Selection And Application Of Aptamers Against Human Pancreatic Polypeptide

Pancreatic polypeptide?PP?is a 36 amino acid peptide produced and secreted by PP cells of the pancreas.As the specific marker of non-functional pancreatic neuroendocrine tumors,PP has great clinical significance.The most commonly used method for PP detection is radioimmunoassay?RIA?.RIA is a sensitive and specific technique but still has several disadvantages:radioactive materials,specialized equipment and a laboratory classified for handling radioactive assays.While LC/MS-based method has also been developed for PP detection,this method still requires expensive equipment and complicated operation.Development of rapid and simple methods for PP detection is still urgently needed.Aptamers are a class of emerging probes which could bind to the targets with high affinity and specificity.Aptamers have several advantages,including ease of chemical synthesis and modification,high chemical stability,high affinity and low molecular weight.Therefore,they have been incorporated in the detections for small molecules,proteins and cells.In this thesis,aptamers against PP were screened through SELEX?systematic evolution of ligands by exponential enrichment?technique and characterized in order to develop new strategies for PP detection.The following are the research contents of this thesis:1.In vitro selection of aptamers against PP based on GO-SELEX.The GO-SELEX method could be helpful to obtain aptamers against free targets.In the process of GO-SELEX,high affinitive sequences would bind to targets after incubation and not be adsorbed onto GO any more;whereas,low affinitive sequences in single-stranded state would be adsorbed onto GO via ?-? stacking and removed by centrifugation.After 18 rounds of selections,the enrichment assays were conducted and the PCt products of 13th and 17th rounds were subjected to high-throughput sequencing.2.Characterization of aptamers against pancreatic polypeptide.By the use of two high-throughout and label-free strategies,eight sequences were picked up from 50 candidate sequences of high-throughout sequencing results.Next,the eight sequences were labeled with FAM and explored by a GO quenching assay.Three high affinity aptamers,named 26₁7,10₁3 and 5₁3,were obtained after that.Their Kd values were 33.15 ± 4.08 nM,58.81±3.28 nM and 77.39±3.93 nM,respectively.By dividing the aptamers into three different pairs,fluorescence quenching assays were conducted to determine which sequences could bind to different sites of PP.The results showed that 26₁7 and 10,13 could bind to PP simultaneously.This pair of aptamers would help develop kinds of detection methods for PP.3.Sandwich-type assay for pancreatic polypeptide based on dual-aptamers and dynamic light scattering?DLS?.First,aptamers 26₁7 and 10₁3 were immobilized on the surface of different AuNPs to obtain AuNP-26₁7 and AuNP-10₁3.When PP was present in solution,larger clusters existed because AuNP-26₁7 and AuNP-10₁3 could bind to PP simultaneously,closing the distance of different AuNPs.This process could be characterized by DLS with clear signal changes.Based on this principle,a rapid and simple sandwich assay for PP detection was developed.The linear range and the detection limit of this assay were 1?15 nM and 56 pM,respectively.This assay also possessed high specificity and could be used to detect PP in human serum samples,showing great potential for use in biological samples.4.Detection of pancreatic polypeptide using gold nanoparticle-enhanced surface plasmon resonance?SPR?and dual-aptamers.First,aptamers 26₁7 and 10₁3 were immobilized on the surface of gold film and AuNPs,respectively.When PP was present in solution,the distance of AuNPs and gold film would be closer because 26₁7 and 10-13 could bind to PP simultaneously.This process would result in enhanced SPR singnals according to the electronic coupling interaction between AuNPs and gold film.Based on this principle,a SPR sensor was developed for PP detection.The linear range and the detection limit were 5?150 nM and 101 pM,respectively.This SPR sensor exhibited high selectivity,good recycling properties and could also be used to detect PP in human serum samples.