Simple and selective optical biosensor using Ultrasonicator synthesis of 5-((anthracen-9-ylmethylene) amino)-2,3-dihydrophthalazine-1,4-dione for direct detection of ascorbic acid in vegetables and fruits.

We report an optical biosensor using imine, 5-((anthrcene-9-ylmethylene) amino)-2,3dihydrophthalazine) 1-4-dione (ADD) for direct detection of ascorbic acid (AA) via FRET quenched. The ADD was successfully prepared by using simple ultra - sonication method, which was characterized by various spectroscopic techniques. The fluorescence intensity of ADD probe was drastically quenched in presence of AA, and shown excellent selectivity towards the detection of AA in presence of possible biological active interferences. A wide linear range from 0.25 to 190 µM was achieved towards the detection of AA with a LOD of 10 nM. The occurrence of FRET mechanism is due to intermolecular hydrogen bonding between ADD and AA, which was confirmed by Density Functional Theory calculations. Moreover, the biosensor was successfully applied for the detection of AA in real samples such as fruits and vegetables to demonstrate the practicability. In addition, the developed biosensor could be a simple and economically cheap platform for the detection of AA in food samples.

Cu(II)-Regulated On-Site Assembly of Highly Chemiluminescent Multifunctionalized Carbon Nanotubes for Inorganic Pyrophosphatase Activity Determination.

A novel signal-on chemiluminescence (CL) assay for pyrophosphatase (PPase) activity determination was innovatively developed based on the Cu(II)-regulated on-site assembly of highly chemiluminescent Cu(II), N-(aminobutyl)-N-(ethylisoluminol) (ABEI), gold nanodot, and chitosan multifunctionalized carbon nanotubes (Cu(II)/ABEI-Au/cs-CNTs). First, ABEI-functionalized gold nanodots (ABEI-Au) were assembled on the surface of chitosan-modified carbon nanotubes (cs-CNTs) via the reduction of HAuCl4 with ABEI in a cs-CNT suspension to form ABEI-Au/cs-CNTs. Then, it was found that the catalyst Cu(II) can be selectively, efficiently, and quickly adsorbed onto ABEI-Au/cs-CNTs via the high-affinity interactions between Cu(II) and cs-CNTs to form novel hybrid nanomaterials Cu(II)/ABEI-Au/cs-CNTs. The CL intensity of Cu(II)/ABEI-Au/cs-CNTs was enhanced by about 2 orders of magnitude compared with that of ABEI-Au/cs-CNTs. Furthermore, it was found that in the presence of pyrophosphate ions (PPi), PPi could coordinate with Cu(II) to form a stable PPi-Cu(II) complex and block the assembly of Cu(II)/ABEI-Au/cs-CNTs. After the addition of PPase, PPase could catalyze the hydrolysis of PPi into Pi and release Cu(II) from the PPi-Cu(II) complex. The released free Cu(II) could trigger the on-site assembly of highly chemiluminescent Cu(II)/ABEI-Au/cs-CNTs, resulting in an enhanced CL intensity. The enhanced CL intensity had a good linear relationship with the activity units of PPase ranging from 0.025 to 0.5 U, with a detection limit of 9 mU. The method was employed to monitor the PPase inhibitor efficiently. Cu(II)/ABEI-Au/cs-CNTs with excellent CL may also find more applications in the development of novel CL analytical methods.

Synthesis and Chemiluminescent Properties of Amino-Acylated luminol Derivatives Bearing Phosphonium Cations.

The monitoring of reactive oxygen species in living cells provides valuable information on cell function and performance. Lately, the development of chemiluminescence-based reactive oxygen species monitoring has gained increased attention due to the advantages posed by chemiluminescence, including its rapid measurement and high sensitivity. In this respect, specific organelle-targeting trackers with strong chemiluminescence performance are of high importance. We herein report the synthesis and chemiluminescence properties of eight novel phosphonium-functionalized amino-acylated luminol and isoluminol derivatives, designed as mitochondriotropic chemiluminescence reactive oxygen species trackers. Three different phosphonium cationic moieties were employed (phenyl, p-tolyl, and cyclohexyl), as well as two alkanoyl chains (hexanoyl and undecanoyl) as bridges/linkers. Synthesis is accomplished via the acylation of the corresponding phthalimides, as phthalhydrazide precursors, followed by hydrazinolysis. This method was chosen because the direct acylation of (iso)luminol was discouraging. The new derivatives' chemiluminescence was evaluated and compared with that of the parent molecules. A relatively poor chemiluminescence performance was observed for all derivatives, with the isoluminol-based ones being the poorest. This result is mainly attributed to the low yield of the fluorescence species formation during the chemiluminescence oxidation reaction.

Hydrazide Derivatives of Luminol for Chemiluminescence-Labelling of Macromolecules.

A series of chemiluminescent compounds containing a hydrazide group as a nucleophilic functional group has been synthesized. The syntheses were started from chemiluminescent luminol and isoluminol. The linker moiety was easily introduced onto non-nucleophilic exocyclic amino groups of luminol and isoluminol by gentle heating with cyclic acid anhydrides such as glutaric anhydride. The resulting carboxy group was converted to hydrazide by a simple condensation reaction using carbodiimide. Although majority of the synthesized compounds did not emit strong light, a sufficient chemiluminescence intensity was obtained from luminol-amido-C2-hydrazide (L2H) comprising of luminol scaffold with a dimethylene linker. The ability of L2H to form a covalent bond with a macromolecule was further investigated by incubation with oxidized horseradish peroxidase. The analysis on matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS revealed that the coupling efficiency of L2H was similar to that of commercially available labelling reagent having a hydrazide group. These results suggested that L2H, the luminol hydrazide containing a dimethylene linker, could be useful for the labelling of macromolecules in the sensitive bioassay such as chemiluminescence immunoassay.

Biothiols as chelators for preparation of N-(aminobutyl)-N-(ethylisoluminol)/Cu(2+) complexes bifunctionalized gold nanoparticles and sensitive sensing of pyrophosphate ion.

In this work, chemiluminescence (CL) reagent and catalyst metal ion complexes bifunctionalized gold nanoparticles (BF-AuNPs) with high CL efficiency were synthesized via an improved synthesis strategy. Biothiols, such as cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy), and glutathione (GSH), instead of 2-[bis[2-[carboxymethyl-[2-oxo-2-(2-sulfanylethylamino)ethyl]amino]ethyl]amino]acetic acid (DTDTPA), were used as new chelators. N-(aminobutyl)-N-(ethylisoluminol) (ABEI) was used as a model of CL reagents and Cu(2+) as a model of metal ion. In this strategy, biothiols were first grafted on the surface of ABEI-AuNPs by Au-S bond. Then, Cu(2+) was captured onto the surface of ABEI-AuNPs by the coordination reaction to form BF-AuNPs. The CL intensity of Cu(2+)-Cys/ABEI-AuNPs was 1 order of magnitude higher than that of DTDTPA/Cu(2+)-ABEI-AuNPs synthesized by the previous work. Moreover, strong CL emission of Cu(2+)-Cys/ABEI-AuNPs was also observed in neutral pH conditions. In addition, the present BF-AuNPs synthesis method exhibited advantages over the previous method in CL efficiency, simplicity, and synthetic rate. Finally, by virtue of Cu(2+)-Cys/ABEI-AuNPs as a platform, a simple CL chemosensor for the sensitive and selective detection of pyrophosphate ion (PPi) was established based on the competitive coordination interactions of Cu(2+) between Cys and PPi. The method exhibited a wide detection range from 10 nM to 100 µM, with a low detection limit of 3.6 nM. The chemosensor was successfully applied to the detection of PPi in human plasma samples. It is of great application potential in clinical analysis. This work reveals that BF-AuNPs could be used as ideal nanointerface for the development of novel analytical methods.

Evaluation of lophine derivatives as L-012 (luminol analog)-dependent chemiluminescence enhancers for measuring horseradish peroxidase and H2O2.

8-Amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012) was recently synthesized as a new chemiluminescence (CL) probe; the light intensity and the sensitivity of L-012 are higher than those of other CL probes such as luminol. Previously, our group developed four lophine-based CL enhancers of the horseradish peroxidase (HRP)-catalyzed CL oxidation of luminol, namely 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI), 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole (HPI), 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA), and 4-[4,5-di(2-pyridyl)-1H-imidazol-2-yl]phenylboronic acid (DPPA), and showed that DPPA was suitable for the photographic detection of HRP. In this study, we replaced luminol with L-012 and evaluated these as L-012-dependent CL enhancers. In addition, to detect HRP and/or H2O2 with higher sensitivity, each detection condition for the L-012-HRP-H2O2 enhanced CL was optimized. All the derivatives enhanced the L-012-dependent CL as well as luminol CL; HPI generated the highest enhanced luminescence. Under optimized conditions for HRP detection, the detection limit of HRP was 0.08 fmol. By contrast, the detection limit of HRP with the enhanced L-012-dependent CL using 4-iodophenol, which is a common enhancer of luminol CL, was 1.1 fmol. With regard to H2O2 detection, the detection limits for enhanced CL with HPI and 4-iodophenol were 0.29 and 1.5 pmol, respectively. Therefore, it is demonstrated that HPI is the most superior L-012-dependent CL enhancer.

Synthesis of novel isoluminol probes and their use in rapid bacterial assays.

Rapid assays for bacteria have been developed utilizing novel LysLysLys-isoluminol (14) and GluGlu-isoluminol (16) probes that have been derived from peptides which potentially mimic bacteriophage attachment protein binding regions. Compared to two conventional methods that are widely used, namely nucleic acid probes and polymerase chain reaction (PCR) assays, these types of probes may eventually have certain advantages, such as high sensitivity, and short preparation and assay time.

New electrochemiluminescent biosensors combining polyluminol and an enzymatic matrix.

Performant reagentless electrochemiluminescent (ECL) (bio)sensors have been developed using polymeric luminol as the luminophore. The polyluminol film is obtained by cyclic voltammetry (CV) on a screen-printed electrode either in a commonly used H(2)SO(4) medium or under more original near-neutral buffered conditions. ECL responses obtained after performing polymerization either at acidic pH or at pH 6 have been compared. It appears that polyluminol formed in near-neutral medium gives the best responses for hydrogen peroxide detection. Polymerization at pH 6 by cyclic voltammetry gives a linear range extending from 8 x 10(-8) to 1.3 x 10(-4) M H(2)O(2) concentrations. Based on this performant sensor for hydrogen peroxide detection, an enzymatic biosensor has been developed by associating the polyluminol film with an H(2)O(2)-producing oxidase. Here, choline oxidase (ChOD) has been chosen as a model enzyme. To develop the biosensor, luminol has been polymerized at pH 6 by CV, and then an enzyme-entrapping matrix has been formed on the above modified working electrode. Different biological (chitosan, agarose, and alginate) and chemical (silica gels, photopolymers, or reticulated matrices) gels have been tested. Best performances have been obtained by associating a ChOD-immobilizing photopolymer with the polyluminol film. In this case, choline can be detected with a linear range extending from 8 x 10(-8) to 1.3 x 10(-4) M.

Diazoluminomelanin: a synthetic luminescent biopolymer.

The purpose of this work was to synthesize a water-soluble derivative of 5-amino-2, 3-dihydro-1, 4-phthalazinedione (luminol) that generated sustained high level luminescence under physiologic conditions without the necessity of a catalyst. The derivative was made by a diazotization reaction with luminol and 3-amino-L-tyrosine. The resulting orange-brown anionic polymer has been given the trivial name of diazoluminomelanin (DALM). It was water soluble above and insoluble at or below pH 5.0. DALM luminesced when treated with hydrogen peroxide without the presence of a catalyst at pHs ranging from 6.5 to 12.0. Microgram quantities produced high levels of chemiluminescence for longer than 52 hr. Dried polymer generated a long-term stable electron spin resonance spectrum. The long-term chemiluminescence of DALM at pH 6.8-7.4 makes it a potentially useful reagent for detecting free radicals and peroxides in cellular and biochemical preparations.

Synthesis and chemiluminescence of copolymers of 5-amino-8-vinyl-phthalazine-1,4(2H,3H)-dione with methyl methacrylate or styrene, and of alpha, omega-bis[5-amino-phthalazine-1,4(2H,3H)-dion-]8-yl alkanes [= alpha, omega-bis(6-luminyl) alkanes]: investigations on an intramolecular 'distance effect'.

Oligomers of 5-amino-8-vinyl-phthalazine-1,4(2H,3H)-dione exhibit about 0.05% of the chemiluminescence quantum yield of the corresponding 'monomer unit', i.e. 5-amino-8-ethyl-phthalazine-1,4(2H,3H)-dione which has a similar quantum yield to luminol. The quantum yields of copolymers of 5-amino-8-vinyl-phthalazine-1,4(2H,3H)-dione (1a) with methyl methacrylate or with styrene increase up to 1000-fold, relative to the quantum yield of oligomers of (1a). Thus the monomer units of methyl methacrylate or styrene appear to act as 'spacers' between the lumigenic groups. alpha, omega-Bis[(5-amino-phthalazine-1,4(2H,3H)-dion-)8-yl] alkanes show an analogue 'distance' effect: the chemiluminescence quantum yield increases with increasing alkane chain length. As the fluorescence of the corresponding amino phthalates (which are intermediates in the synthesis of the phthalazine diones) is only slightly influenced by the distance between the lumigenic groups it is suggested that a mainly chemical 'distance effect' is working here: the smaller the intramolecular distance between the hydrazide groups the more inhibition exists in respect of the oxidative reaction producing the luminol-type chemiluminescence.

Chemiluminescence energy transfer: a new technique applicable to the study of ligand--ligand interactions in living systems.

Chemiluminescent molecules can be readily detected in the range 10(-15) to 10(-18) mol, and potentially at least down to 10(-20) mol reacting/s. The chemiluminescent compound aminobutylethylisoluminol (ABEI) and its isothiocyanate derivative have been synthesized. The ABEI was coupled to rabbit immunoglobulin and cyclic AMP. These labeled antigens were stable for at least 9 months and were used to establish chemiluminescent immunoassays. When these chemiluminescent-labeled antigens bound to their respective fluorescein-labeled antibodies, a wavelength shift towards the green was detected in the chemiluminescence. This was due to chemiluminescence energy transfer and used to establish an homogenous immunoassay which could measure these antigens in biological samples at least as sensitively as conventional radioimmunoassays.