Construction Of Biosensing Platform Based On Primer Exchange Reaction Combined With Glucose Meters For Disease Markers Detection

Disease markers refer to biological molecules(including proteins,nucleic acids,sugars and hormones,etc.)with abnormal concentrations resulting from metabolic disorders in the early and middle stages of the disease.At present,disease markers have become a new measure to diagnose early disease in clinical analysis.For example,the content of amyloid-β oligomer(AβO)in cerebrospinal fluid can be used as a marker for the diagnosis of Alzheimer’s disease(AD).Human immunodeficiency virus(HIV)-related gene fragments have been considered as markers for AIDS screening.The diagnosis of disease markers,on the one hand,can detect the disease as early as possible,assess and stratify the risk,improve the treatment plan,and improve the survival rate of patients.On the other hand,it can also have a deeper understanding of the disease,so as to better conduct drug research and development.However,the current detection methods for related disease markers still have some shortcomings:(1)The detection process is complex and requires bulky instruments,which is hard to achieve on-site detection.(2)The sensitivity is low,and difficulty in meeting the demands of trace marker analysis.Therefore,the development of highly sensitive,high accuracy and portability strategies to detect trace disease markers is urgently needed.Recently,the introduction of nucleic acid amplification strategies has enabled researchers to develop biosensing strategies with significant improvements in sensitivity.Among them,primer exchange reaction has broad application prospect because of its excellent amplification performance and high efficiency utilization.Personal glucose meters(PGM),due to its small size,low cost,reliable results,and simple operation,has become increasingly popular for pointof-care detection and on-site analysis.In the thesis,innovative biosensing strategies were designed using primer exchange reaction as signal amplification technology combined with glucose meter and were applied to the detection of amyloid-β oligomer(AβO)and HIV gene fragments.The thesis is divided into the following four chapters:Chapter one is an introduction:This section summarizes the concept,classification,and the current detection methods of disease markers,elaborates on the mechanism of primer exchange reaction and its applications in biological detection,and expound the research purpose and content.In chapter two,cascade primer exchange reaction(PER)based amplification technology was developed for sensitive and portable detection of AβO using personal glucose meters(PGM).Two PER processes were employed here.In the primary PER,the hairpin template 1(HT1)was designed with a primer binding domain,a primer extending domain and a blocking extending domain.The primers were designed to be modified on magbeads surface.Initially,the primer binding domain in HT1 was locked by AβO aptamer.When target AβO was present,aptamer bound with AβO and dissociated from HT1 to initiate the primary PER.The products acted as the primer of the secondary PER to hybridize with another hairpin template 2(HT2),initiating the secondary PER and producing numerous ssDNA with repeated DNA-invertase binding sites.After binding with DNA-invertase,the obtained conjugates were magnetically separation to catalyze the conversion of sucrose to glucose,which were detected by a PGM.The strategy achieved a limit of detection of 0.22 pM with a linear ranged from 1 pM to 250 pM.Satisfactory reproducibility results were obtained in actual samples.This strategy provided a superior tool for sensitive and convenient detection of AβO,displaying a good prospects in the early diagnosis of AD.In chapter three,a magnetic separation assisted cross primer reaction based amplification strategy combined with personal glucose meter for sensitive and convenient detection of HIV gene fragments.The target is first hybridized with auxiliary probes L1 and L2 to form a threeway structure.Then the polymerase triggers the extension reaction of L1 to generate double strands containing the cleavage enzyme recognition sites.When the cleavage enzyme cuts a specific site,new trigger strands T1 and T2 are generated to participate in the subsequent primer exchange reaction,and the cut three-way structure continues to participate in a new round of polymerization and nicking cycle.T1 and T2 hybridize with hairpin templates HT1 and HT2 respectively.With the participation of polymerase,the first primer exchange reaction is completed,so that T1 extends the binding sequence of HT2,T2 extends the binding sequence of HT1,the extended T1 hybridizes with HT2,and the extended T2 hybridizes with HT1.T1 and T2 hybridized templates exchanged repeatedly,and cross PER occurred step by step,resulting in the generation of a large number of single strands DNA(ssDNA)containing repeated sequences.After toehold-mediated strand replacement reaction between these ssDNA and the DNA-invertase bound on the magnetic beads,the DNA-invertase is replaced into the supernatant.After magnetic separation,sucrose was added to the supernatant.Under the action of invertase,sucrose was hydrolyzed to glucose,which was detected by PGM.The linear range of this strategy is 2.5-75 pM,and the detection limit is 0.46 pM.The results of this strategy in serum samples are also satisfactory due to the use of magnetic separation technology.These results indicate that this strategy has a promising application prospect in HIV diagnosis.The chapter four is the summary and outlook of this paper.