The Research And Application Of Biosensor Based On Immunoliposomes In Vitro Diagnosis

In vitro diagnosis(IVD)is a method for diagnosing diseases by testing human body samples such as blood and urine in vitro.Biosensor is a special type of sensor developed between the life sciences and information science,formed under the integration and cross-cutting of various disciplines,including medicine,nanotechnology,informatics,food monitoring and chemistry,etc.In vivo imaging and clinical applications have a huge role and are currently hot researches at home and abroad.At present,the detection methods for various types of markers in clinical practice are tedious in operation,long in detection time,requiring special sites,large-scale equipment,and professional personnel and other factors,so the development of high sensitivity,simple,rapid,suitable for complex environments and conducive to clinical promotion.The biosensing technology is imminent.Proteins are indispensable biomolecules in organisms and involve a wide range of biological functions.Unique protein expression may indicate organisms and cells,and abnormal expression of proteins is often associated with disease,which is of great significance for the diagnosis,treatment,and prognosis of the disease.In the detection of protein,realizing trace,dynamic,real-time and rapid,is the focus of the current scientific research staff.Nanotechnology has formed nano-biotechnology in the biological field.Applying nanomaterials to biosensors can greatly improve the performance of sensors.As a new type of nanodiagnostic particles,liposomes can link different ligands such as antigens,antibodies,biotin,carboxyl,etc.,and can also encapsulate different markers,and detection modes are various.It can be used as a good carrier for signal transduction,signal conversion,and signal amplification,and has a wide range of application prospects.It has its superiority in combination with high sensitivity and multiple detection methods.In this dissertation,we use the commonly used liver cancer,pancreatic cancer and inflammatory biomarkers as protein detection models,combined with LAMP,handheld fluorescence detectors,and microfluidic chips,to construct a series of biosensors based on immunoliposome nanoparticles.And applied with clinical specimens,greatly improving the detection sensitivity and POCT technology.Part1 Magnetic-Bioluminescent-Nanoliposomes for ultrasensitive and portable detection of protein biomarkers in bloodProtein biomarkers play an important role in the diagnosis and treatment of disease.Herein,we report an ultrasensitive magnetic-bioluminescent-nanoliposome based technique using a portable ATP luminometer for the POCT of protein biomarkers in blood.In this study,we use alpha-fetoprotein(AFP,a protein biomarker associated with hepatocellular carcinoma)as a model protein.The bioluminescent nanoliposomes conjugated to magnetic particles(LBM)enabled target protein capture,isolation and detection.In this assay,we use a portable magnetic bead separation pen to simplify the steps.The output(relative light units,RLUs)had a linear correlation with AFP concentration between 0.05 and 1000 ng/mL,with a limit of detection of 0.01 ng/mL.Our LBM assay for AFP based on the portable luminometer exhibited high specificity for AFP,with no cross-reactivity with other proteins tested at 25 ng/mL.Forty clinical samples(twenty AFP positive and twenty AFP negative)were tested by the LBM assay,and the results were in good agreement with those determined by electrochemiluminescence,with relative deviations of less than 10%.The successful application of magnetic-bioluminescent-nanoliposomes with a portable ATP luminometer system to detect protein biomarkers in blood has opened a new avenue for biomarker testing.Thus,our LBM assay holds great potential as a POCT assay for use in clinical diagnostics.Part2 Nano-biotinylated liposome-based immunoassay for the ultrasensitive detection of protein biomarker in urineWith the development of proteomics and the continuous discovery of biomarkers of trace proteins,it is very important to accurately quantify the low abundance protein,especially in urine for clinical diagnostics.In this paper,we reported a novel Nano-biotinylated liposome-based loop-mediated isothermal amplification(LI-LAMP)for the ultrasensitive detection of REG1A(a kind of biomarker for the Pancreatic ductal adenocarcinoma(PDAC)in urine)with high specificity.The detection range was from 1 ?g/mL to 1 fg/mL,with LOD 1 fg/mL,and no cross reactivity occur in this assay.Compared with the amount of REG1 A added,the REG1 A recovery using this method was 130% and 89%.Real sample detection of REG1 A concentrations obtained using LI-LAMP were in good agreement with those determined by the ELISA Kit,and the relative deviations were not more than 10%.Thus,LI-LAMP holds great potential as a clinical diagnostic assay method.Part 3 Microfluidic chips and quantum dot liposome for quantitative iPOCT etection in inflammatory markerIn this section,we used liposomes encapsulated with quantum dots to convert oil-based quantum dots into water-soluble nano-quantum dot liposome microspheres for specific labeling and recognition of protein markers.iPOCT on microfluidic chips is realize rapid and sensitive diagnosis.We named this method LQC(Liposome encapsulated quantum combined with microfluidic chip for iPOCT).In this study,we used procalcitonin(PCT,an inflammatory protein biomarker)as a model protein.The antibody 2 and goat anti-mouse antibody were laid on a microfluidic chip at a certain concentration and volume to form a detection line and a control line.The binding zone was pre-layered with antibody 2-coupled quantum dot liposome to add the sample to be tested.After entering the binding zone,it is driven by capillary force and enters the reaction zone.Combine the self-developed portable UV excitation device and mobile phone application program to perform reading analysis and verify the feasibility.The G channel and Qds concentrations were linear between 0-10 ng/mL and the LOD was 0.5 ng/mL.This method shows high specificity for PCT and no reaction with other proteins at a concentration of 2 ng/mL.Twenty clinical samples(10 positive and 10 negatives samples for PCT)were tested by LQC method.The results were consistent with the Roche electrochemiluminescence assay.This method greatly simplifies the experimental procedure and steps.It takes only 15 minutes to obtain the results.It is very suitable for self-testing in clinics,field,bedside,and home.