Three-Dimensional Inverse Opal Photonic Crystal Substrates toward

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Three-Dimensional Inverse Opal Photonic Crystal Substrates toward Efficient Capture of Circulating Tumor Cells Hongwei Xu, Biao Dong, Qiaoqin Xiao, Xueke Sun, Xinran Zhang, Jiekai Lyu, Yudan Yang, Lin Xu, Xue Bai, Shuang Zhang, and Hongwei Song ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10094 • Publication Date (Web): 22 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017

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ACS Applied Materials & Interfaces

Three-Dimensional Inverse Opal Photonic Crystal Substrates toward Efficient Capture of Circulating Tumor Cells †

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Hongwei Xu , Biao Dong *, Qiaoqin Xiao , Xueke Sun , Xinran Zhang , Jiekai Lyu , §

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Yudan Yang , Lin Xu , Xue Bai , Shuang Zhang , Hongwei Song

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State Key Laboratory on Integrated Optoelectronics, College of Electronic Science

and Engineering, Jilin University, 2699 Qianjin Street, Changchun, 130012, P. R. China ‡

School of Electronic and Information Engineering, South China University of

Technology, Guangzhou 510641, China §

China-Japan Union Hospital, Jilin University, Changchun, China 130033

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ACS Paragon Plus Environment


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ABSTRACT Artificial fractal structures have attracted considerable scientific interest in circulating tumor cells (CTCs) detection and capture, which plays a pivotal role in the diagnosis and prognosis of cancer. Herein, we designed a bionic TiO2 inverse opal photonic crystal (IOPC) structure for highly efficient immunocapture of CTCs by combination of a magnetic Fe3O4@C6@silane nanoparticles with anti-EpCAM and microchannel structure. Porous structure and dimension of IOPC TiO2 can be precisely controlled for mimic to cellular components, and anti-EpCAM antibody (anti-EpCAM，anti-epithelial cell adhesion molecule) was further modified on IOPC interface by conjugating with polydopamine (PDA). The improvement of CTCs capture efficiency reaches a surprising factor of 20 for the IOPC interface compared to that on flat glass, suggesting that the IOPC are responsible for the dramatic enhancement of the capture efficiency of MCF-7 cells. IOPC substrate with pore size of 415 nm leads to the optimal CTCs capture efficiency of 92% with 1 mL/h. Besides the cell affinity, IOPC also have the advantage of light scatting property which can enhance the excitation and emission light of fluorescence labels, facilitating the real-time monitoring of CTCs capture. The IOPC based platform demonstrates excellent performance in CTCs capture, which will take an important step toward specific recognition of disease-related rare cells.

KEYWORDS Circulating tumor cells (CTCs), inverse opal photonic crystal (IOPC), nanostructure, detection and isolation, enhancing fluorescence signal 2


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INTRODUCTION The sensitive detection and screening of circulating tumor cells (CTCs)1 has evoked a lot of interest since FDA-approved CellSearch appeared (2004)2, due to the significance for prognosis and selection of appropriate treatment3. So far, a considerable number of CTCs isolation methods have been developed4, with strategies that involve immunomagnetic beads5 or microfluidic devices6. The former utilizes capture-agent-coated magnetic beads to immunologically recognize CTCs in the blood, followed by magnetic isolation7. Microfluidic method can provide important advantages that enable highly efficient processing of complex cellular fluids, with minimal damage to sensitive cell populations due to shear forces or need for cell fixation8. However, isolation of CTCs has been technically challenging due to the extremely low abundance (a few to hundreds per milliliter) of CTCs among a large number of hematologic cells in the blood (109 mL-1)6. For in-depth study, a critical problem still need to be solved among the methods9, and that is reasonable design of capture layer for enhanced CTCs/substrate contact frequency and duration10. It is known that cellular surface components such as filopodia and extra cellular matrix (ECM) are easily embedded in nanoscale structures11. Many studies12 have proved the efficient interactions between cellular surface and sub-micrometer structures which share similar dimension. In addition, the fractal dimension of cancer cell surface structures is much larger than that of normal cells. Therefore, it is very important to isolate and detect CTCs by a suitable capture layer in microfluidic chip with smart morphology. Several versatile nanostructures have been established for 3



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sensitive and specific detection of CTCs, such as nanofibers12, nanoroughened surfaces13, fractal nanostructure11, which fully utilize the unique interactions between cellular surface components and nanostructured materials, exhibiting outstanding cell capture efficiency. Wang et al.14 firstly developed a three-dimensional (3D) nanostructured substrate, silicon nanopillar (SiNPs) arrays, which allowed for enhancing topographic interactions between the SiNPs substrates and nanoscale components of the cellular surface and resulting in vastly improved cell-capture affinity compared to unstructured substrates. A 3D hierarchical nanostructured graphene platform15 was further employed for CTCs capture, which masterly combined microporosity graphene foam with ZnO nanorod array. Gu et al.16 presented spherical colloidal crystal clusters with aptamer probes and further etched the particles to form a cell-preferred spherical array surface nanostructure for improved CTCs-capture, which led to the improved capture yields by the synergistic effects of higher topography and probe density. Besides, TiO2 nanofiber17, graphene oxide nanosheets18, and nanodots19 have also been explored in CTCs capture. These works have greatly improved the detection sensitivity of rare cancer cells in incubation environment, while the majority of these novel nano-architectures have not been tested within microfluidic devices for enhancing topographic interactions20.

Herein we developed a new TiO2 nanostructured substrate with 3D inverse opal photonic crystal (IOPC) morphology for mimic to cellular components, and anti-EpCAM antibody was further grafted with polydopamine (PDA). The designed 3D IOPC capture substrate served as upside capture layer, not only shows good cell 4


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affinity, but also fluorescence signal enhancement which facilitates the real-time monitoring function, as shown in Figure 1. Besides the 3D IOPC bio-mimic design for the ECM-mimicking topographical environment, this method was also featured with a magnetic Fe3O4@C6@silane nanoparticles (NPs) with anti-EpCAM. Base on this capture system, the influence of pore size of IOPC structure on capture efficiency was explored and monitored with confocal laser scanning microscope (CLSM) within microchip.

Figure 1. The schematic of the IOPC based on microfluidic chip. (a) The IOPC substrate serves as capture interface. External magnet was set on upside of the chamber for generating upward magnetic field. The enlarged image depicts the side view of inside chamber: the IOPC interface was modified with anti-EpCAM antibody. After the magnetic labeled CTCs suspensions are pumped into the chamber, the tumor cell will be pulled up along the magnetic field and touch the IOPC capture layer. The bottom is transparent glass, and the capture process can be monitored on CLSM. (b) Workstation setup for CTCs isolation. The sample is continually pumped through the chip using syringe pump. The amplifying image shows the microfluidic chip with magnet. RESULTS AND DISCUSSION

Compared to the conventional microfluidic device, our capture system was featured by the IOPC substrate, which is employed as the capture surface and set on 5



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the upside, bonding onto a transparent glass slide substrate, as shown in Figure 1. The IOPC structure can not only complement cell filopodia and efficiently enhance CTCs capture yields, but also provide the advantage of improving imaging function by enhancing the diffraction of excitation and emission light. It should be noted that from the previous nanostructure based studies, it is still a challenge for the real time monitoring of the capture process21. In this case, as shown in Figure 1, after labeled with Fe3O4@C6@silane nanoparticles with magnetic and imaging functions (C6), the CTCs were pulled up by the magnetic force from the permanent magnet and collected on the IOPC capture surface. Due to the diffraction enhancement from the IOPC structure, the excitation and emission light of C6 molecules inside the CTCs can be monitored.

The IOPC substrate is made from TiO2 which has superior biological compatibility to biomolecules. The structure has the advantages of large surface-to-volume ratio, well-ordered porous architecture and the easily controlled pore size22. In this work, TiO2 IOPC substrate with three pore sizes were prepared, as depicted in Figure 2. Firstly, opal structure formed of PMMA spheres were prepared, with sizes of 335, 430 and 500 nm, and shown in Figure 2a-c. Then, the corresponding TiO2 inverse opal structure was obtained by a controllable sacrificial template method. All the samples yield a long-range ordered hexagonal arrangement of IO nanostructure with the pore size changing from 250 ， 305 ， 415 nm, as presented in Figure 2d-f. In addition, mesoporous can also be seen in the skeleton of IO nanostructure with an increased mesoporous size as the arrows point out, forming 6


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a double order porosity of IO sample with macroporous hollow thick films combining mesoporous skeleton. The macropore diameters of the IOPC substrate all experienced shrinkage relative to the corresponding PMMA opal PC templates during calcination.

Figure 2. The SEM images of PMMA PC template (a) - (c), and TiO2 IOPC surface (d)-(f). Mesoporous structure appears in IOPC skeleton as the red arrows point out. The size of PMMA microsphere and inverse opal pore can be tuned by changing reaction time. It is known that PC possesses spatial periodicity in their dielectric constants on the length scale of the optical wavelength, and, consequently, respond to electromagnetic waves which is similar to the way of atomic crystals responding to electrons. Because an electronic band gap is created by the periodic arrangement of atoms in a semiconductor, the periodic electromagnetic modulation created by the PC or IOPC can also yield a size-dependent photonic stop band (PSB), in which the light propagation is forbidden in the PC23. Figure 3a-b shows the transmission spectra of the PMMA PC and the corresponding TiO2 IOPC with different PSBs, respectively. The PSB positions can be well described by the Bragg diffraction equation using the 7



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PMMA sphere diameters and the pore size of the TiO2 measured by SEM. In this work, PSB of the well-ordered PC (734, 990 and 1048 nm) and IOPC (518, 650 and 798 nm) means nice and uniform structure, as well as the pore size.

Figure 3. The transmittance spectra of opal PC template (a) and TiO2 IOPC structure (b). PSB of PC and IOPC both red-shifted with the increasing size of the PMMA template. For CTCs capture in microfluidic channels, the IOPC substrate should be stable with the shear force from the flowing liquid. To evaluate the stability, surface morphology and PSB of the TiO2 IOPC was measured before and after flowing water immersing. The SEM images in Figure 4a-b indicate the IOPC keeps the morphology after water immerse and PSB in Figure 4c further proves the stability due to the unchanged position. In addition, various liquid, including phosphate buffer solution and the mixture of culture medium and serum, were also measured. The surface topography still remained the same (Figure S1).

Before the CTCs isolation experiment based on the IOPC structure, an antibody anti-EpCAM was modified for capture specificity. Here, we modified PDA with amino groups on the surface of IOPC structure by adhesion mechanisms24-26, then 8


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followed the introduction of antibody through amino-carboxyl reaction27, which was further verified by the infrared spectroscope (FTIR) in Figure 4d. It can be seen that PDA-IOPC sample exhibits bands at around 3200-3500 cm-1 corresponding to stretching vibration of N-H and O-H. In addition, the peaks at around 1518 cm-1, 1616 cm-1 can be observed, which is assigned to C=C resonance vibrations in the aromatic ring and the N-H bending vibrations of polydopamine28, which also indicates the successful modification of PDA on IOPC.

Figure 4. The SEM images of the origin IOPC nanostructure (a) and that after immersed in flowing water (b). (c) Comparison of the transmitted spectral of IOPC structure before and after immersed in water. (d) The infrared spectroscopy of IOPC (black) and that modified with PDA (red). To evaluate cell capture performance of the IOPC substrate within our CTCs isolation system, the experiments were conducted with CTCs labeled with 9



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Fe3O4@C6@silane, which was fabricated based on amphiphilic silanes modification strategy29. Briefly, Fe3O4 NPs with ultrasmall size serve as core and encapsulate with silanes via hydrophobic interactions. Coumarin 6 (C6) hydrophobic organic molecules were loaded into the hydrophobic interspace between Fe3O4 NPs and silane via the hydrolysis reaction, serving as imaging agent as shown in Figure 5a. To target capture, anti-EpCAM antibody was employed due to its overexpressed in most tumors. Fe3O4@C6@silane was modified by poly-L-lysine (PLL) and obtained positively charged, then, negatively charged antibodies were modified on the surface of composites via electrostatic attraction. The surface Zeta potential of each modification was shown in Figure S2. TiO2 IOPC substrate was employed as the capture surface and set on the upside, bonding onto a transparent glass slide substrate. Figure 5b shows the side view of the IOPC structure. The thickness of the capture layer is about 10 µm which can be controlled by concentration of PMMA microspheres in colloid suspension. For cell capture experiment, the MCF-7 cancer cells labeled with Fe3O4@C6@silane was pumped through the microfluidic chamber with a magnet at the rate of 2 ml/h. In the flowing process, CTCs were imaged with CLSM system to evaluate the capture situation on the upside surface. Besides the magnetic labels (green emission from C6), CTCs were also stained with a blue nuclear fluorescence dye Hoechst 33342.

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Figure 5. (a) The TEM image of nanocomposites Fe3O4@C6@silane, (b) SEM cross sectional image of TiO2 IOPC. The specificity of the IOPC interfaces in a microfluidic channel was evaluated by comparing the capture efficiency with anti-EpCAM-coated flat glass substrate and the influence from the different pore sizes of IOPC capture layers. The capture situation on anti-EpCAM modified flat glass and IOPC are displayed in Figure 6a-b, respectively. The flat glass and IOPC interfaces exhibited vastly different capabilities for catching MCF-7 cells. Even modified with anti-EpCAM molecules, there are very few CTCs can be observed on glass as Figure 6a-c shows. On the contrary, with the same condition of glass, dramatically increasing CTCs can be observed based on the blue and green light, which indicates the high cell-capture efficiency of anti-EpCAM-coated IOPC structure as Figure 6d-f shows. The capture yield of the IOPC capture surface within microfluidic device was measured by hemocytometer and counting, which is input cells number measured with captured cells number percentage. The IOPC interfaces can greatly improve the detection sensitivity and capture efficiency by as much as 20 times for an EpCAM-positive MCF-7 cell line (85%) compared to the flat glass interfaces (4.315%). The similar fractal dimensions 11



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of the IOPC structure and cancer cells can induce topographic recognition in addition to biomolecular recognition, which results in dramatic enhancement of the capture efficiency of cancer cells.

Figure 6. CTCs capture situation on flat glass (a) - (c) and IOPC (d) - (f) interface inside the microfluidic chip. Blue: dye Hoechst 33342, Green: C6 from Fe3O4@C6@silane. The scale bar was 100 µm. Furthermore, we explored the influence from the pore size of IOPC structure on CTCs specific adhesion property by conducting experiments using MCF-7 cells. With the same condition, the IOPC structure based microfluidic chip with different pore sizes shows different capture efficiency. Increasing number can be seen from Figure 7a-c, which correspond to the IOPC structure with pore size of 250 nm, 305 nm and 415 nm and the corresponding cell-capture efficiencies are 38.4%, 50.56%, 85% , respectively, as depicted in Figure 7d. The maximum capture efficiency was achieved with the largest pore size. Flow rate is an essential parameter for CTCs capture efficiency, which influences the duration of cell-nanosubstrate contact. Therefore, capture efficiency was measured 12


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under different flow rate velocity (1, 2 and 3 mL/h) with the 415 nm pore size. As the continuous flow rate was set as 1 mL/h with CTCs concentration (2×103/mL), the capture efficiency can reach 92% in Figure 7e. As the flow rate grew to 2 and 3mL/h, the capture efficiency drops gradually to 85 % and 80.3% (Figure 7e).

Figure 7. The influence of different pore size of upside IOPC capture surface inside the microfluidic chip on CTCs capture efficiency. (a), (b), (c) capture substrate correspond to the pore size of 250, 305, 415 nm TiO2 IOPC nanostructure, respectively. (d) The statistic number of captured cells under different pore size. (e) The capture yield with various flow rate (1mL/h, 2mL/h, 3mL/h). The scale bar is 100 µm. For artificially spiked CTCs into blood samples, with the same experimental condition, capture efficiency also further was measured with different concentration of MCF-7 cells (2×103/mL, 1×103/mL and 2×102/mL). The capture efficiency was obtained more than 60% (Figure 8a) and captured CTCs were imaged under CLSM with cell concentration 2×102/mL (Figure 8b-c). When cell concentration is 2× 13



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103/mL, 1×103/mL, the captured cells also were imaged in Figure S3.

Figure 8. The capture yield with spiked MCF-7 cells into blood. (a) The capture yield with various cell concentration spiked blood (2×103/mL, 1×103/mL and 2×102/mL). Captured cells were imaged under CLSM, (b) Blue: a nucleus dye Hoechst 33342, there are CTCs and white blood cell. (c)Green: C6 inside Fe3O4@C6@silane. Only CTCs were presented. WBCs only present blue light while CTCs present both blue and green light, the cell concentration 2×102/mL with 1mL/h. The scale bar was 100 µm. The images were clipped from a photo within a view of microscopy for clear show. It is reported that cancer cells will stretched out more filopodia on the rough interface than that on flat substrate28, 30. In this case, it also should be the reason for the higher capture efficiency on IOPC interface than that on glass. For the different capture efficiency with IOPC substrate with different pore size, some detailed experiments were performed to explore the working mechanism of the topographic interaction between cancer cells and IOPC surface. The MCF-7 cells were incubated with IOPC substrate to perform static cell capture studies with concentration of 103 cells mL-1. By comparing the filopodia numbers, widths of the filopodia11 on the cell/capture surface interfaces (shown in Table 1), it can be concluded that the main difference is the number of the filopodia. Cells on IOPC with the size of 415 nm stretched out more filopodia (40-46 per cell，Figure 9g) than those on other interface (Figure 9c, e), indicating that fractal dimensions strongly influenced the number of

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filopodia. In contrast, there only were less filopodia (16-21 per cell) on flat glass and the maximum width of the filopodia is 0.45µm (Figure 9a-b). IOPC with size of 305 nm has more filopodia (36-45 per cell, Figure 9e) than those on IOPC with size of 250 nm (25-32 per cell, Figure 9c). From enlarged SEM image of captured cell in Figure 9d, f, h, width of the filopodia can be noticed which shows a variation ranging from 1 µm to 2.5 µm. These results indicate that cancer cells prefer to interact with larger size nanostructures (415 nm) with a shape-matched nanometer-scale topography which can enhance interaction between the substrate and target cells. It is the nature for cancer cells to grip the substrate for better incubation environment, and that is way the cells will stretch out filopodias to catch anything to stabilize itself

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. Rough surface has been proved to be suitable for the incubation

environment, especially for raised architecture with similar dimension with cell filopodia31. While few studies explored the cell surface sensitivity of macroporous structure, such as this IOPC substrate32. From our results, it should be confirmed that the skeleton holds and adhere the filopodias, which, in addition, stretch down to the next layer of IOPC substrate with thinner branches for better grasping stability.

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Figure 9. The SEM of captured cancer cells on flat glass and different size IOPC nanostructure. (a), (c), (e), (g) Captured CTCs on flat glass, 250 nm, 305 nm, 415 nm nanostructure and the amplification of surface filopodia (b), (d), (f), (h).

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Table 1. The statistic data of surface filopodia of captured cells

A comparison of our work and other representative 3D nanostructure based CTCs captured devices is shown on Table 2. Considering similar cell concentration and flow rate, our isolation device is comparable to those of the others in capture yield. Table 2. Comparison of capture efficiency other similar 3D nanosubstrate

Our IOPC based CTCs capture system own an advantage for real-time imaging, due to the C6 molecules in magnetic labels. That’s way the capture situation on the

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IOPC layer can be monitored as above mentioned. The IOPC nanostructure can not only provide three-dimensional nano-topography, but also enhance the emission light due to the scattering property of photon crystal for improving imaging property. Note that, in the microfluidic channel, light signal is very weak due to the very few tumor cells in the blood7, therefore, it is highly necessary to involve a fluorescence enhancement approach. Herein, the IOPC capture substrate was set upside of the microfluidic chamber, and, therefore, it can scatter the excitation and emission light from the CTCs cells to increase the detection signal by CLSM. To support this mechanism, we detected the fluorescence from the cells that incubated on the flat glass substrate and IOPC substrate, as well as the cells in the gap of the IOPC, which has no regular photonic crystal structure. Figure 10a-b shows the incubated cells on the flat glass and IOPC substrate which is made on the same glass substrate as the the inset of Figure 10c. With the same measuring condition on CLSM, such as the same lens magnification and focal length, the emission spectra of Fe3O4@C6@silane labeled cells on different part of the glass were measured under confocal microscope from 470 nm to 550 nm. It is obvious that the cell on IOPC shows highest emission intensity relative to that on the gap of IOPC and flat glass, indicating the light enhancing function of IOPC structure.

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Figure 10. Comparison of cells imaging on flat glass (a) and IOPC substrate (b) with CLSM. The insets of (a) and (b) show SEM images of the interface structure of flat glass and IOPC substrate. The cells on site 2 are in the gap of IOPC, meaning on glass, while the cells on site 3 are all on IOPC structure. The spectra of the C6 from cells in (a) and (b) were measured from 470 nm to 550 nm by CLSM and displayed in (c), which are corresponding to 1 (on glass), 2(on the gap of IOPC) and 3 (on IOPC interface), respectively. The three sites are all on the same substrate as the inset of (c) shows. The scale bar is 100 µm. CONCLUSIONS In summary, we have developed a novel three-dimensional IOPC based microfluidic chip for CTCs capture combined with a magnetic Fe3O4@C6@silane nanoparticles. Anti-EpCAM was conjugated onto the IOPC interface by PDA for specific recognition and detection of CTCs. The as prepared IOPC substrate served as capture layer and showed a pore size dependent CTCs capture efficiency. The optimal efficiency was obtained with the pore size of 415 nm, which was as high as 92% under 1ml/h in culture medium. Meanwhile, with the artificially spiked CTCs into blood samples, the capture yield was obtained above 60% with 1mL/h and 200 cells/mL cell concentration. Besides the bionic structure for cell affinity, IOPC structure could also scatter the excitation light from the CLSM and emission light from fluorescence dye C6, which greatly improved the fluorescence signal by 35% enhancement. The IOPC based platform shows promising potential for dynamically

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manipulating cell-substrate interactions in vitro, and holds great promise for reliable and sensitive detection in a clinical environment. EXPERIMENTAL SECTION Preparation of TiO2 IOPC

The TiO2 IOPC was prepared by using sol-gel method with a polymethyl methacrylate (PMMA) latex sphere template technique. Firstly, monodispersed PMMA latex sphere with controllable size (335 nm, 430 nm, 500 nm) were synthesized22. Then, a thin-film template was self-assembled through the vertical deposition. The colloid suspension of 5% PMMA microspheres was dropped onto a clean glass substrate and placed in a 32 ℃ for 24 h. The PMMA colloidal spheres slowly self-organized into highly ordered colloidal arrays on the glass substrate, driven by surface tension of the liquid in the evaporating process. Following deposition, the opals were sintered for 40 min at 120 ℃ to enhance their physical strength. In the preparation of a TiO2 precursor sol, butyltitanate, ethanol, and nitric acid were mixed and stirred for 1 h to form a transparent solution，with volume ratio 10:10:1. The precursor solution was used to infiltrate into the voids of the opal template through capillary force. After infiltration, the resulting products were dried in air at room temperature. Annealing was carried out by slowly elevating the temperature (1℃/min) up to 500 ℃ for 3 h. By controlling the diameters of PMMA latex spheres during polymerization in the previous step, the size of IOPC was finely tuned.

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The IOPC coated capture surfaces were prepared by monodispersed PMMA latex sphere with controllable sizes, we fabricated various size IOPC with diameters of 250, 305, 415 nm on glass. After preparing the IOPC substrates with PDA to treat with the substrate, IOPC were immersed into 0.2 mg/ml dopamine with Tris buffer solution for 1 h and drying, resulting in a layer of PDA coating onto the IOPC substrate. Finally, we utilized EpCAM antibody onto the surfaces of substrate prior to the cell capture experiments.

Synthesis of Fe3O4@C6@silane The Fe3O4 NPs modified with oleic acid (5mg/ml)33, trimethoxy (octadecyl) silane (7.5mg/ml) and Coumarin 6 (C6, 1mg/ml) were mixed in tetrahydrofuran (THF) under sonication with volume ratio 10: 25: 2. Then, the mixture was swiftly poured water with 5 fold (pH ≈ 9, including poly-L-lysine (PLL) in a conical flask for the hydrolyzing process at room temperature 4 h. Finally, the solution was dialyzed overnight at room temperature. In this case, Fe3O4@C6@silane surface was obtained with positive charge, and anti-EpCAM antibodies (20ul 1mg/mL) were mixed for targeting at 4℃，excess antibodies were removed by centrifugation.

Preparation of the microfluidic device

After preparing the antibody-IOPC substrates, the antibody-IOPC substrates and a clean slide were glued together via acrylate structure adhesive(9900 ergo) with the flow chamber of 1 cm×2 cm×100 µm. The silicone tube as routes was used to inlet and outlet for the microchannel. 21



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Pretreatment of cells for capture experiment

MCF-7 cells were purchased from Shanghai Institute for Biological sciences, Chinese Academy of Science. The culture medium contains RPMI 1640 (GIBCO) and fetal bovine serum, and the ratio was 9:1. MCF-7 Cells were cultured at 37℃ with CO2 (5%). Trypsin (EDTA 0.02%) was used to re-suspend cells before plating.

Before capture experiment, MCF-7 cells were incubated with Fe3O4@ C6@silane for 4h in incubator. Then cells were washed three time by phosphate buffer (PBS, PH=7.4), trypsinized and resuspended in the culture medium, the cell suspension contains 2×104 cells in 1 mL the culture medium. The experiment required concentration of cell suspensions was obtained by dilution. For the artificial sample forming, cancer cells were spiked into the whole blood from healthy people and the red blood cell was removed by lysis buffer. We imaged and counted cells using the CLSM and hemocytometer for calculating capture efficiency, the cell counting was carried out tree time for accuracy.

In order to better determine and observe the cells, fluorescence staining is necessary. PBS was injected to remove no specificity cells. The cells were fixed with 4% (wt/vol) paraformaldehyde for 10 min and washed by PBS for three times. Finally, Hoechst 33342 dye was added for nuclei staining and washed with PBS.

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SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental procedures, characterization, as well as the supporting data.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (Biao Dong)

*E-mail: [email protected] (Hongwei Song) ORCID Hongwei Song：0000-0003-3897-5789 Lin Xu: 0000-0001-5831-430X Author Contributions These authors contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS

This work was supported by the National Key Research and Development Program (2016YFC0207101), the Major State Basic Research Development Program of China (973 Program) (no. 2014CB643506), the National Natural Science Foundation of China (Grant no.11374127, 21403084 ， 11674126 ， 11674127, 11504131, and 61674067), the Jilin Province Natural Science Foundation of China 23



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(NO. 20150520090JH, 20170101170JC) and the Jilin Province Science Fund for Excellent Young Scholars (No. 20170520129JH, 20170520111JH).

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TOC

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Three-Dimensional Inverse Opal Photonic Crystal Substrates toward

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