Study on SERS Immunoassay and Photothermal Immunoassay for Typical Cancer Biomarker Detection

Author:Yang Lin

Supervisor:huang cheng zhi


Degree Year:2018





Cancer which is also known as malignant tumor is one of the major diseases that seriously affect human health.And in recent years,the people suffering from cancer has been getting younger and younger.The representative of the world health organization(WHO)says that 90%to 95%of tumors can survive up to 90%if they are diagnosed early and treated in time.Early detection is the key to solving cancer prevention and treatment.Unfortunately,it is difficult to detect symptoms of discomfort in the early stages of cancer.When the asymptomatic mass develops into the patient’s detectable solid tumor,some of the tumors are in the middle and late stages or even some tumors have metastasized.This situation has led many patients to miss the best treatment period.Most tumors develop from asymptomatic early stages of cancer to detectable solid tumor stages need 5-10 years.If suitable detection technology can be used during this period,early detection,early diagnosis and early treatment of cancer can be performed.Early detection,early diagnosis and early treatment of cancer not only can achieve good therapeutic effect,but also can greatly improve the survival rate.At present,the detection of cancer biomarkers is the only way to detect asymptomatic tumor in early stage.Cancer biomarkers refer to a class of substances that mark the presence and growth of tumors arising from the synthesis and release of tumor cells themselves or from the body’s response to tumor cells during tumorigenesis and proliferation.It mainly includes protein,hormone,enzyme,polypeptide,cancer gene product,etc.These substances do not exist in normal adults or are significantly higher in cancer patients than in normal adults.But in the early stages of cancer the concentration of the cancer biomarkers were very low.At present,the detection methods of cancer markers used clinically have the disadvantages of low sensitivity,complicated operation steps and high detection cost,which can not reach the purpose of early diagnosis of cancer.Concerning the background and existing problems in the detection of cancer biomarkers,basing on the previous works,in order to achieve the purpose of detecting cancer markers with high sensitivity,simple operation and low detection cost.We introduce noble metal nanoparticles,graphene oxide silver nnaocomposites and chalcopyrite semiconductor nanomaterials into immunoassay,using the excellent optical properties of nanomaterials,a series of highly sensitive immunoassay for the detection of cancer biomarkers have been established.The main points of our work in this study are summarized as follows:1.An enzyme-induced Au@Ag core–shell nanoStructure used for an ultrasensitive surface-enhanced Raman scattering immunoassay of cancer biomarker.An ultrasensitive surface-enhanced Raman scattering(SERS)immunoassay is newly developed with the principle of introducing a common enzyme-induced deposition(EID)reaction to coat a silver layer on the surface of gold nanoparticles and to form a core–shell nanostructure of Au@Ag.By using alkaline phosphatase(ALP)to dephosphorylate its substrate,2-phospho-L-ascorbic acid trisodium salt(AAP),to form vitamin C,silver ions could be reduced into silver atoms and coated on the surface of the AuNPs,a greatly enhanced SERS signal was then obtained.As a proof of concept,α-fetoprotein(AFP)was detected as a target,which is a biomarker of liver cancer.Excellent analytical performance of the SERS immunoassay could be achieved in the range from 0.5 to 100 pg mL-1 with a limit of detection of 0.081 pg mL-1(3σ).Identical results could be obtained by using the newly proposed SERS immunoassay for the clinical detection of AFP in serum samples of patients to those clinically obtained by chemiluminescence immunoassays,demonstrating the potential applications of the developed method in clinical diagnosis.2.Silver nanoparticles deposited graphene oxide for ultrasensitive surface-enhanced Raman scattering immunoassay of cancer biomarker.Graphene oxide(GO)exhibits distinctive Raman scattering features,but too weak for sensitive quantitation purpose.By depositing silver nanoparticles on the surface of GO in this contribution,Raman spectra of GO get enhanced.With the dissolution of deposited silver nanoparticles(AgNPs)on the surface of GO through the oxidation of hydrogen peroxide produced from the oxidization of glucose by glucose oxidase(GOx),the enhanced SERS signals of GO get decreased,and subsequently used for ultrasensitive and selective surface enhanced Raman scattering(SERS)immunoassays through streptavidin biotin binding chemistry between the streptavidin labelled glucose oxidase(SA-GOx)and biotin labelled antibody,and the formed sandwich structure of capture antibody and biotin labelled antibody in the presence of the target.This strategy involves in dual signal amplification of enzyme and nanocomposites to improve the detection sensitivity.As a proof of concept,prostate specific antigen(PSA),a biomarker of prostate cancer,is successfully detected as a target by forming a sandwich structure in immunoassays.The SERS immunoassay possesses excellent analytical performance in the range from 0.5 pg mL-11 to 500 pg mL-1 with a limit of detection of 0.23 pg mL-1,making the detection of PSA serum samples of prostate cancer patients satisfactory,demonstrating the AgNPs deposited graphene oxide for ultrasensitive SERS immunoassay of cancer biomarker is successful in clinical diagnosis.3.Plasmonic Cu2-xSySe1-y nanoparticles catalysed azide alkyne reaction for surface-enhanced Raman scattering immunoassay of cancer biomarkers.Natural enzyme based immunoassay has found widely applications in research laboratories and clinical diagnosis,but suffers the limitations such as low physical/chemical stability,susceptibility to protein denaturation.Herein an enzyme-free SERS immunoassay is developed by utilizing plasmonic Cu2-xSySe1-y-y nanoparticles as nanocatalyst to catalyze azide alkyne reaction,wherein C≡C-PEG2-CH2CH2NH2 acts as both catalytic substrate and SERS signal reporter.Since the unique vibration of C≡C is a very good background free Raman tag located in the Raman-silent region(1800-2800 cm-1)and does not overlap other conventional Raman reporters and biological species,the enzyme-free SERS immunoassay thus has high selectivity and sensitivity.As a proof of concept,prostate specific antigen(PSA)in blood was detected as a detecting target with excellent analytical performance of the SERS immunoassay,demonstrating that the proposed enzyme-free SERS immunoassay can find potential applications in clinical diagnosis.4.Preparation of cobalt doped Cu2-xSe NPs and its application in SERS immunoassay for the detection of cancer biomarkerα-fetoprotein(AFP).Doped nanomaterials can not only maintain the excellent properties of the original nano-materials,but also have new characteristics different from the original nanomaterials due to the doping of other elements.The SERS immunoassay is based on the special eaching effect of Co2+for the poly(p-phenylenediamine)microcrystals.In this work,based on the release Co2+property of cobalt doped Cu2-xSe NPs we developed a SERS immunoassay for cancer biomarkerα-fetoprotein(AFP)detection.The new developed SERS immunoassay compared with the traditional method,the sensitivity has been further improved,with potential clinical application value.4.Photothermal immunoassay for cancer biomarkerα-fetoprotein(AFP)detection.A photothermal immunoassay(PTI)detected with a common thermometer was developed herein by taking the detection of cancer biomarker as an example,wherein temperature changes(ΔT)resulted from photothermal labels such as Cu2-xSe nanoparticles(NPs)could be easily measured with the completion of sandwich immunoassay.Via the sandwish structure different cocnentration of target can bring different concentration of Cu2-xSe NPs,which can release different heat under the radiation of 1064 nm laser light.The risen temperature can quantitatively reflect the amounts of target.A common infrared thermal imager was used to capture the images of different temperature to make the sensitive detection realiable.As a proof of concept,α-fetoprotein(AFP)was detected as a model analyte.The results illustrate that the newly developed PTI is effecctive,practical and applicable to a wide range of analytes.In summary,this research establishes a series of new methods for the detection of cancer markers using the excellent properties of noble metal nanomaterials,graphene silver nanocomposites and chalcopyrite semiconductor nanomaterials.The study in this paper not only broadens the types of immunoassay methods,but also realizes ultrasensitive detection of cancer markers and has certain clinical value in the early detection of cancer.