Development Of A New Type Of Electrochemical Immune Analyser And It's Application In Diagnosis Of Acute Leukemia

Immunophenotyping of acute leukemia (AL) plays a very important role in the diagnosis of AL. At present, there are two methods of immunophenotyping of AL:one is immunocytochemistry or immunofluorescence method by means of microcsope and the other is immunofluorescence by means of flow cytometry (FCM). However, the former is subjectivity, poor reproducibility, time consuming,one kind of antigen being detected(ie, low flux) and et al. Because of expensive equipment and reagents, it is difficult to apply FCM in the primary hospitals.Therefore, it is essential to develop a rapid, simple, high-through-put, high sensitivity, economical and more objective new technology for immunophenotyping of AL.In our studies,a new method of cELISA electrochemical analysis was developed by combining cell enzyme-linked immunoassay technique with electrochemical methods,in which enzymatic silver deposition reaction took the place of enzyme-catalyzed color reaction,and the number of antigen was detected by the number of deposition silver. First,we developed a small new electrochemical immunoassay analyzer whose current detection limit was pA level.Secondly, a new methods of cELISA enzymatic silver deposition in the interdigitated electrode array (referred as cELISA interpolation electrochemical) was developed, the feasibility of the new type of electrochemical immunoassay analyzer applied in immunophenotyping of AL was explored. Finally, the feasibility of the new type of electrochemical immunoassay analyzer applied in detecting of MPO in AL with the electrochemical impedance method was explored.Objective:To develope a multi-function small new type of electrochemical immune analyser suitable for detecting of micro-current in micro-electrochemical sensor.Methods:First, the hardware circuitry designed included the circuitry of signal acquisition and conversion amplification, filters and short-circuit alarm, et al. Secondly, software programs designed was made up of micro-controller program, communications software and PC software. Finally, the performance of new electrochemical immunoassay analyzer was tested using linear sweep voltammetry (LSV) and electrochemical impedance impedance method. Results:The current of less than 10pA was detected by the new type of electrochemical immunologic analyser; a impedance waveforms was displayed and recorded by it. Conclusion:The new electrochemical immune analyzer has been developed, its performance meets the design requirements of multi-functional, high sensitivity and small-type.Objective:To explore the feasibility of the new type of electrochemical immune analyser applied in immunophenotyping of AL patients by cELISA interdigitated electrochemistry method established in this work. Methods:Leukemic cells were coated with Poly-L-Lysine (P-L-L) in the wells of microplate,after primary antibody of mouse against human and second antiobody of horse against mouse labled with alkaline phosphatase (ALP) was added one by one,the solution containing silver and ALP substrate was added, after the silver deposition on the surface of interdigitated electrode, the electrical conductance was measured with the new electrochemical immunologic analyzer.First,a new method of cELISA interdigitated electrode electrochemistry was established, which included optimizing the parameters such as the molecular size of P-L-L used, the concentration of antibodies,et al and analyzing the specificity and sensitivity of the cELISA interdigitated electrode electrochemistry method. Subsequently, the expression of 8 cluster of differentiation (CD) antigens on the surface of leukemic cells in 72 AL patients were determined by the method and FCM, and their results compared with each other. Results:The optimal molecular weight of poly-L-lysine was 7-15 million Dalton; The optimal number of cells coated on a microplate was 1.6×105, The optimal concentration of the primary antibody was 20μg/ml, and the optimal concentration of the second antibody was a ratio of 1:500 dilution; The sensitivity of the method of the cELISA interdigitated electrochemistry was about 300 of HL-60 cells. The relative coefficient for the results obtained by the cELISA interdigitated electrochemistry method and FCM analysis was 0.93. The results of 8 CD antigens expression in 72 AL patients, which obtained by cELISA interdigitated electrochemistry method had no significant difference from those of FCM byχ2 test (P=0.373). Conclusion:The results of testing leukemia cell lines and clinical specimens by cELISA interdigitated electrochemical analysis which we created were consistent with those of the FCM analysis, this indicates that there is high consistency in two methods. CELISA interdigitated electrochemical analysis is specificity, high sensitivity and a new way of immunophenotyping of AL in clinic. The new type of electrochemical immune analyser can be applied in immunophenotyping of AL by cELISA interdigitated electrochemistry method.Objective:To explore the feasibility of the new type of electrochemical immunologic analyser applied in the detection of MPO activity in AL patients by electrochemical impedance spectroscopy method. Methods:Cells were lysis with the solution of Tris-HCL (pH 8.7), and added slowly on the surface of gold electrode, POX staining solution of A and B were added respectively. The conductivity before and after catalysis was measured with the new electrochemical immunologic analyzer. Firstly, the specificity of electrochemical impedance spectroscopy method was analyzed with HL-60 cell and Jurket cell. Subsequently, the activity of MPO in AL patients was analyzed by the electrochemical impedance spectroscopy method, and whose results compared with those obtained from the enhanced chemiluminescent assay Results:15 of 25 AL patients were MPO-positive, and the remaining 10 of AL patients were MPO-negative detected by the electrochemical impedance spectroscopy method. For the 14 MPO positive AL patients detected by the enhanced chemiluminescent assay, MPO positive were also obtained by the electrochemical impedance spectroscopy in the 14 AL patients;for the 11 MPO negative AL patients detected by the enhanced chemiluminescent assay,one case was detected as positive by the electrochemical impedance spectroscopy. Results obtained with the new method has no significant difference from the enhanced chemiluminescent assay byχ2 test (P=1).Conclusion:The results of MPO activity in 25 tested AL patients by electrochemical impedance spectroscopy are consistent with those of the enhanced chemiluminescent assay, which indicates that there is high consistency in the two methods. The electrochemical impedance spectroscopy is a specificity, high sensitivity method,which provides a new method for detecting the activity of MPO in AL patients. The new type of electrochemical immune analyser can be applied in detecting the activity of MPO in AL patients by the electrochemical impedance spectroscopy.This study showed that the new type of electrochemical immunoassay analyzer is a multifunction apparatus,which could detect micro-current such as pA level.This instrument could be used not only to detect cell surface antigens, but also to detect intracellular MPO activity. It is simple, portable,and has clinical value and market prospect.