Native state single-cell printing system and analysis for matrix effects

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Native state single-cell printing system and analysis for matrix effects Qi Li, Fei Tang, Xinming Huo, Xi Huang, Yan Zhang, Xiaohao Wang, and Xinrong Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00344 • Publication Date (Web): 31 May 2019 Downloaded from http://pubs.acs.org on May 31, 2019

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Analytical Chemistry

Native state single-cell printing system and analysis for matrix effects Qi Li1, Fei Tang1,*, Xinming Huo1, Xi Huang2, Yan Zhang3, Xiaohao Wang1, Xinrong Zhang4 1

State Key Laboratory of Precision Measurement Technology and Instruments, Department of Precision Instrument, Tsinghua University, Beijing, 100084, China 2 Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China 3 Department of Electrical & Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801, USA 4 Department of Chemistry, Tsinghua University, Beijing 100084, China

Corresponding Author * To whom correspondence should be addressed: Fei Tang, [email protected]

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ABSTRACT Mass spectrometry is subject to matrix effects, which causes severe limitations on the analysis of live single cells in their native state. Here, we propose a three-phase droplet-based single-cell printing analysis system (TP-SCP), which can package, extract, separate, print and analyse live single cells in saline matrices (such as phosphate buffered saline) with a matrix-assisted laser desorption/ionization mass spectrometry. This method can eliminate matrix effects to obtain information on a single cell in their native state. We report that a cell packaging percentage of 44% and single-cell packaging percentage of 88% can be achieved by TP-SCP. The system was capable of processing three to four single cells per second, which was 30 to 40 times higher than the traditional droplet-based micro extraction (about 10s/cell). Additionally, the MCF-7, A2780, 293 and 4T1 cells were screened in our system. The effect of cell viability and heterogeneity analysis was investigated, suggesting that the concentration of monounsaturated phosphatidylinositol and phosphatidylethanolamine both increase in cancer cells. Compared with conventional mass spectrometry, TP-SCP can ensure the accuracy of heterogeneity analysis of live single cells in their native state. Both a principal component analysis and a linear discriminant analysis were used to perform classification and identification of cells with an accuracy of 100%. This method provides an innovative framework for research on cell quality control, cell biology, cancer diagnosis and prevention.


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INTRODUCTION Single cell studies reveal cell information in an accurate and comprehensive manner, thus having great potential for the early diagnosis and prevention of disease. As such, these studies have become a pressing topic in cell biology research1-5. Mass spectrometry (MS) technology, as an unlabelled analysis method, is capable of simultaneously detecting multiple components and even providing unknown molecular structure information. Accordingly, MS is promising for unlabelled multi-component analysis in research on single cells; some previous work has assessed single-cell studies using MS. For instance, Do and colleagues analysed the heterogeneity of single neurons with ionic liquid-assisted enhanced secondary ion MS6. Additionally, Andrea et al studied the drug metabolism of unicellular organisms through matrix-assisted laser desorption/ionization (MALDI) MS7 and Nemes et al conducted intensive research on capillary electrophoresis (CE) single-cell MS8-10. The latter study identified 40 metabolites in three different cell types in distinct tissues with CE-MS, anchoring the interconnected central metabolic networks10. Furthermore, Sweedler et al made a notable contribution to the field of single cell analysis with MALDI MS11-13. They presented a protocol that combines MALDI-MS with immunocytochemistry to assay predominant lipids over thousands of individual rat brain cells11. Vertes and co-authors conducted important single-cell and sub-cellular research with probe MS14-17, using GeO2 fibre probes for various oligosaccharides in epidermal cells of onion and daffodil bulbs and lipids in sea urchin egg cells18. Caprioli’s group used transmission geometry MALDI MS to realize the direct imaging of single cells at a sub-cellular spatial resolution19-22. However, most MS-based single cell research is performed in a non-saline solution environment. Most cells maintain their activity only in isotonic conditions and a fixed pH range. A non-saline environment will lead to deformation and rupture of cells, thus having a negative impact on the MS analysis of live single cells in their native state. This type of analysis is very important for many kinds of cell research, including quantitative analysis, substructure dynamic analysis and decomposable functional group analysis23, 24. Phosphate buffered saline (pbs) is the preferred reagent for cell cultures and live cell analysis owing to its effective buffering of salts and pH. However, MS is very sensitive to micro-molecule salt matrices, especially phosphates, which make the target hard to detect with MS due 3 ACS Paragon Plus Environment


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to matrix effects25-29. While MALDI-MS has an advantage in matrix tolerance, it is still impossible to directly analyse the cells in saline matrices30. Meanwhile, the conventional flushing desalination method can only remove salt in the extracellular solution, but intracellular salts cannot be removed, which negative impacts the MS analysis31, 32. The interference then is more pronounced, especially when small-volume samples of single cells are detected. In that case, the MS peak will be completely covered by the noise, thus restricting the prospect of applying MS to live single-cell analysis. So far, there has been limited research regarding the MS of live single cells (super-small samples; pL); in those studies, the saline interference matrices were reported33,

34.

Present research on

desalination of interferential salt mostly focuses on small-volume samples (μL) such as liquid chromatography (LC)35, capillary electrophoresis (CE)36-38, solid phase microextraction (SPME)39, 40 and droplet-based (solid-liquid) microextraction33, 34, 41. However, liquid chromatography or capillary electrophoresis may lead to sample dilution during the elution42. Though the risk of sample dilution can be reduced by certain procedures when solid phase microextraction is used for single-cell analysis, the operation is relatively tedious and complicated. For instance, with droplet-based (solid-liquid) microextraction, it is hard to realize a high throughput analysis of single cells (traditional high precise detection speed is about 10 seconds /cell) because manual intervention is needed for ensuring experiment precision. Moreover, it is still a major difficulty to accurately control the volume of the extraction phase. Therefore, how to analyse live single cells in saline matrices and obtain reliable and high throughput information urgently requires a solution. This study proposes a three-phase droplet-based single-cell printing analysis system (TP-SCP), which can package, extract, separate and print the cell substance of live single cells in saline matrices (such as pbs) and analyse the cell substance with a MALDI-MS. This method can eliminate matrix effects to obtain single cell information in native state. In this system, three-phase droplet technology was first used to assess cell packaging. Then, octyl alcohol/acetonitrile solution was employed to assess the single-cell disruption and cellular substance extraction within the saline matrices; chemical modification and the pressure balance method were then used to assess the highly effective three-phase separation among the extraction phase of single cells to be analysed (the aqueous phase of cell residual 4 ACS Paragon Plus Environment

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liquid and the partition phase). Subsequently, a high-speed electro-motive stage was used to print the separated phase (extraction phase of single cells to be analysed) on a microarray chip to ensure that each hole on the chip contained only the information of the single cells. Finally, MALDI MS was used for analysis. A three-phase droplet model was established as well in this study to provide a theoretical basis for packaging and separation of single cells. The TP-SCP system was applied to perform contrastive analysis on cell heterogeneity in different solutions. Then, the TP-SCP system was used to analyse the lipids in MCF-7, A2780, 293 and 4T1 cells in the pbs and the PCA and LDA algorithm was used to perform classification and identification of cells. The TP-SCP system provides a feasible method for high throughput MS analysis of live single cells in saline matrices.

EXPERIMENTAL SECTION Materials and Apparatus SU-8 photoresist (2075) and polydimethylsiloxane (PDMS, RTV615) were purchased from Suzhou Wenhao Microfluidic Technology Co., Ltd. Polyethylene glycol (PEG; molecular weight 400 g/mol), Octadecyltrichlorosilane (OTCS), N-(1-naphthyl)ethylenediamine dihydrochloride powder (nedc) and perfluorotripentylamine were purchased from Beijing J&K Scientific Ltd. Polystyrene fluorescent particles with diameter of 19μm were purchased from Tianjin BaseLine Chromtech Research Centre. Phosphatidic acid (PA (34:1)), phosphatidylglycerol (PG (34:1)) and phosphatidylserine (PS (34:1) PS (36:2)) were purchased from Avanti. Superhydrophobic coatings (NC310, NC316) were purchased from Changzhou Nanocoating Co., Ltd. Indium tin oxide (ITO) conductive glasses were purchased from Shenzhen Huanan Xiangcheng Technology Co., Ltd. Mineral oil was purchased from Beijing Solarbio Science & Technology Co., Ltd. Cell balancing liquid (Optiprep) was purchased from Sigma Limited Company). Absolute methanol (99.5%), isopropanol (99.7%), acetonitrile, absolute ethyl alcohol and pbs were purchased from the platform of Tsinghua University. All reagents were used at their original concentrations without secondary purification. The spin coater (SUSS, Germany), stepper (MA6, SUSS, Germany) and drying machine (SAWA, Switzerland) used for preparing a microchip were all provided by the Institute of Semiconductors, Chinese Academy of Sciences. Pumps (PHD ULTRA Programmable) were purchased from Harvard 5 ACS Paragon Plus Environment


Apparatus. A fluorescence microscope (Olympus lx73) was used to observe liquid flow. A superhydrophobic target plate (MTP Anchorchip384, Bruker Corp.) was used to test the effect of extracting agent. Electro-motive stages (SGSP26-50, SIGMA Corp.; purchased from Shanghai Boson Technology Co., Ltd. STM32F407) were used to control the movement of stage. A Dino-Lite digital microscope (AM4815ZT, AnMo Electronics Corporation, Taiwan) was employed to observe the motion of intracellular fluid drop of single cells. A spray pen was purchased from Shenzhen Crab Kingdom Technology Co., Ltd. A MALDI-TOF MS (ultrafleXtreme, Bruker Corp.) and FT-ICR MS (solariX, Bruker Corp.) were all provided by the Institute of Chemistry, Chinese Academy of Sciences. Cell Culture and Preparation MCF-7 cells were cultured in a 10mL DMEM culture medium containing 10% foetal bovine serum (FBS), 100U/mL penicillin and 100μg/mL streptomycin in a humid environment with 5% CO2 at a temperature of 37°C. Cells were revaccinated once every 2 to 3 days. Before the experiment, trypsinEDTA was used to treat cells to obtain the cell suspension solution. Cells were centrifuged and suspended again to gain the cell suspension solution with proper cell concentration. A cell suspension solution was divided equally and tested with a live/dead cell detection kit for cell viability. Cell suspension solution used for the experiment contained 98±1% of live cells. The cell suspension solution was then equally divided into four parts and centrifuged at a speed of 1000 rpm. The upper suspension was removed and pbs, 0.9% aqueous ammonium formate, water, and 50% aqueous methanol solution were added successively to prepare the final cell suspension solution, with approximately 3.375 × 106 cells/mL. A cell suspension solution of 4T1, 293 and A2780 cells were prepared with the same method. Microfluidic Chip Fabrication Microfluidic Chip moulds were prepared by standard soft lithography (Supporting Section 1.1, Table S1). Moulding technology was utilized to get a PDMS-based micro-channel with a depth of 120 μm. The schematic diagram of the microchannel is shown in Figure 1b. Microchannels M1 and M3 were 30 μm wide and the rest of the microchannels were 60 μm wide. The surfaces of microchannels M1, M2, M3 and M4 in the packaging and extraction zones were provided with a complete hydrophobic treatment. In the separation zone, however, channel M5 was hydrophobic and M6 was hydrophilic through a two6 ACS Paragon Plus Environment

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step chemical modification. A schematic diagram of the modification process is shown in Figure 1c-d and a schematic effect diagram and real effect diagram post-modification are shown in Figure 1e and f, respectively (modification methods were shown in Supporting section 1.2 and 1.3, Figure S1). Microarray chip fabrication A spin-coating method was used to apply a superhydrophobic coating on ITO substrate; then, laser processing was applied to remove the coating of partial areas to gain the non-superhydrophobic round array. This method was employed to gain a 20×20 round array with a diameter of 300 μm and centre to centre spacing of 600 μm. After the printed analyte was dried, it was dissolved by spraying the nedc methanol water matrix (1:1, v/v) with a spray pen. After recrystallization, MS was performed. The nedc matrix concentration was 5mg/mL (fabrication and test are shown in Supporting Section 2, Figure S2). System Setup The TP-SCP system can be divided into three modules: sample injection module, lab-on-a-chip, and printing module for the substances to be analysed (Figure 1a; additional image in Figure S3). The microfluidic chip was fixed by a printing module. The microarray chip was placed on electro-motive stages directly below the printing module. The moving speed of the stage was controlled by adjusting the pulse frequency of the drive. Here, the flow rate of the aqueous phase, extract phase and partition phase were Qw=0.3μL/min, Qe=0.6μL/min and Qp =1μL/min, while the controller pulse frequency was 2KHz. Under these conditions, the difference in hydrophobicity of the microarray chip ensures that the phase droplets enter the pores of the microarray chip upon analysis43. MS Analysis A Bruker 9.4 T solarix FT-ICR MS equipped with an Apollo dual-mode electrospray ionization (ESI)/MALDI ion source, with a 355 nm and 200 Hz solid-state Smartbeam Nd:YAG UV laser (Bruker Daltonics, Bremen, Germany) was employed for recording accurate masses from single cells. Mass spectra were acquired over the mass range from 100 to 1000 Da in negative ion mode. For MALDI-MS profiling, the mass spectra were recorded by accumulating 20 times at 200 laser shots per scan. Instrument calibration used a standard calibration procedure. The sodium trifluoroacetate solution was selected as the calibration solution. Calibration error is less than 2 ppm. 7 ACS Paragon Plus Environment


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Data Analysis Background subtraction was conducted by extracting and eliminating all peaks that appeared in all blank experiments. Both a principal component analysis and a linear discriminant analysis were obtained to classify and visualize the single-cell data. The identification of metabolites was achieved through

matching

the

accurate

mass

spectra

with

the

standards

in

the

METLIN

(http://metlin.scripps.edu/) and HMDB (http://www.hmdb.ca/) databases. The data were statistically tested with an unpaired t-test. Values of P < 0.05 was considered significantly different, while P >0.05 was considered to be no significant difference.

RESULTS AND DISSCUSSION Overview of TP-SCP System The lab-on-a-chip mainly consists of the single cell packaging zone, the microextraction zone and the separation zone. The three-dimensional diagram of microchannel is shown in Figure 1a. Microchannels M1 and M3 employ an S-shaped structural design to reduce the disturbance of the injection system and ensure the stability of the sample injection. Microchannel M4 in the microextraction zone also employs an S-shaped structural design to increase the length of channel. This zone increases the effective time of extraction and accelerates the surface flow velocity of liquid at the bends of channel for improving extraction efficiency. A three-phase droplet estimation model and a separation model were established to achieve precise regulation and stable separation of three-phase droplets in a microfluidic system (Supporting Sections 3 and 4: Figure S4-S9, Table S2-S5). In this case, an octyl alcohol/acetonitrile solution (7:3, v/v) was used, while acetonitrile was used to dissolve cell membranes and octanol was used to extract water-insoluble substances in cells (Supporting Section 5.1 for details).


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Figure 1. The TP-SCP system and module design. (a) A schematic diagram of TP-SCP systems. L1, L2, and L3 are the inlets of the partition phase, the extraction phase and the aqueous phase, respectively. The outlets of the phase to be analysed and the cell residual liquid phase are O1 and O2, respectively. (b) A schematic diagram of lab-on-a-chip. Microchannels M1, M2, and M3 belong to the single cell packaging zone, M4 belongs to the microextraction zone, and M5 and M6 belong to the separation zone.(c) A schematic diagram of the laminar flow interface. (d) Hydrophobic modification. (e) A schematic diagram of three phase droplet separation. (f) A CCD photograph of three phase droplet separation.

Single Cell Packaging In the experiment, the flow velocities of the three phases were Qw =0.3μL/min, Qe =0.6μL/min and Qp =1μL/min, respectively. The lengths of the outlet capillary tubes were Lext,tube=50mm and Laqu,tube=78mm, respectively. The packaging effect of the polystyrene fluorescent particles is shown in Figure S10. The same experiment was repeated with the same parameters by changing the fluorescent particles to MCF-7 cells and adjusting the density of the solution to a value equal to that of the cells (cell concentration is about 3.375× 106 cells/mL) with Optiprep. During the experiment, no magnetic stirring was conducted because intense mechanical movement may rupture the cell membrane44, even causing the death of cell. The experimental result of encapsulation of a single cell into a droplet is shown in Figure 2a. Since sample injection is a random process, the rate of packaging single cells into droplets can be estimated with a Poisson distribution P(𝑋 = 𝑥) = 𝑒 ―𝜆(𝜆𝑥/𝑥!). As such, the number of 9 ACS Paragon Plus Environment


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cells packaged per droplet was counted (Figure 2b). Furthermore, optimizing the experimental conditions achieved a total cell packaging rate of 44%, of which the single cell packaging rate is 39% and the rate of packaging two or more cells in a droplet is about 5% (Supporting Section 5.2). Consequently, the single cell packaging rate is about 88% of the cell packaging rate.

Figure 2. Cell distribution of the optimized single-cell packaging. (a) Image of the single cell packaging results with red rectangular box indicating a single cell package unit. (b) Distribution of different cell numbers per droplet (blue bar) and a Poisson fit (red line). Single cell packaging rate is 39% and the rate of packaging two or more cells in a droplet is about 5%.

According to the calculations, experimental results and previous literature, the printing of single cell substance to be analysed at a rate of 3 to 4 single cells per second. This rate is achieved through adjusting the translational speed of the stage to enable the droplets to be entrapped into each hydrophilic hole on the base plate in sequence. System Stability A series of standard substances with the same concentration (PA (34:1), PG (34:1) and PS (34:1)) were used to test the stability of the TP-SCP system under the same experimental conditions. To eliminate the error in absolute strength caused by the difference in crystalline state of the samples to be analysed (Supporting Section 5.3, Figure S11), two groups of mixed solution, prepared with standard substances PAs/PGs and PGs/PSs, were selected to measure the strength ratio of the two samples in the mixed solution and evaluate the stability of the system accordingly. The results of the experiment are shown in Figure 3. For the mixed solution of PAs/PGs and PGs/PSs, the values of RSD are 3% and 4.2%, respectively, indicating that the TP-SCP system shows a good stability (RSD0.05).

Figure 3. Analysis of the stability of TP-SCP system. (a) The MS spectra of the mixed solution of PA (34:1) and PG (34:1), RSD is 3%. (b) The MS spectra of the mixed solution of PG (34:1) and PS (34:1), RSD is 4.2%.

The standard curve of the concentration ratio of phospholipid standard samples was measured by TP-SCP. The PG (34:1) and PS (36:2) values common to cells used in the experiment were selected, and the concentration of PS (36:2) was 1 μM. The concentration of PG (34:1) was 1~5 μM. The R2 was 0.988 (Figure S13). Extracting Agent Analysis The mixture of octyl alcohol and acetonitrile (7:3) was selected to be the cell lysis/extracting agent. This mixed solution can evaporate quickly without imposing any influence on the substances to be analysed. The analysis of an nedc aqueous methanol solution, solution A, solution B and solution C was conducted with MTP Anchorchip384 target plates (results are illustrated in Figure 4a; preparation steps are listed in Supporting Section 5.4). The orange rectangular box corresponds to the spectra within m/z= 700~900. The nedc aqueous methanol solution (blue) does not show obvious characteristic peaks, indicating that it will not cause any interference to the analysis that results during the cell lipid analysis and it is thus a good matrix solution for lipid analysis. Solution A (green, multi-cell suspension solution) shows an obvious lipid characteristic. Solution B (pink, extracted extraction liquid) shows peaks with the same position as pattern as solution A. Solution C (red, extracted cell residual liquid) does not show any similar characteristic with that of solution A. These results imply that this latter extracting agent can separate the cell lipid information from pbs.



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Figure 4. Results of MALDI MS analysis. (a) MS spectra in different solutions. blue standing for matrix solution, green for solution A, purple for solution B and red for solution C. (b) Single-cell lipid analysis of MCF-7 cell pbs suspension solution with TP-SCP system (single droplet packaging single cells). (c)Lipid analysis of droplets without cells packaged with TP-SCP system (single droplet packaging zero cells). (d) Cell culture medium in the control group with TP-SCP system. (e) pbs in control group with TP-SCP system. (f) results of cell lipid analysis of MCF-7 single cells level pbs solution with MALDI MS.

Discussion on Common Solution for Single-cell analysis The pbs reagent provides an ideal environment for cell survival and is thus preferred for biological assays. In this study, the impact of pbs and other common solutions for MS (such as 0.9% aqueous ammonium formate, water and an aqueous methanol solution) was discussed. As shown in Supporting Section 5.5 and Figure S14, the results reveal that pbs can maintain the osmotic pressure of the cells and buffer the environment for a long time, thus representing the best environment for cell survival. These results ensure that the cell structure has not changed and that cell activity can be maintained. To study the difference in phospholipid analysis of cells in different active states, the comparison was conducted by selecting the common solution (water, on-saline solution) and pbs for MS analysis. 12 ACS Paragon Plus Environment

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A multi-cell analysis is provided in Supporting Section 5.6. The results show that both solutions can detect the characteristic phospholipid of the cell and that the main characteristic peaks are essentially the same (Figure S15a and S15b). The results additionally imply that cell activity does not have a clear impact on the multi-cell analysis (Figure S16). Then, TP-SCP was used to analyse the phospholipid content of single cells of MCF-7 in both an on-saline solution and pbs (Figure S15c and S15d). The characteristic phospholipid of the cell in the on-saline solution represents 77% of the multi-cell analysis result, while only 50% of the characteristic signal can be detected in with the pbs solvent. The result of the 4T1 single cell is shown in Figure S17. The number of phospholipid signals gained in on-saline solution conditions is about 1.7 times that gained in pbs conditions, which is basically consistent with the analysis result of MCF-7 single cells. The reason may be that cells are vulnerable to swelling, fracture and rapture in on-saline solution conditions, causing easy interference and stacking of different single cells; the heterogeneity of single cells is thus averaged out of the sample. In pbs conditions, however, cells can effectively maintain their activity and preferably exhibit the heterogeneity of single cells without interference and stacking due to swelling fracture and rapture. Single-Cell MS Analysis MCF-7 cells were used to test the performance of TP-SCP in a single-cell analysis. In this experiment, the solution (for the cell culture medium and pbs) was selected as the control group to exclude the impact of interference signal on the analysis result. The cell pbs suspension solution (experimental group) was changed to the cell culture medium and pbs in sequence, with other conditions unchanged and TP-SCP was used to perform control experiment. The results of the single-cell analysis with TP-SCP are shown in Figure 4b. This analysis showed obvious phospholipid peaks; empty droplets without cells packaged were analysed with TP-SCP and no phospholipid peaks were detected (Figure 4c). The solution of the control group was analysed with TP-SCP and there were no detected effective phospholipid peaks (Figure 4d and 4e). Based on these findings, we conclude that the result of singlecell analysis with TP-SCP is indeed the lipid of cells. Accurate molecular weights were determined with FT-ICR MS to assess the molecular composition (Table S6).



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To verify the sensitivity of TP-SCP in analysing single cells in saline matrices, a traditional analysis method was used to assess the single-cell level in a pbs solution12. The MS peaks are indistinguishable from the noise (Figure 4f). This finding is because single cells inherently contain very few substances and saline interference matrices (pbs) may form crystalline complexes on the surface of the analyte, thus complicating the signal analysis. The comparison between Figure 4b and 4f shows that TP-SCP can accurately analyse liposoluble substances of single cells in interference matrices to gain single cell information that cannot be detected due to matrix effects (the result is same as that for the solvent PG (34:1); Supporting Section 5.7 and Figure S18). Identification of Cell Subset This study also assessed cell classification. Cell suspension of three kinds of cells (4T1, 293 and A2780) in pbs solutions were selected (Figure 5a, 5b and 5c). The signal-to-noise ratio of partial main phospholipids can be two orders of magnitude higher (885.5518, 744.5566, 863.5675 and 887.5668). The information on the composition of phospholipids is shown in Table S6. Both PCA and LDA were used to effectively recognize the type of single cells, according to the MS information. Twenty sets of data for each type of cell (a total of 80 sets of data) were selected (A2780 single cell analysis with TPSCP; Figure S19). The cell types 4T1, 293, A2780 and MCF-7 cells were successfully classified as separate populations (Figure 5d). Another ten sets of data for each cell type (a total of 40 sets of data) were randomly mixed and classified using the same PCA and LDA function to verify the accuracy of the classification. The data of the test group can be precisely assigned to the corresponding cell zone with an accuracy rate of 100% (Figure 5d, Supporting Section 6, Figure S20).


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Figure 5. Results of detection, analysis and classification of the different types of single cells in pbs with TP-SCP. (a) MS spectra of 4T1 single cells. (b) MS spectra of 293 single cells. (c) MS spectra of A2780 single cells (d) Classification of the four types of cells (MCF-7, 4T1, 293 and A2780) with the PCA and LDA algorithms.

Single cell phospholipid differential analysis. After calculation by standard curves, the concentration ratios of PG (34:1)/PS (36:2) in four kinds of cells were obtained (Figure S21). From the results of the T test, there was a significant distinction in the PGs/PSs ratio between the four kinds of cells. Moreover, differences in concentration ratios between cells of the same species can also be found (P

Native state single-cell printing system and analysis for matrix effects

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