Ratiometric Electrochemical Immunosensor Based On Three-Dimensional Platinum-Base Nanomaterials And Its Application

Cancer has been a life-threatening disease throughout the world that aggressive progression is associated with the level and variation of some tumor markers in patient blood,body fluids,feces or tissues.These tumor markers are generally synthesized and released by tumor cells,or produced when the body responds to them.They mainly exist in the form of proteins,enzymes,hormones,etc.,whose concentrations in patients is far beyond the normal individuals.Therefore,highly sensitive determination of tumor markers plays an important role in early diagnosis and treatment of cancer.At present,a variety of immunoassay techniques have been developed for detection of tumor markers,such as enzyme-linked immunoassay,fluorescenceimmunoassay,chemiluminescenceimmunoassay,electrochemiluminescence immunoassay etc.Ratiometric electrochemical biosensors with self-calibration capability,show the advantages of high sensitivity and accuracy,which plays the significant roles in immunoassay of disease biomarkers.Also,Pt-base nanomaterials exhibit many excellent characteristics such as excellent electrical conductivity,good biocompatibility,and superior electrocatalytic activity,which are beneficial for signal amplification in biological analysis and further improve sensitivity.In this study,three-dimensional Pt-base nanomaterials were prepared by facile and controlled strategies with different morphologies,structures and compositions.Based on these,the ratiometric electrochemical biosensors were constructed for ultrasensitive immunoassay of disease biomarkers,and expand their practical application in human serum samples.（1）Ultrasensitive ratiometric electrochemical immunoassay of N-terminal pro-B-type natriuretic peptide based on three-dimensional PtCoNi hollow multi-branches/ferrocene-grafted-ionic liquid and Co-N-C nanosheetsDeveloping a simple and sensitive method for detecting tumor biomarkers is practically important for early diagnosis and clinical treatment of diseases.However,it is still the major challenge to improve the sensitivity and accuracy.Here,“a dual-signal read out mode”ratiometric electrochemical immunosensor was developed based on well-dispersed hierarchical PtCoNi hollow multi-branches（HMBs）/ferrocene-grafted-ionic liquid（Fc-g-IL,synthesized via Schiff-base reaction）and Co-N-C nanosheets（Co-N-C NSs,obtained by a simple pyrolysis method）as signal amplification.Specifically,the PtCoNi HMBs were synthesized by solvothermal treatment and then etched with acetic acid.The PtCoNi HMBs/Fc-g-IL showed improved catalytic property and superior biocompatibility.Meanwhile,Au nanostars/thionine decorated Co-N-C NSs（Au-Thi-Co-N-C NSs）performed as the label of secondary antibody（Ab₂）to greatly enhance the signals.This immunoassay for N-terminal pro-B-type natriuretic peptide（NT-proBNP）was realized by boosting the Thi signals while inversely decaying the Fc responses,in turn showing the linear relationship towards the analyte in the range of 0.001-10.0 ngmL^-1 with the low detection limit down（LOD）to 0.35 pgmL^-1（S/N=3）.This study displays a promising platform for determination of other tumor biomarkers in real medical applications.（2）A facile ratiometric electrochemical strategy for ultrasensitive monitoring HER2 using polydopamine-grafted-ferrocene/reduced graphene oxide,Au@Ag nanoshuttles and hollow Ni@PtNi yolk-shell nanocagesHuman epidermal growth factor receptor 2（HER2）is believed as a clinically important breast cancer marker,and thereby it is extremely imperative to develop a highly sensitive and effective strategy for specific recognition and assay of HER2.In this work,a facile ratiometric electrochemical immunosensor was fabricated for sensitively monitoring HER2 as a model analyte,in which reduced graphene oxide-polydopamine-grafted-ferrocene/Au@Ag nanoshuttles（rGO-PDA-Fc/Au@Ag NSs）performed as electrode material and hollow Ni@PtNi yolk-shell nanocages-thionine（Ni@PtNi HNCs-Thi）as signal tags,which provided two distinct electrochemical signals from Fc and Thi,respectively.The Au@Ag NSs endowed the rGO-PDA-Fc with improved biocompatibility and dramatic magnification of the Fc signals upon sensing interface.Also,by integration of the hollow structure,enlarged specific surface area and superior catalytic ability together,the Ni@PtNi HNCs-Thi achieved substantial amplification in the detection signals from Thi.Benefiting from the above,the as-developed immunosensor exhibited a broad linear range(0.01～100ngmL^-1)and a low detection limit(LOD,3.3pgmL^-1)for HER2（S/N=3）,followed by exploring its application in actual serum samples with acceptable results.It holds great potential for clinical screening of cancer biomarkers and point of-care assays.（3）Facile construction of ratiometric electrochemical immunosensor using hierarchical PtCoIr nanowires and porous SiO₂@Ag nanoparticles for accurate detection of septicemia biomarkerProcalcitonin（PCT）is a sensitive and specific biomarker for sepsis diagnosis.In this paper,a novel ratio-typed electrochemical immunosensor was constructed for reliable and sensitive assay of PCT based on hierarchical PtCoIr nanowires/polyethylene polyamine-grafted-ferrocene（PtCoIr HNWs/PEPA-Fc）and porous SiO₂@Ag nanoparticles-toluidine blue（porous SiO₂@Ag NPs-TB）.Importantly,the PtCoIr HNWs/PEPA-Fc was first modified on the sensing interface,which harvested stable and strong electrochemical signals for readout of Fc due to the enriched anchoring sites created by the PtCoIr HNWs.Meanwhile,porous SiO₂@Ag NPs-TB behaved as the label to conjugate with secondary antibody（Ab₂）,which also provided another strong detection signals stemmed from TB confined in such porous structures.The resulting immunosensor displayed a measurable output of PCT in the dynamic scope of 0.001～100 ngmL^-1 with a low limit of detection（LOD）of 0.46 pgmL^-1（S/N=3）.Moreover,we exploited this strategy for PCT assay in a diluted human serum sample with acceptable results,exhibiting promising applications in the clinical analysis.