合物进行了定性定量研究,得到了各组分的纯物质质谱及浓度变化曲线,且可以得到反应动力学的合理解释。说明借助化学计量学可大大扩展色质联用分析的应用范畴。     

Determination of various hemoglobin species with thermal-lens spectrometry

Desoxyhemoglobin, oxyhemoglobin, carboxyhemoglobin, methemoglobin, cyanohemoglobin, and hemichrome are determined using thermal-lens spectrometry, with the detection limits at the level of 10^?8 M/l (2–5 mcg/l depending on hemoglobin species). Signal behavior and detectability thresholds are in good agreement with theoretical modeling based on the approach proposed earlier by the authors to describe thermal-lens signal generation in complex (inhomogeneous) systems. The thermooptical response for all studied hemoglobin species depends on the power of laser radiation within the range of 1–50 mW (532, 514.5, and 488 nm). Under the conditions of a thermal-lens experiment, the total temperature growth (0.0001 K) due to heating of the studied solution as the radiation of the inducing laser is absorbed by hemoglobin is estimated. Due to the interference with oxyhemoglobin the error in determining desoxyhemoglobin using thermal-lens spectrometry (for a maximum radiation power of 532 nm, 210 mW) does not exceed 3% in the case when the ratio of the species is 10: 1. In the opposite case (determination of oxyhemoglobin in the presence of desoxyhemoglobin), the error does not exceed 5%, the ratio of test and interfering species being the same.     

通过红外光谱结合化学计量学方法研究奥克托今的合成机理

通过红外(IR)光谱在线监测醋酐法合成奥克托今(HMX)的反应过程, 采用渐进因子分析(EFA)结合多元曲线分辨-交替最小二乘法(MCR-ALS)以及直观推导式演进特征投影法(HELP)等化学计量学方法对反应过程的光谱数据矩阵进行解析, 获得了各组分浓度变化曲线和对应的IR光谱; 并采用密度泛函理论(DFT)的B3LYP方法, 在6-31G^*基组水平上得到该化合物的全优化结构, 在振动分析的基础上求得体系的振动频率和IR光谱. 通过对比发现, MCR-ALS和HELP法可得出相互验证的一致结论; 将通过量子化学计算的中间体的IR光谱特征吸收峰与2种方法进行比较, 结果吻合, 从而推导出合理的反应机理. 实验结果表明, 化学计量学方法结合在线红外光谱是研究反应机理的有效手段, 对反应路线的选择具有指导意义.     

Relationship between selectivity and average resolution in comprehensive two-dimensional separations with spectroscopic detection

The average value of the multivariate selectivity (SEL) of randomly positioned peaks in a multi-component separation is shown to equal the average fraction of peaks that are singlets, as predicted by statistical-overlap theory (SOT). This equality is the basis for proposing a useful metric, specifically the average minimum resolution of nearest-neighbor peaks, for the performance of comprehensive two-dimensional (2D) separations. Furthermore this metric was computed both without ancillary spectroscopic information and with the assistance of such help, specifically multi-wavelength UV-vis spectra, acquired during the separation. Separations are simulated with randomly positioned peaks over wide ranges of total number of peaks, first- and second-dimension peak capacity, dimensionless first-dimension sampling time, and spectral diversity. The specific version of the general multivariate selectivity concept that is used here--identified as SEL--gives the relative precision of quantification when using the PARAFAC (parallel factor analysis) method, a popular curve resolution algorithm. The SEL values of all peaks were calculated, averaged, and compared to the predictions of SOT. In the absence of auxiliary spectral data, the SEL-based average minimum resolution required to separate two peaks in a 2D separation is 0.256, compared to resolution of 0.5 if no chemometric assistance is available. This was found to be valid over a wide range of conditions and is essentially independent of peak crowding. With the assistance of the spectral data, the requisite minimum resolution substantially improves, that is, it decreases, especially when peak crowding is severe. The requisite minimum resolution decreases even further, up to a limit, as the spectral diversity is increased. In contrast, the SEL-based average under-sampling correction factor is virtually independent of the presence of the additional spectral data, and additionally is about the same as calculated with SOT from the average number of maxima in closely analogous simulations. The use of selectivity greatly increases the fraction of peaks that are singlets, relative to the number of singlet maxima, especially when spectral assistance is added. The insensitivity of the under-sampling correction factor to either the use of selectivity or added spectral data simplifies optimization of the corrected peak capacity in on-line comprehensive 2D separations.     

Determination of volatile components in peptic powder by gas chromatography-mass spectrometry and chemometric resolution

Gas chromatography-mass spectrometry (GC-MS) coupled with chemometric resolution upon two-dimensional data was proposed as a method for the analysis of volatile components in a traditional Chinese medicinal preparation peptic powder which contains Rhizoma Atractylodis, Pericarpium Citri Reticulatae, Cortex Magnoliae Officinalis and Radix Glycyrrhizae. Ninety-three components were separated and 65 of them were qualitatively and quantitatively analyzed which represented about 90.28% of the total content. With the help of chemometric resolution, the data were resolved into a pure chromatogram and a mass spectrum of each chemical component. The accuracy of qualitative and quantitative results was greatly improved by using the two-dimensional comprehensive information of chromatograms and mass spectra. The example showed that chemometric resolution could greatly enhance separation ability. This makes it possible to analyze complicated practical systems like traditional Chinese medicinal preparations with the help of coupled instruments and chemometric resolution methods.     

The aqueous cationic system sodium undecenoate-dodecyltrimethylammonium bromide at low concentration

The aqueous sodium undecenoate (SUD) –dodecyltrimethylammonium bromide (DTAB) catanionic system was studied at low concentration. The system did not precipitate, even at a 1:1 SUD:DTAB proportion, but showed the formation of a coacervate in a range of surfactant mixture compositions. Micelles have a preferential composition of 0.37 mole fraction of SUD. This behavior is attributed to the presence of the double bond at the distal extreme of the SUD molecule, which can form hydrogen bonds with water. Consequently, the –CH=CH₂ group is situated at the interface between the hydrocarbon micelle core and water, reducing the interfacial free energy. Structural computations demonstrate that the mentioned SUD proportion produces complete coverage of the micelle surface by the double bonds.     

Interactions of Hemoglobin with Vesicles and Tubes Formed from Mixtures of Histidine-Derived Bolaamphiphile and Conventional Surfactants

The interactions between protein and surfactant aggregates have been the subject of intensive studies due to their potential applications in biological systems. Here we report the interactions of hemoglobin (Hb) with vesicles and tube-like aggregates formed from mixtures of a histidine-derived bolaamphiphile and a cconventional surfactant dodecyltrimethylammonium bromide (DTAB). This study was performed using a combination of UV–vis spectroscopy, steady and synchronous fluorescence spectroscopy, circular dichroism, and microcalorimetry measurements. The secondary structure of the protein is disturbed, and then the partially unfolded protein is capable of penetrating the vesicles and tube-like aggregates, this is mainly the result of hydrogen bonding and hydrophobic interactions between Hb and the H₂D/DTAB aggregates. The polar portion of the unfolded protein chains is near to the polar head of the amphiphile in the aggregate’s membrane. Hb is converted to hemichrome in the vesicles, and the heme monomer is solubilized in tube-like aggregates after escaping from the hydrophobic cavity of Hb.     

Alternative moving window factor analysis for comparison analysis between complex chromatographic data

In this investigation, a novel chemometric method is developed for the analysis of five possible relationships of components or spectral features between two correlative but different hyphenated chromatographic systems. It is very helpful for comparison study of components present in different complex systems in both chemistry and systems biology. The proposed method, named alternative moving window factor analysis (AMWFA), could be utilized to determine the number of common components between different samples and then to identify their corresponding spectra half-automatically. AMWFA can alternatively be employed to mind for the selective information hiding in anyone of the two compared data X and Y, and to self-verify the resolution results by changing the extracted target matrices in analysis. From the results of comparison of simulated hyphenated chromatographic data, volatile chemical components in drug pair rhizoma ligustici chuanxiong-radix paeoniae rubra (RLC-RPR) and its single herbal medicines, and analysis of Angelica oral solution and its plasma sample after oral intake to rabbit, powerful ability of the proposed method is shown.     

Comparative determination of phosphate and silicate using molybdenum blue by radial basis function and feed-forward neural networks assisted by principal component analysis.

In the present study, chemometric analysis of visible spectral data of phospho-and silico-molybdenum blue complexes was used to develop artificial neural networks (ANNs) for the simultaneous determination of the phosphate and silicate. Combinations of principal component analysis (PCA) with feed-forward neural networks (FFNNs) and radial basis function networks (RBFNs) were built and investigated. The structures of the models were simplified by using the corresponding important principal components as input instead of the original spectra. Number of inputs and hidden nodes, learning rate, transfer functions and number of epochs and SPREAD values were optimized. Performances of methods were tested with root mean square errors prediction (RMSEP, %), using synthetic solutions. The obtained satisfactory results indicate the applicability of this ANN approach based on PCA input selection for determination in highly spectral overlapping. The results obtained by FFNNs and by RBF networks were compared. The applicability of methods was investigated for synthetic samples, for detergent formulations, and for a river water sample.     

Resolution of overlapped peaks of amino acid derivatives in capillary electrophoresis using multivariate curve resolution based on alternating least squares

The application of chemometric techniques to the resolution of overlapped peaks in capillary electrophoresis (CE) is described. When a physical separation can not be completely accomplished, chemometrics might still resolve the determination of the analytes mathematically. CE with diode array detection can provide a large amount of data consisting of spectra registered over time. In this study, the capillary electrophoretic separation of 1,2-naphthoquinone-4-sulfonate derivatives of amino acids is studied. Most of the common amino acid derivatives can be separated at 30 kV in a fused-silica capillary by using a 40 mM sodium tetraborate + isopropanol (3:1 v/v) solution as background electrolyte. However, peaks of certain derivatives (Phe, His, Leu and Ile) still overlap. A multivariate curve resolution method based on an alternating least squares optimization procedure is used for the resolution of the overlapped electrophoretic peaks. The method takes advantage of spectral and electrophoretic differences of analytes to recover their pure electrophoretic and spectral profiles. In addition, each analyte in the mixture can be quantified using the corresponding standards.     

Online UV–visible spectroscopy and multivariate curve resolution as powerful tool for model-free investigation of laccase-catalysed oxidation

The laccase-catalysed transformation of indigo carmine (IC) with and without a redox active mediator was studied using online UV–visible spectroscopy. Deconvolution of the mixture spectra obtained during the reaction was performed on a model-free basis using multivariate curve resolution (MCR). Thereby, the time courses of educts, products, and reaction intermediates involved in the transformation were reconstructed without prior mechanistic assumptions. Furthermore, the spectral signature of a reactive intermediate which could not have been detected by a classical hard-modelling approach was extracted from the chemometric analysis. The findings suggest that the combined use of UV–visible spectroscopy and MCR may lead to unexpectedly deep mechanistic evidence otherwise buried in the experimental data. Thus, although rather an unspecific method, UV–visible spectroscopy can prove useful in the monitoring of chemical reactions when combined with MCR. This offers a wide range of chemists a cheap and readily available, highly sensitive tool for chemical reaction online monitoring.