Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
www.acsami.org
Graphene-Coated Glass Substrate for Continuous Wave Laser Desorption and Atmospheric Pressure Mass Spectrometric Imaging of a Live Hippocampal Tissue Jae Young Kim,†,‡,⊥ Heejin Lim,†,⊥ Sun Young Lee,† Cheol Song,‡ Ji-Won Park,§ Hyeon Ho Shin,∥ Dong-Kwon Lim,*,∥ and Dae Won Moon*,†
Downloaded via BUFFALO STATE on July 30, 2019 at 03:12:48 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Department of New Biology and ‡Department of Robotics Engineering, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, Republic of Korea § Graduate School of Analytical Science and Technology (GRAST), Chungnam National University, Daejeon 34134, Republic of Korea ∥ KU-KIST Graduate School of Converging Science and Technology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea S Supporting Information *
ABSTRACT: The atmospheric pressure mass spectrometric (AP-MS) imaging technology combined with an inverted optical microscopic system is a powerful tool for determining the presence and spatial distributions of specific biomolecules of interest in live tissues. Efficient desorption and ionization are essential to acquire mass spectrometric (MS) information in an ambient environment. In this study, we demonstrate a new and efficient desorption process using a graphene-coated glass substrate and a continuous wave (CW) laser for highresolution AP-MS imaging of a live hippocampal tissue. We found that desorption of biomolecules in a live tissue slice was possible with the aid of a graphene-coated glass substrate and indirect application of a 532 nm CW laser on the graphene substrate. Interestingly, the desorption efficiency of a live tissue on the graphene-coated substrate was strongly dependent on the number of graphene layers. Single-layer graphene was found to be the most sensitive substrate for efficient desorption and reproducible high-resolution hippocampal tissue imaging applications. The subsequent ionization process using nonthermal plasma generated sufficient amounts of molecular ions to obtain highresolution two-dimensional MS images of the cornu ammonis and the dentate gyrus regions of the hippocampus. Therefore, graphene-coated substrates could be a promising platform to induce an efficient desorption process essential for highly reproducible ambient MS imaging. KEYWORDS: mass spectrometry imaging, graphene-coated substrate, laser desorption, ambient mass spectrometry, hippocampal tissue
1. INTRODUCTION
intuitively analyzing biological specimens containing moisture, and various engineering attempts have been made to achieve mass spectrometric (MS) imaging with high spatial information.14−18 We recently reported a micrometer-resolution AP-MS imaging system for mass-based live hippocampal tissue imaging.19,20 We found that efficient desorption was possible by the use of a focused pulsed laser and rod-shaped gold nanoparticles (AuNPs). When coupled with a subsequent ionization process using nonthermal plasma, high-resolution hippocampal MS imaging was able to identify the chemical
Atmospheric pressure mass spectrometric (AP-MS) imaging technology has been considered as a simple, fast, and accurate chemical imaging method, and many technological developments have been achieved for its use in analyzing biological specimens.1−8 AP-MS relies on charged molecules from the desorption and ionization processes in atmospheric environments (pressure and humidity) to introduce them into a mass analyzer. Because a specimen is not exposed to vacuum conditions, AP-MS can greatly reduce the possibility of deformation of its original state as compared with a vacuumbased MS instrument.3,9−13 Therefore, AP-MS has been investigated for various biotechnology research studies or in situ diagnosis applications. In particular, one of the main challenges is to improve the AP-MS technique for rapidly and © XXXX American Chemical Society
Received: February 14, 2019 Accepted: June 11, 2019 Published: June 11, 2019 A
DOI: 10.1021/acsami.9b02620 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces information of interest.19 More recently, we advanced this method with the use of a continuous wave (CW) laser, in which AuNPs, graphene oxide (GO), or reduced GO solution were used to accelerate and enhance the desorption process.20 In these methods, the use of nanomaterials was found to be critical for the laser-induced desorption process. However, an incubation step for a live tissue of 1.0−3.0 h to evenly distribute nanomaterials on the whole tissue surface is a timeconsuming and delicate step. The failure of uniform distribution of nanomaterials on the tissue surface for any reason causes a failure to obtain the information of interest. In this regard, an alternative method without the incubation step with nanoparticles is required for more straightforward and universal use of an AP-MS system for various biological samples.21,22 In this work, we investigated the use of a graphene-coated substrate for AP-MS imaging of live tissues with a 532 nm CW laser. We found that the use of graphene-coated glass substrates for AP-MS imaging of live tissues is compatible with the previous method reliant upon the nanoparticle incubation step before MS imaging. Interestingly, the desorption efficiency of live tissue with a CW laser was strongly correlated with the number of graphene layers on the glass substrate. The single-layer graphene (SLG)-coated glass substrate was excellent for efficient desorption of live tissue with 200 μm thickness, which enabled micrometer spatial resolution of hippocampal live tissue imaging with the CW laser.
2. EXPERIMENTAL SECTION 2.1. Materials. Polymethylmethacrylate (PMMA, average Mw ≈ 996 000, crystalline) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anisole and ammonium persulfate ((NH4)2S2O8) were purchased from Daejung Chemical & Materials Corporation (South Korea). Graphene-covered Cu foil was purchased from Graphene Platform Corporation (Japan). All materials for sucrose artificial cerebral spinal fluid (sACSF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Poly(ethylenimine) solution [50% (w/v) in H2O] was purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Preparation of Single and Multiple Graphene Layers on a Glass Substrate. For the SLG-coated glass substrate, a PMMA solution [average Mw ≈ 996 000, 4.0% (w/w) in anisole] was spincoated onto the surface of the graphene-covered Cu foil (single layer, Graphene Platform Corp., Japan) at 4000 rpm for 45 s and dried in air. A 0.1 M aqueous solution of (NH4)2S2O8 was used to remove the Cu foil. The PMMA polymer-supported graphene was then rinsed in deionized water several times and transferred onto a glass slide. The sample was dried in air for 30 min and annealed at 180 °C for 3 h. The polymer was then dissolved by immersion in acetone for 30 min and rinsed in methanol for 10 min and in isopropanol for 10 min (Figure 1A).23 For the preparation of the multilayer graphene-coated glass substrate, the PMMA polymer-supporting graphene prepared in the same manner as described above was transferred onto another graphene-covered Cu foil and dried at ∼50 °C for 10 min. Then, a PMMA-supported graphene-layer-covered Cu foil was made to float on a 0.1 M aqueous solution of (NH4)2S2O8 to remove the Cu foil. This transfer process can be repeated to control the number of graphene layers.24,25 The polymer-supported multilayer graphene was then rinsed in deionized water several times and transferred onto a glass slide. In the same manner, the sample was dried in air for 30 min and annealed at 180 °C for 3 h. The polymer was then dissolved by immersion in acetone for 30 min and rinsed with methanol for 10 min and then with isopropanol for 10 min. 2.3. Mouse Hippocampal Tissue Preparation. The live tissue slice has been a well-established experimental model in various
Figure 1. Fabrication of the graphene-coated glass substrate and the CW-laser-based MS system for live hippocampal tissue imaging. (A) Experimental procedures to prepare the graphene-coated glass substrate for hippocampal tissue imaging. (B) Schematic description for the ambient MS system combined with an inverted optical microscopic system equipped with a focused (20×, 0.45 NA objective lens) CW laser (532 nm) for desorption and AP helium plasma for ionization. research areas such as neurophysiology, pathophysiology, and electrophysiology. In particular, hippocampal tissue slices have been utilized for studying stretch-activated ion channels (SACs) and microelectrode arrays (MEAs) of various organotypic tissues. Live hippocampal tissue slices for our study were prepared with the sample preparation protocol used in the SAC and MEA studies.26,27 Male 7 week-old C57BL/6 mice were purchased from Koatech (Pyeongtaek, South Korea). We followed the guidelines for animal use and care in the Daegu Gyeongbuk Institute of Science & Technology (DGIST, South Korea). The primary purpose of developing ambient MS imaging is to observe live or fresh tissues with a minimal sample deformation. Immediately after sacrificing, the brain was extracted, and the isolated hippocampus was sliced transversely with a tissue chopper (McIlwain Tissue Chopper, Cavey Laboratory Engineering, UK) at a thickness of 200−500 μm according to the experimental protocol. It is very difficult to obtain hippocampal tissue slices of less than 200 μm without freezing or paraffin-fixation. In addition, a thickness of more than 200 μm is also required to maintain a short-term living tissue state. Oxygenated sACSF was used to aerate the hippocampal tissue slices (sACSF preparation: a solution containing 124 mM NaCl, 2.5 mM KCl, 24 mM NaHCO3, 5 mM N-(2-hydroxyethyl)piperazine-N′ethanesulfonic acid, 2 mM MgSO4, 2 mM CaCl2, 12.5 mM D-glucose, and 4 mM sucrose was bubbled with 95% O2/5% CO2 using an aquarium bubbler at 32 °C). After 1 h of aeration, the hippocampal B
DOI: 10.1021/acsami.9b02620 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces tissue slice was washed five times and placed on an O2 plasmapretreated graphene-coated substrate for tissue adhesion. For AuNPtreated hippocampal slices, the aerated slices were submerged in an sACSF solution (5 mL) with citrate-AuNPs (0.5 nM, 20 nm) for 3 h. Then, the hippocampal slice was washed 10 times and placed on an O2 plasma-treated graphene-coated glass slide (Figure 1B). 2.4. AP-MS Imaging System Setup. The previously developed AP-MS imaging system was used in this study with a change of the optical path from the laser source.19,20 The AP-MS imaging system consisted of a mass spectrometer, a 532 nm CW laser, a scanning stage, an atmospheric pressure (AP) plasma device, and airflowassisted ion transport equipment, as shown in Figure 2 (schematics) and Figure S1 (picture).
with a 30° angle of incidence. The desorbed neutral molecules produced by the focused laser essentially collide with the helium plasma. The metastable helium species in the plasma readily react with water, oxygen, or other components in the air to generate reactive protonated water clusters. A possible mechanism for ionization is that the desorbed biomolecules with proton affinities greater than water can be ionized by these protonated water clusters.28,29 The airflow-assisted ion transport equipment requires a gas pumping system coupled with the mass spectrometer. The equipment consisted of an ion-transfer tube, a chamber, and a diaphragm pump designed to transfer the ionized molecules to the mass analyzer. Even though a considerable number of biomolecules desorbed by the focused laser were directed to the MS inlet using the ion transport equipment, only a small fraction of the desorbed biomolecules was ionized by reactive protonated water clusters and metastable helium species. Therefore, carryover is not a significant problem in this APMS system. 2.5. Instruments for Raman and Helium Ion Microscopy. Raman spectra were acquired using an inverted Raman microscope (NOST, South Korea) with a 60× objective (NA 0.7) (Olympus, Tokyo, Japan). The sample was excited using a 532 nm laser to characterize the graphene substrate. The scattered Raman signal was detected with a confocal motorized pinhole (100 μm) directed to a spectrometer (FEX-MD, NOST, South Korea) (600 g mm−1 grating). EZScan (NOST, South Korea) software was used for the spectra acquisition and analysis. Helium ion microscopy (HIM) (ORION NanoFab, Carl Zeiss, USA) was used to obtain the surface morphology of the tissue slices. No conductive coating was required for HIM analysis. (Instrument setting for HIM: beam energy: 30 keV, beam current: 1.0−1.98 pA, average scan number: 16, working distance: 10.20 mm, gas field ion sources aperture size: 20 μm, scan dwell time: 2 μs, and field-of-view: 700 μm).
Figure 2. Schematic description of the AP-MS imaging system used in this study. The AP-MS imaging system consists of (i) a mass spectrometer, (ii) a scanning stage, (iii) a 532 nm CW laser, (iv) an AP plasma device, and (v) airflow-assisted ion transport equipment.
3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of GrapheneCoated Glass Substrate. The typical transfer method for graphene onto the glass substrate is suitable to prepare a controlled number of graphene layers on a glass substrate.23−25 Raman analysis showed a characteristic Raman spectrum of graphene on glass as shown in Figure 3A. The G peak at 1580 cm−1 is the signature peak produced by the in-plane phonon mode of all graphite materials.25 The intensity of the G peak is proportional to the number of graphene layers (
