A glucose-selective fluorescence sensor based on boronic acid-diol recognition

A glucose selective diphenylboronic acid fluorescent sensor (10a) with a K(a) of 1472M(-1) has been synthesized and evaluated. This sensor shows a 43- and 49-fold selectivity for glucose over fructose and galactose, respectively. The binding affinity improvement is about 300-fold and the selectivity improvement for glucose over fructose is about 1400-fold compared with the monoboronic acid compound, phenylboronic acid. 1H NMR studies indicate that sensor 10a binds with alpha-D-glucofuranose in a bidentate manner (1:1 ratio).     

Oxidation of dihydronicotinamide adenine dinucleotide by a flavin derivative of polyethylenimine

A modified Polyethylenimine has been prepared that has riboflavin attached to it, as well as hydrophobic groups. The catalytic efficiency toward oxidation of6 dihydronicotinamide adenine dinucleotide (NADH) by this flavopolymer is more than 100-fold greater than observed with small-molecular riboflavin. The products of the reaction in aerobic solution have been established to be the oxidized nicotinamide NAD⁺ and hydrogen peroxide. The kinetics fit a Michaelis-Menten mechanism. Thus, the versatility of modified polyethylenimines as catalysts has been extended from hydrolyses and decarboxylations to oxidation-reduction reactions also.     

Sensitive detection of estradiol based on ligand binding domain of estrogen receptor and gold nanoparticles

With increasing concerns of estrogenic effects of endocrine disrupting compounds, the development of simple detection assay for these compounds is an ongoing need. Herein, a simple, rapid, and highly sensitive assay for estradiol (E2) detection was developed using the ligand binding domain of estrogen receptor α (LBD-ERα), the receptor interacting domain of steroid receptor co-activator 1 (RID-SRC1), and gold nanoparticles (AuNPs). The colloidal AuNPs could be stabilized against a salt-induced aggregation by adding LBD-ERα protein. However, with the presence of E2, the specific binding of LBD-ERα protein and E2 led to a salt-induced aggregation of AuNPs as seeing from a color change from red to blue. This developed assay exhibited a high sensitivity for E2 detection with the limit of detection (LOD) of 2.62 × 10⁻¹⁴ M. When the RID-SRC1 protein was included, the detection sensitivity was increased, which the LOD for E2 was at 1.20 × 10⁻¹⁵ M. This assay was specific for a detection of E2 but not progesterone, the negative control ligand. Results of this work clearly showed the efficiency of developed assay for E2 detection, which possibly further developed for an onsite monitoring of E2.     

Signal-enhanced electrochemiluminescence immunosensor based on synergistic catalysis of nicotinamide adenine dinucleotide hydride and silver nanoparticles

A new metal-organic nanocomposite with synergistic catalysis function was prepared and developed to construct an electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of tumor biomarker CA125. Silver nanoparticles (AgNPs) and nicotinamide adenine dinucleotide hydride (NADH) that can participate and catalyze the ECL reaction of Ru(bpy)(3)(2+) were employed as the metal component and the organic component to synthesize the metal-organic nanocomposite of NADH-AgNPs (NA). The novel ECL immunosensor was assembled via Ru(bpy)(3)(2+)-doped silica nanoparticles (Ru-SiO(2)) modified electrode with the NA as immune labels. First, the chitosan-suspended Ru-SiO(2) nanoparticles were cast on the gold electrode surface to immobilize the ECL probes of Ru(bpy)(3)(2+) and link gold nanoparticles. Then, the primary antibodies were loaded onto the modified electrode via the gold sulfhydryl covalent binding. After immunobinding the analytes of antigen, NA-attached secondary antibodies could be captured as a sandwich type on the electrode. Finally, based on the circularly synergistic catalysis by the silver and NADH for the solid-phase ECL of Ru(bpy)(3)(2+), the proposed immunosensor sensed the concentration of antigen. The synergistic ECL catalysis of metal-organic nanocomposite amplified response signal and pushed the detection limit down to 0.03 U ml(-1), which initiated a new ECL labeling field and has great significance for ECL immunoassays.     

Aqueous zymography screening of matrix metalloproteinase activity and inhibition based on colorimetric gold nanoparticles

An optical gold nanoparticles (AuNPs)-based method was fabricated for the rapid detection of matrix metalloproteinase (MMP) activity and screening potential MMP inhibitors without sophisticated instruments. The diagnosis platform was composed of AuNPs, particular MMP substrates and 6-mercapto-1-hexanol (MCH). The functionalized AuNPs were subjected to specific MMP digestion, and the MMP found the substrate on AuNPs, such that the AuNPs lost shelter and MCH increased the attraction force between AuNPs. Consequently, AuNPs aggregation and a color change from red to purple with increasing MMP concentration were observed. The surface plasmon resonance (SPR) of the formed AuNPs allowed for the quantitative detection of MMP activity. A sensitive linear correlation existed between the absorbance and the activity of the MMPs, which ranged from 10 ng/mL to 700 ng/mL in NTTC buffer and plasma samples. The proposed colorimetric method could be accomplished in a homogeneous solution with one-step operation in 30 min and has been successfully applied to the determination of particular MMP activity in plasma samples, in which the results are consistent with substrate zymography. This technology may become a simple platform for parallel screening a number of inhibitors and offer an alternative method to studying the efficiency of inhibitors for suppressing MMP activity. The absorbance ratio at 625 nm and 525 nm (A(625)/A(525)) confirmed the efficiency of the inhibitors as observed in substrate zymography. The IC(50) of ONO-4817 and galardin for MMP-1, MMP-2 and MMP-7 determined by the proposed colorimetric method was similar to the results of substrate zymography.     

Protein binding of nicotinamide adenine dinucleotide and regulation of nicotinamide adenine dinucleotide glycohydrolase activity in homogenates of rabbit skeletal muscle

Gel-permeation chromatography and ultrafiltration have been used to study the free and bound forms of NAD in crude extracts prepared from rabbit muscle. Both techniques indicate that over 80% of the endogenous NAD is free.Nicotinamide inhibits the destruction of NAD in muscle homogenates (50% inhibition at 1.6 mm nicotinamide). In the absence of nicotinamide, there is a rapid destruction of free NAD, but a more gradual destruction of bound NAD. The latter result confirms earlier findings that bound NAD is protected from the hydrolytic action of NADase. However, this protection is unlikely to constitute an important mechanism for controlling NADase activity in muscle homogenates because such a small proportion of the endogenous NAD is bound.In the absence of nicotinamide, NAD also disappears rapidly from minced muscle. Interestingly, the NAD/NADH ratio remains constant (NAD/NADH = 18.1–18.5) during the disappearance of NAD in minced muscle. Upon homogenization of the mince, the NAD/NADH ratio abruptly decreases, then slowly increases during subsequent incubation. The latter rise in NAD/NADH ratio appears to be independent of absolute changes in NAD concentration brought about by the action of NADase or the addition of exogenous NAD.     

Escherichia coli uridine diphosphate galactose 4-epimerase: circular dichroism of the protein and protein bound dihydronicotinamide adenine dinucleotide.

The circular dichroism spectra of E. coli UDP-galactose-4-epimerase in its native (epimerase-NAD+) and reduced (epimerase-NADH.UMP) forms between 190 and 400 nm are presented. The reduced form exhibits a large positive circular dichroism band at 340 nm attributed to NADH in the complex. Relative to the small negative band exhibited at this wavelength by free NADH itself, the rotational strength of enzyme-bound NADH is some 50 times larger than that of free NADH, while the oscillator strengths and other special characteristics are similar. This enhancement reflects dissymetric interactions involving the 340-nm transition and is most consistent with the dihydropyridine ring of NADH being highly immobilized in the reduced complex. In the 200- to 230-nm region both enzyme forms exhibit a negative band at 220 nm and a negative shoulder at 208 nm. The ellipticities of the reduced form are minimally 7% greater at both band positions than those of the native form. The spectra are interpreted to indicate that conversion of the native to the reduced form is accompanied by an increase in alpha-helix structure at the expense of unordered structure.     

Simple colorimetric sensing of trace bleomycin using unmodified gold nanoparticles

A simple colorimetric sensing platform for trace bleomycin (BLM) was proposed with the unmodified gold nanoparticles (AuNPs) as the sensing element. BLM has multiple N-donor functionality and exhibited strong coordination effect on AuNPs, which made it possible for the occurrence of ligand exchange of BLM with the weakly surface-bound citrate ions on AuNPs. Meanwhile, the positively charged BLM molecules further neutralized the surface charge, leading to increased van der Waals attractive force among AuNPs for rapid aggregation. This was reflected by the obvious color change from wine red to blue and rapid aggregation kinetics within 7.5 min. The BLM sensing based on unmodified AuNPs can be seen with the naked eye and monitored by UV-vis extinction spectra. The linear range of the colorimetric sensor for BLM was from 2 to 150 nM. The as-established colorimetric strategy opened a new avenue for trace BLM determination.     

Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide

The binding of NAD+ to liver alcohol dehydrogenase was studied by stopped-flow techniques in the pH range from 6.1 to 10.9 at 25 degrees C. Varying the concentrations of NAD+ and a substrate analogue used to trap the enzyme-NAD+ complex gave saturation kinetics. The same maximum rate constants were obtained with or without the trapping agent and by following the reaction with protein fluorescence or absorbance of a ternary complex. The data fit a mechanism with diffusion-controlled association of enzyme and NAD+, followed by an isomerization with a forward rate constant of 500 s-1 at pH 8: E E-NAD+ *E-NAD+. The isomerization may be related to the conformational change determined by X-ray crystallography of free enzyme and enzyme-coenzyme complexes. Overall bimolecular rate constants for NAD+ binding show a bell-shaped pH dependence with apparent pK values at 6.9 and 9.0. Acetimidylation of epsilon-amino groups shifts the upper pK to a value of 11 or higher, suggesting that Lys-228 is responsible for the pK of 9.0. Formation of the enzyme-imidazole complex abolishes the pK value of 6.9, suggesting that a hydrogen-bonded system extending from the zinc-bound water to His-51 is responsible for this pK value. The rates of isomerization of E-NAD+ and of pyrazole binding were maximal at pH below a pK of about 8, which is attributable to the hydrogen-bonded system. Acetimidylation of lysines or displacement of zinc-water with imidazole had little effect on the rate of isomerization of the E-NAD+ complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Refinement of a radioreceptor binding assay for nicotinic acid adenine dinucleotide phosphate

The measurement of changes in nicotinic acid adenine dinucleotide phosphate (NAADP) levels in cells has been, and remains, key to the investigation of the functions of NAADP as a Ca2+ -releasing second messenger. Here we provide details of how to isolate NAADP from cells by extraction with perchloric acid and then measure the NAADP using a radioreceptor assay. We demonstrate that NAADP is neither generated nor broken down during sample processing conditions and that radioreceptor assay is highly selective for the detection of NAADP under cell extract conditions. Furthermore, a number of improvements, such as solid-state detection of the radioactivity, are incorporated to enhance the safety of the procedure. Finally, we have developed a new method to prevent the endogenous metabolism of NAADP by chelating Ca2+ with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), thereby reducing the difficulty of catching a small transient rise in NAADP levels. In summary, we have refined and improved a method for measuring NAADP levels and presented it in a manner accessible to a wide range of laboratories. It is expected that this will enhance research in the NAADP field.     

Effect of nicotinamide adenine dinucleotide on the membrane-associated reduced nicotinamide adenine dinucleotide oxidase of Mycobacterium phlei.

NAD+ had a biphasic effect on the NADH oxidase activity in electron transport particles from Mycobacterium phlei. The oxidase was inhibited competitively by NAD+ at concentrations above 0.05 mM. NAD+ in concentrations from 0.02 to 0.05 mM resulted in maximum stimulation of both NADH oxidation and oxygen uptake with concentrations of substrate both above and below the apparent K-M. Oxygen uptake and cyanide sensitivity indicated that the NAD+ stimulatory effect was linked to the terminal respiratory chain. The stimulatory effect was specific for NAD+. NAD+ was also specific in protecting the oxidase during heating at 50 degrees and against inactivation during storage at 0 degrees. NAD+ glycohydrolase did not affect stimulation nor heat protection of the NADH oxidase activity if the particles were previously preincubated with NAD+. Binding studies revealed that the particles bound approximately 3.6 pmol of [14C1NAD+ per mg of electron transport particle protein. Although bound NAD+ represented only a small fraction of the total added NAD+ necessary for maximal stimulation, removal of the apparently unbound NAD+ by Sephadex chromatography revealed that particles retained the stimulated state for at least 48 hours. Further addition of NAD+ to stimulated washed particles resulted in competitive inhibition of oxidase activity. Desensitization of the oxidase to the stimulatory effect of NAD+ was achieved by heating the particles at 50 degrees for 2 min without appreciable loss of enzymatic activity. Kinetic studies indicated that addition of NADH to electron transport particles prior to preincubation with NAD+ inhibited stimulation. In addition, NADH inhibited binding of [14C]NAD+. The utilization of artificial electron acceptors, which act as a shunt of the respiratory chain at or near the flavoprotein component, indicated that NAD+ acts as at the level of the NADH dehydrogenase at a site other than the catalytic one resulting in a conformational change which causes restoration as well as protection of oxidase activity.     

Label-free antibody-antigen binding detection by optical sensor array based on surface-synthesized gold nanoparticles

Gold nanoparticles grown in situ from printed seed particles on a glass substrate have been fabricated into a biosensor array. The light-scattering properties of the resulting surfaces show sensitivity to changes in the local refractive index. Each array spot is functionalized with fibrinogen or bovine serum albumin and scattered radiation is used to monitor the refractive index change on label-free binding of the antibodies to their antigens from whole blood antiserum. Data were collected real-time and the association rate constants for the specific antibody-antigen binding were derived from a kinetic analysis. The minimum antibody concentration detection sensitivity is of 100 nM.     

Thermodynamics of the binding of flavin adenine dinucleotide to D-amino acid oxidase.

The enthalpy of binding, deltaHb, of flavin adenine dinucleotide to the apoenzyme of D-amino acid oxidase was determined by flow calorimetry at pH 8.5 to be +3.8, -4.1 and -11.0 kcal mol-1 at 10 degrees, 25 degrees and 38 degrees, respectively. These values correspond to a heat capacity change, deltaCp, of -530 cal K-1 mol-1. From the binding constant reported by Dixon and Kleppe (1965a) and the above enthalpies, the standard free energy and standard entropy of binding are evaluated. These thermodynamic data are interpreted in terms of hydrophobic and vibrational contributions (Sturtevant, 1977). The product of the assay reaction (Fonda and Anderson, 1967), benzoylformic acid, is a non-competitive inhibitor of the enzyme with a value for KI of 1.4 X 10(-4)M at 25 degrees.     

Characterisation of the flavin adenine dinucleotide binding region of Myxococcus xanthus protoporphyrinogen oxidase

Protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the haem biosynthetic pathway catalysers the six electron oxidation of protoporphyrinogen-IX to protoporphyrin-IX, in the presence of flavin adenine dinucleotide (FAD) and oxygen. In humans, partial defects in PPOX result in variegate porphyria. In this study, the FAD binding region in Myxococcus xanthus PPOX was analysed by engineering and characterising a selection of mutant proteins. Amino acid residues which interact with FAD via their side chains were selected for study. Mutants were characterised and compared with wild type protein. Characterisation included FAD quantitation, analysis of FAD spectra and kinetic assay. Results revealed that Serine 20 mutants could still bind FAD, but polarity in this position is favourable, yet not essential for the integrity of FAD binding. Study of Glutamate 39 mutants suggest that a negative charge at position 39 is clearly favoured for interaction with the ribose ring of FAD, as all non-conservative replacements could not bind sufficient FAD. Asparagine 441 appears not to be directly involved in FAD binding but rather in stabilizing the FAD, and polarity in this position appears important. Tryptophan 408 may play a role in orientating or stabilizing the bound substrate during catalysis, and a non-polar (or slightly polar) residue is favoured at this position; however, aromaticity in this position appears not to be critical. Overall this study sheds further light on how M. xanthus PPOX interacts with FAD.     

Aptamer-based colorimetric biosensing of Ochratoxin A using unmodified gold nanoparticles indicator

This work presents an aptasensor for Ochratoxin A (OTA) using unmodified gold nanoparticles (AuNPs) indicator. The assay method is based on the conformation change of OTA's aptamer in phosphate buffered saline (PBS) containing Mg(2+) and OTA, and the phenomenon of salt-induced AuNPs aggregation. A single measurement took only five minutes. Circular dichroism spectroscopic experiments revealed for the first time that upon the addition of OTA, the conformation of OTA's aptamer in PBS buffer changed from random coil structure to compact rigid antiparallel G-quadruplex structure. This compact rigid G-quadruplex structure could not protect AuNPs against salt-induced aggregation, and thus the color change from red to blue could be observed by the naked eye. The linear range of the colorimetric aptasensor covered a large variation of OTA concentration from 20 to 625 nM and the detection limit of 20 nM (3σ) was obtained.