Novel Surface-enhanced Raman Spectroscopy (SERS) Strategies For Real Biochemical System

With inherent advantages such as ultra-high sensitivity,specificity and abundant fingerprint information,surface-enhanced Raman spectroscopy(SERS)has been widely applied in both qualitative and quantitative studies.Particularly,innate high spectral resolution and powerful multiplexing potential make SERS a perfect candidate for simultaneous multi-target analysis in complex real biochemical systems,with rapid and efficient readout.However,a vacuum still exists in the translation of SERS for real world applications.Particularly for complex biochemical systems,the performance of SERS tags needs to be improved upon,and innovation for SERS sensing mechanisms is also required.Recent advances in nano-synthesis have provided tremendous improvement for novel SERS substrates.However,the development of Raman reporters,an integral element of SERS tags,has generally been overlooked.Indeed,the current abundant fingerprint information of commercial Raman reporters is a limiting factor in SERS multiplexing ability,and is unable to be deployed in complex real systems with high background signal inteferences.As for SERS sensing mechanisms,stable and reproducible signal enhancement is dependent on well controlled hot spots formation.And the critical requirement for accuracy and multiplexing ability in SERS leads to an urgent need for quantification mode innovation.Through the research described in this thesis,progress has been made on both fronts based on the discoveries of superior novel Raman reporters and ground-breaking sensing mechanisms.Explicitly,this thesis research demonstrated novel SERS strategies(as listed below)for in vitro/in vivo muti-target detection in complex biochemical systems,in order to promote practical translation of SERS.1.A sequential ‘on/off' dual mode SERS assay platform for heparin with wider detection window and higher reliability.Generally,the SERS strategy for ultra-sensitive analysis is constructed by hot spots formation based on aggregation of surface functionalized SERS tags,but the study of aggregation modes is always a great challenge.Here,a segregation phenomenon after aggregation is discovered.Based on electrostatic forces,highly protonated chitosan encapsulated Au@Ag nanoparticles undergo sequential aggregation/segregation upon the addition of heparin.This continuous “on/off” SERS sensing platform possesses wider detection range and provides higher reliability.The enhancing substrate Au@Ag combines both the stability of gold and high enhancing ability of silver,along with the specific recognition event based on heparin's highest negative charge.This SERS strategy presents excellent sensitivity with a limit of detection of 43.74 ng/m L(5.69 U/m L)and a continuous concentration range of 50–800 ng/m L(6.5–104 U/m L),realizing a reliable and practical assay for heparin detection.2.Quantitative DNA functionalization of alkyne-coded gold nanoparticles for field and pretreatment-free detection of heavy-metal ions in organic polluted waterField detection of heavy-metal ions is important but still challenging in current water pollution emergency response systems.Here we report a polyadenine(poly A)–DNAmediated approach for a rationally designed alkyne-coded SERS test kit.This approach enables rapid and simultaneous detection of Hg²⁺ and Ag⁺ by a portable spectrometer,and is impervious to organic interferences.With the aid of poly A,DNA functionalization is conducted in a quantitative manner and produces bivalent DNAgold nanoparticles(Au NPs).With the formation of thymine(T)–Hg–T and cytosine(C)–Ag–C,highly recognizable SERS signals from nanochains are rapidly detected when two different alkyne-labeled Au NPs are induced to undergo controllable bridging upon the addition of low-volume targets.For multiplex detection through a portable spectrometer,the limits of detection reach 0.77 and 0.86 n M for Hg²⁺ and Ag⁺,respectively.Of particular significance,the proposed C?C-containing Raman reporters provide an extremely effective solution for multiplex sensing in a spectral silent region,when the hyperspectral and fairly intense optical noises originating from lower wavenumber region(<1800 cm–1)are inevitable under complex ambient conditions.3.Rapid and reliable detection of alkaline phosphatase by a hot spots amplification strategy based on well-controlled assembly on single nanoparticleHighly stable and reproducible hot spot formation in SERS sensing platforms plays a critical role for reliable results.In this alkaline phosphatase(ALP)monitoring method,a well-controlled self-assembly on single nanoparticle has been established as a reliable detection mechanism.Here,ALP mediates the dephosphorylation of enzyme substrate,which induces a redox reaction of Ag⁺ and subsequently dynamic silver coating on Au NPs.“Hot spots” are formed in a uniform way between Au core and Ag shell,leading to amplification of SERS signal.The simple packaged test kit can achieve one-step clinical assay of ALP in human serum within several minutes,while the operation is simple as it directly inputs the sample into the test kit,which potentially decreases the need for time-consuming clinical trials.This work provides a great example for uniform hot spots formation to analyze biomolecules in complex real biosystems.4.Wild-type gene blocking for multiplex fusion gene analysis with triple bond-encoded SERSRecurrent gene fusions resulting from chromosomal rearrangements serve as promising diagnostic and therapeutic biomarkers for cancer aggressiveness.But the high abundance of wild type genes in the clinical background severely affects reliable detection of relative low levels of mutant genes,especially for multiplex sensing of intrinsically analogous fusion genes.Herein,for ternary TMPRESS2:ETS fusion gene variants detection in prostate cancer(PCa),we describe a wild-type gene blocking strategy for clinical samples collected from PCa-related cell lines and patients.Using locked nucleic acid(LNA)to potently block wild type sequences,target fusion gene sequences are specifically accessible to our triple bond-encoded SERS readout.Additionally,from the perspective of spectra output,quantification in Raman-silent region also blocked signal interferences within the complicated biological system.Thus,this combination of wild type sequence and spectra output blocking strategy successfully yields non-invasive triplex fusion gene profiling in PCa patient urine samples.Intriguingly,the competence of this strategy for halfanalogous TMPRESS2:ETS fusion gene sequences demonstrates a promising outlook for universal fusion gene analysis.5.Splicing nanoparticles-based “Click” SERS could aid multiplex liquid biopsy and accurate cellular imagingA completely new readout technique,“Click” SERS,has been developed based on Raman scattered light splice derived from nanoparticle(NP)assemblies.The single and narrow(1–2 nm)emission originating from triple bond-containing reporters undergoes dynamic combinatorial output,by means of controllable splice of SERS-active NPs analogous to small molecule units in click chemistry.Entirely different to conventional “sole code related to sole target” readout protocol,the intuitional,predictable and uniquely identifiable “Click” SERS relies on the number rather than the intensity of combinatorial emissions.Using this technique,10-plex synchronous biomarkers detection under a single scan,and accurate cellular imaging under double exposure have been achieved.“Click” SERS demonstrated multiple single band Raman scattering could be an authentic optical analysis method in biomedicine,and opens new possibilities for high thoughput and highly accurate SERS strategy.