Biomolecule-Functionalized Nanoparticles For Electrochemical Immunoassay Of Tumor Markers

Tumor marker,one of the most threatening diseases of the human being,plays an essential role in modern medicine.The detection of tumor markers including serum tumor markers and potential prognostic factors for tumors plays an important role in clinical diagnoses and cure evaluation for the patients with certain tumor-associated diseases.This includes classical diagnostic applications such as detection,diagnosis,prognosis and monitoring of diseases.In recent years,many immunoassay methods and immunosensors as currently popular techniques are being developed for the detection of tumor-related biomarkers.And great efforts have been made worldwide to develop and improve immunoassays for the detection of biomarkers with the aim of making portable and affordable devices.Despite of many advances in this field,it is still a challenge to exploit new approaches that can improve the simplicity,selectivity,and sensitivity of clinical immunoassay,to meet the requirements of modern medical diagnostics and biomedical research applications.Work specific to noble metal nanoparticles stems from both basic research interests and their potential as interconnects in nanoelectronic systems and optical nanometallic communicating channels,as well as biological spectroscopic enhancers.Gold nanoparticles could provide a large well-defined surface area and the capacity for modification of proteins on the gold layer surface,which would adopt an more flexible orientation and resulted in high amount of proteins.Since metallic core-shell types of iron oxide nanoparticles,i.e.owing to inner iron oxide core with an outer metallic shell of inorganic materials such as silica,gold or gadolinium,provided not only the stability to the nanoparticles in solution but also helps in binding the various biological ligands at the nanoparticle surface for various biomedical applications,the core-shell nanoparticles have been extensively used as a matrix and cytochemical label for the immobilization and study of macromolecules such as drugs, proteins,enzymes,antibodies,nucleotides and organ,tissue,tumor in hyperthermia.Magnetic nanoparticles have attracted much research interest in the past decade due to their potential applications in drug and gene delivery system,enzyme and protein immoibilization, diagnostics,data storge,biolabeling and separation of biomolecules.These applications commonly require for the magnetic nanoparticles with chemical stability and good dispersion in liquid medium. Usually a protection layer is necessary to ensure their chemical stability and improve their dispersing ability.Magnetic controlled molecular electronics and bioelectronics examine the effect of an external magnetic field on electrochemical signals of functionalized magnetic particles associated with electrodes.Attraction of the functionalized magnetic nanoparticles to the probe surface with an external magnet activates the electrical contact between the immobilized proteins and the base electrode,and the sensor's circuit is switched on.Positioning the magnet above the cell retracts the magnetic nanoparticles from the probe surface,and the electrochemical activity of the functional magnetic particles is switched off.This thesis mainly consists of the following issues:First paragraph:PredictionSecond paragraph:Ligands-functioualized gold nanoparticles for immunosensing assay of tumor markers in clinical immunoassays(â…°)Two generic,fast,sensitive and novel electrochemical immunosensors have been developed based on the immobilizatin of HBsAb onto{Au/Co(bpy)₃³⁺}_n multilayer film.Cyclic voltammetry, electrochemical impedance spectroscopy(EIS)were adopted to monitor the regular growth of the multilayer films.The multilayer film-modified immunosensor was used for HBsAg determination via the amperometric and potentiometric immunosensor systems,and both systems provided the same linear ranges from 0.05 to 4.5μg/mL with different detection limits for the amperometric system 0.005μg/mL and for the potentiometric system 0.015μg/mL.(â…±)A new electrochemical immunosensor for the determination of carcinoma antigen 125(CA 125)was developed by means of immobilizing CA 125 antibody(anti-CA 125)on gold nanoparticles(Au)and thionine(Thi)modified carbon paste interface.The fabrication process of the immunosensor was characterized by fourier transform infrared spectroscopy(FTIR)and UV-vis absorption spectroscopy.A direct electrochemical immunoassay format was employed to detect CA 125 antigen based on the current change before and after the antigen-antibody reaction.The current change was proportional to CA125 concentration ranging from 10 to 30 U/mL with a detection limit of 1.8 U/mL(at 3δ).(â…²)A special protein assay system based on a highly hydrophilic,non-toxic and conductive biominetic interface has been demonstrated.To fabricate such assay system,red blood cells of crucian carp(RBC)was initially grown on a glassy carbon electrode surface(GCE)deposited nano-sized gold particles(GPs),a second gold nanoparticle layer(NG)was then absorbed on the RBC surface,and finally mammary cancer 15-3 antibody(anti-CA15-3)was attached on the functional RBC surface.A competitive immunoassay format was employed to detect CA15-3 with horseradish peroxidase(HRP)-labeled CA15-3 as tracer and hydrogen peroxide as enzyme substrate. When the immunosensor was incubated into a mixture solution containing HRP-labeled CA15-3 and CA15-3 sample for 1 h at 37℃,the amperometric response decreased with the increment of CA15-3 sample concentration.AFM images of the modified layer revealed a uniform distribution of protein and nanogold.In situ QCM and electrochemical measurements demonstrated that the wanted antibody-antigen reactions should occur with high specificity and selectivity.The specific immunoassay system can be developed further to yield sophisticated structures for other proteins.Third paragraph:Ligands-functionalized magnetic nanoparticles for immunosensing assay of tumor markers in clinical immunoassays(â…°)This study demonstrates a novel approach toward development of advanced immunosensors based on chemically functionalized core-shell Fe₃O₄@Ag magnetic nanoparticles,and the preparation,characterization,and measurement of relevant properties of the immunosensor useful for the detection of carcinoembryonic antigen(CEA)in clinical immunoassay.The immunosensor based on the combination of a magnetic nanocore and an Ag metallic shell shows good adsorption properties for the attachment of the CEA antibody selective to CEA.The core-shell nanostructure presents good magnetic properties to facilitate and modulate the way while it was integrated into a carbon paste.Under optimal conditions,the resulting composite presents good electrochemical response for the detection of CEA,and allows detection of CEA at concentration as low as 0.5 ng/mL.(â…±)This study demonstrates a novel approach toward development of advanced protein assay systems based on physically functionalized core-shell magnetic CoFe₂O₄/SiO₂ porous composite nanoparticles,and the preparation,characterization,and measurement of relevant properties of the protein assay system useful for the detection of cancer antigen 15-3(CA 15-3,as a model)in clinical immunoassay.The protein assay system based on the combination of a magnetic nanocore and a silica shell shows good adsorption properties for the attachment of the CA 15-3 antibody selective to CA 15-3.The core-shell nanostructure presents good magnetic properties to facilitate and modulate the way while it was integrated into a quartz crystal microbalance(QCM)detection cell with the aid of permanent magnet.Under optimal conditions,the resulting immunoassay system presents good QCM responses for the detection of CA 15-3,and allows detection of CA 15-3 at concentration as low as 1.5 U/mL(units/milliliter).Importantly,the proposed protein assay system could be extended to the detection of other antigens or biocompounds. (â…²)We synthesized magnet core/shell NiFe₂O₄/SiO₂ nanoparticles and fabricated an electrochemical magnetic controlled microfluidic device for the detection of 4 tumor markers.The immunoassay system consisted of 5 working electrodes and an Ag/AgCl reference electrode integrated on a glass substrate.Each working electrode contained a different antibody immobilized on the NiFe₂O₄/SiO₂ nanoparticle surface and was capable of measuring a specific tumor marker using noncompetitive electrochemical immunoassay.Under optimal conditions,the multiplex immunoassay enabled the simultaneous detection of 4 tumor markers.The sensor detection limit was＜0.5μg/L(or＜0.5 kilounits/L)for most analytes.Intra-and interassay imprecision(CVs)were＜4.5%and 8.7%for analyte concentrations＞5μg/L(or＞5 kilounits/L),respectively.No nonspecific adsorption was observed during a series of procedures to detect target proteins,and electrochemical cross-talk(CV)between neighboring sites was＜10%.Forth paragraph:Ligands-functionalized gold/magneto composte nanoparticles for immunosensing assay of tumor markers in clinical immunoassays(â…°)The synthesis of the magnetic gold spheres includes the following procedures:the three-dimensional(3-mercaptopropyl)trimethoxysilane(MPS)sol-gel network was constructed via hydrolyzed reaction;magnetic CoFe₂O₄ particles with average diameter of 250 nm were prepared; three-dimensional silica gel was absorbed onto the magnetic CoFe₂O₄ particles;gold nanoparticles were assembled onto the thiol groups of the sol-gel network.The ligand-functionalized magnetic-core/nanogold-shell spheres,and the preparation,characterization,and measurement of relevant properties of the immunosensor were used for the detection of cancer antigen 15-3(CA 15-3),as a model,in clinical immunoassay.(â…±)A new signal amplification strategy based on thionine(TH)-doped magnetic gold nanospheres as labels and horseradish peroxidase(HRP)as enhancer holds promise to improve the sensitivity and detection limit of the immunoassay for carcinoembryonic antigen(CEA),as a model protein.This immunoassay system was fabricated on a carbon fiber microelectrode(CFME)covered with a well-ordered anti-CEA/protein A/nanogold architecture.Reverse micelle method was initially used for the preparation of TH-doped magnetic gold nanospheres(nanospheres),and the synthesized nanospheres were then labeled on HRP-bound anti-CEA as a secondary antibody(bionanospheres). Sandwich-type protocol was successfully introduced to develop a new high-efficient electrochemical immunoassay with the labeled bionanospheres towards the reduction of H₂O₂.Under optimized conditions,the linear range of the proposed immunoassay without HRP as enhancer was 1.2-125 ng/mL CEA,while the assay sensitivity by using HRP as enhancer could be further increased to 0.01 ng/mL with the linear range from 0.01 to 160 ng/mL CEA.The developed immunoassay method showed good precision,high sensitivity,acceptable stability and reproducibility,and could be used for the detection of real sample with consistent results in comparison with those obtained by the ELISA method.