The Detection Of Biomarkers In Neurological Paraneoplastic Syndrome

BackgroundTumors are divided into benign and malignant types. Generally speaking, the latter is what we often call cancer. In recent years, along with the increase of environmental pollution and food safetythe incidence of and mortality induced by cancer are increasing annually. Cancer has a reputation as a deadly disease.Paraneoplastic syndrome generally refers to the abnormal immune responses caused by unusual product of tumor or some relevant clinical manifestations in digestive, endocrine nervous, hematopoiesis, bone, kidney and skin system with unknown cause. These phenomenaare generally not directly caused by the primary tumor but through indirect routes so these syndrome are called paraneoplastic syndrome.Paraneoplastic neurologic syndromes,(NPS) occurs in some patients with malignant tumor, and can affect any part of the central and peripheral nervous system without tumor metastasis.. PNS is not caused by the tumor direct invasion or its metastasis, or by metabolic disruptions, nutrition, infection or anti-tumor therapy, It is often marked by functional disorder of muscle, peripheral nerve, even spinal cord or brain. PNS is rare, affecting less than1%patients with cancer. However, PNS has a wide range of lesions and diverse clinical manifestations. In most NPS patients, the neurological disorder may develop before or after the cancer becomes clinically overt. According to statistics, about80%of patients, the paraneoplastic neurological syndrome (PNS), antedates the diagnosis of cancer. This provides an opportunity for us to discover the existence of malignant tumor. For those cancers with a serious threat to human life and health early discovery, diagnosis and treatment can significantly improve the survival of patients.Compared to the symptoms of general nervous system tumor the paraneoplastic neurological syndrome (PNS) incidence is relatively low. Compared with complications in nervous system induced by other organ tumor, paraneoplastic syndrome is rare. But because of various factors, it is very important to early discover and treat paraneoplastic neurological syndrome (PNS) clinically. First, PNS may cause more serious damage and function defect, and the disorder may last longer; Secondly, for regular system patients with nervous system disease who show the phenomena of PNS,, it is often characteristic of that the carcinoma in situ is not yet diagnosed.. Therefore, as far as the neurologist is concerned, this is a kind of effective way to diagnose the disease in an early stage. The doctor needs to have a high ability to recognize and diagnose cancer. Again, if PNS could be found and treated in an early stage, the possibility to cure cancer could be greatly improved, and the function of the nervous system could be restored gradually.Currently there are no particularly effective therapeutic agents to cure PNS. The current therapy includes vitamins, corticosteroids, plasma exchange and immunosuppressant treatmentbut their effects remain to be proved. Clinical studies demonstrated that the neurological paraneoplastic syndrome were relieved and improved after the primary tumor was effectively curedThe main innovation points of this research:1. A novel quantum dot nanoparticles was designed in this study. Our study used aptamer DNA as stabilizer in the process of preparation of water-soluble nanoparticles and as the compound that participate in the reaction. This protocol simplifies the cumbersome steps required in the general protocol which needs to separately conjugate aptamer DNA to the surface of nanoparticles.2. The surface of a single quantum dot nanoparticle can have multiple DNA aptamers while in the surface of a single microspheres carrier can be combined with multiple quantum dot nanoparticles. so it has a function of cascade amplification, significantly improving the efficiency and sensitivity of detection. PART I The detection of biomarkers in Neurological paraneoplastic syndromeObjective:To investigate the early detection of the biomarkers in neurological paraneoplastic syndrome (paraneoplastic neurologic syndrome, PNS) and to study the early diagnosis and early preventive treatment.Methods:Information was obtained from our hospital between Sep2010and May2013. The32outpatients include12cases with PNS and20cases without PNS. Twelve PNS patients include7male and5female,,35to72years old with mean age49.3±11.7. old., The time of disease course was from3months to5years. The pathological types are small cell lung cancer SCLC) with5cases, breast cancer with3cases, ovarian cancer with2cases, liver cancer with1, pheochromocytoma with1. The20cases without NPS include12male and8female, aged38to75with mean age50.5±9.1, The time of disease course was from five months-4.5years. There are5cases of gastric carcinoma,5cases of systemic lupus erythematosus (sle),3of rheumatoid arthritis,3cases of prostate cancer,2dermatomyositis cases and2cases of pulmonary tuberculosis. All cases are confirmed by imaging, hematology and pathological examination. Other randomly selected20healthy people who are served as control group include10male and10female, with age from26to65and mean age40.1±9.2.Age, sex, the time of disease course and other laboratory tests (including the electromyography, eeg, etc.) and imaging characteristics (including CT, MRI, enhanced MRI, MRA, etc were analyzed and evaluated. All healthy people and patients were subjected to clinical examination and detection of serum antibodies such as anti-Hu, anti-yo anti-Ri by enzyme-linked immunosorbent assay. We evaluated the relevance between the concentration and titer of specific antibodies and their and the primary lesions of PNS.Results:(1) No difference regarding the regular information The number of casesage, gender, time of disease course between two groups showed no statistically significant difference by T-test.(P>0.05).(2) Enzyme-linked immunosorbent assay results: Enzyme-linked immunosorbent assay results show:The expression of antibodies such as anti-Hu, anti-yo anti-Ri in group PNS was increased significantly than in group non-PNS (P<0.01)(3) anti-Hu antibody in the diagnosis of NPS: According to diagnostic criteria for NPS by Graus,9of12cases were diagnosed as PNS or tumor,2cases (see table1. NO.9and12) without the primary tumor and the other7cases with tumor. The detection of Hu antibody may predict75%of patients with PNS (9/12)(see table2).(4) the relationship between neurons antibody and NPS clinical types: NPS with positive anti-Hu mainly manifested as tumor sensory neuron disease (paraneoplastic chipmaker neuronopathy, PSN) and deputy tumor encephalomyelitis (paraneoplastic encephalomyelitis, P EM), with a few paraneoplastic limbic encephalitis(PL E) and paraneoplastic cerebellar degeneration(PCD) paraneoplastic chipmaker-motor neuropathy,(PSMN), etc.; Anti-Yo is mainly related to PCD yukio okamoto, antibody and Anti-Ri antibody is associated with paraneoplastic opsoclonus myoclonus(POM).Conclusion:1. The main biological markers of neurological paraneoplastic syndrome are anti Hu antibody, anti-yukio okamoto and anti-Ri antibody.2. Enzyme-linked immunosorbent assay results show:The expression of antibodies such as anti-Hu, anti-yo anti-Ri in group PNS was increased significantly than in group non-PNS (P<0.01)3. Detection of anti-Ri antibody,anti-Hu antibody and anti-Yo antibody in the sera of patients, can improve early detection of tumor cells. It can also provide early treatment to diagnostic basis for clinical. PART ⅡElectrochemical biosensor detection based on quantum dots and markers of tumor cellsObjective:By improving the method of preparation of the DNA aptamer modified quantum dot nanoparticles and the probe after using anodic stripping voltammetry to detect specific ion in order to achieve the target tumor cells and tumor cell surface markers detection.Methods:PbS quantum dots and the preparation of CdS quantum dots is in reference to the related literature, the synthetic process for the improvement of synthetic method. First will lead nitrate solid powder and solid powder mixed with a certain concentration of cadmium chloride solution, And then in turn add thioglycolic acid and connection DNA, Half an hour after adding suitable amount of Na2S solution of24h corresponding quantum dot nanoparticles can be got. The whole experiment process was conducted under the condition of nitrogen protection of, the product obtained4°C saved for later use.Preparation of divided into magnetic probes and non magnetic probe. Magnetic microspheres and the non magnetic polystyrene microspheres are cleaned with PBS buffer solution before use. Then prepared beforehand (MUC1) optimal solution and the preparation of the CdS quantum dots in magnetic beads system, Will its ehrs-3aptamer solution and preparation in advance of the PbS system of quantum dots in polystyrene microspheres, Thermostatic incubation can get corresponding nano probe.Eventually adopted by the tumor cell electrochemical detection method for the anodic stripping voltammetry (DPV). With nitric acid solution will be immobilized on the surface of the tumor cells, probe from down and release the Cd2+and Pb2+. Will be prepared of glassy carbon electrode for the electrolytic cell, and then remove the electrode and in the samples are ready for testing system, begin to differential pulse voltammetric scans, set the leave time to15seconds,50mV pulse amplitude, pulse width50ms, potential scan range is0.30to0.80V (vs. Ag/AgCl). The potential of0.65V and0.45V anodic stripping peak current of Cd2+and Pb2+respectively the anodic dissolution of the signal.Results: Through continued optimization of experimental conditions eventually lead sulfide quantum dots and cadmium sulfide quantum dot the best preparation conditions of these two kinds of quantum dots. The optimum condition for synthesis of CdS quantum dots for10ml concentration of1.0×10-3M cadmium chloride solution, concentration of10ml of1.34x10-3M sodium sulphide solution, stabilizer for1mu L thioglycolic acid (C2H4O2S) and300mu L1.0×10-6M connection DNA; The optimum condition for synthesis of PbS quantum dots for10ml concentration for2×10-4M lead nitrate solution, concentration of4ml of6.7×10-4M sodium sulphide solution, stabilizer for1mu L thioglycolic acid (C2H4O2S) and300mu L1.0×10-6M connection DNA.Through continued optimization of experimental conditions the resulting in the best test condition is:in the process of electrochemical detection of DNA aptamer optimal concentration of4.0×10-7M; The optimal pH value of4.7; Probe preparation for30h incubation time.Finally detection of tumor cells in cell concentration1.0x102cells mL-2-1.0×106cells mL-1range, electrochemical detection signal to numerical proportional relationship with cell concentration, The calibration curves for the I (mu)=14.0024+9.7932lgC (cell mL-1), in which I deducted for blank current after the current intensity, C is the concentration of the cells in the cell SAP, linear correlation coefficient is0.995, n=9. Further calculated the detection limit of31.7cells mL-1; A single target cells (MUC1) on the surface of the target protein is about6.41x104, the number of single target cells on the surface of its ehrs-3the number of target protein is about3.47x104. At the same time, the test method has been verified by the experiment in the mixed samples are even more complex environment can still achieve good detection result.Conclusion:This study proposes a new method of detection of tumor cells and tumor markers. Through the setting of biological sensors, combining quantum dots and biological sensors, and then fixed load to the two magnetic beads and polystyrene microspheres carrier, they produced a type of two quantum dots based on CdS and PbS new electrochemical biosensors. Combining biological sensor and DNA aptamer again, it gives the biosensor specificity of certain goals. In this experiment, we connected on the biosensor (MUC1) and its ehrs-3of these two kinds of suitable body and used to analyze the detection of human breast cancer cells (MCF-7). Using connection DNA as a synthesis of quantum dots stabilizer is one of the features of the experiment, so it makes quantum dots solid load to magnetic beads or polystyrene microspheres were possible. Then two kinds of DNA aptamer respectively connected to the quantum dots and modified magnetic beads made of polystyrene microsphere surface nano probe. Then through suitable body on the specificity of the target protein recognition probes immobilized onto the surface of the target cells, with aqueous nitric acid after releasing Cd2+and Pb2+in quantum dots, the anodic stripping voltammetry was developed for the determination of ion and get the corresponding signal, the signal processing after they got the information about target tumour cells and their markers. Experimental detection sensitivity is higher, thanks to the experiment of preparation of nano probe has amplification effect very well.