Development of automated SABRE workstationThe automated SABRE workstation integrates a commercial desktop robotic arm as a core component of the shuttling system for transferring the sample tube (Fig. 1a). The magnetic field for efficient polarization transfer with SABRE is generated by a self-wound solenoid coil positioned either inside or outside of a mu-metal magnetic shield. This component is referred to as the polarization transfer field (PTF) generator. A custom-built controlling system (Fig. 1b) controls the PTF generator, ensuring accurately manipulate both the strength and orientation of the magnetic field required for optimal hyperpolarization conditions. In the subsequent sections, each component of the system will be described in detail to provide a clear understanding of how they contribute to the overall experimental operation.Fig. 1(a) 3D rendered overview of the automated SABRE workstation for benchtop NMR. The workstation consists of a robotic arm-assisted shuttling system, a self-wound solenoid coil placed in the mu-metal shielding as a μT-field PTF generator and a custom controlling system. (b) Explosion view of the controlling system. Several pins on the micro controller (Arduino® UNO R3) are configured to control the bubbling of para-\({\text{H}_2}\) and the gripper on the robotic arm. With the PWM function of Arduino, the H-bridge driver is able to precisely generate the desired magnetic field on the electromagnetic coil.Robotic arm-assist shuttling systemThe 4-axis desktop robotic arm (Dobot MG400, Shenzhen, China) featuring a repeatability of \(\pm 0.05\) mm and a maximum joint speed of \(300^\circ\) per second, enables precise and rapid field cycling of the sample (Fig. 2a). The movement of the robotic arm, including the position coordinates and speed, can be customized through programming in a Python environment. Consequently, the desired position of sample tube can be programmed by inputting the corresponding spatial coordinates for the gripper (Fig. 2b). The gripper is equipped with a digital servo (LDX-335MG, Hiwonder, China) and features a custom 3D-printed finger designed to match the curvature of the sample holder (Fig. 2c). By programming the gripper to open or close, it is capable of seamlessly picking up and dropping off sample tubes at desired positions. In this work, the average speed of movement of the robotic arm was set to \(70\%\) of its maximum capacity to balance the transfer speed with safety considerations. Consequently, the transfer time of the hyperpolarized (HP) sample from the PTF to the detection area of the benchtop NMR was consistently maintained at 3 s across all experiments.Fig. 2(a) The robotic arm-assisted shuttling system for fast magnetic fields cycling. (b) The anti-block gripper with high torque steering gear. The opening and closing of the gripper was independently controlled by a digital servo, allowing the action of grasping or releasing to be completed within 0.5 s. (c) 3D schematic of the finger for the gripper with a curved contour.Bubbling unitAs it mentioned in the introduction, bubbling method is compatible with fields cycling and automated setup. In this work, we built an automated bubbling unit for efficiently delivering para-\({\text{H}_2}\) into the sample solution (Fig. 3).Fig. 3(a) Schematic drawing of the bubbling unit (not shown in real scale). The automated bypass gas valve includes a shut-off valve (b), a digital servomotor (c), and a scaffold (d) for mounting these components together. By controlling the digital servomotor, the automatic bubbling of para-\({\text{H}_2}\) into the sample solution through the capillary tubing is managed.The bubbling unit is comprised of a standard 5 mm NMR tube, to which PTFE tubing (ID: 4.35 mm, OD: 6.35 mm) is affixed at the upper portion. The other end of this PTFE tubing is connected to a Y-type fast pneumatic connector, splitting the gas flow into two pathways: one for bubbling and the other for gas release through a back-pressure regulator (BPR) with a cartridge of 75 psi (P-786, IDEX Health&Science, USA) mounted at the downstream (Fig. 3a). The source pressure of para-\({\text{H}_2}\) in the gas bottle was initially at 30 bar. To adapt this pressure for the experimental requirements, a gas valve was employed to reduce it. The pressure was adjusted down to 78 psi at the inlet of the mass flow controller. The flow rate of para-\({\text{H}_2}\) was controlled by a mass flow controller (MC-500SCCM-D, Alicat Scientific, USA). In the bubbling pathway, a capillary tubing (ID: 0.30 mm, OD: 0.60 mm) is inserted into the outer tubing, with its end submerged in the sample solution. A bypass line (PTFE tubing, OD: 3.2 mm, ID: 1.0 mm), created using two T-connectors, bridges the bubbling and releasing pathways to ensure equal pressure in both. A shut-off valve ( P-782, IDEX Health&Science, USA) controls the state of the bubbling. When this valve is open, bubbling ceases, and the gas is diverted through the bypass line and subsequently released. Closing the valve blocks the bypass, directing the gas through the capillary tubing where it forms bubbles in the sample solution, before being released through the BPR. This setup was previously introduced in earlier research, where the bubbling process was controlled by manually opening and closing the shut-off valve (Fig. 3b)37.Throughout the experiment, the tubing of the bubbling unit remained attached to and moved in conjunction with the NMR tube. This configuration was integral to automating the process. However, it should be note that having the capillary immersed in the sample solution for bubbling could potentially broaden the linewidth of the NMR spectrum due to the additional magnetic field inhomogeneities introduced by the capillary’s presence within the sample.To automate the bubbling procedure, traditional solenoid valves could be employed as replacements for the manual shut-off valve. However, when controlling hydrogen gas, the solenoid valve must be explosion-proof, which increases the cost and requires more installation space. To enhance experimental safety while maintaining automation, a digital servomotor (Amewi AMX Racing Digital Servo HV2060MG) is implemented to automatically drive the handle of the shut-off valve (Fig. 3c). A 3D-printed scaffold, as depicted in Fig. 3d, is utilized to assembly of the servomotor and the shut-off valve. The primary benefit of this mechatronic modification to the manual valve is the automated control of the hydrogen gas valve, achieved in a cost-effective and safe manner. Additionally, leveraging the rapid response capability of the digital servomotor, the bubbling process can be switched on and off by the digital servomotor within 0.5 s, ensuring consistent operation of the bubbling process and enhancing the reproducibility of experiments.PTF generatorSABRE possesses a well-known magnetic field dependence, for which an optimal PTF is essential to achieve spontaneous and efficient spin order transfer of para-\({\text{H}_2}\) to the target substrates. In this study, we constructed two solenoid coils specifically designed for SABRE hyperpolarization of 1H, and heteronuclei, respectively. The first coil is positioned within the stray magnetic field of a benchtop NMR to access fields in the mT range, suitable for 1H SABRE experiments. The second coil, intended for heteronucleus hyperpolarization via SABRE-SHEATH, is capable of generating PTFs ranging from \(-13.5\) to \(13.5\) μT. This coil is housed within a mu-metal magnetic shield (ZG 206, Magnetic Shield Corporation, USA) to shield the Earth’s magnetic field.Fig. 4Schematic diagram of the circuit connections of the PTF generator. By combining the functions of an H-bridge and PWM on Arduino, it is capable to precisely control both strength and direction of the magnetic field generated on the solenoid coil.The core component of the PTF generator is an H-bridge-based drive circuit that incorporates pulse width modulation (PWM). The H-bridge configuration consists of four switches, which can be transistors or relays, arranged in a manner that allows current to flow through the solenoid in either direction. By programming and toggling these switches in various combinations, it is possible to alter the direction of the magnetic field induced in the coil.The schematic diagram of the circuit connections of the PTF generator is detailed in Fig. 4. The process for generating PTFs operates as follows: the micro controller (Arduino® UNO R3) is programmed to emit a square wave signal, which can be modulated using PWM to produce signals with varied duty cycles. Consequently, the H-bridge driver board then outputs an average voltage based on the square wave signal, generating the designated PTF within the electromagnetic coil. More details of the PTF generator can be found in Supporting Information. With a minimum duty cycle change of 1/255 of the full signal, the resolution of the PTF generator achieves a precision of 0.05 milligauss (mG). By programming the corresponding duty cycles into the micro controller, we were able to precisely and automatically sweep the PTF for each SABRE hyperpolarization to determine the optimal PTF for various substrates.In addition, the PTF generator is capable of producing a field pulse sequence that consists of two alternating magnetic fields with different durations. This functionality facilitates investigation of the polarization transfer mechanisms in SABRE.User-friendly GUIA GUI was designed based on the experimental procedures of SABRE and SABRE-SHEATH, and allows users to easily control various parameters, such as the strength and direction of the PTF, the bubbling time, and configure the field pulse sequence. It includes features such as real-time monitoring of system status, automated sequence initiation, and data logging capabilities. The main window of this GUI is depicted in Fig. 5.Fig. 5The main window of the GUI developed for controlling SABRE and SABRE-SHEATH experiments, showing the layout and functional elements. Upper left: general functions for activating the pre-catalyst by clicking the Bubbling On or OFF, and for grasping and releasing the sample tube by clicking the corresponding ON or OFF buttons. Bottom left: state indicator and issuance of instructions. Right column: four categories of SABRE experimental procedures, each of which can be selected by selecting the corresponding radio button.The upper left part of the GUI features the general function module, where users can individually operate the bubbling and gripper functions by clicking the corresponding buttons. This design allows for routine tasks such as activating the SABRE pre-catalyst in a sample solution, or moving the sample tube to a desired position. For instance, the sample tube can be transferred to the benchtop NMR for detection of the thermal equilibrium signal after activation. The real-time status of the SABRE workstation is visualized by different colors displayed at the bottom of the GUI: blue signifies standby, green indicates operating, and red alerts to a bug. Clicking the Experiment Start button without selecting any additional procedures or inputting parameters will automatically initiate the SABRE experiments using a set of default parameters. These default settings involve bubbling at the PTF of \(+0.4\) μT for 40 s with a para-\({\text{H}_2}\) gas flow rate of 85 sccm and a pressure of 90 psi (= 0.621 MPa). Immediately after bubbling, the sample is transferred to a benchtop NMR for detection. Additionally, all ongoing operations can be immediately halted by clicking the Emergency Stop button for ensuring the safety of both the personnel and the workstation in case of any unexpected situations. However, it is important to note that engaging this emergency stop will not result in the depressurization of the system.Within the GUI, users can operate four categories of SABRE experimental procedures: magnetic field sweeping, measurements of polarization relaxation time, measurements of polarization buildup time, and pulse sequence. Users can select the desired experiments and input the necessary parameters displayed in each module to initiate the experiments. By clicking the Set All Parameters button, the parameters in GUI were written into a multi-threading python program. During the experiments, the program will execute the corresponding experimental procedure and call the API of SpinSolveExpert (prospa.exe) to execute the signal acquisition and saving the data. The subsequent sections will detail each procedure and present the corresponding experimental results to demonstrate the capabilities of the constructed system.Chemical sample preparationSample solution for 1H SABRE and 13C SABRE-SHEATH hyperpolarizationAll SABRE sample solutions were prepared under inert gas conditions. For SABRE hyperpolarization of 1H substrates (pyrazine, pyridine, and nicotinamide), each sample solution contained 3 mmol/L of standard homogeneous SABRE pre-catalyst [IrCl (COD) (IMes)] (IMes=1,3-bis(2,4,6- trimethylphenyl)imidazole-2-ylidene; COD=cyclooctadiene)) and 60 mmol/L of each substrate dissolved in degassed methanol-d4.For SABRE-SHEATH and pulsed SABRE-SHEATH of [1-13C] pyruvate, each sample contained 5 mmol/L pre-catalyst [IrCl(COD)(IMes)] (the same catalyst used in \(^1\)H SABRE), 25 mmol/L sodium [1-13C] pyruvate, and 40 mmol/L water-free DMSO dissolved in degassed methanol-d\(_4\). All the chemicals were purchased from Sigma Aldrich and used without further purification.Enrichment of para-\({\text{H}_2}\)
Normal hydrogen gas (\(\hbox {ALPHAGAZ}^\text {TM}\) \({\text{H}_2}\), 99.999%) flows along the pipeline with an appropriate flow rate (0.5 mL/min) controlled by a Ex-proof mass flow controller (F-201CX / F-211CX, Bronkhorst, Germany). A commercial water-cooled helium compressor (Model ARS-4HW, Advanced Research System) offers the cooling source to the custom-tailored cryostat (DE-204AE 9K, Advanced Research System, USA). The enrichment process begins by cooling the cryostat to a stable temperature of 25 K. Subsequently, normal hydrogen gas is introduced into the catalyst chamber inside the cryostat. After contacting the paramagnetic hydrated iron(III) oxide catalyst (\({Fe_{2}O_{3}\cdot H_{2}O}\)) for facilitating the o\({\text{H}_2}\)-p\({\text{H}_2}\) conversion, para-\({\text{H}_2}\) with fraction of 98% was generated and collected within an aluminium gas bottle (DIN477-1, Knautz GmbH & Co. KG, Germany). More details about the para-\({\text{H}_2}\) enrichment apparatus is comprehensively described by Hoevener et al.38 in their previous work.Activation of the pre-catalystThe prepared sample solution was deposited in the modified 5 mm NMR tube with a 10 cm long needle syringe and capped tightly until connected to the tubing of the bubbling unit. Before starting the experiments, the tubing path of the bubbling unit was flushed with fresh para-\({\text{H}_2}\) for 2 minutes with a flow rate of 30 sccm at ambient pressure to ensure no residual air remained. After connecting the sample tube to the pneumatic connector of the bubbling unit, the flow rate was set to 85 sccm until the bubbling unit reached a stable over-pressure of 90 psi. Activation of the sample is initiated by clicking the ON button of the bubbling module in the GUI window. Full activation of the SABRE pre-catalyst typically requires 10 min of bubbling time.Experimental protocolsAll the experiments conducted under the room temperature of \(25\,^\circ\)C. All NMR signals were measured on a Benchtop NMR operating at a 1H frequency of 61.92 MHz (1.45 T, Spinsolve 60 Carbon Ultra, Magritek, Germany). The 1H NMR spectra were obtained with a single pulse excitation experiment, collecting single scan of 32768 points with 200 μs dwell time over a sweep width of 20 ppm using \(90^\circ\) RF pulse with length of 15.8 μs (signal acquisition time 6553.6 ms). The 13C NMR spectra were obtained with a single pulse excitation experiment, collecting single scan of 16384 points with 200 μs dwell time over a sweep width of 240 ppm using \(90^\circ\) pulse length of 81.9 μs (signal acquisition time 3276.8 ms). All the recorded NMR data were processed using MNOVA Software (Mestrelab Research), where the acquired free induction decays (FIDs) were apodized with an exponential filter of 0.3 Hz.Regeneration of 1H HP substrates with SABRE at 6.5 mT1H SABRE hyperpolarization of each sample was performed in three consecutive trials using this automated workstation with the procedure shown in Fig. 6a. The activated sample was bubbled for 20 s with flow rate of 85 sccm at 90 psi at the PTF of 6.5 mT. Immediately after this, the samples were transferred with the robotic arm-assisted shuttling to the benchtop NMR within 3 s to measure the hyperpolarized 1H signal. After each measurement cycle, the sample was transferred back into the solenoid coil with the power off for 60 s. During this period, the signal relaxed back to its thermal equilibrium at Earth’s magnetic field. This process readied the sample for the next batch of hyperpolarization, ensuring that each cycle started from a consistent baseline state.Studying field dependency in SABRE-SHEATH of [1-13C] pyruvateThe experimental protocol of ’Magnetic Field Sweeping’ on the GUI window was chosen for studying field dependency in SABRE-SHEATH (Fig. 5). As the procedure illustrated in Fig. 7c, the activated [1-13C] pyruvate sample was hyperpolarized at different magnetic fields by sweeping the generated PTFs in increments of \(\Delta B_{\text {PTF}} = 0.1\) μT from − 1 to 1 μT. In each cycle, the sample solution was bubbled for 40 s with a flow rate of 85 sccm at 90 psi inside the degaussed magnetic shield at the desired PTF. Upon immediate cessation of the para-\({\text{H}_2}\) bubbling, the sample was transferred to the benchtop NMR for 13C detection. After each acquisition, the sample was transferred back into the magnetic shield with the solenoid coil powered off for 60 s. The 13C polarization then relaxed at near-zero field conditions back to its thermal equilibrium.Measuring 13C polarization buildup time \(T_B\) at optimal PTFThe experimental protocol for ’Polarization Buildup Time’ was selected on the GUI window (Fig. 5). As the procedure illustrated in Fig. 8, the activated [1-13C] pyruvate sample was bubbled for varying durations (from 0 to 75 s in increments of 5 s) at its optimal PTF of − 0.6 μT and at \(25\,^\circ\)C. The sample was transferred to the benchtop NMR for detection immediately after cessation of bubbling. After each acquisition, the sample was transferred back into the magnetic shield with the solenoid coil powered off for 60 s. The 13C polarization then relaxed at near-zero field conditions back to its thermal equilibrium.Measuring 13C polarization relaxation time \(T_1\) at different interesting fieldsThe experimental protocol for ’Polarization Relaxation Time’ was selected on the GUI window (Fig. 5). As the procedures illustrated in Fig. 9a–d, the sample was initially fully polarized by bubbling para-\({\text{H}_2}\) at − 0.6 μT and \(25\,^\circ\)C for 80 s. Subsequently, the sample tube was immediately exposed to various magnetic fields to observe polarization relaxation. The sample was maintained inside the shield at − 0.6 μT, or kept near-zero field (0.026 μT) in situ, or moved outside the magnetic shield to Earth’s field (50 μT), or transferred to a benchtop NMR system at 1.45 T. After varying wait durations at these fields, 13C NMR signals were measured at the benchtop NMR. After each acquisition, the sample was transferred back into the magnetic shield with the solenoid coil powered off for 60 s. The 13C polarization then relaxed at near-zero field conditions back to its thermal equilibrium.Pulse sequence of 13C SABRE-SHEATH hyperpolarizationThe experimental protocol for ’Pulse Sequence’ was selected on the GUI window (Fig. 5). As the procedure illustrated in Fig. 11a, the sample was bubbled at a flow rate of 85 sccm and 90 psi at the set PTF with field pulse sequence, involving two alternating magnetic fields with corresponding durations \(\tau _L\) and \(\tau _H\) for each field. The low and high fields of the pulse sequence were set to near-zero field (0.026 μT) and 13 μT, respectively. Upon immediate cessation of the para-\({\text{H}_2}\) bubbling, the sample was transferred to the benchtop NMR for 13C detection. After each acquisition, the sample was transferred back into the magnetic shield with the solenoid coil powered off for 60 s. The 13C polarization then relaxed at near-zero field conditions back to its thermal equilibrium..
Development of a fully automated workstation for conducting routine SABRE hyperpolarization
