Periodic Reporting for period 1 - NANOBIOME (Gradient NANOcluster Screening Arrays for SERS Analytics of Wound MicroBIOMEs) Reporting period: 2018-05-01 to 2020-04-30 Summary of the context and overall objectives of the project This project addressed the quest for a new nanoparticle-based biosensing platform to study the chemical signaling within bacterial microbiome, bridging the gap from nanotechnology to biodiagnostic application. Healing-impaired and chronic wounds are a snowballing threat to public health and the economy. Their efficient treatment requires insight into the local wound microbiome, namely the composition and temporal evolution of bacterial communities. However, such bacterial communities are still poorly understood. To improve our understanding, we need access to the chemical cell-to-cell communication (quorum sensing, QS), to enable monitoring of changes in biofilms, specifically the competition of bacterial communities for resources and space, and the population density-dependent change from non-virulent to virulent. To realize this goal, NANOBIOME developed a plasmonic 2D screening platform for sensitive, rapid, and robust detection of bacterial QS signaling by surface-enhanced Raman scattering (SERS) spectroscopy. Contrary to other approaches, NANOBIOME followed the concept of (1) well-defined “sensing pixels” in the form of self-assembled plasmonic nanoclusters and anisotropic nanostructures; and (2) their ordered assembly on solid supports with defined interparticle spacing - to avoid plasmonic coupling (crosstalk) between pixels. Consequently, the decoupling of optical properties and assembly structure allowed for optical tailoring and by localized post-modification. (3) The controlled growth of the “sensing pixels” transformed the preassembled array into a “plasmonic particle library” with a gradient in sizes, morphology, and optical properties – ideally suited for rapid screening for highest SERS activity. This SERS screening platform has been used for the detection of different bacterial QS molecules (pyocyanin, violacein). Such hydrophobic biomarkers pose a major challenge for SERS detection because of their low adsorption tendency on gold surfaces. For this purpose, the polymer coating around the “sensing pixels” was used as a molecular trap to chemically harvest and accumulate hydrophobic analyte molecules near the particle surface. We achieved a proof-of-principle for the quantitative detection of pyocyanin (PYO), a QS signaling molecule of P. aeruginosa, with a lower limit of detection between 10^-7-10^-6 M. This is 1-2 orders of magnitude below concentrations of PYO commonly found in clinical samples (10^-5-10^-4 M PYO, HPLC after 24 h). In clinical samples, the concentration of PYO is traditionally determined by extraction with chloroform and subsequently high-performance liquid chromatography (HPLC), which is a time-consuming, cost-intensive, and laborious analysis. Since P. aeruginosa is the only known micro-organism producing PYO, it could serve as a biomarker for infections.We are confident that studies of the intra- and interspecies signaling in bacterial communities will improve our understanding of bacterial competition in wound microbiomes. Our sensing platform may provide guidelines for alternative designs and future studies of bacterial model systems (e.g. pathogens versus commensal bacteria). Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far "The action has led to results in all phases, some of which have already been disseminated by publication in peer-reviewed journals and conference contributions. The results include various ""sensing pixel"" designs, each with specific property profiles in regard to their interaction with light. Strategies have been developed by using the size, shape and composition of particles and their superstructures to adjust the light-matter interaction for specific plasmonic application. This includes the generation of SERS, as the main application, but also the generation of heat (photothermal therapy) or the photocatalysis of chemical reactions on the particle surface. For this, the optical and structural properties were closely correlated, supported by electromagnetic simulations. Methods of advanced optical characterization, this is diffuse-reflection spectroscopy, have been further developed and applied to complex particle systems to reveal information not accessible by standard UV/vis/NIR transmission spectroscopy.A methodology was developed to growth nanostars directly at the surface of solid supports with high kinetical control and tailored optical properties. To this end, a “plasmonic particle library” of nanostars can be fabricated with a continuous gradient in size, morphology, and optical properties – ideally suited for rapid screening of optical/structural properties and high SERS activity. Publication of the results is still pending.The developed sensing platform was applied for the quantitative detection of the bacterial biomarker pyocyanin, a QS signaling molecule of P. aeruginosa. In a proof-of -principle, we evidenced quantitative detection with a lower limit of between about 1-2 orders of magnitude below concentrations of PYO commonly found in clinical samples. Publication of the results is still pending." Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) "This action achieved several results beyond the current state of the art, including:- The first colloidal superstructures with triangular cores have been reported. - The first analysis of the diffuse optical properties of anisotropic superstructures.- Novel particle assemblies that can detect and act, to combine photothermal heating with SERS sensing. - The hypothesis that the anisotropic core of such assemblies controls their diffuse optical properties has been proven. This grants unprecedented insight into light-matter interactions of various plasmonic processes such as SERS generation and light-to-heat conversion, just to name a few.- A competition between SERS enhancement and increased scattering losses in larger assemblies has been identified. - We have developed the first growth of nanostars directly on solid supports with controlled interparticle spacings and high kinetical control.- A new method for the preparation of SERS-active gradient surfaces at the macroscopic level in the form of ""plasmonic particle libraries"".- A microbiome-sensing platform for the sensitive, rapid, and robust SERS detection of bacterial (QS) analytes at proof-of-principle level. Quantitative detection of concentrations 1-2 orders of magnitude below the concentrations of PYO usually found in clinical samples.- The first microbiome sensor platform that does not rely on hotspots formed by interparticle coupling, but on individual particles.In view of the results achieved, we expect that the developed microbiome-sensing platform will have an important socio-economic and societal impact when applied for future studies of biofilm competition and real-time investigation of bacterial infection models. Especially with respect to hard-to-heal and chronic wounds, a better understanding of population density-controlled bacterial behavior would enable more efficient, topologic antibiotic wound care to counteract spreading of antibiotic resistances. The developed sensing array is readily scalable on macroscopic areas and could potentially be integrated in smart wound dressings to allow on-patient detection of bacterial infections. The scaling and translation aspects are commonly recognized as bottlenecks in the development of materials for bio-applications." The picture shows schematically the chemical communication of bacteria using pyocyanin.
