Detection Of Lung Cancer MicroRNA Biomarkers Based On Quantum Dot-magnetic Bead Sensing System

Quantum dots(QDs)are semiconductor nanocrystals which have unique optical properties,such as high quantum yield,high molar extinction coefficients,broad absorption with narrow and symmetric photoluminescence(PL)spectra spanning the UV-visible spectrophotometer(UV)to nearinfrared,large stokes shifts and high photochemical stability.These special optical properties make QDs very useful for biological imaging,sensing and diagnostics.Lung cancer is one of the most malignant tumors in the world.The development of effective lung cancer marker detection technology has important scientific significance and application value.As a new type of lung cancer biomarkers,mi RNA(micro RNA)has attracted the attention of researchers.Mi RNA is a class of non-coding single-stranded RNA(ss RNA)consisting of 18 to 25 nucleotides.Aberrant expression of mi RNA is ubiquitous in tumors and mi RNA has tissue specificity,so that mi RNA can be used as a biomarker for tumor detection.Many studies have shown that mi RNA is involved in almost every stage of lung cancer including tumor development,invasion and metastasis of cancer and so on.Therefore,mi RNA can be used as a biomarker for lung cancer detection.In the early stages of the tumor,the concentration of tumor mi RNA markers in the serum is very low.Hence,it is necessary to design a sensor with high detection sensitivity to detect minimal amount of mi RNA.On the other hand,a particular tumor is not regulated by a single mi RNA,they are regulated simultaneously by multiple highly related mi RNAs,so that the detection of a single mi RNA is still not enough in specificity and sensitivity.Therefore,it is practical to detect multiple mi RNAs simultaneous for the early diagnosit of tumors.In the current work,Quantum dot-Magnetic bead(QD-MB)sensing system was designed to detect multiple lung cancer mi RNA biomarkers with high sensitivity and specificity.1.The construction of QD-MB biosensor and the detection of mi RNA biomarkers of lung cancer.First,using biotinylated single-stranded DNA and streptavidin-functionalized QD and MB to construct mi RNA detection probes: QD-DNA probes and MB-DNA probes.After the target lung cancer biomarker mi RNA was added in the buffer containing the QD-DNA and MB-DNA probes,two probes hybridize with the target mi RNA to form a ‘QD-DNA-mi RNA-DNA-MB' sandwich structure.After magnetic separation,sandwich structures were separated from unpaired single strand QD-DNA probes.With the increase of the target mi RNA,the amount of the sandwich structure increases and the residual QD in the supernatant decreases.By detecting the change of the fluorescence intensity of the residual QD in the supernatant,it shows that fluorescence intensity decreases with the increase of mi RNA concentration.Quantitative detection of mi RNA can be achieved by comparing the fluorescence intensity before the addition of mi RNA target molecules.In this work,we constructed two different QD-DNA probes(QD-DNA-200 b and QD-DNA-21)and two corresponding MB-DNA probes(MB-DNA-200 b and MB-DNA-21).Probes were used to detect two different lung cancer biomarkers: mi RNA-200 b and mi RNA-21.In tris-HCl buffer,the detection limit of mi RNA-200 b is 0.73 fM,and the linear range of detection is from 10 fM to 10 p M.The detection limit of mi RNA-21 is 1.16 fM,and the linear range of detection is between 10 fM to 100 p M.In order to study the anti-interference ability of the sensor,we further studied the detection ability of mi RNA in serum.In 1% serum,the detection limit of mi RNA-200 b reaches 6.09 fM,and the linear range of detection is 10 fM-10 p M.The detection limit of mi RNA-21 reaches 3.90 fM,and the linear range of detection is 10 fM-100 p M.In addition,the detection of single-base mismatched mi RNA and random sequence mi RNA shows that QD-MB sensor has good detection specificity,high detection sensitivity,wide detection range and has good specificity and anti-interference ability.It provides technical support for the early diagnosis of lung cancer.The sensor can achieve simultaneous detection of two lung cancer biomarkers(mi RNA-200 b and mi RNA-21),and provides technical support for the early diagnosis of lung cancer.2.The construction of a QD-MB nucleic acid sensor and its detection application based on the duplex-specific nuclease(DSN).DSN can selectively recognize and cleave DNA from perfectly matched DNA duplexes or RNA/DNA hybrid duplexes.However,DSN can not cleave singlestranded DNA,RNA,or double-stranded RNA.Rely on the specific degradation ability of DSN,we designed a QD-MB fluorescence sensing system based on DSN enhanced signal amplification strategy.First,we constructed a QD-MB nucleic acid sensor using mi RNA-21 as a target molecule based on DSN amplification.In the presence of target mi RNA,mi RNA-21 hybridizes with MB-DNA probe to form DNA-RNA double strands,then DNA in DNA-RNA heteroduplexes is specifically cleaved by DSN,and mi RNA-21 will be released and further hybridize with other MB-DNA probe for next circle.This assay allows for quantitative and sensitive detection of mi RNA-21.Different concentrations of target mi RNA-21 hybridize with MB-DNA probes to form DNA-RNA double strands.DNA in double strands can be specifically cut by DSN and cause the mi RNA-21 to be released.mi RNA continues to hybridize with the DNA in the probe and causes cyclic cleavage by the DSN enzyme,then uncleaved MB-DNA probes hybridize with QD-DNA probes.When we detect the supernatant fluorescence after magnetic separation,we can find that the fluorescence intensity increases gradually with the increase of mi RNA-21 concentration.The detection range of the sensor is 1 p M-10 nM.The detection range is 4 orders of magnitude for mi RNA-21.Detection limit is about 120 fM.