An Accurate And Ultrasensitive SERS Sensor Based On Au-Se Bond For In-situ Imaging And Quantification Of Intracellular MMP-2

Achieving accurate imaging and quantification of biomolecules in complex biological systems is a key challenge in the fields of biology and analytical chemistry.Therefore,the development of ultrasensitive and accurate biomolecule detection methods is what we have been focusing on.Surface-enhanced Raman spectroscopy(SERS)has provided a promising solution for highly sensitive biodetection,as it can achieve a Raman signal enhancement of 10⁶-10¹⁴ through electromagnetic and chemical enhancement.In addition,ratio detection can be performed in SERS biosensing by comparing the characteristic peaks and a constant internal standard peak,and this technique has valuable applications for in situ quantitative analysis.Regarding the sensitivity and accuracy of SERS biosensing,the use of enhanced substrates and the way in which the substrates and reporter molecules are connected are particularly crucial.Among a variety of materials,Au is most commonly used as an enhanced substrate to construct SERS nanoprobes due to its advantages of efficient SERS enhancement,good biocompatibility,controllable size and ease of modification.Currently,various kinds of Au substrates have shown a wide range of applications in SERS sensing of biological molecules.In such SERS nanoprobes,Raman reporter molecules are universally connected to the Au substrates through Au-sulfur(Au-S)bonds.However,abundant glutathione(GSH)and other biothiols in biological environments can easily replace the thiolate ligands connected to the Au substrates through ligand exchange,leading to destruction of the SERS nanoprobes,and resulting in detection signal distortion and inaccurate quantification.Aside from chemical bonding,physical adsorption is another method by which Raman signal molecules can be attached to Au nanoparticles(Au NPs),but it is difficult to control the amount that adsorb on the Au nanoparticles.Furthermore,nanoprobes constructed by adsorption are not stable in complex environments.Therefore,it is extremely necessary to develop new ways of connecting Au substrates and reporter molecules to construct optimized SERS probes,which can resist interference from biothiols and improve the efficiency of SERS enhancement.Compared with Au-S bonds,Au-Se bonds exhibit higher bonding energy.Recently,fluorescence assays using nanoprobes with Au–Se bonds to reduce thiol interference have been demonstrated.However,ultrasensitivity and absolute quantification are still great challenges for such fluorescence nanoprobes.We also developed an electrochemical analysis platform with Au-Se interface,but in situ imaging was difficult to perform using electrochemical methods.Taking into account the advantages of SERS in highly sensitive quantitative detection and in situ imaging,as well as the irreplaceable role of the Au substrate in SERS biosensing,using Au-Se bonds to construct stable SERS probes represents a breakthrough in the high-fidelity detection of biomolecules.On the other hand,Au-Se bonds possess a metallic nature with easier charge transfer from the Au substrate to the coupled molecules.SERS nanoprobes with Au-Se bonds will make a great contribution to SERS chemical enhancement of reporter molecules by Au substrates,resulting in ultrahigh sensitivity.Hence,we established a new kind of SERS nanoprobe based on covalent Au-Se bonding between the Au substrate and the Raman reporter molecules(Au-Se SERS nanoprobes)to perform SERS bioimaging and in situ quantitation.Matrix metalloproteinase-2(MMP-2)SERS analysis was used to illustrate the performance of the Au-Se SERS nanoprobes.Within the nanoprobes,peptide chains containing-Se H at the end and labeled with TAMRA(P1)for specific MMP-2 recognition were attached to the surface of gold nanoparticles(Au NPs)by Au-Se bonding.Due to the SERS effect,TAMRA showed a strong Raman signal.In the presence of MMP-2,the P1 chains were recognized and cleaved by activated MMP-2,leading to TAMRA dissociation from Au NPs and a decrease of the Raman signal,which was related to the amount of target MMP-2.Meanwhile,Au-S SERS nanoprobes were constructed in a similar way,with peptide chains containing-SH at the end and labeled with TAMRA(P2),for performance comparison.The Au-S SERS nanoprobes were inevitably destroyed by biothiols due to ligand exchange,generating a distorted signal corresponding to TAMRA dissociation along the Au-S bond break.Thus,the Au-Se SERS nanoprobes can be used to perform accurate cell imaging.In order to further achieve high-fidelity absolute quantitative detection of biomolecules,we constructed a ratio Au-Se SERS nanoprobe with a core–shell structure,which was modified with the constant internal standard molecule,1,4-BDT,at the core-shell gap and TAMRA-modified P1 for responding to intracellular MMP-2.Through the Raman signal ratio of TAMRA to 1,4-BDT,we successfully achieved high-fidelity quantitative analysis of MMP-2 in living cells.The Au-Se SERS nanoprobes exhibit excellent performance for accurate,high-sensitivity bioimaging and in situ quantification.This work will provide a favorable new method for biological analysis and clinical detection.