the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
3D-printed microcell for protein NMR at high ionic strengths and small sample volumes
Abstract. Standard solution NMR measurements use 5-mm outer diameter (OD) sample tubes that require ca 0.5 mL of solvent to minimize “end effects” on magnetic field homogeneity in the active volume of the sample. Shigemi cells reduce the solvent requirement to ca 0.29 mL. At high ionic strength, or at ultrahigh magnetic fields, smaller OD samples are needed to study samples in conductive, radiofrequency absorbing solvents such as water. We demonstrate an effective and inexpensive alternative for reducing the active sample volume to 0.13 mL by 3D printing of ellipsoidal shaped cells that are inserted into 5-mm OD NMR tubes. Static magnetic susceptibility, χ, of printer resin was measured using a simple slice-selection pulse sequence. We found that the χ of water increases linearly with NaCl concentration, from -9.05 ppm to -8.65 ppm for 0 to 2 M NaCl. The χ of D2O was measured to be -9.01 ppm. The susceptibility difference between the resin (χ= -9.40 ppm) and water can be minimized by paramagnetic doping of the resin. Such doping was found unnecessary for obtaining high quality protein NMR spectra when using ellipsoidal shaped cells that are insensitive to susceptibility mismatching. The microcells offer outstanding RF and good Bo homogeneities. Integrated 600-MHz HSQC signal intensities for the microcell sample in PBS buffer were 6.5±4 % lower than for 0.5 mL of the same protein solution in a regular 5-mm sample tube. The cell is demonstrated for N-acetylated α-synuclein in PBS buffer, and for observing tetramerization of melittin at 2 M NaCl.
Competing interests: AB is a member of the editorial board of Magnetic Resonance.
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims made in the text, published maps, institutional affiliations, or any other geographical representation in this preprint. The responsibility to include appropriate place names lies with the authors.- Preprint
(1885 KB) - Metadata XML
- BibTeX
- EndNote
Status: final response (author comments only)
-
CEC1: 'Editorial Comment on mr-2025-5', Geoffrey Bodenhausen, 14 Mar 2025
Our American colleagues may be glad to hear that Copernicus, who produce our journal, will accept payment of the modest Author Page Charges (merely 80 € / page) AFTER their papers are published in their final form, unlike some American publishers who decided to withhold papers that are ready to be published until payment has been received. This may help in these troubled times.
Citation: https://doi.org/10.5194/mr-2025-5-CEC1 -
AC1: 'Reply on CEC1', Ad Bax, 14 Mar 2025
Thanks Geoffrey. The flexibility of Copernicus is much appreciated and I hope that all our colleagues appreciate that not-for-profit publishing in MR benefits our entire community
Citation: https://doi.org/10.5194/mr-2025-5-AC1
-
AC1: 'Reply on CEC1', Ad Bax, 14 Mar 2025
-
CC1: 'Comment on mr-2025-5', Tom Barbara, 19 Mar 2025
This paper is a fine effort in the progression of "small volume NMR", a familiar topic I was introduced to during my days at Varian Associates. In 2009 I wrote up a small review on the history and progress for the eMagRes Journal:
"NMR Probes for Small Sample Volumes" 2009 Thomas M. Barbara https://doi.org/10.1002/9780470034590.emrstm1084
Throughout that history, the issues of dielectric loss, background signals and magnetic susceptibility have always played critical roles. At one time, there was a great effort by a group to etch an approximately elliptical cavity into a glass capillary and this effort reminded me of that work. It would be of some interest to compare this effort with that one (I reference that effort in my article). Background signals in that case are much smaller. This is an aspect of 3D printing materials that can cause headaches for probe builders.Citation: https://doi.org/10.5194/mr-2025-5-CC1 -
CC2: 'Comment on mr-2025-5', Tom Barbara, 19 Mar 2025
Section 2.1 might perhaps appear rather obscure to a neophyte. For those who are curious, "Cylindrical Demagnetization Fields and Microprobe Design in High Resolution NMR" Journal of Magnetic Resonance A109, 265-269 (1994) will bring satisfaction. It is a real advantage to recognize that magnetization can be viewed as equivalent to a current source whenever there is a gradient or a discontinuity. Back then, it was AOK to use Gaussian units in magnetostatics, but the conversions to SI are actually very simple.
Citation: https://doi.org/10.5194/mr-2025-5-CC2 -
EC1: 'Comment on mr-2025-5', Gottfried Otting, 21 Mar 2025
Copernicus asks to make data available for the long term. Typically, this is by providing the doi of files stored in a repository (CERN's Zenodo is popular).
Besides the pulse program which is already in the Appendix, it would be nice to make the STL file used for 3D printing available.
Citation: https://doi.org/10.5194/mr-2025-5-EC1 -
AC2: 'Reply on EC1', Ad Bax, 21 Mar 2025
Our apologies for the omission. The STL file is available at
https://doi.org/10.5281/zenodo.15064675
and this info will be included in the revision
Citation: https://doi.org/10.5194/mr-2025-5-AC2
-
AC2: 'Reply on EC1', Ad Bax, 21 Mar 2025
-
RC1: 'Comment on mr-2025-5', Anonymous Referee #1, 06 Apr 2025
In the manuscript by Bax et al., the authors presented a nice demonstration of 3D-printed microcell as an effective and inexpensive alternative for reducing the sample volume needed for protein NMR at high ionic strengths. Fully taking advantage of the versatility of SLA 3D printing, the authors created a 3D printed ellipsoidal polymer plug that could be inserted into regular NMR tubes, thus enabling high quality NMR acquisition without considering mismatching susceptibility. The authors further demonstrated the application of the 3D printed microcell for the observation of the tetramerization of melittin, which is challenging to perform in regular NMR tubes. The concept of employing 3D printing to create freeform vessels for NMR is novel and the NMR experiments were conducted rigorously. This manuscript will be of great interest to the readers of Magnetic Resonance and this reviewer would like to recommend its publication. The following comments extend beyond the major findings of this work but may enhance the discussion section at the editor’s discretion.
1. It is interesting that authors observed leaching impurities from the 3D-printed microcells as they noted in Figure 5. The authors were using Formlabs Clear V4 resin, which is mainly composed of methacrylate monomers, bismethacrylate crosslinker, and TPO (diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide) photo-initiator. Telling from the chemical shifts of the leaching impurities, they are likely to be methacrylate (hydroxypropyl methacrylate, HPMA) monomers or oligomers. HPMA and its oligomers have reasonable water solubility and that would be problematic if this material were to be used for protein NMR.
2. To remove water-soluble components, this reviewer noticed that the authors used rigorous post-printing processing, including IPA wash, UV curing, and Milli-Q water wash at an elevated temperature. Water-soluble polymers with hydrophobic nature like poly(HPMA) tend to exhibit lower critical solution temperature (LCST) behaviors (Macromolecules 2008, 41(14), 5132-5140) meaning they become less soluble in water as temperature increases. Therefore, washing with cold water rather than hot water would likely be more effective at removing these impurities, as they would remain dissolved at lower temperatures. This reviewer suggests the authors try cold water washing of the printed microcells, which might help reduce the leaching problems observed in the NMR experiments.
3. If poly- or oligo- HPMA is indeed the leaching impurities, another phenomenon the authors might have observed is when the ionic strength is increased, the resonances of the impurities might broaden and disappear. This reviewer would be interested, and it would be much appreciated if the authors could comment on that.
4. In Method Section, a different resin, ELEGOO ABS-like Resin V3 was used for doping study. However, experiments using V3 were not mentioned elsewhere in the manuscript. It would be valuable if the authors could include results from these experiments and comment on the NMR performance of this material compared to Formlabs Clear V4 resin. Based on the material specifications, ELEGOO ABS-like Resin V3 is relatively more hydrophobic than Clear V4, which might result in less leaching of water-soluble components. However, this potential advantage might be offset by its high titanium oxide content, which could potentially interfere with magnetic susceptibility matching despite the optimized shape design. Including those comparative results would be helpful in understanding material selections.
5. The observations regarding different resin materials lead to a more general consideration that is beyond the scope of this work but worth noting. Commercial 3D printing resins are formulated in a way that try to meet various performance specifications, such as material strength, printing resolution, and printing speed. As such, they typically contain multiple components and are not necessarily suitable for high precision work such as protein NMR. This work provided a great proof of concept and would motivate material chemists to develop specialized resins for analytical applications.Citation: https://doi.org/10.5194/mr-2025-5-RC1 -
CC3: 'Reply on RC1', Tom Barbara, 10 Apr 2025
This is a great comment on the problems inherent in current 3D printing materials. Background signals and chemical leaching have always been a problem with polymeric materials. Back in the day susceptibility plugs were offered by Varian, Doty and others, with only limited success.
Citation: https://doi.org/10.5194/mr-2025-5-CC3
-
CC3: 'Reply on RC1', Tom Barbara, 10 Apr 2025
-
RC2: 'Comment on mr-2025-5', Anonymous Referee #2, 07 Apr 2025
General comments:
The manuscript describes the construction of NMR microcells using 3D printing. The authors thoroughly measured the magnetic susceptibility of water containing NaCl, D2O and cobalt-doped Clear V4 resin. The authors also constructed an ellipsoidal-shaped microcell using Clear V4 resin and successfully observed HSQC signals of N-acetylated alpha-synuclein in PBS buffer and tetramerized melittin at 2M NaCl using the microcell. As the experimental magnetic susceptibility data are useful for designing NMR microcells with a wide variety of sample chamber shapes, I recommend publication of this manuscript.
Specific comments:
When an NMR tube made of glass is used, there is a case where a protein adsorbs to the surface of glass. Was such adsorption to the surface of the Clear V4 resin observed for synuclein and melittin?
The authors state that H2O appears to diffuse into the Clear V4 resin (in Line 260). If a protein sample is placed in the microcell and left it for a long time, will the protein leach from the sample space into the printer resin?
Technical correction:
Figures 1 to 4: While each panel is labeled in lowercase letters in the artwork, capitals are used in the figure legends.
Citation: https://doi.org/10.5194/mr-2025-5-RC2 -
EC2: 'Comment on mr-2025-5', Gottfried Otting, 11 Apr 2025
The bioengineering group of Prof. Adam Perriman at the ANU happens to have the 3D printer and printing material described in this article, and his postdoc Dr Mark Shannon kindly printed a few ellipsoidal shaped microcells. Our preliminary observations:
- printing using the STL file provided was very easy;
- washing with isopropanol needs to be thorough to avoid non-smooth inner surfaces of the microcells from residual resin (perhaps the authors can give more details on the extent of washing);
- degassing in vacuum made a significant difference to field homogeneity, even when air bubbles are not visible to the naked eye;
- the microcells are designed for thin-wall NMR tubes (and don't fit into inexpensive tubes with slightly thicker glass walls) but scaling by 5% in the x-y plane would be easy; as they are, they glide easily down and back out the NMR tube (if not, they can probably be fished out with some chicken wire);
- the microcells tend to bend slightly during UV curing at 60 oC (the Perriman group proposes to cure by leaving in sunlight at room temperature first, but this has not been tested for this application).
It would be nice to know, whether 3-axis pulse field gradient probes are a significant advantage over single-axis gradient probes. Preliminary experiments suggest that the non-axis shims can be quite different from the z-axis ones.
Bruker also sells shaped tubes with an approximately rectangular cross-section that are claimed to provide 86% of the sensitivity of a 5 mm tube in the absence of salt, equivalent sensitivity at 20 mM salt, and 20% better sensitivity at 100 mM salt. A direct comparison may be impossible as it requires access to a specifically equipped probe head, but a comment would be welcome. Certainly, the micro-cell proposed is far less costly.
Citation: https://doi.org/10.5194/mr-2025-5-EC2 -
RC3: 'Comment on mr-2025-5', Marcel Utz, 12 Apr 2025
Kakheshpour et al present a study of susceptibility matching for aqueous samples. They show an ingeniously simple method for measuring the susceptibility difference between a solid material (eg, glass) and a liquid, by comparing the observed chemical shift of signals stemming from the solid/liquid interface to those observed for a glass/liquid interface of the same geometry. They also show that 3d printed inserts can be susceptibility-matched by doping of the 3d printing resin with a suitable paramagnetic dopant. Finally, they demonstrate the use of a 3d printed insert with an ellipsoidal sample chamber to reduce the impact of susceptibility differences. The resulting lower filling factor also makes the probe tuning less sensitive to sample conductivity, and therefore enables the study of samples that require high salt concentrations.
The manuscript is well written, with figures that clearly illustrate the concepts and results. This is a strong contribution to the field, and should definitely be published in Magnetic Resonance. There are a few minor issues to address, as listed below:
1. L120: the approach based on eq. 4 implicitly neglects all other interfaces. However, the experimental setup has a glass/air interface at the bottom end of the shigemi tube that, while further away, represents a much larger step in susceptibility (of order 10ppm). I suspect that it is correct to ignore it due to the initial shimming on a filled NMR tube, which has the same interface. This should be more clearly explained in the text.
2. Figure 1: the method relies on the shift difference between the maximum of the h=0 slice spectrum and one that has been taken at h=13mm. While the latter is a single peak, the former has a strong downfield shoulder due to the lateral inhomogeneity. To what extent does this affect the precision of the resulting shift difference? An error estimate based on the accuracy with which the peak positions can be determined should be carried out.
3. L 151: the extensive work of Wapler et al (10.1016/j.jmr.2014.02.005) to determine the susceptibility of a range of construction and microfabrication materials should be cited. Their method was based on susceptibility-induced MR image distortions in water, and therefore measured susceptibility differences with respect to water.
4. L197: the units are not clean in this expression. the concentration needs to be divided by 1M, and it should be -9.01 ppm rather than -9.01
5. We have proposed a tangentially related approach to compensate for solvent/container susceptibility differences in the context of microfluidic NMR. (Ryan et al, 10.1039/C3LC51431E, Hale et al, 10.1039/C8LC00712H) Maybe the authors could consider citing this work for the benefit of the interested reader?Citation: https://doi.org/10.5194/mr-2025-5-RC3 -
EC3: 'Comment on mr-2025-5', Gottfried Otting, 13 Apr 2025
Looking forward to the revised version! When preparing the revision, please go through the references with a fine comb to check for completeness (e.g., doi, page numbers), correctness (Copernicus does not have the means to check for typos in references) and format (it is recommended that titles of articles do add a capital to every noun).
Citation: https://doi.org/10.5194/mr-2025-5-EC3
Viewed
HTML | XML | Total | BibTeX | EndNote | |
---|---|---|---|---|---|
240 | 69 | 14 | 323 | 2 | 0 |
- HTML: 240
- PDF: 69
- XML: 14
- Total: 323
- BibTeX: 2
- EndNote: 0
Viewed (geographical distribution)
Country | # | Views | % |
---|
Total: | 0 |
HTML: | 0 |
PDF: | 0 |
XML: | 0 |
- 1