Construction Of Biodetection Building Blocks: Ultrabright Nanospherical Polyelectrolyte Brushes For Signal Amplification And Dual Dye-doped Barcodes For Multiplex Biodetection

In life science and clinical medicine,with the rapid development of genomics,proteomics and metabonomics etc.,increasing demand for biodetection in field of disease diagnostics and therapeutic treatment has led to the development of a fast,ultrasensitive and multiplexed analysis in one assay.Therefore,higher requirements are put forward for the performance of signal labels and biocarriers which are two essential building blocks of existing detection systems.Fluorescent signal-labeled materials are the most commonly used kind of labeling materials due to its strong and stable fluorescent signal that is essential for an ultrasensitive biodetection.Likewise,bead-based biocarriers play a crucial role for target recognizing,capturing and identifying because of rapid reaction kinetics and large specific surface area.Among them,suspension array based on encoding microspheres has been established as a good candidate for multiplexed assays,which display several significant advantages including high flexibility,superior detection sensitivity and fast binding kinetics.Recently,ultrabright fluorescent particles(UFPs)have been required enormously owing to their excellent signal amplification functions.Nevertheless,there is still an importunate demand for designing novel UFPs with new structures or components as the existing ones cannot satisfy the practical requirements because of their inherent disadvantages including:risks of quenching,low preparation scale and wide size distribution.An ultrabright fluorescent particle platform was developed by doping dyes of 5-aminofluorescein(5-AF)into silica core-based spherical poly(acrylic acid)brushes(Si O₂@PAA@5-AF)and their fundamental structure-fluorescence tuning principles were discussed.A series of spherical brushes with different polymer chain lengths were successfully synthesized and then filled with 5-AF by chemical binding.The main reasons to imply the ultrabrightness of Si O₂@PAA@5-AF are high loading amount,proper density,and enhanced quantum yield(QY)of 5-AF.Altogether,in this study,1)fluorescent signal was enhanced through replacing conventional scaffold with spherical PAA brush to immobilize 5-AF as a label and further obtained 5-AF doped brushes(Si O₂@PAA@5-AF)to achieve term of ultrabrightness.2)Fundamental principles of fluorescent properties were investigated by tuning particle size and controlling dye distribution on PAA chains systematically and further optimized size and dye density to avoid fluorescence quenching.The results of this study demonstrate a quite encouraging potential of novel Si O₂@PAA@5-AF as an ultrabright tool for amplifying signal in biodetection,labeling,and sensing applications.Further,a sensitive immunoassay system by utilizing Si O₂@PAA@5-AF as signal amplification label was developed,demonstrating stronger detection signal than traditional fluorescent labeling.Moreover,the multiplexed assays have been required enormously,owing to the increasing demand for high-throughput multiplexed assays.This demand can be realized by employing encoded bead-based suspension arrays,and as the core biocarrier of suspension array,the preparation technology of optically encoded beads has attracted much attention.Among many fluorescent candidate elements,organic dyes and quantum dots(QDs)as two main groups of commonly used encoding agents have been widely used.Although QDs are utilizable for barcoding but,several issues hinder their broad utilization such as poor solvent tolerance,heavy metal pollution and,so on.Up to now frequently reported bead-based optical suspension arrays are composed of beads with doped fluorescent dyes since they are relatively ideal fluorophore benefit from high fluorescence intensity and biocompatibility.Despite plenty of success in fabrication of dye-doped barcodes,several issues such as the occurrence of self-quenching effect which is happened by enclose packed of dye molecules and complex encoding process still need to be addressed.However,most of the previously reported methods are focused on enhancing encoding capacities,more flexible and controllable encoding system in a simple and reproducible way remains a challenge for dye-doped encoding platforms.In this study,a new polymer mediate method was proposed to improve encoding controllability by generating dual-fluorescent-doped beads with unique spectral codes through flexible and reproducible process steps.By this strategy,the doping of dyes and functionalization of the bead surface are integrated,resulted in improving the encoding efficiency.Herein,fluorescein isothiocyanate(FITC)and Rhodamine B isothiocyanate(RITC)as two kinds of intense green and red fluorescent molecules,respectively,were chosen as model organic dyes.Both types of dyes were covalently linked with Polyethyleneimine(PEI)initially.Then FITC-PEI,RITC-PEI,and blank PEI solutions were combined at a certain stoichiometric ratio,resulting in spectrally tuned fluorescence intensity levels.Then PEI mixtures adsorbed onto the internal and external interfaces of the mesoporous silica microspheres(MSs).Therefore,the proposed polymer mediate method,took advantage of the simplified one-step fluorescent tuning by pre-modification of dye molecules onto the chain segments of PE.Ultimately a 101 encoded beads library by a superior encoding capacity was developed and successfully decoded by flow cytometry.Flow cytometry analysis showed high and tuned fluorescence.High encoding resolution and capacity is due to the broad adjustable linear range of fluorescence intensities.Encoding capacity was extended to achieve term of ultrahigh by controlling three practical functions:Size of beads,the emission wavelength of two different dyes,and the concentration of doped dyes(three different size levels of beads doped with FITC or RITC by a systematic tuned concentration range).Based on the successful preparation of barcode library,the primary purpose of last part of this research was trying to establish a model pre-study of the multiplex digital detection via dual fluorescent encoded beads I prepared.However,research in this part still needs to continue and reach the aim of single molecule sensitivity in multiple targets simultaneously which is promising to detect and quantify low concentrations of multiplexed samples in clinical applications.