Cross-SERS · SERS ultrasensitive universal sensing of proteins through cross-reactive sensor arrays

SERS ultrasensitive universal sensing of proteins through cross-reactive sensor arrays

The Cross-SERS project is a true reflection of the increasingly interdisciplinary character of modern science and lies at the frontier of scientific innovation. This proposal built on the genuinely original idea of developing rapid, inexpensive and sensitive methods for label-free analysis, based on cross-reactive sensor arrays (CRSA) and surface-enhanced Raman scattering spectroscopy (SERS) to be used for the accurate and ultrasensitive fast detection of proteins, especially those related with pathogenic states such as prions (bovine spongiform encephalopathy, Creutzfeldt-Jakob disease but also related to Alzheimer and Parkinson diseases), prostate-specific antigen or/and drug abuse in sports. As an intermediate goal, the project aimed at the multiplex ultrasensitive detection and quantification of metallic cations within environmental and biological fluids. The Cross-SERS sensor is a collection of hybrid composite beads comprising a discrete colloidally stable polystyrene or silica microparticle that supports plasmonic materials. Each of these sensing elements is functionalized with organic ligand coordinated with cations. These surface complexes are designed to interact with pathogenic and drug-abuse related proteins leading to an ensemble of different molecular interactions which constitute the fingerprint response of the whole array to the target biomolecule. During the first part of his IEF grant, Dr. Guerrini successfully fabricated the hybrid plasmonic microbeads, investigating also in detail the effects that metallic nanoparticles of different size, composition and shape have on the overall SERS performance of such materials (Fig.A).[1] This also included the development of new fabrication methods for the synthesis of asymmetric gold nanostructures with high optical activity (Fig.B,C).[2] The metallic surfaces needed then to be functionalized with organic ligands (such as terpyridines and porphyrins) able to coordinate cationic species and undergo a significant electronic redistribution upon their complexation to be revealed by SERS. Initially, thiolated terpyridines were successfully tested on silver colloids and applied to the SERS multiplex detection of Co(II) and Cu(II) ions in water (Fig.D).[3] However, due to the unexpected impossibility to be supplied with metal ion-free thiolated porphyrins and (more) terpyridine, Dr. Guerrini decided to explore other organic chemoreceptors for metal ion sensing. Firstly, he successfully employed 4-mercaptopyridine self-assembled onto the hybrid plasmonic beads to design a sensing platform capable of delivering the first example of a highly selective SERS-based sensor for chemical speciation of inorganic mercury and methylmercury ions in water at trace levels (Fig.E).[4] Moreover, mercatpobenzoic acid (MBA) was selected as an effective chelating agent of a different variety of cationic species, forming surface complexes with characteristic vibrational patterns.[5] In particular, the metallorganic surface complex comprising MBA and Al3+ was exploited as chemoreceptor for the SERS detection of misfolded proteins in buffered media (Fig. F).[5] These toxic proteins are responsible for neurodegenerative disorders such as Alzheimer’s, Parkinson’s, Creutzfeldt−Jakob’s and different non-neurophatic amyloidosis. On the other hand, Dr. Guerrini decided to pursue a second detection strategy for the identification and quantification of proteins in complex media. As an alternative to the indirect SERS sensing of biomolecules via organic chemoreceptors (such as those previously described), he installed a Raman label at one extremity of a protein chemoreceptor to indirectly monitor its interaction with the target protein. Specifically, he exploited the ability of the oncoprotein c-Jun to heterodimerize with its native protein partner, c-Fos, and therefore he designed a c-Fos peptide receptor chemically modified to incorporate a thiophenol group (TP) at the N-terminal site. The TP functionality anchors the c-Fos protein onto the metal substrate and works as an effective Raman spring to sense the structural rearrangements associated with the c-Fos/c-Jun heterodimerization (Fig. G). Such strategy was applied to develop a SERS-based sensor for the detection of the oncoprotein c-Jun at nanomolar levels in biological fluids.[6] Dr. Guerrini devoted more efforts to the translation of this approach onto hybrid plasmonic microparticles for the construction of the CRSA as well as the design of different protein chemoreceptors for the effective SERS detection of other biologically relevant target proteins. However, some reproducibility issues have been encountered during this process which have eventually hampered the implementation of the individual sensing units into final fabrication of the CRSA device. In addition to the Cross-SERS project, Dr. Guerrini was also involved, as a SERS expert, in external collaborations of the Zeptonic group which resulted in several published [7,8] and few others were recently submitted. More importantly, Dr. Guerrini independently designed a novel method for direct SERS analysis of DNA, which paved the way for successful translation of the analytical potential of SERS to the direct study of DNA in its native duplex state (Fig. H). Two articles were already published,[9,10] few more are either submitted or in preparation, and a patent has been applied (EP14382415.9). Overall, despite the final goal of the Cross-SERS project has not been reached (i.e. the fabrication of a CRSA for proteins detection in biological media), the successful achievement of most intermediate objectives has provided significant advances in several research fields. Among others, the work carried out by Dr. Guerrini has proven the analytical potential of the SERS technique in the practical application for metal ion sensing, overcoming the intrinsic limitations of most common analytical techniques employed for these purposes. Quantitative detection of metal ions in solution at trace levels is a topic of widespread interest, spanning from environmental sensing in natural waters through industrial process monitoring to biomedical diagnostics. On the other hand, a prompt, reliable and quantitative detection of oncoprotein such as c-Jun in its complex biological media is expected to have great diagnostic and therapeutic impact, and aid in further studies on its biological role. Finally, regarding the development of a fast, high-throughput, low-cost, direct SERS analysis of DNA in its native state, Dr. Guerrini anticipates these findings of key importance in a number of different fields including diagnosis, genetic engineering, biotechnology, drug discovery (antibiogram in microbiology), agriculture, forensic science or molecular electronics. For this reason, a patent has been applied EP14382415.9. References: [1] Journal of Optics 2015. Just accepted [2] ACS Photonics 2014, 1, 1237. [3] Nanoscale 2013, 5, 5841. [4] Nanoscale 2014, 6, 8368. [5] ACS Appl. Mater. Interfaces 2015, 7, 9420. [6] J. Am. Chem. Soc. 2013, 135, 10314. [7] Chem. Mat. 2015, 27, 950. [8] J. Phys. Chem. Lett. 2015, 6, 868. [9] Angew. Chem.-Int. Edit. 2015, 54, 1144. [10] J. Am. Chem. Soc. 2015, 137, 469.

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