Patent Grant
11289319
The present invention generally relates to the analysis of particles, and particularly to measuring the mass of particles.
There are different technologies to analyze particles. One technology is known as mass spectrometry. In mass spectrometry, in essence, the particles to be analyzed are ionized. The ionized particles travel through an electromagnetic field. Their deflection by the electromagnetic field depends on their mass-to-charge ratio. By determining their position after the electromagnetic field, one can thus determine the mass-to-charge ratio of the particles.
In other words, conventional mass spectrometry (MS) enables identification of analytes by first charging them within an ion source, and then by tracking their induced motion in applied electromagnetic fields. This enables deduction of their mass-to-charge ratio. Since its first applications to organic compounds, MS has assumed an increasingly important role in the life sciences and medicine. Recently, it has become a preeminent technology for proteomic analysis. The increased resolution and mass range of modern mass analyzers now enable MS measurements of high-mass protein complexes and even virus capsids—in the mass range up to 1-50 MDa. These high-mass analytes are introduced into MS ion optics using nanospray at so-called native conditions (i.e. at a pH close to that of physiological conditions). For example, it was shown in U.S. Pat. No. 8,791,409 that the Orbitrap mass spectrometry enables detection of individual ions of protein complexes, with mass resolving power in the thousands range. However, conventional MS typically shows rapidly decreasing performance at higher masses, especially because of overlapping charge distributions of heterogeneous MDa analytes.
A new approach of mass spectrometry is based on systems of nanotechnology. This approach uses nanoelectromechanical systems (NEMS) resonators. NEMS resonators are electronically controllable, submicron-scale mechanical resonators, that enable analyte mass detection with very high sensitivity. Upon adsorption onto a NEMS resonator, an individual analyte can precipitously and measurably downshift the resonant frequency of the device. Embedding the NEMS resonator in specialized electronic circuitry permits real-time readout of individual molecular adsorption events. The frequency change induced by individual molecular adsorption events is proportional to both the mass of the molecule and the specific location the analyte adsorbs upon the resonator. Technical solutions enabling this technology can be found, for example, in U.S. Pat. Nos. 6,722,200, 7,302,856; 7,330,795; 7,552,645; 7,555,938; 7,617,736; 7,724,103; 8,044,556; 8,329,452, 8,350,578; 9,016,125.
These developments have been applied to ultra-sensitive mass detection of biomolecules as initially described in U.S. Pat. Nos. 6,722,200 and 8,227,747. They have recently culminated in mass resolution at the single-molecule level as shown in US2014/156224, where simple spectra have been assembled by statistical analysis from only a few hundred molecular adsorption events. One challenge for single-molecule analysis is the fact that the resonant frequency shift induced by analyte adsorption depends upon both the mass of the analyte and its precise location of adsorption upon the NEMS resonator. This may be solved by continuous tracking, that is, exciting and detecting two vibrational modes of the resonator. Analysing the different frequency shifts an analyte induces upon two vibrational modes permits both unknowns—mass and position-of-adsorption—to be deduced; this methodology is described in US2014/156224. A mass resolution of 50-100 kDa has already been demonstrated.
In contrast to conventional MS, the technology based on NEMS has sensitivity and resolving power that continues to improve with increasing analyte mass. This can prove useful for analysis of high-mass, intact, and native species.
In U.S. Pat. No. 9,347,815, a nanospray ion source and MS atmosphere-to-vacuum interface were used together to facilitate NEMS-based detection and mass analysis. An alternative method for analyte introduction was also demonstrated, involving the use of a matrix-assisted laser desorption and ionization source. Cooling the NEMS sensor enhances non-specific physical adsorption of the arriving analytes onto the surface of the devices. In U.S. Pat. No. 9,347,815 it is shown that a mass spectrum representing the entire heterogeneous sample can be constructed by individually measuring the mass of sequentially-arriving analyte particles.
In other words, in the described approach NEMS (nanoelectromechanical system) resonators are used for the detection of mass spectra, and the NEMS resonators are electronically controllable, submicron-scale mechanical resonators. Due to the adsorption of molecules a downshift of the resonant frequency of the device can be observed. Aspects of these technology are described in U.S. Pat. Nos. 6,722,200 and 8,227,747. US 2014/0156224 and WO2016/118821A1 describe further details of this technology. The mass of single molecules can be determined for any adsorption event of a molecule and mass spectra can be assembled by statistical analysis from a few hundred adsorption events. With the analysis of different frequency shifts in two vibrational modes for each molecule the mass of each molecule can be deduced. The investigated sample are ionized, e.g., by a nanospray ion source and transferred via an atmosphere-to-vacuum interface into a vacuum chamber, where they are adsorbed on a NEMS detector. Thus, a mass spectrum representing the entire heterogeneous sample can be constructed by individually measuring the mass of sequentially arriving analyte particles.
While the use of NEMS systems to analyze samples and to detect particles in a sample may be promising, there are still certain drawbacks and limitations related to the use of NEMS systems. While an individual NEMS sensor may in principle detect a single particle, the detection limit in NEMS systems may be a lot higher, i.e., one may need substantially more than one particle in a sample to detect the presence of such a particle. That is, when embedded in known NEMS systems, the sensitivity of the technology is far from optimal.
In light of the above, it is an object of the present invention to overcome or at least alleviate the shortcomings and disadvantages of the prior art. More particularly, it is an object of the present invention to provide a NEMS system, method and corresponding use being improved with regard to sensitivity and detection threshold.
These objects are met by the present invention.
In a first embodiment, the present invention relates to a system. The system is configured for analyzing particles. The system comprises a NEMS device comprising at least one NEMS sensor for detecting particles impacting the at least one NEMS sensor, each NEMS sensor comprising a NEMS sensor area. That is, the NEMS device comprises at least one NEMS sensor area. The system further comprises a particle lens assembly, the particle lens assembly comprising at least one particle lens for focusing particles onto a NEMS sensor area of the at least one NEMS sensor. The particle lens assembly is spaced from the at least one NEMS sensor area by a separation distance. Further, the system is configured to maintain a space defined between the particle lens assembly and the NEMS device at a pressure where a mean free path for a reference particle is greater than the separation distance.
It will be understood that the particles may be ions. It will further be understood that NEMS denotes nanoelectromechanical system.
That is, as discussed, in particular, the system comprises a particle lens assembly, e.g., an ion lens assembly. The ion lens assembly is typically configured and located to focus the ions onto each of the NEMS sensor areas and more particularly onto each of the NEMS target areas.
It has been found that the NEMS sensors only have a limited target area allowing for ideal detection of particles. Only if particles land on such a limited target area, will they be detected. Typically, the target area only is a fraction of the area of the NEMS sensor area, e.g., only a few percent or even less. If not focusing the particles onto the NEMS target area, the majority of the particles will not impact on the target area and will thus not be detected. By means of the proposed technology, it is possible to suitably focus the particles onto the NEMS target area. Thus, a substantially higher portion of the particles may impact on the NEMS target area, e.g., this may increase the fraction impacting onto the NEMS target area by one or more orders of magnitude. Thus, the sensitivity is substantially increased and the detection limit is substantially reduced.
Further, it will be understood that such a particle lens assembly is typically located before or “upstream” of the NEMS device, i.e., the particles are first guided through the particle lens assembly and then to the NEMS device. Further, the NEMS device and more particularly the NEMS sensor area is spaced from the particle lens assembly by a certain separation distance.
Furthermore, in the present system, the space defined between the particle lens assembly and the NEMS device may also be held at a reduced pressure fulfilling certain requirements. More particularly, the pressure may be chosen so that a mean free path of a reference particle is greater (and preferably substantially greater) than the separation distance.
The reference particle may be one particle of the class of particles to be analyzed. For example, when one intends to analyze proteins, the reference particle may be a protein and preferably a protein with a large size (and thus a comparatively low mean free path).
When reference is herein made to the mean free path, the momentum dissipation mean free path is meant, which can be calculated as
where
MFP is the mean free path;
M is the mass of the reference particle;
m is the mass of the gas particle;
n is the concentration of the gas; and
S is the collisional cross section of the reference particle with one particle of the gas.
It will be understood that the gas particle refers to the type of gas present in the space between the particle lens assembly and the NEMS device. An exemplary gas is air or N2. When the gas, which may also be referred to as the background gas, is known, the mean free path depends on the collisional cross section of the reference particle with the gas particle (i.e., S=π(rref+rgas)2 where rref is the radius of the reference particle and rgas is the radius of the gas particle (in the approximation of spherical shape of both types of particles) and further on the concentration and thus on the pressure of the gas.
The pressure is set so that the mean free path for the reference particle is larger and preferably substantially larger than the separation distance. Thus, it is ensured that (at least most of) the particles travel freely from the particle lens to the NEMS device and focusing follows laws of ion optics in vacuum. This further contributes to the particles being guided to the relatively small target area, as the particles do not collide with the gas particles and do thus not change their direction in response to such collisions.
Further, the reference particle will typically be chosen to be a large particle of the respective class of particles (e.g., a large protein in case the particles to be detected are proteins). This ensures that the mean free path is sufficiently long for all particles to be detected.
It has been found that the low sensitivity of known NEMS detectors is caused by an extremely small collection efficiency for incident particles, such as ions. The invention is addressing this problem by applying a particle lens assembly, e.g., an ion optical lens assembly in front of the NEMS detectors. The beam of the incident particles, e.g., ions may be splitted into narrow beamlets and focused on the NEMS detector area by positioning a microlens in front of each NEMS detector. More particularly, an array of microlenses may be arranged in front of an array of NEMS detectors. One parameter of the technology is the distance between the micro lenses and the NEMS detector surface, which may be only a few to a few tens of micrometers. This kind of focusing upon nanotechnical structures by micro lenses may be particularly advantageous.
In other words, embodiments of the invention may greatly increase a nanosensor's capture efficiency for analytes, in particular for charged analytes. An ion optical micro-lens, which may be fabricated by methods including nano/micro system technologies, may be placed in front of a nanoelectromechanical sensor (NEMS) for single-molecule measurements, thereby increasing the capture rate of ionized molecules incident from an external ion source.
In still other words, although NEMS-based inertial mass sensors have a good ability to directly detect the inertial mass of physisorbed analytes, they typically provide only an extremely small capture cross section for species incident upon them. For example, a biomolecular ion beam within quadrupole ion guides of a conventional mass spectrometer will have cross sectional diameter typically of order 1 mm. An individual NEMS sensor, with capture area of order several μm2, when placed in-line with such a source will only capture approximately one in 105 of the analytes incident upon it. However increasing the physical size of the devices generally results in decreased performance for inertial mass sensing. However, the proposed technology, by focusing the particles onto the NEMS target area, massively increases the portion of particles that can be analysed.
Generally, one concept discussed herein is that a greatly increased capture cross section can be realized by using a particle lens assembly, e.g., by using electrostatics: incoming analyte flux is deflected away from areas on the sensor that are insensitive and, instead, focused precisely upon active areas of the device that provide highest mass sensitivity. This can be accomplished by ion optics at the “sensor pixel” level—in effect, splitting the incoming broad beam into narrow beamlets, each focused by micro-lenses positioned in front of the NEMS sensor. According to one concept, one micro-lens per individual NEMS sensor is used. The micro-lenses can be fabricated with the processing steps of nano- and micro-systems technologies known in the art.
The particle lens assembly may include parallel particle lenses, such as parallel ion lenses. Such parallel particle lenses have already been described, e.g., in WO2007/055756, U.S. Pat. Nos. 7,772,564, 7,411,187, 6,762,406, 5,206,506, and U.S. Pat. No. 7,365,317.
The at least one NEMS sensor may be a plurality of NEMS sensors.
Thus, a plurality of measurements may be done in parallel, increasing the efficiency of the system.
The at least one particle lens may be a plurality of particle lenses and each particle lens may be configured and located to focus particles onto a NEMS sensor associated with the particle lens.
Each NEMS sensor of the at least one NEMS sensor may comprise a NEMS substrate, at least one NEMS resonator, and a circumferential section, wherein the NEMS substrate and the NEMS resonator are comprised by the NEMS sensor area.
Particles travelling from the particle lens assembly to the NEMS device may define a z-direction.
The circumferential section may be located in front of the NEMS substrate and the NEMS resonator in the z-direction and may be distanced from these elements by a height.
The height may be in the range of 0.5 μm to 6 μm, preferably 1 μm to 4 μm, such as 1.5 μm to 3.5 μm.
The NEMS sensor area may have an area, the area preferably being in the range of (2 μm)2 to (15 μm)2, further preferably (5 μm)2 to (11 μm)2, such as (8 μm)2 to (9 μm)2.
The NEMS sensors may be arranged in a NEMS array.
The NEMS array may comprise (2×2) to (1000×1000) NEMS sensors, preferably (3×3) to (100×100) NEMS sensors, such as (4×4) to (10×10) NEMS sensors.
This may allow for a suitable parallelization of simultaneous measurements.
The NEMS array may have an area in the range of (10 μm)2 to (1,000 μm)2, preferably (20 μm)2 to (300 μm)2, such as (50 μm)2 to (100 μm)2.
The particle lens assembly may be an electrostatic particle lens assembly and may contain a preliminary lens for the entire beam followed by a particle lens array corresponding to the NEMS array.
The electrode forming the particle lens array may comprise a circumferential portion of the preliminary lens and mesh portions.
The NEMS device may be separated from the particle lens assembly by a distance in the range of 1 μm to 20 μm, preferably 2 μm to 10 μm, such as 3 μm to 7 μm.
The preliminary particle lens assembly may have a length along the z-direction, which length preferably is in the range of 50 μm to 500 μm, further preferably 100 μm to 300 μm, such as 150 μm to 250 μm.
The electrodes may be formed of metal such as gold, molybdenum, gold-coated aluminum, etc.
The preliminary particle lens assembly may further comprise a insulating Si3N4 layer, wherein the Si3N4 layer is located between the NEMS device and the electrode closest to the NEMS device.
The Si3N4 layer may comprise a circumferential portion and mesh portions.
The thickness of the Si3N4 layer may be in the range of 20 nm to 300 nm, preferably 50 nm to 150 nm, such as 100 nm.
The electrodes of the preliminary lens may define an aperture, wherein, in a cross-sectional view, the aperture has a maximum extension and a minimum extension.
Thus, the particles coming from a previous stage may be focused from an initial maximum extension to a minimum extension and further towards the NEMS device.
The maximum extension of the preliminary lens may be in the range of 200 μm to 1,000 μm, preferably 300 μm to 500 μm, such as 400 μm to 450 μm, and wherein the minimum extension may be in the range of 20 μm to 200 μm, further preferably 40 μm to 150 μm, such as 50 μm to 80 μm.
The aperture may be successively reduced from the first to the last electrode, i.e., along the z-direction.
In a cross-sectional view, each electrode of the preliminary lens may be protruding, with respect to the preceding electrode, by a distance of 5 μm to 50 μm, preferably 10 μm to 30 μm, such as 15 μm to 25 μm.
The complete system may comprise 3 to 25, preferably 5 to 15, such as 8 to 12 electrodes.
Each electrode may comprise a thickness along the z-direction of 50 nm to 500 nm, preferably 100 nm to 300 nm, such as 150 nm to 250 nm.
The system may further comprise at least one insulator located between the electrodes.
The at least one insulator may be formed of parylene or SU8.
Each insulator may comprise a thickness along the z-direction of 5 μm to 50 μm, preferably 10 μm to 30 μm, such as 15 μm to 25 μm.
The shape of each insulator may correspond to an electrode in contact with the insulator.
The particle lens assembly may further comprise a support located between the NEMS device and the electrode closest to the NEMS device.
The support may have a length along the z-direction, wherein the length is in the range of 10 μm to 200 μm, preferably 50 μm to 250 μm, such as 100 μm.
The support may be formed of Si.
The support may widen along the z-direction.
The particle lens assembly may comprise a tapering electrode.
The tapering electrode may taper along the z-direction.
The tapering electrode may comprise an overall ring shape, i.e. the tapering may generate an overall conus shape.
The tapering electrode may comprise an overall square shape, i.e. the tapering may generate an overall frustum shape.
The tapering angle, with respect to the z-direction, of the tapering electrode may be in the range of 30° to 60°, preferably 40° to 50°, such as 45°.
All electrodes and all of the at least one insulator may comprise respective circumferential portions and respective mesh portions.
The electrodes may have the same shape and size in a plane perpendicular to the z-direction.
The system may comprise a forward electrode, located more forward in the z-direction than the electrode forming the particle lens array.
The forward electrode may comprise a length in the z-direction.
The length of the forward electrode may be in the range of 1 μm to 10 μm, preferably 1.5 μm to 5 μm, such as 3 μm.
The forward electrode may comprise a peripheral portion and mesh portions.
The peripheral portion of the forward electrode may taper in the z-direction at an angle in the range of 30° to 60°, preferably 40° to 50°, such as 45°.
The mesh portions, in a cross-sectional view, may be provided at an angle in the range of 30° to 60°, preferably 40° to 50°, such as 45°.
That is, in a cross-sectional view, the mesh portion follows a general V-shape.
The thickness of the insulator may be in the range of 0.1 μm to 5 μm, preferably 0.2 μm to 0.5 μm, such as 0.25 μm.
The electrodes may be supplied with different voltages.
The system may be configured to focus a particle beam onto the at least one NEMS sensor such that at least 50% of particles reaching the NEMS sensor impact the NEMS sensor in an area of (2 μm)2, preferably in an area of (1 μm)2, such as in an area of (0.5 μm)2.
Thus, a substantial portion of the particles may be focused onto the target area, substantially increasing the portion of particles that can be detected by the system. This may thus substantially increase the sensitivity and decrease the detection limit.
The system may be configured to focus a particle beam onto the at least one NEMS sensor such that at least 50% of particles reaching the NEMS sensor impact the NEMS sensor in an area that is smaller than 20% of a cross-sectional area of the particle lens assembly, preferably smaller than 10% of this cross-sectional area, most preferably smaller than 1% of this cross-sectional area.
The system may further comprise an ion source for ionizing the particles.
It may be particularly advantageous that the particles are ionized, as this may facilitate the focusing of the particles by means of an electrostatic particle lens assembly.
The system may further comprise an atmosphere-to-vacuum interface to transfer particles from atmospheric pressure to a reduced pressure.
The system may further comprise a first mass analyzer.
The first mass analyzer (20) is selected from a group consisting of an ion-trap mass analyzer (such as an Orbitrap™ analyzer, a cylindrical ion trap, or a quadrupole ion trap mass analyzer); an ion cyclotron resonance mass analyzer, e.g., a Fourier transform ion cyclotron resonance mass analyzer; a time-of-flight mass analyzer; an electrostatic trap mass analyzer; a quadrupole mass analyzer; a magnetic sector mass analyzer; and an electrostatic sector mass analyzer. Also ion mobility spectrometer of trapping or continuous-beam type could be used.
The system may further comprise a second mass analyzer.
The second mass analyzer may be selected from the group recited above.
The system may further comprise a third mass analyzer.
The third mass analyzer may be selected from the group recited above.
The system may also comprises at least one further, and preferably at least 3 further mass analyzers.
The system may further comprise an ion storage device such as a C-trap.
The system further may comprise a collision cell.
The atmosphere-to-vacuum interface may be located downstream of the ion source; the C-trap may be located downstream of the atmosphere-to-vacuum interface; the mass analyzer may be located downstream of the C-trap, thus defining a first branch downstream of the C-trap; the collision cell may be located downstream of the C-trap, thus defining a second branch downstream of the C-trap; and the particle lens assembly and the NEMS device may be located downstream of the collision cell.
The system may be configured to maintain the space defined between the particle lens assembly and the NEMS device at a pressure where a mean free path for the reference particle is more than 5 times greater than the separation distance, preferably more than 10 times, further preferably more than 50 times, and still further preferably more than 100 times.
Such mean free paths may be particularly advantageous so that particles will not collide with particles in the background gas after the lens assembly. This will again increase the portion of particles that are focused onto the target area.
The separation distance may be in the range of 1 μm to 20 μm, preferably 3 μm to 15 μm, such as 5 μm to 10 μm.
The quotient of the height of the NEMS sensor and its size across the sensor area (or to the square root of the area of the NEMS sensor area) may be in the range of 0.1 to 1, preferably 0.2 to 0.5, such as 0.3 to 0.5.
The quotient of the separation distance and the size of NEMS sensor across its area may be in the range of 0.3 to 50, preferably 0.5 to 2, such as 0.8 to 1.2.
The quotient of the separation distance and the square root of the area of the NEMS sensor area may be in the ranges defined in the preceding paragraph. This quotient may also be in the range of 0.5 to 20, such as 0.5 to 2.
The quotient of the maximum extension and the minimum extension may be in the range of 2 to 20, preferably 4 to 10, such as 5 to 8.
The quotient of the length of the particle lens assembly and the separation distance may be in the range of 2 to 50, preferably 10 to 40, such as 20 to 30.
The system may be configured to maintain the space defined between the particle lens assembly and the NEMS device at an absolute pressure not higher than 10−3 mbar, preferably not higher than 10−4 mbar, further preferably below 10−5 mbar, such as below 10−9 mbar. That is, some embodiments of the present technology may employ ultra high vacuum.
The system may further comprise an alignment mechanism configured to alter the position of the particle lens assembly relative to the NEMS device.
The alignment mechanism may have an accuracy below 2 μm, preferably below 1 μm, such as in the range of 0.01 μm to 1 μm.
The alignment mechanism may be configured to alter the position of the particle lens assembly relative to the NEMS device in 2 or 3 directions.
The alignment mechanism may comprise piezoelectric and/or mechanical translational elements.
Generally, the described technology may combine micro lenses with a hybrid instrument based on NEMS devices in combination with ion-optics based MS instrumentation.
The present invention also relates to a method to analyze particles, the method comprising utilizing the system according to any of the preceding embodiments.
The method may have advantages corresponding to the advantages discussed above with regard to the system.
The method may comprise focusing particles onto the at least one NEMS sensor by means of the particle lens assembly.
The method may comprise focusing particles onto each of the plurality of NEMS sensors by means of the plurality of particle lenses.
For each NEMS sensor, at least 50% of the particles may be focused onto an area of (2 μm)2, preferably onto an area of (1 μm)2, such as onto an area of (0.5 μm)2.
The focusing of particles may comprise at least 50% of particles reaching the NEMS sensor impacting the NEMS sensor in an area that is smaller than 20% of a cross-sectional area of the particle lens assembly, preferably smaller than 10% of this cross-sectional area, most preferably smaller than 1% of this cross-sectional area.
The method may comprise applying a first combination of voltages to the electrodes, resulting in a first focal point on the at least one NEMS sensor area; and applying a second combination of voltages to the electrodes, the second combination of voltages being different from the first combination of voltages, resulting in a second focal point on the at least one NEMS sensor area, the second focal point being spatially shifted relatively to the first focal point.
That is, the present method also allows the focal points to be changed and to be set by means of the voltages. As different modes of the NEMS pixels may have different ideal impact areas, this may result in still better results.
In a plane perpendicular to the z-direction, the first focal point and the second focal point may be separated by a distance between 0.1 μm and 10 μm, preferably 0.5 μm and 5 μm. First and second focal points might reside on the same NEMS sensor or they could belong to physically different NEMS sensors.
The method may comprise applying a voltage combination to the plurality of electrodes and thereby decreasing a velocity of particles along the z-direction.
The particles may be decelerated to an average velocity along the z-direction below 10,000 m/s, preferably below 5,000 m/s, such as below 1,000 m/s.
The particles may be decelerated to a kinetic energy which is smaller than 2,000 times the thermal energy per charge, preferably smaller than 1,000 times the thermal energy per charge, further preferably smaller than 500 times the thermal energy per charge, such as smaller than 100 times the thermal energy per charge.
The method may comprise applying a voltage to the electrodes and thereby removing particles located on the at least one NEMS sensor area.
That is, the present technology also allows particles that have impacted the NEMS sensor to be subsequently removed from the NEMS sensor and to be used in further detection techniques. In other words, one aspect of the present technology also relates to the use of the particle lens assembly (e.g., the microlens array). Here, by applying another voltage to the electrodes of the microlenses the investigated particles (e.g., molecules) can be desorbed from the NEMS detector by electrostatic desorption.
The method may comprise analyzing the particles by an additional instrument different from the NEMS device after removing the particles from the NEMS sensor.
The additional instrument may be a mass analyzer.
The method may comprise defining a reference particle; and maintaining the space defined between the particle lens assembly and the NEMS device at a pressure where a mean free path for the reference particle is greater than the separation distance.
The space defined between the particle lens assembly and the NEMS device may be maintained at a pressure where a mean free path for the reference particle is more than 5 times greater than the separation distance, preferably more than 10 times, further preferably more than 50 times, and still further preferably more than 100 times.
The method may further comprise altering the position of the particle lens assembly relative to the NEMS device.
The position of the particle lens assembly with regard to the NEMS device may be altered in 2 or 3 directions (i.e., dimensions).
Altering the position of the particle lens assembly relative to the NEMS device may result in an increase of particles per time reaching the NEMS sensor area.
The increase may be at least 10-fold, preferably at least 100-fold, such as at least 1,000-fold.
In other words, embodiments of the present technology thus also relate to calibrating the particle lens assembly relative to the NEMS device to maximize the number of particles incident on the “sweet spot” of the NEMS device.
The present invention also relates to a use of the discussed system for the discussed method.
In the use, the mass of particles may be determined on the basis of measurements by the NEMS device and measurements by the mass analyzer measurements.
The discussed system may be configured to carry out the discussed method accordingly.
That is, generally, the present technology relates to a system, a method and a corresponding use. The technology is for analyzing particles, such as molecules. The technology may comprise creating a stream of molecules, and analyzing said molecules using an electromechanical device at pressures substantially below atmospheric, so that said device measurably changes one of its characteristics upon adsorption of a single molecule to be analyzed. The capture efficiency of the incoming stream of molecules may be substantially improved by locating a microscale electrostatic lens, which focuses molecules onto the desired adsorption site on the device.
The molecules may be charged in an ion source. The ion source may utilize a electrospray.
An inner size of the particle lens assembly, e.g., the electric lens, may be at least a) 5, b) 10, or c) 50 times bigger than the size of the adsorption site. Again, this exemplifies how much the particles may be focused by the present technology.
The present invention is also defined by the following numbered embodiments.
Below, system embodiments will be discussed. System embodiments are abbreviated with the letter “S” followed by a number. Whenever reference is herein made to system embodiments, these embodiments are meant.
S1. A system (1) for analyzing particles, the system (1) comprising
It will be understood that the particles may be ions. It will further be understood that NEMS denotes nanoelectromechanical system.
S2. The system (1) according to the preceding embodiment, wherein the at least one NEMS sensor (104) is a plurality of NEMS sensors (104).
S3. The system (1) according to the preceding embodiment, wherein the at least one particle lens (2060) is a plurality of particle lenses (2060) and wherein each particle lens (2060) is configured and located to focus particles onto a NEMS sensor area (1044) associated with the particle lens (2060).
S4. The system (1) according to any of the preceding embodiments, wherein each NEMS sensor (104) of the at least one NEMS sensor (104) comprises
S5. The system (1) according to any of the preceding embodiments,
S6. The system according to the preceding embodiment and with the features of the penultimate embodiment, wherein the circumferential section (1043) is located in front of the NEMS substrate (1046) and the NEMS resonator (1048) in the z-direction and distanced from these elements by a height (H).
S7. The system (1) according to the preceding embodiment, wherein the height (H) is in the range of 0.5 μm to 6 μm, preferably 1 μm to 4 μm, such as 1.5 μm to 3.5 μm.
S8. The system (1) according to any of the preceding embodiments, wherein the NEMS sensor area (1044) has an area, the area preferably being in the range of (2 μm)2 to (15 μm)2, further preferably (5 μm)2 to (11 μm)2, such as (8 μm)2 to (9 μm)2.
S9. The system (1) according to any of the preceding embodiments and with the features of embodiment S2, wherein the NEMS sensors (104) are arranged in a NEMS array (105).
S10. The system according to the preceding embodiment, wherein the NEMS array (105) comprises (2×2) to (1000×1000) NEMS sensors, preferably (3×3) to (100×100) NEMS sensors, such as (4×4) to (10×10) NEMS sensors.
S11. The system according any of the 2 preceding embodiments, wherein the NEMS array (105) has an area in the range of (10 μm)2 to (1,000 μm)2, preferably (20 μm)2 to (300 μm)2, such as (50 μm)2 to (100 μm)2.
S12. The system according to any of the preceding embodiments, wherein the particle lens assembly (200) is an electrostatic particle lens assembly.
S13. The system according to any of the preceding embodiments, wherein the particle lens assembly (200) comprises an electrode system.
S14. The system according to the preceding embodiment, wherein the electrode system comprises a plurality of electrodes.
S15. The system according to any of the 2 preceding embodiments and with the features of embodiments S3 and S9, wherein the electrode system comprises an electrode (2328) forming a particle lens array (206) corresponding to the NEMS array (105).
S16. The system according to the preceding embodiment, wherein the electrode (2328) forming the particle lens array (206) comprises a circumferential portion (2330) and mesh portions (2332).
S17. The system according to any of the preceding embodiments, wherein the NEMS device (100) is separated from the particle lens assembly (200) by a distance (d1) in the range of 1 μm to 20 μm, preferably 2 μm to 10 μm, such as 3 μm to 7 μm.
S18. The system according to any of the preceding embodiments and with the features of embodiment S5, wherein the particle lens assembly (200) has a length along the z-direction, which length preferably is in the range of 50 μm to 500 μm, further preferably 100 μm to 300 μm, such as 150 μm to 250 μm.
S19. The system according to any of the preceding embodiments with the features of embodiment S14, wherein the electrodes are formed of metal such as gold, molybdenum, and/or gold-coated aluminum.
S20. The system according to any of the preceding embodiments with the features of embodiments S14, wherein the particle lens assembly (200) further comprises a Si3N4 layer, wherein the insulating Si3N4 layer is located between the NEMS device (100) and the electrode closest to the NEMS device.
S21. The system (1) according to the preceding embodiments, wherein the Si3N4 layer (2329) comprises a circumferential portion (2330) and mesh portions (2332).
S22. The system (1) according to any of the 2 preceding embodiments, wherein the thickness of the Si3N4 layer (2329) is in the range of 20 nm to 300 nm, preferably 50 nm to 150 nm, such as 100 nm.
S23. The system (1) according to any of the preceding embodiments with the features of embodiment S14, wherein the electrodes define an aperture, wherein, in a cross sectional view, the aperture has a maximum extension and a minimum extension.
S24. The system according to the preceding embodiment, wherein the maximum extension is in the range of 200 μm to 1,000 μm, preferably 300 μm to 500 μm, such as 400 μm to 450 μm, and preferably wherein the minimum extension is in the range of 20 μm to 200 μm, further preferably 40 μm to 150 μm, such as 50 μm to 80 μm.
S25. The system (1) according to the any of the 2 preceding embodiments and with the features of embodiment S5, wherein the aperture is successively reduced from the first to the last electrode (232), i.e., along the z-direction.
S26. The system (1) according to the preceding embodiment, wherein in a cross-sectional view, each electrode (232) is protruding, with respect to the preceding electrode (232), by a distance (D5) of 5 μm to 50 μm, preferably 10 μm to 30 μm, such as 15 μm to 25 μm.
S27. The system according to any of the preceding embodiments and with the features of embodiment S14, wherein the system comprises 3 to 25, preferably 5 to 15, such as 8 to 12 electrodes.
S28. The system according to any of the preceding embodiments with the features of embodiment S5 and S14, wherein each electrode comprises a thickness along the z-direction of 50 nm to 500 nm, preferably 100 nm to 300 nm, such as 150 nm to 250 nm.
S29. The system according to any of the preceding embodiments with the features of embodiment S14, wherein the system further comprises at least one insulator (234) located between the electrodes.
S30. The system according to the preceding embodiment, wherein the at least one insulator is formed of parylene or SU8.
S31. The system according to any of the 2 preceding embodiments and with the features of embodiment S5, wherein each insulator comprises a thickness along the z-direction of 5 μm to 50 μm, preferably 10 μm to 30 μm, such as 15 μm to 25 μm.
S32. The system according to any of the 3 preceding embodiments, wherein the shape of each insulator corresponds to an electrode in contact with the insulator.
S33. The system (1) according to any of the preceding embodiments with the features of embodiment S14, wherein the particle lens assembly (200) further comprises a support (240) located between the NEMS device (100) and the electrode closest to the NEMS device.
S34. The system (1) according to the preceding embodiment and with the features of embodiment S5, wherein the support (240) has a length (d6) along the z-direction, wherein the length (d6) is in the range of 10 μm to 200 μm, preferably 50 μm to 250 μm, such as 100 μm.
S35. The system according to any of the 2 preceding embodiments, wherein the support (240) is formed of Si.
S36. The system according to any of the 3 preceding embodiments and with the features of embodiment S5, wherein the support (240) widens along the z-direction.
S37. The system (1) according to any of the preceding embodiments with the features of embodiment S14, wherein the particle lens assembly (200) comprises a tapering electrode (2327).
S38. The system (1) according to the preceding embodiment and with the features of embodiment S5, wherein the tapering electrode (2327) tapers along the z-direction.
S39. The system (1) according to the preceding embodiment, wherein the tapering electrode (2327) comprises an overall ring shape, i.e. the tapering generates an overall conus shape.
S40. The system (1) according to the penultimate embodiment, wherein the tapering electrode (2327) comprises an overall square shape, i.e. the tapering generates an overall frustum shape.
S41. The system according to the 4 preceding embodiments, wherein the tapering angle, with respect to the z-direction, of the tapering electrode (2327) is in the range of 30° to 60°, preferably 40° to 50°, such as 45°.
S42. The system (1) according to embodiment S16 and including the features of embodiment S14 and S29, wherein all electrodes (232) and all of the at least one insulator (234) comprise respective circumferential portions (2330) and respective mesh portions (2332).
S43. The system (1) according to the preceding embodiment and with the features of embodiment S5, wherein the electrodes (232) have the same shape and size in a plane perpendicular to the z-direction.
S44. The system (1) according to any of the preceding embodiments and with the features of embodiments S5 and S15, wherein the system (1) comprises a forward electrode (2335), located more forward in the z-direction than the electrode (2328) forming the particle lens array (206).
S45. The system (1) according to the preceding embodiment, wherein the forward electrode (2328) comprises a length (d7) in the z-direction.
S46. The system (1) according to the preceding embodiment, wherein the length (d7) of the forward electrode (2328) is in the range of 1 μm to 10 μm, preferably 1.5 μm to 5 μm, such as 3 μm.
S47. The system (1) according to the preceding embodiment, wherein the forward electrode (2328) comprises a peripheral portion (2337) and mesh portions (2339).
S48. The system (1) according to the preceding embodiment, wherein the peripheral portion (2337) of the forward electrode (2335) tapers in the z-direction at an angle in the range of 30° to 60°, preferably 40° to 50°, such as 45°.
S49. The system (1) according to any of the two preceding embodiments, wherein the mesh portions (2339), in a cross-sectional view, are provided at an angle in the range of 30° to 60°, preferably 40° to 50°, such as 45°.
That is, in a cross-sectional view, the mesh portion (2339) follows a general V-shape.
S50. The system (1) according to the preceding embodiment and including the features of embodiments S29, wherein the thickness of the insulator (234) is in the range of 0.1 μm to 5 μm, preferably 0.2 μm to 0.5 μm, such as 0.25 μm.
S51. The system (1) according to any of the preceding embodiments with the features of embodiment S14, wherein the electrodes (232) are supplied with different voltages.
S52. The system (1) according to any of the preceding embodiments, wherein the system (1) is configured to focus a particle beam onto the at least one NEMS sensor (104) such that at least 50% of particles reaching the NEMS sensor impact the NEMS sensor (104) in an area of (2 μm)2, preferably in an area of (1 μm)2, such as in an area of (0.5 μm)2.
S53. The system (1) according to any of the preceding embodiments, wherein the system (1) is configured to focus a particle beam onto the at least one NEMS sensor (104) such that at least 50% of particles reaching the NEMS sensor impact the NEMS sensor (104) in an area that is smaller than 20% of a cross-sectional area of the particle lens assembly (200), preferably smaller than 10% of this cross-sectional area, most preferably smaller than 1% of this cross-sectional area.
S54. The system (1) according to any of the preceding embodiments, wherein the system (1) further comprises an ion source (2) for ionizing the particles.
S55. The system (1) according to any of the preceding embodiments, wherein the system further comprises an atmosphere-to-vacuum interface (30) to transfer particles from atmospheric pressure to a reduced pressure.
S56. The system (1) according to any of the preceding embodiments, wherein the system further comprises a first mass analyzer (20).
S57. The system according to the preceding embodiment, wherein the first mass analyzer (20) is selected from a group consisting of
S58. The system according to any of the 2 preceding embodiments, wherein the system further comprises a second mass analyzer.
S59. The system according to the preceding embodiment, wherein the second mass analyzer is selected from the group recited in the penultimate embodiment.
S60. The system according to any of the 2 preceding embodiments, wherein the system further comprises a third mass analyzer.
S61. The system according to the preceding embodiment, wherein the third mass analyzer is selected from the group recited in S57.
S62. The system according to any of the 2 preceding embodiment, wherein the system comprises at least one further, and preferably at least 3 further mass analyzers.
S63. The system (1) according to any of the preceding embodiments, wherein the system further comprises an ion storage device, such as a C-trap (14).
S64. The system (1) according to any of the preceding embodiments, wherein the system further comprises a collision cell (16).
S65. The system according to any of the preceding embodiments and with the features embodiments S54, S55, S56, S63, and S64, wherein
S66. The system according to any of the preceding embodiments, wherein the system is configured to maintain the space defined between the particle lens assembly (200) and the NEMS device (100) at a pressure where a mean free path for a reference particle is more than 5 times greater than the separation distance (D), preferably more than 10 times, further preferably more than 50 times, and still further preferably more than 100 times.
S67. The system according to any of the preceding embodiments, wherein the separation distance (D) is in the range of 2 μm to 20 μm, preferably 3 μm to 15 μm, such as 5 μm to 10 μm.
S68. The system according to any of the preceding embodiments with the features of embodiments S6 and S8, wherein the quotient of the area of the NEMS sensor area (1044) and the height (H) is in the range of 5 μm to 50 μm, preferably 10 μm to 30 μm, such as 15 μm to 20 μm.
S69. The system according to any of the preceding embodiments with the features of embodiments S8, wherein the quotient of the area of the NEMS sensor area (1044) and the separation distance (D) is in the range of 2 μm to 20 μm, preferably 3 μm to 15 μm, such as 5 μm to 10 μm.
S70. The system according to any of the preceding embodiments and with the features of embodiment S23, wherein the quotient of the maximum extension and the minimum extension is in the range of 2 to 20, preferably 4 to 10, such as 5 to 8.
S71. The system according to any of the preceding embodiments with the features of embodiment S18, wherein the quotient of the length of the particle lens assembly (200) and the separation distance is in the range of 2 to 50, preferably 10 to 40, such as 20 to 30.
S72. The system according to any of the preceding embodiments, wherein the separation distance (D) is in the range of 1 μm to 20 μm, preferably 2 μm to 15 μm, such as 5 μm to 10 μm.
S73. The system according to any of the preceding embodiments, wherein the system is configured to maintain the space defined between the particle lens assembly (200) and the NEMS device (100) at an absolute pressure in the range of not higher than 10−3, preferably not higher than 10−4 mbar, further preferably below 10−5 mbar, most preferably below 10−9 mbar.
S74. The system according to any of the preceding embodiments, wherein the system further comprises
S75. The system according to the preceding embodiments, wherein the alignment mechanism has an accuracy below 2 μm, preferably below 1 μm, such as in the range of 0.01 μm to 1 μm.
S76. The system according to any of the 2 preceding embodiments, wherein the alignment mechanism is configured to alter the position of the particle lens assembly (200) relative to the NEMS device (100) in 2 or 3 directions.
S77. The system according to any of the 3 preceding embodiments, wherein the alignment mechanism comprises piezoelectric and/or mechanical translational elements.
Below, method embodiments will be discussed. Method embodiments are abbreviated with the letter “M” followed by a number. Whenever reference is herein made to method embodiments, these embodiments are meant.
M1. A method to analyze particles, the method comprising utilizing the system (1) according to any of the preceding embodiments.
M2. The method according to the preceding embodiment, wherein the method comprises
M3. The method according to any of the preceding method embodiments, wherein the system (1) comprises the features of embodiment S3, the method further comprising
M4. The method according to any of the preceding method embodiments, wherein, for each NEMS sensor (104), at least 50% of the particles are focused onto an area of (2 μm)2, preferably onto an area of (1 μm)2, such as onto an area of (0.5 μm)2.
M5. The method according to any of the preceding method embodiments with the features of embodiment M2, wherein the focusing of particles comprises at least 50% of particles reaching the NEMS sensor impacting the NEMS sensor (104) in an area that is smaller than 20% of a cross-sectional area of the particle lens assembly (200), preferably smaller than 10% of this cross-sectional area, most preferably smaller than 1% of this cross-sectional area.
M6. The method according to any of the preceding method embodiments, wherein the system comprises the features of embodiment S14, the method comprising
M7. The method according to the preceding embodiment, wherein the system comprises the features of embodiment S5, wherein in a plane perpendicular to the z-direction, the first focal point and the second focal point are separated by a distance between 0.1 μm and 10 μm, preferably 0.5 μm and 5 μm.
M8. The method according to any of the preceding method embodiments, wherein the system comprises the features of embodiment S5 and S14, the method further comprising applying a voltage combination to the plurality of electrodes and thereby decreasing a velocity of particles along the z-direction.
M9. The method according to the preceding embodiment, wherein the particles are decelerated to an average velocity along the z-direction below 10,000 m/s, preferably below 5,000 m/s, such as below 1,000 m/s.
M10. The method according to any of the 2 preceding embodiments, wherein the particles are decelerated to a kinetic energy which is smaller than 2,000 times the thermal energy per charge, preferably smaller than 1,000 times the thermal energy per charge, further preferably smaller than 500 times the thermal energy per charge, such as smaller than 100 times the thermal energy per charge.
M11. The method according to any of the preceding method embodiments, wherein the system comprises the features of embodiment S14, the method further comprising
M12. The method according to the preceding embodiment, wherein the method further comprises analyzing the particles by an additional instrument different from the NEMS device (100) after removing the particles from the NEMS sensor.
M13. The method according to the preceding embodiment, wherein the additional instrument is a mass analyzer.
M14. The method according to any of the preceding embodiments, wherein the method comprises
M15. The method according to the preceding embodiment, wherein the space defined between the particle lens assembly (200) and the NEMS device (100) is maintained at a pressure where a mean free path for the reference particle is more than 5 times greater than the separation distance (D), preferably more than 10 times, further preferably more than 50 times, and still further preferably more than 100 times.
M16. The method according to any of the preceding method embodiments, wherein the system comprises the features of embodiment S74, wherein the method further comprises
M17. The method according to the preceding embodiment, wherein the system comprises the features of embodiment S76, wherein the position of the particle lens assembly (200) with regard to the NEMS device (100) is altered in 2 or 3 directions.
M18. The method according to any of the 2 preceding embodiments, wherein altering the position of the particle lens assembly (200) relative to the NEMS device (100) results in an increase of particles per time reaching the NEMS sensor area (1044).
M19. The method according to the preceding embodiment, wherein the increase is at least 10-fold, preferably at least 100-fold, such as at least 1,000-fold.
Below, use embodiments will be discussed. Use embodiments are abbreviated with the letter “U” followed by a number. Whenever reference is herein made to use embodiments, these embodiments are meant.
U1. Use of the system according to any of the preceding system embodiments for the method according to any of the preceding method embodiments.
U2. Use of the system according to the preceding embodiment, wherein the system comprises the features of embodiment S56, wherein mass of particles is determined on the basis of measurements by the NEMS device and measurements by the mass analyzer.
S74. The system according to any of the preceding system embodiments, wherein the system is configured for the method according to any of the preceding method embodiments.
The present technology will now be described with reference to the accompanying drawings.
The ion source 2 may be located at atmospheric pressure. The ion source 2 may be of electrospray type. In some embodiments, the ion source 2 may be configured for atmospheric pressure ionization (API) and may thus also be referred to as an API source 2. It will be appreciated that other ion sources could be used. For example, the present technology could also be used for analysis of ions produced by matrix-assisted laser desorption/ionization (MALDI), laserspray or any other inlet ionization, or indeed any other techniques capable of producing high-m/z ions.
Ions from the electrospray pass through the atmosphere-to-vacuum interface 30. More particularly, the ions pass through a transfer capillary 3 to a stacked ring ion guide (S-lens) or ion funnel 4, through its exit lens 5, and then through an injection flatapole 6, an inter-flatapole lens 7, and a bent flatapole 8. The pressure in the region of the S-lens 4 to bent flatapole 8 is typically 1-10 mbar (e.g. 1.6 mbar). The bent flatapole 8 may have 2 mm gaps between its rods. Collisional cooling occurs in the bent flatapole 8 at pressures of 10−3 to 10−2 mbar.
In some embodiments, the system 1 may also comprise other elements, e.g., ion gate 10, quadrupole mass filter 12, an ion storage device (e.g., a C-trap 14), Z-lens 15, mass analyzer 20, and collision cell 16, details of which are also described in US2014/027629A1, which is incorporated by reference herein in its entirety. Such elements may allow the system 1 to also analyze particles by means of the mass analyzer 20, which may be, e.g., an Orbitrap™ mass analyzer.
In some embodiments, the system 1 may be based on an Q Exactive™ Plus instrument (Thermo Fisher Scientific (Bremen), Bremen, Germany) and may utilize an electrostatic trap in the form described in WO96/30930, which is realized in the Orbitrap™ mass analyzer provided by ThermoFisher Scientific Inc., Waltham, USA and its affiliates. In such embodiments, an ion gate 10 in the form of a fast split lens controls the entry of the ions into an RF-only transport multipole 12, which may be also replaced by a quadrupole mass filter and typically is held at a pressure less than 10−4 mbar. From the transport multipole the ions enter a C-trap 14 typically with a pressure therein of (0.1-4.0)×10−3 mbar (for example 0.5×10−3 mbar). For further cooling, the ions may be passed into a collision cell 16, e.g., a gas-filled HCD cell 16 comprising RF multipole rods typically with a pressure of (1-20)×10−3 mbar (e.g. 5×10−3 mbar). The HCD cell 16 may be separated from the C-trap 14 by a single diaphragm, which allows easy tuning of the HCD cell.
In some embodiments, the RF and axial field applied to the HCD cell 16 can be set to provide for fragmentation of ions therein. The HCD cell 16 allows better trapping while maintaining a certain pressure in the C-trap 14 and thus the Orbitrap™ mass analyzer, because the HCD cell 16 is i) longer and ii) at a higher pressure than the C-trap 14. Ions are injected from the C-trap 14 via a Z-lens 15 into the Orbitrap™ mass analyzer 20. The vacuum in the Orbitrap compartment is preferably below 7×10−10 mbar although it is dependent on the pressure in the HCD cell 16. For some large proteins, pressures in excess of 2×10−9 mbar could be used
That is, in some embodiments, ions may be produced by an ion source 2 (e.g., a nanospray source) at atmospheric pressure preferably at native conditions, and are then transported through atmosphere-to-vacuum interface 30 into vacuum. Optionally, the ions may be mass selected by a multipole mass filter 12 (e.g., a quadrupole mass filter), and then introduced into a mass-to-charge analysis system, where a mass-to-charge spectrum is acquired by mass spectrometer as is known in the art. In the example illustrated in
While in the above, an embodiment comprising a mass spectrometer 20 has been described, it will be understood that such a mass spectrometer 20 may be independent from the other features described below, and that other embodiments may not comprise such a mass spectrometer 20.
In general words, the exemplary system 1 may comprise an atmospheric-pressure ion source 2 and an atmosphere-to-vacuum interface 30. The atmosphere-to-vacuum interface 30 may comprise a capillary 3, an S-lens 4, an S-lens exit lens 5, an injection flatapole 6, an inter-flatapole lens 7, and a bent flatapole 8. Further, the system 1 may comprise an ion gate 10, a multipole mass filter 12 (e.g., a quadrupole mass filter), and an ion storage device, such as a C-trap 14. Further still, the system 1 may also comprise an MS analyser 20 (e.g., an Orbitrap™ mass analyzer). In addition to these components, the system according to the depicted embodiments of the present technology also comprises a NEMS device 100 in a differentially-pumped chamber behind the collision cell 16. By applying voltages to apertures of the C-trap 14, one may control the flow of particles to the MS analyser 20 and/or to the collision cell 16 and the NEMS device 100 containing one NEMS array.
The NEMS resonator 1048 defines a NEMS effective area 1050, which may be approximately 2.5 μm×2.5 μm (it is noted that
It will be understood that the exact preferred landing area or sweet spot 1052 may typically vary in dependence of the mode of the NEMS that is to be changed by the particle. In that regard, reference can be made to U.S. Pat. No. 8,227,747 B2 and particularly
It will be understood that particles that fly towards the NEMS sensor 104 depicted in
Further, considering a NEMS die 103 (corresponding to a NEMS sensor 100) in
Each NEMS sensor area 104 has a NEMS detection area 1052 of approximately 10−12 m2. Thus, the NEMS well array 105 has an NEMS well array effective area of approximately 25×10−12 m2. The same applies for an individual NEMS die 103 (or NEMS sensor 100), as there is one NEMS well array 105 per NEMS die 103 (or NEMS sensor 100). Further, the NEMS die 103 (or NEMS device 100) has an overall area of 25×10−6 m2.
The ratio between the NEMS well effective area and the NEMS device is 10−6. It should be understood that other configurations resulting in different ratios are also possible. For example, it is also possible that the NEMS sensors 104 are packed more densely, side by side. The fill ratio will then be limited by the fill ratio of an individual sensor. Each NEMS sensor has area 104 of approximately 25×10−12 m2 and a NEMS detection area 1052 of approximately 10−12 m2. Therefore, the fill ratio may also be 1/25=4%, or, more generally in the range of 0.5% up to 5%, preferably in a range of 1% up to 4.5%.
Further, it has been found that particles (e.g., ions) landing on the oxide/metallization section 1043 may in fact distort the particle current and thus are therefore considered undesirable, while particles (e.g., ions) passing through the substrate 1046 cannot be investigated (and are thus “wasted”), but do not distort the particle current.
Embodiments of the present technology therefore seek to concentrate the particles (e.g., ions) onto the NEMS detection area 1052 in each NEMS well (a NEMS well being defined by an individual NEMS sensor 104 and the associated section of the membrane 1030). This is very conceptually illustrated in
Reference is again made to the NEMS device 100 as depicted in
In this regard, it is also noted that the spot of the external ion beam generated by the ion source 2 may have a diameter from many tens to hundreds of micrometers even after focusing by traditional ion optical lenses, which is substantially larger than the dimensions of the NEMS detection area 1052, which is approximately 1 (μm)2. That is, the ion beam has to be focused and to be guided towards the NEMS detection area 1052. Following this rationale and assuming the best-case extension of the ion beam of 100 μm×100 μm, it will be understood that only approximately 1 in 10,000 ions would normally land on the NEMS detection area 1052.
Again with reference to
With particular reference to the embodiment of
As discussed, the preferred NEMS detection area 1052 typically has a capture area in the range of one square micrometer. To increase the yield of particles, such as ions, captured and analyzed by an individual NEMS pixel 1044 (also referred to as a NEMS sensor pixel 1044) in the NEMS array 100, the present technology directs or focuses the incoming ion flux onto the preferred detection area 1052 of the NEMS device 100. This can be realized by an electrostatic micro-lens assembly 200 positioned in front (preferably directly in front) of each of the NEMS sensor pixels, as is further illustrated in
A separation distance D between the micro lenses 202 and sensor surface of the NEMS sensor 104, i.e., the NEMS sensor area 1044, will be only a few to a few tens of micrometers, dimensions that are typically comparable with both the internal size of the micro-lens elements and the dimensions of the NEMS pixel itself. Thus, precise alignment between the NEMS sensor 104 (and thus, also the complete NEMS device 100) and the micro lens 202 (and thus, also the lens assembly 200) may be advantageous to permit the concentration of ions onto the active “sensing” region on each NEMS pixel 1044—and to avoid landing of ions onto inactive areas of the sensor, especially the dielectric surfaces, e.g., the oxide/metallization section 1043 (which can accumulate charge and cause distortion of the focusing fields).
It will be understood that particles travelling from the micro lens assembly 200 to the NEMS sensor area 1044 may generally collide with gas particles present between the micro lens assembly 200 and the NEMS sensor area 1044, and that this may impact the collection and therefore detection of particles (as the particles may no longer be on their focussed trajectory after such collisions). To reduce such defocusing due to collisions, a reduced pressure may be present between the micro lens assembly 200 and the NEMS sensor 100. The reduced pressure may allow the particles to be analysed to travel relatively freely from the micro lens assembly 200 to the NEMS sensor 100.
More particularly, a reference particle may be defined, the reference particle being representative for the class of particles to be analysed. For example, if it is intended to analyse proteins, a reference protein may be defined. Typically, the reference particle would be an extreme case of the class, i.e., a large particle of the class.
By setting the pressure in the space between the micro lens assembly 200 and the NEMS sensor 104, one can also set a free mean path for the reference particle. It will be understood that the free mean path typically depends on the reference particle, the gas present in the space between the micro lens assembly 200 and the NEMS sensor 104 and the concentration of said gas. More particularly, when reference is herein made to the mean free path, the momentum dissipation mean free path is meant, which can be calculated as
where
MFP is the mean free path;
M is the mass of the reference particle;
m is the mass of the gas particle;
n is the concentration of the gas; and
S is the collisional cross section of the reference particle with one particle of the gas.
It will be understood that the collisional cross section is S=π(rref+rgas)2, where rref is the radius of the reference particle and rgas is the radius of the gas particle in the approximation of spherical shape for both particles. As the radius of the reference particle rref may typically be much greater than the radius of the gas particle rgas, the collisional cross section is typically much greater than the cross-section of the gas molecule.
It will be understood that once the reference particle and the gas (which may also be referred to as the background gas) are known, the mean free path mostly depends on the concentration of the gas, and thus its pressure.
Generally, in embodiments of the present technology, the pressure in the space between the micro lens assembly 200 and the NEMS sensor 104 is set so that the mean free path is greater (and preferably substantially greater) than the separation distance D, i.e., the way the particles have to travel from the micro lens 2002 to the NEMS sensor area 1044. This may ensure that the impact location of the particles is not strongly affected by any collisions of the particles with the background gas.
It will further be understood that residual misalignment and irreproducibility of micro lens shape may further require individual fine adjustment of alignment between axis of the lens and desired ion deposition spot. This could be facilitated by splitting the lens into several segments, e.g. 2-4, with adjustable voltages applied across the cross-section of the lens to steer the ion beam.
As the accuracy of mutual positioning of lenses and NEMS sensors within each array may be very high (e.g., better than 1 micron), it may be advantageous to ensure that the lens array and the NEMS array (or NEMS device) are aligned to each other with a similar precision. Such an alignment may be provided by additional piezoelectric or mechanical translational elements, and a calibration procedure may be used to activate these elements and shift lens array relatively to NEMS array in 2 or 3 directions until optimum alignment is reached. For example, the calibration procedure could be tuned to maximize detected ion particles from the “sweet spot” of detection of each NEMS sensor.
Such adjustment also allows flexibility in design: any layout of NEMS sensors could be accommodated, whether 1 or many resonators per sensor, in-center or off-center zones of preferable deposition, etc. Particular settings of steering could be optimized for each microlens individually during the initial calibration process that takes place at the initial installation and then possibly at some intervals as appropriate for a particular application. Further, there may be several NEMS devices served by one micro-lens.
Manufacturing of the micro-lenses 202 could be realized by micro- or nano-systems fabrication technologies, either co-integrated with the NEMS device 100, thus enabling precise alignment of the lens assembly 200 in front of the NEMS device 100, or fabricated as a separate structure and assembled in a controlled post-process step using specially-designed guides to insure proper alignment of the separate components. It may be advantageous for operation of both lens 202 and NEMS device 100 that a mean free path of residual gas around them is larger, and preferably substantially larger than the largest characteristic dimension of either lens 202 or individual NEMS sensor 104, preferably more than 104 to 105 times larger.
Correspondingly,
In
Once the particle has surpassed the longitudinal center 220′ of the micro lens 202, the electric field along the x-direction generally corresponds to the one depicted in
Thus, an arrangement as depicted in
The micro lens assembly or micro lens module 200 in
The electrodes 232 may be formed of gold, molybedenum, gold-coated aluminum, etc. and/or they may have a thickness of approximately 200 nm. They could be thickened by electroplating.
The electrodes 232 may be separated by insulators 234. The shape of the insulators 234 may correspond to the shape of the electrodes 232. E.g., the electrodes 232 (except for the last or rear electrode 2328) and insulators 234 may form pairs, and the electrode 232 and the respective insulator 234 of one pair may have corresponding shapes, particularly aperture sizes. The insulators 234 may have a thickness D4 of approximately 20 μm. The insulators may be formed of parylene or SU8.
The last or rear electrode 2328 may be followed by a layer 2329 of Si3N4.
In the embodiment depicted in
It will be understood that the electrodes 232 may be supplied with different voltages, e.g., the last electrode 2328 may be supplied with a last electrode voltage V1, and the second last electrode 2326 may with a second last electrode voltage V2.
As also discussed in connection with
As discussed, the last electrode 2328 may not only comprise a circumferential portion 2330, but also the web portions 2332, such that the apertures of the last electrode correspond to the apertures in the membrane 1030 of the NEMS array 105. Thus, the setup depicted in
The micro lenses and micro lens assemblies 200 may have different ion optical shapes as is further shown in
Throughout
The micro-lens assembly 200a in
In other words, the embodiment depicted in
This allows to locate ions into areas 1048 of NEMS sensor that are especially favourable for multi-physics measurements (e.g. shape and size in addition to mass) using multiple harmonics of sensor oscillations as described in WO2016/118821A1.
The embodiment depicted in
In this regard, it is noted that rearward and forward as used herein denote orders of elements along the z-direction. With general reference to
The Si-support 240 may have a length d6 of approximately 100 μm along the z-direction. Further, the Si-support 240 may taper towards the electrode 2328 and the Si3N4 layer 2329, i.e., it may have an aperture increasing with increasing distance from the electrode 2328 and the SiN Si3N4 layer 2329. The provision of the Si support 240 may simplify the construction of the system 1.
Further, in the cross-sectional view of
Again, the rear electrode 2328 may be at a first voltage or electric potential V1 and the forward electrode 2355 may be at a second voltage or electric potential V2, different from the first electric potential V1. The peripheral portions and the web portions (of the forward electrode 2335, the rear electrode 2328, the Si3N4 layer 2329, and the insulator 234) again define apertures acting as individual lenses for particles, such as ions. Due to the tapering of the peripheral portion 2357 of the forward electrode 2355 and the V-shape of the web portions 2359 of the forward electrode, the individual apertures (“the lenses”) may also be referred to as conical lenses. Again, this design may be used to focus particles, such as ions onto the individual NEMS sensors 104, and more particularly onto the NEMS detection area 1052 of the individual NEMS sensors 104.
That is, generally, the present technology allows focusing of the ion beam onto the NEMS sensor 104 at the end of a transfer line with a beam cross-sectional profile that is optimized to the size of the “receiving” sensor array.
That is, with reference to
For analysing the particles, the system 1 further comprises a NEMS device 100 comprising a NEMS array 105 of NEMS sensors 104 (see, e.g.,
Further, the system 1 also comprises a micro lens assembly 200 for focusing the particles (e.g., ions) onto the NEMS pixels 1044, particularly onto the preferred NEMS detection areas 1052. The micro lens assembly 200 forms a plurality of micro lenses, and generally one micro lens per NEMS sensor 104. Thus, the micro lens assembly 200 may focus the particles onto the individual NEMS pixels 1044, and thus, the system 1 may be more effectively used to analyse particles by means of a NEMS device 100.
More particularly, in embodiments where an individual NEMS pixel 1044 comprises a plurality of NEMS resonators 1048 (e.g., two NEMS resonators 1048 as depicted in
In the above, it has been described that a micro lens assembly 200 comprising a plurality of micro lenses 202 can be used to focus particles (such as ions) onto the preferred NEMS detection areas 1052. This may allow a far more sensitive detection of particles.
In some embodiments, the present technology may also be used to desorb (charged) molecules from the NEMS sensor, which may also be referred to as a NEMS sensor 104. The molecules may undergo “soft landing” upon a NEMS resonator 1048, may be desorbed and transported back into the MS instrument 20 for subsequent analysis by the MS instrument 20, including dissociative analysis protocols. Here, “soft landing” means adsorption onto the NEMS pixel 1044 where important attributes of the incoming analyte are preserved; such attributes may include the analyte's charge state, its primary molecular structure (i.e. by adsorption of the intact species, avoiding its fragmentation), and its higher-order structure (i.e., by avoiding denaturing of the protein upon adsorption.) To achieve this, the lens assembly 200 may switch between different potentials to provide electrostatic focusing and analyte deceleration upon deposition (to mitigate the effects of impact upon adsorption) versus optimal acceleration and focusing towards mass spectrometer following desorption experiments.
As regards the basic rationale for deceleration of ions, reference can be made to
Further, after a particle has been deposited on the NEMS sensor 104, the lens assembly 200 may also be used to generate an electric field to desorb the particle from the NEMS sensor 104. It will be understood that there may generally be a force that needs to be overcome in order to separate a particle from the NEMS sensor 104, i.e., to “desorb” a particle from the NEMS sensor 104, and that this force may be provided by an electric potential gradient generated by the micro lens assembly 200. Further rationales of this will be provided below.
If wanting to remove a particle 2000 that is deposited on a surface 2002, one needs to overcome the adhesion force Fad between these two. Applying the Johnson-Kendall-Roberts (JKR) model of the contact between the particle 2000 and the surface 2002, the adhesion force Fad can be calculated as follows:
See, Israelachvili, J. N. Intermolecular and surface forces: revised third edition (2011), and herein equation (12.10), where R is the radius of the particle and γs is the energy per unit area. It is noted that equation (12.10) in this book relates to two spheres, but the above equation can be derived from equation (12.10) in this book when considering the flat surface to be a sphere with an infinite radius. The particle size is assumed to be approximately 15 nm and γs is assumed to be between 5 to 150 mN/m. The latter assumption is based on assuming that the interaction between the surface and the particles is dominated by van-der-Waals forces (see the above book and Ruz Tamayo Pini et al. Physics of Nanomechanical Spectrometry of Viruses. Sci Reports 2014, in that regard). Thus, the force required to absorb a particle from a NEMS sensor 104 can be estimated to be between 0.7 and 21 nN.
The mechanical force from routine NEMS operation can be expressed as follows:
F
mech
=ma
0ω2 sin(ωt)
Fmech,max≈(800 kDa)·(50 nm)·(2π·20 MHz)2=0.001 nN
Herein, the following notation was used:
m: mass of the particle;
a: acceleration;
s: location;
a0: amplitude of the oscillation; and
ω: oscillation frequency.
For the estimation, typical values for these parameters were used.
An electric field needed for removing particles (of a charge z=70) from the surface can thus be estimated as follows:
A very strong electric field generated by a micro lens 202 is determined by simulating such fields, as was done in
More particularly,
Thus, the z component of the electric field is also changed. In
That is, in the vicinity of the surface 2002 (corresponding to the NEMS sensor), the electric field has approximately the following z-components, which is able to desorb particles from a NEMS senor area 1044:
Again, the z-component of the resulting electric field is depicted by electric field lines at corresponding values. In the vicinity of the surface 2002, the following approximate z-components of the electric fields apply:
It will be understood that such values for the electric field may be too high for long-term operation and therefore could be applied only for a short time as nanosecond-long pulses. Such high values of field strengths might be reduced by other means to reduce adhesion, e.g. IR laser for heating or usage of sacrificial layer on the NEMS device with much lower adhesion than ions of analyte (e.g. self-assembled monolayers of polymers).
Again with reference to
Again, with reference to
More particularly,
It should be understood that the relative voltage between the surface 2002 (corresponding to the NEMS assembly) and the portion 2004 (corresponding to the microlens) matters. Practically, it may be the micro lens (corresponding to portion 2004) that is lowered in voltage, with 2002 staying around ground for simplicity of NEMS operation.
More particularly, in all of the
It can thus be observed that the spread of the particles i.e. the spot size of the desorbed particles in a distance of 5 mm from the surface of the NEMS sensor 104 is smaller the higher the voltage is.
Furthermore, for a voltage of 10 V, different distances of the portion 2004 from the surface 2002 were assessed, i.e., the spot size of the desorbed particles at a distance of 5 mm was simulated for a distance of 10 μm, 5 μm (as above) and 2 μm. For all these distances, which may also be referred to as electrode spacing, the spot size was 430 μm, i.e., the electrode spacing did not have any measurable effect in the simulations.
In particular at higher voltages, the generated beam thus has diameter below 100 μm facilitating further usage in the system.
Based on the above discussed simulations, it will be understood that the present technology can also be used to desorb particles from the NEMS sensor 104 and focus these particles for further inspection of such particles.
Whenever a relative term, such as “about”, “substantially” or “approximately” is used in this specification, such a term should also be construed to also include the exact term. That is, e.g., “substantially straight” should be construed to also include “(exactly) straight”.
Whenever steps were recited in the above or also in the appended claims, it should be noted that the order in which the steps are recited in this text may be accidental. That is, unless otherwise specified or unless clear to the skilled person, the order in which steps are recited may be accidental. That is, when the present document states, e.g., that a method comprises steps (A) and (B), this does not necessarily mean that step (A) precedes step (B), but it is also possible that step (A) is performed (at least partly) simultaneously with step (B) or that step (B) precedes step (A). Furthermore, when a step (X) is said to precede another step (Z), this does not imply that there is no step between steps (X) and (Z). That is, step (X) preceding step (Z) encompasses the situation that step (X) is performed directly before step (Z), but also the situation that (X) is performed before one or more steps (Y1), . . . , followed by step (Z). Corresponding considerations apply when terms like “after” or “before” are used.
While in the above, preferred embodiments have been described with reference to the accompanying drawings, the skilled person will understand that this embodiment was provided for illustrative purpose only as examples of the invention and should by no means be construed to limit the scope of the present invention, which is defined by the claims.
The invention can be realized by each embodiment alone or by a combination of several or all features of the described embodiments without any limitations.
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