Construction Of Fluorescent Nanomaterial-Based Ratiometric Sensors For The Detection Of Colorectal Cancer-Associated MicroRNA

The early diagnosis of cancer is of great significance to gain valuable treatment time and reduce mortality.As a non-invasive diagnostic technique,liquid biopsy has merits in sensitivity,safety,prospectiveness and subsequent treatment guidance compared with traditional cancer diagnosis methods.Circulating RNAs are a class of targets of liquid biopsy.MicroRNAs(miRNAs)are a class of single-stranded noncoding RNAs of 18-24 nucleotides.The miRNA system is primarily responsible for the regulation of expression of target genes by participating in the posttranscriptional inhibition.The aberrant expression of specific miRNAs is associated to diseases including cancer.Therefore,these miRNAs are identified as biomarkers.Exosomes are a class of extracellular vesicles;they mediate intercellular communication and regulate the function of recipient cells by transferring their internal substances from donor cells to recipient cells.Exosomes are the main carriers of miRNAs,and the change of exosomal miRNA concentration can be triggered by physiological changes during disease progression.In addition,exosomal miRNAs have high stability against RNase degradation.Hence,the sensitive detection of exosomal miRNAs shows enormous potential in cancer liquid biopsy.The poor portability,complicated procedures and expensive instruments associated with traditional RNA detection methods propel the emergence of new detection technologies.Fluorescence method has been widely used due to its advantages of high sensitivity,convenient operation and high reproducibility based on the determination of homogeneous liquid.Due to its self-referencing capability,ratiometric fluorescent strategy is capable of counteracting the influence of environmental factors by calculating the fluorescence intensity ratio of distinct fluorescent substances with different emission wavelengths,thus making the detection results more reliable.Nanomaterials exhibit unique interfacial,mechanical and optical properties,enabling it possible to develop new strategies based on nanomaterials for sensitive detection of exosomal miRNAs.Colorectal cancer(CRC)ranks high among all cancers in terms of morbidity and mortality.The onset of CRC is insidious and often difficult to detect during its early stages.Over 50%of CRC patients are already undergoing metastasized at diagnosis.Therefore,it is urgent to develop new strategies for CRC diagnosis.In this thesis,a series of ratiometric sensors based on fluorescent nanomaterials were constructed to achieve sensitive detection of miRNA.The detection performance of these sensors was evaluated with CRC-associated miRNA as the target.The main research contents are as follows.(1)A ratiometric fluorescent sensor based on fluorescence resonance energy transfer(FRET)between carbon dots and acridine orange was constructed for miRNA detection.Fluorescent carbon dots and acridine orange were used as the donor and receptor of the FRET system.Silica microspheres modified with complementary DNA sequence of target miRNA were used for extracting target miRNA from sample.When the target miRNA is absent,cationic dye acridine orange is adsorbed on the phosphate side of the DNA probe,and carbon dots with negative surface charge are adsorbed on the base side of the DNA probe.The close distance results in the FRET between acridine orange and carbon dots.As a result,the fluorescence intensity of acridine orange decreases,while the fluorescence intensity of carbon dots increases.When the target miRNA is presented,DNA probe and target miRNA form the double strand by complementary base pairing.In the case of double-stranded nucleic acids,the bases are wrapped and the phosphate ions are exposed to exhibit a negative charged surface.Acridine orange is inserted into the base pair gap of the double strand,while the carbon dots are far away from the double strand due to the base being wrapped and charge repulsion.In this case,the decrease or disappearance of FRET leads to the increase of the fluorescence of acridine orange and the decrease of the fluorescence of carbon dots.The acridine orange/carbon dots fluorescence intensity ratio is proportional to the concentration of the target miRNA.The occurrence of FRET was proved by the fluorescence spectra and fluorescence lifetime analysis,and the feasibility of detection was proved by the fluorescence test.This sensor showed a linear detection range of 19 nM and a limit of detection of 0.14 nM.The high selectivity for the target miRNA was exhibited.The detection results for target miRNA with the same concentration(1,5 and 9 nM)in serum and water were close.In addition,the detection results of this sensor could reflect exosomal miRNA concentration difference between healthy individuals and CRC patients.(2)A ratiometric fluorescent sensor based on multi-walled carbon nanotubes@gold nanoclusters(MWCNTs@Au NCs)and duplex-specific nuclease(DSN)-assisted signal amplification was constructed for miRNA detection.The water solubility of the MWCNTs was improved by carboxyl modification.Thiol groups were further modified onto MWCNTs to achieve the connection between MWCNTs and fluorescent Au NCs through Au-S bonds.MWCNTs@Au NCs showed the dual functions of fluorescence emission and fluorescence quenching.In the absence of target miRNA,Atto-425-modified single-stranded DNA probes are adsorbed on the surface of MWCNTs@Au NCs,resulting in the quenching of Atto-425 via energy transfer and charge transfer.In the presence of target miRNA,the double strand is formed by the hybridization of probe and target miRNA and leaves MWCNTs@Au NCs,resulting in the fluorescence recovery of Atto-425.DSN can specifically cleave DNA probe in double strand and result in the release of target miRNA.The released target miRNA can hybridize with other DNA probes,resulting in more Atto-425 returning fluorescence emission.The stable fluorescence of MWCNTs@Au NCs and the changing fluorescence of Atto-425 constitute a ratiometric fluorescence system.The concentration of target miRNA is positively correlated with the fluorescence intensity ratio of ΔAtto-425 to MWCNTs@Au NCs.The detection feasibility was demonstrated by the fluorescence test.Experimental conditions such as the amount of MWCNTs@Au NCs,the mixing time of MWCNTs@Au NCs and DNA probe,and incubation temperature were optimized.A linear detection range of 0.1-10 pM and a limit of detection of 0.031 pM were obtained under optimized experimental conditions.The specific cleavage characteristic of DSN endows this sensor with high selectivity.The detection results of this sensor were close to those of RT-qPCR,demonstrating that this sensor possesses high accuracy.(3)A ratiometric fluorescent sensor based on fluorescent metal-organic framework(MOF-525)and target-triggered rolling circle amplification(RCA)was constructed for miRNA detection.The fluorescence property of MOF-525 was used to construct a sensor for the first time.MOF-525 served as both a fluorescent reference and a quencher of the fluorescence of single-stranded reporter.When the target miRNA is absent,MOF-525 can adsorb and quench the fluorescence of reporter(a single-stranded DNA modified with fluorescent dye FAM)through photo-induced electron transfer.In the presence of target miRNA,the RCA reaction can be triggered and the periodic long strand is formed.The sequence of reporter is complementary to the partial sequence of periodic long strand,resulting in the formation of double strand.The double strand cannot be adsorbed by MOF-525,which prevents the fluorescence of FAM from being quenched.This effect is significantly enhanced by RCA.The concentration of target miRNA is positively correlated with the fluorescence intensity ratio of Δreporter to MOF-525.The feasibility of the detection was verified by non-denaturing polyacrylamide gel electrophoresis(PAGE)and fluorescence test.A linear relationship was obtained in the target miRNA concentration range of 0.1-10 pM,and the limit of detection was 0.03 pM.This sensor could differentiate target miRNA from mismatched RNA sequences,showing good selectivity.The detection results of this sensor were close to that of RT-qPCR,showing its potential for miRNA detection.(4)A ratiometric fluorescent sensor based on a nanoplatform composed of DNA probe(probe P1)-templated Ag-Au nanoparticles(Ag-Au NPs/P1)and Au nanoparticles/g-C3N4(Au NPs/g-C3N4),catalytic hairpin assembly(CHA)and sequence-induced fluorescence enhancement was constructed for miRNA detection.Au NPs were hybridized with g-C3N4 to form Au NPs/g-C3N4 with enhanced fluorescence quenching ability.Meanwhile,the fluorescence of g-C3N4 acted as fluorescence reference.Ag-Au NPs/P1 with excellent fluorescence stability was used as the fluorescent probe.In addition,probes P2 and P3 were designed for CHA reaction.In the absence of target miRNA,Ag-Au NPs/P1 can be adsorbed on the surface of Au NPs/g-C3N4 and the fluorescence of the former is quenched via photo-induced electron transfer.In the presence of target miRNA,the CHA reaction can be triggered and the Ag-Au NPs/Pl-P2-P3 double strand that cannot be adsorbed by Au NPs/g-C3N4 is formed.The released miRNA is next able to hybridize with other Ag-Au NPs/P1,forming a CHA-based signal amplification cycle and causing more Ag-Au NPs/P1 to restore fluorescence emission.Furthermore,the bespoke nucleic acid sequence design makes the guanines of P2 adjacent to the consecutive cytosines of P1 in the double strand,resulting in the fluorescence enhancement of Ag-Au NPs/P1.The detection signal is thus further enhanced.The ratiometric fluorescence system composed of the stable fluorescence of Au NPs/g-C3N4 and the varying fluorescence of Ag-Au NPs/P1 indicates the concentration of target miRNA.The feasibility of the detection was verified by non-denaturing PAGE and fluorescence test.Experimental conditions including the pH of solution,the amount of Au NPs/g-C3N4,and incubation time were optimized.This sensor showed a linear detection range of 0.1-50 pM and a limit of detection of 0.047 pM.In addition,this sensor could differentiate target miRNA from mismatched sequences.The detection of exosomal miRNA extracted from clinical samples showed that the detection results of this sensor were close to those of RT-qPCR,demonstrating this sensor can be used for miRNA detection.