A Novel Fluorescence Immunoassay Based on Two-time Amplified Fluorescence Signal by Hemin and Magnetic Nanoparticles for the Detection of Antigen

Abstract

A novel, selective, and sensitive magnetic-mimetic enzyme fluorescence immunoassay method for antigen detection has been developed by taking advantage of a magnetic separation process and the amplification feature of the hemin label. This method is based on a twice amplified fluorescence signal. The signal is first amplified due to the ultrasmall size and the high surface-to-volume ratio of the silica-coated magnetite nanoparticles, which enable the nanoparticles to carry much more antibodies. Second, the mimetic enzyme (hemin) as a labeling reagent catalyzes the reaction of p-hydroxyphenyl acetic acid and H₂O₂ can further amplify the fluorescence signal. This protocol was also evaluated for a sandwich-type immunoassay of human IgG, and the calibration graph for human IgG was linear over the range of 0–100 ng mL^?1 with a detection limit of 9.8 ng mL^?1. This method can easily separate magnetic nanoparticles from the solution, which simplified the process and played a promising role for various applications in immunoassay.     

Studies on the Phenol Oxidation by H2O2 Catalyzed by Metallomicelle Made from Crowned Schiff Base Co(II) Complexes Containing Benzoaza-15-crown-5

Three novel cobalt(II) complexes of the benzoaza-15-crown-5 Schiff base, CoL¹, CoL², and CoL³ were synthesized and characterized. Metallomicelles made from CoL and surfactants (CTAB, LSS, and Brij35) were used as mimetic peroxidase in the catalytic oxidation of phenol by H₂O₂. For comparison, the catalytic activity of the complexes (CoL¹, CoL², and CoL³) were also investigated. The mechanism and a kinetic mathematic model of the phenol catalytic oxidation were studied. The acid effect of reaction system, structural effect of the complexes, and effect of temperature on the rate of the phenol catalytic oxidation by the mimetic peroxidase were discussed. The results show that the Schiff base cobalt(II) complexes and their metallomicelles as peroxidase mimics exhibit good catalytic activity and similar catalytic character to natural enzyme.     

Study On The Catalytic Reaction Of β-Cd-Hemin Using 2, 3, 4-Trichlorophenol As Substrate And Analytic Application

ABSTRACT|The catalytic activity of various mimetic enzymes instead of the peroxidase have been investigated by 4-aminoantipyrine (4-AAP) and 2, 3, 4-trichlorophenol (TCP) to form a dye utilizing hydrogen peroxide as hydrogen acceptor. The different Chlorophenolic derivatives, which act as a substrate in β-CD-hemin-H₂O₂-4-AAP catalytic reaction, have been systematically studied.|Meanwhile, the relationship of structure-effect for the β-CD-hemin as catalyst, and chlorphenols as substrate has been respectively discussed. The mechanism of catalytic reaction has been investigated. The results showed that β-CD-hemin was the best mimetic enzyme for peroxidase among those tested and TCP was a good substrate for the determination of hydrogen peroxide with β-CD-hemin. The method for the determination of hydrogen peroxide was proposed using 4-AAP-TCP system with β-CD-hemin as catalyst. A linear calibration graph was obtained over the H₂O₂ concentration of 4.8×10-^?8-7.7×10-^?5M, and the relative standard deviation at a H₂O₂ concentration of 2.8×10-^?5M was 2.5%. The apparent molar absorptivity of the chromogenic reaction for H₂O₂ was 1.54× 10⁴ L.mol-^?1.cm^?1. Satisfactory results were obtained in the determination of H₂O₂ in synthetic samples by this method.

Also, the method was coupled with the glucose oxidation reaction to determination glucose in human serum.     

Studies on Phenol Oxidation with H2O2 Catalyzed by Schiff Base Cobalt(II) Complexes in Micellar Solution

The two Schiff base cobalt(II) complexes, CoL¹ and CoL², were synthesized and characterized. The metallomicelle made up of the cobalt(II) complexes and surfactants (CTAB, LSS and Brij35), as mimic peroxidase metalloenzyme, were used in the catalytic oxidation of phenol by H₂O₂. The mechanism and a kinetic mathematic model of the phenol catalytic oxidation were studied. The acid effect of reaction system, structural effect of the complexes, and effect of temperature on the rate of the phenol oxidation catalyzed by the mimetic peroxidases have been discussed. The results showed that the schiff base cobalt(II) complexes and their metallomicelles as peroxidase mimics exhibit good catalytic activity and similar catalytic character to natural enzyme.     

A Kinetic Study of Phenolic Oxidation by H2O2 Using the Schiff Base Complexes As Mimetic Peroxidases

The Schiff base complexes containing a transition metal ion, Co^II and Cu^II, were used as mimetic peroxidase in the catalytic oxidation of phenol by H₂O₂. The characteristic spectra of the Schiff base complexes in H₂O₂-buffered solution were recorded and analyzed, respectively. The mechanism and the kinetic mathematic model of the phenol catalytic oxidation were studied. The results showed that the Schiff base complexes containing the transition metal ion, Co^II and Cu^II, as peroxidase mimics exhibited good catalytic activity and the character of the peroxidase in the catalytic oxidation of phenol by H₂O₂ under different conditions.     

Studies on the Phenol Oxidation with H2O2 Catalyzed by Metallomicelle Made from Crowned Schiff Base Mn(III) Complexes

Metallomicelles made from two Schiff base manganese(III) complexes (MnL¹ and MnL²) and surfactants (CTAB and Brij35) were used as mimetic peroxidase in the catalytic oxidation of phenol by H₂O₂. The catalytic activity of the complexes (MnL¹ and MnL²) were investigated. The mechanism and a kinetic mathematic model of the phenol catalytic oxidation were also studied. The results show the optimum acidity of the enzyme-like system in the paper is ca. pH 7.0, the optimum temperature which is ca. 35°C and the optimum molar ratio of H₂O₂ to the complex is ca. 30 in the complexes-H₂O₂-buffered solution; the Schiff base manganese(III) complexes and their metallomicelles as peroxidase mimics exhibit good catalytic activity and similar catalytic character to natural enzyme.     

Synthesis of tri-layer hybrid microspheres with magnetic core and functional polymer shell

Tri-layer magnetite/silica/poly(divinylbenzene) (Fe₃O₄/SiO₂/PDVB) core-shell hybrid microspheres were prepared by distillation precipitation polymerization of divinylbenzene (DVB) in the presence of magnetite/3-(methacryloxyl)propyl trimethoxysilane (MPS) modified silica core-shell particles as seeds. The polymerization of DVB was performed in neat acetonitrile with 2,2′-azobisisobutyronitrile (AIBN) as initiator to coat magnetite/MPS-modified silica particles through the capture of DVB oligomers with the aid of vinyl groups on the surface of inorganic seeds in absence of any stabilizer or surfactant. Other magnetite/silica/polymer tri-layer hybrid particles, such as magnetite/silica/poly(ethyleneglycol dimethacrylate) (Fe₃O₄/SiO₂/PEGDMA) and magnetite/silica/poly(ethyleneglycol dimethacrylate-co-methacrylic acid) (Fe₃O₄/SiO₂/P(EGDMA-co-MAA)) with various polarity and functionality, were also prepared by this procedure. Magnetite/silica/poly(N,N′-methylenebisacrylamide-co-methacrylic acid) (Fe₃O₄/SiO₂/P(MBAAm-co-MAA)) were synthesized with unmodified magnetite/silica particles as seeds. The resultant tri-layer hybrid particles were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectra (FT-IR), dynamic light scattering, and vibrating sample magnetometer (VSM).     

3-Amino-5-mercapto-1,2,4-triazole-functionalized Fe3O4 magnetic nanocomposite as a green and efficient catalyst for synthesis of bis(indolyl)methane derivatives

A core–shell Fe₃O₄@silica magnetic nanocomposite functionalized with 3-amino-5-mercapto-1,2,4-triazole (Fe₃O₄/SiO₂/PTS/AMTA) was prepared using Fe₃O₄ with silica layer, and its surface was modified with 3-amino-5-mercapto-1,2,4-triazole. The novel synthesized magnetite nanocomposite was characterized using various techniques. The catalytic activity of Fe₃O₄/SiO₂/PTS/AMTA was demonstrated in the synthesis of bis(indolyl)methane derivatives under solvent-free conditions. Some of the bis(indolyl)methane derivatives were synthesized through one-pot, three-component reaction of 1 mol of various benzaldehydes or ketones with 2 mol of indole in the presence of Fe₃O₄/SiO₂/PTS/AMTA in good to excellent isolated yields. In addition, the catalyst could be recovered and used for several reaction runs without loss of catalytic activity. The stability of recycled catalyst was investigated. This method has some advantages including experimental simplicity, good to excellent yields, solvent-free conditions and stability and reusability of the catalyst.     

Synthesis and catalytic evaluation of Fe3O4/MWCNTs nanozyme as recyclable peroxidase mimetics: Biochemical and physicochemical characterization

In this research, the nanocomposite of multiwalled carbon nanotubes and magnetic metal oxide nanoparticles (Fe₃O₄/MWCNTs), as enzyme mimetic, was synthesized using an in situ chemical reduction method. The structure, composition and morphology of the prepared Fe₃O₄/MWCNT nanocomposite materials were characterized using X‐ray diffraction, FT‐IR and scanning electron microscopy with energy dispersive X‐ray spectroscopy, respectively. The magnetic properties of the nanocomposite were investigated by the vibrating sample magnetometer. A colorimetric system involving nanozyme, phenol/4‐aminoantipyrine and H₂O₂ was utilized for the determination of peroxidase mimetic catalytic assay. The obtained results confirmed that the synthesis of Fe₃O₄/MWCNTs nanostructures was successful. It was found that Fe₃O₄/MWCNTs nanohybrid exhibited peroxidase‐like activity without any pH limitation. Colorimetric data demonstrated that the prepared nanocatalyst had higher catalytic activity toward H₂O₂ than MWCNTs. The kinetic parameters of the nanozyme, K_m and V_max, were estimated to be 8.3 mm and 1.4 mm min^?1, respectively. The Fe₃O₄/MWCNTs nanostructures were also successfully applied for glucose detection. In addition, peroxidase‐like activity of the nanozyme increased in the presence of butyl‐imidazolium bromide ionic liquid. These biomimetic catalysts have some advantages, such as simplicity, stability, reusability and cost effectiveness, which makes them great candidates to be used in various fields of biotechnology applications.     

Vitamin B3 as a high acid-alkali tolerant peroxidase mimic for colorimetric detection of hydrogen peroxide and glutathione

Small-molecule enzyme mimics as biocatalysts have been extensively applied in diverse colorimetric sensors fabrication. However, excavating potential organic enzyme mimics with high catalytic activity still remains challenging. In this study, the peroxidase mimicking activity of nicotinic acid (VB3) was demonstrated for the first time through chromogenic substrate 3, 3′, 5, 5′-tetramethylbenzidine (TMB) at the existence of hydrogen peroxide (H₂O₂). The catalytic activity of VB3 kept more than 80% of its optimum activity in a broad pH range of 3.0–9.0. In addition, the kinetic parameter (Michaelis constant, K_m = 0.037 mM) of VB3 catalysis to H₂O₂ is smaller than natural horseradish peroxidase (HRP) and previously reported peroxidase mimics. The catalytic mechanism of VB3 is mainly attributed to the active species of hydroxyl radical (OH) and partially attributed to the superoxide free radicals (O₂^?). A convenient and sensitive colorimetric method based on VB3-H₂O₂-TMB chromogenic system for H₂O₂ and glutathione detection was fabricated with the linear ranges of 5.0–100.0 μM and 5.0–50.0 μM, respectively. In short, this work will not only bring new enlightenment on the physiological functions and practical applications in the analytical field of VB3, but also provide a new type of structural reference for small-molecule enzyme mimics.     

Amelioration of H4[W12SiO40] by nanomagnetic heterogenization: For the synthesis of 1H–pyrazolo[1,2‐b]phthalazinedione derivatives

We conveniently coated silicotungstic acid (STA, H₄[W₁₂SiO₄₀]) on amino‐functionalized Si–magnetite nanoparticles, as surface functionalization of magnetic nanoparticles is an excellent way for green and efficient catalysis. The nanoparticles were structurally characterized using various techniques. The catalytic activity and recyclability of the STA–amine–Si–magnetite nanoparticles were probed through synthesis of 1H–pyrazolo[1,2‐b]phthalazinedione derivatives. The reaction proceeds smoothly to provide products in excellent yields and short reaction times. The catalyst could be readily recovered using a simple external magnet and reused several times without any significant loss in activity. Herein, we report a comparison of the activity of H₄[W₁₂SiO₄₀] as a homogeneous and heterogeneous catalyst, the latter being found to be more efficient. The findings offer effective methods for environmentally friendly synthesis of pyrazolo[1,2‐b]phthalazinedione derivatives.     

Artificial Enzyme Mimics for Catalysis and Double Natural Enzyme Co-immobilization

This work presents a new chemiluminescent (CL) probe array assay. The new type CL probe array is based on enzyme mimics of Co₃O₄–SiO₂ mesoporous nanocomposite material, which not only have an excellent catalytic effect on the luminol–H₂O₂ CL reaction in an alkaline medium but also can be used for the immobilization of enzymes. The linear range of the lactose concentration is 3.0?×?10^?7 to 1.0?×?10^?5 g mL^?1 and the detection limit is 6.9?×?10^?8 g mL^?1. β-Galactosidase and glucose oxidase were selected as a model for enzyme assays to demonstrate the applicability of Co₃O₄–SiO₂ mesoporous nanocomposite material in multienzyme immobilization. The novel bifunctional CL probe array has been successfully applied to the determination of lactose in milk.     

Oxidation Reaction of Phenol with H2O2 Catalyzed by Metallomicelles Made of Co(II) and Cu(II) Complexes of Imidazole Groups and Micelle as Mimic Peroxidase

The imidazole derivatives (N,N‐bis(2‐ethyl‐5‐methyl‐imidazole‐4‐ylmethyl) amino‐propane (biap)) and its complexes containing cobalt or copper ion were synthesized in this study. The oxidation reaction of phenol with oxidant H₂O₂ catalyzed by the metallomicelle made of the complexes of imidazole groups and micelle (CTAB, Brij35, LSS) as the mimetic peroxidase was studied. The results show that the reaction rate for the catalytic oxidation of phenol increases by a factor of approximately 1×10⁵ in the metallomicelle over that in the simple micelles or the pure buffer solution at pH=6.9 and 25°C. The catalytic effects changed with H₂O₂, temperature, pH, and surfactant kind in the catalytic reactive process are discussed. A kinetic mathematic model of the phenol oxidation catalyzed by the metallomicelle is proposed.     

Insight into How Telomeric G‐Quadruplexes Enhance the Peroxidase Activity of Cellular Hemin

The toxic oxidative damage of G‐quadruplexes (G4), linked to neurodegenerative diseases, may arise from their ability to bind and oxidatively activate cellular hemin. However, there have been no precise studies on how telomeric G4 enhances the low intrinsic peroxidase activity of hemin. Herein, a label‐free and nanopore‐based strategy was developed to explore the enhancement mechanism of peroxidase activity of hemin induced by telomeric G4 (d(TTAGGG)_n). The nanopore‐based strategy demonstrated that there were simultaneously two different binding modes of telomere G4 to hemin. At the single‐molecule level, it was found that the hybrid structural telomeric G4 directly binds to hemin (the affinity constant (K_a)≈10⁶ m ^?1) to form a tight complex, and some of them underwent a topological change to a parallel structure with an enhancement of K_a to approximately 10⁷ m ^?1. Through detailed analysis of the topology and peroxidase activity and molecular modeling investigations, the parallel telomere G4/hemin DNAzyme structure was proven to be preferable for high peroxidase activity. Upon strong π–π stacking, the parallel structural telomere G4 supplied a key axial ligand to the hemin iron, which accelerated the intermediate compound formation with H₂O₂ in the catalytic cycle. Our studies developed a label‐free and single‐molecule strategy to fundamentally understand the catalytic activity and mechanism of telomeric DNAzyme, which provides some support for utilizing the toxic, oxidative‐damage property in cellular oxidative disease and anticancer therapeutics.     

Spectrophotometric Determination of Hydrogen Peroxide and Glucose Based on Hemin Peroxidase-Like Catalyzed Oxidation of Bromopyrogallol Red

In an ammonium buffer medium at pH 8.9–9.5, hemin exhibits mimetic peroxidase activity, and has a catalytic effect on the oxidative decoloration of bromopyrogallol red (BPR) with hydrogen peroxide. On this basis and in presence of ethanol as an effect-enhancing agent, a spectrophotometric determination of hydrogen peroxide is described with an apparent molar absorptivity of 4.00×10⁴?l?mol^?1?cm^?1 and a linear range from 3.2×10^?7 to 3.2×10^?5?mol?l^?1. BPR has advantages over some of widely used chromogenic substrates in aspects of sensitivity, simplicity and detection wavelength, while hemin has better stability than peroxidase. The system can be easily coupled with a glucose oxidase-catalyzed reaction, and glucose in the concentration range of 6.0×10^?7? 3.2×10^?5?mol?l^?1 is spectrophotometrically determined. The method has been applied to the analyses of synthetic water and human serum samples. The Michaelis parameters and the mechanism of the mimetic peroxidase reaction are also investigated.     

Catalytic conversions of chloroolefines over iron oxide nanoparticles 3. Electronic and magnetic properties of γ-Fe2O3 nanoparticles immobilized on different silicas

Catalytic properties of superparamagnetic γ-ferric oxide nanoclusters, which are uniform in terms of size and magnetic properties were studied. The catalysts were supported on the activated silica gel matrix (AGM) prepared from the KSK-2 silica gel of globular structure and on the activated silica matrix (ASM) prepared from layered natural vermiculite. The clusters are active in some reactions of chloroolefin conversions: isomerization of dichlorobutenes and alkylation of benzene with allyl chloride. Their activity in these reactions is many times higher that of usual supported catalysts based on α-ferric oxide. Analysis of the Mössbauer spectra of the 2.5 wt.% Fe/AGM and 2.5 wt.%Fe/ASM samples before and after the reaction at T = 3–300 K shows that during the reaction some Fe^III ions arranged in ～2–3-nm γ-Fe₂O₃ nanoclusters magnetically ordered at 6 K are reduced to form a high-spin Fe^II complex in the paramagnetic state. According to the macroscopic magnetization data (SQUID) of the initial clusters, curves with hysteresis are observed at 2 K in the plots of forward and backward magnetization, while the 2.5 wt.%Fe/ASM catalyst after the reaction at T = 2 K demonstrates a linear field dependence of the magnetization passing through the coordinate origin. Analysis of the Mössbauer spectra and magnetic properties suggests that during the catalytic reaction the Fe^III ions in the γ-Fe₂O₃ nanoclusters interact with chloroolefin with the allylic structure to be partially reduced to the Fe^II ions that are bound in a complex containing chloride ions and O^II ion(s) of the silicate matrix as ligands. This is a reason, probably, for the high catalytic activity of γ-Fe₂O₃ nanoparticles.     

Synthesis of nanostructured magnetic photocatalyst by colloidal approach and spray-drying technique

Nanostructured particles with a magnetic core and a photocatalytic shell are very interesting systems for their properties to be magnetically separable (and so reusable) in photocatalytic water depuration implant. Here, a robust, low time-consuming, easily scale up method to produce Fe₃O₄/SiO₂/TiO₂ hierarchical nanostructures starting from commercial precursors (i.e. Fe₃O₄, SiO₂) by employing a colloidal approach (i.e. heterocoagulation) coupled with the spray-drying technique is presented. In particular, a self-assembled layer-by-layer methodology based on the coagulation of dissimilar colloidal particles was applied. First, a passive layer of silica (SiO₂, amorphous) was created on magnetite in order to avoid detrimental phenomena arising from the direct contact between magnetite and titania, then the deposition of titania onto silica-coated-magnetite was promoted. TiO₂, SiO₂ and Fe₃O₄ nanosols were characterized in terms of zeta potential, optimized and a self-assembled layer-by-layer approach was followed in order to promote the heterocoagulation of silica onto magnetite surface and of titania onto silica coated magnetite. Once optimized the colloidal route, the mixture was then spray-dried to obtain a granulated powder with nano-scale reactivity, easier to handle and re-disperse in comparison to starting nanopowders with the same surface properties. The nanostructured particles have been characterized by different techniques such as SEM, TEM, XDR and their magnetic properties have been investigated. Moreover, preliminary photocatalytic texts have been performed.     

Nano‐Fe3O4@SiO2‐SO3H: A magnetic,reusable solid‐acid catalyst for solvent‐free reduction of oximes to amines with the NaBH3CN/ZrCl4 system

In this study, the immobilization of sulfonic acid on silica‐layered magnetite was carried out by the reaction of ClSO₃H with silica‐layered magnetite. The prepared magnetic nanoparticles of Fe₃O₄@SiO₂‐SO₃H were then characterized using scanning electron microscopy, energy dispersive X‐ray spectroscopy, X‐ray diffraction, Fourier transform infrared spectroscopy, vibrating sample magnetometry, and transmission electron microscopy. The sulfonated nanocomposite exhibited excellent catalytic activity and reusability in the reduction of various aldoximes and ketoximes with NaBH₃CN in the presence of ZrCl₄. All reactions were carried out under solvent‐free conditions (r.t. or 75–80°C) within 3–70 min to afford amines in high to excellent yields.     

Investigation of 3,3′,5,5′-tetramethylbenzidine as colorimetric substrate for a peroxidatic DNAzyme

In this work, the suitability of 3,3′,5,5′-tetramethylbenzidine sulfate (TMB) as the substrate of a DNAzyme catalytic system composed of a guanine-quadruplex DNA molecule and hemin was investigated. In the presence of H₂O₂, the hemin-DNA complex catalyzes the oxidation of TMB to produce two colored products, much like a peroxidase. The color-generating activity of this system could be influenced by several factors such as buffer type, pH value, DNA sequence, reaction time, and concentrations of both the hemin and H₂O₂. To illustrate the utility of this catalytic system, we designed a colorimetric assay, in which a synthetic oligonucleotide with a sequence complementary to the G-quadruplex DNA was used as the target. A detection limit of 1.86 nM was obtained. Our data have shown that TMB was an excellent colorimetric indicator that reported the peoxidase activities of the widely studied hemin-G-quadruplex DNAzyme system.