Preparation Of High-Performance Ferritin-based Electrochemiluminescence Probes For Immunoassay Of Disease Markers

Early diagnosis of major human diseases has become one of the most challenging problems in clinical medicine nowadays,which is of great significance to realize precision medicine and ensure people’s life and health.In vitro detection of disease biomarkers is one of the most used analytical techniques in the early clinical diagnosis of major diseases.Electrochemiluminescence（ECL）immunoassay technology is developed by integrating the advantages of electrochemistry,chemiluminescence and immunoassay.ECL owns characteristics of high sensitivity,strong spatio-temporal controllability and specificity,fast response,and simple operation,which has become an important technological means in the research and development of disease early diagnosis technology.Ferritin（Ft）is a kind of soluble protein with iron-storage ubiquitous in nature,composed of a ferric nanocore and a protein shell named apoferritin（apo Ft）.Ferritin has a variety of biological functions,unique nanostructures,and excellent physical and chemical properties,showing important applications in biological analysis and imaging,drug delivery and disease diagnosis and treatment.To explore the potential application of functional ferritins in immunoassay for analyzing disease biomarkers,this study was conducted by the comprehensive application of ECL,immunosensing and nanomaterials related basic theories and technologies.By the research on preparation of ECL functionalized ferritins,construction of specific biosensing interfaces,design of signal-amplification strategies and sensor development,this paper presented a variety of ECL immunosensing methods for the early and highly-sensitive detection of disease markers,including the following five sections:（1）N-（4-aminobutyl）-N-ethylisoluminol（ABEI）molecules were modified on the outer-surface of ferritin,the formed ABEI-Ft was used to fabricate an ECL probe for developing an immunoassay for the sensitive detection of procalcitonin（PCT）,a biomarker of sepsis.The ferric nanocore of ferritin owned a good peroxide-like activity which could efficiently catalyze the oxidative decomposition of H₂O₂ to produce more reactive oxide species（ROSs）,which significantly enhanced the ECL signals.Then,the HWRGWVC（HWR）functionalized polyaniline nanowire array/reduced graphene oxide composite was prepared as a specific sensing substrate,which realized the site-oriented immobilization of antibodies.Based on the advances of natural enzyme-like catalytical activity of ferritin and the site-oriented strategy for antibody,this immunoassay performed a low detection limit of 54 fg/m L（S/N=3）for PCT detection.This work not only revealed the feasibility of immobilizing ECL molecules on the outer surface of ferritin and designing signal amplification strategies using the natural enzyme activity of ferritin nanocore,but also provided a new research idea for the development of immunoassay for the early diagnosis of sepsis.（2）Gold nanoparticle（Au NP）was in-situ reduced on the surface of ABEI-Ft to form ABEI-Ft@Au,based on which an ECL probe was proposed to develop an immunoassay for PCT detection.Both the ferric nanocore and Au NP in ABEI-Ft@Au structure exhibited peroxidase-like activity,which formed synergistic effects to catalyze the oxidative decomposition of H₂O₂ into more ROSs radicals for producing more excited[ABEI_ox]^*.Due to the overlap between the ECL emission spectrum and UV-vis absorption spectrum of ABEI-Ft@Au,the ECL emission was quenched（～44%）when the excited[ABEI_ox]^*returned to the ground sate.Combined with the theoretical calculation of RET,we confirmed that the quenching process belonged to the nanometal surface energy transfer（NSET）mechanism using ABEI as the energy donor and Au NP as the energy receptor.This NSET efficiency followed a1/d⁴ dependence rule to the distance.The ABEI-Ft@Au was conjugated with HWR to act as an ECL probe to develop an immunosensor for PCT detection,achieving a low detection limit of41 fg/m L（S/N=3）.This work not only revealed the feasibility of grafting noble-metal NPs on the outer surface of ECL functionalized ferritin,but also provided a new research idea for the preparation of multifunctional ECL biological probes with intra-structural characteristic of co-catalysis or energy-transfer.（3）Using apoferritin（apo Ft）as a biocompatible carrier,two kinds of ECL active iridium complexes included tri-（2-phenylpyridine）iridium[Ir（ppy）₃]and diacetylpyruvate（2-phenylpyridine）iridium[Ir（ppy）₂（acac）]were encapsulated in the apo Ft cavity through p H-dependent disassembly and assembly,forming two fabulous soluble iridium-complex nanodots1 and 2.The encapsulation ratios for above nanodots were separately determined to be 14.5%and 10.7%.Using TPr A as a coreactant,the oxidative-reductive mechanism and ECL quenching behaviors induced by high concentration of TPr A free radicals（TPr A·）were separately investigated for each nanodot.Using Ru（bpy）₃²⁺-TPr A as the benchmark,the ECL efficiencies for nanodots 1 and 2 were determined to be 22%and 57%,respectively.Finally,the feasibility for lung cancer biomarkers was discussed and proved by the prepared iridium nanodot-based ECL probe.This chapter provided a simple and reliable strategy for the rapid preparation of ECL iridium nanodots with water solubility and high luminous efficiency,which was conducive to broaden the immunoassay application range of iridium complexes in the early diagnosis of disease markers.（4）By virtue of the p H-dependent disassembly and assembly of apo Ft,the AIE-active Ir（ppy）₃ was encapsulated in apo Ft cavity to form the Ir（ppy）₃@apo Ft,based on which an ECL probe was prepared to develop an ECL immunoassay for detecting CYFRA 21-1 antigen,a lung cancer marker.This chapter mainly investigated the aggregation-induced emission enhancement（AIEE）property of the aggregated Ir（ppy）₃ assemblies in apo Ft cavity,which was found 5.3 times stronger than Ir（ppy）₃ monomers.The gold functionalized iron nitride/graphene composite was prepared as the sensing substrate which can efficiently catalyze the generation of more TPr A^·to realize signal-amplification.The proposed immunoassay showed a low detection limit of 0.43 pg/m L（S/N=3）for CYFRA 21-1.This work revealed the feasibility of preparing biocompatible AIE-based ECL probe using apo Ft cavity as carrier and provided a sensitive immunoassay method for clinical detection of lung cancer markers.（5）Using pegylated active ester（NHS-PEG-NHS）modified ferritin（PEG-apo Ft）as the carrier,Ir（ppy）₂（acac）was encapsulated in PEG-apo Ft cavity（Ir@PEG-apo Ft）through p H adjustmnet,based on which an ECL probe to develop an immunoassay for detecting neuron-specific enolase（NSE）,a biomarker for non small cell lung cancer（NSCLC）.The modification of NHS-PEG-NHS not only improved the water dispersion and stability of Ir@PEG-apo Ft,and the encapsulation efficiency of Ir（ppy）₂（acac）in apo Ft was found 1.4 times increased.Ti₃C₂T_x/Ti O₂ composite was prepared as the sensing substrate,which can effectively accelerate the electron transfer during the electrooxidation processes of TPr A and Ir（ppy）₂（acac）to promote the formation of excited state[Ir（ppy）₂（acac）]*,thus achieving signal amplification.The proposed immunoassay owned a a low detection limit of 35 fg/m L（S/N=3）for NSE detection.This work not only revealed the feasibility of preparing iridium ECL luminescent cells using PEG modified apo Ft as water-soluble carrier,but also provided important references for developing immunoassay of NSCLC markers.