Ultrasensitive and Simultaneous Detection of Two Cytokines Secreted

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Ultrasensitive and simultaneous detection of two cytokines secreted by single cell in microfluidic droplets via magnetic-field amplified SERS Dan Sun, Fanghao Cao, Weiqing Xu, Qidan Chen, Wei Shi, and Shuping Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05892 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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Analytical Chemistry

Ultrasensitive and Simultaneous Detection of Two Cytokines Secreted by Single Cell in Microfluidic Droplets via Magnetic-Field Amplified SERS Dan Sun,§ Fanghao Cao,§, ‡ Weiqing Xu,§ Qidan Chen,§, ‡ Wei Shi,# Shuping Xu§* State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, Jilin University, Changchun 130012, PR China ‡ School of Chemical Engineering and New Energy Materials, Zhuhai College, Jilin University, Zhuhai 519041, PR China # Key Lab for Molecular Enzymology & Engineering of Ministry of Education, Jilin University, Changchun 130012, PR China §

ABSTRACT: A surface-enhanced Raman scattering (SERS)-microfluidic droplet platform for the rapid, ultrasensitive and simultaneous detection of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) secreted by a single cell is presented. The high throughput water-in-oil droplets containing individual cell along with four kinds of immune-particles (antibodyconjugated silver nanoparticles or magnetic beads, AgNPs@Ab1 and MNs@Ab2) in each were achieved by a cross-typed microfluidic chip, and then they were captured by a collection channel array for SERS measurements. In the appearance of cytokines secreted by one cell, AgNPs@Ab1 can be linked onto the surface of MNs@Ab2 through the immune-recognition to form an immune-sandwich, which makes the “turn on” SERS signal of the Raman reporters previously laid on the surface of MNs due to the adjacent AgNPs. Furthermore, the second SERS signal amplification is from the magnetic field-induced spontaneous collection effect, which brings 75 times enhancement for SERS signal. Additionally, the encapsulation of cytokines in an isolated droplet permits an accumulation effect of targets with time. Owing to the dual signal enhancement and the accumulation effect, such few cytokines secreted by single cell become detectable and a limit of detection is achieved as 1.0 fg/mL in one droplet. By using this ultrasensitive SERS-microdroplet method, the VEGF and IL-8 secretions from several cells in one droplet were explored and the data show that the cell–cell interactions may promote angiogenesis of cancer cells through the up-regulation of VEGF and IL-8.

activated flow cytometry is a standard tool for high-throughput collection, sorting and detection of cells based on many developed fluorescent reporters responding to the components of the cell surface or intracellular complexes. However, this method is unavailable for tracing secreted molecules.11 Droplet microfluidics gained more and more attention and have widely used for cell-related studies.12-14 The cutting of the aqueous phase with the oil phase to form water-in-oil microdroplets in the T-shaped or cross-typed microfluidic chips.15 This technique can acquire the encapsulation of a single cell in one picoliter droplet by adjusting the size of chip channel and the flow rate of two-phase, which avoids interference from surrounding environments.16 Therefore, microdroplets are particularly suitable for monitoring single cell secretion. Beside the development of microdroplet techniques, various real-time detection techniques have been developed to realize on-chip or off-chip detections, e.g. electrochemistry,17 absorption spectroscopy,18 mass spectrometry19 and Raman spectroscopy.20-22 These dropletbased sensing methods become flexible and powerful in biosensing, and single cell analysis in particular. Surface-enhanced Raman spectroscopy (SERS) as a fingerprint analytical technique has attracted more and more

The increased expression of the pro-angiogenic factors is associated with tumor vascularization and growth.1, 2 Vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) are two potent angiogenic factors secreted by cancer cells, and the functions of which are to generate and expand tumor neovasculature.3-5 So, tracing VEGF and IL-8 secreted by cancer cells is of great importance for understanding the tumor evolution and transfer. Previous methods for determining these two cytokines are mainly based on the enzyme-linked immunosorbent assay (ELISA), which can achieve the overall results of a large population of cells. For the cytokine secretion at the single-cell level that is crucial for early inspection and precise treatment,6,7 it is still challenging due to the extremely high demand on detection sensitivity to a pg/mL level. Except for the high sensitivity, the high throughput is another desired nature for the detection of secreted products, if we want to define cell-to-cell variation for better understanding of the role of the individual cancer cell on controlling the global responses.8 Conventional approaches for single-cell cytokine detection including the cell-based microarrays9 and the enzyme-linked immunospots10 can arrive the detection limits of 10 pg (Table S1), but they show insufficient in detection throughput. The fluorescence-

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configurations. We modified the MNs@Ab2 with Ramanactive reporters of 4-amnobiphenyl (4-ABP) and acetamide (AAD) respectively to trace VEGF or IL-8 secreted from one cell encapsulated in a single droplet. The highly sensitive and selective SERS sensing of VEGF/IL-8 were achieved based on the dual effects (immune identification and magnetic force) driving aggregation of AgNPs@Ab1 and MNs@Ab2, which causes the “turn on” phenomenon of SERS signals of the Raman reporters that were previously located on the MNs@Ab2. The superiorities of this sensing method can be found in two aspects: (1) extremely low SERS detection limit to 1.0 fg/mL was achieved. Such high sensitivity of this sensing method is contributed from amplification effects of the metal plasmon enhancement and the magnetic field-induced aggregation. (2) Simultaneous detection of VEGF and IL-8 secreted from a single cell can be realized. This single-cell SERS-microdroplet analysis technique is applied for the dynamic monitoring of two cytokines, the cell sorting based on their different excretive capability and the exploration on the cell-to-cell heterogeneity in releasing cytokines from individual cell.

attention due to its merits of nondestructive data acquisition26 and single-molecule sensitivity,27 which gains potential applications in biological systems.23-25,28 Such remarkable enhancement on Raman signals is originated from strong plasmonic resonance effect, which can achieve million times amplification of signal. Thus, lots of efforts have been paid to composite metallic nanostructures for well plasmonic coupling.25 One of a successful example is to combine the magnetic particles with the metal nanoparticles, called magnetic SERS. The external magnetic field induced dynamical aggregation, decreasing the gap distance and increasing the detection sensitivity.29 Besides the contribution of magnetic materials for high SERS performances, the magnetic separation is also feasible for complicate sample extraction, which simplifies the sample pretreatment and separation,30 as well as the analysis of SERS data. The combination of SERS and microdroplet provides a practical strategy for achieving high sensitive detections and reproducible measurements at defined detection spots.31-33 The integrated platform of both can precisely control the aggregation time of metal nanoparticles and adjust the mixing efficiency of metal colloids and analytes via the regulation of flow rate and the design of channel structure, which can solve the low reproducibility of SERS in quantitative analysis.34 Thus, the SERS-based droplet microfluidics have been considered as a promising platform for highly sensitive, quantitative detection of biosamples.35,36 Herein, we presented a SERS-microfluidic droplet platform to detect secreted cytokines at the single-cell level based on a magnetic-field amplified SERS strategy. We first prepared four kinds of immune-particles, the cytokines-specific monoclonal antibody-conjugated silver nanoparticles (AgNPs@Ab1) and the anti-VEGF/IL-8 polyclonal antibodyconjugated magnetic beads (MNs@Ab2), for the purpose of constructing the target-bridged immune-sandwich

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EXPERIMENTAL SECTION Preparation of AgNPs@Ab1 and MNs@Ab2@4ABP/AAD. Four types of immune-particles were prepared in present study (Scheme 1A) and they can respond to VEGF and IL-8 to form immune-sandwich configurations as shown in Scheme 1B. The AgNPs used in the experiment were synthesized by the citrate reduction reaction according to Lee's method.37 The achieved AgNPs have a plasmonic band located at 425 nm and the transmission electron microscopic (TEM) image shows they are all in a quasi-spherical shape with an average size of 36 nm (Figure S4). To prevent the aggregation

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Scheme 1. A) Schematic illustration of the fabrication processes of four types of immune-particles, the polyclonal VEGF/ IL-8 antibody-conjugated MNs modified with the Raman reporter 4-ABP/AAD and the monoclonal VEGF/IL-8 antibodyconjugated AgNPs. B) The workflow of the droplet-based microfluidics for single-cell encapsulation and the SERS detection of VEGF and IL-8 secreted by one cell. centrifuged at 1500 rpm for 3 min and the growth medium was carefully removed. They were re-suspended in 840 μL of the ice-cold complete DMEM, accompanying with 160 μL of the OptiPrep medium. In our experiment, by controlling the cell density at 3.0 × 106 cells per mL, we obtained roughly 20% of droplets with a single cell, while less than 6% of droplets have two or more cells (Figure S6 and related discussion are found in the ESI†). According to Weitz’s study,38 the single cell encapsulation rate in the droplets is roughly 22%. Fluorinert HFE-7500 fluorocarbon oil (3 M, St. Paul, MN) was used to suspend the droplets. To stabilize the droplets, a PFPE–PEG block-copolymer surfactant (Ranbiotech) was added to the suspending oil with a concentration of 2.0% (w/w). The surfactant can protect the droplets against coalescence. For droplets formation, the outer, carrier-oil flow rate was 1040 μL/h and the inner, cell suspension solution flow rate was set as 58 μL/h.

of AgNPs during surface modification, 1.0 mL of AgNPs (0.29 nM) and 10 μL of Tween 20 (74 μM) were gently mixed for 30 min. The carboxylate terminal groups on the surfaces of AgNPs were activated by adding 5.0 μL of N-ethyl-N0-(3(dimethylamino)propyl) carbodiimide (EDC) and Nhydroxysuccinimide (NHS) (both 2.5 mM) for next chemical bonding to antibodies. After 1 h activation, the unreacted molecules were removed by centrifugation at 5000 rpm for 10 min. Next, 2.0 μL of 0.05 mg/mL monoclonal anti-VEGF antibody were mixed with the AgNPs solution (1.0 mL, 0.29 nM) for 2 h at room temperature and unreacted molecules were removed by centrifugation (5000 rpm for 10 min). Finally, unreacted sites on the surfaces of the AgNPs were deactivated by 1.0 μL of ethanolamine (15.0 mM) for 20 min. Nonspecific binding chemicals and antibodies were washed through centrifugation, and the remaining antibody-conjugated AgNPs were resuspended in the PBS buffer solution. Thus, the prepared AgNPs@Ab1-VEGF was ready for use. Next, we conjugated polyclonal anti-VEGF antibody and SERS reporter 4-ABP onto the surfaces of magnetic beads. 400 μL of 0.5 mg/mL carboxylic group-functionalized magnetic beads were activated by 5.0 μL of 0.1 M EDC and NHS dissolved in distilled water for 1 h. Then, the magnetic beads were separated using a magnet and washed with PBS buffer solution to remove unreacted molecules. After the magnetic beads were resuspended with PBS buffer, 5.0 μL of polyclonal anti-VEGF antibodies (0.5 mg/mL) and 20 μL of 4ABP (5.0 mM) were added to the magnetic bead solution and reacted for 2 h under stirring at room temperature. Magnetic beads were washed three times to remove nonspecifically bound antibodies and 4-ABP, followed by re-suspending in the PBS buffer solution to achieve MNs@Ab2-VEGF@4-ABP. The preparations of the monoclonal anti-IL-8 antibodyconjugated AgNPs (AgNPs@Ab1-IL-8) and the Raman-reporter of AAD labelled immune magnetic beads (modified with polyclonal anti-IL-8 antibody, noted as MNs@Ab2-IL-8@AAD) are as same as the above methods, except that AAD is used as a Raman signal reporter. TEM (JEOL, Tokyo, Japan, JEM-2100F), ultraviolet-visible (UV-Vis, Ocean Optics, USB4000) and dynamic light scattering (DLS, Malvern Zetasizer Nano ZS) spectroscopies were used to measure the size, morphology, plasmonic property and zeta potential of the obtained immune-particles.

Detection of VEGF and IL-8 secreted by single cells. Prior to analyze the cytokines secreted by the cells, we first verified the feasibility of simultaneously detecting two cytokines VEGF and IL-8 with SERS in the droplets. The mixed solution including AgNPs@Ab1-VEGF, MNs@Ab2VEGF@4-ABP, AgNPs@Ab1-IL-8 and MNs@Ab2-IL-8@AAD and different concentrations of VEGF and IL-8 (1.0 fg/mL to 100 pg/mL in pH=7.4 Tris-HCl buffer solution) were coencapsulated into water-in-oil droplets. The generated droplets are directly flowed into a collection chamber array channels. Then, the SERS measurement at defined location points of the droplets was carried out by a Horiba J-Y Aramis spectrometer at 632.8 nm from the laser power of ~ 7 mW, and accumulation time of 40 s /1 time. To evaluate two cytokines expressed on a single cancer cell, different cell lines stimulated with phorbol myristate acetate (PMA, 10 μL, 1.0 μg/mL,) for 6 h mixing with AgNPs@Ab1MNs@Ab2-VEGF@4-ABP, AgNPs@Ab1-IL-8 and VEGF, MNs@Ab2-IL-8@AAD to form discrete, water-in-oil droplets respectively (Scheme 1B). By tuning the pumping rate and cell concentration, we obtained roughly 20% achievement for single cell in one droplet while less than 6% of droplets have two or more cells (Figure S6) when the cells density was 3.0×106 cells/mL and the carrier-oil flow rate was 1040 μL/h, the cell suspension solution flow rate was set as 58 μL/h. Then, cyto kines secreted by a single cell in each droplet were

Droplets formation and cell encapsulation. The breast cancer (MDA-MB-231), lung cancer (A549) and gastric cancer (SGC) cells lines were bought from Shanghai ATCC cell bank. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibcos) supplemented with 10 % fetal bovine serum (FBS, GIBCO) along with penicillin/streptomycin (1 %, 1.0 g/mL) and incubated in a humidified atmosphere with 5 % CO2 for 37 ℃ . Cells were digested with the trypsase (wt 0.25%) for 3 min to achieve a cell-suspended solution. Then trypsase were pulled out and the cells were re-suspended in a fresh medium. They were

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with four types of immune-particles in the zigzag mixing region and each cytokine simultaneously binds to its respective monoclonal antibody on the silver nanoparticles and polyclonal antibody on the magnetic beads to form an immune-sandwich structure. Finally, these droplets were captured in a collection chamber array channels including about 100 droplets (Figure S1). The droplets containing single cell were easily found under a bright field microscope. The SERS signals were collected by focusing the laser on the collective immune-sandwiches in the droplets containing a single cell, and the cytokines (VEGF and IL-8) secreted by individual cells were measured according to the signal intensity value of Raman reporters (4-ABP and AAD).

Magnetic-field amplified SERS. The feasibility of our designed sensing system was proved first. The SERS spectra of AgNPs@Ab1, MNs@Ab2@4-ABP and immune-sandwich before and after reacting with VEGF (1.0 ng/mL) were compared, as shown in Figure 2a. Several SERS bands can be observed for the AgNPs@Ab1, attributed to monoclonal antiVEGF antibody (1 in Figure 2a). For the spectrum of the MNs@Ab2@4-ABP (2 in Figure 2a), a stronger peak at 664 cm-1 identified as a characteristic peak of magnetic beads is found. Next, we tried to spike VEGF in buffer solution to test the feasibility of our sensing design. Before VEGF was added, AgNPs@Ab1 and MNs@Ab2@4-ABP keep a distance from each other and no distinguishable peaks from 4-ABP are observed from (3) in Figure 2a, which provides a zerobackground for our sensing strategy. In the presence of VEGF, the sandwich immune-complexes formed and we can observe almost all the characteristic peaks of 4-ABP as (4) in Figure 2a and Figure S7(a). The band assignments are provided in Table S2. Also, by comparing (1)-(4), we can confirm that the signals of the magnetic beads and anti-VEGF antibody have no interference in the determination of VEGF since their signals have no overlapped with the SERS signal of 4-ABP.

RESULTS AND DISCUSSION Characterization of AgNPs@Ab1 and MNs@Ab2@4-ABP/AAD. By conjugating monoclonal

anti-VEGF antibody with AgNPs, a 5 nm red-shift is observed in their UV-vis spectra (Figure 1a) compared with the plasmonic band of AgNPs, along with a 12.5 nm increase in the average hydrodynamic size (from 35.8 to 48.3 nm) and a change of Zeta-potential from −32.5 to −20.2 mV (Figure 1b). In addition, the Zeta potential of the MNs@Ab2@4-ABP varies from −54.8 to −29.1 mV relative to MNs, proving the successful modification of the polyclonal anti-VEGF and 4ABP on its surface. Figure 1c and 1d demonstrate the TEM images of the sandwich immune-complex without/with VEGF (a concentration of VEGF was 10 pg/mL). When the targets (VEGF) are absent, almost no AgNPs@Ab1 are bound on the

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Figure 3. (a) Anti-interference experiment. SERS spectra for samples containing (1) AgNPs@Ab1-VEGF + MNs@Ab2-VEGF@4-ABP + VEGF and IL-8 (both 10 pg/mL). (2) AgNPs@Ab1-IL-8 + MNs@Ab2-IL-8@AAD + VEGF and IL-8 (both 10 pg/mL). (3) Four kinds of immune-particles + VEGF and IL-8 (both 10 pg/mL). (b) Concentration-dependent SERS spectra of four kinds of immuneparticles in response to different concentrations of VEGF and IL-8 (1:1, from 1 fg/mL to 10 pg/mL). The calibration curves for parallel detections of VEGF (c) and IL-8 (d). detection of cytokines compared to many published methods (Table S1).

Magnetic field can easily facilitate the aggregation of the sandwich immune-complexes, which is expected to further amplify the SERS signal of Raman reporters. To prove this, we monitored the dynamic process of aggregation from 0 to 25 min (photos taken at every 5 min) and found more and more sandwich immune-complexes in the droplet spontaneously gather along time due to their magnetic effect. In addition, we collected the SERS spectra of the sensing system with aggregation time at 0 and 25 min. SERS intensity of sandwich immune-complexes (Figure 2b) in the aggregated state (Figure 2c, Ⅵ ) can be improved about 75 times relative to that in a loose state (Figure 2c, Ⅰ), which further guarantees a higher SERS detection sensitivity. Based on exploration above, the quantification of VEGF in droplets was carried out by the present sensing method at different VEGF concentrations ranging from 1.0 fg/ml to 1.0 ng/mL (Figure 2d). The SERS intensity of 4-ABP increases as the VEGF concentration. By using the 664 cm-1 peak from MNs as an internal standard, the ratios of I1143 cm-1/I664 cm-1 with the VEGF concentrations are plotted (Figure 2e), which can be fitted by a linear model. The limits of detection are calculated as 1.0 fg/mL (signal/noise=3), indicating that this dropletbased SERS method features an ultrahigh sensitivity in the

Simultaneous detection of VEGF and IL-8. In order to target two analytes, we chose 4-ABP and AAD as Raman reporters to trace VEGF and IL-8 (Scheme 1B), respectively, due to their distinguishable and strong Raman peaks. More importantly, 4-ABP and AAD both have amino group as one terminal that attach to MNs, while the other terminal is blocked to avoid non-specific binding for antigen or antibody and guarantee high sensing specificity. We investigated the anti-interference experiment of our device. Firstly, we spiked IL-8 (10 pg/mL) in droplets to test whether such a concentration of IL-8 can interference the VEGF (10 pg/mL) sensing when the VEGF-specific immune-particles existed (AgNPs@Ab1-VEGF + MNs@Ab2-VEGF@4-ABP + VEGF + IL8). The testing results are shown in Figure 3a(1), in which we only observed the characteristic Raman peaks of 4-ABP. For comparison, another anti-interference experiment was carried out by using VEGF (10 pg/mL) for the system containing IL-8 (10 pg/mL) and IL-8-specific immune-particles (AgNPs@Ab1IL-8 + MNs@Ab2-IL-8@AAD + VEGF + IL-8). In this case, we only observed the Raman peaks of AAD (in Figure 3a(2)), which is in consistence with the SERS spectrum of pure AAD (Figure S7b & Table S3). Figure 3a(3) shows the SERS spec

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tra of droplets containing four kinds of immune-particles plus VEGF and IL-8 (both 10 pg/mL). As we expected, SERS

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Figure 4. Scattered plots of the SERS intensities of (a) 4-ABP at 1143 cm-1/664 cm-1 and (b) AAD at 1178 cm-1/664 cm-1 from the different single-cell lines encapsulated microdroplets. (c) Single cell encapsulated in individual droplet. The cell-bearing droplets are highlighted by the yellow arrows. Scale bar represents 100 μm. (d) The histogram of the secreted VEGF and IL-8 by different cell lines. of discrete, water-in-oil droplets (Figure 4c). The SERS measurements were performed after 25 min culture time (the data reproducibility are shown in Figure S8). The scatter plots of Figure 4a and 4b clearly present the cell-to-cell differences in secreting VEGF and IL-8. We can find that the VEGF levels secreted by three kinds of cells are very close (Figure 4a), while the amounts of the IL-8 expression are distinct. (Figure 4b). Figure 4d compares the secreted VEGF and IL-8 by different cell lines. It is interesting that MDA-MB-231 cells show the highest level in the IL-8 expression among three cell lines, while the IL-8 secreted by SGC cells are very few (also presented in Figure 4b). Both A549 and SGC cells express much higher VEGF levels rather than IL-8, whereas MDAMB-231 cells present an opposite situation (Figure 4d). This observation is consistent with previous studies in which they traced VEGF and IL-8 expression in different cell lines by the enzyme-linked immunosorbent assays (ELISA).3, 43, 44

1178 cm-1) simultaneously appear, demonstrating that our approach is practical for simultaneous detection of two targets. Further, the concentration-dependent SERS spectra (Figure 3b) of the designated concentrations of VEGF and IL-8 (1:1, from 1.0 fg/mL to 10 pg/mL) were measured by the SERSmicrodroplet device with four kinds of immune-particles. The detection data show that SERS intensities at 1143 cm-1 (4-ABP) and 1178 cm-1 (AAD) increase with the concentrations of cytokines (VEGF and IL-8). This microdroplet-based SERS sensing method shows high detection sensitivity and the concentration as low as 1.0 fg/mL can also be traceable (Figure 3b2). The coefficient of determination (R2) of 0.992 (VEGF) and 0.917 (IL-8) are achieved for the data of Figure 3c and 3d, respectively.

Determination of VEGF and IL-8 secreted from different single cell lines. The elevated expression of proangiogenic cytokines is associated with aggressive tumor growth.39-41 The applicability of the SERS-microfluidic droplet platform for the detection of VEGF and IL-8 in single living cell was evaluated. Three kinds of cancer cells including breast cancer (MDA-MB-231), lung cancer (A549) and gastric cancer (SGC) were activated by PMA (10 μL, 1.0 μg/mL) in order to stimulate cytokine secretion.42 Then each cell was mixed with four kinds of immune-particles in a great number

Regulation of VEGF and IL-8 secretions by cell– cell interactions. This platform can also be used for exploring the cell–cell interactions on the angiogenic potential of cancer cells. We quantified VEGF and IL-8 secretion by MDA-MB-231 cells when different numbers of cells were encapsulated in one droplet, respectively (Figure 5a). It is interesting that our measurements (200 droplets for each) based on the SERS-microdroplet method show that the VEGF

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the adjacent AgNPs and the magnetic field-induced aggregate amplification effect. SERS acted as fingerprint-distinguishable spectroscopy enables specific and simultaneous detection of various cytokines. We have detected VEGF and IL-8 secreted by different types of cell lines at the single-cell level and the results present cell- to-cell differences in the expression of IL8. Both A549 and SGC cells expressed higher VEGF levels than IL-8 whereas MDA-MB-231 cells present opposite. Moreover, the comparison of the VEGF and IL-8 secretions of one to four cells in one droplet shows that cell–cell interactions may promote angiogenesis of cancer cells through up-regulation of VEGF and IL-8. The ultrasensitive performance of our SERS-microdroplet platform makes it powerful in investigation of cell-to-cell heterogeneity in releasing cytokines at the large scale, promising revelation of new knowledge to understand the biological role of various cytokines in tumor vascularization and aggressive tumor growth.

secretion by cells increases sharply and tends to be gentle when the cell number arrives three or four in single droplet. VEGF secretion was up-regulated from four cells by 2.34-fold as compared to single cells (5.76 vs. 2.46 fg/mL). For IL-8 secreted by cells, it showed a tendency of increasing slowly first and

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Supporting Information. 1) Experimental section. 2) The construction of microfluidic system. 3) The sensing performance of other methods. 4) The TEM image of AgNPs, 5) The statistic particle size of droplets. 6) Poisson distribution for 3 different cell densities. 7) Raman and SERS spectra of 4ABP and AAD. 8) Band assignments of 4-ABP and AAD. 9) Expression of VEGF and IL-8 on different cell lines. The Supporting Information is available free of charge on the ACS Publications website.

VEGF IL-8

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AUTHOR INFORMATION

2 1

2

3

Number of cells

Corresponding Author

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* Email: [email protected] (S. X.)

Figure 5. (a) SERS spectra for probing VEGF and IL-8 by the SERS-microdroplet method when the droplet contains 1-4 cells. Inserts show the optical images of the cell contained droplets. Scale bar represents 50 μm. (b) Plots of the VEGF and IL-8 secretion with the cell number in one droplet.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (Grant Nos. 21873039, 21573087 and 21573092).

then rising sharply with the increase of the number of cells. More specifically, IL-8 secretion was up-regulated from four cells by 3.36-fold as compared to single cells (7.52 vs. 2.24 fg/mL). These results suggest that the cell–cell interactions lead to an increase in the secretion of VEGF and IL-8. It has been reported that VEGF and IL-8 expression regulate tumor angiogenesis.45 Therefore, we speculate that cell–cell interactions may promote angiogenesis of cancer cells through up-regulation of VEGF and IL-8.

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CONCLUSIONS In summary, we presented a method for sensitively detecting VEGF and IL-8 secretion simultaneously at the single-cell level using the SERS-microdroplet platform combined with magnetic amplification. The droplets are ideal micro-reactors and can be precisely sized to contain one individual cell. The introduction of immune-MNs/AgNPs into the microdroplets greatly improves the sensitivity of the detection due to the metal plasmon enhancement effect from

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Ultrasensitive and Simultaneous Detection of Two Cytokines Secreted

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