Sensitive Detection of MMP9 Enzymatic Activities in Single Cell

Mar 24, 2017 - Encapsulated Microdroplets as an Assay of Cancer Cell Invasiveness. Zhao Yu,. † .... detect the enzymatic activities of MMP9 in singl...
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Sensitive Detection of MMP9 Enzymatic Activities in Single CellEncapsulated Microdroplets as an Assay of Cancer Cell Invasiveness Zhao Yu,† Lu Zhou,† Ting Zhang,† Rui Shen,† Chenxi Li,† Xu Fang,† Gareth Griffiths,‡ and Jian Liu*,† †

Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-based Functional Materials & Devices, Soochow University, Suzhou, Jiangsu Province 215123, China ‡ Imagen Therapeutics Ltd, Suite 4D Citylabs, Nelson Street, Manchester M13 9NQ, United Kingdom S Supporting Information *

ABSTRACT: Matrix metalloproteinases (MMPs) are typically up-regulated in cancer cells, and play a critical role in assisting metastasis by the breakdown of the extracellular matrix. Here we report an effective strategy for cell invasiveness assay by integrating MMP9 functional activity analysis with single cellencapsulated microdroplets. A flow focusing capillary microfluidic device has been assembled using “off-the-shelf” fluidic components for high-throughput generation of microdroplets. Tumor cells, MMP9 specific peptides, and other cofactors can be loaded into the device and encapsulated into individual droplets as dynamic microreactors for proteolytic cleavage of the substrate. This design allows for rapid and robust detection of MMP9 enzymatic activities by fluorescent signals in a few minutes. It represents the first demonstration of quantifying MMP9 enzymatic activities at the single cell level with a high throughput performance. This new technique promises functional evaluation of cancer cell invasiveness for important diagnostic or prognostic applications. KEYWORDS: cell invasiveness, cancer heterogeneity, matrix metalloproteinases, microfluidic technology, fluorescent probes

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MMPs, ideally at the single cell level, for cancer diagnosis and potential treatments in clinics.20−23 There are mainly two approaches to detect MMP9, depending on the different focuses, on either the structure or function of this enzyme. The epitopes on the MMP9 protein structure can selectively be recognized by antibodies, which is the foundation of the antibody-based approach for MMP9 detection such as enzyme-linked immunosorbent assay (ELISA).24−28 Although the format of ELISA is generally popular, the assay performance may be variable because of the different choices of host species or batches of the antibodies. ELISA assay focuses on the affinity binding between the antibodies and epitopes of MMP9, but these epitopes are not necessarily the active sites of MMP9 enzymatic function. An alternative approach for MMP9 detection focuses on evaluating the proteolytic activities of the enzyme on its substrate, which is particularly interesting because of its closer relevance to the cell invasion by breaking ECM molecules.29,30 Recently, micropatterned surface or hydrogels31 have been integrated with cleavable peptides for MMP9 detection. Either the electrochemical32 or fluorescent method33 can be employed in these designs to detect MMP9 secreted by cancer cells with the improved detection limit, ranging from hundreds down to a dozen cancer cells. These are important advances in sensitive detection of MMP9 as research tools.31−33 However, it remains

etastasis is the process of spreading malignant tumor cells throughout the body and forming secondary tumors by invasion into different organs/tissues, which causes the majority of deaths (nearly 90%) associated with cancers.1−3 Circulating tumor cells (CTCs) have become a promising biomarker of cancer metastasis as a liquid biopsy. During cancer metastasis, malignant tumor cells have to break physiological barriers surrounding them for migration.4,5 Among many factors involved with this process, there is a prerequisite for the tumor cells to secrete the matrix metalloproteinases (MMPs) for proteolytic cleavage of extracellular matrix proteins (ECM).6−9 MMPs, a family of zinc-dependent endopeptidases, have been verified to be associated with tissue remodeling, degradation of extracellular matrix proteins or basement membrane components, and induction of angiogenesis.8,10,11 MMP9 is a gelatinase subgroup of the MMPs, with a molecular weight of 92 kDa. Elevated expression and activation of MMP9 in cancer cells are able to assist metastasis.12 Therefore, detecting the functional activities of MMP9 provides an important clue to evaluate the invasiveness of cancer cells. The traditional cell invasion assays13,14 using Matrigel15,16 suffer from the limitations of low reproducibility, long experimental procedure, and only the average results based on large cell populations. However, the population-averaged results tend to mask many important details in the biological functions of individual cells. Unfortunately cancer cells are known for the complicated cell-to-cell heterogeneity.17−19 Emerging needs have been raised to develop a sensitive detection method of © XXXX American Chemical Society

Received: November 12, 2016 Accepted: March 15, 2017

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Scheme 1. Scheme of Detecting MMP9 Enzymatic Activities in Single Cell-Encapsulated Microdroplets for Cancer Cell Invasiveness Assaysa

a (a) Setup of the microfluidic device and the loading of mineral oil, tumor cells, and the peptide solution. The two PEEK chromatography T-shaped connectors are shown in orange. (b) Schematic drawing of the double pipe-like alignment of the glass capillaries. The tip ends of the inner glass capillaries are flamed round. Two different liquid phases are indicated by the colors of grey and cyan, respectively, with the arrows for the flow directions. (c) Detection of the enzymatic activities of MMP9 secreted by the cells in the microdroplet, as a new assay to evaluate tumor cell invasiveness.

PMA for cell activation (final concentration 100 ng mL−1) to the master PEGDA solution. The dispersed phase components A and B were separately transferred into two microsyringes, then pumped simultaneously into the microfluidic device through a small Y-shaped plastic hose nipple. The continuous phase of mineral oil containing Abil EM 90 surfactant (2 wt %) was loaded into the device through the perpendicular inlets of the T-shaped connectors. The liquid flow rates were described in the previous section. Different cell loading concentrations were tested to titrate the cell number encapsulated by the microdroplets. The cell nuclei were stained by DAPI molecules. The images of cell-encapsulated microdroplets were visualized by a confocal laser scanning microscope (CLSM, Leica TCS SP5) with zaxis scanning. It allowed us to determine the cell number in individual microdroplets with a good spatial resolution. Detection of MMP9 Enzymatic Activities in the Microdroplets. The microdroplets generated by our approach provided volume well-defined microreactors for the detection of MMP9 enzymatic activities. The cells were stimulated in situ by PMA, secreting up-regulated MMP9 for proteolytic cleavage of the peptides in the microdroplets rapidly. After flowing through the microfluidic device, the microdroplets were collected into the glass-bottom cell culture dishes or plastic microtubes. In parallel, the recombinant MMP9 enzymes at a series of concentrations were used to replace the cell samples, and encapsulated into the microdroplets in identical conditions for the purpose of calibration. The recombinant MMP9 enzyme samples were activated by following the protocol of the supplier (incubation with 1 mM APMA at 37 °C for 18 h before using). The fluorescence of the microdroplets was saturated in 25 min. Images were acquired using an inverted fluorescence microscope (Olympus IX71) mounted with a multispectral imaging CCD camera (Nuance, CRI). An objective (4×) was used to monitor the fluorescence of the microdroplets. The excitation/emission filter set was selected to match the fluorophore probe of FITC (488 nm/520 nm). More details of the experimental procedures are available in the Supporting Information.

a challenge to measure MMP9 enzymatic activities at the single cell level. Recent advances in microfluidic technologies have attracted many researchers by offering unique advantages,34−39 including small volume of biological samples, high sensitivity, and largescale integration.40−42 Herein, we report a new method to detect the enzymatic activities of MMP9 in single cellencapsulated microdroplets with a flow focusing capillary microfluidic device (Scheme 1). This device is assembled to regulate the flow mode of the continuous/dispersed fluid phase, using two poly(ether ether ketone) (PEEK) chromatography T-shaped connectors and a set of coaxially aligned glass capillaries.43 We have demonstrated that size-tunable microdroplets can be generated in a high-throughput manner. The device enables encapsulation of cancer cells down to the single cell level. Once a single cell, peptides and other cofactors together are encapsulated in the droplet, MMP9 secreted by the cell will cleave the peptides modified with a pair of fluorophore/quencher molecules (FITC and DABCYL). Therefore, a fluorescence light-up assay can be performed to measure the MMP9 enzymatic activities of the cell in the microdroplet. This design allows for rapid detection of MMP9 as fast as in a few minutes. The proteolytic activities of MMP9 secreted by a single cancer cell can be quantitatively analyzed with excellent reproducibility, thus promising functional evaluation of cancer cell invasiveness. To the best of our knowledge, it represents the first demonstration of quantifying MMP9 enzymatic activities at the single cell level with a highthroughput performance. This microfluidic approach of cell invasiveness assays featured with ultrahigh sensitivity will have important applications in cell-based diagnostics and highthroughput screening for cancer research.

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RESULTS AND DISCUSSION Design of a Flow Focusing Capillary Microfluidic Device. This study aimed to develop a highly sensitive method for cancer cell invasiveness assays using microfluidic technologies. As shown in Scheme 1, a capillary microfluidic device was assembled to produce microdroplets as the small volume vessels for biochemical assays with “off-the-shelf” fluidic components. In the previous report,43 a specialized capillary with a square-shaped cross section was proposed for the alignment of the inner capillaries to generate microdroplets.

EXPERIMENTAL SECTION

Single Cell-Encapsulated Microdroplets. The cell sample was centrifuged and washed with PBS one time, then resuspended in serum and Phenol Red free RPMI-1640 or DMEM. The concentration of cell suspension was determined by direct counting using a standard bright-line hemocytometer (Sigma-Aldrich). The cell suspension was mixed with the master PEGDA solution to prepare component A of the dispersed phase, in a concentration of 6 × 105 cells mL−1 or any other specified cell densities. Component B of the dispersed phase was prepared by adding the peptides (final concentration 160 μM) and B

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Figure 1. Generation of different types of microdroplets and investigation of tuning their sizes by the flow rates. (a) Optical micrographs of the microfluidic device and the double pipe-like glass capillaries. (b−d) Optical micrographs of the different types of microdroplets, including oil-inwater emulsion, water-in-oil emulsion, and PEGDA-in-oil emulsion. Scale bar:100 μm, 500 μm, 500 μm, respectively. (e) Calibration curve of the microdroplet size versus the flow rate ratio Qc/Qd of different liquids. (f) Snapshot micrographs from the video clips during the experiments of microdroplet production at a series of flow rates of Qc including 10, 20, 40, 60 μL min−1, the flow rate of Qd 10 μL min−1. Scale bar: 400 μm.

demonstrated the effect of flow rates on the microdroplet sizes (Figure 1f). The microdroplets were stable enough to be collected into different kinds of containers after they flowed out of the microfluidic device. They were very uniform in the size for each specified flowing condition. The microdroplets generated in different batches were measured for their diameters. As shown in Figure S1, the sizes of the microdroplets were approximately centered at 200 μm, with a standard deviation of 5 μm from 7 different batches (n = 7). It suggested a well-controlled size distribution for the microdroplets with high reproducibility using our devices. Encapsulation of Single Cells in the Microdroplets. A series of cell concentrations (104−106 cells mL−1) were titrated to determine the cell number encapsulated by the microdroplets. The cell nuclei were prestained with DAPI for easier visualization in the microdroplets. A z-axis scanning with CLSM was performed on all the samples of microdroplets in order to count the encapsulated cells accurately. As shown in Figure 2a−c, the cell nuclei can be identified clearly for counting in individual microdroplets. When the loading concentration was 6 × 106 cells mL−1, the number was 1−15 cells per droplet. Dilution of the loading concentration allowed us to push the limit of cell encapsulation to the single cell level. For example, when the concentration was reduced to 6 × 105 cells mL−1, one cell per droplet turned out to be the prevailing fraction. In the lower cell concentration (6 × 104 cells mL−1), only a small percentage of microdroplets were able to capture the cells at the single-cell level, where nearly 85% of the microdroplets were empty without cell encapsulation. After analysis of all the images, we obtained a histogram to show the percentagewise distribution of the cell number per droplet (Figure 2d). It confirmed that different loading concentrations produced distinct profiles of the cell number per droplet. It reached the highest possibility to encapsulate one single cell in each microdroplet when the cell concentration was 6 × 105

This clever design allowed for quick alignment, but it suffered from leakage of the liquid phases around the mouthpiece or high risks of overstress during assembly (capillary breakdown), due to the difference between the square-shaped and roundshaped capillaries. In this work, we made a successful improvement to bypass the specialized capillary by designing customized adapters at the two ends for each chromatography T-shaped connector. These adapters were paired to each other for sealing between the capillaries and the micro ferrules. They not only prevented leakage of the liquid phases, but also simplified the alignment task by conveniently screwing the two ends of a T-shaped connector. The continuous/dispersed phases were introduced into the central region of the device through these glass capillaries respectively, interfacing each other when they flowed through the narrow gap. Immediately, microdroplets were generated and introduced to the downstream inner glass capillary in a flow focusing mode. Generation of Size-Controllable Microdroplets. We demonstrated that the microfluidic device was able to generate different types of microdroplets, including oil-in-water, waterin-oil, or PEGDA-in-oil emulsion (Figure 1a−d). The capillaries with flamed-tips (Figure 1a) allowed for injection of the different types of liquids with smooth interfacing profiles. It facilitated the generation of microdroplets in a highthroughput manner (2.4 × 103 droplets min−1), without the requirement of chemically modifying the surface of glass capillaries. The size of microdroplets can be controlled by adjusting the flow rates of the continuous/dispersed phases (Figure 1e). With the increase of the flow rate ratio (Qc/Qd), the size of the microdroplets decreased in a range from 400 to 100 μm. This trend was consistent with the previous reports on microdroplets using chip-based microfluidic technologies. The process of microdroplet generation in different flowing conditions was monitored by using a high-speed video recorder. The snapshot micrographs from the video clips also C

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lymphoma (U937) cells or invasive breast ductal carcinoma (MCF7) cells. In addition, the mouse embryonic fibroblast cells (NIH3T3) was also included in the experiments as the negative control cell line, due to the wide use of mouse models for tumor implantation in cancer research. As shown in Figure 3a,b, single cell-encapsulated microdroplets allowed in situ stimulation of the tumor cell (U937 or MCF7) by PMA, and rapid detection of peptide cleavage by MMP9 with the fluorescent method. We observed a significant increase of fluorescence in these microdroplets in the time course of 0−25 min. There was no fluorescent signal for the NIH3T3 cell-encapsulated microdroplets. The additional negative control experiments included single U937 (or MCF7) cell-encapsulated microdroplets without stimulation of PMA, or microdroplets containing PMA but without the cell. These control samples did not produce fluorescent signals, suggesting that the peptide containing the FITC/DABCYL pair was kept intact. These results also suggested that stimulation by PMA or by other biochemical signals was required for up-regulated expression of MMP9 in tumor cells. The fluorescent intensities of the samples were quantitatively analyzed after the image acquisition.47,48 On the right column of Figure 3b, quantitative analysis verified that the averaged fluorescence intensities of the cancer cells (U937 and MCF7) after stimulation were tremendously higher than the negative control cell line (NIH3T3) or the tumor cells without stimulation. Analysis of the images also suggested a very low background noise. These cancer cells exhibited remarkable MMP9 enzymatic activities for peptide cleavage after PMA stimulation. There was an intensity difference by more than 1 order of magnitude between the microdroplets of positive and negative samples. MCF7 cell-encapsulated microdroplets produced stronger fluorescence than U937, consistent with the direct observation on the images. Therefore, these experimental results supported that our approach using microdroplets can be applied to measure MMP9 activities in different cell lines as an effective assay of cell invasiveness. However, in the current cell loading concentration (6 × 105 cells mL−1), there was a possibility that individual microdroplets might encapsulate more than one cancer cell. Further dilution of the cancer cells in the loading procedure would reduce this risk/concern for single-cell analysis. MMP9 Enzymatic Titration Experiments. Titration experiments were performed in the identical conditions of generating microdroplets, using the commercial recombinant MMP9 enzyme to replace the cell suspension. The MMP9 enzyme solutions in different concentrations were loaded into the device, in situ mixed with the peptides after the generation of microdroplets, then monitored for the fluorescence change. Due to the dynamic mixing effect in the microdroplets, the fluorescent signals produced by the MMP9 enzyme can be read out quickly. Fluorescence saturation of individual microdroplets was present in 25 min (Figure 4a). The fluorescent signals of microdroplets and recombinant MMP9 concentrations were fitted into a linear equation very well. The limit of detection using recombinant MMP9 enzyme was determined to be around 4.0 nM in the current experimental setup (Figure 4b). A further improvement in the limit of detection can be realized using an objective of higher magnification. Based on the calibration curve described as above, the concentrations of tumor cell-secreted MMP9 enzyme were determined, around 7.0 nM for a U937 cell and 17.0 nM for a MCF7 cell, respectively. In the literature, a sensitive method was previously

Figure 2. Fluorescent, bright field, and merged images of the microdroplets encapsulating the DAPI prestained cells in three different cell loading concentrations: (a) 6 × 106 cells mL−1. (b) 6 × 105 cells mL−1. (c) 6 × 104 cells mL−1. Scale bar: 400 μm. (d) Calibration curve of probability versus the number of cells per droplet, loaded in different cell concentrations such as 6 × 106 cells mL−1 (blue), 6 × 105 cells mL−1 (red), and 6 × 104 cells mL−1 (black). The cell numbers encapsulated in the microdroplets were accurately counted by using a z-axis scanning with CLSM.

cells mL−1. There were some microdroplets containing more than one cell, but overall, it was still consistently matching the concept of the single-cell level in the literature.44−46 Therefore, we successfully demonstrated that single cell-encapsulated microdroplets can be generated using the “off-the-shelf” capillary microfluidic device. Quantitative Analysis of MMP9 Enzymatic Activities Using Microdroplets. Detection of MMP9 enzymatic activities of tumor cells was performed in the microdroplets using a MMP9-specific peptide. The peptide was modified with a pair of fluorophore/quencher molecules (FITC and DABCYL), so that the proteolytic cleavage of the peptide by MMP9 would bring down the quenching effect. The “turn-on” fluorescent signals by MMP9 enzymatic activities were monitored using an inverted fluorescence microscope mounted with a Nuance CCD camera. Sensitive detection of MMP9 enzymatic activities was demonstrated with histiocytic D

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Figure 3. (a) Fluorescence turn on after the specific enzymatic cleavage of the peptide by MMP9. (b) Fluorescent images of the single cellencapsulated microdroplets for the MMP9 enzymatic cleavage reaction of the peptide substrates. U937, MCF7, and NIH3T3 cells were tested using the microdroplets, including three additional negative control experiments: no cell encapsulated in the microdroplets, nonactivated U937 and nonactivated MCF7 cell without the PMA molecules in the microdroplet. Cell loading concentration: 6 × 105 cells mL−1. The fluorescent intensities of microdroplets were quantified and displayed on the right column. The bar with asterisks indicates a statistical significance difference (P < 0.001). Error bar: standard deviation (n = 5). Ex/Em: 488 nm/520 nm, scale bar: 400 μm.

reported to detect MMP9 using dozens of tumor cells,32,33 suggesting that the averaged concentration of MMP9 enzyme by U937 cells was also in the range of nanomolar. Unfortunately it did not provide the quantitative data for the MCF7 cell line. According the designed geometry in this report, there was a limitation of one-way diffusion for the

MMP9 enzyme because of the separation between the cell location and sensing molecules. In contrast, our method using microdroplets was featured with the dynamic mixing between the fluorescent probe molecules and MMP9 enzyme, which should contribute to an improved sensitivity. While U937 cells are immature cells derived from a diffuse histocytic lymphoma, E

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Figure 4. Quantitative analysis of MMP9 enzymatic activities using the microdroplets. (a) Fluorescence changes of the microdroplets containing different concentrations of recombinant MMP9 in the course of time. (b) Calibration curve from fluorescence intensities by using a series of recombinant MMP9 concentrations in microdroplets at the time point of 25 min. (c) The titrated MMP9 enzyme molecules from a single U-937 cell (black) or a single MCF7 cell (red) in the microdroplet. The bar with asterisks indicates a statistical significance difference (P < 0.001). Error bar: standard deviation. (d) Schematic drawing of the localized effect onto the concentrations of MMP9 enzyme and fluorophores within the small volume of a microdroplet, compared with a bulk solution.

Figure 5. Fluorescent images of the microdroplets encapsulating either MCF7 or blood cells at the single cell level. MCF7 cells were spiked and mixed with the blood cells from a healthy donor to mimic the clinical blood sample of patients. The blood cells were collected and counted before mixing by a standard procedure. Cell loading concentrations: (a) MCF7: 1.5 × 105 cells mL−1, blood cells: 4.5 × 105 cells mL−1. (b) MCF7: 0.75 × 105 cells mL−1, blood cells: 5.3 × 105 cells mL−1. (c) MCF7: 0.37 × 105 cells mL−1, blood cells: 5.6 × 105 cells mL−1. (d) No MCF7 cells, blood cells: 6 × 105 cells mL−1. Ex/Em: 488 nm/520 nm, scale bar: 400 μm.

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either a single blood cell or none. The well-separated boundaries between the positive/negative microdroplets clearly differentiated the target cancer cells from the normal blood cells. The results provided strong evidence supporting that MMP9 activities were detected inside the microdroplets at the single-cell level using the spiked blood sample. Before this work, there had not been such a method of detecting MMP9 activities at the single-cell level. Our platform represents the first microfluidic demonstration for evaluation of cancer cell invasiveness at the single-cell level. Using this platform, we were able to detect different MMP9 activities between cell lines or perform droplet-by-droplet analysis for single-cell studies. The cell-to-cell heterogeneity has been investigated in the genetic or transcript levels in the literature,52−54 showing important and detectable differences. It would be even more interesting to disclose single-cell signature profiles in the protein level or protein functional activity level. The current study is based on cultured tumor cell lines, which may not represent a practically larger level of cancer cell heterogeneity in clinics. More tests directly using the samples from clinical practice will be necessary to understand cancer heterogeneity and invasiveness. As an attractive proof-ofconcept for clinical applications, abnormal metabolic behaviors of cancer cells have been proposed to differentiate rare tumor cells or CTCs in the patient-derived sample using a single-cell analysis technique.55 Rapid evaluation of cancer cell invasiveness will definitely assist the development of personalized medicine for cancer patients.

studies have suggested an overexpression of MMP9 in MCF7 cells as a response to interact with ECM components.49,50 Therefore, a higher level of MMP9 enzymatic activities in MCF7 cells was reasonable, which was supported by our experimental data. In addition, another report in the literature suggest that the levels of MMP9 secreted by the cultured cancer cells might be comparable to those by patient-derived tumor cells, although not very quantitatively yet.51 Furthermore, after taking the ultrasmall volume of a droplet (4.2 nL) into consideration, we could estimate that the level of MMP9 molecules was around 29 attomol by a U937 cell, and 71 attomol by a MCF7 cell, respectively (Figure 4c). In the future, further optimization is definitely worthy of testing microdroplets with even smaller volume for sensitivity enhancement. Microfluidic confinement of the cell into a small volume plays a key role to the high sensitivity of our method. When a tumor cell was successfully isolated in a microdroplet, the stimulation of PMA in situ led to elevated MMP9 enzymatic activities of the cell. Importantly, the microdroplet provided a volume well-defined microreactor for the enzyme reaction. In our experiments, the volume of a droplet was as small as 4.2 nL. The MMP9 enzyme molecules secreted by the tumor cell were confined within the small volume of a microdroplet, resulting in a valid high concentration. Fast mass transfer in the microdroplet allowed for efficient cleavage of the MMP9 specific peptides by the enzyme. The cleaved peptides containing FITC fluorophores were accumulated in the microdroplet. Therefore, it was the localized high concentrations of MMP9 enzyme and the cleaved fluorescent probes in the microdroplets that enabled strong signals for easier detection (Figure 4d). In contrast, if the enzyme molecules or the fluorophores were not isolated in the microdroplet, but distributed in a bulk solution instead, it would tremendously increase the difficulty to detect the fluorescent signals diluted by orders of magnitude. The conventional microtiter plate reader was employed to measure MMP9 enzymatic activities of the cells without using the capillary droplet generator. The U937 and MCF7 cells were respectively titrated from a density range from 2 × 105 cells to 2 × 103 cells per well in the microplate, with three independent assays (repetition n = 3). As shown in Figure S2a,b, using the conventional microplate assays, the fluorescent signals by MMP9 of the cells became indistinguishable from the background noise of the blank control, when the cell density was below 2 × 104 cells/assay. In contrast, encapsulating the cells in the microdroplets allowed for detection of the fluorescent signals at the level of single cell/ assay (Figure S2c,d). The sensitivity of single-cell analysis by microdroplets was hardly achieved with the conventional assay format using the microplate. Detection of MMP9 Activities at the Single-Cell Level Using the Spiked Blood Sample. We attempted to test the platform using cancer cell-spiked blood sample. In the experiments, MCF7 cells were carefully spiked and mixed with the blood cells from a healthy donor in a series of percentages (25%, 12.5%, 6%). We also included a control sample using only blood cells (6 × 105 cells mL−1) without MCF7 spiking. The fluorescent images of the microdroplets after cell encapsulation were acquired for analysis (Figure 5 and Figure S3). Each microdroplet with the fluorescent signal was determined to encapsulate only a single MCF7 cell due to the significant dilution effect of the spiking procedure. In contrast, the microdroplets without fluorescence, as highlighted by the white dashed circles in Figure 5, were likely to encapsulate

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CONCLUSION In summary, we have developed an approach featured with high sensitivity and high throughput for evaluation of cancer cell invasiveness. Sensitive detection of MMP9 enzymatic activities in single cell-encapsulated microdroplets has been achieved with a flow focusing capillary microfluidic device. In each microdroplet, up-regulated MMP9 by the tumor cell can rapidly cleave the peptides modified with a pair of fluorophore/ quencher molecules, therefore producing a fluorescence “turnon” signal. This new approach is fast and reproducible. In the manual mode of operation, hundreds of microdroplets can be monitored for the analysis of MMP9 enzymatic activities at the single cell level in several minutes. The throughput performance can further be raised by orders of magnitude when this approach is assisted by the high speed scanning facilities. Interestingly, it is able to differentiate cancer cells from normal cells, or different cancer cell lines, but also offers a tool to reveal the potential cell-to-cell heterogeneity in the protein functional level. Isolation of single cells within the microdroplets under the mild conditions may maintain cell viability and allow for subsequent cell culture or function-based cell screening. Our work has provided a powerful platform for single cell analysis by integrating the microfluidic technology, protein functional activity assay, and fluorescence image processing. As a sensitive assay of cancer cell invasiveness, it can be applied to the research of metastasis and cancer prognostics.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.6b00731. G

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Detailed experimental procedures, size distribution of the microdroplets from the different batches, assays using conventional microtiter plate, scatter plot of individual single-cell encapsulated microdroplets using the spiked blood sample, viscosity measurements (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jian Liu: 0000-0002-0095-8978 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the Major State Basic Research Development Program (2013CB932702), and by the National Natural Science Foundation of China (21275106, 21575095); a project supported by Collaborative Innovation Center of Suzhou Nano Science and Technology, the Priority Academic Program Development of Jiangsu Higher Education Institutions, and a Doctoral Fund of Ministry of Education of China (20123201120025). We thank Prof. Jian Chen of Soochow University for reviewing this manuscript and helpful suggestions. J.L. is supported by the “1000 Youth Talents” plan of the Global Expert Recruitment Program.

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DOI: 10.1021/acssensors.6b00731 ACS Sens. XXXX, XXX, XXX−XXX