A Novel Ratio-fluorometric Method For Biosensing Study Based On Dual-emissive Ultra-small Gold Nanoparticles

With the continuous improvement and further development of nanotechnology and nanomaterials,more and more new types of nanomaterials from zero to three dimensions have sprung up.Glutathione coated AuNPs（GS-AuNPs）are a type of ultra-small luminescent noble metal nanoparticles which are approximately 2.5 nm.Differring from the AuNPs reported before,GS-AuNPs have two different luminescent center existed on the same nanoparticle.Furthermore,they have many excellent fluorescent properties including their tunable fluorescence emission and fluorescence intensity,a large Stokes shift,excellent resistance to photobleaching,smaller size,nontoxic and good biocompatibility.The AuNPs have been widely applied in biosensing and bioimaging of biological small molecules,heavy metal ions,enzymes,and nucleic acids detection.Therefore,based on the high sensitivity,good selectivity,fast response,high performance-to-price ratio and simple operation,using dual-emissive luminescent AuNPs as a fluorescent probe,the following biosensor research has been carried out:1.As the crucial merit of ratiometric assay is impregnable of potential ly interfering processes,a novel ratiometric method for pesticide detection was developed based on charge transfer of dual-emissions luminescent ultra-small gold nanoparticles（AuNPs）.Ultra-small AuNPs with a high emission at 800 nm and a low emission at600 nm was firstly synthesized.Interestingly,the postaddition of thiol compounds results in completely opposite changes to strengthen the emission of 600 nm and weaken the emission of 800 nm.In the presence of acetylcholinesterase（AChE）,acetylthiocholine can be hydrolyzed into thiocholine containing a newly generated thiol group to interact with AuNPs,resulting in the opposite change of the dual emissive fluorescence intensity.While adding pesticide as AChE inhibitor,the catalytic activity of AChE is inhibited and less thiocholine was produced.The biosensing system shows an obvious sensitivity to the pesticide with an LOD of 0.2nM and 0.07 nM for aldicarb and chlopyrifos,respectively.Therefore,this simple assay is suitable for AChE activity and pesticide detection,even in the complex samples.2.Based on the good stability of trypsin activity and its low price for purchasing,a biosensing system with dual-emission luminescent gold nanoparticles for enzyme activity detection and pesticide screening was obtained further development.The gold nanoparticles used in this section have a higher fluorescence ratio F₈₀₀/F₆₁₅ which was successfully synthesized in ratio of 0.9:1 between glutathione and HAuCl₄.While adding trypsin and its substrate,a negatively charged peptide,the fluorescence intensity of GS-AuNPs changes oppositely.However,adding pesticides as trypsin inhibitor,the enzyme activity was inhibited and less short peptide with a thiol group was produced,resulting in no obvious fluorescence change of GS-AuNPs.Therefore,the biological sensing system can be improved for the screening of pesticides due to the reduced cost and increased stability.3.Most molecular beacons use organic molecules or nanomaterials as quenchers.Since many transition metal ions also have excellent fluorescence quenching ability,we aim to recruit them as very small quenchers.Cr³⁺has a very slow ligand exchange rate,forming stable adducts with DNA.With its strong fluorescence quenching ability,we herein explored site-specific labelling of Cr³⁺on DNA to form a new type of molecular beacon.The quenching kinetics by Cr³⁺were measured for single-and double-stranded DNA as a function of salt concentration,pH,and Cr³⁺concentration.The goal was to achieve selective reaction with the single-stranded but not double-stranded region.The reaction mechanism was also probed by adding ATP,showing both a fast but unstable,and a slow but stable binding mode.By designing a partially complementary duplex with a short poly-guanine overhang under the optimal condition,we selectively labelled Cr³⁺to the overhang.The resulting sequence was tested as a molecular beacon with a detection limit of 0.3 nM DNA and a saturated fluorescence enhancement of 5-fold.With a 13-mer target DNA,the single mismatch discrimination of the beacon reached 22-fold.This study has demonstrated the possibility of forming useful Cr³⁺adducts with DNA.Such adducts are not only useful for developing biosensors but also for constructing new materials.