JP2007114209A - Nuclear magnetic resonance analyzer for solution - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new detection method which is independent of the magnetic field strength, for improving the detection sensitivity, by increasing the central magnetic field of NMR, since the improvement in the sensitivity of conventional methods has reached a limit, and according to the development of a protein research in recent years, increasing the need for the structure analysis of a complicated compound, having a large molecular weight, thus, enhancing the need for NMR performance each year. <P>SOLUTION: The shape of a detecting coil is modified from a conventional bird cage type 27 to a solenoid type 4, having higher sensitivity. A superconducting magnet is constituted of a split magnet using the superconducting magnets 1, 2 and 3 and is divided into left and right sections having the magnetic field 11T horizontally, preferably, 14.1T or higher, instead of the superconducting magnet, comprising the conventional multilayered solenoids 28, 29 and 30, and it is required that the magnetic field homogeneity be 0.001 ppm or smaller, and time stability be 0.001 ppm or smaller. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a nuclear magnetic resonance analyzer suitable for analyzing in a solution the structure and interaction of organic molecules such as proteins and substrates interacting with proteins and ligands.

  Analytical methods for organic substances using nuclear magnetic resonance have been making rapid progress in recent years. In particular, by combining with powerful superconducting magnet technology, it has become possible to efficiently analyze the structure of organic compounds such as proteins having a complex molecular structure at the atomic level. An object of the present invention is an NMR spectrometer necessary for analyzing the structure and interaction at the atomic level of protein molecules in an aqueous solution in which a minute amount of protein is dissolved, and requires so-called millimeter-class image resolution. A medical MRI diagnostic imaging system intended for tomographic imaging of a human body is required to have a magnetic field strength of one digit or higher, a magnetic field uniformity of four digits, and a stability of three digits higher. It is a special energy spectrometer that requires device fabrication technology. Details on conventional high-resolution nuclear magnetic resonance analyzers are described in “NMR of proteins” on pages 33 to 54, written by Yoji Arata, Kyoritsu Shuppan, 1996. Recent inventions related to typical apparatus configurations when using NMR for protein analysis include inventions related to superconducting magnets, and typical configurations of multilayer air-core solenoid coils such as JP 2000-147082. In addition, as an invention related to signal detection technology, US Pat. No. 6,121,776 which disclosed a birdcage type superconducting detection coil, a signal detection technology using a conventional saddle type coil or birdcage type coil, JP 2000-266830, JP-A-6-237912, and the like. According to these reports, the conventional high-sensitivity nuclear magnetic resonance analyzer for protein analysis uses a superconducting magnet device composed of a combination of solenoid coils that generate a magnetic field in the vertical direction, and uses an electromagnetic wave of 400 to 900 MHz. Is irradiated to the sample, and a resonance wave emitted from the sample is detected using a saddle-shaped or birdcage-type detection coil. In addition, as in the example of US Pat. No. 6,121,776, there is a case where a detector cooled to a low temperature is used to reduce the thermal noise during reception and the S / N sensitivity ratio is improved. .

  Historically, high-sensitivity nuclear magnetic resonance apparatuses have basically improved the sensitivity by a method of increasing the central magnetic field strength of a superconducting magnet while keeping the basic configuration of the system such as an antenna and a magnet the same. Therefore, the highest NMR measurement sensitivity reported so far is obtained by a 900 MHz NMR apparatus, and a large superconducting magnet having a central magnetic field of 21.1 Tesla is used, but the basic configuration of the apparatus is disclosed in JP-A-2000-147082. This is no different from the conventional technology. In protein analysis using a solution, the improvement of the central magnetic field has the effect of improving sensitivity and clarifying the separation of chemical shifts.

  As for the sensitivity improvement effect by the detection coil shape, as described in Yoji Arata, “NMR book” 2000, Maruzen, p326, conventionally, if a solenoid coil is used as the detection coil, a saddle shape or a bird It was known that there are various advantages compared to the saddle type. For example, it is excellent in terms of impedance controllability, filling factor, RF magnetic field efficiency, and the like. According to the same book, however, the conventional superconducting magnet configuration uses a sample tube placed perpendicular to the magnetic field when sensitivity is important, such as for measuring proteins dissolved in trace amounts in an aqueous solution. It is actually impossible to wind a solenoid coil around and is not generally used. In exceptional cases, it may be used only when measuring a small amount of sample solution with high sensitivity, and a specially designed micro sample tube is used to measure using a special probe. It was.

  As a special example, a method of magnetizing a high-temperature superconducting bulk magnet in the horizontal direction and detecting an NMR signal with a solenoid coil has recently been devised, as disclosed in JP-A-11-248810. Japanese Patent Application Laid-Open No. 7-240310 discloses a method of constructing a superconducting magnet and a cooling container suitable for general NMR applications for removing restrictions on the ceiling height of the apparatus. However, detection necessary for protein analysis is disclosed. There are no known methods for improving the sensitivity, the magnetic field uniformity, the technical response to the temporal stability of the magnetic field, or the like.

  In recent years, with increasing needs for protein research, there has been an increasing need for analysis of samples with low protein solubility in water, and the need to improve NMR measurement sensitivity has arisen. In order to adapt the nuclear magnetic resonance analyzer to such needs, it is necessary to improve the measurement sensitivity while maintaining the same sample space as before, and to stabilize the superconducting magnetic field during a long data integration time. Ensuring sex is also essential. The improvement in measurement sensitivity is not only shortening the measurement time but also reducing the amount of sample, especially if the sample has the same degree of solubility, and it is possible to analyze proteins with low solubility. There is. Therefore, NMR analyzers used for protein analysis require particularly excellent detection sensitivity and stability compared to conventional NMR, and are accurate and stable over a long period of time of one week or more. Detection of the NMR signal is necessary. This is because if the magnetic field fluctuates during measurement, the peak of the NMR signal moves. Especially in the measurement of interaction, the peak movement is due to interaction or due to instability of the magnetic field. This is because it is impossible to determine whether it is an object. In addition, if the magnetic field is not uniform, desired peaks are overlapped, causing problems such as difficulty in discriminating the interaction. Therefore, it is necessary to first note that future NMR techniques aimed at various analysis of proteins will require new technology development that is not a simple extension of conventional NMR apparatus. .

  As an example, the specifications of the magnetic field uniformity of a general NMR apparatus are 0.01 ppm in the sample space and 0.01 ppm / h in the time stability. When this is converted by 600 MHz proton NMR for general use, an allowable error of 6 Hz is obtained. However, in the case of the above-described protein interaction analysis, at least a space of 1.0 Hz or less and a temporal resolution are required, and preferably 0.5 Hz or less. It is necessary to optimally configure the superconducting magnet and the detection coil by a method capable of realizing the uniformity of the magnetic field and the temporal stability of the magnetic field. Therefore, the performance of the NMR apparatus generally used conventionally is insufficient, and the stability and the magnetic field uniformity higher by one digit or more than before are required.

  The conventional technology mainly improved the sensitivity by relying on the improvement of the magnetic field strength, resulting in a new installation problem such as a large-sized device and the need for a dedicated building due to the problem of leakage magnetic field and floor strength. It was. Furthermore, problems such as an increase in the cost of the superconducting magnet occurred. In addition, the sensitivity improvement by this method reaches the upper limit of about 21T due to the limitation due to the critical magnetic field of the superconducting material, and in order to further improve the sensitivity, a detection sensitivity improvement technique using a new means that does not depend on the magnetic field strength is used. It was desired.

  As described above, the high-sensitivity measurement method using the solenoid coil can be used with a very small amount of a special sample tube and a special detection probe. Could not be applied. In the method of generating a magnetic field in the horizontal direction with a strong magnet and detecting the NMR signal with a solenoid coil as in the example of JP-A-11-248810, a magnetic field of less than 10T is generated on the surface of the high-temperature superconductor. The magnetic field of the sample portion is at most several Tesla, and it is possible to generate a magnetic field of 11 Tesla or higher, preferably 14.1 Tesla or higher necessary for protein analysis in a desired sample space. This method was not possible. In this method, it has been substantially difficult to achieve the time stability of 1.0 Hz / hour or less necessary for protein analysis due to the effect of magnetic flux creep of the high-temperature superconductor. As for the magnetic field homogeneity necessary for protein analysis, achieving a magnetic field homogeneity within 1.0 Hz at a proton nuclear magnetic resonance frequency in a space of 10 mm in diameter and 20 mm in length is the high temperature superconducting bulk material. It was difficult due to inhomogeneities due to the manufacturing process.

  As described above, the conventional technology is required to develop a breakthrough technology to meet the needs of protein analysis, but has reached the limit of sensitivity improvement by magnetic field. There was a need for a solution.

In the case of analyzing efficiently and accurately the interaction between a protein, a substrate, a low molecule such as a ligand in a solution that is expected to increase in the future, empirically 600 to 900
It is desirable that the measurement can be performed with an appropriate sample amount at about 14 to 21 T in MHz and the central magnetic field, and it is desired to increase the measurement sensitivity and increase the throughput from the current level. In general, an apparatus of 800 MHz or higher is operated by depressurizing 4.2K liquid helium and supercooling to 1.8K in order to use the superconducting characteristics to the limit. For this reason, the complexity of operation of the apparatus is increased, and maintenance is also difficult. Moreover, since a magnet apparatus becomes large, a leakage magnetic field is large and usually a dedicated building is required. In particular, from the standpoint of device installation, the conventional method increases the leakage magnetic field in the vertical direction as the central magnetic field increases. For example, a 900 MHz class device generates a leakage magnetic field of 5 m in the height direction. I need a tall building. Therefore, there has been a problem that the construction cost increases. In addition, the size of the conventional 900 MHz superconducting magnet is the same as that of I. Triple-Transactions Upride Superconductivity IEEE. Transactions on Applied Superconductivity Vol. 11 No. 1 p2438, the width diameter was 1.86 m and the height was several meters only by the size of the magnet portion.

  In the present invention, the measurement sensitivity of the NMR signal is increased by at least 40% or more at about 600 MHz (14.1 T) in a state where the sample solution is mainly filled with a sample solution of about 30 mm height using a normal 5-10 mm diameter sample tube. Another object of the present invention is to provide a novel nuclear magnetic resonance analyzer capable of providing temporal stability and spatial uniformity of a superconducting magnet necessary for protein analysis. The configuration of the present invention does not define the system operating temperature as 4.2K. In addition, it is also possible to aim at the ultimate performance by applying the present invention, and depending on the use, there may be operation at 1.8 K at 21.1 T, that is, 900 MHz, which is a conventional magnetic field limit. In that case, the sensitivity can be improved by 40% as compared with the conventional method, and it became possible for the first time to greatly break the detection sensitivity limit due to the magnetic field intensity, which was impossible in the past.

  The inventors have devised a problem common to the current nuclear magnetic resonance apparatus and a countermeasure for it by conducting earnest research. The current nuclear magnetic resonance apparatus is a method in which a solution sample is placed in the center of a multilayer air-core solenoid coil with excellent magnetic field uniformity and detected with a saddle type or birdcage type antenna in order to achieve both cost and installation. Has developed. Historically, as NMR has evolved from low magnetic fields of less than 400 MHz due to advances in measurement technology and analysis methods, measurement sensitivity has been improved by increasing the central magnetic field while maintaining this basic form. Recently, an example using a superconducting birdcage antenna to reduce thermal noise has been reported. We have been diligently investigating methods for significantly increasing the signal strength compared to the conventional method while maintaining the same magnetic field strength. As a result, the inventors have found that this problem can be solved according to the features of the present invention described below.

One feature of the present invention is that the sample space has a diameter of 5 to 10 mm and a magnetic field of 400 MHz or more suitable for solution NMR having a height of 20 mm, preferably about 600 MHz to 900 MHz, and the detection coil is a sample tube for ordinary NMR research. The sensitivity is improved by applying a solenoid type detection coil with a height of about 5 to 10 mm and a height of about 20 mm. In principle, the sensitivity can be improved by at least 1.4 (√2) times due to the difference in the shape factor of the detection coil, and further improvement can be expected by other factors, and the data integration time is reduced to less than 1/2. can do. Samples in solution form a sample tube with a diameter of 5-10 mm and a height of 20-20
Inserted about 30mm and inserted vertically from the top. In order to detect NMR signals with high sensitivity using a solenoid coil with the vertical axis as the winding axis, the magnetic field generated by the superconducting magnet is placed in the horizontal direction, and a solution sample that can be easily attached and detached can be placed at the center of the magnetic field. There is a need. Therefore, unlike the conventional simple solenoid magnet, the superconducting magnet needs to be composed of a pair of split magnets divided into left and right. Here, in order to deal with a special analysis application of analysis of protein dissolved in a solution, as described above, time stability, proton nuclear magnetic resonance frequency of 1.0 Hz / hour (h), sample It is necessary to optimally design and manufacture superconducting magnets with a spatial uniformity of 1.0 Hz or less. This is a design that is one or more orders of magnitude stricter than in the past, and is not within a range that can be configured by simply combining known techniques. Therefore, each split magnet has a limit of 0.00 on the number of significant digits on the computer.
After a sufficient design study to generate a high-precision magnetic field of ppm, the optimal arrangement and combination of a coil composed of high-field superconducting wires such as Nb ₃ Sn and a superconducting coil for low-field composed of NbTi superconducting wires Constitute. There is no example of a general-purpose NMR apparatus configuration for solution protein analysis using a split magnet. The inventors have conducted intensive research and are the world's first time and spatial stability that can be applied to protein analysis, that is, proton nuclear magnetic resonance frequency within 1.0 Hz within the sample space for 1 hour. We found that this device configuration can achieve within 1.0 Hz per unit. The magnet optimization technology that we have accumulated eagerly enabled us to design a uniform magnetic field for complex split coil system systems, which was difficult in the past. The size of the magnet part including the cryogenic container can be gathered with a width of about 1 m and a height of about 1 m per unit, and a space-saving and highly integrated experimental device can be achieved while keeping the leakage magnetic field low. It was found that a high-throughput solution NMR apparatus configuration can be provided that can be configured and has approximately twice the data integration time.

  That is, a sample of protein or the like dissolved in a solution is inserted from the top in the vertical direction, and the sample is inserted into a sample tube having a diameter of 5 to 10 mm with a height of about 10 to 30 mm. A superconducting magnet, a high-frequency transmission coil, and a reception coil In the nuclear magnetic resonance analyzer for solution, the stationary magnetic field generated by the superconducting magnet is 11T or more, preferably 14.1T or more, the direction of the magnetic field generated by the superconducting magnet is horizontal, and is stationary. The fluctuation of the proton nuclear magnetic resonance frequency per hour due to the fluctuation of the magnetic field is 1.0 Hz or less, and the magnetic field uniformity in the sample space is 1.0 Hz or less at the proton nuclear magnetic resonance frequency. Is inserted and arranged in the vertical direction from the upper part, and the receiving coil is a solenoid coil installed near the center of the magnetic field from the lower part of the apparatus, so that the diameter is 5 to 10 m. A small amount of protein inserted in m sample tubes can be analyzed with high sensitivity.

  Further, in the present invention, a sample such as a protein dissolved in a solution is inserted from the upper part in the vertical direction, and the sample is inserted into a sample tube having a diameter of 5 to 10 mm with a height of about 10 to 30 mm. And a nuclear magnetic resonance analyzer comprising a receiving coil, the stationary magnetic field generated by the superconducting magnet is 11T or more, preferably 14.1T or more, the magnetic field direction generated by the superconducting magnet is horizontal, and The fluctuation of the proton nuclear magnetic resonance frequency per hour due to the fluctuation of the stationary magnetic field is 1.0 Hz or less, and the magnetic field homogeneity in the sample space is 1.0 Hz or less at the proton nuclear magnetic resonance frequency. The sample is installed in the center of the magnetic field from the upper part in the vertical direction, the receiving coil is a solenoid coil made of a superconducting material, and is arranged from the lower part of the device. It can be achieved by cooling below the onset temperature.

  Further, it is preferable that the organic sample is a polymer organic compound, protein, or ligand.

  Further, it is preferable that the superconducting magnet is a pair of split magnets that generate a magnetic field in the horizontal direction.

  Further, this can be achieved by using a toroidal magnet in which the superconducting magnet is disposed horizontally.

  Further, in the present invention, a sample such as a protein dissolved in a solution is inserted from the upper part in the vertical direction, and the sample is inserted into a sample tube having a diameter of 5 to 10 mm by a height of about 10 to 30 mm. In a nuclear magnetic resonance analyzer comprising a receiving coil, a stationary magnetic field generated by the superconducting magnet is 11T or more, preferably 14.1T or more, and the superconducting magnet is a toroidal magnet arranged in a horizontal direction, and The fluctuation of the proton nuclear magnetic resonance frequency per hour due to the fluctuation of the stationary magnetic field is 1.0 Hz or less, the magnetic field uniformity of the sample space is 1.0 Hz or less at the proton nuclear magnetic resonance frequency, and a plurality of the solutions Solenoids in which a cylindrical sample is arranged at substantially equal intervals around the circumference of the toroidal coil, and the receiving coil corresponding to each sample is made of a superconducting material A coil, which is arranged from the lower part of the apparatus and can be achieved by cooling to a temperature lower than the superconducting expression temperature.

  In addition, the superconducting magnet is a horizontal toroidal magnet, and the magnetic field strength applied to the sample is adjusted so that the nuclear magnetic resonance signals generated from a plurality of adjacent samples can be clearly distinguished. Can be achieved.

  Further, the configuration of the present invention is characterized in that in the nuclear magnetic resonance analyzer for solution, the solution sample is arranged from the upper part of the apparatus to the magnetic field center, the detection coil is arranged from the lower part of the apparatus to the magnetic field center, and the magnetic field direction Is in the horizontal direction, and the superconducting magnet is in the nuclear magnetic resonance analyzer for solution, which is divided into left and right.

  The above and other features of the present invention are not limited to the above description, but will be further described below.

According to the present invention, the measurement sensitivity of the NMR signal is at least 40% or more of the conventional measurement at about 600 MHz (14.1 T) in a state where the sample solution is mainly filled with a sample solution of about 30 mm in height using a normal 5-10 mm diameter sample tube. It is possible to provide a novel nuclear magnetic resonance analyzer that can increase the temporal stability and spatial uniformity of a superconducting magnet necessary for protein analysis. further,
When operating at 21.1T, that is, 900 MHz at 1.8K, the sensitivity can be improved by 40% compared to the conventional method, and the detection sensitivity limit due to the magnetic field strength, which was impossible in the past, can be greatly broken. It became possible for the first time.

Example 1
A first embodiment of the present invention is shown in FIG. In the superconducting magnets 1, 2 and 3, the coil is formed of a material having a higher superconducting critical magnetic field toward the inside closer to the sample. For example, although 1 is Nb ₃ Al, 2 is Nb ₃ Sn, and 3 is NbTi, an optimal combination may be made so that the generated magnetic field and the uniformity of the combination coil become a desired value as necessary. For example, a Bi-based material such as Bi ₂ Sr ₂ CaCu ₂ O ₉ or a superconducting material such as Y ₁ Ba ₂ Cu ₃ O ₇ -based material or MgB ₂ may be used. A superconducting magnet composed of these combinations has a horizontal magnetic field generation direction. In FIG. 1, a protein sample 4 dissolved in an aqueous solution filled with a 30 mm height in a glass sample tube having a diameter of 5 to 10 mm and having magnetic properties equivalent to water is inserted from the upper part of the apparatus into the center of the magnetic field in the vertical direction. The magnetic field is applied to the sample from the lateral direction. Accordingly, each superconducting magnet is wound in a solenoid shape with the horizontal direction as a winding axis, and is arranged symmetrically. The maximum width of the magnet is 400 mm, and the maximum height is 700 mm. At the center of the magnet, the uniformity of the magnetic field is adjusted to 0.001 ppm or less and 0.5 or less when expressed in terms of proton nuclear magnetic resonance frequency, and the time stability is 0.001 ppm / h or less, or 0.001 ppm or less when expressed in terms of proton nuclear magnetic resonance frequency. 5 Hz / hour or less. In this case, if necessary, a coil for adjusting the uniformity may be disposed near the center of the magnetic field. The adjustment may be performed by using a lead wire in the normal temperature part, or by using another superconducting wire in the low temperature part, or by combining both. For example, when used as NMR of proton nuclear magnetic resonance frequency of 600 MHz, the central magnetic field is approximately 14.1 T, the magnetic field uniformity is 18 mm sphere, and the proton nuclear magnetic resonance frequency is 0.5 Hz or less. Under these conditions, the operating temperature of the coil is 4.2 K, and liquid helium pumping is not necessary and can be operated easily. The sample is inserted from the vertical direction.

According to this method, since the magnetic field is generated in the horizontal direction and the sample is inserted from the upper part in the vertical direction, there is no fear of spilling the solution stored in the test tube. In addition, since the detection coil is inserted from the bottom, a sufficient sample space can be secured, and the sample space can be utilized to the maximum extent possible for measurements that require sensitivity for protein analysis. . Further, the sample may be rotated depending on the measurement conditions. In addition, even when the detection coil system is cooled to a low temperature, the sample concentration and additives can be continuously changed by this arrangement, making it easy to change various conditions in protein interaction studies. Can be implemented. For the detection of the NMR signal, a copper solenoid coil kept at room temperature or a solenoid coil made of Y-based or MgB ₂ cooled to 10 to 20K is used. It is inserted in the center, arranged around the glass tube containing the sample solution, and is configured to send the detected signal to the outside via the signal cable 6. By inserting from the bottom of the device, the sample space can be increased and vibration noise from the measurement system to the sample can be reduced. The superconducting magnet is held in the permanent current mode by the permanent current switch 9, and the temporal fluctuation of the magnetic field is adjusted to 0.5 Hz or less per hour. The superconducting magnet is immersed in liquid helium 7 and held at a low temperature, and has a low temperature capacity that saves helium consumption by forming a double structure in which the outside is covered with liquid nitrogen 8. Instead of cooling with liquid helium, the superconducting magnet may be directly cooled using a refrigerator having no vibration problem such as a pulse tube refrigerator. Note that reducing the leakage magnetic field around the magnet is important from the standpoints of installation and safety of the apparatus, and a magnet configuration provided with a magnetic shield adapted to the installation conditions can be obtained.

  As described above, in this embodiment, with such a configuration, a central magnetic field was 14.1 T and a proton nuclear magnetic resonance frequency of 600 MHz was obtained. However, a conventional NMR system as shown in the comparative example of FIG. Compared with the signal-to-noise ratio (S / N) ratio, it can be improved by a factor of approximately 1.4, and when compared by NMR signal integration measurement of a sample in which a protein with a molecular weight of 20K of the same sample concentration is dissolved The data integration time of the same level has been doubled. This was the same measurement time performance as the case where the same experiment was observed with an NMR apparatus equivalent to 840 MHz. Moreover, since the interference with other analytical instruments around the apparatus is reduced by applying the magnetic shield, the effect of improving the installation density of the instruments has been recognized.

  In the case of 600 MHz, the 5 gauss line of the leakage magnetic field from the apparatus was 2 m in the vertical direction and 3 m at the maximum in the horizontal direction. As a result, NMR data having the same quality as 840 MHz can be installed without using a special dedicated building.

(Example 2)
A second embodiment of the present invention is shown in FIG. In this embodiment, the configuration is substantially the same as that of the first embodiment, but the cryogenic container is divided by the left and right superconducting magnets so that the user's use space has releasability. In other words, unlike the conventional sealed sample space, there is an open space around the sample chamber. For example, dynamic protein behavior can be measured while easily irradiating the sample with light or laser light. it can. Since such a dynamic NMR signal can be observed, for example, protein signal transduction and photosynthetic reaction can be examined. In the case of such a special experiment, the superconducting magnet can be operated with a central magnetic field (21.1 T) of about 900 MHz by pumping liquid helium and cooling it and operating at 1.8 K. it can. The detection sensitivity in this case is equivalent to 1.26 GHz NMR when converted to the conventional NMR apparatus of FIG. 7, is comparable to the strong magnetic field of 29.6 T, and greatly exceeds the critical magnetic field of the conventional superconducting material. For this reason, such a detection sensitivity cannot be achieved with the conventional method, but according to the present invention, a high level of detection sensitivity that cannot be achieved with the prior art can be realized with a magnetic field strength of 900 MHz (21.1 T). Even in this case, there is a large difference in the leakage magnetic field in the vertical direction of the apparatus compared with the conventional 900 MHz class NMR apparatus. In the present invention, the vertical direction is 3 m, the horizontal direction is the maximum in the coil axis direction. It was 4.5 m.

(Example 3)
A third embodiment of the present invention is shown in FIG. In the present embodiment, eight pairs of superconducting magnets 11 are arranged in a horizontal toroidal shape, whereby the leakage magnetic field can be reduced and the degree of integration of the device is increased. That is, eight NMR analyzers are installed side by side in one cryogenic container cooled to liquid helium. The details of the configuration of each of the eight pairs of split magnets may be the same as those in the first embodiment. For example, approximately 600 MHz is generated, but preferably, each of the adjacent NMR apparatuses is based on 600 MHz. It is desirable to change the frequency by 10 Hz. For example, 610, 620, 630 MHz, etc. Thereby, even if the sample 12 is the same, there exists an effect which can distinguish each other's NMR resonance signal between different apparatuses. With such a configuration, highly integrated NMR analyzers can be aggregated in a space with a diameter of 5 m to 10 m. Therefore, it is possible to provide a high-throughput NMR analysis system having a large economic effect and low installation property and maintenance cost.

Example 4
A fourth embodiment of the present invention is shown in FIG. In this example, an example of the NMR system configuration of the present invention is shown. The sample is configured so that an automatic supply device 13 for solution sample can be used, and for example, an arbitrary concentration or an additive such as a substrate, a ligand, or a metal element can be applied using a flow cell or the like. Thereby, it is possible to make an apparatus configuration suitable for protein interaction studies. The NMR signal is sent to the analyzer 15 via the preamplifier 14 and controlled by the workstation 16. Various pulse sequences are applied for NMR signal acquisition. If necessary, pulses are emitted and the pulse sequence can be combined with a gradient magnetic field. In order to reduce the thermal noise of the signal, the preamplifier 14 can be cooled to about the liquid nitrogen temperature and used.

(Example 5)
A fifth embodiment of the present invention is shown in FIG. FIG. 5 shows details of the detection probe configuration of the present invention. Sample 4 is a protein dissolved in an aqueous solution. Sample 4 is inserted into a glass tube with a diameter of 5 to 10 mm having the same properties as water, and is located at the center of the magnetic field from the lower part of the apparatus. The solenoid-shaped detection coil 17 is made of copper or superconducting wire. In the case of a superconducting wire, it is cooled to about 10 to 20 K with helium gas 25. In the case of copper wire, it is kept at ordinary temperature. The sample is rotated as necessary.

  An arbitrary magnetic field gradient can be applied in an arbitrary direction by a combination of the three-axis gradient magnetic field coils 19, 20, 21, 22, 23, and 24, and is used for analysis in combination with a pulse sequence. The detected signal is sent to the preamplifier via the lead wire 18.

  According to the present invention, the sensitivity can be improved without using a detection coil using a superconducting wire, so that it is excellent in maintainability, and the light irradiation experiment described in the second embodiment is also possible. . Further, by making the detection coil solenoid-shaped and superconducting, the sensitivity can be improved by 40% or more. For example, when applied at 900 MHz (21.1 T), it corresponds to the conventional 1.26 GHz (29.6 T). Since the detection sensitivity can be obtained, there is an effect that it is possible to realize the detection sensitivity that has been impossible to reach in the past.

(Example 6)
A sixth embodiment of the present invention is shown in FIG. The basic configuration is the same as that of the second embodiment of the present invention. In order to reduce the leakage magnetic field, it is combined with an iron magnetic shield. By combining with the return yoke 26 using iron, there is an effect of reducing the leakage magnetic field to the periphery within 2 m. In addition, you may utilize the active shield using a superconducting magnet as needed for a magnetic shield. There is an advantage that the weight is lighter than iron. Also, iron and active shield can be used in combination.

(Comparative Example 1)
As an NMR apparatus configuration of a comparative example of the present invention, a configuration of a 600 MHz class NMR apparatus is shown in FIG. The central magnetic field is 14.1T. The birdcage-shaped receiving coil 27 and superconducting magnets 28, 29, and 30 are multilayer air-core solenoid coils. The only way to improve the sensitivity equivalent to the present invention with this detection method is to increase the central magnetic field, but at present, if 21T is the limit and the same frequency (central magnetic field strength), the present invention and In comparison, the detection sensitivity is inferior by about 40%. The uniformity and temporal stability of the magnetic field were 0.5 Hz or less necessary for protein analysis.

(Comparative Example 2)
As a configuration of the NMR apparatus of the comparative example of the present invention, the configuration of the birdcage probe 27 is shown in FIG. Although a saddle-shaped coil may be used, it is impossible to obtain the same detection sensitivity as that of Example 1 of the present invention by the method in combination with Comparative Example 1 in terms of sensitivity. In order to improve the sensitivity by this detection method, there is no method other than lowering the temperature. At present, a method using superconductivity has been proposed, but there are problems of cost increase and maintainability. Further, the detection sensitivity is approximately 40% lower than that of the solenoid type detection coil of the present invention.

(Comparative Example 3)
A comparative example of the present invention is shown in FIG. Using a general-purpose NMR solenoid coil that generates a uniform magnetic field in the horizontal direction, combined with the birdcage type detection coil of Comparative Example 2 as a horizontal magnetic field type arrangement, an aqueous solution of protein is measured from a horizontal communication hole and a test tube is measured Inserted into. A nuclear magnetic resonance signal of approximately 400 MHz was obtained by the magnetic field at the measurement site, but when the sample space had a diameter of 10 mm and a length of 20 mm, an error of 4 Hz caused a spatial non-uniformity corresponding to the error. Moreover, the nonuniformity of the magnetic field per hour was 3 Hz / h. These values are standard values for a general-purpose NMR apparatus, but are insufficient for the interaction analysis of the target protein of the present invention. In addition, the liquid level of the solution in the test tube moved in the horizontal direction during the measurement, and it was often impossible to perform stable measurement.

(Comparative Example 4)
A comparative example of the present invention is shown in FIG. Using a general-purpose NMR solenoid coil that generates a uniform magnetic field in the horizontal direction, the horizontal magnetic field type arrangement is combined with the birdcage type detection coil of Comparative Example 2 through the communication hole from the top of the apparatus to the measurement site. Inserted into test tube. Because the measuring instrument was inserted from the top, the maximum diameter of the communication hole was increased to 200 mm. As a result, the central magnetic field strength decreased. Even if the magnetic field intensity was tuned, only a 300 MHz nuclear magnetic resonance signal could be obtained at the center of the magnetic field. Further, when the sample space had a diameter of 10 mm and a length of 20 mm, an error of 5 Hz was non-uniform in magnetic field corresponding to the error. These values are standard values for a general-purpose NMR apparatus, but are insufficient for protein interaction analysis.

(Comparative Example 5)
A comparative example of the present invention is shown in FIG. Two general-purpose 18T-class NMR solenoid coils that generate a uniform magnetic field in the vertical direction were used in combination with a horizontal magnetic field type arrangement. Then, from the upper part of the apparatus to the measurement site, a 70 mm diameter communication hole was passed through and combined with the solenoid type detection coil of the present invention, and an aqueous protein solution was inserted into the test tube. From the top, all instrument and solution samples were inserted. As a result, the effective space of the sample was narrowed to 1 mm in diameter and 20 mm in length. Compared with a sample tube having a diameter of 5 to 10 mm, which is used in a normal solution NMR apparatus, the sample volume can only take 1/25 to 1/100, and the signal intensity from the sample is 1 /
Although it decreased to 25 to 1/100 and aimed at improving the sensitivity, conversely, a significant decrease in sensitivity was caused. On the other hand, in the direction of the central axis of the two magnet systems, the maximum magnetic field of each magnet was 18T. The temporal stability of the magnetic field was about 0.001 ppm / h. However, the steady magnetic field strength of the sample space is 7.5T, and the uniformity of the sample space magnetic field is 100.
It was below ppm, and it was a level that could not be used for measurement as NMR that could be used for protein analysis.

(Comparative Example 6)
A comparative example of the present invention is shown in FIG. This is an example in which NMR measurement of an aqueous protein solution is configured using a solenoid-wound detection coil 31 combined with a high-temperature superconducting bulk magnet 32 that generates a magnetic field in the vertical direction. The feature is that the solenoid coil is largely wound out of the magnetic field uniform region, and it captures changes in a wide range of magnetic flux, but lacks measurement accuracy compared to the case where the detection coil is placed in a uniform magnetic field. . In the bulk magnet, the maximum magnetic field of the magnet was 10T, but the maximum magnetic field in the sample space was 4T. The uniformity of the magnetic field was 200-500 ppm or less due to the non-uniformity of the high temperature superconductor. The temporal stability of the magnetic field was about 20 to 100 ppm / h 2 due to the magnetic flux creep phenomenon of the high-temperature superconductor. By these, as NMR which can be used for protein analysis, it was a system which cannot be used for measurement at all.

As described above, with recent progress in protein research, there is an increasing need for structural analysis of complex compounds having a large molecular weight. Therefore, the performance required for NMR is increasing year by year, and the central magnetic field of NMR is increasing to improve detection sensitivity. In principle, the detection sensitivity increases in proportion to the 1.7th power of the magnetic field, but actually increases in proportion. Existing superconducting materials are reaching the limit of sensitivity improvement, and new detection methods and detection devices that do not depend on the magnetic field strength have been demanded. In the present invention, in solution NMR for protein analysis, the detection sensitivity is improved by at least 40% compared with the conventional one without increasing the central magnetic field strength. As the method, the solution sample was placed in the center of the magnetic field from the upper part of the apparatus, the detection coil as a solenoid coil from the lower part of the apparatus, and the magnetic field direction was the horizontal direction. In addition, the magnetic field was set to a proton nuclear magnetic resonance frequency required for protein analysis of 1.0 Hz per hour or less, and the uniformity in the sample space was 1.0 Hz or less. The superconducting magnet was divided into left and right. That is, the shape of the detection coil is changed from the conventional birdcage type 27 to the solenoid system 4 with higher sensitivity. For this purpose, the superconducting magnet is not divided into the conventional multilayer air-core solenoids 28, 29 and 30, but is composed of split magnets composed of superconducting magnets 1, 2 and 3 which are divided into left and right and generate 11T in the horizontal direction, preferably 14.1T or more. . The magnetic field uniformity was set to 0.001 ppm or less and the time stability was set to 0.001 ppm or less. By using a solenoid coil for the detection coil, it is possible to perform high-sensitivity measurement 1.4 times higher than the conventional one with the same central magnetic field (resonance frequency). , 29.6T equivalent NMR detection sensitivity can be realized at 900 MHz (21.1T).

  According to the present invention, it becomes possible to analyze the interaction between proteins and to perform NMR measurement with ultra-high sensitivity that is 40% increase compared to the conventional method. For example, if the central magnetic field is operated at 900 MHz (21.1 T), which is the highest level of the current superconducting technology, detection sensitivity equivalent to the conventional 1.26 GHz (29.6 T) can be obtained. For the first time, it is possible to reach the ultra-high detection sensitivity that could not be achieved with the improved sensitivity method.

The present invention is also suitable for protein interaction screening studies. In the post-genomic era, it is considered that competition for structural analysis of proteins will progress, and when the era of using proteins whose structures have been clarified is reached, the need for mutual relationships among known proteins, that is, interaction screening will increase. It is expected. Specifically, it is widely applied in tailor-made drug discovery, bioindustry, food and medical fields. In the near future 5 to 10 years later, under the circumstances where the elucidation of the three-dimensional structure of proteins has progressed, it is predicted that new drug development (so-called drug discovery research) that actively utilizes the structural information will proceed. By utilizing the NMR measurement technology of the present invention, in the development of new drugs in such an era, the discovery of unknown interactions between proteins and ligands or low-molecular compounds whose structures have been known is efficiently promoted. it can. For the creation of new drugs, based on new interactions found in NMR, the development of optimal new drugs using advanced biotechnology will be promoted through molecular design support combined with computational simulation. These methods will greatly reduce the development cost and duration of new drugs that effectively act on the human body. The ripple effect of such ripple effects on Japan and the world's humanity is immeasurable. From the technical viewpoint, according to the NMR technique of the present invention, the detection sensitivity is improved to 40% or more as compared with the conventional technique, so that the integration time can be reduced to ½. Therefore, in addition to the above-described interaction detection, it is possible to efficiently study the influence of trace metals on the living body. As a specific example, trace elements and proteins in the living body can be traced in real time by monitoring the effects of metal elements at the same concentration in the living body on the presence of proteins and the dynamics of labeled proteins and trace elements in the living body. There is a possibility that it can be applied to the development of therapeutic methods such as Alzheimer's disease and early diagnosis of chronic / refractory diseases (such as diabetes and Creutzeft Jacob disease) before the onset of the disease. Moreover, according to this measurement technique, the maintenance property and installation property as a measurement device can be greatly improved, and the introduction merit is great. In particular, even in a relatively small experimental facility, it is possible to acquire high-quality data that is close to a large experimental device of 900 MHz class.

1 is a structural example of an NMR analysis system of the present invention. 1 is a configuration example of an open NMR analysis system of the present invention. 1 shows a configuration example of a toroidal NMR analysis system of the present invention. The NMR signal detection system structural example of this invention. The NMR detection coil system structural example of this invention. The magnetic shielding structural example of the NMR analysis system of this invention. The NMR analysis system configuration of a comparative example. The detection coil structure of a comparative example. The NMR system combination structure of a comparative example. The NMR system combination structure of a comparative example. The NMR system combination structure of a comparative example. The NMR system combination structure of a comparative example.

Explanation of symbols

1 ... magnetic field generator direction horizontal direction Nb ₃ Al superconducting magnets, 2 ... magnetic field generating direction horizontal direction Nb ₃ Sn superconducting magnets, 3 ... magnetic field generator direction horizontal direction NbTi superconducting magnet, 4 ... in an aqueous solution Dissolved protein sample, 5 ... solenoid type NMR signal detection coil, 6 ... signal line, 10 ... open space, 11 ... toroidal superconducting magnet, 12 ... sample and detection probe, 14 ... preamplifier, 15 ... analyzer, 17 ... solenoid type Detection coil, 26 ... Leakage magnetic shield.

Claims (8)

  A sample such as a protein dissolved in a solution is held in a sample tube having a diameter of 5 to 10 mm and is inserted from a substantially vertical direction, and is composed of a superconducting magnet, a high-frequency transmitting coil, and a receiving coil. In the analyzer, the stationary magnetic field generated by the superconducting magnet is 11 T or more, the direction of the stationary magnetic field generated by the superconducting magnet is horizontal, and 1 of the proton nuclear magnetic resonance frequency due to the fluctuation of the stationary magnetic field. The fluctuation per time is 1.0 Hz or less, and the uniformity of the stationary magnetic field in the sample space is 1.0 Hz or less at the proton nuclear magnetic resonance frequency. A nuclear magnetic resonance analyzer for solution, wherein the apparatus is a solenoid coil inserted into the center of the magnetic field and the receiving coil is inserted into the center of the magnetic field from the lower part of the apparatus.   A nuclear magnetic resonance analyzer comprising a superconducting magnet, a high-frequency transmitting coil, and a receiving coil, in which a sample such as a protein dissolved in a solution is held in a sample tube having a diameter of 5 to 10 mm and is inserted from a vertical direction. , The stationary magnetic field generated by the superconducting magnet is 11 T or more, the direction of the stationary magnetic field generated by the superconducting magnet is horizontal, and the fluctuation of the proton nuclear magnetic resonance frequency per hour due to the fluctuation of the stationary magnetic field Is 1.0 Hz or less, and the homogeneity of the stationary magnetic field in the sample space is 1.0 Hz or less in proton nuclear magnetic resonance frequency, and the solution-like sample is inserted into the center of the magnetic field from the upper part in the vertical direction. The receiving coil is a solenoid coil made of a superconducting material, and is inserted into the center of the magnetic field from the lower part of the apparatus and cooled to a temperature lower than the superconducting expression temperature. The solution for nuclear magnetic resonance analyzer for.   3. The nuclear magnetic resonance analyzer for solution according to claim 1, wherein the organic sample is a polymer organic compound, protein, or ligand.   3. The nuclear magnetic resonance analyzer for solution according to claim 1, wherein the superconducting magnet is a pair of split magnets that generate a magnetic field in a horizontal direction.   3. The nuclear magnetic resonance analyzer for solution according to claim 1, wherein the superconducting magnet is a toroidal magnet arranged horizontally.   A sample such as a protein dissolved in a solution is held in a sample tube having a diameter of 5 to 10 mm and is inserted from the upper part in the vertical direction, and is composed of a superconducting magnet, a high-frequency transmitting coil, and a receiving coil. In the analyzer, a steady magnetic field generated by the superconducting magnet is a toroidal magnet in which the superconducting magnet is arranged in the horizontal direction, and fluctuations in proton nuclear magnetic resonance frequency per hour due to fluctuations in the steady magnetic field. Is 1.0 Hz or less, the magnetic field homogeneity of the sample space is 1.0 Hz or less at the proton nuclear magnetic resonance frequency, and a plurality of the solution-like samples are arranged at substantially equal intervals around the toroidal coil. The receiving coil corresponding to each sample is a solenoid coil made of a superconducting material, and is inserted into the center of the magnetic field from the lower part of the device. The solution for nuclear magnetic resonance analysis apparatus characterized by being cooled below expression temperature.   7. The toroidal magnet in which the superconducting magnet is horizontally arranged according to claim 6, wherein the magnetic field strength applied to the sample is adjusted so that nuclear magnetic resonance signals generated from a plurality of adjacent samples can be clearly distinguished. A nuclear magnetic resonance analyzer for solution.   In the nuclear magnetic resonance analyzer for solution, the solution sample is arranged from the upper part of the apparatus to the magnetic field center, the detection coil is arranged from the lower part of the apparatus to the magnetic field center, the magnetic field direction is horizontal, and the superconducting magnet is left and right. A nuclear magnetic resonance analyzer for a solution having a divided configuration.

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