An alternative "signal on" immunosensor for ultrasensitive detection of apurinic/apyrimidinic endonuclease 1 (APE-1) was designed utilizing the self-enhanced electrochemiluminescence (ECL) of a novel Ru(II) complex functionalized coil-like nanocomposite as signal labels. The desirable self-enhanced ECL luminophore was achieved by combining the coreactant of poly(ethylenimine) (PEI) and the luminophor of bis(2,2'-bipyridine)-5-amino-1,10-phenanthroline ruthenium(II) [Ru(bpy)2(5-NH2-1,10-phen)(2+)] to form one novel Ru(II) complex, which exhibited significantly enhanced ECL efficiency and stability. Moreover, the carbon nanotubes (CNTs) were employed as nanocarriers for self-enhanced Ru(II) complex loading via π-π stacking to obtain the coil-like nanocomposite to act as signal probe. Compared with traditional ECL immunoassay, our proposed strategy is simple and sensitive, avoiding the adding of any coreactant into testing solution for signal amplification, and shows a detection limit down to subfemtogram per milliliter level under the optimized experimental condition.
A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.
This work described a new electrogenerated chemiluminescence (ECL) aptasensor for ultrasensitive detection of thrombin (TB) based on the in situ generating self-enhanced luminophore by β-lactamase catalysis for signal amplification. Briefly, a ruthenium complex (Ru-Amp), including two regions of [Ru(phen)2(cpaphen)](2+) and ampicillin (Amp), was synthesized as a self-enhanced ECL luminophore, which can produce an ECL signal through intramolecular interactions. Then, carbon nanotubes (CNTs) were used for immobilization of Ru-Amp via π-π stacking interactions to form the Ru-Amp@CNTs nanocomposite. Using poly(ethylenimine) (PEI) as a linkage reagent, Au nanocages (AuNCs), owing to their electronic property and large surface areas, were decorated to the CNTs to form the Ru-Amp@CNTs-PEI-AuNCs nanocomposites, which were further used to immobilize thrombin binding aptamer II (TBA II) to form a signal probe (Ru-Amp@CNTs-PEI-AuNCs-TBA II). Through "sandwich" tactics, TBA II bioconjugates, TB and TBA I were immobilized onto the gold nanoparticles modified electrode. Then, with the enzyme catalysis of β-lactamase, a novel self-enhanced ECL luminophore (Ru-AmpA) was in situ produced, which could exhibit a significant enhancement of ECL signal, due to the structure transformation of an amide bond into a secondary amine. A sandwich ECL assay for TB detection was developed with excellent sensitivity of a concentration variation from 1.0 fM to 1.0 pM and a detection limit of 0.33 fM. Therefore, the self-enhanced ECL luminophore, combining the further enhancement by in situ enzymatic reaction, is expected to have potential applications in biotechnology and clinical diagnosis.
A dual signal amplification strategy was designed to construct a cathodic peroxydisulfate-based electrochemiluminescence (ECL) aptasensor for the ultrasensitive detection of thrombin (TB) as a model analyte. The signal was amplified by the employment of two kinds of co-reactant, (1) in situ generated dissolved O2 from the cascade catalysis of GOD and HRP as co-reactants and (2) intercalation of a new co-reactant into the grooves of the dsDNA polymers based on the hybridization chain reaction (HCR). Femtomolar levels of TB could be detected using the developed aptasensor, with high sensitivity and good stability. Thus the S2O8(2-)-O2 ECL system has great potential for development and application in clinical diagnostics, immunology and biomedical research.
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