Study On The Novel Methods For Aptamers In The Detection Of Proteins And Small Moleculers

Protein moleculars such as IgE and small moleculars such as adenosine and other biological species play vital role in the maintenance of biological activity of individual life.Quickly and effectively monitoring these life substances has a direct and practical significance in maintaining health life.Biosensors hold unique advantages comparing with many other analytical methods. They not only have the advantages of conventional analysis methods, but also their analysis processes associates with life activities and can provide in situ,real-time analysis.The aptamers are chemically stable, small in size, inexpensive and can bind almost any target existing in nature with desirable affinity and specificity, making them promising candidates for the detection of a broad range of target molecules.In this thesis,our work is focused on the following areas:1. Fluorescent oligonucleotide probe based on G-quadruplex scaffold for signal-on ultrasensitive protein assay(chapater 2). In This presentation,we reported the G-quadruplex structure of pyrene-labeled G-rich DNA probe and its application in the immunoglobulin E (IgE) detection, providing plausibly an insight into the biological function of human telomere. Based on the intermolecular G-quadruplex, a terminal single pyrene-labeled oligonucleotide signaling probe was developed and a novel protein assay strategy was proposed via combining specific DNA cleavage by S1 nuclease with target-recognizing aptamer. This assay platform not only circumvented the optimization of specific sites for reporter attachment and the pyrene monomer fluorescence quenching by flanking nucleotide bases but also presented a signal-on mechanism. Thus, ultrasensitive homogeneous detection of IgE was successfully conducted. A linear dynamic range of 4.72×10-12 to 7.56×10-9 M, a regression coefficient of 0.9941 and a detection limit of 9.45×10-14 M were given.2. Background current-free electrochemical aptameric sensing platform for ultrasensitive signal-on detection of small molecule(chapater 3).In this contribution, a versatile electrochemical aptameric sensing scheme for the ultrasensitive detection of small molecular species is proposed using adenosine as target model. A ferrocene (Fc)-functioned thiolated aptamer probe is delicately adapted and immobilized onto an electrode surface. Introducing a recognition site for EcoRI into the aptamer sequence not only eliminates the background current but also provides a signal-on response mechanism. In the absence of target adenosine, the aptamer can fold into a hairpin structure and form a cleavable double-stranded region. Fc species are capable of being removed from the electrode surface by treatment with endonuclease, and no peak current is observed. The adenosine/aptamer binding induces the conformational transition of designed aptamer, dissociating the cleavable double-stranded segment. Therefore, the integrated aptamer sequence is maintained when exposing to endonuclease, generating a peak current of Fc. Utilizing the present sensing scheme, target species even at substantially low concentration can give a detectable current signal. Thus, a detection limit of 10-13 M and a linear response range from 3.74×10-12 to 3.74×10-8 M is achieved. The proposed proof-of-principle of a novel electrochemical sensing is expected to extend to establish various aptameric platforms for the analysis of a broad range of target species of interest.3. Fluorescent probe for highly selective detection of adenosine based on molecular beacon aptamer(chapater 4).In this work,based on aptamer binding target configuration change, DNA hybridization competition,a new signal-off detection method of adenosine was proposed.We design the molecular beacon based on adenosine aptamer sequences.In the absence of adenosine, the complementary DNA sequence and molecular beacon hybridized to form double-helix structure and the molecular beacon was opened,resulting in strong fluorescence.In the presence of adenosine,the target molecular of adenosine and molecular beacon binded together,then configuration change occured.when added the cDNA sequence,fluorescence intensity was little changed.With the lowering of adenosine concentration, the corresponding fluorescence response of the increases and provides specificity detection and precise quantification of adenosine.