The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

A Hybrid Orbitrap-Nanoelectromechanical Systems Approach for the Analysis of Individual, Intact Proteins in Real Time.

Nanoelectromechanical systems (NEMS)-based mass spectrometry (MS) is an emerging technique that enables determination of the mass of individual adsorbed particles by driving nanomechanical devices at resonance and monitoring the real-time changes in their resonance frequencies induced by each single molecule adsorption event. We incorporate NEMS into an Orbitrap mass spectrometer and report our progress towards leveraging the single-molecule capabilities of the NEMS to enhance the dynamic range of conventional MS instrumentation and to offer new capabilities for performing deep proteomic analysis of clinically relevant samples. We use the hybrid instrument to deliver E. coli GroEL molecules (801 kDa) to the NEMS devices in their native, intact state. Custom ion optics are used to focus the beam down to 40 µm diameter with a maximum flux of 25 molecules/second. The mass spectrum obtained with NEMS-MS shows good agreement with the known mass of GroEL.

Real-Time Sensing with Multiplexed Optomechanical Resonators.

Nanoelectromechanical resonators have been successfully used for a variety of sensing applications. Their extreme resolution comes from their small size, which strongly limits their capture area. This leads to a long analysis time and the requirement for large sample quantity. Moreover, the efficiency of the electrical transductions commonly used for silicon resonators degrades with increasing frequency, limiting the achievable mechanical bandwidth and throughput. Multiplexing a large number of high-frequency resonators appears to be a solution, but this is complex with electrical transductions. We propose here a route to solve these issues, with a multiplexing scheme for very high-frequency optomechanical resonators. We demonstrate the simultaneous frequency measurement of three silicon microdisks fabricated with a 200 mm wafer large-scale process. The readout architecture is simple and does not degrade the sensing resolutions. This paves the way toward the realization of sensors for multiparametric analysis with an extremely low limit of detection and response time.

Requirements and attributes of nano-resonator mass spectrometry for the analysis of intact viral particles.

When studying viruses, the most prevalent aspects that come to mind are their structural and functional features, but this leaves in the shadows a quite universal characteristic: their mass. Even if approximations can be derived from size and density measurements, the multi MDa to GDa mass range, featuring a majority of viruses, has so far remained largely unexplored. Recently, nano-electromechanical resonator-based mass spectrometry (NEMS-MS) has demonstrated the ability to measure the mass of intact DNA filled viral capsids in excess of 100 MDa. However, multiple factors have to be taken in consideration when performing NEMS-MS measurements. In this article, phenomena influencing NEMS-MS mass estimates are listed and discussed, including some particle's extraneous physical properties (size, aspect ratio, stiffness), and the influence of frequency noise and device fabrication defects. These factors being accounted for, we could begin to notice subtler effects linked with (e.g.) particle desolvation as a function of operating parameters. Graphical abstract.

Enhanced all-optical cavity-tuning using graphene.

All-optical tuning of the resonance of an optical cavity is used to realise optical signal-processing including modulation, switching, and signal-routing. The tuning of optical resonance is dictated by the two primary effects induced by optical absorption: charge-carrier-generation and heat-generation. Since these two effects shift the resonance in opposite directions in a pure silicon-on-insulator (SOI) micro-ring resonator as well as in a graphene-on-SOI system, the efficiency and the dynamic range of all-optical resonance-tuning is limited. In this work, in a graphene-oxide-silicon waveguide system, we demonstrate an exceptional resonance-tuning-efficiency of 300 p m/m W (0.055 π/m W), with a large dynamic range of 1.2 n m (0.22 π) from linear resonance to optical bistability. The dynamics of the resonance-tuning indicates that the superior resonance-tuning is due to large linear-absorption-induced thermo-optic effect. Competing free-carrier dispersion is suppressed as a result of the large separation between graphene and the silicon core. This work reveals new ways to improve the performance of graphene-on-waveguide systems in all-optical cavity-tuning, low-frequency all-optical modulation, and switching.

VHF NEMS-CMOS piezoresistive resonators for advanced sensing applications.

This work reports on top-down nanoelectromechanical resonators, which are among the smallest resonators listed in the literature. To overcome the fact that their electromechanical transduction is intrinsically very challenging due to their very high frequency (100 MHz) and ultimate size (each resonator is a 1.2 µm long, 100 nm wide, 20 nm thick silicon beam with 100 nm long and 30 nm wide piezoresistive lateral nanowire gauges), they have been monolithically integrated with an advanced fully depleted SOI CMOS technology. By advantageously combining the unique benefits of nanomechanics and nanoelectronics, this hybrid NEMS-CMOS device paves the way for novel breakthrough applications, such as NEMS-based mass spectrometry or hybrid NEMS/CMOS logic, which cannot be fully implemented without this association.

High frequency top-down junction-less silicon nanowire resonators.

We report here the first realization of top-down silicon nanowires (SiNW) transduced by both junction-less field-effect transistor (FET) and the piezoresistive (PZR) effect. The suspended SiNWs are among the smallest top-down SiNWs reported to date, featuring widths down to ~20 nm. This has been achieved thanks to a 200 mm-wafer-scale, VLSI process fully amenable to monolithic CMOS co-integration. Thanks to the very small dimensions, the conductance of the silicon nanowire can be controlled by a nearby electrostatic gate. Both the junction-less FET and the previously demonstrated PZR transduction have been performed with the same SiNW. These self-transducing schemes have shown similar signal-to-background ratios, and the PZR transduction has exhibited a relatively higher output signal. Allan deviation (σA) of the same SiNW has been measured with both schemes, and we obtain σ(A) ~ 20 ppm for the FET detection and σ(A) ~ 3 ppm for the PZR detection at room temperature and low pressure. Orders of magnitude improvements are expected from tighter electrostatic control via changes in geometry and doping level, as well as from CMOS integration. The compact, simple topology of these elementary SiNW resonators opens up new paths towards ultra-dense arrays for gas and mass sensing, time keeping or logic switching systems on the SiNW-CMOS platform.

Compact and modular system architecture for a nano-resonator-mass spectrometer.

Mass measurements in the mega-to giga-Dalton range are essential for the characterization of natural and synthetic nanoparticles, but very challenging to perform using conventional mass spectrometers. Nano-electro-mechanical system (NEMS) based MS has demonstrated unique capabilities for the analysis of ultra-high mass analytes. Yet, system designs to date included constraints transferred from conventional MS instruments, such as ion guides and high vacuum requirements. Encouraged by other reports, we investigated the influence of pressure on the performances of the NEMS sensor and the aerodynamic focusing lens that equipped our first-generation instrument. We thus realized that the NEMS spectrometer could operate at significantly higher pressures than anticipated without compromising particle focusing nor mass measurement quality. Based on these observations, we designed and constructed a new NEMS-MS prototype considerably more compact than our original system, and which features an improved aerodynamic lens alignment concept, yielding superior particle focusing. We evaluated this new prototype by performing nanoparticle deposition to characterize aerodynamic focusing, and mass measurements of calibrated gold nanoparticles samples. The particle capture efficiency showed nearly two orders of magnitude improvement compared to our previous prototype, while operating at two orders of magnitude greater pressure, and without compromising mass resolution.

Optomechanical mass spectrometry.

Nanomechanical mass spectrometry has proven to be well suited for the analysis of high mass species such as viruses. Still, the use of one-dimensional devices such as vibrating beams forces a trade-off between analysis time and mass resolution. Complex readout schemes are also required to simultaneously monitor multiple resonance modes, which degrades resolution. These issues restrict nanomechanical MS to specific species. We demonstrate here single-particle mass spectrometry with nano-optomechanical resonators fabricated with a Very Large Scale Integration process. The unique motion sensitivity of optomechanics allows designs that are impervious to particle position, stiffness or shape, opening the way to the analysis of large aspect ratio biological objects of great significance such as viruses with a tail or fibrils. Compared to top-down beam resonators with electrical read-out and state-of-the-art mass resolution, we show a three-fold improvement in capture area with no resolution degradation, despite the use of a single resonance mode.

Optomechanical resonating probe for very high frequency sensing of atomic forces.

Atomic force spectroscopy and microscopy are invaluable tools to characterize nanostructures and biological systems. State-of-the-art experiments use resonant driving of mechanical probes, whose frequency reaches MHz in the fastest commercial instruments where cantilevers are driven at nanometer amplitude. Stiffer probes oscillating at tens of picometers provide a better access to short-range interactions, yielding images of molecular bonds, but they are little amenable to high-speed operation. Next-generation investigations demand combining very high frequency (>100 MHz) with deep sub-nanometer oscillation amplitude, in order to access faster (below microsecond) phenomena with molecular resolution. Here we introduce a resonating optomechanical atomic force probe operated fully optically at a frequency of 117 MHz, two decades above cantilevers, with a Brownian motion amplitude four orders below. Based on Silicon-On-Insulator technology, the very high frequency probe demonstrates single-pixel sensing of contact and non-contact interactions with sub-picometer amplitude, breaking open current limitations for faster and finer force spectroscopy.

Neutral mass spectrometry of virus capsids above 100 megadaltons with nanomechanical resonators.

Measurement of the mass of particles in the mega- to gigadalton range is challenging with conventional mass spectrometry. Although this mass range appears optimal for nanomechanical resonators, nanomechanical mass spectrometers often suffer from prohibitive sample loss, extended analysis time, or inadequate resolution. We report on a system architecture combining nebulization of the analytes from solution, their efficient transfer and focusing without relying on electromagnetic fields, and the mass measurements of individual particles using nanomechanical resonator arrays. This system determined the mass distribution of ~30-megadalton polystyrene nanoparticles with high detection efficiency and effectively performed molecular mass measurements of empty or DNA-filled bacteriophage T5 capsids with masses up to 105 megadaltons using less than 1 picomole of sample and with an instrument resolution above 100.

Single-particle mass spectrometry with arrays of frequency-addressed nanomechanical resonators.

One of the main challenges to overcome to perform nanomechanical mass spectrometry analysis in a practical time frame stems from the size mismatch between the analyte beam and the small nanomechanical detector area. We report here the demonstration of mass spectrometry with arrays of 20 multiplexed nanomechanical resonators; each resonator is designed with a distinct resonance frequency which becomes its individual address. Mass spectra of metallic aggregates in the MDa range are acquired with more than one order of magnitude improvement in analysis time compared to individual resonators. A 20 NEMS array is probed in 150 ms with the same mass limit of detection as a single resonator. Spectra acquired with a conventional time-of-flight mass spectrometer in the same system show excellent agreement. We also demonstrate how mass spectrometry imaging at the single-particle level becomes possible by mapping a 4-cm-particle beam in the MDa range and above.

Frequency fluctuations in silicon nanoresonators.

Frequency stability is key to the performance of nanoresonators. This stability is thought to reach a limit with the resonator's ability to resolve thermally induced vibrations. Although measurements and predictions of resonator stability usually disregard fluctuations in the mechanical frequency response, these fluctuations have recently attracted considerable theoretical interest. However, their existence is very difficult to demonstrate experimentally. Here, through a literature review, we show that all studies of frequency stability report values several orders of magnitude larger than the limit imposed by thermomechanical noise. We studied a monocrystalline silicon nanoresonator at room temperature and found a similar discrepancy. We propose a new method to show that this was due to the presence of frequency fluctuations, of unexpected level. The fluctuations were not due to the instrumentation system, or to any other of the known sources investigated. These results challenge our current understanding of frequency fluctuations and call for a change in practices.

Neutral particle mass spectrometry with nanomechanical systems.

Current approaches to mass spectrometry (MS) require ionization of the analytes of interest. For high-mass species, the resulting charge state distribution can be complex and difficult to interpret correctly. Here, using a setup comprising both conventional time-of-flight MS (TOF-MS) and nano-electromechanical systems-based MS (NEMS-MS) in situ, we show directly that NEMS-MS analysis is insensitive to charge state: the spectrum consists of a single peak whatever the species' charge state, making it significantly clearer than existing MS analysis. In subsequent tests, all the charged particles are electrostatically removed from the beam, and unlike TOF-MS, NEMS-MS can still measure masses. This demonstrates the possibility to measure mass spectra for neutral particles. Thus, it is possible to envisage MS-based studies of analytes that are incompatible with current ionization techniques and the way is now open for the development of cutting-edge system architectures with unique analytical capability.

Modal control of mechanically coupled NEMS arrays for tunable RF filters.

A novel tuning strategy of nanoelectromechanical systems (NEMS)-based filters is proposed based on the modal control of mechanically coupled NEMS arrays. This is done by adjusting separately addressed distributed actuation and detection configurations proportionally to desired modal vectors. This control scheme enhances the global output signal, raising the power handling of the filter on all channels. Although the modal control of 1-D arrays exhibits narrow-band responses with adjustable resonance frequency, its application to 2-D arrays produces filters with both adjustable bandwidth and central frequency. One possible realization scheme is suggested by using electrostatically driven coupled NEMS arrays whose transduction gains are adjusted by changing the electrodes¿ bias voltages. Dispersion effects on both 1-D array and 2-D array frequency response are analytically expressed using eigenvalues perturbation theory. Based on these results, we show how to reduce their impact by appropriately choosing the coupling stiffness and the number of resonators.