Apparatus for generating shear flow

Abstract

To provide an instrument which generates a shear flow in a sample and enables NMR lock without affecting the composition of the sample to be measured.SOLUTION: The shear flow generator 10 is an instrument for generating a shear flow in a sample S to be measured in a state of being inserted into a sample introduction hole 91 of a magnetic resonance device 90, and includes a sample tube 1 for storing the sample S to be measured, a stirring tube 2 which is a bottomed tube for storing a reference sample for NMR lock, has a bottomed tip 2a side inserted into the sample tube 1, and applies a shear force to the sample S by rotating around a long axis, and a holding portion 3 for holding both the sample tube 1 and the stirring tube 2.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to an apparatus for generating shear flow in a sample within a sample tube located within a magnetic resonance instrument. [Background technology]

[0002] Rheo-NMR (hereinafter referred to as Rheo-NMR) is a measurement technique that performs nuclear magnetic resonance while generating a shear flow in a sample containing a measurement target.

[0003] Rheo-NMR measurements enable the investigation of samples under shear flow, enabling the identification and analysis of dynamic phenomena such as the shear rate, shear viscosity, and shear-thinning of the sample. Most previous Rheo-NMR measurements have focused on polymers and food samples. In recent years, Rheo-NMR has been used to analyze the fluidity of blood within blood vessels, for example, in the field of biorheology, a field that studies the function and shape of living organisms from the mechanical aspects of deformation and fluidity.

[0004] A rheology unit used in rheo-NMR measurements is described in the following Patent Document 1. The following Non-Patent Documents 1 and 2 describe a rheometer or Couette cell used in rheo-NMR measurements.

[0005] As shown in Non-Patent Document 3, rheo-NMR measurements can be performed using existing NMR instruments. The sample to be measured is placed in a sample tube called an NMR tube. The sample tube is attached to a rotating device called a spinner and placed at a predetermined measurement position in the NMR instrument. A stirring rod, thinner than the sample tube, is inserted into the sample introduction hole, which is located vertically in the NMR instrument, from the upper open end, and the tip of the stirring rod is inserted into the sample in the sample tube. Many existing NMR instruments are equipped with a spinner and a drive mechanism that rotates the spinner. This drive mechanism, installed on the NMR instrument, is used to rotate the spinner and sample tube relative to the stirring rod, generating shear flow in the sample. By performing NMR measurements in this environment, rheo-NMR measurements of the sample can be performed. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2016-529517 [Non-patent literature]

[0007] [Non-Patent Document 1] Paul T. Callaghan and Elmar Fischer, "Rheo-NMR: a New Application for NMR Microscopy and NMR Spectroscopy", [online], 2001, Bruker Corporation, [Retrieved March 29, 2019], Internet <URL:https: / / www.bruker.com / fileadmin / user_upload / 8-PDF-Docs / MagneticResonance / NMR / Rheo-NMR_Report2001.pdf> [Non-patent document 2] Patrick JB Edwards, Motoko Kakubayashi, Robin Dykstra, Steven M. Pascal, and Martin AK Williams, “Rheo-NMR Studies of an Enzymatic Reaction: Evidence of a Shear-Stable Macromolecular System”, Biophysical Journal, Volume 98, Issue 9, 5 May 2010, Pages 1986-1994. [Non-patent document 3] Daichi Morimoto, Erik Walinda, Naoto Iwakawa, Mayu Nishizawa, Yasushi Kawata, Akihiko Yamamoto, Masahiro Shirakawa, Ulrich Scheler, and Kenji Sugase, “High-Sensitivity Rheo-NMR Spectroscopy for Protein Studies”, Analytical Chemistry, Volume 89, Issue 14, June 30, 2017, Pages 7286-7290. Summary of the Invention [Problem to be solved by the invention]

[0008] In an NMR instrument, a sample tube is placed in a generally uniform static magnetic field generated by a superconducting magnet. However, the magnitude of the static magnetic field generated by the superconducting magnet gradually drifts over time. This causes the resonance frequency of the NMR signal to also drift over time, resulting in a decrease in the resolution of the spectrum obtained by integrating the NMR signal over a long period of time.

[0009] NMR locking is performed to prevent such a decrease in resolution. NMR locking is performed by observing the frequency of a specific NMR signal as a reference signal and correcting the amount of resonance frequency drift based on this observed frequency. Deuterium, for example, is used as a reference sample for observing the reference signal. Deuterium as a reference sample is added to the sample to be measured, for example, in the form of various compounds. In Rheo-NMR measurements, as in regular NMR measurements, a reference sample is added to the sample to be measured, for example, in the form of a deuterated solvent.

[0010] One of the targets of measurement by Rheo-NMR is protein stability. However, when a heavy solvent (deuterium) used for NMR locking is added to a protein solution, the composition of the protein solution changes.

[0011] It is an object of the present invention to provide an apparatus for generating shear flow in a sample, which allows for NMR locking without affecting the composition of the sample being measured. [Means for solving the problem]

[0012] In order to achieve the above object, the present invention includes, for example, the following aspects. (Section 1) An apparatus for generating a shear flow in a sample to be measured while being inserted into a sample introduction hole of a magnetic resonance apparatus, a sample tube for storing a sample to be measured; a stirring tube which is a bottomed tube for storing a reference sample for NMR lock, the tip side of which is inserted into the sample tube and rotates around its longitudinal axis to apply shear force to the sample; a holder that holds both the sample tube and the stirring tube. (Section 2) Item 1. The apparatus according to item 1, wherein the holding portion holds the sample tube and the stirring tube coaxially. (Section 3) The holding portion is a cylindrical stirring tube holder that holds the rear end side of the stirring tube; a cylindrical sample tube holder for holding the sample tube; Item 1. The apparatus according to item 1, comprising a cylindrical housing that holds the stirring tube holder and the sample tube holder. (Section 4) the housing has a first cavity and a second cavity communicating with the first cavity along a longitudinal axis direction; the stirring tube holder is fitted into the second cavity in the housing, Item 4. The apparatus according to item 3, wherein the rear end of the sample tube holder fits into the first cavity in the housing. (Section 5) the agitation tube holder rotatably holds the rear end side of the agitation tube inserted therein; Item 4. The device according to item 3, wherein the sample tube holder holds the sample tube inserted therein. (Section 6) Item 4. The apparatus described in Item 3, wherein the stirring tube holder has a third cavity at the front end side that accommodates the rear end side of the sample tube, and a fourth cavity at the rear end side that accommodates a bearing that rotatably holds the stirring tube. (Section 7) a main shaft connected to the stirring tube and transmitting rotational power around the long axis to the stirring tube; a cylindrical spindle housing portion that houses the spindle, Item 7. The instrument according to any one of Items 1 to 6, wherein the spindle housing has a circumferential groove on its side, and a ring-shaped elastic member is disposed in the groove, protruding from the groove and elastically deforming to fit the opening end of the sample introduction hole. (Section 8) Item 8. The instrument according to item 7, wherein the main shaft housing has a hole extending along the longitudinal axis on its side. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide an apparatus for generating shear flow in a sample, which allows NMR locking without affecting the composition of the sample being measured. [Brief explanation of the drawings]

[0014] [Figure 1]1 is a schematic diagram illustrating a shear flow generating device according to one embodiment. FIG. [Figure 2] 1 is a schematic cross-sectional view illustrating a state in which a shear flow generating device according to one embodiment is used. FIG. [Figure 3] FIG. 3 is a partial enlarged view of the lower region of the two regions enclosed by the dashed dotted lines in FIG. 2. [Figure 4] FIG. 3 is a partial enlarged view of the upper region of the two regions surrounded by dashed lines in FIG. 2, and is a schematic view for explaining the state in which a spindle housing part according to one embodiment is inserted into a sample introduction hole of a magnetic resonance apparatus. [Figure 5] FIG. 2 is a cross-sectional view of a holding portion according to one embodiment. [Figure 6] FIG. 2 is a cross-sectional view of an agitation tube holder according to one embodiment. [Figure 7] FIG. 2 is a cross-sectional view of a sample tube holder according to an embodiment. [Figure 8] FIG. 2 is a cross-sectional view of an assembled state of a spindle, a spindle housing, and a tip position adjustment mechanism according to one embodiment. [Figure 9] FIG. 2 is a cross-sectional view of a spindle housing according to one embodiment. [Figure 10] FIG. 10 is a cross-sectional view of a tip position adjustment mechanism according to one embodiment. [Figure 11] 10A and 10B are diagrams illustrating a cylindrical portion that constitutes the tip position adjustment mechanism. [Figure 12] 10A and 10B are diagrams showing a handle used to adjust the position of the cylindrical portion. [Figure 13] 2 is a diagram illustrating an electric motor mounting portion and an electric motor according to an embodiment. FIG. [Figure 14] 5 is a schematic cross-sectional view for explaining a state in which the electric motor mounting portion and the electric motor are used. FIG. [Figure 15] 1 shows test results regarding performance evaluation of Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description and drawings, the same reference numerals will denote the same or similar components, and therefore, redundant descriptions of the same or similar components will be omitted.

[0016] Fig. 1 is a schematic diagram showing a shear flow generating device according to one embodiment. Fig. 2 is a schematic cross-sectional view illustrating a state in which the shear flow generating device according to one embodiment is in use. Fig. 3 is a partially enlarged view of a lower region of two regions enclosed by dashed lines in Fig. 2. Fig. 4 is a partially enlarged view of an upper region of two regions enclosed by dashed lines in Fig. 2, and is a schematic diagram illustrating a state in which a spindle housing part according to one embodiment is inserted into a sample introduction hole of a magnetic resonance spectrometer.

[0017] [Outline of the equipment] As shown in FIGS. 1 to 3 , an instrument 10 for generating a shear flow in a sample (hereinafter referred to as shear flow generating instrument 10 or simply instrument 10) according to one embodiment of the present invention is an instrument that generates a shear flow in a sample S while inserted into a sample introduction hole 91 of a magnetic resonance spectrometer 90. As shown in the partially enlarged view of FIG. 3 , the shear flow generating instrument 10 includes a sample tube 1 for storing a sample S to be measured, a stirrer tube 2, which is a bottomed tube for storing a reference sample R for NMR locking, the bottomed tip of which is inserted into the sample tube 1 and rotates about its longitudinal axis to apply a shear force to the sample S, and a holder 3 for holding both the sample tube 1 and the stirrer tube 2. In the following description, the longitudinal axis direction of the instrument 10 is the Z-axis direction shown in the drawings. For each component of the instrument 10, the tip side is the Z1 direction and the rear side is the Z2 direction.

[0018] In a shear flow generating device 10 according to one embodiment, a sample S to be measured is stored at the bottom of a bottomed sample tube 1. A reference sample R is also stored at the bottom of a bottomed stirring tube 2. As shown in the partially enlarged view of FIG. 3 , the sample S and the reference sample R are separated by the wall and bottom of the stirring tube 2, and the sample S and the reference sample R do not mix. As a result, the shear flow generating device 10 according to one embodiment enables NMR locking without affecting the composition of the sample S to be measured in a rheo-NMR measurement in which nuclear magnetic resonance is performed while generating a shear flow in the sample S.

[0019] Furthermore, the shear flow generating device 10 according to one embodiment can provide accurate alignment between the sample tube 1 and the stirring tube 2.

[0020] Furthermore, the shear flow generating device 10 according to one embodiment can provide accurate alignment of the sample tube 1 and the stirring tube 2. Generally, high-resolution NMR instruments for sample analysis use superconducting magnets to improve measurement sensitivity. To generate high magnetic fields of several tens of tesla or more, a refrigerant such as liquid helium or liquid nitrogen is disposed around the superconducting magnet. As a result, NMR instruments using superconducting magnets are large, exceeding two meters in height. The position of the sample tube cannot be seen from outside the NMR instrument. Meanwhile, the sample tube, which contains the sample and rotates with the spinner, is cylindrical with an outer diameter of approximately 5 mm, and the inner diameter, into which the tip of the stirring rod is inserted, is approximately 4 mm. The stirring rod is even thinner than the sample tube, with an outer diameter of approximately 3 mm.

[0021] Thus, in a typical high-resolution NMR instrument for sample analysis, the gap between the inner diameter of the sample tube and the outer diameter of the stirrer rod is extremely narrow, about 0.5 mm. The vertical position within the NMR instrument where the sample tube is placed is also several meters away from the open end of the vertically extending sample introduction hole. It is difficult to insert the tip of a stirrer rod from outside the NMR instrument through the open end of the sample introduction hole into a sample tube placed at the measurement position within the NMR instrument with an error of within about 0.5 mm from a position several meters away. This task is even more difficult because it requires not only the insertion of the tip of the stirrer rod into the sample tube but also the precise alignment of the center of the diameter of the sample tube with the center of the diameter of the stirrer rod, which increases the difficulty even further. Furthermore, in some NMR instrument models, the sample introduction hole has a reverse tapered shape, with its diameter increasing slightly from the upper open end to the lower end. This reverse tapered shape makes it even more difficult to precisely align the center of the diameter of the sample tube with the center of the diameter of the stirrer rod at the position where the sample tube is placed.

[0022] According to one embodiment of the shear flow generating device 10, in rheo-NMR measurements, not only can NMR lock be performed without affecting the composition of the sample S to be measured, but also precise alignment of the sample tube 1 and the stirring tube 2 can be provided.

[0023] [Fixture details] The following describes each component of the shear flow generating device 10. For each component of the shear flow generating device 10 described below, it is preferable to use a non-paramagnetic material, and it is more preferable to use a non-magnetic material (for example, a material with a relative permeability close to 1) that does not adversely affect the inhomogeneity of the magnetic field B0 generated by the superconducting magnet.

[0024] The sample tube 1 is a cylindrical member with a bottom that stores the sample S to be measured and is also called an NMR tube. The stirring tube 2 is a cylindrical member with a bottom that stores the reference sample R for NMR lock. The stirring tube 2 has its bottomed tip 2a inserted into the sample S in the sample tube 1, and rotates around its longitudinal axis to apply a shear flow to the sample S to be measured. The reference sample R for NMR lock can be, for example, a heavy solvent (deuterium). The sample tube 1 and stirring tube 2 are made of, for example, glass or resin. The sample tube 1 and stirring tube 2 may be either hydrophilic or hydrophobic, but hydrophobicity is preferred. In terms of magnetic permeability and hydrophobicity, the sample tube 1 and stirring tube 2 are preferably made of glass, such as borosilicate glass, and more preferably PYREX GLASS (registered trademark).

[0025] The holder 3 holds both the sample tube 1 and the stirring tube 2. Preferably, the holder 3 holds the sample tube 1 and the stirring tube 2 coaxially. That is, the holder 3 holds the sample tube 1 and the stirring tube 2 so that their longitudinal axes are roughly aligned. The detailed structure of the holder 3 will be described later with reference to FIG. 5. Each part of the holder 3 is made of a resin such as polyoxymethylene (POM) or a metal such as aluminum.

[0026] In this embodiment, the shear flow generating instrument 10 further includes a main shaft 4, a main shaft housing 5, a tip position adjustment mechanism 6, and an electric motor 7 that applies rotational power to the main shaft 4. The electric motor 7 is attached to an electric motor mounting part 8, which will be described later, and the electric motor mounting part 8 is attached to an open end flange 94 of the magnetic resonance apparatus 90.

[0027] The main shaft 4 is, for example, a rod-shaped or cylindrical member that is connected to the rear end 2b of the stirring tube 2 and transmits rotational power around the long axis to the stirring tube 2. The holding part 3 is connected to a main shaft housing part 5 that rotatably houses the main shaft 4, and is connected to a tip position adjustment mechanism 6 via the main shaft housing part 5.

[0028] 2 to 4, a superconducting magnet (not shown) for generating a magnetic field B0 and a probe (not shown) for signal measurement are provided around a predetermined measurement position of the magnetic resonance spectrometer 90. In this embodiment, the longitudinal axis of the sample introduction hole 91 is oriented vertically and along the magnetic field B0. The magnetic field B0 is a magnetic field for defining the quantization axis of the magnetic moment of a substance contained in the sample S, and is a high magnetic field of, for example, several tens of tesla or more.

[0029] An open-end flange 94 is provided at the upper end 91a of the sample introduction hole 91. The shear flow generating device 10 is inserted into the sample introduction hole 91 from the tip of the holder 3 that holds both the sample tube 1 and the stirring tube 2, through the open-end flange 94. The tip position adjustment mechanism 6 is engaged with the upper end 91a of the open-end flange 94, thereby positioning the tips 1a and 2a of the sample tube 1 and the stirring tube 2 at predetermined measurement positions in the magnetic resonance spectrometer 90.

[0030] Illustratively, the dimension of the shear flow generating device 10 from the tip of the sample tube 1 to the rear end of the tip position adjustment mechanism 6 is approximately 1 m to 2 m. The outer diameter of the stirring tube 2 is approximately 3 mm, and the inner diameter of the sample tube 1 is approximately 4 mm. The outer diameter of the sample tube 1 is approximately 5 mm. This creates a gap of approximately 1 mm in the radial direction (direction perpendicular to the Z-axis) between the sample tube 1 and the stirring tube 2. The sample S is accumulated in this approximately 1 mm gap and is subjected to shear force. The rotation speed of the main shaft 4, which is rotated by the electric motor 7, is approximately 0.0167 Hz to approximately 66.7 Hz (approximately 1 rpm to approximately 4,000 rpm). In this embodiment, the sample tube 1 has a solid portion 1c at the tip 1a of the hollow portion. Illustratively, the length of the solid portion 1c along the longitudinal axis (Z-axis) of the sample tube 1 is approximately 8 mm, and the distance between the solid portion 1c of the sample tube 1 and the tip 2a of the stirring tube 2 is approximately 1 mm. In this embodiment, the sample S can also enter a gap of about 1 mm that occurs in the axial direction between the sample tube 1 and the stirring tube 2.

[0031] The precise positional relationship between sample tube 1 and stirring tube 2 is maintained by holder 3 before they are inserted into sample introduction hole 91 of magnetic resonance instrument 90. Rotational power for stirring tube 2, which applies shear force to sample S, is transmitted to stirring tube 2 from motor 7, which is located outside magnetic resonance instrument 90, via main shaft 4. This makes it possible to place sample tube 1 and stirring tube 2 in a precisely aligned state at a predetermined measurement position of magnetic resonance instrument 90, and to apply shear force to sample S stored in sample tube 1 in this accurately aligned state within magnetic resonance instrument 90.

[0032] Furthermore, as will be described later, a circumferential groove can be provided on the side surface of the spindle housing part 5. A ring-shaped elastic member 56 can be placed in the groove, protruding from the groove and elastically deforming to have an outer diameter that fits the open end 91a of the sample introduction hole 91. The ring-shaped elastic member 56 can further reduce radial displacement of the spindle housing part 5 within the sample introduction hole 91 when the shear flow generating device 10 is inserted into the sample introduction hole 91.

[0033] In addition, a magnetic resonance apparatus 90 for performing rheo-NMR measurements is normally equipped with a rotating member called a spinner and a drive mechanism for rotating the spinner. However, when performing rheo-NMR measurements using the shear flow generating device 10, these spinners and drive mechanisms are not used.

[0034] 5A and 5B are cross-sectional views of the holding unit according to one embodiment. (A) is an exploded cross-sectional view showing the engagement between the stirring tube holder 31 and the housing 33, and (B) is an exploded view showing the engagement between the sample tube holder 32 and the housing 33. (C) is a cross-sectional view of each part constituting the holding unit 3.

[0035] The holding unit 3 according to one embodiment comprises a stirring tube holder 31, a sample tube holder 32, and a housing 33 as a structure for holding both the sample tube 1 and the stirring tube 2.

[0036] The stirring tube holder 31 is a cylindrical member that holds the rear end 2b of the stirring tube 2. The sample tube holder 32 is a cylindrical member that holds the sample tube 1. The housing 33 is a cylindrical member that holds the stirring tube holder 31 and the sample tube holder 32. The housing 33 has a first cavity 331 and a second cavity 332 that communicates with the first cavity 331 along its longitudinal axis. The stirring tube holder 31 fits into the second cavity 332, which is located closer to the rear end than the first cavity 331, and the rear end 32b of the sample tube holder 32 fits into the first cavity 331, which is located closer to the front end than the second cavity 332. This secures the stirring tube holder 31 to the housing 33. The stirring tube holder 31 and housing 33 may be secured together with screws (not shown). The rear end 1b side of the sample tube 1 is accommodated in the third cavity 311 of the stirring tube holder 31. Note that "engaged" also includes a state in which male and female threads are engaged (screwed).

[0037] More specifically, the first cavity 331 of the housing 33 is a cavity with a circular cross section that has a diameter larger than the diameter of the stirring tube holder 31 and slightly larger than the diameter of the rear end 32b of the sample tube holder 32. The second cavity 332 of the housing 33 is a cavity with a circular cross section that has a diameter slightly larger than the diameter of the stirring tube holder 31 and smaller than the diameter of the rear end 32b of the sample tube holder 32. Therefore, when the cylindrical stirring tube holder 31 is placed in the second cavity 332 and the male thread at the rear end 32b of the sample tube holder 32 is fitted (screwed) into the female thread of the first cavity 331, the front end 31a of the stirring tube holder 31 comes into contact with the rear end 32b of the sample tube holder 32, thereby restricting movement of the stirring tube holder 31 toward the front end. Furthermore, rear end 31b of stirring tube holder 31 abuts against a step at the rear end of second cavity 332, restricting movement of stirring tube holder 31 toward the rear end. Therefore, stirring tube holder 31 is fixed to housing 33. Furthermore, sample tube holder 32 is fixed to housing 33 by screwing. As a result, with sample tube 1 and stirring tube 2 aligned, stirring tube holder 31, sample tube holder 32, and housing 33 are fixed together.

[0038] The housing 33 has a male thread on its outer peripheral surface 333 at its rear end, which threads into the female thread on the inner peripheral surface 51 of the hollow portion at the front end of the spindle receiving portion 5. Preferably, the housing 33 has a hole 334 extending along the longitudinal direction on its side surface at its rear end. The hole 334 is connected to the hollow portion of the cylindrical housing 33. In this embodiment, the rear end of the protective tube 21 (described later) and the spindle 4 are connected, for example, by a cylindrical coupler 48 (see FIG. 3 , made of, for example, rubber). When the rear end of the protective tube 21 and the spindle 4 are connected by the coupler 48, the interior of the housing 33 can be visually observed through the hole 334. The coupler 48 is attached to the rear end of the protective tube 21 in advance, and can be brought into the hollow portion of the housing 33 and connected to the spindle 4 when the stirring tube holder 31 is attached to the housing 33.

[0039] 6 is a cross-sectional view of an agitator tube holder according to one embodiment, where (A) to (C) are exploded views or exploded cross-sectional views showing the assembly of the parts constituting the agitator tube holder in order, and (D) is a cross-sectional view of the assembled state of the parts constituting the agitator tube holder.

[0040] The stirring tube holder 31 rotatably holds the stirring tube 2 with the rear end 2b of the stirring tube 2 inserted inside. The stirring tube holder 31 is a cylindrical member, and has a third cavity 311 on the front end 31a side that houses the rear end 1b of the sample tube 1, and a fourth cavity 312 on the rear end 31b side that houses the rotation holder 22 that rotatably holds the stirring tube 2.

[0041] As shown in (A) to (D), the agitating tube 2 is connected such that its rear end 2b fits into the hollow portion of, for example, a cylindrical protective tube 21 (made of, for example, aluminum). The agitating tube 2 and the protective tube 21 can be connected in a variety of ways, such as by bonding with an adhesive, by adhering with a sticky substance, or by plastically deforming and crimping the protective tube 21. The agitating tube 2 is further inserted into a plurality of bearings 221 arranged via a cylindrical spacer 222. The plurality of bearings 221 and the spacer 222 form the rotation holder 22, which rotatably holds the agitating tube 2. The rotation holder 22 is housed in the fourth cavity 312 of the agitating tube holder 31.

[0042] An annular lid 313 and a bearing 314 are attached to the rear end 31b of the stirring tube holder 31. The rear end 2b of the stirring tube 2, i.e., the rear end of the protective tube 21, is inserted into the lid 313 and the bearing 314. The bearings 221, 314 are formed using a non-magnetic material such as resin, glass, or ceramic. Preferably, the bearings 221, 314 are radial deep groove ball bearings made of a resin housing and glass or ceramic balls.

[0043] 7A and 7B are cross-sectional views of a sample tube holder according to one embodiment, in which (A) is an exploded view of the components that make up the sample tube holder, and (B) is a half-sectional view of the assembled components that make up the sample tube holder.

[0044] The sample tube holder 32 holds the sample tube 1 inserted therein. As shown in (A) and (B), the sample tube holder 32 is a generally cylindrical member, and the sample tube 1 is inserted into the hollow portion of the sample tube holder 32. The sample tube 1 is further inserted into the hollow portion of an annular elastic member 321 and a cylindrical fixing screw 322. In this embodiment, the inner surface of the hollow portion at the rear end 32b of the sample tube holder 32 is female-threaded, and the outer surface 322a at the tip end of the fixing screw 322 is male-threaded. When the tip end of the fixing screw 322 threads into the hollow portion at the rear end 32b of the sample tube holder 32, the annular elastic member 321 is crushed and deformed, and the elastic member 321 holds the sample tube 1. The annular elastic member 321 is, for example, an O-ring made of Viton (registered trademark).

[0045] The process for assembling the sample tube 1 and stirring tube 2 to the housing 33 will be described below. As will be described below, both the sample tube 1 and stirring tube 2 are fixed to the housing 33. As a result, the positional relationship between the sample tube 1 and stirring tube 2 is determined with reference to the housing 33 before the holder 3 is inserted into the sample introduction hole 91 of the magnetic resonance spectrometer 90.

[0046] First, the assembly of the stirring tube 2 and the housing 33 will be described. As shown in order in FIGS. 6A to 6D, the stirring tube holder 31 that supports the stirring tube 2 rotatably around its longitudinal axis is assembled. The rotary holder 22 that rotatably holds the stirring tube 2 is assembled, and the positional relationship between the stirring tube 2 and the rotary holder 22 is fixed. The assembled rotary holder 22 is placed in the fourth cavity 312 of the stirring tube holder 31, and the positional relationship between the rotary holder 22 and the stirring tube holder 31 is fixed. Then, as shown in FIGS. 5A and 5C, the stirring tube holder 31 with the assembled rotary holder 22 is inserted and fitted into the second cavity 332 in the housing 33, and the positional relationship between the stirring tube holder 31 and the housing 33 is fixed. By fixing the stirring tube holder 31 inside the housing 33 in this way, the stirring tube 2 is fixed to the housing 33, and the positional relationship between the stirring tube 2 and the housing 33 is fixed.

[0047] Next, the assembly of the sample tube 1 to the housing 33 will be described. As shown in Figures 7A and 7B, the sample tube holder 32, which holds the sample tube 1 inserted therein, is assembled to fix the relative positions of the sample tube 1 and the sample tube holder 32. Then, as shown in Figures 5A to 5C, with the stirring tube holder 31 first fixed inside the housing 33, the rear end 32b of the sample tube holder 32 is inserted and fitted into the first cavity 331 in the housing 33, fixing the relative positions of the sample tube holder 32 and the housing 33. By attaching the sample tube holder 32 to the housing 33 in this way, the sample tube 1 is fixed to the housing 33, and the relative positions of the sample tube 1 and the housing 33 are fixed.

[0048] As described above, both sample tube 1 and stirring tube 2 are fixed to housing 33, and the positional relationship between sample tube 1 and stirring tube 2 is thereby defined with housing 33 as the reference. In this way, with holding unit 3 of shear flow generating instrument 10 according to one embodiment, the positional relationship between sample tube 1 and stirring tube 2 can be defined with housing 33 as the reference, and the accurate positional relationship between sample tube 1 and stirring tube 2 can be defined and fixed before instrument 10 is inserted into sample introduction hole 91 of magnetic resonance spectrometer 90.

[0049] Fig. 8 is a cross-sectional view of an assembled spindle, spindle accommodating section, and tip position adjustment mechanism according to one embodiment, Fig. 9 is a half-sectional view of a spindle accommodating section according to one embodiment.

[0050] The spindle receiving portion 5 is a cylindrical member that rotatably receives the spindle 4. The inner peripheral surface 51 of the hollow portion on the front end side is female-threaded, and the outer peripheral surface 52 on the rear end side is male-threaded. The inner peripheral surface 51 of the hollow portion is threadedly engaged with the outer peripheral surface 333 on the rear end side of the housing 33 or the outer peripheral surface 52 on the rear end side of another spindle receiving portion 5, both of which are male-threaded.

[0051] Preferably, the spindle accommodation portion 5 has a hole 53 formed in its side surface, extending along the longitudinal direction. The hole 53 is connected to the hollow portion of the cylindrical spindle accommodation portion 5. Generally, high-precision machining can be performed using a machine tool for precision small-scale machining. In this embodiment, by providing the hole 53 in the side surface of the spindle accommodation portion 5, the length of the hollow portion (through hole) of the spindle accommodation portion 5 that is machined in one machining run is shortened and divided into smaller portions. This allows the hollow portion of the spindle accommodation portion 5 to be machined with high precision using a machine tool for precision small-scale machining.

[0052] Preferably, a bearing 54 is disposed in the hollow portion of the spindle accommodating portion 5. The spindle 4 is inserted through the bearing 54. The bearing 54 is formed using a non-magnetic material, similar to the bearings 221 and 314. Preferably, the bearing 54 is a radial deep groove ball bearing, similar to the bearings 221 and 314.

[0053] Preferably, a plurality of annular elastic members 56 are disposed in the spindle housing 5 via a plurality of annular spacers 55. Each annular spacer 55 has a circumferential groove formed on its side, and the annular elastic member 56 is disposed in the groove. The annular elastic member 56 is, for example, an O-ring made of Viton (registered trademark). The annular elastic member 56 has an outer diameter that fits the opening end 91 a of the sample introduction hole 91 by protruding from the groove of the annular spacer 55 and elastically deforming. More preferably, the annular elastic member 56 has an outer diameter that fits both the diameter of the upper opening end 91 a and the diameter of the lower end 91 b of the sample introduction hole 91, which has an inverse tapered shape whose diameter increases slightly from the upper opening end 91 a toward the lower end 91 b.

[0054] Fig. 10 is a half-side cross-sectional view of a tip position adjustment mechanism according to one embodiment. Fig. 11 is a diagram showing a cylindrical part constituting the tip position adjustment mechanism. (A) is a half-side cross-sectional view from the side, and (B) is a top view.

[0055] The tip position adjustment mechanism 6 adjusts the position of the holder 3 within the sample introduction hole 91, at a position along the longitudinal direction of the sample introduction hole 91. Preferably, the holder 3 is positioned so that the tapered portion 32c of the sample tube holder 32 included in the holder 3 fits into a predetermined position within the sample introduction hole 91 (for example, a tapered cavity provided at a predetermined measurement position of an NMR apparatus (not shown)). The tip position adjustment mechanism 6 includes an annular base 61, a cylindrical portion 62, and a fixing ring 63.

[0056] The annular base 61 and the fixing ring 63 are both annular members, and in this embodiment, the inner peripheral surfaces of both are internally threaded. The fixing ring 63 functions as a so-called double nut, preventing the tubular portion 62 from loosening.

[0057] The cylindrical portion 62 is a cylindrical member. An inner peripheral surface 621 of the hollow portion on the tip side is an internal thread, and an outer peripheral surface 622 on the rear end side is an external thread. The inner peripheral surface 621 of the hollow portion is threadedly engaged with the outer peripheral surface 52 on the rear end side of the spindle accommodation portion 5, thereby connecting the spindle accommodation portion 5 and the cylindrical portion 62. The outer peripheral surface 622 is threadedly engaged with the inner peripheral surfaces of the annular base portion 61 and the fixing ring 63. This threaded connection makes it possible to change (adjust) the position of the cylindrical portion 62 along the longitudinal axis direction with the annular base portion 61 as a reference.

[0058] 12A and 12B are diagrams showing a handle used to adjust the position of the cylindrical portion, where (A) is a top view and (B) is a side view.

[0059] Preferably, a hole 623 is provided in the surface of the solid portion on the rear end 62b side of the cylindrical portion 62. A pin 691 and a hole 692 are provided in the handle 69. When the handle 69 is rotated around the longitudinal axis of the cylindrical portion 62 with the pin 691 of the handle 69 engaged with the hole 623 of the cylindrical portion 62, the position of the cylindrical portion 62 relative to the annular base portion 61 can be changed.

[0060] The diameter of hole 692 provided in handle 69 is larger than the diameter of main shaft 4. As a result, when handle 69 is arranged on the rear end 62b side of cylindrical portion 62, main shaft 4 passes through hole 692. As a result, main shaft 4 does not interfere with handle 69, and handle 69 can be rotated around the longitudinal axis of cylindrical portion 62.

[0061] Fig. 13 is a diagram showing an electric motor mounting portion and an electric motor according to one embodiment, and Fig. 14 is a schematic cross-sectional view for explaining the state of use of the electric motor mounting portion and the electric motor.

[0062] In the shear flow generating instrument 10, the tip position adjustment mechanism 6 is fixed to an upper end 91a of an open end flange 94 of the magnetic resonance apparatus 90. An electric motor mounting part 8, to which an electric motor 7 is attached, is also fixed to the upper end 91a of the open end flange 94. In this embodiment, the electric motor mounting part 8 is disposed above the open end flange 94 provided at the upper end of the open end 91a so as to cover the tip position adjustment mechanism 6. The electric motor 7 is disposed on a top plate 81 above the electric motor mounting part 8, and the rotational power of the electric motor 7 is applied to the main shaft 4 that passes through the hollow portion of a cylindrical portion 62 of the tip position adjustment mechanism 6. In this embodiment, the rotating shaft 71 of the electric motor 7 and the rear end 4b of the main shaft 4 are connected by, for example, a cylindrical coupler 49 (made of, for example, rubber).

[0063] As described above, the shear flow generating device according to one embodiment of the present invention can provide a device that generates a shear flow in a sample and enables NMR locking without affecting the composition of the sample being measured.

[0064] In a shear flow generating instrument 10 according to one embodiment, a sample S to be measured is stored at the bottom of a bottomed sample tube 1. A reference sample R is also stored at the bottom of a bottomed stirring tube 2. As shown in the partially enlarged view of FIG. 3 , the sample S and the reference sample R are separated by the wall and bottom of the stirring tube 2, preventing the sample S from mixing. This allows the shear flow generating instrument 10 according to one embodiment to perform NMR locking without affecting the composition of the sample S in a rheo-NMR measurement, which performs nuclear magnetic resonance while generating a shear flow in the sample S. One example of a measurement target for rheo-NMR is protein stability. For example, with the shear flow generating instrument 10 according to one embodiment, when performing rheo-NMR measurement on a protein solution, NMR locking can be performed without adding a heavy solvent (deuterium) used for NMR locking to the protein solution, i.e., without affecting the composition of the protein solution. This allows for repeatable measurement of good NMR signals from protein solutions with high reproducibility.

[0065] Furthermore, the shear flow generating instrument 10 according to one embodiment can also provide accurate alignment of the sample tube 1 and the stirring tube 2. The accurate positional relationship between the sample tube 1 and the stirring tube 2 is maintained by the holder 3 before they are inserted into the sample introduction hole 91 of the magnetic resonance instrument 90. Rotational power for the stirring tube 2 to apply a shear force to the sample S is transmitted to the stirring tube 2 from an electric motor 7 disposed outside the magnetic resonance instrument 90 via the main shaft 4. This allows the sample tube 1 and the stirring tube 2 to be placed in an accurately aligned state at a predetermined measurement position of the magnetic resonance instrument 90, and shear force can be applied to the sample S stored in the sample tube 1 in such an accurately aligned state within the magnetic resonance instrument 90.

[0066] When the electric motor 7 is used to transmit rotational power to the stirring tube 2, the rotation speed of the main shaft 4 and stirring tube 2, which are driven to rotate by the electric motor 7, is approximately 0.0167 Hz to approximately 66.7 Hz (approximately 1 rpm to approximately 4,000 rpm). According to the shear flow generating device 10 of one embodiment, the sample tube 1 and stirring tube 2 are precisely aligned, and good NMR signals can be measured in rheo-NMR measurements even when the stirring tube 2 is rotated at a relatively high rotation speed of approximately 30 Hz or higher. Furthermore, when the electric motor 7 is used to drive the stirring tube 2, a relatively high rotational torque can be transmitted to the stirring tube 2 by the electric motor 7. This allows good NMR signals to be repeatedly measured in rheo-NMR measurements, even for samples S with relatively high viscosity.

[0067] In rheo-NMR measurements, if good NMR signals can be measured repeatedly with high reproducibility, it becomes possible to quantitatively evaluate the physical properties of, for example, proteins as samples. This makes it possible to quantitatively evaluate, for example, the stability of proteins. Furthermore, if it becomes possible to measure samples with higher viscosity in rheo-NMR measurements, it becomes possible to measure, for example, the process of making butter from fresh cream at the atomic level in real time. In the shear flow generating device 10 according to one embodiment, the sample S and the reference sample R are separated by the wall and bottom of the stirring tube 2, and the sample S and the reference sample R do not mix. Therefore, after measuring the process of making butter from fresh cream using rheo-NMR, it is possible to taste the produced butter and perform a sensory evaluation test of the butter.

[0068] Examples of the present invention will be described below to clarify the features of the present invention. [Example]

[0069] In Example 1, a shear flow generator was fabricated and its performance evaluated. For the performance evaluation, the same shear flow generator as described in the above embodiment was used, except that a stub without the tip position adjustment mechanism was used instead. Fresh cream was used as the sample, and heavy water (DO) was used as the reference sample for NMR lock. Fresh cream was placed in the sample tube of the fabricated shear flow generator, and heavy water was placed in the stirring tube. The shear flow generator was inserted into the sample inlet of the NMR instrument, and rheological NMR measurements were performed while rotating the stirring tube to generate shear flow in the fresh cream in the sample tube. The measurements were performed with the NMR lock function enabled. The results of the rheological NMR measurements (proton NMR measurements) are shown in Figure 15. Note that the fresh cream used as the sample was stirred during the rheological NMR measurements, and some of it became butter.

[0070] Two signal peaks, designated S and R, were observed in the NMR spectrum shown in Figure 15, between approximately 4.5 ppm and approximately 5.0 ppm. The peak position of the signal designated S was approximately 4.7 ppm, confirming that the signal originated from water (HO) in the sample. The signal designated R originated from a small amount of water (HO) contained in heavy water (DO) used as the reference sample for NMR locking. The measurement results shown in Figure 15 confirmed that the shear flow generating device we fabricated performed well. [Explanation of symbols]

[0071] 1 sample tube 2 stirring tubes 3 Holding part 4 spindle 5 Spindle housing 6 Tip position adjustment mechanism 7 Electric motor 8 Motor mounting section 10 Shear flow generating device 21 Protection tube 22 Rotation holding part 31 Stirring tube holder 32 Sample tube holder 33 Case 48,49 Cylindrical coupler 51 Inner surface 52 Outer surface 53 holes 54 bearings 55 spacer 56 Annular elastic member (O-ring) 61 Annular base 62 Cylindrical part 63 Fixing ring 69 Handle 71 (electric motor) rotating shaft 81 Top plate 90 Magnetic Resonance Device 91 Sample introduction hole 91a Open end 94 Open end flange 221 Bearing 222 Spacer 311 Third Cavity 312 Fourth Cavity 313 Circular Lid 314 Bearing 321 Annular elastic member (O-ring) 322a outer surface 331 First Cavity 332 Second Cavity 333 Outer surface 334 holes 621 Inner surface 622 Outer surface 623 holes 691 pins 692 holes B0 static magnetic field Reference sample for R NMR lock S: Sample to be measured

Claims

1. An apparatus for generating a shear flow in a sample to be measured while being inserted into a sample introduction hole of a magnetic resonance apparatus, a sample tube for storing a sample to be measured; a stirring tube which is a bottomed tube for storing a reference sample for NMR lock, the tip side of which is inserted into the sample tube and rotates around its longitudinal axis to apply shear force to the sample; a holder that holds both the sample tube and the stirring tube.

2. The apparatus of claim 1 , wherein the holder holds the sample tube and the stirring tube coaxially.

3. The holding portion is a cylindrical stirring tube holder that holds the rear end side of the stirring tube; a cylindrical sample tube holder for holding the sample tube; The apparatus according to claim 1 , further comprising a cylindrical housing that holds the stirring tube holder and the sample tube holder.

4. the housing has a first cavity and a second cavity communicating with the first cavity along a longitudinal axis direction; the stirring tube holder is fitted into the second cavity in the housing, 4. The apparatus of claim 3, wherein the sample tube holder has a rear end that fits into the first cavity in the housing.

5. the agitation tube holder rotatably holds the rear end side of the agitation tube inserted therein; 4. The instrument according to claim 3, wherein the sample tube holder holds the sample tube inserted therethrough.

6. The apparatus of claim 3, wherein the stirring tube holder has a third cavity at the front end side that accommodates the rear end side of the sample tube, and a fourth cavity at the rear end side that accommodates a bearing that rotatably holds the stirring tube.

7. a main shaft connected to the stirring tube and transmitting rotational power around the long axis to the stirring tube; a cylindrical spindle housing portion that houses the spindle, 7. The instrument according to claim 1, wherein the spindle accommodating portion has a circumferential groove on its side, and a ring-shaped elastic member is disposed in the groove, protruding from the groove and elastically deforming to fit the opening end of the sample introduction hole.

8. The instrument of claim 7 , wherein the main shaft receiving portion has a hole formed in a side surface thereof extending along the longitudinal direction.

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