Method Article

Fabrication of Micro-Patterned Chip with Controlled Thickness for High-Throughput Cryogenic Electron Microscopy

DOI:

10.3791/63739

⸱

April 21st, 2022

 ,  ,  , 
1Department of Biomedical-Chemical Engineering, The Catholic University of Korea, 2Department of Biotechnology, The Catholic University of Korea, 3School of Chemical and Biological Engineering, and Institute of Chemical Processes, Seoul National University, 4Center for Nanoparticle Research, Institute of Basic Science (IBS), 5Institute of Engineering Research, College of Engineering, Seoul National University, 6Advanced Institutes of Convergence Technology, Seoul National University

In This Article

Summary

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A newly developed micro-patterned chip with graphene oxide windows is fabricated by applying microelectromechanical system techniques, enabling efficient and high-throughput cryogenic electron microscopy imaging of various biomolecules and nanomaterials.

Abstract

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A major limitation for the efficient and high-throughput structure analysis of biomolecules using cryogenic electron microscopy (cryo-EM) is the difficulty of preparing cryo-EM samples with controlled ice thickness at the nanoscale. The silicon (Si)-based chip, which has a regular array of micro-holes with graphene oxide (GO) window patterned on a thickness-controlled silicon nitride (SixNy) film, has been developed by applying microelectromechanical system (MEMS) techniques. UV photolithography, chemical vapor deposition, wet and dry etching of the thin film, and drop-casting of 2D nanosheet materials were used for mass-production of the micro-patterned chips with GO windows. The depth of the micro-holes is regulated to control the ice thickness on-demand, depending on the size of the specimen for cryo-EM analysis. The favorable affinity of GO toward biomolecules concentrates the biomolecules of interest within the micro-hole during cryo-EM sample preparation. The micro-patterned chip with GO windows enables high-throughput cryo-EM imaging of various biological molecules, as well as inorganic nanomaterials.

Introduction

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Cryogenic electron microscopy (cryo-EM) has been developed to resolve the three-dimensional (3D) structure of proteins in their native state1,2,3,4. The technique involves fixing proteins in a thin layer (10-100 nm) of vitreous ice and acquiring projection images of randomly oriented proteins using a transmission electron microscope (TEM), with the sample maintained at liquid nitrogen temperature. Thousands to millions of projection images are acquired and used to reconstruct a 3D structure of the protein by computational algorithms5,6. For successful analysis with cryo-EM, cryo-sample preparation has been automated by plunge-freezing the equipment that controls the blotting conditions, humidity, and temperature. The sample solution is loaded onto a TEM grid with a holey carbon membrane, successively blotted to remove the excess solution, and then plunge-frozen with liquid ethane to produce thin, vitreous ice1,5,6. With the advances in cryo-EM and the automation of sample preparation7, cryo-EM has been increasingly used to solve the structure of proteins, including envelope proteins for viruses and ion channel proteins in the cell membrane8,9,10. The structure of envelope proteins of pathogenic viral particles is important for understanding viral infection pathology, as well as developing the diagnosis system and vaccines e.g., SARS-CoV-211, which caused the COVID-19 pandemic. Moreover, cryo-EM techniques have recently been applied to material sciences, such as for imaging beam-sensitive materials used in battery12,13,14 and catalytic systems14,15 and analyzing the structure of inorganic materials in solution-state16.

Despite noticeable developments in cryo-EM and relevant techniques, limitations exist in cryo-sample preparation, hindering high-throughput 3D structure analysis. Preparing a vitreous ice film with optimal thickness is especially important for obtaining the 3D structure of biological materials with atomic resolution. The ice must be thin enough to minimize background noise from electrons scattered by the ice and to prohibit overlaps of biomolecules along the electron beam path1,17. However, if the ice is too thin, it can cause protein molecules to align in preferred orientations or denature18,19,20. Therefore, the thickness of vitreous ice should be optimized depending on the size of the material of interest. Moreover, extensive effort is typically needed for the sample preparation and manual screening of ice and protein integrity on the prepared TEM grids. This process is extremely time-consuming, which hinders its efficiency for high-throughput 3D structure analysis. Therefore, improvements in the reliability and reproducibility of cryo-EM sample preparation would enhance the utilization of cryo-EM in structural biology and commercial drug discovery, as well as for material science.

Herein, we introduce microfabrication processes for making a micro-patterned chip with graphene oxide (GO) windows designed for high-throughput cryo-EM with controlled ice thickness21. The micro-patterned chip was fabricated using microelectromechanical system (MEMS) techniques, which can manipulate the structure and dimensions of the chip depending on the imaging purposes. The micro-patterned chip with GO windows has a microwell structure that can be filled with the sample solution, and the depth of the microwell can be regulated to control the thickness of the vitreous ice. The strong affinity of GO for biomolecules enhances the concentration of biomolecules for visualization, improving the efficiency of the structure analysis. Furthermore, the micro-patterned chip is composed of an Si frame, which provides high mechanical stability for the grid19, making it ideal for handling the chip during sample preparation procedures and cryo-EM imaging. Therefore, a micro-patterned chip with GO windows fabricated by MEMS techniques provides reliability and reproducibility of cryo-EM sample preparation, which can enable efficient and high-throughput structure analysis based on cryo-EM.

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Protocol

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1. Fabrication of micro-patterned chip with GO windows (Figure 1)

  1. Deposit the silicon nitride.
    1. Deposit low-stress silicon nitride (SixNy) on both sides of the Si wafer (4 inch diameter and 100 µm thickness) using low-pressure chemical vapor deposition (LPCVD) at 830 °C and a pressure of 150 mTorr, under a flow of 170 sccm dichlorosilane (SiH2Cl2, DCS) and 38 sccm ammonia (NH3).
    2. Using a deposition rate of ~30 Ã…/min, control the SixNy thickness to be within 25-100 nm by varying the deposition time.
      NOTE: Extreme care should be taken when handling the Si wafer because the wafer is very thin and fragile. Take care not to bend the wafer during its handling or loading in the equipment.
  2. Pattern the photoresist.
    1. Apply a hexamethyldisilazane (HMDS) solution on the SixNy-deposited Si wafer with enough volume to cover the entire surface of the wafer, spin coat with a spin coater at 3,000 rpm for 30 s, and bake at 95°C for 30 s on a hot plate to render the wafer surface hydrophobic and thus ensure a good coating performance with photoresist (PR).
    2. Apply positive PR (Table of Materials) with enough volume to cover the entire surface of the wafer, spin coat at 3,000 rpm for 30 s, and bake at 100 °C for 90 s on a hot plate. Spin-coated PR has a thickness of 500 nm.
    3. Expose the PR-coated wafer with ultraviolet light (365 nm wavelength and 20 mW/cm2 intensity) for 5 s through a chromium mask (Figure 2A-D) using an aligner.
    4. Develop the PR for 1 min using a developer (Table of Materials) and rinse the wafer by immersing it in deionized (DI) water 2x. Fully dry the PR-patterned wafer by blowing N2 gas onto the wafer surface.
      NOTE: Extreme care should be taken while blowing N2 gas onto the Si wafer because the wafer is very thin and fragile. Do not blow N2 gas with high pressure in a direction perpendicular to the wafer, as this may cause the wafer to fracture.
  3. Pattern the SixNy.
    1. Etch the exposed SixNy following the patterning of the PR using a lab-built reactive ion etcher (RIE), with 3 sccm sulfur hexafluoride (SF6) gas at a radiofrequency (RF) power of 50 W. The etching rate with these settings is ~6 Ã…/s. Set the etching time depending on the thickness of the SixNy layer deposited.
      NOTE: The etching rate may vary and need in-lab optimization depending on the specifications of the RIE equipment used.
    2. Eliminate the PR by immersing the SixNy patterned wafer in acetone at room temperature for 30 min, followed by rinsing the wafer by immersing it in DI water 2x. Fully dry the wafer by blowing N2 gas onto the wafer surface.
      NOTE: Extreme care should be taken while immersing or taking out the wafer from the solutions because the wafer can be fractured by the surface tension of the solution. Do not immerse or take out the wafer parallel to the surface of the solution. Use precision wafer handling tweezers with carbon fiber tips. Do not strongly grab the wafer with the tweezer; lift one side of the wafer until the wafer tilts to an angle, where it can be taken out from the solution. The wafer may fracture when it bends due to the firm grip during lifting.
  4. Etch the Si.
    1. Prepare a 1.5 M potassium hydroxide (KOH) solution by dissolving KOH powder in DI water at 80 °C.
    2. Immerse the SixNy patterned wafer in KOH solution to etch the exposed Si. Leave the wafer in the solution with stirring until the free-standing SixNy windows can be observed at the opposite side of the patterned SixNy.
      NOTE: The wet etching time may differ depending on the thickness of the Si; for a 100 µm thick wafer, wet etching normally takes several hours. Do not set the stirring speed too high during Si etching because the free-standing SixNy windows are very thin and can be fractured by the flow of the fluid. In this experiment, the stirring rate was set to 250 rpm.
    3. Clean the etched wafer by dipping it several times in a DI water bath to eliminate etching residues. Dry the wafer in the air.
      NOTE: Extreme care should be taken while immersing or taking out the Si patterned wafer from the solutions because the free-standing SixNy windows are very thin and fragile and can be fractured by the surface tension of the solution. The wafer should be immersed or taken out at an angle, such that the edge of the wafer enters and exits the solution first.
  5. Eliminate the KOH etching residues.
    1. Lightly press the boundaries of the chip array with a tweezer to obtain an array of chips that will be micro-patterned (Figure 1B).
    2. Prepare 1.5 M KOH solution at 80 °C with stirring.
    3. Immerse the chip array in KOH solution for 30 s and rinse it by dipping it in DI water 2x. Fully dry the chips by blowing N2 gas.
      NOTE: Extreme care should be taken while dipping the chips in solutions and blow-drying them with N2 gas because the free-standing SixNy windows are very thin and fragile. While the chip is immersed in KOH solution, stirring should be stopped. The chips should be dipped with their edges first in the direction perpendicular to the solution and blown with N2 gas in the parallel direction.
    4. Fully dry the chip array in the air for at least 1 h.
  6. Pattern the PR.
    1. Prepare a blank 525 µm Si wafer as solid support. Spin coat the Si wafer with HMDS and positive PR, as described above, but attach the chip array (with the free-standing SixNy window side upward) on the Si wafer before baking the PR. The PR acts as an adhesive between the wafer and the chip array. Bake the Si wafer attached with the chip array at 100 °C for 90 s on a hot plate.
    2. Spin coat the chip set with HMDS and positive PR, as described above.
    3. Expose the chip set with ultraviolet light (365 nm wavelength; 20 mW/cm2 intensity) for 5 s through a chromium mask (Figure 2E,F) using an aligner.
    4. Develop the PR using a developer for 15 s, rinse the chip set by dipping it in DI water 2x, and fully dry the PR patterned chip set by blowing N2 gas.
  7. Prepare the micro-patterned SixNy.
    1. Etch SixNy following the PR patterning using a lab-built RIE, with 3 sccm SF6 gas at RF power of 50 W. Control the etching time depending on the thickness of the SixNy layer.
  8. Eliminate the PR.
    1. Eliminate the PR by immersing the patterned chip set in 1-methyl-2-pyrrolidinone (NMP) solution at 60 °C and leaving it overnight. Rinse the chip set by dipping it in DI water 2x, and fully dry the patterned chip set by blowing N2 gas.
    2. Eliminate the PR residues with an O2 plasma process using 100 sccm O2 gas at RF power of 150 W for 1 min with the lab-built RIE.
  9. Rinse the micro-patterned chip.
    1. Prepare 1.5 M KOH solution at 80 °C.
    2. Immerse the micro-patterned chips in KOH solution for 30 s to fully eliminate the PR residues and rinse the chips by immersing them in DI water 2x. Fully dry the chips by blowing N2 gas.
    3. Fully dry the chips in the air for at least 1 h.
  10. Transfer graphene oxide (GO) by the drop-casting method.
    1. Dilute GO solution (2 mg/mL) to 0.2 mg/L with DI water and sonicate for 10 min to break up aggregates of GO sheets. Centrifuge the diluted GO solution at 300 x g for 30 s.
    2. Glow discharge the Si-etched side of the micro-patterned chip to render the chip surface with positive charge using a glow discharger (Table of Materials) at 15 mA for 1 min.
    3. Drop 3 µL of the GO solution onto the glow discharged side of the micro-patterned chip and leave the drop on the chip for 1 min. After 1 min, blot away the excess GO solution on the chip with filter paper.
    4. Wash the GO-transferred chip with DI water droplets prepared on paraffin film and blot away the DI water on the chip with filter paper. Repeat this procedure 2x on the GO transferred side and 1x on the opposite side. Dry the GO-transferred chip at room temperature overnight.
    5. Wash the micro-patterned chip with GO windows by immersing it in the DI water and blow-dry the chip with N2 gas.

2. Cryo-EM imaging

  1. Prepare the cryo-sample.
    1. Prepare the cryo-sample using a mechanical cryo-plunging machine (Table of Materials), which controls the temperature, humidity, blotting time, and force. After loading the blotting pad onto the blotters, ensure that the humidity and the temperature in the chamber are maintained at 100% and 15 °C, respectively.
    2. Pick up the micro-patterned chip with a typical cryo-tweezer and load the tweezer to the cryo-plunging machine. Pipet 3 µL of sample solution onto the micro-patterned chip at the hole-patterned side, with GO windows on the bottom. Control the blotting time and force depending on the sample solution.
      NOTE: Here, biological specimens, namely human immunodeficiency virus (HIV-1), ferritin, proteasome 26S, groEL, apoferritin protein particles, and tau filament proteins were used for cryo-EM imaging. In addition, diverse types of inorganic materials, such as Fe2O3 nanoparticles (NP), Au nanoparticles, Au nanorods, and silica nanoparticles, were used for cryo-EM imaging. The desired blotting time and force were set on the cryo plunger for different types of samples.
    3. After the blotting process, plunge-freeze the sample-loaded chip immediately in liquid ethane. Transfer the chip to the grid box in liquid nitrogen (LN2) and store it in LN2 before cryo-EM imaging.
  2. Carry out cryo-EM imaging.
    1. Load the cryo-sample to a cryo-EM holder with the temperature maintained at -180 °C.
    2. Load the cryo-EM holder into a TEM and observe the samples with the minimum dose system (MDS) mode.

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Results

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A micro-patterned chip with GO windows was fabricated by MEMS fabrication and 2D GO nanosheet transfer. Chips for micro-patterning were mass-produced, with about 500 chips produced from one 4 in wafer (Figure 1B and Figure 2A,B). The designs of the micro-patterned chips can be manipulated using different designs of the chromium mask (Figure 2) during the photolithography procedure. The fabricated micro-patterned chi...

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Discussion

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The microfabrication processes for producing micro-patterned chips with GO windows are introduced here. The fabricated micro-patterned chip is designed to regulate the thickness of the vitreous ice layer by controlling the depth of the micro-hole with GO windows depending on the size of the material to be analyzed. A micro-patterned chip with GO windows was fabricated using a series of MEMS techniques and a 2D nanosheet transfer method (Figure 1). The major advantage of using the MEMS fabric...

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Disclosures

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The authors have no conflicts of interest.

Acknowledgements

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M.-H.K., S.K., M.L., and J.P. acknowledge the financial support from the Institute for Basic Science (Grant No. IBS-R006-D1). S.K., M.L., and J.P. acknowledge the financial support from Creative-Pioneering Researchers Program through Seoul National University (2021) and the NRF grant funded by the Korean government (MSIT; Grant Nos. NRF-2020R1A2C2101871, and NRF-2021M3A9I4022936). M.L. and J.P. acknowledge the financial support from the POSCO Science Fellowship of POSCO TJ Park Foundation and the NRF grant funded by the Korean government (MSIT; Grant No. NRF-2017R1A5A1015365). J.P. acknowledges the financial support from the NRF grant funded by the Korean government (MSIT; Grant No. NRF-2020R1A6C101A183), and the Interdisciplinary Research Initiatives Programs by College of Engineering and College of Medicine, Seoul National University (2021). M.-H.K. acknowledges the financial support from the NRF grant funded by the Korean government (MSIT; Grant No. NRF-2020R1I1A1A0107416612). The authors thank the staff and crew of the Seoul National University Center for Macromolecular and Cell Imaging (SNU CMCI) for their untiring efforts and perseverance with the cryo-EM experiments. The authors thank S. J. Kim of the National Center for Inter-university Research Facilities for assistance with the FIB-SEM experiments.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1-methyl-2-pyrrolidinone (NMP)Sigma Aldrich, USA443778
Acetone
AFMPark Systems, South KoreaNX-10
AlignerMidas System, South KoreaMDA-600S
AZ 300 MIF developerAZ Electronic Materials USA Corp., USA184411
Cryo-EM holderGatan, USA626 single tilt cryo-EM holder
Cryo-plunging machineThermo Fisher SCIENTIFIC, USAVitrobot Mark IV
Focused ion beam-scanning electron microscopy (FIB-SEM)FEI Company, USAHelios NanoLab 650
Glow dischargerTed Pella Inc., USAPELCO easiGlow
Graphene oxide (GO) solutionSigma Aldrich, USA763705
Hexamethyldisizazne (HMDS), 98+%Alfa Aesar, USA10226590
Low pressure chemical vapor deposition (LPCVD)Centrotherm, GermanyLPCVD E1200
maP1205 positive PRMicro resist technology, GermanyA15139
Potassium hydroxide (KOH), flakeDAEJUNG CHEMICALS & METALS Co. LTD., South Korea6597-4400
Raman SpectrometerNOST, South KoreaConfocal Micro Raman System HEDA
Reactive ion etcher (RIE)Scientific Engineering, South KoreaLab-built
SEMCarl Zeiss, GermanySUPRA 55VP
Si waferJP COMMERCE, South Korea4" Silicon wafer, P(B)type, (100), 1-30ohm.c m, DSP, T:100um
Spin coaterDong Ah Trade Corp., South KoreaACE-200
TEMJEOL, JapanJEM-2100F

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Cryogenic Electron MicroscopyMicro Patterned ChipControlled Ice ThicknessGraphene Oxide WindowSilicon Nitride MembraneUV PhotolithographyChemical Vapor DepositionReactive Ion EtchingHigh Throughput ImagingBiomolecule Structure Analysis

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