| Aptamers are single-stranded DNA (ssDNA) or RNA oligonucleotide fragments screened by Systematic Evolution of Ligands by Exponential enrichment (SELEX).Aptamers can bind to proteins or other target substance specifically, and have the characteristics of high selectivity and high affinity. Compared to antibodies, aptamers have a more stable chemical property and have the advantages of low-toxicity and no-immunogenicity, which make them have great promising applications in protein detection. Aptamers can be obtained easily and rapidly by SELEX technology, which show great prospect in diagnostics and therapeutics of clinical diseases.Progastrin-releasing peptide 31-98 fragments (ProGRP31-98),is a highly reliable,specific and sensitive tumor marker of small cell lung cancer (SCLC). Measurement of ProGRP31-98 levels in serum enables diagnosis, prediction of prognosis, and treatment monitoring in SCLC. The main detection methods of serum ProGRP31-98 levels are based on immune principle, including enzyme-linked immunosorbent assay(ELISA), chemiluminescence immunoassay (CLIA), etc. To our knowledge, there are no aptamers against ProGRP31-98 have been selected currently. Our lab obtained eight sequences bound with ProGRP31-98 by SELEX.Among these eight sequences, we selected highly specific aptamers. We established a lable-free electrochemiluminescence (ECL) measurement method to investigate the binding specificity of aptamers to ProGRP31-98,with a DNA light-switch molecular, [Ru (bpy)2dppz] 2+,as the ECL probe. One DNA sequence (89 nt) and its random region (48 nt) were identified as specific aptamers for ProGRP31-98.Based on the secondary structures of the two obtained DNA aptamers, two other truncated DNA aptamers, 40 nt and 15 nt long, respectively, were also identified as specific aptamers for ProGRP31-98.The four aptamers have strong affinities to ProGRP31-98, with a dissociation constant (Kd) low to 16 nM, and can detect ProGRP31-98 with a detection limit of 17 nM using the label-free ECL measurement.Because the levels of serum ProGRP31-98 are very low,the sensitivity of the label-free ECL measurement is not enough to detect the content of target. To get a more sensitive detection method to ProGRP31-98, we designed an electrochemical aptasoensor.Firstly, 3-Mercaptopropionic acid (3-MPA) were self-assembled to the gold electrode surface by Au-S bond and streptavidin was immobilized after the N-(3-dimethylaminopropyl)-N'-ethylcar-bodiimide hydrochloride (EDC) and N-hydroxysuccinimide (sulfo-NHS) activated surface carboxyl group. The aptamer,forming hairpin structure with both ends modified with biotin and sulfydryl respectively, and another hairpin DNA modified with sulfydryl were together connected to Au nanoparticals (AuNPs) to form double hairpin co-modification AuNPs composite (hpDNA-AuNPs-Aptamer). The composite, as a signal amplification element, after incubated with ProGRP3i-98 to lead to the structure of aptamer changing to expose biotin, was fixed to the electrode surface. A bifunctional electrochemical indicator, [Ru (NH3) 5L]2+, was embedded into the hairpin DNA of hpDNA-AuNPs-Aptamer bylinear sweep voltammetry (LSV) and then differential pulse voltammetry (DPV) was used to detect the oxidation current signal of Ru (?). L represents 3-(2-phenanthrene-9-Methyl-vinyl) pyridine, which can embed into double-stranded DNA, while Ru (?) is the redox center. We characterised the assembly process of the electrode and studied the primary detective performance, and optimized part of conditions. |
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