Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

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.

In situ generation of self-enhanced luminophore by ß-lactamase catalysis for highly sensitive electrochemiluminescent aptasensor.

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 ß-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 ß-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.

Ultrasensitive apurinic/apyrimidinic endonuclease 1 immunosensing based on self-enhanced electrochemiluminescence of a Ru(II) complex.

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.

The Ru complex and hollow gold nanoparticles branched-hydrogel as signal probe for construction of electrochemiluminescent aptasensor.

In this work, a novel Ru complex and hollow gold nanoparticles branched-poly(N-(3-aminopropyl)methacrylamide) hydrogel composites (pNAMA-Ru-HGNPs) were prepared and used as electrogenerated chemiluminescence (ECL) signal probe to construct aptasensor for ultrasensitive detection of thrombin (TB). Herein, [Ru(phen)2(cpaphen)](2+) linked N-(3-aminopropyl)methacrylamide and hollow gold nanoparticles functionalized N-(3-aminopropyl)methacrylamide were used as two polymer monomers to prepare pNAMA-Ru-HGNPs composites via free-radical polymerization. The obtained hydrogel composite, containing amount of Ru complex and HGNPs, were used as effective tag-carriers for the immobilization of thrombin binding aptamer II (TBA II) to form the pNAMA-Ru-HGNPs labeled TBA II (pNAMA-Ru-HGNPs-TBA II). For building the interface of the aptasensor, dendritic gold nanoparticles reduced by poly(ethyleneimine) (PEI@DGNPs) were modified on the carbon nanotube-nafion (CNTs-Nf) coated electrode through electrostatic adsorption, which was used not only as matrix for immobilization of thrombin binding aptamer I (TBA I) but also as enhancer to amplify the ECL signal because PEI is an efficient co-reactant of Ru complex. Target TB was sandwiched between pNAMA-Ru-HGNPs-TBA II and TBA I, resulting in the ECL signals relevant to the TB concentrations. Combining the novel pNAMA-Ru-HGNPs containing amount of Ru complex as the ECL signal probe and PEI@DGNPs as the enhancer for signal amplification, the sandwich ECL aptasensor was constructed for the detection of TB with a wide range of 1.0 fM to 10 pM and a low detection of 0.54 fM.

A novel ECL biosensor for ß-lactamase detection: Using RU(II) linked-ampicillin complex as the recognition element.

In this work, Ru(phen)2(cpaphen)(2+) linked-ampicillin (Ru-Amp), as the novel specific recognition element, was proposed to construct a simple and sensitive electrogenerated chemiluminescence (ECL) biosensor for the determination of ß-lactamase. Here, Ru-Amp complex acted not only as a novel specific recognition element for ß-lactamase but also as the ECL Luminescent reagent. Through electrostatic adsorption and the intermolecular π-π interactions, a large amount of Ru-Amp was immobilized to gold nanoparticles (TA@AuNPs) prepared by thiophenemalonic acid (TA) to obtain Ru-Amp/TA@AuNPs nanocomposites. The nanocomposites, which can produce very stable films exhibiting excellent ECL behaviors, were self-assembled on the CNTs-Nf modified glassy carbon electrode surface. The presence of the target ß-lactamase resulted in autonomous hydrolysis reaction of Amp, achievement of the efficient ECL emission and highly sensitive detection of ß-lactamase. The biosensor for ß-lactamase detection was developed with excellent sensitivity of a concentration variation from 50 pg mL(-1) to 100 ng mL(-1) with a low detection limit of 37 pg mL(-1). An ECL assay offers the proposed method opportunities for designing new Ru-based ECL luminophores for biosensing applications.

A reagentless electrochemiluminescent immunosensor for apurinic/apyrimidinic endonuclease 1 detection based on the new Ru(bpy)3(2+)/bi-arginine system.

Apurinic/apyrimidinic endonuclease 1 (APE-1), a kind of multifunctional protein widely-distributed in the body, plays an essential role in the DNA base excision repair and serves as multiple possible roles in the response of human cancer to radiotherapy and chemotherapy. In this work, an ultrasensitive solid-state electrochemiluminescence (ECL) immunosensor is designed to determine APE-1 based on the new Ru(bpy)3(2+)/bi-arginine system. The bi-arginine (bi-Arg) is decorated on the Au nanoparticles functionalized magnetic Fe3O4/reduced graphene oxide (bi-Arg/Au@Fe3O4-rGO) according to the self-assembling and covalent cross-linking interaction to obtain the functionalized nanocomposite of bi-Arg/Au@Fe3O4-rGO. Herein, the bi-Arg/Au@Fe3O4-rGO plays not only an amplification label to enhance the ECL signal of Ru(bpy)3(2+) due to the coreactant of bi-Arg but also an ideal nanocarrier to load numerous secondary antibody. Based on sandwich-type immunoassay format, this proposed method offers a linear range of 1.0fgmL(-1)-5.0pgmL(-1) and an estimated detection limit of 0.3fgmL(-1) for the APE-1. Moreover, the reagentless ECL immunosensor also exhibits high sensitivity, excellent selectivity and good stability, which has greatly potential development and application in clinical diagnostics, immunology and biomedical research.