RU2189086C2 - Macromolecule mass-spectrometer with cryogenic particle detector - Google Patents

Abstract

FIELD: studying macromolecules for determining their mass. SUBSTANCE: mass-spectrometer is designed to determine mass of macromolecules including proteins, large peptides, long DNA fragments, and polymers. Kinetic energy of electrostatically accelerated and charged particle are absorbed in cryogenic particle detector ; in the action phonons are emitted and detected by phonon sensor. Macromolecules are detected in the course of counting each particle irrespective of their mass. One of mass-spectrometer alternatives has single- channel cryogenic particle detector and other one, spatially distributed multichannel cryogenic particle detector. Mass-spectrometer has vacuum bowl accommodating magnet, mass separator, conducting member, and multicomponent phonon-sensing cryogenic particle detector. The latter has absorbent and definite number of phonon sensors. Mass separator is disposed in magnetic field of magnet together with conducting member; magnet-isolated electrical potential U1 is applied to the latter. Multicomponent cryogenic detector is grounded and placed at electrical potential U2. Macromolecules are accelerated due to potential difference U1 U2 and arrive at multicomponent cryogenic particle detector with kinetic energy proportional to this potential difference. Macromolecule mass is determined basing on space-time information coming from multicomponent cryogenic particle detector. EFFECT: enhanced time and space resolving power. 30 cl, 25 dwg

Description

 The invention relates to a mass spectrometer for macromolecules.

 Previously, various techniques for the evaporation and ionization of biological macromolecules were successfully used in mass spectrometry: fast atom bombardment (FAB) [1], electron beam ionization (ESI) [2, 3], and laser desorption / crystal lattice ionization (MALDI) [4 -7]. In the MALDI method, low concentration macromolecules are embedded in the crystal lattice of a material with high photon absorption. When exposure to a high-intensity laser occurs, the crystal lattice quickly heats up and turns into a plasma. In the process of evaporation, the pulse is transmitted to macromolecules, which are subsequently ionized in the plasma. Because the plasma in the crystal lattice cools quickly, most macromolecules remain intact. Earlier in mass spectrometers, the mass of these ionized macromolecules was determined by the method of measuring the time of flight (TOF) [3, 7, 8], by the Fourier method of transforming ion-cyclotron resonance (FT-ICR) [2, 5, 6], as well as using single- or multiquadrupole mass filtration method [9, 10].

 A disadvantage of the available ionized particle detectors used in macromolecule mass spectrometry is a strong decrease in the ionization efficiency for large macromolecules due to a decrease in particle velocity [11, 12]. In existing detectors for mass spectrometry, an accelerated macromolecule emits an electron at the moment of collision with a detector, which is subsequently amplified by an electron multiplier. The emission efficiency of the above electron depends on the speed of the acting particle, which is small for large macromolecules. This lack of detector efficiency can be compensated for in mass spectrometers by increasing the flow of macromolecules, but at the cost of reducing the overall sensitivity of the system. Usually in the above mass spectrometers, the detection of macromolecules with a mass greater than 50,000 A.E.M. is ineffective. Using FT-ICR techniques, significantly larger masses can be detected, although at the cost of longer integration time of the order of one second, excluding applications where high performance is required.

 Mass spectrometry is used in biology in the design and determination of proteins by measuring the distribution of mass of protein fragments [9, 13]. Mass spectroscopy is considered a promising technique for increasing the speed and reducing the cost of constructing DNA molecules [2, 3, 5-8, 14]. The standard procedure for constructing a DNA sequence is to separate multiple parts of DNA fragments prepared according to the Maxam-Gilbert and Sanger strategies using pulsed gel electrophoresis [15, 16]. With this technique, DNA fragments are separated by their lengths according to their properties to move in a gel to which an electric field is applied. Spatially separated bands of DNA fragments are usually recorded by autoradiographic and fluorescence techniques.

 A disadvantage of the above gel electrophoresis technique is the slow ordering rate and poor resolution for very large DNA fragments. The disadvantage of mass spectrometers for the high rate of DNA sequencing is the low sensitivity of the ionization detector of DNA fragments consisting of more than 100 bases, making it impossible to increase the rate of DNA sequencing, which is possible in mass spectrometry.

SUMMARY OF THE INVENTION
The subject of the invention is a mass spectrometer for large macromolecules.

 Further, the subject of the invention is a device for measuring the mass of macromolecules with effective detection of independent mass, i.e. also allowing to measure macromolecules with a very large mass.

 Further, the subject of the invention is a device for measuring the mass of macromolecules with effective detection of independent mass, i.e. also allowing to measure macromolecules in the mode of counting a single particle.

 Further, the subject of the invention is a device for measuring the mass of macromolecules with a detector system having both high temporal and spatial resolution.

 Further, the subject of the invention is a device having high performance and high sensitivity to the main sequence of short and long DNA fragments.

 Further, the subject of the invention is a device having high performance and high sensitivity to amino acids and small and large proteins.

 Further, the subject of the invention is a device having high performance and high sensitivity for the determination of small and large proteins.

 Further, the subject of the invention is a device having high performance and high sensitivity for the determination of small and large polymers.

 These capabilities are achieved through the use of a phonon-sensitive cryogenic particle detector. In a cryogenic particle detector, the absorbed kinetic energy of accelerated macromolecules is converted into phonons (i.e., vibration of the detector lattice), which are converted into electrical signals using phonon sensors. The above phonon sensor is sensitive only at cryogenic temperatures (i.e., temperatures of several degrees Kelvin), where the background of thermal phonons is negligible.

 In accordance with the foregoing and in the following, the subject of the invention is one of the implementations of the device - a mass spectrometer for determining the flight time of particles, when the macromolecules are separated by a mass-dependent velocity, which is proportional to the square root of m, where m denotes the mass of the macromolecule. For one emission time, heavier macromolecules reach the detector later than lighter macromolecules. The above cryogenic particle detectors determine the time the macromolecule arrives at the detector in the single particle counting mode. The kinetic energy of all accelerated macromolecules is the same.

 In another implementation, single or multiquadropule mass spectrometers separate and analyze the mass of macromolecules using the quadropule filtration method, in which a cryogenic particle detector measures the emerging macromolecules. Such an implementation requires a very small number of macromolecules, because the above cryogenic particle detector is used in the single-particle counting mode.

 In another implementation, the macromolecules are spatially separated by a constant magnetic field in accordance with their mass-to-charge ratio, are sequentially accelerated, and then determined by a cryogenic particle detector.

 In another implementation, the macromolecules are desorbed in the order of the pulses, spatially separated by a constant magnetic field in accordance with their mass-to-charge ratio, accelerated sequentially, and then detected by a cryogenic particle detector with spatial and temporal resolution. Based on spatial and temporal information about the macromolecule and knowledge of the emission structure, the emission time of the macromolecule can be restored and mass determined on the basis of the flight time of the particles. This leads to parallel actions of the mass spectrometer with high sensitivity and performance.

 In accordance with the foregoing and further subject of the invention is one of the implementations of a cryogenic particle detector using a crystalline substrate of both absorbents and superconducting tunnel junctions operating in the Giaever mode of phonon sensors. In another implementation of the above cryogenic particle detector, the superconductivity of the Giaever-type tunnel junctions is used in both absorbents and phonon sensors.

 In another implementation of the aforementioned cryogenic particle detector, the phonon sensitivity of the Giaever-type tunnel junctions is expanded by depositing a superconducting tunnel junction onto the surface of a large region of the superconducting film that has a superconducting energy gap larger than the corresponding film in order to exploit the quasiparticle trap effect.

 In another implementation of the above cryogenic particle detector, the crystal absorber is coated with a superconductor at the edges of the phonon sensors.

 In another implementation of the above cryogenic particle detector, microcalorimeters are used, consisting of a crystalline absorber with low heat capacity and high thermistor sensitivity, like a phonon sensor.

 In another implementation of the aforementioned cryogenic particle detector, microcalorimeters are used, consisting of a crystalline absorber with low heat capacity and highly sensitive kinetic thermometers as phonon sensors.

 In another implementation of the above cryogenic particle detector, microcalorimeters are used where the crystal absorber and the thermal phonon sensor are identical.

 In another implementation of the aforementioned cryogenic particle detector, the crystal absorber is coated with superconducting granules, which are used as phonon sensors at ultra-low temperatures.

 In another implementation of the above cryogenic particle detector, the crystal absorber is coated with superconducting granules that are used as absorbers and phonon sensors.

From the foregoing, it can be understood that the mass spectrometer, which is the subject of the invention, has several advantages:
1) it allows you to determine the mass of a macromolecule regardless of its mass, i.e. also very large macromolecules;
2) allows you to determine the mass of the macromolecule in the mode of counting a single particle, i.e. including very high sensitivity;
3) thin-film technology for the production of phonon sensors, taking into account the high spatial resolution of the cryogenic detector, that is, high performance is possible due to the spatial separation of macromolecular flows and the production of measurements parallel in time;
4) the sensitivity and performance of determining DNA sequences can be improved by several orders of magnitude;
5) the sensitivity and performance of determining protein sequences can be improved by several orders of magnitude;
6) the sensitivity and performance of protein determination can be improved by several orders of magnitude;
7) the sensitivity and performance of the determination of polymers can be improved by several orders of magnitude.

Brief Description of the Drawings
In accordance with the foregoing and in the future, the subject of the invention will be understood and described in detail with reference to the accompanying drawings, in which
figure 1 shows a schematic representation of a phonon cryogenic particle detector;
in FIG. 2 schematically shows an electrical circuit of a phonon cryogenic particle detector of FIG. 1;
figure 3 schematically shows an invention using a single-channel cryogenic particle detector, the same as in a MULDI-TOF mass spectrometer;
figure 4 is a schematic representation of a mechanical damper (chopper) as in the MULDI-TOF mass spectrometer of figure 3;
in FIG. 5 - synchronization of the laser trigger and the mechanical damper (chopper) of FIG. 4;
in FIG. 6 shows synchronization in the case of three macromolecules for the implementation shown in FIGS. 3 and 4;
7 is a schematic representation of the spatial region of a cryogenic particle detector in which macromolecules are absorbed directly by the phonon sensor;
on Fig schematically presents an alternative implementation of a cryogenic particle detector in which macromolecules are absorbed by the substrate and indirectly affect the sensor;
Fig. 9 schematically shows an alternative to Fig. 8 an implementation of a cryogenic particle detector in which the phonon sensor is a capture layer as a superconducting tunnel junction;
figure 10 presents the calculated trajectory of macromolecules of different masses with an alternative implementation using a multi-element cryogenic detector;
in FIG. 11 is a schematic representation of a mass spectrometer that provides the paths of the macromolecules shown in FIG. 10; the separation of macromolecules by mass occurs under the influence of a constant magnetic field and is determined using a multi-element cryogenic detector;
in FIG. 12 is a calculation result illustrating the dependence of spatial resolution power on accelerating voltage for implementation in FIG. 11;
in FIG. 13 presents calculation results illustrating the dependence of the spatial separation of mass on the molecular weight of the macromolecules of FIG. 11;
in FIG. 14 presents calculation results illustrating the dependence of resolution upon separation of mass on the molecular weight of the macromolecules of FIG. 11;
in FIG. 15 presents calculation results illustrating the dependence of the magnetic field on the accelerating voltage for determining macromolecules, according to the embodiment of FIG. 11 in accordance with the mass difference of FIG. 10;
in FIG. 16 presents calculation results illustrating the dependence of the post-accelerating voltage on the initial accelerating voltage for determining the macromolecules of FIG. 11 and FIG. 10;
on Fig presents the results of the calculation, according to the Monte Carlo method illustrating the resolution of the proposed equipment of Fig.7, 8 or 9, used in the embodiment of Fig.11;
on Fig presents the results of the calculation, according to the Monte Carlo method illustrating the resolution in determining mass, as determined in Fig;
Fig.19 is a pulse mode of operation, the embodiment of Fig.11;
on Fig presents the results of the calculation, according to the Monte Carlo method illustrating the travel time of particles, according to the embodiments depicted in Fig.7, 8 or 9, using the embodiment of Fig.11;
on Fig presents the results of the calculation, according to the Monte Carlo method, illustrating the possibility of determining the mass of particles based on Fig;
in FIG. 22 shows the calculation results illustrating the dependence of the particle mass determination efficiency depending on the pulsed emission mode shown in FIGS. 19 and 11;
in FIG. 23 shows the calculation results illustrating the dependence of the particle mass determination efficiency using the spatial cryogenic detector shown in FIG. 11;
in FIG. 24 shows the calculation results illustrating the dependence of the particle mass determination efficiency using the pulsed emission mode shown in FIG. 19;
on Fig schematically presents a sample used to determine the DNA sequence in the mass spectrometer shown in Fig.11.

Detailed description
The cryogenic particle detector circuit shown in FIG. 1 contains a particle absorber, indicated by the number 1, on which the phonon detector 2 is located. In the implementation shown in FIG. 1, this phonon sensor is a superconducting tunnel junction consisting of an upper layer 3 a thickness of about 100 nm, a separating oxide layer 4 of several nm and a lower layer 5 of a thickness of about 100 nm. The superconducting tunnel junction is well known as a photon detector of q particles and x-rays [17–20]. There are other implementations of a cryogenic phonon detector [19]: superconducting transition thermometers close to semiconductor thermistors that close at a critical temperature T _c , superconducting kinetic induction thermometers, and superconducting superheated granular and point ones. Basically, phonon sensors are required to be sensitive to an exposure energy of about 10 Kev and an exposure time of not more than 100 nsec. Cryogenic particle detectors operate at temperatures of several degrees Kelvin, where the background of thermally excited phonons is negligible. The principle of operation of a phonon-sensitive cryogenic particle detector in mass spectrometry of macromolecules is as follows (see figure 1.). Macromolecule 6, which is accelerated to a kinetic energy of the order of 10 Kev due to the electric field of the mass spectrometer, generates phonons 7. Phonons pass through the particle absorber 1 and are ultimately converted into an electric signal in the phonon sensor. The sensitivity of cryogenic particle detectors to absorbed ionized particles with an energy of several keV was shown in [21] by other authors [19]. The novelty of the invention is the possibility of using cryogenic particle detectors in mass spectrometry of macromolecules. The cryogenic particle detector shown in FIG. 1 has the unique property of detecting not only ionized particles, but also determining non-ionized particles with the same or even greater sensitivity, transmitting kinetic energy to the crystal lattice of the particle absorber. The phonon capture efficiency increases when a silicon single crystal, for example, is embedded deep into the substrate to a depth of about 10 μm, in order to localize the phonon density in the vicinity of the phonon sensor.

 Figure 2 shows an electronic circuit in which a superconducting tunnel junction is used as a phonon sensor. Phonons 7, arising from the absorption of macromolecules 6, propagate through the absorber 1, which includes superconducting layers 3 and 5. Phonons with an energy higher than the Cooper pair break up the Cooper pair and produce excess quasiparticles (an electrical signal of an excited state in a superconductor ) Two superconducting layers 3 and 5 are at different energy levels, which leads to a change in current i, which is supported by source 8. The flow of excess quasiparticles passes through the insulating layer 4. Since phonons 7 and the associated excess quasiparticles are destroyed in a time of the order of several microseconds, then the flow of excess quasiparticles di will move through the capacitor 9. With a change in the sensitivity of the preamplifier, which consists of an operational amplifier 10, a capacitor 11 and resistance 12, the flow of excess quasiparticles di int It is integrated, and the changes will be proportional to the number of phonons 7 absorbed by the phonon detector 2. Unfortunately, macromolecules can also be absorbed by the phonon detector and cause a signal that will be opposite to that of the cryogenic phonon detector.

Figure 3 shows the implementation of a macromolecular mass spectrometer with a cryogenic particle detector, using mainly MALDI-TOF technology. A vacuum of about 10 ^-8 mbar is created in the chamber 13 of the turbo pump of system 14, which is controlled by a vacuum measuring system 15. A cryostat 16 with a cryogenic particle detector 17 is connected to a cold tube connected to the vacuum receiver 13 through element 18. The flow of macromolecules 19 passes in the vacuum receiver 13 cold region, falls into the cryostat 16 in the form of a series of small portions 20 in the cold regions of the cryostat. The flow of macromolecules 19 is produced by ejecting macromolecules from sample 21 due to a high-voltage device 22, which is connected to a high-voltage power source 23 and by irradiation with a laser stream 29 emanating from a laser source 24. The laser may be a UV laser or an infrared laser. The laser beam 29 coming from the laser source 24 is separated by a divider 25. One part of the beam is used to measure the laser power using the meter 26, and the other part is sent to the vacuum receiver 13 through the window 27. In the vacuum receiver, the laser beam 29 is directed to the sample 21 using a mirror 28. The sample contains a crystal lattice of a photosensitive crystal solution (for example, cyanic acid or -cyano-4-hydroxycyanic acid [12]), in which the macromolecules are dissolved in a ratio of 10 ⁴ : 1. Laser power, usually several MJ / nsec, is absorbed by a grating, which breaks and turns around, turning into an electric plasma. The expansion of the atomic lattice transmits momentum to macromolecules, which evaporate and subsequently charge depending on the characteristics of the plasma. Due to the electric field in the vacuum receiver, to create which the current is supplied through the high-voltage probe 22, the macromolecules are charged with the same charge as on the probe. Then the macromolecules are accelerated towards the transfer tube 32, the cooling valve 20 passes through the cryostat 16 and reaches the cryogenic particle detector 17. The time difference between the start of the laser pulse and the arrival of the signal at the cryogenic particle detector characterizes the mass of the macromolecule. Since the cryogenic particle detector is a very sensitive device, the light and residual flux of macromolecules should not enter the cryostat. To prevent this, a mechanical shutter 30 is installed at the inlet of the transfer tube 32. In one implementation of the shutter, there is a motor 31 that rotates the slotted disc. There is also a light diode, a light detector system 33 and an electronic control system 35, which determines the position of the slot and sends a clock signal 36 to the laser 24.

Figure 4 shows the practical implementation of such a mechanical barrier: the motor 38 rotates the disk 37 with a slot at an angle alfa (usually 5-10 ^o ) and a speed f (usually 50-100 Hz). The position of the rotating slot is controlled by the transducer 39. Accelerated macromolecules 41 from the source 21 will fall into the tube 40, which connects the vacuum receiver to the creostat only when the slot 37 is opposite the tube entrance. With the right timing of the laser and the screen, the cryogenic particle detector 17 only gets a certain size of the macromolecule. As shown in FIG. 5, the electronic control system of the mechanical barrier must be such that the trigger signal t _laser arrives at the laser for the time interval Δ t _laser before opening the barrier 37 at the tube 40. The detector is tuned to a certain range of molecular weights depending on the angle alfa on the disk screen and the rotation speed f of the disk. FIG. 6 shows the time intervals for activating the screen for the movement of macromolecules to the detector for installation in FIG. 3. Since the kinetic energy is proportional to mv ² , where m is mass and v is the speed of the macromolecule, the time of flight is proportional to m ^1/2 . The shaded area in FIG. 6, which defines a time period of 10 μs in this example, is associated with a range of macromolecular masses that do not reach the detector.

 To increase the efficiency in another implementation of the invention, a cryogenic detector matrix is used, which allows determining the interaction time and the contact point of the accelerated macromolecule with the surface of the detector. Since many implementations of cryogenic detectors use film coating and lithographic techniques, the small-scale coating structure, of the order of microns, will provide spatial resolution to determine the contact position. Such an implementation of the invention is preferred. Macromolecules are spatially separated in a magnetic field in accordance with their specific charge, and the mass is based on the position of the collision of the macromolecule with a cryogenic detector matrix. If you use another embodiment of the invention using pulsed emission technology, the mass spectrometer becomes a highly parallel instrument that allows you to simultaneously determine a wide range of masses in a short time of several microseconds. Such an implementation of the cryogenic detector matrix is presented for the first time and will be discussed in detail later.

 The Cryogenic Detector Array (CDA) used in the invention consists of N phonon sensors 43 D1 ... DN with electronic channels 45 located on the substrate 42 (see Fig.7-9). In the implementation of the multi-element cryogenic detector shown in FIG. 7, accelerated macromolecules 46 are absorbed directly by the phonon sensor 43, in which phonons 47 are formed and are immediately converted into an electrical signal. The advantage of this implementation is the high efficiency of phonon-charge conversion and signal processing speed. However, a phonon sensor with a large surface that is required is technologically difficult to manufacture (for example, in the case of a superconducting tunneling conductor). An alternative to the above implementation will be the use of a large number of small phonon sensors, to which, however, a large number of electronic channels will be supplied. Therefore, the implementation of the cryogenic detector matrix of FIG. 8 is preferable to the previously described. Accelerated macromolecules 46 are absorbed by the particle absorber, produce photons 47, which propagate through the absorber and are converted into electronic signals by the phonon sensor 43. When a pure single crystal, for example, a silicon crystal is used, the phonons propagate ballistically over a large distance and will be detected by more than one phonon sensor . In the above implementation, superconducting tunnel junctions are used as phonon sensors in which the electronic signal is proportional to the number of absorbed phonons and the pulse height is proportional to the distance between the tunnel junctions. Therefore, an exact determination of the absorption point of a macromolecule can be made by calculating the centroid at a different pulse height. An alternative to the above implementation of FIG. 8 will be the implementation shown in FIG. 9, in which an additional superconducting layer 44 is located above the superconducting tunnel junction. The material of this superconducting layer is chosen so that the energy threshold Δ of the superconducting layer is greater than the threshold of the corresponding transition in order for the capture of quasiparticles to occur [22]. The quasiparticles arising under the influence of phonons 47 will propagate in the superconducting layer 44 due to diffusion and will eventually penetrate the lower layer of the superconducting tunnel junction (see Fig. 1). They will then penetrate the oxide baffle and produce a detector signal, as described above. The implementation of FIG. 9 improves the phonon detection efficiency compared to the implementation of FIG. 8. The calculations presented below show that the space-time resolution requirements for a cryogenic detector matrix (CDA) are technically feasible: the implementation of the invention in the form of a specific model is presented in the next section. The model provides a spatial resolution of dx = 0.1 mm and a temporal resolution of dt = 100 nsec, which allows determining the mass with an accuracy of 100 A.E.M. with a total weight of 600,000 A.E.M. In these calculations, a cryogenic detector matrix with a length of 10 cm and containing 100 phonon sensors was used. Other implementations of phonon sensors have been mentioned above in the discussion of FIG.

The preferred implementation of the invention is a mass spectrometer with a cryogenic detector matrix, discussed above and with the possibility of calculating the trajectories of macromolecules. Consider figure 10: macromolecules are ionized, knocked out of the sample, first accelerated to 51, then passing through a magnetic field 50 are separated along the trajectories. The trajectories are circles, the radius of which depends on the specific charge (the ratio of the charge of the macromolecule to its mass) of each molecule. Subsequently, the macromolecule is electrostatically accelerated due to the potential difference U1-U2 and collides with the cryogenic detector matrix 53. The collision point and collision time of the macromolecule and the sensor are determined for each molecule (single molecule counting mode). We draw attention to the fact that one of the main improvements of the invention is the ability to determine the mass of high molecular weight macromolecules. In the future, special calculations performed on the proposed implementation are presented and discussed. In accordance with these calculations, the mass is about 10 ⁶ A.E.M. was determined with an accuracy of 100 A.E.M. In addition, the mass spectrometer could operate in a quasi-continuous mode with a cycle power of 10%. Since the mode of single counting of molecules was used, a small amount of macromolecules was required, usually no more than ^10-15 moles.

 The proposed implementation of a high-performance mass spectrometer with a cryogenic detector matrix is shown schematically in FIG. 11. There are two main structural elements: a magnet, consisting of two rectangular superconducting coils 48, and a cryogenic detector matrix 53, enclosed in a superconducting shell 52. The magnet creates a constant magnetic field B parallel to the Z axis. All structural elements are in a vacuum system and are cooled by a combination cryogenic installation 60. T1 - operating temperature of the superconducting magnet, T2 - operating temperature of the cryogenic detector, cooled by an additional cryostat 58, T3 - operating temperature of the preamplifier.

 The analyzed sample is placed in the chamber 51, where the macromolecules are ionized and knocked out using a mechanism 67, supplied through the interlayer connection 66. The mechanism 67 may be a laser beam in the case of using the MALDI technique or capillaries in the ESI technique. Ionized macromolecules are first accelerated to kinetic energy of about 100 eV and fall into a mass separator 50, which is located inside a magnet with a magnetic field of several Tesla. The magnetic field strength depends on the range of values of the shared masses. The chamber 51 and the mass separator 50 are at a high voltage voltage U1 supported by the source 65 through the interlayer connection 64. The superconducting magnet consists of two rectangular superconducting coils 48, separated by a gasket 49 of superconducting material for magnetic fields, in the position when the mass separator is parallel to the z axis. The magnet is cooled by a cryostat 60 connected through a connection 61 and has an operating temperature T1. The device 73 provides a cryogenic fluid to the creostat through channel 72. The current for the superconducting magnet is supplied from source 63 via cable 62.

Under the action of the magnetic field B of the magnet, the macromolecules move in circles with radii proportional to the specific charge of the macromolecule. After half the circumference, the separated molecules fall into the electric field and are then introduced into the cryogenic detector matrix 53. The kinetic energy of the macromolecules at the moment of contact with the cryogenic detector matrix e • (U2-U1), where e is the unit of charge, U1 is the electric separator potential and U2 is the electrical potential of the cryogenic particle detector, which is usually grounded. In order to protect the cryogenic particle detector from a strong magnetic field, it is placed in a magnetic chamber 52, also made of a superconducting material. The cryogenic detector matrix 53 is located on a cold panel 54, which is cooled by the cryostat 58 to the operating temperature T2 of the matrix 53. The cryostat 58 is connected to the main cryostat 60, if both use ³ He. The cryostat is controlled by a temperature control system and a pump 69 connected through a device 68. The cryogenic detector matrix transmits signals to an electronic preamplifier 56, which can be cooled to operating temperature T3 through a cooling channel 57 from cryostat 60. The preamplifier output is connected via data link 70 to a data assimilation system 71.

Subsequently, the response of the proposed high-performance system to various control parameters is evaluated, which are illustrated by the calculation results. Basically, a two-dimensional machine code will be used [23], which allows one to calculate electric and magnetic fields and determine the trajectories of macromolecules in these fields. The paths of a macromolecule are calculated from spatio-temporal information at any time step. Unless otherwise specified, the following parameters are used in the calculations: geometric — as shown in FIG. 10, where the most convenient dimensions are the internal dimensions of the mass separator 50 (64 cm • 32 cm), the mass of macromolecules - from M = 400000 A.E.M. up to M = 800000 A.E.M., magnetic field strength B = 6.5 Tesla, voltage for primary acceleration U _pre = 200 V, voltage for secondary acceleration U _post = 50 kV. The spatial resolution of the detector is dx = 0.1 mm, the time resolution is 100 nsec. The last two values are typical for cryogenic particle detectors and technically feasible. For calculations in which the mass of the macromolecule was fixed, we used the value M = 600000 A.E.M.

The separation of macromolecules by mass is due to the magnetic field B and voltage for primary acceleration U _pre . The voltage for secondary acceleration U _post is required for the macromolecules to provide a sufficiently high kinetic energy of the order of 10 kV so that they can be fixed with a cryogenic detector matrix, an increase in U _post facilitates the determination of macromolecules, however, it is obvious from Fig. 12 that the macromolecules are focused in a small area cryogenic detector matrix, thereby reducing the spatial resolution of the detector. On Fig shows the ranges of values for different values of the intensity of the magnetic field. For a given value of the magnetic field, the mass range of macromolecules reaching the cryogenic detector matrix is limited due to the limited input window of the mass separator, FIG. 10. As can be seen from FIG. 13, a larger magnetic field produces a wider range of mass values that can be determined simultaneously by a cryogenic detector matrix. However, due to the fact that these masses are distributed at a height of 10 cm, the resolution of the spatial separation of the masses is reduced to increase the masses, as shown in Fig. 14. However, as can be seen from FIG. 14, with increasing masses of macromolecules, mass separation improves. A magnetic field of 6.5 T can be easily achieved, however, in this case, a relatively large region of the detector will be technologically inactive. A good uniform magnetic field is required in order to correct small inhomogeneities, given two important calibration curves: the spatial calibration curve M _{x-cal (x)} and the temporary calibration curve M _{t-cal (t)} . In order to get a good resolution, it is important to have a time-stable magnetic field for a short and long period.

Another critical factor on which the good resolution of a high-performance mass spectrometer depends is the quality of ionization of the macromolecular flow entering the separator (see 65 in FIG. 10). This stream should have good collimation and monoenergy. It is desirable that a high-quality flow is easily obtained at high U _pre . Three parameters B, U _prе, and U _post controlling the magnetic field cannot, however, be selected independently of one another for a given range of measured masses and a certain arrangement of the cryogenic detector matrix. On Fig shows the dependence of the magnetic field B on U _pre for the range of measured masses from 400000 A.E.M. up to 800000 A.E.M. On Fig shows the voltage dependence for the secondary acceleration U _post as a function of voltage for the primary acceleration U _pre for the same range of measured masses. If, for example, the voltage for primary acceleration U _pre = 400 V, then the magnetic field strength B = 8 T (Fig. 15) and the voltage for secondary acceleration U _post = 100 kV (Fig. 16) make it possible to measure masses in the range from 400,000 A .EAT. up to 800000 A.E.M. at the above location of the cryogenic detector matrix. An advantage of the implementation of the invention is that without changing the location of the matrix, it is possible to measure the mass of macromolecules, relatively speaking, without limiting the mass range, changing only the magnetic field. Additionally, the voltage for primary and secondary acceleration can be selected so as to optimize performance.

The mass of the macromolecule can be determined directly by the spatial resolution power of the cryogenic detector matrix (see Fig. 14). In this specific calculation model, the spatial separation ability of a macromolecule by a cryogenic detector matrix is 0.2 μm / A.E.M. (see Fig. 14). If it is necessary to determine the mass of a macromolecule with a resolution of, say, 100 A. Ye.M. (this will be required if the mass of large DNA fragments of the DNA sequence is measured, where the base mass of the macromolecules is mainly 300 A. EM), the spatial resolution of the cryogenic-detector matrix of about 25 μm will be required. About 4000 individual detectors will also be required for a 10 cm cryogenic detector matrix, if we use the implementation of Fig. 7. When the implementation shown in Figs. 8, 9 is used, it is possible to obtain a spatial resolution dx = 0.1 mm with a distance between the phonon sensors of 1 mm. Further, only 100 phonon sensors per 10 cm area will be required. The Monte Carlo method was used to study the resolution of various implementations of the invention. "True" values of X _stop 2000 macromolecules of mass M ₀ = 600000 A.E.M. were randomized to a Gaussian distribution (FWHM) with a resolution of dx = 0.1 mm, simulating the output information X _out . In FIG. 17 shows the x-coordinate distribution of the output signal X _{out of the} cryogenic detector matrix. The calculated trajectories of the corresponding molecules were used to calculate the calibration curve M _{x-cal (x)} . Based on the obtained data, the mass of the macromolecule M ₁ was determined. In FIG. 18 shows the distribution of the error in calculating the mass M ₁ -M ₀ . The resulting mass determination error of about 500 A.E.M. will not be enough to determine the DNA sequence. However, an increase in the accuracy of mass determination can be obtained through the application of the “flying time” strategy and the implementation of the pulsed emission mode of a high-performance mass spectrometer. The following paragraph will be devoted to this.

To determine the “flight time” in the mass spectrometer, the difference between the moment of the start of particle emission t _start and the time of collision of the particle with the detector t _{stop is} measured. Since usually in our calculation model the flight time is 5 ms, particles can be emitted every T ₂ = 10 ms with a pulse duration of, say, T ₁ = 100 nsec. Under such conditions, the cyclic load of the detector will be t ₁ / (T ₁ + T ₂ ) of the order of 10 ^-5 and, accordingly, the low productivity of the device. The idea of an on-line particle counting mode is to carry out several measurements of the flight time in parallel, contributing to the determination of spatial trajectories in a magnetic field. Emission pulses are shown in Fig. 19. The collision time of a particle with a t _stop detector is fixed by a cryogenic-detector matrix, however, the moment of start of particle emission t _start must be determined. This is done using spatial particle motion paths as follows. As shown in FIG. 19, macromolecules are emitted during T ₁ and not emitted during T ₂ . Such an emission pulse can come either from a laser (MALDI circuit) or from electron-optical systems. In the future, knowledge of two curves will be required: the spatial calibration curve M _{x-cal (x)} and the temporary calibration curve M _{t-cal (t)} . The starting point of the calculation is the two output signals of the cryogenic detector matrix:
X _out = X _stop + dX,
t _out = t _stop + dt,
where X _stop , t _stop is the location and contact time of the macromolecule and cryogenic particle detector, respectively,
dX, dt - deviations from the true position of the macromolecule in space and time, respectively, depending on the spatio-temporal resolution of the cryogenic detector matrix. From the Xout value, the first approximation of the mass Mi is determined using the spatial calibration curve M _{x-cal (x)} :
M ₁ = M _{x-cal (Xout)} .

Substituting this value of M ₁ into the inverted M ^-1 _{tc al (t)} ^- time calibration curve, we obtain the first approximation t _{1 of the} beginning of particle emission:
t ₁ = t _out -M ^{- 1} _{t-cal (M1)} .

Now, knowing that the macromolecule must be emitted from each pulse, you can set the corresponding number of emission cycles N (see Fig. 19):
N = 1 + t ₁ / (T ₁ + T ₂ )
and get the second approximation t _{2 the} start of particle emission:
t ₂ = (N-1) (T ₁ + T ₂₎ + T _2/2.

The value of t ₂ , unfortunately, is determined accurate to the value of T ₁ . Using the time calibration curve again, we finally find the mass of the M ₂ macromolecule using the TOF method
M ₂ = M _{t-cal (tout-t2)} .

In the presented method (TOF), the accuracy of determining the mass of a macromolecule is determined by the duration of the emission pulse T ₁ and the time resolution dt of the cryogenic detector matrix. In the pulsed mode, the spatial resolution dx of the cryogenic detector matrix is used only to determine the number of pulses N. FIG. 20 shows the Monte Carlo distribution for the particle collision time t _stop obtained with the emission pulse duration T ₁ = 0.5 μs and cryogenic temporal resolution -detector matrix dt = 100 nsec.

In FIG. Figure 21 shows the distribution of the reconstructed mass values by the TOF method. This distribution is obtained as follows: in the Monte Carlo calculation, each case was characterized by the corresponding values of X _out (Fig. 17) and t _stop (Fig. 20). Using the mass recovery method (TOF) described above, the mass value of the M ₂ macromolecule was determined from two calibration curves. The distribution of the error in determining the mass of M ₂ -M ₀ has a central peak in the region of 100 A.E.M. (taking into account the FWHM conditions) and is associated with the accuracy of determining the number of pulses N. In addition, two peaks at the edges of the graph are associated with a false determination of the confidence limits of emission cycles, which leads to a “leak” of t ₁ values. The cause of these "leaks" is the limited spatial resolution dx of the cryogenic detector matrix and the small pause value T ₂ in the pulse sequence. Increasing T ₂ reduces the number of error cases. However, the cyclic load of the detector will be reduced. In FIG. 22, the efficiency of mass determination depending on the cyclic load of the detector at two spatial resolutions of the detector dx is presented as the ratio of the cases of mass determination to cases of incorrect mass determination.

The accuracy of the mass measurement M ₁ can be improved by increasing the spatial resolution of the cryogenic detector matrix (i.e., by determining the corresponding dt values less) (see Fig. 23). The accuracy of determining the mass distribution of M ₂ can be improved by reducing the duration of the emission pulse T ₁ (see Fig. 24). However, in the latter case, the accuracy of mass measurement will strongly depend on the time resolution of the cryogenic detector matrix. Calculations of the accuracy of the mass calculation described above were obtained under the assumption that ionization of the initial flow of macromolecules entering the separator leads to the fact that all macromolecules have a kinetic energy of 200 eV. Unfortunately, this is not entirely true for a real device and this will be one of the main technological problems in the construction of the above proposed invention.

 In particular, consider the use of the invention for the study of DNA sequences. The DNA sequence chain is determined by the four basic sequences of DNA alpha-beta, A (adenine), G (guanine), T (thymine), C (cytosine). In molecular biological laboratories, samples containing DNA sequences are prepared according to Sanger or Maxam-Gilbert techniques. For example, using the Sanger technique, four samples of identical parts of DNA can be labeled A, G, T, and C, each of which contains information related to the location of the main specific parts of the DNA molecule. Typically, macromolecules are sorted according to their lengths for each of the four samples using gel electrophoresis techniques. The macromolecules are mixed in a gel, which is placed in an electric field. For a given chain length, short fragments mix faster than long fragments that are sent to the separator. The main disadvantage of gel electrophoresis is the large time (of the order of several hours) required for mixing the macromolecules in the gel. Typically, good laboratory equipment can prepare about 10,000 samples per day.

The fastest method for separating DNA fragments involves the use of mass spectrometry. As shown above, the present invention can analyze large DNA fragments weighing 600,000 A.E.M. with a resolution of about 100 A.E.M. Subsequently, samples equivalent to the human genome are analyzed, containing 3.10 ⁹ samples, which are evaluated by the proposed method. Since a cryogenic detector matrix is used operating in the unit particle counting mode, the intensive load of the detector will be prevented. Therefore, only a small number of macromolecules will reach the multi-element detector in a given time. The particle detector allows you to identify each set of values (X _stop , t _stop ), unique to the pulse emission cycle. Thus, heavy molecules in the case of early emission will reach the cryogenic particle detector later than light molecules in the case of late emission. They will never reach the same position on the detector, since the unique trajectory depends on the specific charge. One of the improvements of the proposed implementation is that charged macromolecules can be separated using control parameters. If you place the DNA fragments for analysis, as shown in Fig.25, then the emission of each sample can be performed synchronously with the emission pulse of the mass spectrometer. Each mass value measured by the cryogenic detector matrix is associated with a specific breakdown. We will again select a mass measurement range between 400,000 A.E.M. and 800000 A.E.M. and accept the accuracy of determining the mass for the selected samples Δm <300 A.E.M. Due to the available resolution of the system, we assume that 100 cases will be required to determine the mass uniquely. Within a given mass range, the selected sample contains 1300 bases, which can be reconstructed for the four selected samples A, G, T, and C shown in FIG. 25. In order to satisfactorily determine the mass from the values (X _stop , t _stop ), only one macromolecule must reach the detector at a given point in a certain time. The given sample will be analyzed 100 times. If you choose the emission pulse duration of 100 μs, then you can restore the sequence of 1300 bases of the given standard DNA for 100 • 4 • 100 μsec = 40 ms. The next four samples will be associated with a population of macromolecules displaced by 1300 bases. It will take 40 ms to determine the 1300 base sequences. In 1 sec, the machine has the ability to determine 3.25 • 10 ⁴ bases, or, in other words, the number of bases of the human genome containing 3 • 10 ⁹ bases can be set in 9.2 • 10 ⁴ sec, i.e. in 25 hours. If one of the sequences of the human genome with one single gel electrophoresis apparatus has the ability to create 10,000 bases per day, it will take 800 years for such calculations. However, sequence separation is only the first step to determine DNA sequences. The biological production of human genome sequences in a laboratory environment will require more than a few hours. Nevertheless, an array of samples can be produced and stored, as shown in FIG. 25, by various laboratories in parallel and analyzed using the invention in a very short time.

A complete DNA count will also require a little: a simple array, as shown in FIG. 25, should consist of 3 • 10 ⁹ /1300=2.3•10 ⁴ samples, which can be represented as an array of 1500 • 1500 samples. In our example, 4 samples, defined as A, G, T, and C, contain completely 1300 macromolecules with an average mass of 600000 A. E. M. The total mass of macromolecules reaching the cryogenic-detector matrix in one emission cycle is (1300/4) • (600000 AE M. Since the analysis of each sample is performed 100 times to obtain a good estimate of the mass value, the previous value must be multiplied by 100. In addition, since the efficiency of the output and ionization of macromolecules is not more than 10 ^-4 , the resulting number to be multiplied per 10 ^4. Hence, each floor ennaya sample (1 amu = 1.66 • 10 ^-24 g) comprises:
the amount of DNA in the sample = 10 ⁴ • 100 • (1300/4) • 600000 A.E.M. = 0.32 • 10 ^-9 g.

The product of the number of samples and the reference array (2.3 • 10 ⁶ ) gives the total number of DNA:
total number of DNA = 2.3 • 10 ⁶ • 0.32 • 10 ^-9 g = 0.74 • 10 ^-3 g

Now let's estimate the size of the model array. Each sample contains a solution of DNA with a concentration of 10 ^-4 , i.e. the dissolved mass will be 3.2 • 10 ^-4 g. If, for example, we take the density of the solution equal to the density of water, then the mass will correspond to a volume of 3.2 • 10 ^-6 cm ³ , or, alternatively, the radius of the sample will be 91 microns. Samples may be spaced 0.2 mm apart. The total size of the sample array will therefore be:
total size = (1500 • 0.2 mm) • (1500 • 0.2 mm) = 30 cm • 30 cm.

The total number of DNA accumulated by the cryogenic detector in the analysis of the human genome described above will be 4 x 10 ^-10 g, which corresponds to a film thickness of 7.4 nm for a cryogenic detector 10 cm long and a molecular beam 1 mm thick. A polymer film of this thickness will not be an obstacle to the phonon sensitivity of the cryogenic detector.

 The invention was used for the above calculations. The described invention can also be applied to proteins, peptides, polymers and other macromolecules.

 The structural forms of the invention presented herein may have other uses. The presented implementation does not mean to note all possible equivalent forms of the invention. It is understood that the terms used herein are more descriptive than restrictive, and that various changes can be made without prejudice to the invention.

Claims (30)

 1. Mass spectrometer, including a means for evaporation and charge of macromolecules from a condensed solution; a vacuum receiver in which said macromolecules are vaporized and charged with said agent; an electro-optical device placed in said receiver for accelerating said charged macromolecules; high voltage power source and electrical connections with the specified electro-optical device; a mass separator for separating said macromolecules in accordance with their mass / charge ratio; a detection system for detecting said macromolecules, characterized in that the detection system comprises a cryogenic particle detector comprising at least one absorber and at least one phonon sensor and configured to determine the interaction time and contact point of the accelerated macromolecule with the detector surface as a result of which the absorbed kinetic energy of the colliding macromolecules is converted into phonons excited in the specified absorber, and as a result of which these phonons sensors convert said phonons into an electrical signal; a cryostat with a cold tube to which said cryogenic particle detector is thermally coupled.  2. The mass spectrometer according to claim 1, characterized in that the mass separator for separating said macromolecules according to their mass / charge ratio is a vacuum receiver around which a magnet is located to form a uniform magnetic field in which said macromolecules are separated according to their mass / charge depending on the trajectories in the indicated uniform magnetic field.  3. The mass spectrometer according to claim 2, characterized in that the detector system is a cryogenic detector matrix enclosed in a superconducting shell, while the mass separator into which the ionized molecules enter is located inside the magnet and has an electric potential different from the electric potential cryogenic detector matrix.  4. Mass spectrometer according to any one of paragraphs. 1-3, characterized in that the aforementioned absorber and phonon sensor of the cryogenic detectors of the part are identical and the excited phonons are directly converted into an electrical signal.  5. Mass spectrometer according to any one of paragraphs. 1-3, characterized in that the above absorber of the cryogenic particle detector is a single silicon crystal.  6. Mass spectrometer according to any one of paragraphs. 1-5, characterized in that the above phonon sensors are superconducting tunnel junctions, operating in the Jayer mode; excited phonons break the Cooper pair in the superconducting layers of the above superconducting tunnel junctions and quasiparticles are ejected; quasiparticles penetrate the dividing wall of the superconducting tunnel junction and quasiparticles are released, which forms an electrical signal; The magnetic field is directed parallel to the superconducting tunnel junctions using a magnet to suppress Josephson flux.  7. The mass spectrometer according to claim 2 or 3, characterized in that the magnet is a superconducting magnet.  8. The mass spectrometer according to claim 6, characterized in that the superconducting tunnel junction is formed by niobium or a niobium alloy.  9. The mass spectrometer according to claim 6, characterized in that the superconducting tunnel junction is formed by aluminum or an aluminum alloy.  10. The mass spectrometer according to claim 6, characterized in that the superconducting tunnel junction is formed by tantalum or an alloy of tantalum.  11. The mass spectrometer according to claim 6, characterized in that the superconducting tunnel junction is formed by tin or an alloy of tin.  12. The mass spectrometer according to claim 6, characterized in that the superconducting tunnel junction is formed by indium or an indium alloy.  13. The mass spectrometer according to claim 6, characterized in that the superconducting tunnel junction is formed by lead or a lead alloy.  14. Mass spectrometer according to any one of paragraphs. 1-6, characterized in that the phonon sensors are semiconductor thermistors; the above semiconductor thermistors are controlled by electric current; phonons heat the absorber; heating leads to an increase in temperature; an increase in temperature leads to a change in the resistance of the semiconductor thermistor and, as a consequence, to a change in the controlled electrical signal.  15. Mass spectrometer according to any one of paragraphs. 1-14, characterized in that it further introduced a preamplifier placed on the substrate of a cryogenic particle detector.  16. The mass spectrometer according to claim 15, characterized in that the preamplifier has a superconducting structure.  17. Mass spectrometer according to any one of paragraphs. 1-16, characterized in that the means for the evaporation and charge of the macromolecules of the condensed solution is based on the technique of multigrid laser desorption or ionization (MSLDI).  18. Mass spectrometer according to any one of paragraphs. 1-16, characterized in that the means for evaporation and charge of the macromolecules of the condensed solution is based on electroaerosol ionization (EAI).  19. Mass spectrometer according to any one of paragraphs. 1-16, characterized in that the means for the evaporation and charge of the macromolecules of the condensed solution is based on fast atom bombardment (BBA).  20. Mass spectrometer according to any one of paragraphs. 1-16, characterized in that the means for the evaporation and charge of the macromolecules of the condensed solution is based on plasma desorption (PD).  21. Mass spectrometer according to any one of paragraphs. 1-16, characterized in that the means for the evaporation and charge of the macromolecules of the condensed solution are based on surface-enhanced net desorption (PPRP).  22. Mass spectrometer according to any one of paragraphs. 1-16, characterized in that the means for evaporation and charge of the macromolecules of the condensed solution are based on the evaporation of macromolecules by thermal heating of the substrate on which the macromolecules are located; the charge of macromolecules is produced in a separate way.  23. The mass spectrometer according to claim 22, characterized in that the means for the evaporation of macromolecules by thermal heating is high-frequency phonon emission.  24. A mass spectrometer according to claim 22 or 23, characterized in that photoionization is used to charge the emitted macromolecules.  25. Mass spectrometer according to any one of paragraphs. 3-24, characterized in that the mass spectrometer operates in a pulsed mode, the spatial position of the particle collision point gives the first approximation of the macromolecule mass, the first mass approximation together with the collision time determines the emission time of the macromolecule, the first approximation of the emission time and the collision time gives the second mass approximation the macromolecule is finer, the determined mass of the macromolecule is calculated by the difference between the collision time and the emission time.  26. Mass spectrometer according to any one of paragraphs. 1-25, characterized in that the samples of macromolecules are a two-dimensional array.  27. Mass spectrometer according to any one of paragraphs. 1-26, characterized in determining the mass of DNA fragments in the DNA sequence.  28. Mass spectrometer according to any one of paragraphs. 1-27, characterized in determining the mass of proteins or protein fragments in the protein sequence.  29. Mass spectrometer according to any one of paragraphs. 1-26, characterized in determining the mass of proteins or protein fragments for determining protein.  30. Mass spectrometer according to any one of paragraphs. 1-26, characterized in determining the mass of polymers or polymer fragments for determining the polymer.

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