Studies On Electrochemiluminescence Immunosensors Based On Luminescent Nanomaterials For Cardiac Troponin I

With the rapid development of nanomaterials, electrochemiluminescent function-alized nanomaterials and luminous nanomaterials including semiconductor quantum dots, carbon dots, and metal nanoclusters, have been used as analytical probe to constitute sandwich immunoassay. Electrochemiluminescence immunosensors as a useful and available detection tool have been the focus of recent investigations, due to the merits of high sensitivity, good selectivity, low back ground. Recent years, acute myocardial infarction（AMI） has been listed as the leading cause of morbidity and mortality among cardiovascular diseases. The cardiac troponin I（c Tn I） is a kind of cardiac contraction muscle to regulate protein. It is reported c Tn I has been recognized as the gold standard cardiac biomarkers in the detection of AMI, because of its superior cardiac specificity and selectivity. Therefore, highly efficient and reliable analytical techniques are necessary for measuring c Tn I present at ultralow levels during early stages of disease progress. Thus, combining the advantages of the ECL immunosensor and nanomaterials plays an important theoretical and practical value for ultrasensitive detection of c Tn I. The application of the luminous nanomaterials not only avoided the addition of any coreactant into testing solution for simplifying the operation, but also achieved the intramolecular reaction for improving the ECL signal due to shorter electron transfer path and less energy loss. It is expected to provide a promising approach for the detection of a wide range of molecular analytes.In our paper, research work is carried out from the aspects as follows: Part 1 Study on ultrasensitive electrochemiluminescent detection of cardiac troponin I based on a self-enhanced Ru（II） complexTo promote the luminous efficiency of luminophore, traditional electrochemilumines-cence（ECL） immunoassay usually adopts the adding of coreactant into testing solution. However, many adverse micro-environmental factors in the solution are a limiting factor in ECL analytical techniques and received extensive attention. In our work, a self-enhanced ECL luminophore was synthesized by combining the coreactant（L-cysteine） and the luminophor(Tris（4,4′-dicarboxylicacid-2,2′-bipyridyl） ruthenium（II） dichloride(Ru（dcbpy）₃²⁺)) to form one Ru（II） complex and wasapplied to fabricate a reagentless immunosensor for the detection of cardiac troponin I（c Tn I） for the first time.The application of the self-enhanced Ru（II） complex not only avoided the addition of any coreactant into testing solution for simplifying the operation, but also achieved the intramolecular reaction for improving the ECL signal due to shorter electron transfer path and less energy loss. And then gold nanorods（Au NRs）, due to their high specific surface area and good electrocatalytic ability, were used as carriers for the immobilization of Ru（II） complex and c Tn I antibody to obtain the Ab2 bioconjugates as signal labels. In view of these advantages, the proposed immunosensor achieved a wide linear range from 0.25 pg/m L to 0.1 ng/m L with an impressive detection limit of 0.083 pg/m L for c Tn I（S/N = 3）. The immunosensor exhibited advantages of simple preparation and operation, high sensitivity. Part 2 Study on ultrasensitive immunoassay based on pseudobienzyme amplifying system of choline oxidase and luminol-reduced Pt@Au hybrid nanoflowersLuminol serves as one of the most extensively studied ECL luminophore, which has low oxidation potential, high emission yields as well as strong luminescence. Currently, great efforts have been made toward the applications of solid-state luminol ECL in sensors by employing the appropriate immobilization methods.This work described an alternative “signal on” immunosensor for ultrasensitive detection of cardiac troponin I（c Tn I） by combining multi-functional luminol-reduced Pt@Au hybrid flower-like nanocomposite（luminol-Pt@Au NFs） and pseudobienzyme signal amplification strategy.Herein, the luminophor of luminol was integrated with the Pt@Au hybrid nanoflowersto form one novel nanocomposite, which not only acted as a signal probe with significantly promoted ECL efficiency, but also exhibited an effective platform for anchoring larger amounts of secondary antibody（Ab2） and choline oxidase（Ch Ox）. Meanwhile, Ch Ox and Pt@Au NFs constructing pseudobienzyme amplifying system could in situ generate coreactant with high local concentrations, the ECL of luminol involving enzymatic reaction of Ch Ox to generate H₂O₂ in situ, then H₂O₂ subsequently was catalyzed by mimicking enzyme of Pt@Au NFs, thus dramatically enhanced the luminous intensity of luminol. The sensing interface was established by assembling c Tn I antibody（anti-c Tn I） on the Mn O₂ functional multiwalled carbon nanotubes（Mn O₂@MWNTs）, which exhibited higher electrical conductivity and larger surface area than pure MWNTs. With the cascade signal amplification, our proposed strategy provided an ultrasensitive detection of c Tn I down to the femtogram level（17 fg m L-1） with a linear range of 4 orders of magnitude（from 50 fg m L-1 to 0.1 ng m L-1）. The immunosensor would extend the applications in the field of immunoassay. Part 3 Study on dual-wavelength ratiometric electrochemiluminescence immunosensor based on resonance energy transfer between silver nanocluster and Ru（bpy）₃²⁺ for c Tn I detectionIn this work, we first observed that electrodeposited silver nanocluster（Ag NCs） exhibited remarkable cathodic ECLemission spectrum at peak wavelength of 458 nm for the first time. Herein, the morphological characteristics andoptical properties of Ag NCs were investigated by field emission scanning electron microscopy（FE-SEM）, X-ray diffraction（XRD）, as well as photoluminescence（PL）. Finally, peroxydisulfate(S₂O₈^2-) was employed to promote the ECL of the Ag NCs, and then the mechanism was elucidated incoreactant path. Compared with traditional ECL based on Ag NCs, our proposed Ag NCs were synthesized using a simple and fast electrodeposition method and obtained the high luminous efficiency. However, the anodic ECL intensity of Ag NCs is much lower than that of conventional luminescent reagents such as luminol or Ru（bpy）₃²⁺, and its application of biological detection is limiteddue to Ag NCs with a high excited potential in ECL processes. A dual-wavelength ratiometric electrochemi-luminescence immunosensor for sensitive detection of cardiac troponin I（c Tn I） was first designed based on resonance energy transfer（RET） from Ag NCs to Ru（bpy）₃²⁺. Herein, the ECL emission of the Ag NCs at 458 nm shows good spectral overlap with the UV-vis absorption peak of Ru（bpy）₃²⁺, indicating that Ag NCs and Ru（bpy）₃²⁺ will produce ECL-RET with high efficiency. Thus, a dual-wavelength ratiometric ECL-RET system was achieved based onthe ECL signals quenching at 458 nmand increasing at 650 nm. Finally, carboxylated MWNTs were employed as nanocarriers for Ru（bpy）₃²⁺ loading via the amide reaction to remain excellent ECL activity of Ru（bpy）₃²⁺. By measuring the ratio of ECL650 nm/ECL458 nm, we could accurately quantify the concentration of c Tn Iin a wide range from 1.0 pg/m L to 1.0 ng/m L. This work provides an important application for the biodetection of luminous Ag NCs and also expands ECL-RET analytical system.