Integrated multienzyme electrochemical biosensors for monitoring malolactic fermentation in wines

Integrated amperometric biosensors for the determination of l-malic and l-lactic acids were developed by coimmobilization of the enzymes l-malate dehydrogenase (MDH) and diaphorase (DP), or l-lactate oxidase (LOX) and horseradish peroxidase (HRP), respectively, together with the redox mediator tetrathiafulvalene (TTF), on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +100 mV (vs. Ag/AgCl), and the reduction of TTF⁺ at −50 mV were used for the monitoring of the enzyme reactions involved in l-malic and l-lactic acid determinations, respectively. Experimental variables concerning the biosensors composition and the detection conditions were optimized for each biosensor. Good relative standard deviation values were obtained in both cases for the measurements carried out with the same biosensor, with no need of cleaning or pretreatment of the bioelectrodes surface, and with different biosensors constructed in the same manner. After 7 days of continuous use, the MDH/DP biosensor still exhibited 90% of the original sensitivity, while the LOX/HRP biosensor yielded a 91% of the original response after 5 days. Calibration graphs for l-malic and l-lactic were obtained with linear ranges of 5.2 × 10⁻⁷ to 2.0 × 10⁻⁵ and 4.2 × 10⁻⁷ to 2.0 × 10⁻⁵ M, respectively. The calculated detection limits were 5.2 × 10⁻⁷ and 4.2 × 10⁻⁷ M, respectively. The biosensors exhibited a high selectivity with no significant interferences. They were applied to monitor malolactic fermentation (MLF) induced by inoculation of Lactobacillus plantarum CECT 748^T into a synthetic wine. Samples collected during MLF were assayed for l-malic and l-lactic acids, and the results obtained with the biosensors exhibited a very good correlation when plotted against those obtained by using commercial enzymatic kits.     

Recent developments, characteristics, and potential applications of electrochemical biosensors.

The objective of this study is to analyze the technical importance, performance, techniques, advantages, and disadvantages of the biosensors in general and of the electrochemical biosensors in particular. A product of reaction diffuses to the transducer in the first generation biosensors (based on Clark biosensors). The mediated biosensors or second generation biosensors use specific mediators between the reaction and the transducer to improve sensitivity. The second generation biosensors involve two steps: first, there is a redox reaction between enzyme and substrate that is reoxidized by the mediator, and eventually the mediator is oxidized by the electrode. No normal product or mediator diffusion is directly involved in the third generation biosensors, direct biosensors. Based on the type of transducer, current biosensors are divided into optical, mass, thermal, and electrochemical sensors. They are used in medical diagnostics, food quality controls, environmental monitoring, and other applications. These biosensors are also grouped under two broad categories of sensors: direct and indirect detection systems. Moreover, these systems could be further grouped into continuous or batch operation. Therefore, amperometric biosensors and their current applications are focused on more in detail since they are the most commonly used biosensors in monitoring and diagnosing tests in clinical analysis. Problems related to the commercialization of medical, environmental, and industrial biosensors as well as their performance characteristics, their competitiveness in comparison to the conventional analytical tools, and their costs determine the future development of these biosensors.     

Label-free electrochemical monitoring of vasopressin in aptamer-based microfluidic biosensors

Vasopressin is an indicating biomarker for blood pressure in the human body and low vasopressin levels can be indicative of late-phase hemorrhagic shock or other traumatic injuries. In this paper we have developed an aptamer-based label-free microfluidic biosensor for the electrochemical detection of vasopressin. The detection area consists of aptamers immobilized on carbon nanotubes which specifically capture the vasopressin molecules in solution resulting in changes in conductivity across the sensor. We report a limit of detection of 43 pM in standard solutions and demonstrate high detection specificity toward vasopressin when different interferents are present. The miniaturized microfluidic biosensor offers continuous monitoring of different vasopressin levels with good potential for portability. Ultimately such a system could serve as a point-of-care diagnostics tool for patients with excessive bleeding when standard medical infrastructure is not available.     

Graphene as signal amplifier for preparation of ultrasensitive electrochemical biosensors

Early diagnosis of diseases with minimal cost and time-consumption has become achievable due to recent advances in the development of biosensors. These devices use biorecognition elements for the selective interaction with an analyte and the signal read-out is obtained via different types of transducers. The operational characteristics of biosensors have been reported as improving substantially when a diverse range of nanomaterials is employed. This review presents the construction of electrochemical biosensors based on graphene, atomically thin 2D carbon crystals, a nanomaterial currently the subject of intensive studies. Here, the most attractive directions for graphene applications in biosensor preparation are discussed, including novel detection and amplification schemes exploiting graphene’s unique electrochemical, physical and chemical properties. There is probably a very bright future for graphene-based biosensors, but much further work is required to fulfill the high expectations.     

Structural diversity in phosphoramidate’s chemistry: Syntheses,spectroscopic and X-ray crystallography studies

Novel phosphorus compounds of bisphosphoramidate, phosphoramidate and phosphoric triamide derivatives were synthesized using the starting materials PCl₅ and POCl₃. The products were then characterized by ¹H, ¹³C, ³¹P, ¹⁹F NMR, IR spectroscopy and CHN elemental analysis. It is noticeable that the reaction of 4-aminobenzamide with PCl₅ in different molar ratios yields different products, bisphosphoramides and phosphoric triamides. Moreover, we were taken by surprise that the interaction of POCl₃ with the first-type aromatic amines gave bisphosphoramidates with P–N–P linkages that exhibited ²J(P,P) ≈ 20.0 Hz in the ³¹P NMR spectra. In fact, two simple one-pot pathways are presented here for the synthesis of new bisphosphoramidates, and to the best of our knowledge these are the first instances of bisphosphoramidates that have been obtained up until now. The structures of compounds I (4-OCH₃–C₆H₄–CH₂–C₉H₁₃–NH₂Cl), 34 and 44 were further determined by X-ray crystallography. All of these structures produced three dimensional polymeric chains through strong- and weak hydrogen bonds. The presence of chiral aminoacidester moieties in the phosphoric triamides lead to chiral molecules that showed two sets of signals for the two groups. Interestingly, in phosphoric triamides containing cyanoacetamide moieties, the existence of aromatic amine substituents on the P atoms created central chiral phosphorus atoms, i.e. the two aromatic groups revealed two sets of peaks in the ¹H and ¹³C NMR spectra, while compounds with aliphatic moieties did not display this effect.     

Bromoiodinanes with an I(III)-Br bond: preparation, X-ray crystallography and reactivity as electrophilic brominating agents

Bromoiodinanes--conveniently and directly prepared from iodobenzenecarbinols and N-bromosuccinimide, and characterised for the first time crystallographically--act as electrophilic bromine donors.     

用于半胱氨酸检测的电化学生物传感器研究进展

半胱氨酸(Cys)作为人体重要的非必需氨基酸之一,对蛋白质合成、渗透调节、解毒过程、神经系统功能和抗氧化过程均发挥着重要作用,近年来广泛应用于医学临床、食品加工和生化研究领域。因此,发展快速、准确检测半胱氨酸浓度的方法具有重要意义。本文简要介绍了半胱氨酸的基本知识以及半胱氨酸的传统检测方法,对半胱氨酸定量检测的电化学传感器的研制与应用进行了重点阐述,并对其发展前景进行了展望。     

用于褪黑素检测的电化学生物传感器研究进展

褪黑素是人体松果体分泌的一种重要的神经递质,近年来其在控制昼夜节律和提供免疫抗炎特性等生理调节作用方面备受关注。因此,发展可靠、快速检测体内和体外样本中褪黑素浓度的方法,对于探索褪黑素的临床应用和生物学特性具有重要的意义。本文对褪黑素及其传统检测方法进行简要介绍,重点阐述了近几年报道的用于生物样本和药物样本中褪黑素定量检测的电化学传感器,并对褪黑素传感器的未来发展方向进行展望。     

A nanoscale DNA-Au dendrimer as a signal amplifier for the universal design of functional DNA-based SERS biosensors

The use of a nanoscale DNA-Au dendrimer as a signal amplifier was proposed for the universal design of functional DNA-based ultra-sensitive SERS biosensors. This novel design combines the high specificity of functional DNA with the high sensitivity of surface-enhanced Raman scattering (SERS) spectroscopy, resulting in sensitivity superior to that of previously reported sensors.     

Molecular simulations of DD-peptidase,a model ß-lactam-binding protein: Synergy between X-ray crystallography and computational chemistry

Using computer model building, the three-dimensional structure of an enzyme from Streptomyces R61 that is inhibited by ß-lactam antibiotics has been constructed starting from incomplete X-ray crystallographic data for this 37.4 kDa protein. The so-called DD-peptidase catalyzes transpeptidation and hydrolysis of peptides terminating in D-Ala-D-Ala and is a model for bacterial transpeptidases and carboxypeptidases essential in the biosynthesis of the peptidoglycan layer of the cell wall. The structure, which was completed with the SYBYL molecular modeling package, has been refined by energy minimization and molecular dynamics using Quanta/CHARMm software. A simulation of 105 ps was run with waters of solvation in the active site. From these computations, the interatomic distances between the active serine and key residues around the active site were determined. Inadequacies at reproducing geometric details of the ß-lactam ring of a cephalosporin are pointed out which are typical of most commercially available force fields.     

Recent advances on the development of graphene-based field-effect transistors,electrochemical biosensors and electrochemiluminescence biosensors

Graphene, a honeycomb lattice of carbon material with single-atom-layer structure, demonstrates extraordinary mechanical, thermal, chemical and electronic properties. Thus, it has sparked tremendous interests in various fields, such as energy storage and conversion devices, field-effect transistors (FET), chemical sensors and biosensors. In this review, we will first focus on the synthesis method of graphene and the fabrication strategy of graphene-based materials. Subsequently, the construction of graphene-based biosensors are introduced, in which three kinds of biosensors are discussed in details, including the FET, electrochemical biosensors and electrochemiluminescence (ECL) biosensors. The performances of the state-of-the-art biosensors on the detection of biomolecules are also displayed. Finally, we also highlight some critical challenges remain to be solved and the development in this field for further research.     

电化学生物传感器测定甲胎蛋白的研究进展

人体血清中甲胎蛋白(AFP)含量已作为肝癌检测的重要指标,快速而准确地检测血清中的AFP含量对肝癌的早期诊断和预后都有极为重要的作用。传统的酶联免疫法存在分析时间长、前处理繁琐等不利因素。利用免疫技术与电化学检测技术结合起来的电化学免疫传感器,由于具有操作简单、灵敏度高、特异性强及成本低等特点,而得到广泛关注。本文将根据所采用的不同检测方式及修饰材料等方面对近年来电化学免疫传感器检测AFP的研究与应用进行评述,并对其发展趋势进行了展望。     

The mitochondria from beef heart as biosensors for the selective monitoring of phenols

We propose a simple and rapid procedure which allows for the selective monitoring in solution of toxic compounds which behave as uncouplers of the oxidative phosphorylation. Since all phenols, are uncouplers of the oxidative phosphorylation, the procedure allows for the selective monitoring in solution of phenols even in presence of other toxic compounds. The biological sensor are the mitochondria from beef heart. This biosensor is easily available without a stabular and therefore the biosensor and the whole procedure is very simple and not expensive. By linear regression analysis, it results that the procedure well predicts the response of the standard fish method to phenols. Therefore the procedure can be utilized as prescreening analysis for the monitoring the phenols in aqueous samples.     

5-Thio-d-glycopyranosylamines and their amidinium salts as potential transition-state mimics of glycosyl hydrolases: synthesis,enzyme inhibitory activities,X-ray crystallography,and molecular modeling

The synthesis of new glycosidase inhibitors, namely, the glycosylamines of 5-thioglucose and 5-thiomannose and their corresponding amidinium salts are described. We report also the crystal structures of 5-thio-d-mannopyranosyl amine 1 and 5-thio-d-mannopyranosylamidinium bromide 2 bound in the enzyme active site of Golgi α-mannosidase II (GM II). Compounds 1 and 2 have been found to be inhibitors with IC₅₀ values of 0.07 and 0.9 mM, respectively. We also report the docked structures of 5-thio-d-glucopyranosylamine 3 and 5-thio-d-glucopyranosylamidinium bromide 4 in the active site of glucoamylase G2, derived by molecular modeling. Compounds 3 and 4 were found to be inhibitors with K_i values of 0.015 and 0.098 mM, respectively. The results led to conclusions about the nature of the transition state and strategy for the inhibition of glycosyl hydrolases in general.     

Smart functionalized thin gel layers for electrochemical sensors,biosensors and devices

Combining hydrogels sensitive to external stimuli with conducting surfaces opens new possibilities in electrochemistry. Thin hydrogel layers as unique electrode-modifying materials provide highly permeable matrix for easy diffusion of analytes. In addition, larger individuals, for example, nanoparticles and enzymes, can be straightforwardly immobilized in the polymeric networks at electrode surfaces. Such properties are strongly desired for construction of sensors and biosensors. In addition, sensitivity to external stimuli allows to significantly enhance or weaken the electroanalytical signal. Recently, a significant number of articles concerning switchable sensors/biosensors, switchable electrochemical systems and signal–responsive interfaces have been published. This report is also focused on the construction of various devices based on electrode surfaces modified with smart hydrogel layers, for example, logic gates and electroresponsive hydrogel layers as potentially advanced drug delivery systems, artificial muscles and electrochemical valves.     

Dimeric W3SO3 cluster complexes: synthesis, characterization, and potential applications as X-ray contrast agents

Our continued research on the use of heavy metal cluster complexes as a new class of X-ray contrast agents in medical diagnostic imaging is described. A series of 2:3 cluster-ligand complexes, [(W(IV)3SO3)2L3]4- (L = linear polyaminopolycarboxylate ligands), were isolated from the reaction of aqua ion [W(IV)3SO3(H2O)9]4- (prepared in large quantities through an improved literature process) with respective ligands in refluxing DMF. The salts of [(W(IV)3SO3)2L3]4- complex anions were fully characterized using routine techniques such as elemental analysis, MS, HPLC, UV-vis, IR, and NMR. The solid structures of two complex anions, [(W(IV)3SO3)2(PDTA)3]4- and [(W(IV)3SO3)2(HO-PDTA)3]4-, were determined by X-ray crystallography. They are the first examples wherein two W(IV)3SO3 clusters are complexed and linked by three ligands that contain two terminal iminodiacetate (bis-IDA) groups. Complexation of the unstable aqua ion [W(IV)3SO3(H2O)9]4- with ligands has imparted desired biological compatibility to the tungsten metal cluster. These complexes are stable and highly soluble in H2O. The potential utility of such tungsten cluster complexes as X-ray contrast agents was evaluated in both in vitro and in vivo animal studies. In addition, the syntheses of several new linear polyaminopolycarboxylate ligands used in this study are reported.     

Preparation,X-ray crystallography,and thermolysis of phenylenediammonium dibromide salts

Phenylenediammonium dibromide (PDADBr) salts have been prepared and characterized by X-ray crystallography. The thermal decomposition of PDADBr has been studied by thermogravimetry (TG) and differential thermal analysis (DTA). Kinetic parameters have been evaluated using model fitting and isoconversional methods. The thermolytic pathways have also been suggested which involve proton transfer as a primary step to regenerate parent amine and HBr. Interaction between amine and HBr at higher temperature yields gaseous products.     

Synthesis, X-ray crystallography, and computational analysis of 1-azafenestranes

The tandem [4+2]/[3+2] cycloaddition of nitroalkenes has been employed in the synthesis of 1-azafenestranes, molecules of theoretical interest because of planarizing distortion of their central carbon atoms. The synthesis of c,c,c,c-[5.5.5.5]-1-azafenestrane was completed in good yield from a substituted nitrocyclopentene, and its borane adduct was analyzed through X-ray crystallography, which showed a moderate distortion from ideal tetrahedral geometry. The syntheses of two members of the [4.5.5.5] family of 1-azafenestranes are also reported, including one with a trans fusion at a bicyclic ring junction which brings about considerable planarization of one of the central angles (16.8 degrees deviation from tetrahedral geometry). While investigating the [4.5.5.5]-1-azafenestranes, a novel dyotropic rearrangement that converts nitroso acetals into tetracyclic aminals was discovered. Through conformational analysis, a means to prevent this molecular reorganization was formulated and realized experimentally with the use of a bulky vinyl ether in the key [4+2] cycloaddition reaction. Finally, DFT calculations on relative strain energy for the 1-azafenestranes, as well as their predicted central angles, are disclosed.     

Synthesis, glycosidase activity and X-ray crystallography of 3-amino-sugars

The cleavage of two sugar epoxides, methyl 2,3-anhydro-alpha-D-mannopyranoside and 2,3-anhydro-alpha-D-allopyranoside, with amines is presented as a method for preparing a library of 3-amino-sugars (methyl 3-amino-3-deoxy-alpha-D-altropyranosides and methyl 3-amino-3-deoxy-alpha-D-glucopyranosides) as potential glycosidase inhibitors. Several of the altropyranosides were micromolar inhibitors of bovine liver beta-galactosidase and almond beta-glucosidase. X-ray crystal structures were determined for one of the methyl 3-amino-3-deoxy-alpha-D-altropyranosides, 4t, and one of the methyl 3-amino-3-deoxy-alpha-D-glucopyranosides, 6d.