State-of-the-art in structure-function analysis

The recent advances in genomics and proteomics revealed a list of molecular constituents that build the cell. However, the knowledge about how these constituents (i.e. different proteins) interact and are spatially arranged within the ‘functional modules’ remains superficial.1 Achieving such knowledge is certainly beyond the scope of classical crystallography and similar established techniques. Therefore, during the recent years there has been an emphasis on combining crystallography with other experimental methods, e.g. NMR, SAXS, electron tomography, etc., as well as bioinformatics methods. The goal then is the integration of structural information from different sources into a structural model through computation. Despite of progress in this area, yet another challenge is in vivo verification of such structural models and, most importantly, establishment of clear mechanisms behind the functional activity of the cellular superstructures.
Ideally, the analysis of structure-function relations in the dynamic, highly complex cellular architectures has to be carried out on a single cell level with high spatiotemporal resolution. An important recent technical advance for single-cell analysis is the introduction of different super-resolution imaging techniques such as photoactivation localization microscopy (PALM), stochastic optical reconstitution microscopy (STORM), or stimulated emission depletion (STED) microscopy. When combined with switchable fluorophores and advanced labeling technologies2  super-resolution microscopy can reveal the sub-cellular structures at a resolution down to 10-20 nm.

Protein labeling technologies

Modern life science employs a large variety of different labeling techniques. Fluorescent proteins facilitate protein localization and can be used to detect even single molecules in the cell. Polyhistidine and streptavidine tags are used for the protein purification. Magnetic resonance probes and bioluminescence probes are useful for whole organism imaging. The EPFL partner has developed a pair of proteins tags, namely SNAP-tag and CLIP-tag which permit the specific and covalent labeling of fusion proteins in living cells. They are based on the reaction of SNAP-tag and CLIP-tag with benzylguanine and benzylcytosine derivatives, respectively. These approaches have recently been exploited to establish a general concept for the creation of fluorescence sensor for analytes of interest, so-called Snifits. Snifits are semisynthetic fluorescent sensor proteins consisting of SNAP-tag, CLIP-tag, and an analyte-binding protein. SNAP- and CLIP-tag are labeled with a synthetic fluorescent ligand and a secondary synthetic fluorophore, respectively. The ligand binds to the binding protein in an intramolecular fashion and thereby keeps the sensor protein in a closed conformation. Free analyte can compete for binding to the binding protein and can shift the equilibrium to the open conformation. This shift can be detected by a change in the Förster resonance energy transfer (FRET) efficiency between the two fluorophores. It has been shown that Snifits can be generated for extra- and intracellular metabolites. This type of sensing approach together with Single-Cell-on-a-Chip platform represents a powerful combination to study metabolite dynamics in living cells. Full control over the shape of the cell, localized stimulation and real-time fluorescent sub-cellular visualization of metabolites using super-resolution techniques will greatly contribute to the understanding of cellular processes.

Opportunities for nanotechnology

In parallel to the here mentioned advances in microscopy techniques, recent developments in nanobiotechnology have enabled sensing, analysis and manipulation on the single cell level. Nanofabrication and functional nanomaterials can be employed for precise actuation and mapping of cellular processes in situ. For example, Charles Lieber’s group introduced an ultrasensitive technique that is based on semiconductor nanowires, brought as electrodes for recording of physiological processes in individual cells.3 A nanobiochip platform4 has been employed to study intracellular signalling.5
However, despite of the tremendous interest and the demonstrated advantages of such nanotechnology-enabled innovative techniques, so far they have rarely been exploited to address real biological problems. Clearly, overcoming the barrier for introducing these approaches into the biological and medical community has been difficult. Exceptions are different micro- and nanofluidic techniques, although they have been limited mostly to cell sorting .

Toward single cell metabolomics

The field of metabolomics is investigating cellular mechanisms at the level of the products of biochemical reactions. Combined with the data from genomic, transcriptomic and proteomic profiling, measurements of the metabolic activity help to create a complete picture of different cellular functions.
Single cell-level metabolic profiling is a new development in this field.6 For example, heterogeneity of a cell population in terms of functional activity has been demonstrated at the level of genome in prokaryotic cells.7 Similarly, in stem cells heterogeneity is important in the development of their final functional role. Thus, profiling of individual cells at the “-omics” level is meaningful and a highly relevant task both from both fundamental knowledge and application point of view. For example, quantitative profiling and identification of phenotypes via a set of metabolites is becoming a very instrumental part for functional genomic analysis.
Currently, the standard techniques for profiling of the metabolic activity of individual cells are based on non-invasive methods such as NMR, or on different invasive techniques such as chromatography, electrophoresis and, mainly, mass spectrometry.6 For the latter, sample preparation, cell identification and handling have been recognized as the most demanding and critical step. This is due to the susceptibility of cells to external stimuli, non-specific interactions, presence of toxic materials, and other environmental factors.

The benefits of the METASENS platform

The proposed METASENS chip platform opens up new ways for spatially-resolved in situ measurements of the metabolic activity of enzymes and cellular organelles under physiological conditions. Molecular nanolithography techniques4  mastered at the CPST group is a powerful tool for designing the sub-cellular array of the sensor elements. This technology permits the positioning of sensors with high spatial precision (the DPN fabrication process currently allows to place the functional entities with 50 nm precision) with respect to a cell of interest.
Our platform synergistically combines several of the above-discussed state-of-the-art developments to open up new possibilities for single cell analytics. The major advantages of the METASENS platform are the following:
Time and resource consuming cell culture experiments are replaced with a multi-functional tool (a chip) in which each cell is defined and addressable and its microenvironment can be controlled to mimick the conditions specific for a particular cell type in a tissue. Tissue conditions can be mimicked by docking the cell on a flat or curved docking zone, that is coated by a solid (up to 5 nm-thin) or soft (more than 50 nm thick) biocompatible layer (short PEG, hydrogel, etc), including ECM proteins and adhesion-promoting peptides. The importance of biocompatibility and full control over protein specific vs. nonspecific binding in single cell analytics is frequently underestimated. The CPST group has an excellent track record in the design of biocompatible surfaces and specific immobilization platforms for different types of proteins (for examples see papers on the multivalent NTA technology by Valiokas, Liedberg and Piehler et al. in the publication list).
The docking zone will function as a support for an array of sensor elements. We will include in the biocompatible layer fluorescent reporters for detection of secreted metabolites and also for proximate sensing/localization of membrane pores and other secretion machineries at the cell membrane. At the same time the chip will be used in super-resolution microscopy and/or electrochemistry, iSPR and SPM.
The chip platform ultimately will allow to simultaneously probing in real time the metabolic activity and structural organization of a living cell. Depending on the task, it will be possible to describe the phenotypes via a set of defined metabolites, and also to relate their concentrations to spatial rearrangements in the cell skeleton, redistribution of organelles, or cell polarization, division or apoptosis.