Construction Of Novel Ultra-stable,highly Fluorescent Water-soluble Quantum Dot Fluorescent Nanobeads And Its Application In In Vitro Diagnostics

Semiconductor quantum dots(QDs)have become important fluorescent probes in biological fields during the past few decades due to their tunable fluorescence wavelength,broad excitation spectra,sharp and symmetrical fluorescent peak,strong and stable emission,and high quantum yield.In particular,high level of brightness and extraordinary photostability of QDs enable high sensitivity detection in the field of in vitro diagnostics.Increasing demand for an efficient,precise and sensitivity detection has promoted the fast development of QDs with high fluorescence and good biocompatibility.Among various methods,QDs-microspheres have received increasing attention,which display the advantages of signal amplification,good biocompatibility,and convenient manipulation,may have great potential to be used as fluorescent probes for improving sensitivity of the assay.Unfortunately,the primary challenge in fabricating of QDs-microspheres is the fluorescence instability of QDs,such as quantum yield loss,fluorescence attenuation and longstanding instability in biological environments,which seriously affects their application for accurate quantitative analysis in in vitro diagnosis.Therefore,this article focuses on the development of novel ultra-stable,high-fluorescence water-soluble quantum dot fluorescent nanobeads,and builds new platforms of in vitro diagnostic technology for high sensitivity detection of relevant disease markers.The main contents are as follows:1.Quantum dot fluorescent nanobeads were synthesized by chemical bond embedding method and used for the quantitative detection of C-reactive protein(CRP).Si O₂ spheres were synthesized with good monodispersity and uniform structures,which as a carrier template so that multiple hydrophilic QDs can be embedded onto the surface of silica sphere by forming amide bonds after silica spheres were amino-functionalized.The Si O₂@QDs nanobeads were further coated by silica shell and functionalization with hydrophilic layers to form Si O₂@QDs@Si O₂-COOH structure for enhancing the stability and biocompatibility.This new convenient method of preparing nanoscale beads had high repeatability,which can control precisely the loading density of QDs in a single Si O₂ sphere and minimize the loss of QDs.The synthesized Si O₂@QDs@Si O₂-COOH nanobeads exhibited strong luminescence(over 50 times higher than single QDs)and excellent stability in complex matrix(p H,temperature).Si O₂@QDs@Si O₂-COOH nanobeads were proposed as signal amplification probe for the detection of CRP.This new developed Si O₂@QDs@Si O₂-COOH nanobead-based quantum dot-based fluorescent linked immunosorbent assay(QLISA)technology can be used to quantitatively detect CRP with a broad detection range between 0.5 ng/m L and 1000ng/m L successfully.The more conjugate antibody proportion resulted in the stronger intensity of the fluorescence signal,the smaller the LOD,and the higher the sensitivity of assay.The minimum sensitivity of this detection method was 0.32 ng/m L.The result of real clinical sample test indicated that the QLISA method had excellent concordance with the Roche immunoturbidimetry.2.Combining the microemulsion technique and dually-protected strategy,quantum dot fluorescent nanobeads were synthesized and used for the quantitative detection of cardiac troponin I(c Tn I).In microemulsion system,hydrophobic QDs were confined to micelles formed by cetyltrimethylammonium bromide(CTAB)under the action of emulsifier.Then,polyvinylpyrrolidone(PVP),which was a non-toxic and biocompatible amphiphilic polymer,was wrapped around the outer layer of QDs to form quantum dot beads(QBs).The QBs were further coated by silica shell and hydrophilic layers to enhance their stability and biocompatibility.In this method,Cd Se/Zn S QDs with suitable size(14 nm)were used to construct nanobeads to eliminate energy transfer among QDs,and the original fluorescence properties of QDs were retained because the organic ligands on the surface of QDs had not been destroyed.Compared with the corresponding QDs,the fluorescence intensity of resultant QBs@Si O₂-COOH(235±15 nm)increased by approximately 1,967 times.And the dual protection strategy with both PVP and Si O₂ could render QBs@Si O₂-COOH less sensitive to surface damage,which showed strong photostability under various complex conditions(p H,salt solution,and thermal treatment).The constructed QBs@Si O₂-COOH-based lateral-flow immunoassay(LFIA)technology had a sensitive response to c Tn I in a wide linear range of 0.12 ng/m L to 125 ng/m L with a low detection limit of36 pg/m L.Moreover,this QBs@Si O₂-COOH-based LFIA showed accurate concordance with a high correlation coefficient value(R²=0.983)compared with the results from direct chemiluminescent immunoassay(ADVIA Centaur Tn I-Ultra assay)for a total of 103 samples.Importantly,the detection method owned good reproducibility(CV<8%)and predominant stability during different temperature and storage times for clinical samples.3.Quantum dot fluorescent nanobeads were synthesized by biomolecular modification and used for quantitative detection of glycosylated hemoglobin(Hb A1c).Under the ultrasonic condition,chloroform solution containing QD was dispersed in aqueous solution of bovine albumin(BSA)to form microemulsion droplets and achieve dynamically stable state.BSA is an important blood protein containing one single cysteine and eight pairs of disulfide bonds,which can preferentially replace the original hydrophobic ligands on the surface of QD outside the droplet.The chloroform of droplet was rapidly volatilized under reduced pressure,so that multiple quantum dots were wrapped in BSA to form nanobeads.The nanobeads were dissolved in water by the hydrophilic group of BSA.In order to further improve the stability and biocompatibility,the QDs@BSA nanobeads were wrapped with silica shell and carboxyl groups by silylation hydrolysis.The QDs@BSA@Si O₂-COOH nanobeads showed admirable stability due to the dual protection strategy with both BSA and Si O₂.The new developed QDs@BSA@Si O₂-COOH nanobead-based LFIA technology can be used to quantitatively detect Hb A1c(4.2%-13.6%).The clinical results showed that detection platforms based on QDs@BSA@Si O₂-COOH nanobeads had good concordance with the corresponding standard method.In summary,in order to improve the quantitative analysis ability and sensitivity of in vitro diagnostic system,three new methods have been successfully developed to synthesize novel ultra-stable,high-fluorescence water-soluble quantum dot fluorescent nanobeads.The nanobeads were used as fluorescent probes to achieve highly sensitive,accuracy and stability detection of related biomarkers by QLISA and LFIA technologies,which not only provides support for the detection of clinical related diseases,but also provides a new platform for the detection of other markers in vitro diagnosis.