Plasmonic Colloidosome-Based Single Cell Detector: A Strategy for

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Plasmonic Colloidosome-Based Single Cell Detector: A Strategy for Individual Cell Secretion Sensing Xinjun Wang, Ji Ji, Tingting Liu, Yujie Liu, Liang Qiao, and Baohong Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04850 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019

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Analytical Chemistry

Plasmonic Colloidosome-Based Single Cell Detector: A Strategy for Individual Cell Secretion Sensing Xinjun Wang,† Ji Ji,*，† Tingting Liu,‡ Yujie Liu† Liang Qiao† and Baohong Liu*，† †Department of Chemistry, Shanghai Stomatological Hospital, Fudan University, Shanghai 200000, China ‡Department of Laboratory Diagnosis, Changhai Hospital, The Naval Military Medical University, Shanghai 20043, China

ABSTRACT: Phenotyping single cells based on molecules that they secrete or consume is a key bottleneck in many biotechnology applications. Here we demonstrate a new approach for detecting secretions from individual single cells, namely “plasmonic colloidosome-based single cell detector” (PCSD). This strategy uses colloidosomes to encapsulate single cells and to aid molecular detection. Colloidosomes are constructed by emulsifying lactate probe and hydrophobic ligand dual-functionalized silver nanoparticles on an immiscible liquid–liquid interface. The established colloidosomebased platform exhibits negligible surface-enhanced Raman scattering (SERS) background interference, ultrasensitive SERS response, and excellent signal reproducibility in response to acidification of the medium inside the colloidosomes. Taking lactate as a model molecule, the acidification induced by single cells confined in the colloidosomes is detected. The approach shows promising applicability in single cell analysis based on extracellular metabolites production or consumption.

Probing the extracellular environment and metabolite secretion of a single cell is very important but challenging in biological and medical research. Studying single cell extracellular metabolite secretion can, for example, recognize circulating tumor cells,1 select strains with special phenotypes,2 and monitor cellular response to environmental changes.3 A key challenge in single cell metabolite secretion analysis is how to acquire the trace target molecules which present in a trace and complex extracellular medium containing high concentrations of interfering molecules.3 To date, a large variety of advanced microsystems, such as wells,4 traps,5 patterns,6 and droplets7, have been used to achieve single cell compartmentalization and manipulation.8 Many analytical methods, such as electrochemical analysis,9,10 mass spectrometry,11,12 quantitative PCR,13,14 and fluorescence spectrophotometry,15,16 have been used to obtain information at single-cell level. However, these strategies usually require prior labeling and/or addition of reagent(s) to obtain target signal from a complex testing environment, which leads to changes in cell states, loss of target information, and limitations in analyte selection.17,18 Therefore, a nondestructive and label-free detection protocol is still urgently required for the analysis of single cell metabolite secretions. Surface-enhanced Raman scattering (SERS) benefits from intense electromagnetic enhancement upon incident excitation to reach high sensitivity for the detection of molecules adsorbed on the surface of noble metal materials. In appropriate experimental conditions, the sensitivity of SERS can even reach single-molecule

detection.17 Furthermore, SERS provides an additional benefit from the unique vibrational fingerprint of molecules to allow identification of analytes.3 Although several SERS sensors have been developed to locally probe intracellular contents, usage of SERS technique to characterize the extracellular metabolites of a single cell remains at its early stage. Endowing confined single cells with an endogenous hot spot factor for signal enhancement can be an effective approach. Herein, we report an approach, namely “plasmonic colloidosome-based single cell detector” (PCSD), which can achieve simultaneously single cell confinement, target recognition, and plasmon-enhanced signal amplification. The PCSDs are formed by self-assembly of hydrophobic silver nanoparticles at an emulsion interface. The liquid volume in each PCSD is only ~ 100 pL, and 1 microliter of dispersed liquid can be emulsified into tens of thousands of micrometer-sized liquid core-solid shell structures to meet the requirement for single-cell encapsulation in a PCSD. The surface of the PCSDs constructed from noblemetal nanoparticles can be adjusted to form a closelypacked monolayer shell, which displays intense electromagnetic coupling upon incident excitation. Furthermore, compared with conventional single-cell analyses, the 3D morphology of the PCSDs allows cells to be observed in an optimal physiological state. Hence, the application of PCSDs as cell-encapsulating chambers for single-cell metabolite secretion sensing can provide a new strategy to acquire and sense target molecules in a nondestructive and label-free way. In this study, lactate is selected as a model molecule due to its abnormal

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extracellular secretion appearing in early tumorigenesis and further increasing with the degree of cancer invasion and metastasis.18

EXPERIMENTAL SECTION Chemicals. Silver nitrate (AgNO3, ≥99%), ethanol (C2H5OH, ≥99.7%), acetone (C3H6O, ≥99.5%), ethylene glycol (EG, ≥99%) and n-hexane (C6H14, ≥97%) were purchased from Shanghai Chemical Corp (Shanghai, China). Isopropyl alcohol (C3H8O, ≥99.7%), 4mercaptobenzoic acid (MBA, ≥99%), poly(vinylpyrrolidone) (PVP, Mw = 55,000), and 1H,1H,2H,2H-perfluorodecanethiol (PFDT, ≥97 %) were purchased from Sigma-Aldrich (St. Louis, Missouri, United States). HFE-7500 (or HFE, for short) was purchased from 3M Corp (Maplewood, Minnesota, United States). All chemicals were used without further purification. Synthesis of Ag nanoparticles. The preparation of Ag nanoparticles was carried out following the method of Xia et al.19 Initially, 0.125 g of AgNO3 and 0.625 g of PVP were dissolved in 50 mL of ethylene glycol. Then, the mixture was heated to 130 °C in 20 min under vigorous stirring. After that, the solution was kept for 1 h at 130 °C without any stirring. Finally, the Ag nanoparticles were obtained by purification with copious amounts of acetone and ethanol. Synthesis of PFDT-Ag-MBA nanoparticles. The prepared Ag nanoparticles were re-dispersed in 40 mL of isopropanol/n-hexane mixture solution (1:1, v:v). Then 2.4 mL of 20 mM MBA in ethanol was added to the above mixture with magnetic stirring for 10 h. After centrifuging and re-dispersing in 40 mL of isopropanol/n-hexane mixture, 344 μL of PFDT was introduced and kept stirring for another 10 h. Finally, the acquired PFDT-Ag-MBA suspension was thoroughly washed with ethanol and nhexane, and dispersed in 5 mL of HFE. The concentration of PFDT-Ag-MBA in HFE was 16.2 mg mL−1.

experiment was performed by cutting off the glucose supply in the final stage of cell culture, and the rest of experiment steps were the same as above. Toxicity of materials. The toxicity of PFDT-Ag-MBA composite nanoparticles was detected for A549 cells by CCK-8. Briefly, A549 cells were cultured overnight under a 5% CO2 atmosphere at 37 °C. Then, 100 μL cell dispersions were seeded into a PFDT-Ag-MBA pre-loaded 96-well plate at approximately 5.0×104 cells/well, in which the amount of sterilized PFDT-Ag-MBA in each well was 10 μL of 25, 50, 100, 125, 250, 500 and 1620 μg mL-1. After 24 h of culture, 10 μL of CCK-8 solution were added to each well and continue culturing for 2h. Next, the optical density of each well was carefully measured at 450 nm by using a microplate reader. SERS measurement of plasmonic colloidosomes. The as-prepared plasmonic colloidosomes were dipped in a polytetrafluoroethylene dish full of n-hexane, and then the dish was transferred onto a Raman sample stage for SERS measurement. All SERS characterization was performed at room temperature (25 °C) with an excitation wavelength of 532 nm (laser power of 2.0 mW). For x–y SERS imaging, the Raman spectra were collected using a 50× objective lens (N.A. 0.50) with 3.6 μm resolution and 1 s accumulation time. For other SERS analyses, a 10× objective lens (N.A. 0.25) was used with 10 s exposure time. Material characterization. Field emission scanning electron microscopy (FESEM) characterization was performed using a Zeiss-Ultra 55 microscope (Germany). X-ray photoelectric spectroscopy (XPS) was performed using a Perkin-Elmer-PHI 5000C (USA). Microscopic imaging and SERS measurements used a Horiba JY XploRA microscope (France). All SERS spectra and peak intensities were recorded by averaging at least 10 individual spectra of the entire scanned area.

Construction of plasmonic colloidosomes. 15 μL of the PFDT-Ag-MBA dispersion was diluted 10-fold by HFE and sonicated to obtain a brown transparent suspension. Next, 5 μL of sample solution was added into the dispersion and violently shaken to prepare plasmonic colloidosomes. A549 cell cultivation. The A549 cell line was obtained from the Naval Military Medical University, Shanghai, China. Cell cultivation was carried out following the procedures of the Agilent Seahorse Glycolysis Stress Test Kit. Typically, the A549 cells were first cultured overnight on a culture flask in Dulbecco’s modified Eagle’s medium (DMEM, supplemented with 10 % fetal bovine serum) at 37 °C in a 5% CO2 incubator, and then seeded into 96-well plates at circa 6.0×105 cells/well. After 2 h of culture, the DMEM was replaced with 200 μL of glycoprival DMEM (no glucose, no phenol red) and continued to culture for 1 h at 37 °C in a non-CO2 incubator. Finally, 20 μL of 100 mM glucose was added into each well and cultured for 30 min to obtain the final cell dispersion. A control

Figure 1. Fabrication and characterization of PCSDs. (A) Schematic illustrating the formation of PCSDs. (B) Microscopic image and (C) field emission scanning electron microscope image of the established PCSDs. (D) Magnified segment of the surface of a PCSD.


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Analytical Chemistry

RESULTS AND DISCUSSION The Ag nanoparticles used as building blocks were respectively functionalized with MBA (a pH-responsive SERS probe), and PFDT to endow hydrophobic properties for self-assembly at the oil/water interface (Figure 1A). Via XPS analysis, the molecular number ratio of PFDT to MBA on the original Ag surface was circa 5:1 (Figure S1 and Supporting Information). The prepared PFDT-AgMBA nanoparticles were then dispersed in fluorinated solvent HFE, a superhydrophobic oil with great cellular compatibility.20 By violently emulsifying the internal fluids (water or cell suspensions) and external fluids (HFE containing silver nanoparticles), the PCSDs can be quickly formed. As shown in Figure 1B and 1C, both optical micrograph and FESEM images clearly showed hollow shell morphology of PCSDs. The magnified segment of a PCSD surface confirmed that its shell consisted of PFDT-Ag-MBA nanoparticles with diameters of 60 ± 10 nm (Figure 1D and Figure S2). It is important to note that these Ag nanoparticles are closely packed together to form the shell of the PCSDs, which will generate abundant SERS “hot spots” with enormous local enhancement in electromagnetic field,21 and further result in high sensitivity of SERS measurements. To obtain the optimal PCSDs with suitable volume and strong plasmonic coupling, the appropriate ratio of functionalized Ag nanoparticles to internal fluids (“the Ag/water ratio”) was investigated. FESEM images were used to track the surface morphology variation of PCSDs with the Ag/water ratio. PCSDs could be readily formed with the Ag/water ratio ranging 18 to 72 mg Ag per mL water, while there were clear differences in the size and morphology of the PCSDs with varied Ag/water ratio (Figure 2 and S3). At low Ag/water ratio (i.e., 18 mg mL−1), there were not enough Ag nanoparticles to cover the entire interface and the nanoparticles tended to form local aggregates on the liquid–liquid interface. The surface morphology of these PCSDs showed many cracks, and the whole skeleton collapsed after the freeze-drying process. Meanwhile, the diameter of these PCSDs had a wide distribution, range from 20 to 110 μm (Figure S3A). Some colloidosomes formed at this Ag/water ratio were unstable and coalesced occasionally. On increasing the Ag/water ratio to 36 mg mL−1, the PCSDs exhibited a narrower size distribution and enhanced structural stability, but some small cracks on the colloidosome surface could still be clearly observed (Figure 2B and Figure S3B). When the Ag/water ratio was increased to 54 mg mL−1, a complete colloidosome structure was successfully formed with close-packed Ag nanoparticles (Figure 2C). The diameter of the PCSDs was essentially at 30–40 μm (Figure S3C), which is ideal for encapsulating an individual living cell. At an Ag/water ratio of 72 mg mL−1, the surface of PCSDs had almost no voids, but excess PFDT-Ag-MBA nanoparticles were distributed around the PCSDs (Figure 2D). The size distribution of colloidosomes formed under this Ag/water ratio was also stable at 30-40 μm (Figure S3D), with no significant changes compared with the case of Ag/water ratio at 54

mg mL−1. The background interference and SERS response of PCSDs with different Ag/water ratios was also examined (Figure S4-S6 and Supporting Information). It was found that the PCSDs exhibited negligible SERS background and the SERS signals showed the best performance when the Ag/water ratio was above 54 mg mL−1. Consequently, an Ag/water ratio of 54 mg mL−1 was selected to perform further experiments, unless specified.

Figure 2. FESEM images of PCSDs formed with (A) 18, (B) 36, (C) 54 and (D) 72 mg mL-1 of Ag/water ratio. (i) The overview of an entire colloidosome, (ii) their magnified section of the shell, and (iii) their fracture surface on the shell. The green dotted parts indicate the break sections of colloidosome shell.

It should be noted that all the PCSDs (at each Ag/water ratio mentioned above) were formed from a monolayer of functionalized Ag nanoparticles (Figure 2Aiii to 2Diii). This is an essential characteristic of colloidosomes formed using the immiscible emulsion system.22-25 The closelystacked PCSD monolayer not only creates strong plasmonic coupling, but also ensures that all “hot spots” can be exposed to the exciting laser.26 Moreover, this monolayer structure allows the exciting laser to easily reach the solid–liquid interface inside the PCSD, thus effectively avoiding potential light shielding effects.27 To determine the uniformity of the SERS active sites on the surface of the PCSDs, x–y SERS mapping was performed by using phosphate buffered saline (PBS)encapsulated PCSDs (0.1 M, pH 7.5). Selecting the SERS band at 1082 cm−1 as a reference peak, we carefully mapped the top-plane and mid-plane of PCSDs with about 3.5 μm resolution. By focusing the confocal plane on the top of a PCSD (Figure 3A and 3B), the SERS imaging of the entire upper surface was brightened with intensity of about 1200 counts s−1. In contrast, the SERS imaging of the external n-hexane was almost dark, with SERS response 0.6. The sensitivity can meet the requirement of detecting extracellular acidification of single cells, wherein the delta(pH) is normally > 1.5.1 It should be noted that our proposed colloidosomes-based SERS approach requires only ~100 pL of encapsulated samples and don’t involve any process that may lethal to cells, which are advantageous over other methods including conventional colloid or interface based SERS approach.8,29

Figure 3. SERS imaging of the PCSDs. (A) x–y SERS imaging and (B) corresponding microscopy image of a PCSD when the exciting laser was focused on its top-plane. (C) SERS imaging of the PCSD when the laser was focused on its midplane. (D) Corresponding SERS spectrum along the white dotted line in figure C.

Inspired by the strong SERS response, we next tried to verify the sensitivity of the PCSDs to variation of pH by encapsulating PBS buffer solution with pH ranging from 4.5 to 8.5. As shown in Figure 4A, the Raman peak of MBA probe at 1720 cm−1 was very sensitive to pH change. This Raman peak arises from C=O bond symmetric stretching vibration of the –COOH group. When pH decreased, the concentration of protonated MBA increased and the intensity of this peak also increased. However, the Raman peak at 1082 cm−1, which is attributable to ν12 aromatic ring vibrations, is insensitive to pH, and can be used as a reference. The behaviors of the two peaks were consistent with previous reports.28 We determined the change of the Raman peak intensity ratio I1720/I1082 as a function of pH.

Figure 4. SERS response to pH variation detected by using PBS encapsulated PCSDs. (A) Typical SERS spectrum of PCSDs with PBS (pH from 4.5 to 8.5) encapsulated. (B) The normalized Raman peak intensity ratio I1720/I1082 as a function of pH. (C) Variation of normalized intensity between


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Analytical Chemistry individual PCSD, the lines labeled with pH 7.3 pH 7.5, and pH 7.9 indicate the I1720/I1082 values corresponding to pH values calculated with the calibration curve in Figure 4(B).

As demonstrated in previous studies, cancer cells show anomalous secretion of lactate and acidification of the tumor environment,1,30 and the metabolic behavior is more significant in more aggressive phenotypes.18 Therefore, it is possible to detect cancer cells based on pH change due to the secretion of lactate using the PCSDs. In this study, the A549 cell line (adenocarcinomic human alveolar basal epithelial cells) was chosen as a model to demonstrate this application, which can induce the acidification of extracellular medium when Dulbecco’s modified eagle medium with 10 mM glucose was used. A control was performed by cutting off the glucose supply. According to the Warburg effect,31 the cancer cells metabolize glucose to lactate, which was impossible to be produced in the control experiment.

Figure 5. Detection of extracellular acidification by single A549 cells. (A) Epifluorescence image of captured single cell transfected with green fluorescent protein. (B) Distribution of cell occupancy in PCSDs. (C) Quantification of SERS intensity ratio (I1720/I1082) from PCSDs encapsulated with cells in the presence (Experimental group) and absence (Control group) of glucose.

The in vitro cytotoxicity of the PFDT-Ag-MBA building blocks on the A549 cell was evaluated by using the CCK-8 assay. After culturing for 24 h with PFDT-Ag-MBA, more than 80% of cell viability can be observed even with 1620 μg mL-1 of the nanoparticles (Figure S7), indicating a low toxicity of PFDT-Ag-MBA to A549 cells. On the other hand, as the dispersed phase of colloidosomes, HFE also has a great biocompatibility, which has been wildly used for the preparation of cells encapsulated micro-droplet.1 Therefore, our proposed colloidosome based platform is suitable for short-term cell culture and in-situ detection. Single cells encapsulation in the PCSDs was then examined by using green fluorescent protein-transfected cells. As shown in Figure 5A, epifluorescence images successfully confirmed the encapsulation of single cells in PCSDs. Next, the experiments were carefully repeated by using larger number of colloidosomes to calculate the encapsulation efficiency of A549 cells. By analyzing the fluorescence images for more than 100 colloidosomes in an emulsified cell suspension, the efficiency of single-cell and double-cell encapsulation was 11.54% and 2.56%, respectively (Figure 5B). The result was consistent with the solution of Poisson equation (see the Supporting Information for the detailed solving process). Afterwards, SERS experiments were performed by using a large number of PCSDs. As shown in Figure 5C, after incubation for 30 min, a distinct population distribution could be observed in terms of the pH-sensitive I1720/I1082 ratio between the experimental group (DMEM with 10 mM glucose as the suspension of single cell) and the control group (DMEM without glucose as the suspension of cells). The experimental group showed a higher degree of acidification (I1720/I1082 ratio circa 0.06). The control group showed lower acidification (relative SERS intensity circa 0.04). Based on the previous report, the lactate is the only pH-affecting substance secreted by the cells.1 So, this obvious difference in acidification of the extracellular medium robustly demonstrated that PCSDs can be used to monitor the extracellular secretion of lactate by just encapsulating a single cell.

CONCLUSIONS In conclusion, we have demonstrated a versatile technique for monitoring single cell metabolite secretion by using picoliter scale plasmonic colloidosomes, which enable single-cell confinement, target recognition and plasmon-enhanced signal amplification. The colloidosomes were constructed by emulsifying MBA and PFDT dual-functionalized silver nanoparticles on an immiscible liquid–liquid interface. The resultant colloidosomes have negligible SERS background interference, stable dimensions, ultrasensitive SERS



response, and excellent signal reproducibility. Lactate was chosen as a model molecule to examine metabolite secretion by an encapsulated single cell, and acidification of the extracellular space was successfully detected. Since the anomalous acidification of extracellular microenvironment is an important characteristic of cancer-cell, our proposed technique can be further applied to the identification of rare tumor cells and circulating tumor cells. Meanwhile, the identification of other disease-related metabolites can also be studied at single cell level by using our colloidosomes-based platform.

ASSOCIATED CONTENT Supporting Information Details on FESEM images on particle size, experimental condition optimization, and supplementary figures. This information is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21874025, 21775028, 21575030) and the Science and Technology Commission of Shanghai Municipality (16391903900, 17JC1401900). In addition, Xinjun Wang especially wishes to thanks Prof. Chang Sun for her guidance regarding the SERS-related experimental, as well as Dr. Xiuli Wang for her contribution in cytotoxicity assay.

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Plasmonic Colloidosome-Based Single Cell Detector: A Strategy for

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