Biosensing Methods Based On Fluorescent Microspheres For Biomarkers Detection

The detection of biomarkers is of great significance for the assessment of human health.In recent years,many diseases tend to occur at younger ages,the health problem has gradually attracted the attention of the society.The emergence of diseases is extremely harmful to the development of individuals,families and even society.Therefore,it is particularly important to detect the relevant biomarkers to realize the early prediction/diagnosis of the disease,the stage of the disease and the monitoring of the disease prognosis.However,the detection processes of most biomarkers are cumbersome,time-consuming,and the detection equipments are usually expensive,which limits real-time detection and real-time monitoring of biomarkers in the human body.Therefore,the development of more convenient,sensitive,and low-cost detection methods has very important practical significance and social value.Fluorescent microsphere(FM)is a kind of widely used fluorescent nanomaterial with good optical properties,such as high fluorescence quantum yield,absorption spectrum bandwidth,narrow emission band,and good photobleaching resistance.In addition,some FMs with active groups on the surface can also be coupled with a variety of biomolecules.Therefore,FMs are often used to construct specific biosensors.FMsbased biosensors have the advantages of high sensitivity,high selectivity,and low cost.It is significant for the development of efficient biomarker analysis and disease diagnosis methods,and is a research hotspot in the field of biomedicine in recent years.This paper combines FMs with a variety of materials to construct a variety of new biosensors based on FMs,which have been successfully applied to the sensitive detection and quantitative analysis of concanavalin A(Con A),alkaline phosphatase(ALP)and breast cancer cells(MCF-7)exosomes.The main contents are as follows:(1)Based on functionalized FMs and boric acid-modified fluorescent carbon dots(BCDs),we constructed a low-cost,meristic fluorescent biosensor,achieving highly sensitive detection of Con A.Firstly,dextran was used to modify the surface of NH2FMs(FMs@PEI@Dextran),and then functionalized BCDs were prepared by a onestep hydrothermal method.FMs and BCDs have different fluorescence emission peaks under the same wavelength of excitation light.Both BCDs and Con A can specifically bind to dextran on FMs@PEI@Dextran,but the state of the binding products is different.the binding product of BCDs and FMs@PEI@Dextran aggregated,while the binding product of Con A and FMs@PEI@Dextran did not aggregate.Combined with the above characteristics and the changes of the two fluorescence signals in the supernatant after centrifugation,we realized the highly sensitive ratio fluorescence detection of Con A with the detection limit of 0.089 μg/mL.In addition,it also provides a new strategy for monitoring the dynamic changes of Con A in serum samples.(2)In order to further develop a convenient biosensor,this work introduced chromogenic reaction that can be observed with naked eyes,and constructed a colorimetric-fluorescent dual-mode biosensor that combines FMs(without surface modification)and phosphomolybdic heteropolyacid(PMA).It provided a strategy to realize low-cost,high-sensitivity detection of ALP.Based on the mechanism of ALP could catalyze L-ascorbic acid-2-trisodium phosphate(AAP)to produce ascorbic acid(AA),AA could reduce PMA to produce phosphomolybdenum blue,which quenched the fluorescence of FMs through the fluorescence internal filtration effect,and output the colorimetric-fluorescence dual-mode signal realized highly sensitive detection of ALP in the buffer solution system.The limit of detection is 0.068 U/L.The colorimetric-fluorescence dual-mode signal of the sensor reduces the interference of environmental factors and improves the accuracy of detection.The raw materials are easy to obtain and the procedure is simple.In addition,a paper-based mobile phone ultra-fast biosensing system based on color reaction was constructed.The system has the advantages of biodegradability and low cost,which can realize the real-time detection of ALP in poor areas where materials are scarce.At the same time,this system also realized the sensitive detection of ALP in serum samples.(3)In order to further develop practical and more convenient fluorescent biosensors,we introduced immunochromatography combined with test strips and established nucleic acid aptamer(MUC1 aptamer)functionalized FMs and MnO2 sheets-based fluorescence quenching immunochromatographic biosensor,providing a strategy for the low-cost,ultra-sensitive and convenient detection of MCF-7 exosomes.MUC1 aptamer functionalized FMs realized the recognition and capture of MCF-7 exosomes.Exosomes(volume 30-150 nm)increased the distance between FMs and MnO2 sheets,limited the fluorescence resonance energy transfer(FRET)between them and reduced the degree of fluorescence quenching,realizing the highly sensitive detection of MCF-7 exosomes.The limit of detection is 2.5×103 particles/mL.In addition,regardless of the presence or absence of MCF-7 exosomes,the MnO2 sheets could be assembled on the Test line by electrostatic action,and the color could be observed with naked eyes,without the introduction of control lines,which simplifies the preparation processes of test strips.This method also realized the detection of MCF7 exosomes in serum,confirming the application value of this immunochromatographic sensor in clinical sample analysis.