Construction Of Fluorescent Probes Based On Multicolor Emitting Carbon Dots And Their Application In The Detection Of Bioactive Molecules

As a new luminescent nanomaterial,carbon dots（CDs）are favored by researchers because of their simple preparation method,adjustable spectrum,good optical stability and good biocompatibility.They are widely used in the field of analytical detection,bioimaging,drug delivery and so on.Bioactive molecules such as polypeptides and microRNAs play important roles in maintaining the normal physiological function of the human body.Their levels are closely related to the occurrence and development of diseases.They can be used as disease markers to provide reference for disease screening,diagnosis and treatment.In consideration of the characteristics of low concentration of bioactive molecules and complex detection environment,it is still necessary to develop detection methods with high sensitivity,good selectivity and simple operation.In this paper,fluorescent CDs with different colours were prepared and used as signal molecules,and MnO₂nanosheets,organic dyes/graphene oxide（GO）and gold nanoparticles（AuNPs）were used as quenchers to construct fluorescent biosensors,which were applied to the detection of glutathione and microRNA in complex matrix samples.The main research contents are as follows:1.A fluorescence and colorimetric dual-mode sensing platform was constructed with blue light emitting CDs as fluorophore and MnO₂ nanosheets as quencher.MnO₂nanosheets were directly synthesized in the solution of CDs to form CDs-MnO₂ mixture.The fluorescence of carbon dots was quenched by MnO₂ nanosheets through internal filtration effect（IFE）.When GSH existed in the system,MnO₂ nanosheets were reduced to Mn²⁺,resulting in a lighter color of the solution,which could be used for the naked eye qualitative detection of GSH.At the same time,the fluorescence of CDs recovered gradually,which could be used for the quantitative detection of GSH.The naked eye distinguishable concentration of GSH was 1.0×10^-5mol/L.The linear range of quantitative assays ranged from 1.0×10^-7 mol/L to 4.0×10^-4 mol/L with a detection limit of 6.6×10^-9mol/L.This strategy shows good selectivity for GSH and can be used for the detection of GSH in human serum and urine samples with a linear range of 5.0×10^-7～3.0×10^-4 mol/L.The detection limits were 1.5×10^-8mol/L and 6.9×10^-9mol/L,respectively.2.MicroRNAs（miRNAs）,a new type of tumor markers,are closely related to the occurrence,development and metastasis of tumors.However,the expression of miRNAs is low and the sequence similarity is high.It is necessary to develop highly sensitive and selective miRNAs detection methods.A molecular beacon sensor based on CDs and organic dye/GO double quencher was constructed for the detection of miRNA-21.Green light emitting CDs were used as fluorophores and organic dye modified molecular beacon sequence（MB-BHQ1）was attached on the surface of CDs.The distance between CDs and BHQ1 is shortened,and the fluorescence of CDs is quenched.When GO was added as a co-quenching agent,and the ring portion of MB was assembled with GO byπ-πstacking to further quench the fluorescence of CDs.When miRNA-21 exists,miRNA-21 hybridizes with the MB sequence to open the hairpin structure and increase the distance between CDs and BHQ1.At the same time,the formation of double stranded nucleic acid separates CDs-MB-BHQ1 from the surface of GO and the fluorescence of CDs is restored.The introduction of double quencher can increase the fluorescence quenching efficiency of CDs,improve the signal-to-noise ratio,and then effectively improve the detection sensitivity.The sensor was used for the detection of miRNA-21 with a linear range of 5.0×10^-13～4.0×10^-10 mol/L.Moreover,the sensor is highly selective to miRNA-21 and can be used for the detection of miRNA-21 in human serum.The recovery was ranged from 88.0%to105%with the RSDs no more than 4.4%.3.Due to the small size of CDs and many kinds of surface functional groups,the surface modification and purification steps of CDs are cumbersome and time-consuming,which limits the development and application of fluorescent sensors based on CDs.The sensing strategy of DNA induced CDs assembled on gold nanoparticles was constructed for the ultrasensitive detection of miRNA-21.Polyethyleneimine（PEI）modified CDs（PEI-CDs）were used as fluorophore,gold nanoparticles（AuNPs）were used as quencher.Positively charged PEI-CDs and negatively charged cDNA modified AuNPs（AuNPs-cDNA）were assembled together through electrostatic interaction,and the fluorescence of CDs was quenched.When miRNA-21 was present,the hybridization between miRNA-21 and cDNA released PEI-CDs from the surface of AuNPs,and the fluorescence of CDs was restored.The assembly of CDs and AuNPs is realized by electrostatic adsorption without surface modification of CDs,which greatly simplifies the operation steps.The sensor can realize the sensitive detection of miRNA-21 with a linear range of 1.0×10^-15～1.0×10^-12mol/L.In addition,the sensor can be used to detect miRNA-21 in human serum.The detection results are in good agreement with the detection results of q RT-PCR.The recoveries range from 88.4%to113%and RSDs were lower than 18.3%.