The Fabrication Of Sensitive And Portable Immunosensor And Its Application In Biomarkers Detection

Biomarkers are a class of biochemical indicator that used to mark the normal biological processes.Biomarkers are commonly used to judge the occurrence and classification of certain diseases,surveille the severity of the disease,predict the individual risk of certain diseases,screen high-risk group of incidences and evaluate effectiveness and safety of new drugs,etc.Therefore,seeking specific biomarkers and developing sensitive and fast detection methods for assay biomarkers are becoming a hot research topic in recent years.Immunosensor is a kind of biosensor by using antigen or antibody as the sensitive biological materials,which measures the concentration of target by combining high sensitive biological sensing technology and high specificity immune analysis.Comparing with other detection technology,the immunosensor has advantages such as high specificity,high sensitivity,does not need complex sample pretreatment and so on,which has grown to be the most important detection method for biomarkers.However,most of them are still in the experimental stage and cannot meet the requirements of low detection limit and high stability in clinical application.In addition,the immunosensor cannot fully realize the fast,real-time and super-sensitive detection of complex samples.Therefore,in the future,the immunosensor will be developed in the following direction:(1)combining with new type of functional nanoparticle to improve its stability in complex samples and enhance the binding efficiency with biological molecules,thus can improve the detection sensitivity;(2)combining with 3D printing technology,smartphones or other new technology or platform to realize the miniaturization,intelligent,portable,commercial and on-site detection of biomarkers.In this thesis,two kinds of new nanomaterials were prepared and applied to the detection of biomarkers to improve the sensitivity of immunosensors.Then the sensor platform was moved to a portable device to assay the biomarkers online.The details are described as follows:1.A novel fluorescent and electrochemical dual-mode immunosensor for sensitive and accurate detection of biomarkers based on cation-exchange reactionSensitive and accurate detection of biomarkers is of vital importance in tumor early detection and clinical therapy.A novel fluorescent/electrochemical dual-mode immunosensing platform for accurate and sensitive quantification of biomarkers was designed based on cation-exchange reaction.To construct such a versatile platform,the model analyte,carcinoembryonic antigen(CEA),was captured by magnetic Fe3O4 nanoparticles bound with primary antibodies(Fe3O4-Ab1)and then recognized by the detection antibodies conjugated complex containing poly(amidoamine)(PAMAM),carbon nanotube(CNT)and carboxyl functionalized CdSe nanocrystals(CNT-PAMAM-CdSe NCs-Ab2).Via ligand exchange,the CdSe nanocrystals(NCs)were functionalized with carboxyl groups and showed high hydrophilicity.The functionalized CdSe NCs were effectively and densely conjugated to CNT coated dendrimer PAMAM that possesses large specific surface area.Finally,the target CEA was detected based on cation-exchange reaction(CER)by adding Ag+to release thousands of cations Cd2+,which were detected by fluorescence and electrochemistry simultaneously.The electrochemical measurement was performed by directly detecting Cd2+through square wave voltammetry(SWV),which displayed an excellent correlation with CEA from 5 pg/mL to 50 ng/mL,with a limit of detection(LOD)of 1.7 pg/m L.The fluorescence detection was implemented since free Cd2+could trigger the weak fluorescence metal-sensitive dyes(Rhod-5N)to generate extremely high fluorescence signal.The fluorescence results showed the LOD for CEA detection was 0.25 pg/mL with a calibration curve range from 1 pg/mL to 20 ng/mL.The dual signal outputs showed an attractively self-correcting ability,which provides the capability of avoiding false positive signal and making the detection result more accurate.The proposed dual-mode platform holds great promises for biomarkers detection in clinical diagnostics and therapy.2.The fabrication of colorimetric and electrochemical dual mode cytosensors using highly concave PtSn NCs and Fe3O4 NPs as synergistical signal amplifier for detection of A549 cellsIn this study,a nanoprobe with ultrahigh catalytic activity was prepared for accurately and sensitively detect non-small lung cancer cells A549.The nanoprobe was synthesized by assembling the PtSn nanocrystals with high energy crystals(PtSn NCs)onto the surface of Fe3O4 NPs to form Fe3O4-PtSn nanocomposite.Since the nanoprobe can not only exhibit extremely good electrocatalytic activity,but also show excellent enzymatic-like activity,we developed an electrochemical and colorimetric dual-mode immunoassay for detection A549 cells.For electrochemical assay,the reduction graphene oxide,Au NPs and hyaluronic acid nanocompounds(rGO/Au NPs/HA)were adopted to modify glassy carbon electrode(GCE).The A549 cells were captured by the GCE due to the interaction between HA and its receptor CD44 which is widely distributed on the surface of A549 cells,and then recognized by anti-CD44 antibody modified Fe3O4-PtSn NPs nanoprobe.The concentration of A549 cells were proportional to the electrical signal generated by electrocatalytic reaction.For colorimetric assay,the A549 cells were first seeded onto the plate and then incubated with anti-CD44/PtSn-Fe3O4 nanoprobe.The nanoprobe can catalyze the substrate to produce color signal which can be recognized by naked eye or microplate reader.This method is not only easy to implement but also show stable and reliable results.The synergistic catalytic effect of Fe3O4 NPs and PtSn NCs can significantly enhance the detection signal,which make the two methods become extremely sensitive and show the limit of detection of three to eight cells.Overall,the proposed sensing strategy showed great application potential in early detection of tumor cells.3.Glucose encapsulating liposome for signal amplification for quantitative detection of biomarkers with glucometer readoutA new technology was developed to quantitatively detect a broad range of disease biomarkers and proven to be portable,economical,and conveniently accessible.Measurements were performed based on releasing encapsulated glucose from antibody-tagged liposomes and subsequently detecting the released glucose using a commercial personal glucose meter(GM).The innovative aspect of this approach lies in the quantification of target biomarkers through the detection of glucose,thus expanding the applicability of the GM by broadening the range of target biomarkers instead of detecting only one analyte,glucose.Because of the bilayer membrane of liposomes,which can accommodate tens of thousands of glucose molecules,the sensitivity was greatly enhanced by using glucose encapsulating liposomes as a signal output and an amplifier.Here,the model analyte,protein 53 phosphorylated on Serine 15(phospho-p5315),was captured by primary antibodies bound on magnetic Fe3O4 nanoparticles and then recognized by reporting antibodies conjugated to glucose encapsulating liposomes.Finally,the target phospho-p5315 was detected by lysing the bound liposomes to release the encapsulated glucose(4+105 glucose molecules per liposome),which is detected with the GM.This approach was demonstrated to be a universal technology that can be easily produced to quantify a wide variety of biomarkers in medical diagnostics,food safety,public health,and environmental monitoring.In the near future,it is expected that these sensors,in combination with a portable GM,can be used in many fields such as physicians' laboratories,hospitals and the common household.4.A nanozyme-and ambient light-based smartphone platform for simultaneous detection of dual biomarkers from exposure to organophosphorus pesticidesA transparent,lateral-flow test strip coupled with a smartphone-based ambient light sensor was first proposed for detecting enzymatic inhibition and phosphorylation.The principle of the platform is based on the simultaneous measurement of the total amount of the enzyme and enzyme activity to biomonitor exposure to organophosphorus(O)P)pesticides.In this study,butyrylcholinesterase(BChE)was adopted as the model enzymeand ethyl paraoxon was chosen as an analyte representing OP pesticides.The total amount of BChE was quantified by a sensitive colorimetric signal originating from a sandwich immunochromatographic assay utilizing PtPd nanoparticles as an enzyme-mimicking probe.In the sandwich immunoassay,only one antibody against BChE was simultaneously utilized as the recognition antibody and the labelling antibody due to the tetrameric structure of native BChE.The BChE activity was estimated by another colorimetric signal using the Ellman assay.Both colorimetric signals were detected by the smart phone-based ambient light sensor.The proposed sensor operated with an LED in a 3D-printed substrate,which emitted excitation light and transmitted it through a transparent,lateral-flow test strip.With the increase in the colorimetric signal in the test line of the test strip,the intensity of the transmitted light decreased.The smart phone-based sensor showed excellent linear responses for assaying the total amount of BChE and active BChE ranging from 0.05 to 6.4 nM and 0.1 to 6.4 nM,respectively.A high portability and low detection limit were simultaneously realized in the common smartphone-based device.This low-cost,portable,easy-operation and sensitive immunoassay strategy shows great potential for on-line detection of OP exposure and monitoring other disease biomarkers.