ionization mass spectrometry

Author’s Accepted Manuscript Inkjet automated single cells and matrices printing system for matrix-assisted laser desorption/ionization mass spectrometry Ahikito Korenaga, Fengming Chen, Haifeng Li, Katsumi Uchiyama, Jin-Ming Lin www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(16)30804-9 http://dx.doi.org/10.1016/j.talanta.2016.10.055 TAL16975

To appear in: Talanta Received date: 10 August 2016 Revised date: 8 October 2016 Accepted date: 12 October 2016 Cite this article as: Ahikito Korenaga, Fengming Chen, Haifeng Li, Katsumi Uchiyama and Jin-Ming Lin, Inkjet automated single cells and matrices printing system for matrix-assisted laser desorption/ionization mass spectrometry, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.10.055 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Inkjet automated single cells and matrices printing system for matrix-assisted laser desorption/ionization mass spectrometry Ahikito Korenagaa, Fengming Chena, Haifeng Lia, Katsumi Uchiyamab, Jin-Ming Lina,* a

Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Department of

Chemistry, Tsinghua University, Beijing 100084, China b

Department of Applied Chemistry, Graduate School of Urban Environmental Sciences, Tokyo

Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan *

Corresponding author: Tel/fax: +86 10 62792343, E-mail: [email protected]

Abstract The ability of single or several cells introduction onto substrate simply would be a useful tool for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this study, we aimed to establish a sample introduction method for pattering cells to the substrate by inkjet technology. Inkjet driving, substrate surface and relative humidity were optimized for single or several cells introduction. Single type cell solution and MALDI matrix solution were automatically printed onto ITO glass substrate which was hydrophobic modified under humidity controlled condition. Then the substrate was inserted to MALDI-MS and cells sample solution provided several peaks from phospholipids. The inkjet technique enables us to print single and subcellular on the substrate with the range of a few hundred micrometers. This diameter would be useful for targeting by laser of MALDI-MS. Our technique provides a new platform for MALDI-MS analysis in single or several cells to get a wide information from one sample.

Graphical abstract

Inkjet cell introduction for matrix assisted laser desorption/ionization (MALDI) mass spectrometry. Single type cells were printed on the MALDI applicable substrate, the substrate was directly inserted to mass spectrometer after sample and matrix printing. 2, 5dihydroxybenzoic acid was selected as a matrix for lipid detection.

Keywords: inkjet, mass spectrometry, laser desorption/ionization, single cells.

1. Introduction Qualitative and quantitative analysis in the area of single or several cells level have great importance to elucidate biological, chemical and medical science. Information provided from single and subcellular studies promotes understanding of inter- and intracellular communication [1, 2], drug screening [3], cell differentiation [4, 5] and chemical responses [6]. In order to

approach these information, various analytical techniques to monitor chemical compositions associated with cellular events dynamically were estimated such as fluorescent observation using organisms exhibiting fluorescence or quantum dots [7, 8], surface plasmon resonance imaging [9], and microfluidic device for mimicking bio-structure [10]. These are actually very powerful method for detecting a specific target by its highly selectivity and especially fluorescent observation using organisms exhibiting fluorescence method is widely used in single and subcellular analysis. Although these have high sensitivity and selectivity for specific quantitative analysis, it provides a narrow information from one sample in single analysis procedure. Mass spectrometry is focused on applying to cellular analysis because it can provide qualitative and quantitative information in a wide variety of bio-compounds [11]. The molecular mass calculated from mass-to-charge ratio value is used for identification and peak intensity in mass

spectrum

can

be

contributory to

quantitation.

Notably matrix-assisted

laser

desorption/ionization mass spectrometry (MALDI-MS) is useful method for detecting large organic molecule such as peptide [12, 13], protein [14, 15], gene [16, 17] and phospholipid [18]. It has possibility to apply for single or several cells analysis, direct analysis of cellular materials at the single-cell level using MALDI-MS have been reported [19, 20]. However the throughput of introduction for single or several cells was low. Because normally the diameter of laser for sample ionization is the range of 50 to 100µm, single or several cells introduction corresponding to laser diameter requires troublesome procedure or a special kind of instrument. Therefore, single and several cells introduction with high throughput is required to promote its analysis by MALDI-MS. Until now, several techniques such as cell capturing by micro structure [21], micro manipulating [22], droplet based separation [23] and drop-by-drop introduction by inkjet device [24] have been applied to single or several cells introduction. Particularly drop-by-drop

introduction method by inkjet device is respectively suitable for MALDI-MS analysis of them because it doesn’t require any dedicated platform or strict condition. Thus inkjet introduction system can directly print sample onto any substrate including the plate for MALDI-MS analysis. In addition, inkjet device can provide sub-nanoliter droplets with high throughput controllably. On the other hand, a number of researchers have reported that inkjet printers could be employed as automatic pipettes to create microarrays [25], manufacture three dimensional parts [26], print electrical devices [27, 28] and facilitate combinatorial chemistry [29]. This technology has been focused on applying to biological field especially in cell or tissue study because of its ability to make micro scale droplets with high reproducibility [30-32]. Sakurai et al. have reported the introduction of cells immobilized on patterning Poly-deoxyribonucleic acid (DNA) using inkjet technology [33]. In this case, printed DNA on the substrate captured complimentary DNAmodified cells. Cells were immobilized on the substrate by DNA binding. Besides, several works have been reported on applying inkjet device to sample preparation process for MALDI-MS analysis. Delaney et al. have reported inkjet introduction method of several MALDI matrices in multiple concentration on single tissue being used as a substrate [34]. They have shown the probability of inkjet technology for small site analysis in MALDI-MS. Inkjet technology has not been gotten attention widely in analytical area yet, but analytical study has trended in the attention of small site analysis recent. Inkjet technology would take the place of batch method or droplet-based method as an introduction platform. The objective of this paper is to provide a new platform as simple and high reproducibility sample introduction system using inkjet technology to get a wide range of information from Single or several cells levels in MALDI-MS analysis. Single or several cells solution and MALDI matrix solution were printed on ITO glass substrate modified hydrophobic. In addition,

the high relative humidity kept constant by saturated salt method to control the marangoni distribution which is dominant fluid phenomenon inside small droplet. Printed sample were directly inserted to MALDI-MS and it provided several peaks from phospholipids.

2. Experimental 2.1. Chemicals and materials Octadecyltrimethoxysilane (ODS) was purchased from TCI chemicals (Shanghai, China). Phosphate buffered saline (PBS) buffer and 2, 5-dihydroxybenzoic acid were purchased from Sigma-Aldrich Co., LLC. (St. Louis, MO, USA). Trifluoroacetic acid was purchased from J&K scientific Ltd. (Beijing, China). Dulbecco's Modified Eagle's Medium and trypsin−EDTA were purchased from Gibco (Grand Island, NY, U.S.A.). Purified water was produced by Wahaha Group (Hangzhou, China). All other reagents used in this experiment were of analytical reagent grade and used without further purification. 2.2. Apparatus Inkjet device was purchased from Fuji Electrics Systems Co., Ltd. (Tokyo, Japan). A 75-mm glass tube used to contain and supply solutions to inkjet was provided by Funakoshi, Co. (Tokyo, Japan). The electromotive x-y stage MMU-30X was Chuo Precision Industrial Co., Ltd. (Tokyo, Japan). Laboratory-made software controlled the inkjet waveform, voltage volume, adding voltage time and the number of droplet. The x-y stage movement was controlled by other laboratory-made software as well. The distance between ITO glass substrate and inkjet head was fixed at 10 mm. The dropped cells were visualized and recorded using a microscope (Leica DMI 4000 B, Wetzlar, Germany) equipped with a CCD camera (Leica DFC 300 FX, Wetzlar,

Germany). MS analysis was performed using an AXIMA Performance MALDI-TOF/TOF mass spectrometer (Shimadzu Co. Ltd. Kyoto, Japan). 2.3. Pre-treatment of ITO glass slides ITO coated glass slides for MALDI measurements were firstly immersed in piranha solution (3: 1 sulfuric acid to hydrogen peroxide) for 1h. Then slides were rinsed in deionized water and dried by nitrogen gas. After that, slides were immersed in ODS for 24h. Then slides were rinsed in deionized water and acetonitrile each with ultrasonic. At last, slides were dried by nitrogen gas quickly. All treated slides were used within 2days from cleaning up. 2.4. Humidity control All introduction process using inkjet device were operated under humidity controlled environment. KCl saturated solution was filled in 60mm diameter petri dish. The cap of petri dish for sealing was thick plastic film with entrance for inkjet head (laboratory made). The relative humidity inside petri dish was maintained constant at 86 %. 2.5. Matrix pre-treatment Matrix solution was 200 mM 2, 5- dihydroxybenzoic acid in 50 % acetonitrile, 0.1 % trifluoroacetic acid and 10mM NaI. The matrix was agitated in ultrasonic bath for 30min immediately before introduction to inkjet device. This matrix solution was used for all MALDI measurement. 2.6. Cell culture and pre-treatment HUVEC cell were grown in culture dishes in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum, 100 mg/mL penicillin, and 100 mg/mL streptomycin. Cells were

incubated in humidified air with 5% CO2 at 37 ℃. The medium was removed 24 h after and fresh medium was added. Cells were detached from the petri dish with 0.25% trypsin to micro tube. Micro tube was centrifuged at 1200 rpm for 3 min and supernatant was carefully removed. Then 1 ml of PBS buffer solution was added in micro tube and cells were washed by several times of pipetting. This washing process was repeated twice. After washing, PBS buffer solution was added to condition cell concentration at 6.6 × 106 cells per ml. 2.7. Inkjet injection and sample introduction All sample introductions using inkjet were driven at optimized condition for single cell per single droplet as follow. Driving voltage which is the value of adding voltage to piezo element to change its shape was 30V. Driving time which is adding time of voltage to piezo element was 25us. Figure.1 (a) shows the scheme of double sample injection method by using inkjet technology. In this method, introduction process was divided to cell sample solution and matrix solution correspond to traditional introduction method on hand operating. Prepared cell and matrix solutions were introduced a channel of inkjet device each. On the first dropping, cell solution was dropped onto ITO glass substrate. Cells were printed with 6 by 6 array in 1.5 mm square. After drying the solution at room temperature, matrix solution was dropped onto the same position of last dropped solution as the second dropping. Fig. 1b shows the scheme of single sample injection method. In this method, cells were removed from PBS buffer solution and mixed in matrix solution before introducing to inkjet device. Cells were printed with 6 by 6 array in 1.5 mm square as well. The scheme of this method is shown in Fig. 2. Cells and matrix solutions were dropped onto ITO glass plate which was modified hydrophobic using inkjet device at controlled high humidity. After sample solution dried out, it was inserted to MALDIMS and some peaks derived from phospholipids were detected.

2.8. MALDI-MS measurement Prepared glass slides were fixed onto dedicated MALDI plate by carbon conductive tape and it was inserted into MALDI-MS. MALDI-MS was equipped with a 337 nm nitrogen laser. The data were collected in the positive linear mode and signals from 400 to 1200 m/z were collected. For each mass spectrum, mass spectra resulting from 20 laser shots at 5 Hz were accumulated to obtain an average mass spectrum for each sample data.

3. Results and discussion ITO coated glass plate has been widely used alternative to MALDI plate to apply for tissue imaging mass spectrometry and it enables mass spectrometry combine optical microscopy due to its conductivity and transparency. The surface of ITO glass slide was modified by ODS because ITO glass has hydrophilic surface and droplets expand. Contact angle measurement was first gathered to estimate surface hydrophobicity. The angle against ITO glass slide surface modified by ODS was 84 degrees, which was enough to make marangoni flow in droplet for gathering cells to facultative point (Fig.3). Relative humidity of introduction part was controlled on 86% by saturated salt method to keep the marangoni flow constant in small droplet which was introduced from inkjet head. In addition, high relative humidity cut down on sample solution evaporation from inkjet head. Sample and matrix solutions were then dropped onto ITO glass plate which surface was modified hydrophobic before dropping. Fig. 4 shows the optical images of dropped cell solution after first injection of double introduction method. Cell solution and matrix solution were completely dropped to same point on substrate and cells were covered with matrix through double introduction method. Accordingly the reproducibility loss caused by position gap was

avoided. On the other hand, matrix crystal wrapped cells through single introduction method Fig. 4b. Matrix crystal was grew up with droplet drying and cells were gathered on convergence point of droplet drying by marangoni flow, thus matrix crystal including cells was organized on convergence point. Condensed droplet helped the ionizing laser hit sample in MALDI-MS, peaks from cells were obtained easier than expanded sample. Moreover, the introduction system could control the number of cell by optimizing inkjet driving condition respectively. 32 % of droplet has one cell and others have 0, 2, 3 and 4 cells in one droplet. The number of cells in one droplet varied from each droplet because cells were individually immersed in solution. Therefore the variation in the number of cells was corresponded to normal distribution (Fig.3c). Although 32% of droplet had one cell in one droplet, inkjet device should be useful method as a single or several cells sampler. Observing number of cells in solution by specific camera and manipulating using dedicated instrument, droplet without including one cell could be removed. The number of cell itself is not main issue in this work so now the ability of controlling cell number in our inkjet introduction system is mentioned. Unfortunately, PBS buffer is not suitable for MALDI-MS analysis due to sodium and potassium which prevent effective ionization in solution. So, purified water was used as solvent instead of PBS buffer in this work. But cells could not be present stably and should be easily burst because of difference of osmotic pressure between inner cells and purified water. Glutaraldehyde can cross link the proteins within the cells and its structural and component information could be maintained strictly. Cells were modified with glutaraldehyde and immersed in purified water before inkjet introduction. In order to confirm the usage of this method for mass spectrometry measurement, phosphatidylcholine (PC) was focused on due to its abundant distribution in cell membrane.

Shown in Fig. 5, characteristic peaks could be shown in the MALDI mass spectra acquired on PC assisted by DHB as a matrix. For example, m/z782 which is the highest peak in the figure indicates the PC (34:1) (a phosphatidylcholine with 34 fatty acid and one double bond) with Na adduct. The second m/z808 is identified PC (36:2) with Na adduct. Other peaks related with phospholipids were described in Table 1. These results indicate inkjet introduction system is able to take a place of batch method. Inkjet device provides high reproducibility for sample introduction, which has relied on operator technique of pipetting. In addition, small droplets dried under humidity control which provided from inkjet device made condensed solution by marangoni flow. The combination of inkjet introduction system and marangoni flow control is respectively useful for single or several cells analysis in MALDI-MS.

4. Summary Inkjet can be applied to MALDI-MS analysis by optimizing the driving condition, substrate surface condition and humidity of introduction. Sample condensing provided from marangoni flow control is suitable not only single or several cells analysis but also microscale analysis. The wide mass information provided from single type cells could promote bio-analytical studies, such as cell-cell interactions, drug screening, chemical responses and high throughput assays in functional analyses of genes.

Acknowledgements This work was financially supported by National Natural Science Foundation of China (Nos. 81373373, 21227006, 21621003) and CERS-China Equipment and Education Resources System

(No. CERS-1-75). Korenaga highly acknowledges the Chinese Government Scholarship for the award of Doctorial studies.

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Figure 1. The scheme of inkjet automated injection system. (a) Matrix solution and cells solution were individually introduced onto ITO glass slide. (b) Pretreated matrix including cells solution was introduced onto ITO glass slide at one time. Figure 2. Total scheme of this method. Sample and matrix solutions were introduced by suitable introduction way under controlled condition. After drying, ITO glass slide was inserted to MALDI-QTOF-MS and peaks derived from phospholipids were collected. Figure 3. The photo image of contact angle measurement. Droplet has 42 degrees against clean ITO glass surface. (a) Droplet has 86 degrees against SAM modified ITO glass surface. Figure 4. The optical image of .dropped cell solution from inkjet device. (a) Droplets dried under humidity control. The scale bars are 200 um. (b) The graph of number of dropped cells per each droplet (N=36). 32% of droplet has one cell in one droplet. The distribution corresponds to normal distribution. (c). Figure 5. the obtained mass spectra from HUVEC cells; (a) and the assigned phosphatidylcoline list from mass spectra. (b).

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Table 1. Effect of free radical scavengers with and without the presence of N-CDs and NaOH m/z

Assigned lipid

716

PC(O-32:1)

726

PC(30:2) + Na

732

PC(32:1)

742

PC(O-34:3)

754

PC(34:4)

758

PC(34:2)

770

PC(O-36:2)

782

PC(34:1) + Na

786

PC(36:2)

808

PC(36:2) + Na

824

PC(40:3)

Highlights  





Inkjet device was used as an automated sampler for matrix assisted laser desorption/ionization mass spectrometry. Inkjet introduction system was used for single type cells printing. Matrix solution was printed on the same point as well. These samples were automatically printed and it provided high reproducibility. In the range of sub-nano liter droplet on hydrophobic surface created convection flow in it. Droplets were dried under constant humidity to control the convention flow. It helped droplets condense on the arbitrary point. Phospholipids were focused on this method because of its high distribution on cell membrane. Some peaks derived from phosphatidylcoline were obtained in linear positive mode detection.