Supporting Information

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1 Supporting Information Multiplex analysis on a single porous hydrogel bead with encoded SERS nanotags Bing Liu,,, Di Zhang,, Haibin Ni,, Delong Wang,, Liyong Jiang, Degang Fu,, Xiaofeng Han,,, Chi Zhang, Hongyuan Chen, # Zhongze Gu, and Xiangwei Zhao *,, State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, National Demonstration Center for Experimental Biomedical Engineering Education, and Key Laboratory of Environmental Medicine Engineering of Ministry of Education, Southeast University, Nanjing , China Department of Physics, School of Science, Nanjing University of Science and Technology, Nanjing , China Nanjing Institute of Product Quality Inspection, Nanjing , China # State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 21009, China * xwzhao@seu.edu.cn Table of contents Materials S2 Fabrication of PHB carriers...s2 Preparation of SERS nanotags...s SERS measurement and instrumentation..s4 Sandwich immunoassay for the selection of excitation wavelength of SERS nanotags.s4 Quantitative analysis.s5 Multiplexed analysis..s5 Clinical samples analysis...s6 Spectral deconvolution..s6 Electromagnetic simulations.s6 Surface area to volume ratios (SVR) calculation..s6 Figure S1 S7 Figure S2 S8 Figure S S8 Table S1 S9 Table S2 S9 References.S9 S1

2 Experimental Method Section Materials Hydrogen tetrachloroaurate (III) trihydrate (HAuCl 4 H 2 O), trisodium citrate (Na C 6 H 5 O 7 ), (-Glycidoxypropyl)-trimethoxysilane (GPTMS, 98 %) and bovine serum albumin (BSA) were obtained from Sigma-Aldrich. Thiolated-carboxylated PEG (HS-PEG-COOH, MW ~ 5 kda) and thiolated PEG (PEG-SH, MW ~ 5 kda) were commercially available from Laysan Bio, Inc. (USA). Crystal violet (CV), Nile blue A (NBA), ethyl dimethylaminopropyl carbodiimide (EDC), silver nitrate (AgNO ), ascorbic acid (AA) and sulfo-n-hydroxysuccinimide (NHS) were purchased from Alfa Aesar. Acrylamide, bisacrylamide, 2-hydroxy-2-methylpropiophenone (HMPP), and glutaraldehyde were purchased from Aladdin Reagent Company (China). All chemical reagents were of analytical grade. All solutions were prepared with Millipore Milli-Q quality water. Glassware was first immersed in the freshly prepared aqua regia solution (HCl/HNO, :1) for 12 h, and next rinsed thoroughly with water before use. Mouse IgG and anti-mouse IgG functional fragments (Fab) were received from Biodee Biotechnology Co., Ltd. (China). Human AFP and CEA, a pair of mouse monoclonal anti-human AFP antibodies and a pair of mouse monoclonal anti-human CEA antibodies, were commercially purchased from Beijing Key-bio Biotech Co., Ltd (China). All protein samples were directly used without further purification and diluted in the phosphate-buffered saline buffer (PBS, 0.05 M, ph=7.4) prior to multiplex bioassays. Monodisperse colloidal silica nanoparticles with diameter of 26 nm were obtained from Nanjing Nanorainbow Biotechnolgoy Co., Ltd. (China). Fabrication of PHB carriers Silica colloidal crystal beads (CCBs) were first fabricated as porous hydrogel beads (PHBs) templates with a homemade co-flow microfluidic, which proposed by Zhao et al. S1 Briefly, 26 nm silica nanoparticles suspension (15 %, w/w) and silicone oil were used as the dispersed phase and the continuous phase, respectively. Next, the aqueous silica suspension could be broken into monodisperse droplets by the oil flows with different speed of the two phases in the microfluidic channel, and then the droplets were heated at S2

3 70 overnight in order to self-assembled into ordered lattices. After solidification at 100 for 2 h, silica CCBs were thoroughly rinsed by hexane and sintered at 700 for h. The fabrication of PHBs was performed according to previously published research. S2 Briefly, a polyacrylamide (PAM) pregel solution composed of 17.4 g acrylamide and 2.6 g bisacrylamide in 80 ml ultrapure water was prepared. Then, HMPP was added to the pregel solution (1.5:100, v/v). Next, silica CCBs with different reflection peaks were dispersed into the mixture for 2 h. Afterward, The pregel solution containing CCBs was cured under UV light for 0 s, and then the polymerized hydrogel was immersed in 4 ethanol and 60 water solution alternately for removing CCBs from the polymerized hydrogel. Finally, silica templates were removed by 1 % hydrofluoric acid etching. Capture antibodies conjugated to PHB by Schiff base reaction. S Briefly, PHBs were disperse into glutaraldehyde solution (5 %, w/w) for 4 h. Next, PHBs were rinsed several times by ultrapure water, and then PHBs were immersed in 1 mg ml -1 Fab at 4 overnight. Afterward, 0.5 % BSA solution was used to block nonspecific binding sites of PHBs, and the mixture was incubated at room temperature for 2 h. Then, PHBs were washed with PBS several times by vortex. Finally, PHBs were stored at 4 for further use. Preparation of SERS nanotag We have reported on the synthesis of SERS nanotags based on gold-silver core-shell nanoparticles (Au@Ag NPs). S4 First, gold nanoparticles (AuNPs) with the diameter of about 60 nm were synthesized followed by Frens method. S5 Briefly, 100 ml HAuCl 4 solution (0.01 %, w/w) was heated to boiling under vigorous stirring. Next, 0.75 ml of sodium citrate solution (1 %, w/w) was quickly injected into HAuCl 4 solution, and then the mixture was allowed to boil for 10 min until the color did not change. Finally, the resultant solution gradually cooled to room temperature under stirring. 45 ml AuNPs colloidal solution was mixed with two kinds of Raman dye (RD) solutions, NBA (5 ml, 2.0 μm), 5 ml CV (1.5 μm) for 20 min under magnetic stirring, respectively. Then, the RDs conjugated AuNPs were centrifuged three times at 10,000 rpm for 10 min, and the sediment was S

4 resuspended in 10 ml ultrapure water. After that, 2 ml AA (0.1 M) was added to the functionalized nanoparticles under stirring. Afterward, 1 mm AgNO was dropwise added by pipet while vigorously stirring the mixture until the finally concentration was 2.5 μm. Then, the resultant silver continuously grew on the surface of functionalized AuNPs because of the reduction of AgNO by AA. After the color of the solution changed from pink to yellow-orange, the solution was stirred for 0 min. Then, the mixture was centrifuged three times and resuspended in 10 ml ultrapure water. Next, 1.5 ml HS-PEG-COOH (10 μm) was added to the bimetal core-shell nanostructure solution and mixed for 20 min under magnetic stirring. Subsequently, 10 ml PEG-SH (50 μm) was added to the mixture and allowed to incubate for an additional h. After the removal of excess PEG by centrifugation, 40 mg ml -1 freshly prepared EDC (12 μl) and 110 mg ml -1 NHS (12 μl) were added to the PEG encapsulated bimetal nanoparticles and mixed vigorously for 20 min at 25 in order to activate carboxyl groups of PEG for effective antibodies binding. Then the mixture was centrifuged three times and resuspended in 2 ml PBS. Afterward, 100 μl detection antibody functional fragments (80 μg ml -1 ) were added to the activated nanoparticles and incubated for 2 h at 25. Next, the mixture kept overnight at 4, and then the mixed solution was centrifuged three times. Finally, SERS nanotags were obtained and stored at 4 for further use. SERS measurement and instrumentation The SERS measurements were carried out using an InVia Renishaw Raman microscope (Renishaw), and the data acquisition time was 10 s. The Raman system was calibrated using silicon wafer with Raman shift at 520 cm -1. Spectral acquisition, baseline correction, and removal of fluorescence band in the spectra were performed using WIRE 4.2 software. For each sample measurement, five independent PHBs were measured. High-resolution transmission electron microscopy (TEM) images were obtained by a JEM-2100 transmission electron microscopy (JEOL). Scanning electron microscopy (SEM) images were captured using an Ultra Plus scanning electron microscope (Zeiss). UV-vis absorption spectra were recorded using a Hitachi 5000 UV/Vis/NIR spectrophotometer. S4

5 Sandwich immunoassay for the selection of excitation wavelength of SERS nanotags PHBs immobilized with anti-mouse IgG Fab were mixed with mouse IgG (10 μl/bead, 10 ng ml -1 ) under shaking 1 h at 7. Then, PHBs were rinsed five times with PBS by vortex for removing nonspecific adsorption mouse IgG. Next, PHBs and SERS nanotags were incubated for 1 h under the same conditions. Subsequently, PHBs were also washed five times with PBS by vortex. Afterward, SERS measurements of PHBs were performed using an InVia Renishaw Raman microscope equipped with 50 objective lens (numerical aperture, NA = 0.75), and the wavelengths of excitation light were 52, 6, and 785 nm with a laser power of 5 mw, respectively. Quantitative analysis In order to evaluate the quantitative performance of the as-proposed multiplex assay, we performed quantitative analysis for CEA detection. A series of CEA concentrations ranging from 0.1 fg ml -1 up to 200 μg ml -1 were prepared. PHBs and NBA-SERS nanotags were decorated with CEA capture and detection antibodies, respectively. First, PHBs were incubated with CEA (10 μl/bead) in test tubes for 1 h at 7 under shaking, and then PHBs were thoroughly rinsed with PBS by vortex. Afterward, PHBs were mixed with SERS nanotags under same conditions and washed again. Finally, SERS measurements were achieved using a Raman microscope equipped with 20 objective lens (NA = 0.4), and the wavelength of incident light was 785 nm. The laser power at the sample was 2.5 mw. Multiplexed analysis To demonstrate the quantitative and multiplex detection of biomarkers, a multiplex assay was performed with a single PHB and two kinds of SERS nanotags (CV and NBA). PHB was immobilized with anti-afp and anti-cea capture antibodies simultaneously (molar ratio, 1:1). CV- and NBA-SERS nanotags were modified with AFP and CEA detection antibodies (molar ratio, 1:1), respectively. First of all, the mixture of AFP and CEA was mixed with PHB for 1 h at 7 under shaking. Next, PHB was washed several times with PBS by vortex. Then, PHB and the mixture of SERS nanotags were S5

6 incubated under same conditions. Finally, Raman spectral measurement of PHB was measured using a Raman microscope with 785 nm excitation laser and equipped with 5 objective lens (NA = 0.12), and the laser power was set to 5 mw. Clinical samples analysis To test whether the as-proposed multiplex assay was suitable for the reliable detection of clinical samples, human serum samples from five clinical patients provided by Zhongda Hospital affiliated with Southeast University were examined, and their concentrations were also measured with electrochemiluminescent immunoassay (ECLIA) kits (Roche Diagnostics Ltd.) in Zhongda Hospital. Raman measurements were conducted by a Raman microscope equipped with 5 objective lens, and the wavelength and power of laser were set to 785 nm and 5 mw, respectively. Spectral deconvolution Data analysis was performed with the help of Origin 2016 analysis software (OriginLab Corp.). By weighted least-squares residuals method, S6 the experimental data were fitted as a linear combination of Raman spectra of individual analyte with addition of constant background. The Raman spectrum of the mixed SERS nanotags can be easily deconvoluted into their characteristic Raman shift of individual nanotag, which allow quantitative comparison of spectral components associated with specific target analyte. Electromagnetic simulations A commercial software (FDTD Solutions, Lumerical Inc.) was employed to FDTD simulation. Three Au@Ag NPs were modeled on hydrogel substrate with different distances of 0.2, 0., 0.4, 0.5 nm, and. The diameter of core is 60 nm, and the thicknesses of NBA monolayer and silver shell are 0.8 and 6 nm, respectively. A linearly polarized plane waves with the wavelengths of 52 and 785 nm incident along the z-axis were used as the excitation lasers, respectively. Surface area to volume ratios (SVR) calculation S6

7 The self-assembled silica colloidal crystal bead we used as the template of PHB is according to the principle of Face-Centered Cubic (FCC). The atomic packing factor of the FCC is 74 %. The radii of the silica nanoparticle in a CCB and the PHB are r = 118 nm and R = 100 μm, respectively. The effective volume ( V ( 1+2+ ) ) of the top three layers of the CCB and surface area ( S ( 1+2+ ) ) of the top three layers of the PHB are determined by the following formulas: V 1+2+ R 2 2 r 4 = 74% R (1) ( ) (12) 2 ( 1+2+) = 4 (2) S V 4 r r The effective volume ( V ( PHB ) ) of the whole PHB is determined by the following formula: 4 V PHB = 1 74% R () ( ) Therefore, the SVR of the top three layers of the PHB is calculated to be S / 1.9 ( 1+2+ ) V( PHB). The SVR of the no nonporous hydrogel bead (NHB) is determined by the following formula: SVR 4 = 4 R R 2 (NHB) (4) Where SVR (NHB) represents the SVR of the whole NHB, and is calculated to be 0.0, thus the ratio of the calculated SVR of the top three layers of the PHB and the NHB is about / 0.0. Figure S1. TEM images of AuNPs (a) and gold-silver core-shell SERS nanotag S7

8 (b). Figure S2. Raman spectra (black line) of both AFP and CEA concentration are 10 ng ml -1. Raman spectra deconvolute into the two distinctive multiple peaks. Red line represents Gauss Fit of the overall measured spectra (black line). Blue dotted lines stand for Gauss Fit of the characteristic Raman shifts of the measured spectra. Figure S. Raman spectra of obtained from the multiplex assays with a solution of CV-labelled anti-afp targeting AFP (a) and NBA-labelled anti-cea targeting CEA (b) among a mixture of AFP and CEA. The concentration of analyte was 20 ng ml -1, and the control samples (0 ng ml -1 ) were obtained in the absence of AFP (a) and CEA (b). (c) Statistical analysis chart of the assay. Ratio is the coefficient between the intensity of identification Raman shift in the presence (20 ng ml -1 ) and absence (0 ng ml -1 ) of analyte. Error bar is calculated from five repeats. S8

9 Table S1. Comparison of linear dynamic range and limit of detection between different methods. Literature Method AFP CEA Linear dynamic Limit of Linear dynamic Limit of Reference range detection range detection ACS Appl. Mater. Interfaces 201, 5, DPV ng ml pg ml -1 S Anal. Chem. 2016, 88, ECL pg ml pg ml -1 S8 Angew. Chem., Int. Ed. 2014, 5, PL ng ml pg ml -1 S Sens. Actuators, B 2015, 221, EIS ng ml -1 1 pg ml -1 S10 Biosens. Bioelectron. 2016, 79, DPV ng ml pg ml -1 S This work PHB + core-shell 10 pg ml μg.6 fg ml pg ml μg 1.9 fg ml -1 SERS nanotag ml -1 ml -1 Abbreviations: DPV, differential pulse voltammetry. ECL, electrochemiluminescence. PL, photoluminescence. EIS, electrochemical impedance spectroscopy. Table S2. Detection and recovery test of AFP and CEA in the human serum samples. Samples AFP concentration (ng ml -1 ) CEA concentration (ng ml -1 ) Referenced method Proposed method a Recovery (%) Referenced method Proposed method a Recovery (%) ± ± ± ± ± ± ± ± ± a The average value was calculated based on five repeats for each sample ± References (S1) Zhao, X. W.; Zhao, Y. J.; Hu, J.; Xu, M.; Zhao, W. J.; Gu, Z. Z. Sintering photonic beads for multiplex biosensing. J. Nanosci. Nanotechnol. 2010, 10 (1), (S2) Zhao, Y. J.; Zhao, X. W.; Hu, J.; Li, J.; Xu, W. Y.; Gu, Z. Z. Multiplex label-free detection of biomolecules with an imprinted suspension array. Angew. Chem. Int. Edit. 2009, 48 (40), (S) Mu, Z. D.; Zhao, X. W.; Huang, Y.; Lu, M.; Gu, Z. Z. Photonic crystal hydrogel enhanced plasmonic staining for multiplexed protein analysis. Small 2015, 11 (45), (S4) Liu, B.; Ni, H. B.; Zhang, D.; Wang, D. L.; Fu, D. G.; Chen, H. Y.; Gu, Z. Z.; Zhao, X. W. Ultrasensitive detection of protein with wide linear dynamic range based on core-shell SERS nanotags and photonic crystal beads. ACS Sens. 2017, 2 (7), S9

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