Single-Cell Analysis Using Drop-on-Demand Inkjet Printing and Probe

Mar 25, 2016 - Hua Zhang , Wei Kou , Aisha Bibi , Qiong Jia , Rui Su , Huanwen Chen ... Hua Zhang , Aisha Bibi , Haiyan Lu , Jing Han , Huanwen Chen...
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Single-Cell Analysis Using Drop-on-Demand Inkjet Printing and Probe Electrospray Ionization Mass Spectrometry Fengming Chen,† Luyao Lin,† Jie Zhang,† Ziyi He,† Katsumi Uchiyama,‡ and Jin-Ming Lin*,†,§

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Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China ‡ Department of Applied Chemistry, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan § Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in University of Shandong, Shandong Normal University, Jinan 250014, China S Supporting Information *

ABSTRACT: This study describes a novel method for singlecell analysis and lipid profiling by combining drop-on-demand inkjet cell printing and probe electrospray ionization mass spectrometry (PESI-MS). Through inkjet sampling of a cell suspension, droplets with single cells were generated, precisely dripped onto a tungsten-made electrospray ionization needle, and immediately sprayed under a high-voltage electric field. Lipid fingerprints of single cells were obtained by a mass spectrometry (MS) detector. A homemade magnetic stirring device was applied to the cell suspension reservoir, which controlled the homogeneous distribution of cells in liquid and improved the single-cell-droplet percentage by 43.8%. Eight types of single cells were screened in our platform and further differentiated by principal component analysis based on cellular surface phospholipids. Thus, this study successfully provides a facile method for the direct MS profiling of single-cell lipids by PESI-MS.

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capture technique17,18 and has been extensively applied in drug testing against individual cell response19,20 and cell heterogeneity screening.21,22 Although the microarray technique enables the direct observation of cell morphology by microscopy and ensures fine compatibility with a fluorescence probe, it is limited by several drawbacks, such as sample crosscontamination and laborious reagent addition procedures. Direct deposition of the cell microarray onto the matrixassisted laser desorption ionization (MALDI) target plate by inkjet nozzle enables a label-free method for cell profiling with minimum sample pretreatment, but a fully automatic system is yet required to enhance the throughput.23,24 In addition, several derivatization procedures may be necessary to improve detection sensitivity, particularly for analytes with trace content inside cells.25−27 Other methods, such as flow cytometry and microfluidic droplets for cell sorting using fluorescence labeling of cell surface markers and metabolites, establish a highthroughput screening platform with processing capacity of up to ∼200 Hz.28,29 However, multiplexing identification is restrained by the lack of highly specific and purified antibodies

ajor differences of multiple cell types have been generally profiled by the expression of specific biomarkers, such as CD133, CD31, and nestin;1−4 however, in the same cell population, the heterogeneities of individual cells are poorly investigated. The function and fate of cells, including gene and protein expression, cell proliferation, selfrenewal, and apoptosis, are always diverse at the single-cell level.5−7 Profiling individual heterogeneity in a cell population is important when investigating cellular drug metabolism8,9 and deciphering the pathogenesis of certain severe deceases,10 as one or a few mother cells are typically regarded as essential to tumor development.11,12 Moreover, the identification of cell subpopulations provides a hint for better understanding of cell−cell communications and innate hierarchy within a cellular microenvironment.13,14 Conventional cellular experiments employ a large amount of cells for drug stimulation studies and metabolite analysis, which arbitrarily lyses and collects cell samples into one pot. These methods assess the overall response of the cell population, but fail to consider the heterogeneity at the single-cell level. Recently, numerous methods have been developed to disperse and confine single cells in discrete compartments for further fluorescence and mass spectrometry (MS)-coupled analysis. One important method is single-cell microarray, which is facilely fabricated by microcontact printing15,16 or microwell © 2016 American Chemical Society

Received: December 15, 2015 Accepted: March 25, 2016 Published: March 25, 2016 4354

DOI: 10.1021/acs.analchem.5b04749 Anal. Chem. 2016, 88, 4354−4360

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as well as effective chromophores to evade fluorescence interference. Since its discovery, MS has been developed as a powerful tool for the qualitative and quantitative detection of analytes ranging from inorganic elements to small organic molecules to macromolecules, such as proteins and nucleic acids. MS has been extensively applied to the study of drug cytotoxicity,30 cell−cell interaction,31,32 and cellular metabolism.33 Multiplexing analysis can be achieved in one scan, benefiting from the diverse information on ion fragments in the entire spectrum. However, the vacuum condition required by conventional methods, such as MALDI and SIMS, makes them less applicable for on-site live cell analysis. The development of the ambient ionization method further enhances the analytical capacity of MS for complex samples, particularly biological samples, such as blood34 and tissue slice,35,36 and spares the tedious sample pretreatment steps. Beginning with desorption electrospray ionization37 and direct analysis in real time,38,39 several variants of the ionization method have been established, for example, desorption atmospheric chemical ionization,40 dielectric barrier discharge ionization,41 and paper spray,42 which are extensively used in material surface mapping and biosample MS imaging. The probe electrospray ionization (PESI)43 method employs a sharp tungsten needle as probing tip and spray emitter for direct sampling. With an auxiliary nozzle for solvent deposition, analytes can be sprayed and ionized under a high-voltage electric field. Zhang’s group developed a PESI method for subcellular-scale molecule profiling in onion cells, with the resolution to differentiate the nucleus and cytoplasm chemically.44 Yang’s group developed a new single-probe MS technology capable of real-time, in situ metabolomics analysis of individual living cells.45 The deposition of solvent enhancing analyte ionization requires precise control of nozzle position to exactly above the needle probe and a precise quota of solvent to reduce interference and embrace reproducibility. The inkjet nozzle combined with the micromotor X−Y platform perfectly meets the demands of profiling, which is capable of producing large-scale arrays in a fully automatic manner at the single-cell level.46,47 Taking the advantage of direct MS sampling of PESI and inkjet cell manipulation, a single-cell MS screening platform can be easily established. Herein, we report a single-cell lipid profiling platform combining an inkjet nozzle cell manipulator with probe electrospray ionization mass spectrometry (PESI-MS). Lipids are often involved in several vital cell physiological processes, and abnormal fluctuation of their contents may lead to certain diseases, such as obesity and diabetes.48 A homemade magnetic stirring device was applied to the cell suspension reservoir to control the homogeneous distribution of cells in liquid. Through inkjet sampling of the cell suspension, droplets containing single cells were generated, precisely dripped onto the tungsten tip of the ESI needle, and immediately sprayed under a strong electric field. Cellular lipid fingerprints were obtained by the MS detector. By adjusting the cell concentrations, the single-cell-droplet fraction was improved to as high as 43.8%. Eight types of cells were screened in our platform and further differentiated by principal component analysis (PCA). This platform provides a facile method for the direct MS profiling of single-cell lipid species without derivatization or labeling procedure and presents a significant potential for future applications of cell subpopulation identification and related disease diagnosis.

Article

EXPERIMENTAL SECTION

Reagents. RPMI medium 1640 and trypsin−EDTA were purchased from Gibco (Grand Island, NY, U.S.A.). Live/Dead assay kit (Calcein AM/EthD-1), penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, U.S.A.). Phospholipid standards were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). All other reagents used in this experiment were of analytical reagent grade and used without further purification. Apparatus. A laboratory-made high-voltage power supply (0−30 kV) was used for electrospray ionization. A 75 mm hematocrit tube used to contain and supply the inkjet solutions obtained from Funakoshi, Co. (Tokyo, Japan). The electromotive X−Y stage MMU-30X was purchased from Chuo Precision Industrial Co., Ltd. (Tokyo, Japan). Laboratory-made software controlled the inkjet driving waveform and the X−Y moving stage. Then, images of the tungsten tip were obtained using the Dino-Lite digital microscope (AM4815ZT, AnMo Electronics Corporation, Taiwan). ESI-Q-TOF was conducted on the Bruker microTOF-Q mass spectrometer (Bruker Daltonics Inc., Billerica, MA, U.S.A.), and the mass spectra were obtained in the negative mode and analyzed using the DataAnalysis software package provided by Bruker. Probe Preparation. The probe was designed with a tip diameter of 2 μm and controlled by a three-dimensional manipulator (SS-40-T, purchased from Semishare Electronic Co., Ltd.) of the MS inlet (shown in Figure S1, Supporting Information). The tungsten-made probes (99.9% tungsten, ST20-2, purchased from GGB Industries Inc.) were initially washed with methanol/water (1:1) to clean its surface and then soaked in 5% HNO3 for 5 min. Afterward, the probes were washed with ultrapure water, methanol/water (1:1), and ethanol. Regeneration of the probes was quite simple. The used probes were washed with methanol/water (1:1), ultrapure water, and ethanol. Afterward, the probes could be used again. By applying a high voltage, an electrospray cone could be formed from the liquid droplet on the probe tip. Cell Culture and Sample Preparation. U87, U251, HepG2, MCF-7, 293, Caco-2, HUVEC, and 3T3 cells (Cancer Institute and Hospital, Chinese Academy of Medical Science, Beijing, China) were cultured with RPMI medium 1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin in the cell incubator with 5% CO2 at 37 °C. The cells were trypsinized and reseeded every 2 to 3 days. Before the experiment, the cells were treated with trypsin− EDTA and removed from the dishes after reaching 80% confluence. Then, the cells were centrifuged and resuspended to obtain a cell suspension of appropriate cell concentration. The cell suspension was aliquoted, and a viability test was conducted using the Live/Dead cell assay kit. The cell suspension used for the experiments had an average of 98 ± 1% viable cells prior to the experiment. Then, the cells were centrifuged at 1000 rpm, fixed with paraformaldehyde, washed three times with PBS solution and pure water, and finally diluted in 50% methanol/water at a density of ∼2.1 × 106 cells/ mL. The cell suspension was kept in a centrifuge tube. A 2 × 3 mm magnetic stick was placed in the tube, which could be driven by an electromotor to prevent cells from sinking or aggregating. The cell suspension was connected to the printing channels of the inkjet head by PE tubes to supply a homogeneous density of cells. 4355

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aggregation. The tungsten probe was fixed in a high-precision X−Y stage at the appropriate position to the inlet of ESI-MS, whereas the inkjet device was moved to the target position by mechanical movement of the X−Y stage and subsequently printed droplets onto the tungsten tip for MS detection. The distance between the tungsten probe tip and the inlet of the mass spectrometer and the distance between the inkjet printing outlets and the tungsten probe tip were critical parameters in this experiment. After careful optimization, the distances of 1.5 and 0.5 cm were utilized to produce the best droplet spraying and ionizing performances. For clearance of nonvolatile cell debris, a different channel in inkjet was used to perform probe washing by printing blank droplets to the needle. Steps of cell profiling and probe washing were carried out alternatively by controlling inkjet head back and forth to direct the different channels to probe. The switching of different channels was controlled by programmed X−Y stage movement automatically and took about 20s for each cycle of profiling and washing. Mass spectra were acquired over the mass range of 0 to 1500 in the reflective positive ion mode.

Operation of the Inkjet Device. The inkjet device was pretreated according to the method described elsewhere.49 Prior to use, the inkjet printing head was washed with Extron washing solution and treated by ultrasonic wave for 15 min. Before printing of the cells, the inkjet channels were washed thoroughly three times following the sequence of washing solution, pure water, ethanol, and PBS solution to remove residuals and air bubbles, which would disturb the printing of droplets, completely. Afterward, the inkjet head was integrated into the electric system composed of voltage and position control components as well as a computer with controlling software. After examination of the entire system and confirmation of its functions, the cell suspension reservoir was connected to the printing channels of the inkjet head by the PE tubes. The position of the inkjet could be adjusted by a precise automatic X−Y stage, and the droplet volume could be changed by adjusting the voltage and pulse time (shown in Figure S2, Supporting Information). Under optimal conditions, a voltage of 38 V and a pulse width of 20 μs were applied to the piezoelectric ceramic on the inkjet. The optimal conditions of the driving waveform and pulse width for inkjet printing dropon-demand droplet volume are listed in Table S1, Supporting Information. The volume of each droplet for the cell suspension solution was 486 pL. Single-Cell Inkjet Printing Droplet Combined with PESI-MS. The design of the platform is shown in Figure 1. The

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RESULTS AND DISCUSSION Inkjet Printing of Single Cells. Before integrating inkjet cell printing with PESI-MS, we validated the single-cell ratio of the inkjet printing system. Cells were printed onto glass slides, and the number of cells per spot was observed under a microscope. The glass slides were soaked in NaOH (1 M) solution for 30 min, then washed thoroughly at least three times with pure water, and finally dried using nitrogen gas. The X−Y-axis system was applied to produce arrays of printed spots at 100 μm center-to-center distance. The cell suspension was loaded into the reservoir as described previously, and droplets with single cells were printed automatically using the hardware and algorithm. The glass slides were inspected under a brightfield microscope, and the cells in each printed spot were counted manually. The printing conditions and solution concentrations were optimized to obtain a high percentage of single-cell spots in the array. The theoretically highest probability of single-object dispensing for a random dispersion was estimated to be approximately 37%, whereas the best results obtained were significantly higher than this value by approximately 43.8% (as shown in Figure 2). Before printing of

Figure 1. Schematic diagram of the experimental setup: (a) inkjet printing of cells onto the tungsten tip for MS analysis and (b) image of the inkjet printing of cell-containing droplets to the tungsten tip applied with a high voltage.

piezoelectric inkjet printing technology could adjust droplet volume by variation of the driving voltage and pulse width for the piezo, and the number of ejected droplets could also be controlled to increase single-cell efficiency. Drop-on-demand techniques are preferred because of their relative simplicity and capability for precise noncontact deposition, yet these techniques have been hindered by several critical limitations. Cell settling and aggregation within printer reservoirs obstruct the nozzles and lead to nonuniform cell distribution such that cell output significantly decreases or fails when printing over long time periods. A magnetic stick was placed in the cell suspension tube to solve this problem, and a permanent magnet was fixed in the electromotor at a uniform rotation rate to drive the magnetic stick in the cell suspension tube, ensuring homogeneous distribution of the cells in the liquid without

Figure 2. (A) Cell distribution of the optimized single-cell dispensing experiment. (B) Distribution of different cell number per spot and a Poisson fit. Marked red spots contain single cell. Scale bar 100 μm.

single cells, we removed the first 1000 drops. In this manner, the dead volume in the inkjet channel was effectively removed because the first set of dripped droplets were always the blank droplets (shown in Figure S3, Supporting Information). PESI-MS Detection of Inkjet-Printed Cells. The nozzle of the inkjet is aligned with the tungsten tip under the microscope and then fixed on the X−Y stage with frames. 4356

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Figure 3. Investigation of the detection capability of different cell numbers: (a) 10−20 cells and (b) a single cell.

Position control of the X−Y stage used in our experiment had a high precision, with tolerance of less than 0.5 μm. Therefore, by adjusting the X−Y stage, the nozzle of the inkjet can be moved on top of the tungsten tip such that the generated droplets can fall onto the tip. Considering that a narrow probe tip provides a more focused Taylor cone and increases the fraction of ionized analyte in the MS detector, we designed the probe tip to be 2 μm. Besides, to aim the droplet to the very tip of ESI needle can be difficult. Therefore, we adjusted the relative position of inkjet to needle, and made the impingement happen in the inner area about 500 um to the tip. Under the high voltage applied on needle, the droplet attachments would be driven to the tip, sprayed, and ionized to MS detection. The contact area (200 um) is larger in diameter than droplet (100 um), and therefore, the droplet hit the needle entirely. The schematic setup is illustrated in Figure 1. The probe was controlled by a three-dimensional manipulator and precisely placed in front of the MS inlet. A high voltage of 2500 V was applied to the probe. The inkjet nozzle was controlled by a two-dimensional manipulator to print droplets containing cells onto the tip precisely, ionize directly, and transfer into the MS for detection. The duration for each electrospray lasted for seconds, and with the washing step, nonvolatile components can be efficiently removed in mass spectra (shown in Figure S4, Supporting Information), thus preventing the interference between different droplets. Although the droplet generation frequency in inkjet nozzle could be as high as 1 kHz, the relatively longer time electrospray limited the analysis throughput to 0.05 Hz. By adjusting the cell density, the number of cells in each printed droplet could be controlled. In this study, we compared a high cell density with an ordinary experimental cell density. For the high cell density, each droplet contained approximately 10−20 cells. Meanwhile, for the ordinary experimental cell density, a relatively high single-cell fraction was achieved to realize single cell analysis with the minimum switches of profiling and washing channels. A droplet containing more cells produced a higher signal intensity while the peak positions of the mass spectra were almost the same (shown in Figure 3). This result proved that the developed system could be applied

to detect cells in droplets and could distinguish different cell densities. Then, the optimized cell density was utilized to achieve the highest single-cell occupation with an acceptable fraction of multiple-cell droplet which is no more than 30%. The MS signals were inspected for different cell densities because distinguishing single-cell droplets with empty droplets is essential. In this study, two different kinds of cells were used, and their components were carefully analyzed on the basis of the signal outputs. As shown in Figure 4, for the case of single U251 and MCF-7 cells, characteristic peaks could be observed. Meanwhile, in the case of empty droplets, the signal intensity was lower, and most characteristic peaks were lost. As for the residual signals in the condition of empty droplets, we determined them from the impurities inside the spectrometer by checking the blank water/methanol solution (data not shown). Considering the relatively lower intensity of residual signal, it did not interfere with our analysis. This result indicated that the inkjet cell printing PESI-MS system was capable of single-cell MS analysis. Determination of Major Cell Subpopulations by PESIMS Using PCA. After validation of the single-cell detection capability of the developed system, a cell classification experiment was conducted. Eight different kinds of cells (U87, U251, HepG2, MCF-7, 293, Caco-2, HUVEC, and 3T3 cells) were analyzed by this system, and the mass spectra with their particular characteristic peaks were obtained (shown in Figure S5 and Figure S6, Supporting Information). In this experiment, the PCA was utilized for the classification of different kinds of cells. The PCA is a statistical procedure that uses an orthogonal transformation to convert a set of possibly correlated variables into a set of values of linearly uncorrelated variables. The PCA is one of the most frequently used tools in exploratory data analysis and for making predictive models. The PCA can be implemented by eigenvalue decomposition of a data covariance (or correlation) matrix or singular value decomposition of a data matrix, usually after mean centering (and normalizing or using Z-scores) the data matrix for each attribute. The molecular weights of several major phospholipids 4357

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Figure 4. Investigation of the detection capability of a single cell.

that could be detected on the cell surface are shown in Table 1. HepG2, HUVEC, 3T3, and MCF-7 cells were selected as examples to demonstrate the PCA process. The phospholipid

measurement data are shown in Figure 5. The heterogeneity between cells was evidently revealed through PCA based on the signal intensities, and the four kinds of cells were successfully classified into separate colonies. This result indicated that the developed system achieved high-throughput single-cell analysis and had the potential for cell population identification, which was significant and could be applied to diagnose diseases within minutes. Detection of Cell Markers by PESI-MS. In addition to the aforementioned experiments, the detection of cell markers was also conducted. Rhodamine 6G was selected as a model compound for cell labeling to examine the detection capability of cell markers. Rhodamine 6G (2 ppm) was used to mark MCF-7 cells for PESI-MS detection. Afterward, the mass spectra of an empty droplet, a single MCF-7 cell, and a single MCF-7 cell marked with Rhodamine 6G were analyzed and compared. From the results obtained (Figure 6), single MCF-7 cells and single MCF-7 cells marked with Rhodamine 6G had almost the same mass spectra, except for the peak of Rhodamine 6G at m/z 443.3304. Meanwhile, for the empty

Table 1. Identified the Phospholipids in Single Cells by PESI-MS Analysis compound PC(32:1) PC(32:1) PC(32:0) PC(32:0) PC(34:2) PC(34:2) PC(34:1) PC(34:1) PC(34:0) PC(36:2) PC(36:2)

m/z

formula +

[M + H] [M + Na]+ [M + H]+ [M + Na]+ [M + H]+ [M + Na]+ [M + H]+ [M + Na]+ [M + Na]+ [M + H]+ [M + Na]+

732.5543 754.5357 734.5694 756.5514 758.5700 780.5514 760.5856 782.5670 784.5827 786.6013 808.5827 4358

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04749. Additional information as noted in the text (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail for J.-M.L: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (No. 81373373, 21435002, 21227006).

Figure 5. PCA of different cell types based on the results of PESI-MS analysis.

sample droplet, no specific phospholipid peaks were observed. This result indicated that the developed system was capable of cell marker detection at the single-cell level and further improved cell classification ability combined with cell marking techniques.

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CONCLUSIONS In summary, a novel method for single-cell analysis combining drop-on-demand inkjet cell printing and PESI-MS was successfully developed. Using a homemade inkjet cell printing device with magnetic stick, a relatively high single-cell fraction was obtained. The PESI-MS was proven to be capable of singlecell analysis, cell classification, and cell marker detection and showed a significant potential for application in different areas, such as cell identification, metabolic detection, and disease diagnosis.

Figure 6. Detection of single cell marked with Rhodamine 6G. 4359

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DOI: 10.1021/acs.analchem.5b04749 Anal. Chem. 2016, 88, 4354−4360