The Construction Of Signal-on And Signal-off Electrochemiluminescence Aptasensors Based On Ru（bpy）₃²⁺ Composite Nanomaterial

Electrochemiluminescence（ECL） is a means of combination of electrochemistry as well as chemiluminiscence, which possessed distinct advantages of high sensitivity and wide concentration response range. Owing to good selectivity, high sensitivity, simple operation and convenient micromation, ECL biosensor has been widely used in many fields. Recently, ECL aptasensor is constructed based on aptamer as recognition element, which has been a very active area of research. Among a variety of ECL luminophores, Ru（bpy）₃²⁺is extensively studied due to many virtues. However, it is difficult to immobilize Ru（bpy）₃²⁺ on electrode surface because of its water-solubility. Based on this, we synthesized novel Ru（bpy）₃²⁺composite nanomaterials with high luminescence efficiency as substrate or signal probe to construct signal-on and signal-off ECL aptasensors for TB detection. The detail contents are as follows:1. Highly efficient electrogenerated chemiluminescence quenching of PEI enhanced Ru（bpy）₃²⁺nanocomposite by hemin and Au@Ce O₂nanoparticlesHere, we prepared Ru-PEI-PAA nanomaterial as substrate in order to overcome the drawbacks of Ru（bpy）₃²⁺, which could obtain high initial signal to construct signal-off ECL aptasensor. Based on high polymer of PEI with amino groups as a co-reagent of Ru（bpy）₃²⁺, the carboxyl groups of AA combined with the amino of PEI via electrostatic interaction to form homogeneous solution.Ru（bpy）₃Cl₂.6H₂ O was injected into the homogeneous solution to form Ru-PEI-PAA nanomaterial with the initiator and cross-linked effect of K₂S₂O₈ and GA. Therefore,we designed a signal-off aptasensor for thrombin detection by the dual quenching of hemin and Au@CeO₂ NPs based on Ru–PEI–PAA composite as substrate. As a result, the aptasensor showed a wider linear range response from 1×10^-13 to 1×10^-8 M with lower detection limit of 0.03 pM.2. A regenerable electrochemiluminescence aptasensor incorporating poly（ethylenimine）and thiosemicarbazide as dual co-reactants for signal amplificationReproducibility as an important indicator was proposed to evaluate the practical value of biosensor, but conventional regeneration reagents of acid, alkali, or electrolyte solution with high concentration may damage the activity of capture probe as well as the functional modification surface. On the basis of the previous work, we synthesized Ru-PEI-PAA nanosphere as signal probe according to the experimental condition change. Based on sandwich-type assay, we constructed a signal-on aptasensor using TB as target detection. with a wider linear range response from 1×10^-5 to 0.1 nM. Furthermore, the proposed aptasensor could be regenerated many times due to the conformation change of aptamer. Thus the method described here might bear some advantages in simple procedures for sensor regeneration and aptasensor development.3. A simple and direct “signal-on” electrochemiluminescence aptasensor based on Au nanoparticles decorated SnO₂@Fe₃O₄ microsphere as multifunctional matrixPorous microsphere with enlarge specific surface area could be used as substrate to absorb abundant aptamers. Based on the enhancement of stannous composite, we designed and synthesized a porous three-layer composite microsphere which was composed of a Fe₃O₄ core, a stannic oxide（SnO₂） interlayer and Au nanoparticles（AuNPs） shell（it can be abbreviated as AuNPs@SnO₂@Fe₃O₄microsphere）. The porous three-layer AuNPs@SnO₂@Fe₃O₄microsphere as multifunctional matrix not only produced a high initial ECL signal, but also offered a great deal of active sites owing to enlarge specific surface area to capture aptamers on electrode surface. The prepared Fe₃O₄ nanoparticles as templates, the urea hydrolysis reaction provided an alkaline environment which could etch the surface of Fe₃O₄ nanoparticles and many SnO₂ nanoparticles were generated on the surface of porous Fe₃O₄ nanoparticles. A simpe and sensitive ECL aptasensor for TB detection was developed using porous three-layer composite microsphere of AuNPs@SnO₂@Fe₃O₄ microsphere as multifunctional matrix, which displayed a wide linear range response for TB from 1×10^-5 to 10 nM with a low detection limit of 3.0 fM. This fabrication strategy as well as detection method of TB-specific conformational change can open a realm of application in an extensive range of other targets in diagnostics and proteomics.