Research On The Detection Of Chronic Myeloid Leukemia K562 Cells And MicroRNA-21 Using Biosensors

With the deepening research of CML,a large number of CML markers and new detection methods continue to be discovered.These findings provide a new theoretical basis for the diagnosis and treatment of the disease.They also bring new hopes for the individualized treatment of the disease.This study integrates molecular biology,cell biology,analysis chemical technology,which developed a simple and practical electrochemical method for direct,rapid,in situ and ultra-sensitive detection of CML K562 cells.A surface plasmon resonance sensor based on high performance hairpin stacking cycle(HSC)and logic gate was designed to realize the rapid and sensitive detection of the CML miRNA-21.This study mainly includes the following two parts: 1.Electrochemical aptamer cytosensor for fast direct detection of chronic myelogenous leukemia K562 cellsBy combining the aptamer technology,cell biology technology,biotin-avidin system and electrochemical detection technology,this method developed an alkaline phosphatase signal amplification strategy for rapid and ultrasensitive detection of chronic myeloid leukemia K562 cell.Aptamers are labeled on the electrode surface.It can specifically combine with the K562 cell surface receptor,thereby immobilizing the cells on the electrode surface.Though the cell surface chitosan antibody bio-concanavalin A(Con A),the biotin-avidin system was introduced in the detection.The alkaline phosphatase as signal recognition element transformed cell recognition events into enzyme signal amplification for electrochemical detection.The established signal amplification strategy has strong signal amplification ability,less nonspecific adsorption and low background signal.This method is able to produce signal response down to 79 cells/mL in biological sample;the detection linear range is 102-107 cell/mL,up to six orders of magnitude;the method can quantitative detection of K562 cells in complex venous blood samples.The developped signal amplification strategy is expected to be a powerful tool for CML K562 cell detection and to be applied to the detection of other related cells.It may play an important role in cell biology research.2.Detection of chronic myeloid leukemia potential marker MicroRNA-21 by surface plasmon resonance sensor based on high performance hairpin stacking cycle and logic gatesSeveral studies have shown that mi RNA-21 is highly expressed in patients with chronic myeloid leukemia.MiRNA-21 can increase the migration and decrease the apoptosis of CML K562 cells.The antisense oligodeoxynucleotide chain can inhibit the migration and promote the apoptosis of K562 cells and up-regulate the expression of PDCD4.In conclusion,miRNA-21 plays an important role in the development of CML,which can be used as a potential marker of the disease.In this study,a novel surface plasmon resonance(SPR)sensor based on high performance hairpin stacking cycle(HSC)and logic gates was designed to detect the new CML marker miRNA-21.HSC is a circulatory system based on the kinetic control mechanism of nucleic acid molecular hybridization.In HSC system,three metastable DNA hairpins are prevented from reacting with one another by ensconcing the complementary sequences within hairpin structures.A short single stranded oligonucleotide “catalyst” that can interact with a toehold on one of the hairpins leads to strand displacement cascade reaction,the formation of a branched junction complex and the recycling of the catalyst.This cycle can be applied to the sensor for producing signal amplification effect.It has a significant impact on enhancing sensitivity and reducing matrix effect.In this study,we introduced a novel strategy of substituting two nucleotides in a specific hairpin,which we termed ?loop-domain substitution‘ to minimize asymptotic leakage.Through these processes,the S/B ratio of the DNA circuit was improved to 6-fold.Finally,the high-performance DNA circuit was successfully applied to detect miRNA-21 down to 1.5 p M and found to be robust against circuit leakage.By designing different DNA input strands,the ―INHIBIT‖,―AND‖ logic gates could also be activated to achieve the output signal based on SPR platform.We believe these achievements not only offer design guidelines in the development of DNA circuitry but also provide a simple,label-free and enzyme-free platform for disease diagnosis by the detection of related gene.