Construction And Application Of SERS-based Ion-Mediated Cascade Amplification Biosensing Platform

Surface enhanced Raman spectroscopy(SERS)is a powerful vibrational spectroscopy technique that allows for highly sensitive structural detection of low concentration analytes on plasmons metal surface of nanostructures.It provides enhanced Raman intensities as well as rich molecular information.SERS as a highly sensitive detection method has become a feasible,versatile tool in analytical chemistry.In the field of biomedical analysis,great needs exists in the detection of important biomarkers and bioactive molecules with low abundance and short life;Meanwhile,the complexity of the biological system has further spawned the demand for high-intensity signal outputs,thus triggering the study of SERS amplification strategies.Researchers have proposed different amplification strategies to construct SERS sensors with high sensitivity.One of them is the exquisite fabrication of SERS-active substrates which have many gaps and tips and high density of hot spots.Another strategy aims at amplifying the signal output by target mediated assembly of numerous reporter-encoded nanoparticles.However,these strategies rely on direct conversion of target recognition events into signal readouts merely in one single step which limits the further improvement of the sensitivity of SERS-based sensors.The challenge could be addressed by employing amplification intermediates,through which multiple steps magnified signal outputs could be produced from one target recognition event.In our study,we discovered that silver ions can induce the aggregation of aminobenzenethiol(4-ABT)encoded gold nanoparticles,leading to the aggregation of 4-ABT nanoparticles to produce strong SERS signal.We reasoned that if silver ions be obtained from dissolution of silver nanoparticle introduced by target,such assays can be extended to detect various species other than silver ions.Therefore we proposed a SERS sensing Platform Based on ion-mediated cascade amplification(IMCA)and realized its application in the detection of mitochondrial DNA,telomerase activity,microRNA and exosomes.The platform also demonstrated its performance in the complex biochemical environment and the ability to detect the target from clinical samples.1)Construction of ion-mediated cascade amplification(IMCA)platform.A SERS amplified platform,featuring "ions-mediated cascade amplification(IMCA)",was proposed by utilizing the dissolved silver ions(Ag+)from silver nanoparticles(AgNPs).We found that using Ag+ as linkage agent can control the gaps between neighboring 4-aminobenzenethiol encoded gold nanoparticles(AuNPs@4-ABT)to form "hot-spot",and thus produce SERS signal output,in which the SERS intensity was proportional to the concentration of Ag+.Therefore,such assays can be extended to detect various species other than silver ions when silver ions are obtained from dissolution of silver nanoparticle introduced by target.We prepared the nanoparticle and Raman reporter in which the synthesis method and particle size of the nanoparticles were optimized,and the properties of the different reporter molecules were investigated.The mechanism of the platform were also studied.Then the selectivity,dynamic range,sensitivity of the platform were investigated and the reported molecular concentration,incubation time,pH and operating procedure were optimized.In addition,we observed the performance of the platform in complex environment.This IMCA platform is expected to be a universal strategy for ultrasensitive quantitative analysis of analytes and these work laid the foundation for its subsequent application.2)Detection of single nucleotide polymorphism in Human mitochondrial DNA based on ligase reaction utilizing IMCAIn this work,the IMCA was utilized for ultra-sensitive detection of single nucleotide polymorphism in human mitochondrial DNA(16189T?C).Combing with the DNA ligase reaction,each target DNA binding event could cause the successful introduction of one AgNP.By detecting the dissolved Ag+ from AgNPs using IMCA,low to 3.0 ×10-5 fm/?L targeted DNA could be detected,which corresponds to extractions from 200 nL cell suspension containing carcinoma pancreatic ?-cell lines from diabetes patients.The result demonstrated that the IMCA approach could effectively discriminated 16189T?C polymorphism in human mitochondrial DNA at low concentration combing with the fidelity of DNA ligase,suggesting its capacity as another alternative for SNP detection.This work demonstrates the applicability of the IMCA platform and provides a cornerstone for further exploration of the application of this platform in other targets.3)SERS assay of telomerase activity at single-cell level and colon cancer tissues via quadratic signal amplificationComparing with last work where one single target binding event introduce one silver nanoparticle,in this work,one target can employ multiple silver nanoparticles and thus realize ultrasensitive analysis.Herein,we develop a quadratic signal amplification strategy for ultrasensitive SERS detection of telomerase activity,which is an important biomarker and therapeutic target.Each telomerase-aided DNA sequence extension could trigger the formation of a long double-stranded DNA(dsDNA),making numerous AgNPs assembling along with this long strand through specific Ag-S bond,to form a primary amplification element.Afterwards,IMCA was utilized for secondary amplification.Through quadratic amplification,a limit of detection down to single HeLa cell was achieved.More importantly,this method demonstrated good performance when applied to tissues from colon cancer patients,which exhibits great potential in the practical application of telomerase-based cancer diagnosis in early stages.To demonstrate the potential in screening the telomerase inhibitors and telomerase-targeted drugs,the proposed design is successfully employed to measure the inhibition of telomerase activity by 3'-azido-3'-deoxythymidine.4)An enzyme-free quadratic SERS signal amplification approach for circulating microRNA detection in human serumIn this work,we introduced DNA-based hybridization chain reaction to provide more binding sites for silver nanoparticles and thus further increased the sensitivity of our IMCA approach.It is a new enzyme-free quadratic SERS signal amplification approach for ultrasensitive detection of circulating miRNA in human serum.Combining with miRNA-triggered hybridization chain reaction and IMCA,a limit of miRNA detection as low as 0.3 fM could be achieved.More importantly,the proposed method is suitable for the direct detection of circulating miRNAs in human serum collected from patients of different stages of chronic lymphocytic leukemia(CLL).We believe that this PCR-free,enzyme-free strategy would add a new concept for the design of simultaneous high sensitive quantitative assay for multiple biomarkers.5)Aptamer-based SERS amplification assay for detecting cancerous exosome from cell and bloodWe tried to expand the scope of application of IMCA from biological macromolecules(nucleic acids,enzymes)to the level of organelle.Exosomes are nanoscale membrane vesicles circulating in biofluids which show potential as cancer biomarker.Herein we developed a mobile aptamer-conjugated micro-captor for capture cancerous exosomes.Exosomes can be capture through specific binding between surface protein marker and aptamer through molecular recognition.Then we employed anti-CD63 aptamer encoded silver nanoparticles(AgNPs)as amplification mediators to binding exosomes captured on beads and applied them to IMCA.The detection limit can be as low as 167 exosomes/?L.The assay show excellent selectivity and sensitivity in serum and plasma,proving its potential for clinical use through the application to patient samples and biofluids.In the scope of real sample application,this work realized the detection of target from untreated body fluid which promote the capacity of IMCA platform in practical applications.