Immobilization-free electrochemical homogeneous aptasensor for highly sensitive detection of carcinoembryonic antigen by dual amplification strategy.

Electrochemical aptasensor has been widely studied, while its practical application is limited by the unavoidable variations of aptamer loading densities and low signal amplification efficiency. To overcome these restrictions, an immobilization-free and label-free electrochemical homogeneous aptasensor was constructed for carcinoembryonic antigen (CEA) assay by combining RecJf exonuclease-mediated target cycling strategy and rolling circle amplification technology. In this system, the pre-immobilization of aptamers or other relevant signal elements on the electrode substrate is no longer necessary, thus the electrochemical homogeneous aptasensor shows good versatility on different transducers. Moreover, the whole recognition and signal amplification process are activated instantaneously by a non-professional operation of the solution mixture. This strategy can not only increase the stability (95.1% after 30 days of storage) and reproducibility (2.12% among five independent electrodes), but also further improve the sensitivity (detection limit of fg mL-1 level) due to the free target recognition and dual signal amplification in the homogeneous solution phase. The proposed immobilization-free electrochemical homogeneous aptasensors on different electrode substrates both achieve satisfactory results in actual sample tests, which has the potential for commercial applications and the establishment of other target platforms in the future.

Electrochemiluminescence aptasensor for vascular endothelial growth factor 165 detection based on Ru(bpy)32+/Au nanoparticles film modified electrode and double signal amplification.

Vascular endothelial growth factor (VEGF165) is a signal protein that plays a central role in the regulation of angiogenesis and can stimulate angiogenesis. The development of highly sensitive and selective detection method for VEGF165 is very important for disease diagnosis and follow-up treatment monitoring. In this study, an electrochemiluminescence (ECL) aptasensor for VEGF165 has been developed based on quench of H2O2 toward Ru(bpy)32+/TPrA ECL system and RecJf exonuclease induced target recovery and hybridization chain reaction (HCR) as amplification strategy. The presence of VEGF165 makes a large number of glucose oxidase (GOD) fixed on the electrode surface through the double signal amplification strategies. The present of GOD cause the production of a large amount of H2O2 near the electrode surface under excess amount of glucose, resulting in the inhibition of the ECL signal of Ru(bpy)32+/Au nanoparticles (Ru(bpy)32+/AuNPs) film fixed on the electrode surface. The ECL response of the designed biosensor has a good linear relationship with the logarithm of the concentration of VEGF165 in the range of 0.5 pg/mL to 500 ng/mL with a detection limit of 0.2 fg/mL. The VEGF165 in serum samples has been detected by the proposed aptasensor with satisfactory results.

Electrochemiluminescence biosensor for thrombin detection based on metal organic framework with electrochemiluminescence indicator embedded in the framework.

Ru(dcbpy)32+-polyethyleneimine-L-lysine (Ru-PEI-L-lys) had been immobilized on metal organic frameworks (ZIF-8) to form an electrochemiluminescent(ECL) indicator (Ru-PEI-L-lys-ZIF-8). In this ECL indicator, PEI-L-lys is used as a co-reactant. Platinum nanoparticles (PtNPs) has been mixed with Ru-PEI-L-lys-ZIF-8 to form a thin film to increase the electron transfer rate and enhanced the ECL response of the system. The prepared material had been characterized carefully and been combined with high selectivity of aptamer to develop a ECL biosensor for thrombin detection. RecJf exonuclease (an ssDNA specific exonuclease) assistant target recycling amplification has been adopted to enhance the sensitivity of the system. The ECL response of the system has a linear relationship with logarithm of thrombin concentration in the range of 1 fM to 10 pM with a detection limit of 0.02 aM. This work not only provides a new strategy for the design and synthesis of high performance and stable ECL indicator, but also opens up a new approach for the development of highly sensitive ECL sensors for biological analysis.

Amplified oxygen reduction signal at a Pt-Sn-modified TiO2 nanocomposite on an electrochemical aptasensor.

In this work, a metallic composite with strong electrocatalytic property was designed by uniformly decorating Pt and Sn nanoparticles on the surface of TiO2 nanorods (Pt-Sn@TiO2). A detection scheme was then developed based on a dual signal amplification strategy involving the Pt-Sn@TiO2 composite and exonuclease assisted target recycling. The Pt-Sn@TiO2 composite exhibited an enhanced oxygen reduction current owing to the synergistic effect between Pt and Sn, as well as high exposure of Pt (111) crystal face. Initially, a Pt-Sn@TiO2 modified glassy carbon electrode produced an amplified electrochemical signal for the reduction of dissolved oxygen in the analyte solution. Next, a DNA with a complementary sequence to a streptomycin aptamer (cDNA) was immobilised on the Pt-Sn@TiO2 modified electrode, followed by the streptomycin aptamer that hybridised with cDNA. The corresponding oxygen reduction current was diminished by 51% attributable to the hindrance from the biomolecules. After a mixture of streptomycin and RecJf exonuclease was introduced, both the streptomycin-aptamer complex and the cDNA were cleaved from the electrode, making the Pt-Sn and Pt (111) surface available for oxygen reduction. RecJf would also release streptomycin from the streptomycin-aptamer complex, allowing it to complex again with aptamers on the electrode. This has then promoted a cyclic amplification of the oxygen reduction current by 85%, which is quantitatively related to streptomycin. Under optimal conditions, the aptasensor exhibited a linear range of 0.05-1500 nM and a limit of detection of 0.02±0.0045 nM streptomycin. The sensor was then used in the real-life sample detection of streptomycin to demonstrate its potential applications to bioanalysis.

An electrochemical aptasensor for multiplex antibiotics detection based on metal ions doped nanoscale MOFs as signal tracers and RecJf exonuclease-assisted targets recycling amplification.

An ultrasensitive electrochemical aptasensor for simultaneous detection of oxytetracycline (OTC) and kanamycin (KAN) has been developed based on metal ions doped metal organic frame materials (MOFs) as signal tracers and RecJf exonuclease-catalyzed targets recycling amplification. The aptasensor consists of capture beads (the anti-single-stranded DNA Antibody, as anti-ssDNA Ab, labeled on Dynabeads) and nanoscale MOF (NMOF) based signal tracers (simplified as Apts-MNM, the NMOF labeled with metal ions and the aptamers). Particularly, the MOF (UiO-66-NH2), with large internal surface areas, ultrahigh porosity and abundant amine groups in the pores, was employed as substrates to carry plenty of metal ions (Pb2+ or Cd2+) and label aptamers of OTC or KAN. Thus, the aptasensor is formed by the specific recognition between anti-ssDNA Ab and aptamers. In the presence of targets (OTC and KAN), aptamers prefer to form targets-Apts-MNM complexes in lieu of anti-ssDNA Ab-aptamer complexes, which results in the dissociation of Apts-MNM from capture beads. With the employment of RecJf exonuclease, targets-Apts-MNM in supernatant was digested into mononucleotides and liberated the target, which can further participate in the next reaction cycling to produce more signal tracers. After magnetic separation, the enhanced square wave voltammetry (SWV) signals were produced from signal tracers. The aptasensor exhibited a linear correlation in the range from 0.5pM to 50nM, with detection limits of 0.18pM and 0.15pM (S/N=3) toward OTC and KAN respectively. This strategy provides specificity and sensitive approach for multiplex antibiotics detection and has promising applications in food analysis.