| Cancer is the second leading cause of death in the world,seriously affecting social and economic development.According to the latest statistics released by the national cancer center of China in 2019,there were about 3.929 million new cases of malignant tumors and 2.338million deaths from malignant tumors.Among them,the incidence of hematological malignancies is consistently in the top 10.In order to reduce the death rate of cancer patients and prevent the spread of cancer,early diagnosis and treatment are necessary.The current gold standard for tumor diagnosis is usually a tissue biopsy.Given the character of biopsy,it has many limitations,including patient risk,sample preparation,low sensitivity and accuracy,high cost,and invasive testing,which make this method incompatible with early clinical trial monitoring.Other auxiliary detection methods are not only widely used in clinical applications,but also provide a great scientific and intuitive basis for the diagnosis of diseases,which can better match with clinical symptoms and play an irreplaceable role in the final accurate diagnosis of diseases.Such as medical imaging diagnosis,mainly include fluoroscopy,X-ray,CT,MRI,ultrasound,angiography and PET-CT.However,due to the high cost,false-negative/positive susceptibility,and high dose of radiation in some of these mentioned imaging methods,early screening and diagnosis in populations are also not appropriate.Therefore,the development of new detection methods,reducing the economic burden and the detection time,enhancing the sensitivity and specificity,and improving the early detection and diagnosis rate for cancer is one of the main tasks of the current healthcare and plays a key role in secondary prevention of cancer.The current hot concept of liquid biopsy has great potential to overcome these existing limitations.Liquid biopsy is the sampling and analysis of non-solid biological tissues,mainly blood.Like traditional biopsies,this type of technology is mainly used as a diagnostic and monitoring tool for diseases.Such as detecting specific targets or markers of cancer in the blood.The main advantage of this technology is non-invasive.As a result,it can better track the process and mutations of tumors over time,and it is possible to monitor the recurrence for patients after treatment.Especially,this method is suitable for patients with hematological malignancies.Commonly used methods of liquid biopsy include enzyme-linked immunosorbent assay(ELISA),fluorescence spectroscopy,radioimmunoassay and immunohistochemistry.However,these methods often require dedicated laboratory instruments and complex protocols,which make them expensive,laborious,and time-consuming.Therefore,these disadvantages limit their use in early clinical diagnosis.In contrast,biosensors are excellent tools for liquid biopsy,due to portable,sensitive,specific,and easy to use.Biosensors often use biological receptors to specifically recognize and capture targets-including proteins,DNA/RNA,and nucleic acids-then convert the generated biological signals into analyzable physicochemical signals.At present,biosensors,such as electrochemical sensors and optical sensors,can amplify the detected biological signal in the samples.This greatly reduces the consumption of samples and the cost of testing,and improves the sensitivity of detection,which is of great significance for the early screening and diagnosis of cancer patients.Based on the above background,this dissertation respectively utilized electrochemical biosensor and optical biosensor to screen and identify target markers in hematological malignancies.Thereafter,the clinical application value and prospect of detection in patients with hematological malignancies by biosensors were verified via comparing with the results of classical methods used in clinical practice.The specific assignments were divided into the following three parts:1.Diagnosis and preliminary screening of hematological malignancies based on electrochemical biosensorTo develop and evaluate an electrochemical biosensor that,screen-printed electrodes wrere desigened to identify healthy individuals,patients with malignant hematologic tumors and patients with solid tumors by detecting the leukocytes in whole blood.Of note,a cell is constantly changing its charge whether it is in an excited state or a resting state.Many physiological activities are accompanied by the directional transfer or conduction of charge.The biochemical reactions of cells are very similar to the electrochemical reactions occurring on electrodes,so electrochemical biosensor can be used to detect the electrochemical properties of cells in blood.Owning to different cellular redox reactions in different kinds of blood cells,normal cells and cancer cells presenting different responses can be sensitively identified.Hence,we will divide the blood samples collected from the hospital into three groups:healthy individuals,hematological malignancies,and solid tumors.Based on the different cellular electrochemical behaviors in blood samples of patients with different diseases,differential pulse voltammetry(DPV)was used to detect the peak potential of leukocytes in a blood sample dripped on a screen-printed electrode using an electrochemical biosensor.Then compared the potential curve to distinguish the diseases among three groups according to the difference peak potential and peak shift.Based on the above mentioned experiments,a tiny of peripheral blood each person only needs can initially diagnose whether or not they have a tumor through the analysis of electrochemical biosensor.At the same time,it can also strengthen the secondary prevention of tumors(early detection,early diagnosis and early treatment),and enhance the early detection rate and population screening.With increasing the early diagnosis rate of tumors,the survival rate of cancer patients could be improved.2.Detection and clinical application of optical biosensor based on surface-enhanced Raman scattering(SERS)in resistant proteins of leukemiaP-glycoprotein(P-gp)is an important regulator of multi-drug resistance(MDR)in leukemia,so the detection the expression of P-gp is of great significance for detecting drug-resistant patients and adjusting chemotherapy regimens.Recently,optical biosensors are rapidly developing in the field of tumor marker recognition owning to their high efficiency,fast speed and simple operation.Therefore,in this study,we chose an optical biosensor,Surface enhanced Raman scattering(SERS)method to evaluate the expression level of P-gp in leukemia cell lines and blood samples from patients with leukemia.Compared with conventional detection methods,Raman spectroscopy was performed in SERS to detect the samples adsorbed on the surface of rough metal particles,which can make the Raman signal of adsorbed sample increase10~3-10~6 times.The strong signal can realize the qualitative and quantitative detection of samples in clinical application,and has great significance for effectively solving the detection of multiple tumor markers owning to its advantages,such as high sensitivity,narrow spectrum,and multiple detection.In this part,we used to antibody-modified magnetic beads(MBs)to capture target cells and antibody-modified SERS probes to recognize P-gp in the cells,then finally formed a"MB-cell-probe"sandwich structure.The Raman spectroscopy was utilized to detect the SERS signal intensity of the sandwich structure,in order to evaluate the expression level of P-gp.This study is not only performed at the cell lines,but also used to evaluate clinical samples from patients with leukemia.Based on the excellent sensitivity,specificity,reliability and application potential of SERS technology,we expect that this SERS-based detection method could be very helpful for the clinical diagnosis of early multidrug resistance in leukemia.3.Multi-detection and clinical monitoring of minimal residual markers in B-cell line hematological tumors by optical biosensor based on SERS technologyThe measurement of minimal residual disease(MRD)is essential for the diagnosis and prognosis of hematological malignancies in the clinic.Therefore,there is an urgent need for a sensitive and accurate method to monitor specific tumor surface markers for early diagnosis and treatment guidance.In this experiment,we also used SERS-based optical biosensor to detect surface markers of MRD in patients with hematological malignancies.Due to the lack of specific marker,the detection of a single marker often cannot accurately diagnose the character and category of the tumor,so the development of multiple detection technology for tumor marker is particularly important.Compared with the traditional single-parallel detection technology,the multiple detection technology can reduce sample consumption,increase the sample throughput,shorten the detection time and reduce the cost of detection.Thus,this study was designed to detect two surface markers of leukemia/lymphoma of the B cell line simultaneously,and then the results of samples detected by SERS were compared with clinical flow cytometry data to evaluate the specificity,sensitivity and repeatability of SERS technology in multiple detection.Under the above conditions,this experiment combined the most troublesome topic of multiple detection in clinical practice with the monitoring of MRD in patients with hematological malignancies.This new method is expected to reduce the limit of detection(LOD)and improve the early positive rate of MRD,which will provide a strong technical support for diagnosis of refractory/recurrence and adjustment of personalized chemotherapy regimen.Meanwhile,the multiple detection of our project can also be extended to other types of tumor markers,which has important value in basic scientific research and clinical application.In summary,the different types of biosensors in our present study have advantages of sensitivity,specificity,and repeatability,which can be used not only to screen and identify cancer patients from healthy people,to distinguish sensitive/resistant cells,and also to recognize the residual tumor cells.It is of great significance to improve the applicability and positive rate of clinical sample,increase the accuracy of classification,benefit for evaluation of cancer progress.This method is convenient and fast,only need tiny samples,without positioning,and can be quantificational and dynamical monitored.In particular,the samples can be centrally processed,so the method only needs tiny samples,saves time and effort compared to other methods,and it should be deserved much attention for its high clinical significance and commercial value. |
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