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High-throughput vibrational fingerprinting by nanoplasmonics for disease biology

Periodic Reporting for period 3 - VIBRANT-BIO (High-throughput vibrational fingerprinting by nanoplasmonics for disease biology)

Reporting period: 2020-01-01 to 2021-06-30

Devastating diseases such as Alzheimer’s, Parkinson’s and cancer are still without cures and they continue to undermine the quality of life for millions of people worldwide and impose a significant economic burden. Misfolded protein aggregates constitute the molecular basis of these diseases. For example, in Alzheimer’s disease, aggregation of toxic amyloid-beta proteins and associated plaque formation in the brain are the major hallmarks. Despite their scientific importance and potential impact on curing diseases, little is known about the structure of misfolded proteins, the triggers that lead to their conformational changes and the mechanisms affecting cell fate. This is due to the fact that current technologies are severely limited for molecular-level identification of protein structures and high-throughput analysis of protein interactions with major biomolecules. To address these challenges, this project aims to introduce and demonstrate a breakthrough mid-infrared (Mid-IR) spectroscopy technology that can be used for complete profiling of biomolecules from identification of their structure and composition to their biological function. VIBRANT-BIO also investigates how to overcome the shortcomings of infrared spectroscopy itself. For instance, state of the art infrared spectrometers are bulky and come with a hefty price tag, which limits the use of IR spectroscopy to lab-based environments. Besides, the light-matter interaction in this spectral range is prohibitively weak, therefore studies of low analyte concentrations are extremely challenging. The objective of VIBRANT-BIO is to overcome these limitations by employing nanophotonics, which will allow to significantly enhance the light-matter interaction, and will also lead to new functionalities, such as miniaturized and sensitive chemical sensors well-suited for point-of-care applications.

Specific objectives of the VIBRANT-BIO include the development of:
• An innovative technology that can perform high-resolution, label-free and real-time structural analysis (e.g. transitions from α-helix to β-sheet conformation) of ultra-small volumes of proteins by directly accessing chemical specific IR fingerprints.
• An extremely surface-sensitive and molecular specific characterization technique that can measure in real time interactions of proteins with biological interfaces (i.e. lipid cell membranes).
• The first high-throughput and ultra-sensitive spectroscopy microarray that can rapidly screen chemical composition, molecular structure and interaction kinetics of biomolecules.
VIBRANT-BIO explores the ultimate limits of light-matter interaction to develop biosensors with extreme sensitivity, throughput and functionality, well beyond the state-of the art. For this aim, the project exploits novel nanophotonic phenomena with (I) plasmonics and (II) dielectric metasurfaces in the Mid-IR, which is an extremely rich spectral window for bioscience research.

I) Using plasmonic metasurfaces, we have introduced mid-infrared biosensors, which are able to track minute quantities of proteins and resolve their secondary structure as well as distinguish multiple analytes in heterogeneous biological samples non-destructively, in real-time and with high sensitivity.
One of our plasmonic sensors is based on multi-resonant metasurfaces and by allowing access to extract distinct chemical fingerprint information of proteins, lipids, peptides, or other biochemical it enables simultaneous and independent monitoring of their interaction dynamics []. In this work we showed that the sensors can spectroscopically resolve the pathological structure of disease-related proteins and the interaction of biomimetic lipid membranes with different peptides as well as the dynamics of vesicular cargo release. These are biologically important mass-preserving processes that are inaccessible to standard label-free techniques, regardless of their sensitivity.
Strikingly, the sensors can resolve the interaction of lipid membranes with a toxic pore-forming peptide such as melittin, both in supported membranes and surface-tethered vesicles loaded with neurotransmitter molecules. The study shows monitoring of melittin-induced membrane disruption and neurotransmitter cargo release from such synaptic vesicle mimics in real time, with monolayer sensitivity, and without labeling. These important proof of concept experiments pave the way for applying these biosensors to investigate the molecular mechanisms underpinning important processes that have been linked to human diseases, such pore formation and membrane disruption induced by protein aggregation in neurodegenerative diseases such as Alzheimer's and Parkinson's disease.
The new biosensors represents a powerful tool for the differentiation, identification and simultaneous investigation of the interactions between different biological species in complex samples, which addresses the clear shortcomings of current label-free techniques. Furthermore, it can be implemented to analyze a multitude of multi-analyte biological systems, opening exciting avenues of applications in diverse fields ranging from fundamental biology to pharmaceutical drug development. We further describe how our sensor can be expanded to simultaneously detect more molecular vibrations from additional spectral ranges (
Using plasmonics we have been also opening up new frontiers for label-free spectroscopic identification of secondary structure conformation of nanometric thin protein layers in aqueous solution. In 2017, [] we used α-synuclein, a protein associated with plaque formation in Parkinson’s diseases. By leveraging plasmonically enhanced amide-band of proteins, we were able to measure a random coil to cross β-sheet conformational change as a result of α-synuclein aggregation. We showed high structural sensitivity of our biosensor by distinguishing the characteristic β-sheet conformations between a native protein and pathological fibrillar aggregates. In our recent work, we advanced the device and presented the first experimental demonstration of real-time label-free spectroscopic monitoring of secondary structure changes in a protein monolayer [].
We have studies a wafer-scale array of resonant coaxial nanoapertures with sub-10 nm gaps as a practical platform for surface-enhanced infrared absorption spectroscopy and performed proof of principle experiments with protein samples []. We have been also contributing to the fundamental understanding of SEIRA and determining its limitations. Since SEIRA is a new field, the general notion on sensitivity parameters is still not well developed. In a recent work, we address this need by introducing methodologies in order to quantify the limit of detection which can provide a valuable reference points for determining biosensor performance metrics []. Within this context we are also investigating the schemes that can allow us to explore the ultimate limits of light-matter interactions at Mid-IR. We have recently reported the study of molecular vibrations through configurable optical interactions of a nanotip with an infrared resonant nanowire that supports tunable bright and nonradiative dark modes. We have demonstrated electromagnetically induced scattering through phase controlled near-field interactions, which presents a new regime of IR spectroscopy for applications of vibrational coherence from quantum computing to optical control of chemical reactions. (

II) Using dielectric metasurfaces, we have recently invented a compact and sensitive nanophotonic system that can identify a molecule’s absorption characteristics without using conventional bulky and expensive spectrometry instruments [DOI: 10.1126/science.aas9768]. We have used our system to detect polymers, pesticides and organic compounds. What’s more, it is compatible with fabrication technology used for mass-production of computer chips.
The system consists of an engineered surface covered with hundreds of tiny sensors called metapixels where each one of them resonates at a different frequency. When a molecule comes into contact with the surface, the way the molecule absorbs light changes the behavior of all the metapixels it touches. Importantly, the metapixels are arranged in such a way that different vibrational frequencies are mapped to different areas on the surface. This creates a pixelated map of light absorption that can be translated into a molecular bar code – all without using costly and cumbersome spectrometers. These barcodes can be massively analyzed and classified using advanced pattern recognition and sorting technology such as artificial neural networks. This research – which sits at the crossroads of physics, nanotechnology and big data – has been published in Science Magazine and opens the door to large-scale, image-based detection of materials aided by artificial intelligence.
There are a number of potential applications for this new system. For instance, it could be used to make portable medical testing devices that generate bar codes for each of the biomarkers found in a blood sample. This new technology is also well-suited for environmental monitoring and new emerging artificial intelligence approaches could be used in conjunction with this new technology to create and process a whole library of molecular barcodes for compounds ranging from protein and DNA to pesticides and polymers. That would give researchers a new tool for quickly and accurately spotting minute amounts of compounds present in complex samples.
Most recently, using Mid-IR dielectric metasurfaces we have developed a complementary method for detecting molecular absorption fingerprints over a broad spectrum, which combines the device-level simplicity of state-of-the-art angle-scanning refractometric sensors with the chemical specificity of infrared spectroscopy (DOI: 10.1126/sciadv.aaw2871). This method provides an extremely broad spectral coverage by using smartly the polarization and angle dependence of the designed metasurfaces.
Detection at the monolayer level and below of the secondary structure of proteins and interactions of major biomolecules, such as lipids, proteins, and nucleic acids in mixtures is crucial for understanding a multitude of biological mechanisms in health and disease. For instance, in cells, signaling and molecular transport are fundamentally governed by association and insertion of proteins with the cell membrane. However, current label-free techniques such as quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) struggle to differentiate protein structure, insertion, chemical release and membrane disruption processes. This is due to the fact that the competing but otherwise undistinguishable signal contributions of protein association (with mass/refractive index increase), and the lipid disruption (with mass/refractive index decrease) cancel each other out. For this reason, it is essential to develop new biosensors with high sensitivity and selectivity that can chemically distinguish different biomolecular species involved in complex multi-analyte interactions. We have achieved this by developing novel nanoplasmonic designs which push sensitivity and functionality of plasmon-enhanced infrared absorption spectroscopy using combined signal enhancement strategies, nanogaps and fractal-like multiresonant approaches.
Our plasmonic biosensors by enabling differentiation and identification of biological species in complex multi-analyte biological systems can addresses the clear shortcomings of current label-free techniques. Furthermore, our techniques can be implemented to analyze a wide range of multi-analyte biological systems (e.g. proteins, carbohydrates, nucleic acids…), opening exciting avenues of research in diverse fields ranging from fundamental biology to pharmaceutical drug development. This represents a timely and major advancement in the fields of nanotechnology, metasurfaces, and nanophotonic biosensing.

In a concurrent effort, our pixelated metasurface-based sensing approach can be combined with an IR imaging detector such as a high resolution microbolometer array in a miniaturized sensor device on a single chip, which is capable of measuring molecular barcodes without the need for bulky spectroscopic equipment. Such an integrated spectroscopy device will constitute a significant breakthrough over existing FTIR spectrometers, overcoming the associated size and cost constraints in the process. Compared to sensors based microelectromechanical systems (MEMS), which is a leading technology for complex on-chip devices, our metasurface approach requires no movable parts and will therefore enable a more robust and reliable sensors for field-deployable applications. Additional efforts for on-chip spectroscopic sensing have utilized chip-integrated quantum cascade laser (QCL) elements coupled to photonic waveguides. However, the spectral operating range of such systems is commonly limited to several 10s of wavenumbers in the IR, allowing them to target, e.g. only a single vibrational band of interest. In contrast, our approach operates with broadband light sources/detectors, allowing it to target multiple vibrational bands over a broad spectral range (e.g. 900-1800 cm-1). This enables the detection of more classes of biomolecules, which leads to a much higher specificity of the sensor.
Our proposed sensor device disrupts the way chemical and biological analysis is carried out by bringing the sensitivity and versatility of infrared spectroscopy to a portable sensing tool that can potentially fit into the palm of your hand. This is in strong contrast to previous spectroscopy approaches, which are limited by size, cost, and sensitivity. Therefore, we believe our technology will have a large scientific and economic impact by providing spectroscopic sensor at a more affordable cost for existing point-of-care applications opening new application areas where no current point-of-care devices exist due to technology limitations.
Molecular barcode generation with a sensor based on a metasurface of dielectric resonators
Dielectric metasurface consisting of an array of elliptical resonators used to detect biomolecules
Metasurface‐based molecular biosensing aided by artificial intelligence
Multiresonant plasmonic sensor used for unraveling complex interactions in biomimetic lipid membrane
Fractal-like metasurface for molecular bond detection over an ultrawide infrared spectral range