Construction Of Gold Nanoprobes And Development Of Novel Optical Methods For Protein Detection

The development of highly sensitive and specific detection methods for protein biomarker is crucial for the early diagnosis of diseases and clinical treatment.Nucleic acid aptamers have emeraged as promising alternatives to antibodies for achieving the protein detection.Particularly,the combination of aptamer and nanomaterials permits the design of versatile hybrid sensing materials with great target specificity and selectivity and tunable signal outputs.By coupling the molecular recognition properties of aptamers with the physicochemical properties of AuNPs,aptamer-gold nanoparticle(Apt-AuNPs)conjugates provide a powerful biosensing platform for various protein bioassays.Our research work mainly focuses on the design and construction of gold nanoprobes with different structures,and their applications in protein detection.Taking full advantage of the unique optical properties of AuNPs including surface plasmon resonance and fluorescence quenching,and their excellent catalytic properties,we developed some novel colorimetric and fluorescent methods for protein detection with high sensitivity and specificity.1.Enzyme-free amplified colorimetric bioassays based on gold nanoparticle-catalyzed reductive decolorization of azo dyesTraditional amplified colorimetric bioassays relied heavily on the use of natural proteases and artificial enzymes.To solve this problem,we developed a novel,enzyme-free amplified colorimetric bioassay for protein detection,which took advantage of aptamer-functionalized paramagnetic microparticles(PMPs)for target capture,enrichment and separation,and aptamer-conjugated gold nanoparticle(AuNP)-catalyzed reductive decolorization of azo dye—methyl orange(MO)to generate the colorimetric signals.It was demonstrated that this colorimetric bioassay could enable simple,cost-effective,sensitive and specific thrombin detection,which exhibited a linear detection range of 45 pM to 455 pM with a detection limit of 30 pM.Importantly,this novel method did not require the use of any enhancing solutions and enzyme-substrate systems,and thus provide new avenues for constructing enzyme-free bioassays.2.Enzyme-free amplified colorimetric bioassays based on gold nanoparticle-catalyzed reductive decolorization of methylene blue and 4-nitrophenolTo further broaden the applications of the above colorimetric strategy using reductive decolorization reaction catalyzed by nanoparticle,herein we mainly focus on its fundmental principle and the influence factors on detection sensitivity and selection of colored substrates.A theoretical analysis showed that the detection sensitivity was favored by a high binding affinity between analyte and receptor(Kd),a high amplification factor(kcatAt),and a high extinction coefficient(ε)of the used colored substrate.With 4-nitrophenol(4-NP)as the non-dye colored substrate,colorimetric bioassay of thrombin was achieved by the endpoint method with a linear detection range of 182 pM to1090 pM and a detection limit of 91 pM.In particular,when using methylene blue(MB)as the substrate,a more convenient and efficient kinetic-based colorimetric thrombin bioassay was achieved without the steps of the acidification termination and magnetic removal of particles,which exhibited a linear detection range of 68 pM to 455 pM with a detection limit of 10 pM.Importantly,to some extent,such novel colorimetric methods overcome the heavy dependence on the limited enzyme-substrate systems.3.Fabrication of PolyA-tailed aptamer-gold/silver nanoprobes and their applications in colorimetric bioassaysDue to no requirement of thiol modification,PolyA-tailed DNA-gold nanoparticle conjugates are emerging as promising hybrid sensing materials for fabricating convenient and cost-effective bioassays.Herein,based on the colorimetric principle of target-induced cross-linking assembly,we demonstrated that PolyA-based AuNPs and AgNPs conjugates could act as the detection probe for homogeneous protein bioassays.Experimental results showed that these colorimetric bioassays using PolyA-based nanoparticle probes have better detection sensitivity than those using thiolated counterparts.More interestingly,over the traditional absorbance-ratio method of dual wavelength,it was found that AgNPs-based plasmonic protein sensing merely required the measurement of absorbance at only one wavelength for quantitative protein detecion and thus provided a more convenient analytical strategy.4.Activatable PolyA-tailed aptamer-gold nanoprobe for fluorescent turn-on bioassays using iodide-induced ligand displacementDepending on the strong AuNPs(or AgNPs)-PolyA interaction coming from its multipoint attachment nature and collective binding affinity,we demonstrated that PolyA-tailed DNA-AuNPs(or AgNPs)conjugates could serve as detection probe for heterogeneous protein bioassays.Moreover,we designed a new activatable PolyA-tailed,fluorophore-labeled AuNP probe,which could be turned on by destroying the fluorescence resonance energy transfer(FRET)between AuNPs and fluorophores.Interestingly,it was found that iodide and thiosulfate could act as the ligand displacing reagents to effectively detach PolyA-tailed and fluorophore-labeled DNA strands,from AuNP surface via a divide-and-conquer strategy,leading to the fluorescence recovery of AuNP-quenched fluorophores.Based on the unique fluorescence "OFF-to-ON" properties of activatable PolyA-based AuNP probe,we developed a novel and cost-effective fluorescence turn-on aptasensing platform for highly sensitive and specific thrombin detection.The proposed fluorescent bioassay exhibited a wide dynamic range from 0.174 nM to 17.4 nM with a detection limit of 0.089 nM.More importantly,employment of iodide as the displacing reagent offers many outstanding advantages over those thiolated compounds,including non-toxic,non foul-smelling,environmentally-benign,low cost and ease of storage.5.Oxidative dissolution-enabled fluorescent turn-on bioassays based on activatable gold nanoprobeWe prepared a novel AuNP-based fluorescence-activatable probe by co-assembling the aptamers and small fluorescein isothiocyanate(FITC)molecules onto the AuNP surface,which carried thousands of fluorescent reporter molecules,and meanwhile possessed the target recognition ability.Moreover,it was found that the mixture of iodide and hexacyanoferrate(HCF(Ⅲ))could be qualified as the efficient fluorescence-activated reagent toward the fluorescence recovery of AuNP-quenched FITC molecules by oxidative dissolution of AuNP quencher.Based on this new discovery,we developed a novel and cost-effective fluorescence turn-on aptasensing platform for highly sensitive and specific thrombin detection.The proposed fluorescent bioassay exhibited a wide dynamic range from 21.7 pM to 3478 pM with a detection limit of 4.7 pM.More importantly,this efficient,general gold dissolution-enabled fluorescence-activated strategy not only avoids the use of toxic cyanide-based etching solution and foul-smelling thiolated compounds,but also possesses many outstanding advantages,including environmentally-benign,low cost and ease of storage.