A DNA Nanomachine On Quantum Dots-Encoded Microbeads For The Detection Of Circulating MicroRNAs

MicroRNAs(miRNAs)are a class of tumor markers that can be used for early and prognostic diagnosis.Circulating micro RNAs in body fluids provide ideal tumor markers for non-invasive detection.Therefore,detection of circulating micro RNAs is of great significance for early diagnosis of tumors.DNA nanomachines are selfassembled superstructures that convert chemical energy into mechanical energy.Among them,DNA walker,which can realize DNA walking in one-dimensional,twodimensional or three-dimensional tracks,has drawn intense attention in recent years.The microsphere suspension array provides an advanced platform for multiplexed nucleic acid analysis which is superior to the typical planar array.In this thesis,we constructs a quantum dots(QDs)encoded microsphere(Qbead)platform,and further implements target-triggered and DNA breathing-mediated DNA walking on Qbead surface.Further,an integrated analytical method based on Qbead-DNA walking has been developed,which has been successfully applied for the detection of multiplex circulating micro RNAs in serum samples of normal people and non-small cell lung cancer patients.The main contents of this paper are as follows:1.Controlled construction of Qbead with "core-shell-shell" structure.The construction is accomplished by five steps: the preparation of silica core by the St?ber method and seed growth method;adsorption of green oil-soluble QDs;the coating of the first layer silica shell by the St?ber method;adsorption of red oil-soluble QDs;the encapsulation of the second layer silica shell.Five fluorescent barcodes of Qbead are prepared by adjusting the ratio of green to red QDs.The core-shell-shell Qbead is established to have high fluorescence stability.2.Cascaded DNA circuit triggered by target miRNA under homogeneous conditions.Circuit I: target micro RNA opens the stem-loop structure of DNA walker,resulting in hybridization of the latter with track,and triggering strand displacement amplification(SDA)catalyzed by Bst polymerase.The circuit I can produce large quantities of double-stranded DNA(ds DNA),and realizes the recycling of target micro RNA.Circuit II: the ds DNA product can breathe at 60 C,producing unstable DNA hybrid,which lead to hybridization with primer and subsequent SDA reaction.Circuit III: when primer Pe is added,the unstable DNA hybrid can also hybridize with it and initiate another SDA reaction.3.DNA walking on Qbead and its use for micro RNA detection.DNA tracks are fixed on the surface of Qbead as walking tracks.DNA walker and primer Pe are labeled with biotin.In the presence of target micro RNA and Bst polymerases,the labeled DNA including DNA walker and primer Pe can walk along the three-dimensional tracks.As a result,a large quantity of biotin-labeled ds DNA is produced on Qbead,which can be fluorescently labeled for quantification.Fluorescence microscopy results showed that DNA walking on Qbead was fast and efficient,and the optimal walking time was defined to be 30 minutes.DNA walking on Qbead was found to be closely related to target concentration.Flow cytometry results showed that the detection limit of micro RNA by Qbead-DNA walking method was 5 copies/?L,and the linear dynamic range was 4 orders of magnitude.4.Rapid,sensitive and multiplexed detection of circulating micro RNAs in serum samples.Based on the five barcodes,the Qbead-DNA walking method was applied to flow cytometric analysis of five micro RNAs in serum samples of normal persons and patients with non-small cell lung cancer(NSCLC).The results showed that the expression levels of micro RNA29 a and micro RNA151-5p were up-regulated and that of micro RNA26 b and micro RNA15 a were down-regulated in NSCLC patient,compared with normal human serum.The expression of micro RNA15 b remained nearly unchanged for serum samples of normal person and NSCLC patient.