Thrombin Linked Aptamer Assay For Protein Detection

Aptamers are artificial oligonucleotides that can specifically bind to metal ions,small molecules,proteins and even whole cells.Recent years,aptamers have been widely used as molecular recognition probes in the domains of diagnostics and therapeutics,chemical analysis,environmental monitoring and food safely analysis.Thrombin and its aptamer are the most commonly used model in aptamer-based assays.The main reasons are that two aptamers of thrombin are short oligonucleotides,and it is relatively easy and inexpensive to synthesize these short aptamers;the two aptamers can bind on two distinct binding sites of thrombin and is easy to establish detection methods using a sandwich format;the binding sites of aptamers are different from the enzyme catalytic sites,and the enzymatic activity of thrombin can be maintained.However,most research focused on the detection of thrombin.In this thesis,taking advantage of aptamer binding affinity and the enzymatic activity of thrombin in terms of cleaving peptide substrates,we developed novel thrombin linked aptamer assays(TLAA)for the detection of proteins.This TLAA contributed a new application of thrombin and its aptamer in bioanalysis,and provide a new way for signal generation.TLAA converted detection of target proteins to the detection of thrombin by using a DNA sequence that consists of two aptamers with the first aptamer binding to the target protein and the second aptamer binding to thrombin,the thrombin was labeled on the protein target,and thrombin catalyzed the peptide substrate into product,generating signals for quantification.To demonstrate the proof of principle of TLAA,we showed TLAA for a model protein target,platelet derived growth factor-BB(PDGF-BB).This thesis includes six parts:Chapter 1 This part briefly introduced the vitro selection and properties of aptamer,reviewed aptamer-based assays for targets and described the research background,main research work,and innovation on the thesis.Chapter 2 We developed a thrombin-linked aptamer assay(TLAA)on magnetic beads for the detection of protein in a sandwich format.PDGF-BB was sandwiched by an antibody coated on magnetic beads and an affinity DNA probe containing target-binding aptamer sequence and thrombin-binding aptamer sequence.Thrombin was labeled to the sandwich complex through specific aptamer-thrombin binding.Thrombin catalyze the hydrolysis of a peptide substrate into a detectable product.Taking advantage of preconcentration by magnetic beads and the thrombin catalysis amplification,the assay showed high sensitivity,and the detection of limit was 16 p M when fluorogenic substrate was used.Simple and commonly absorbance analysis was applied to detect PDGF-BB when chromogenic substrate was used.As the generated product was colored,the use of chromogenic substrate in TLAA also made semi-quantitative analysis feasible by observing the color change of the solution.Chapter 3 Based on the work in Chapter 2,we demonstrated a sandwich thrombin-linked aptamer assay(TLAA)for the detection of protein on microplates taking advantage of high-throughput analysis and rapid sample handling of microplate.The microplate was coated by antibody.PDGF-BB and a DNA probe containing a PDGF-BB-binding aptamer and a thrombin-binding aptamer were successively added to microplate,forming a sandwich structure.Then thrombin bound to the DNA porbe and catalyzed fluorogenic peptide substrate to produce fluorescence signal.A good linear relationship between the signal intensity and PDGF-BB concentration was obtained in a certain range.We also achieved the detection of PDGF-BB in complex samples.Chapter 4 We described a sensitive aptamer-based sandwich assay for protein detection by rolling circle amplification(RCA)coupled with thrombin-linked aptamer assay(TLAA)(called RCA-coupled TLAA).Taking advantage of RCA production of long DNA oligonucleotides with repeat thrombin binding aptamer sequence by RCA,specific aptamer affinity binding to achieve multiple thrombin labeling,and enzyme activity ofthrombin for signal generation,this assay achieved two signal amplification.PDGF-BB was specifically captured by antibody coated microplate.Then,an oligonucleotide containing an aptamer for protein and a primer sequence was added to form a typical sandwich structure.Following a template encoded with complementary sequence of aptamer for thrombin,RCA reaction extended the primer sequence into a long oligonucleotide containing tandem aptamer sequences for thrombin.Many thrombin molecules bound with the RCA product,achieving multiple thrombin labeling in the sandwich complex.Thrombin catalyzed the conversion of its chromogenic or fluorogenic peptide substrates into detectable products for final quantification of protein targets.Due to double signal amplifications from RCA and thrombin catalysis,this assay enabled the detection of PDGF-BB as low as 3.1 p M when a fluorogenic peptide substrate was used.Compared with single thrombin labeling,RCA caused 62-fold signal amplification by multiple thrombin labeling.Chapter 5 We described a competitive thrombin-linked aptamer assay(competitive TLAA)for protein detection.In this strategy,PDGF-BB in solution competed with the PDGF-BB coated on the microplates to the binding with a DNA probe containing a PDGF-BB aptamer sequence and a thrombin aptamer sequence.The DNA probe attached to the coated PDGF-BB then bound with thrombin through interaction between aptamer and thrombin.Thrombin catalyzed the cleavage of a fluorogenic peptide substrate to a fluorescent product.With increasing of PDGF-BB in solution,the obtained fluorescence signal decreased as the labeled thrombin on PDGF-BB coated on microplate decreased.Under the optimal conditions,0.125 n M of PDGF-BB was detected.This assay was applied to detect PDGF-BB in 1% human serum sample.The assay only required one affinity ligand probe instead of two ligands in sandwich assay,which reduced the incubation time.Furthermore,the assay showed promise for detection of other targets with thrombin as a label by using the corresponding aptamers sequence in the DNA probe used.Chapter 6 We summarized the main work and innovation of this thesis,and gave the plan for further work.