Fabrication of Luminol and Lucigenin Bifunctionalized Gold Nnanoparticles/Graphene Oxide Nanocomposites with Dual-Wavelength Chemiluminescence

A facile, fast, and reliable method was proposed to prepare luminol and lucigenin bifunctionalized AuNPs/GO nanocomposites via absorption of AuCl₄^– by positively charged lucigenin functionalized GO and the subsequent reduction of AuCl₄^– by luminol at room temperature for the first time. The morphology and surface composition of the nanocomposites were characterized by transmission electron microscopy, X-ray powder diffraction, and mass and fluorescence spectra. The results indicated that AuNPs with a uniform size are fairly well monodispersed on the surface of GO. The size of AuNPs in the nanocomposites was tunable from 8 to 18 nm by changing the amount of AuCl₄^–. Moreover, it was found that luminol and lucigenin coexisted on the surface of the nanocomposites. A formation mechanism of AuNPs/GO nanocomposites is proposed. It is suggested that lucigenin molecules and AuNPs were located at the surface of GO by π–π stacking and electrostatic force respectively, and luminol existed on the surface of AuNPs by virtue of Au–N covalent interaction in the as-prepared nanocomposites. Because luminol and lucigenin were attached to the surface of the nanocomposites, the obtained nanocomposites could react with hydrogen peroxide resulting in a good dual-wavelength chemiluminescence activity. Also, the nanocomposites could also react with silver nitrate giving rise to chemiluminescence emission. Besides, the nanocomposites exhibited fluorescence properties. The nanocomposites could be considered as not only functionalized materials but also as a promising platform for multipurpose sensing and bioassays

Assembly of Multifunctionalized Gold Nanoparticles with Chemiluminescent, Catalytic, and Immune Activity for Label-Free Immunoassays

In this study, we report a universal label-free immunoassay to detect antigen based on multifunctionalized gold nanoparticles (MF-GNPs), which were obtained by successive assembly of <i>N</i>-aminobutyl-<i>N</i>-ethylisoluminol functionalized gold nanoparticles (ABEI-GNPs) with antibody, bovine serum albumin (BSA) and Co²⁺. MF-GNPs exhibited excellent chemiluminescent (CL), catalytic and immune activity. It was demonstrated that the CL signal of MF-GNPs decreased in the presence of antigens via antigen–antibody specific binding using human immunoglobulin G (hIgG) and corresponding antibody goat anti-human IgG (anti-hIgG) as a model system, due to that immunoreaction led to the aggregation of GNPs. According to the decreased CL intensity, hIgG could be determined in the range of 1.0 fM to 1.0 nM with a low detection limit of 0.13 fM. Furthermore, this CL strategy was also confirmed to be a general one by replacing hIgG with heart-type fatty acid-binding protein (H-FABP), which is a biomarker of early acute myocardial infarction (AMI). The CL strategy could be employed to detect H-FABP ranging from 10.0 fM to 10.0 nM, and the detection limit is 7.8 fM. The CL strategy also showed good selectivity. It might be extended to detect other antigens if their corresponding antibodies are available

Coreactant-Free and Label-Free Eletrochemiluminescence Immunosensor for Copeptin Based on Luminescent Immuno-Gold Nanoassemblies

In this work, the eletrochemiluminescence (ECL) behavior of Cu²⁺/cysteine complexes and <i>N</i>-(aminobutyl)-<i>N</i>-(ethylisoluminol) (ABEI) functionalized gold nanoparticles combined with chitosan (Cu²⁺-Cys-ABEI-GNPs-CS) were studied by cyclic voltammetry and a double-step potential, which exhibited excellent ECL properties without any coreactant. It was found that the ECL intensity of Cu²⁺-Cys-ABEI-GNPs-CS could increase at least 1 order of magnitude compared with that of Cu²⁺-Cys-ABEI-GNPs. Furthermore, a coreactant-free and label-free ECL immunosensor has been established for the determination of early acute myocardial infarction biomarker copeptin based on luminescent immuno-gold nanoassemblys consisting of Cu²⁺-Cys-ABEI-GNPs-CS and immuno-gold nanoparticles prepared by connecting copeptin antibody with trisodium citrate stabilized gold nanoparticles. In the presence of copeptin, an obvious decrease in ECL intensity was observed due to the formation of antibody–antigen immunocomplex, which could be used for the determination of copeptin in the range of 2.0 × 10^–14–1.0 × 10^–11 mol/L with a detection limit of 5.18 × 10^–15 mol/L. The detection limit of the ECL immunosensor is at least 2 orders of magnitude lower than that of sandwich immunoassays based on labeling technology. Also, the ECL immunosensor does not need any coreactant and avoids complicated labeling and purification procedure. It is ultrasensitive, simple, specific, and low-cost. This work reveals that the proposed luminescent immuno-gold nanoassemblies are ideal nanointerfaces for the construction of immunosensors. The proposed strategy may be used for the determination of other antigens if corresponding antibodies are available

Highly Chemiluminescent Magnetic Beads for Label-Free Sensing of 2,4,6-Trinitrotoluene

Until now, despite the great success acquired in scientific research and commercial applications, magnetic beads (MBs) have been used for nothing more than a carrier in most cases in bioassays. In this work, highly chemiluminescent magnetic beads containing <i>N</i>-(4-aminobutyl)-<i>N</i>-ethyl isoluminol (ABEI) and Co²⁺ (Co²⁺/ABEI/MBs) were first synthesized via a facile strategy. ABEI and Co²⁺ were grafted onto the surface of carboxylated MBs by virtue of a carboxyl group and electrostatic interaction. The as-prepared Co²⁺/ABEI/MBs exhibited good paramagnetic properties, satisfactory stability, and intense chemiluminescence (CL) emission when reacted with H₂O₂, which was more than 150 times that of ABEI functionalized MBs. Furthermore, it was found that 2,4,6-trinitrotoluene (TNT) aptamer could attach to the surface of Co²⁺/ABEI/MBs via electrostatic interaction and coordination interaction between TNT aptamer and Co²⁺, leading to a decrease in CL intensity due to the catalytic site Co²⁺ being blocked by the aptamer. In the presence of TNT, TNT would bind strongly with TNT aptamer and detach from the surface of Co²⁺/ABEI/MBs, resulting in partial restoration of the CL signal. Accordingly, label-free aptasensor was developed for the determination of TNT in the range of 0.05–25 ng/mL with a detection limit of 17 pg/mL. This work demonstrates that Co²⁺/ABEI/MBs are easily connected with recognition biomolecules, which are not only magnetic carriers but also direct sensing interfaces with excellent CL activity. It provides a novel CL interface with a magnetic property which easily separates analytes from the sample matrix to construct label-free bioassays

Chemiluminescent Nanogels as Intensive and Stable Signal Probes for Fast Immunoassay of SARS-CoV‑2 Nucleocapsid Protein

It is highly desired to exploit good nanomaterials as nanocarriers for immobilizing chemiluminescence (CL) reagents, catalysts and antibodies to develop signal probes with intensive and stable CL properties for immunoassays. In this work, N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and Co2+ bifunctionalized polymethylacrylic acid nanogels (PMAANGs-ABEI/Co2+) were synthesized via a facile strategy by utilizing carboxyl group-rich PMAANGs as nanocarriers to immobilize ABEI and Co2+. The obtained PMAANGs-ABEI/Co2+ showed extraordinary CL performance. The CL intensity is 2 orders of magnitude higher than that of previously reported ABEI and Cu2+–cysteine complex bifunctionalized gold nanoparticles with high CL efficiency. This was attributed to the excellent catalytic ability of Co2+ and polymethylacrylic acid nanogels, as well as the improved CL catalytic efficiency from a decreased spatial distance between ABEI and the catalyst. The as-prepared nanogels also possess abundant surface reaction sites and good CL stability. On this basis, a sandwich immunoassay for the nucleocapsid protein of SARS-CoV-2 (N protein) was developed by using magnetic bead connected primary antibody as a capture probe and PMAANGs-ABEI/Co2+ connected secondary antibody as a signal probe. The linear range of the proposed method for N protein detection was 3.16–316 ng/mL, and its detection limit was 2.19 ng/mL (S/N = 3). Moreover, the developed immunoassay was performed with a short incubation time of 5 min, which greatly reduced the detection time for N protein. By using corresponding antibodies, the developed strategy might be applied to detect other biomarkers

Dynamically Tunable Chemiluminescence of Luminol-Functionalized Silver Nanoparticles and Its Application to Protein Sensing Arrays

It is still a great challenge to develop an array-based sensing system that can obtain only multiparameters, according to a single experiment and device. The role of conventional chemiluminescence (CL) in biosensing has been limited to a signal transducer in which a single signal (CL intensity) can be obtained for quantifying the concentrations of analytes. In this work, we have developed an dynamically tunable CL system, based on the reaction of luminol-functionalized silver nanoparticles (luminol–AgNPs) with H₂O₂, which could be tunable via adjusting various conditions such as the concentration of H₂O₂, pH value, and addition of protein. A single experiment operation could obtain multiparameters including CL intensity, the time to appear CL emission and the time to reach CL peak value. The tunable, low-background, and highly reproducible CL system based on luminol–AgNPs is applied, for the first time, as a sensing platform with trichannel properties for protein sensing arrays by principal component analysis. Identification of 35 unknowns demonstrated a success rate of >96%. The developed sensing arrays based on the luminol–AgNPs provide a new way to use nanoparticles-based CL for the fabrication of sensing arrays and hold great promise for biomedical application in the future

A Homogeneous Signal-On Strategy for the Detection of <i>rpoB</i> Genes of Mycobacterium tuberculosis Based on Electrochemiluminescent Graphene Oxide and Ferrocene Quenching

Tuberculosis (TB) remains one of the leading causes of morbidity and mortality all over the world and multidrug resistance TB (MDR-TB) pose a serious threat to the TB control and represent an increasing public health problem. In this work, we report a homogeneous signal-on electrochemiluminescence (ECL) DNA sensor for the sensitive and specific detection of <i>rpoB</i> genes of MDR-TB by using ruthenium­(II) complex functionalized graphene oxide (Ru–GO) as suspension sensing interface and ferrocene-labeled ssDNA (Fc–ssDNA) as ECL intensity controller. The ECL of Ru–GO could be effectively quenched by Fc–ssDNA absorbed on the Ru–GO nanosheets. The Ru–GO has good discrimination ability over ssDNA and dsDNA. Mutant ssDNA target responsible for the drug resistant tuberculosis can hybridize with Fc–ssDNA and release Fc–ssDNA from Ru–GO surface, leading to the recovery of ECL. Mutant ssDNA target can be detected in a range from 0.1 to 100 nM with a detection limit of 0.04 nM. The proposed protocol is sensitive, specific, simple, time-saving and polymerase chain reaction free without complicated immobilization, separation and washing steps, which creates a simple but valuable tool for facilitating fast and accurate detection of disease related specific sequences or gene mutations

Potential-Resolved Multicolor Electrochemiluminescence of <i>N</i>‑(4-Aminobutyl)‑<i>N</i>‑ethylisoluminol/tetra(4-carboxyphenyl)porphyrin/TiO₂ Nanoluminophores

Most electrochemiluminescence (ECL) studies involve single luminophore with a unique emission process, which severely limits its applications. Recently, multicolor ECL has attracted considerable interests. Herein, we report a novel nanoluminophore prepared by coating 5,10,15,20-tetrakis­(4-carboxyphenyl)-porphyrin (TCPP) and <i>N</i>-(4-aminobutyl)-<i>N</i>-ethylisoluminol (ABEI) on the surface of TiO₂ nanoparticles (TiO₂-TCPP-ABEI), which exhibited unique potential-resolved multicolor ECL emissions using H₂O₂ and K₂S₂O₈ as coreactants in an aqueous solution. Three ECL peaks, ECL-1 at 458 nm, ECL-2 at 686 nm, and ECL-3 at 529 nm, were obtained with peak potentials of 1.05, −1.65, and −1.85 V, which were attributed to the ECL emission of ABEI, TCPP, and TiO₂ moiety of the nanoluminophores, respectively. Potential-resolved multicolor ECL from a nanoluminophore was observed for the first time in an aqueous solution. It opens a new research area of multicolor ECL of nanoluminophores, which is of great importance in ECL field from fundamental studies to practical applications

Biothiols as Chelators for Preparation of N‑(aminobutyl)-N-(ethylisoluminol)/Cu²⁺ 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 Cu²⁺ as a model of metal ion. In this strategy, biothiols were first grafted on the surface of ABEI-AuNPs by Au–S bond. Then, Cu²⁺ was captured onto the surface of ABEI-AuNPs by the coordination reaction to form BF-AuNPs. The CL intensity of Cu²⁺-Cys/ABEI-AuNPs was 1 order of magnitude higher than that of DTDTPA/Cu²⁺-ABEI-AuNPs synthesized by the previous work. Moreover, strong CL emission of Cu²⁺-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 Cu²⁺-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 Cu²⁺ 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

Highly Chemiluminescent Graphene Oxide Hybrids Bifunctionalized by <i>N</i>‑(Aminobutyl)‑<i>N</i>‑(Ethylisoluminol)/Horseradish Peroxidase and Sensitive Sensing of Hydrogen Peroxide

<i>N</i>-aminobutyl-<i>N</i>-ethylisoluminol and horseradish peroxidase bifunctionalized graphene oxide hybrids (ABEI-GO@HRP) were prepared through a facile and green strategy for the first time. The hybrids exhibited excellent chemiluminescence (CL) activity over a wide range of pH from 6.1 to 13.0 when reacted with H₂O₂, whereas ABEI functionalized GO had no CL emission at neutral pH and showed more than 2 orders of magnitude lower CL intensity than ABEI-GO@HRP at pH 13.0. Such strong CL emission from ABEI-GO@HRP was probably due to that HRP and GO facilitated the formation of O₂^•–, – CO₄^•2–, HO^•, and π-CC^• in the CL reaction, and GO as a reaction interface promoted the electron transfer of the radical-involved reaction. By virtue of ABEI-GO@HRP as a platform, an ultrasensitive, selective, and reagentless CL sensor was developed for H₂O₂ detection. The CL sensor exhibited a detection limit of 47 fM at physiological pH, which was more than 2 orders of magnitude lower than previously reported methods. This work reveals that bifunctionalization of GO by ABEI and HRP leads to excellent CL feature and enzyme selectivity, which can be used as an ideal platform for developing novel analytical methods