Peptide Microarray-Based Metal Enhanced Fluorescence Assay for

May 18, 2017 - Considering time and cost of the assay, MMP-3, MMP-7, and MMP-14 are activated for 2 h (as shown in Figure S8). .... It was performed b...
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Peptide Microarray-based Metal Enhanced Fluorescence Assay for Multiple Profiling Matrix Metalloproteinases Activities Zhen Lei, Hua Zhang, Yaoqi Wang, Xianying Meng, and Zhenxin Wang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 18 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

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Analytical Chemistry

Peptide Microarray-based Metal Enhanced Fluorescence Assay for Multiple Profiling Matrix Metalloproteinases Activities Zhen Lei†,§, Hua Zhang†, Yaoqi Wang‡, Xianying Meng*‡ and Zhenxin Wang*† †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China. ‡ Department of Thyroid Surgery, the First Hospital of Jilin University, Changchun 130021, P. R. China. § University of Chinese Academy of Sciences, Beijing 100049, P. R. China. *Corresponding Author: Fax/Tel: +86 431 85262243 E-mail:[email protected] E-mail: [email protected] ABSTRACT: Matrix metalloproteinases (MMPs) are closely associated with cancer cell invasion and metastasis. Herein, a fluorescence resonance energy transfer (FRET)-peptide microarray-based metal enhanced fluorescence (MEF) assay is proposed for multiple and sensitive profiling MMPs activities on a novel Au/Ag@SiO2 substrate. The Au/Ag@SiO2 substrate is prepared by electroless deposition of silver on gold nanoparticle (GNP) seeds, followed by SiO2 shell coating and surface functionalization. The specific FRET peptides are spotted on the Au/Ag@SiO2 substrate to sensitively detect MMPs (MMP-2, 3, 7, 9, 14) via fluorescence recovery by the MMP cleavage of quenched peptide motifs, and further enhanced by MEF. Under the optimal conditions, the limits of detection are 12.2 fg mL-1 for MMP-2, 60 pg mL-1 for MMP-3, 0.22 pg mL-1 for MMP-7, 102 fg mL-1 for MMP-9 and 0.68 ng mL-1 for MMP-14, respectively. The practicability of the FRET-peptide microarray-based MEF assay is demonstrated by profiling multiplexed MMPs activities of various cell lines and clinical thyroid tissue samples of papillary thyroid carcinoma (PTC) patients and thyroid nodules (TN) patients, and satisfactory results are obtained.

Matrix metalloproteinases (MMPs), with Zn2+ as cofactor, are a family of endopeptidases capable of degrading virtually various kinds of extracellular matrix proteins,1 and their expression and activation have been found to be closely related with multiple kinds of physiological and pathological processes including embryonic development, inflammation and arthritis, especially tumor invasion and metastasis.1-4 A variety of MMPs are overexpressed in various human cancers, such as breast cancer, colorectal cancer, and cervical cancer, and are considered as valuable prognostic factors.4 The sensitive and accurate detection of MMPs is of significant importance for understanding disease mechanism, clinical diagnosis and therapy of cancer at its early stage. For instance, the serum levels of MMP-2 and MMP-9 are significantly higher in breast cancer patients than those in other breast diseases patients and in healthy controls.5 MMP-7 (matrilysin) is correlated with tumor differentiation and metastasis in colorectal cancer patients.6 Therefore, MMPs have the prospect of becoming very promising cancer biomarkers for early cancer diagnosis and prognosis.7 Up to now, several approaches have been developed to detect MMPs which mainly involve enzyme-linked immunosorbent assays (ELISA),8 gelatinase zymography,9 fluorescence resonance energy transfer (FRET) assays,10 surface plasmon resonance (SPR) assays11 and electrochemical biosensors.12 Although these assays have reasonable sensitivity and accuracy, most of them are focused on single protease detection. Recently, barcode-free combinatorial screening platform and droplet-based microfluidics have been employed to simultaneously measure multiple MMPs activities.13,14 Be-

cause there are cross-reactions among FRET peptide substrates and MMPs, sophisticated mathematical techniques (e. g., proteolytic activity matrix analysis (PrAMA)) are normally used to analyze obtained results in these approaches. In order to improve the reliability of results, the mathematical techniques require a mass of data about individual purified MMP cleavage signatures against a huge number of peptide substrates, resulting in extremely costly and time-consuming.15 As an important high-throughput tool, peptide microarray can be the alternative way to simultaneously profile multiple enzymes activities in complex sample.16,17 Fluorescence method is the most commonly used for determining protease activity in the peptide microarray-based assays.17 Several groups demonstrate that the sensitivities of DNA microarray-based fluorescence assays can be improved significantly by increasing the densities of capture probes through employing three-dimensional substrates,18 or enhancing the fluorescence signal through surface enhancement strategies.19 However, oversaturation of probe density on peptide microarray normally leads to decrease the reaction efficiency of enzyme because of steric hindrance effect. Alternatively, surface fluorescence enhancement methods are considered as attractive approaches for increasing the sensitivities of fluorescence-based peptide microarray. Metal-enhanced fluorescence (MEF) can be efficiently achieved by the interactions of localized surface plasmon resonance (LSPR) of metallic (e. g., Ag or Au) nanostructures with the fluorophores on their surfaces.19-23 Dai’s group has demonstrated that the near-infrared fluorescence signal can be enhanced up to 100-fold on the fabricated plasmonic gold substrate-based protein microarray.22 Geddes’s group found

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that the emission intensity of fluorescein-labeled human serum albumin (FITC-HSA) on fractal silver nanostructures was enhanced about 10-fold than the signal from FITC-HSA on unsilvered glass.23 However, because of relative poor chemical stability of silver nanostructures, a protector and/or spacer is required while the silver nanostructure film is used as MEF substrate.24 As a optically transparent and chemically inert material, silica (SiO2) can act as the coating shell on silver nanostructure to fulfill the requirement by traditional Stӧber method.24 Herein, we proposed a FRET-peptide microarray-based MEF assay for multiple and sensitive profiling MMPs activities on a novel Au/Ag@SiO2 substrate. Five specific FRET peptides are spotted on the Au/Ag@SiO2 substrates for sensitive and specific detection of MMPs (MMP-2, 3, 7, 9, 14) via fluorescence recovery by the MMP cleavage of quenched peptide motifs, and further enhanced by MEF. The utility of the assay is further explored to profile patterns of MMPs activities in different cell lines and thyroid tissues.

EXPERIMENTAL SECTION Reagents Tetrachloroaurate (HAuCl4), silver enhancer solution A and B, tetraethyl orthosilicate (TEOS, 98%), glutaraldehyde solution (GA, 25% in H2O), α-Chymotrypsin, phenylmethanesulfonyl fluoride (PMSF, ≥98.5%) and (2aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, ≥97%) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Ammonium hydroxide (28% NH3) was obtained from Alfa Aesar Chemicals Co., Ltd. (Shanghai, China). (3-Aminopropyl)triethoxysilane (APTES, 98%) was received from Aladdin Co., Ltd. (Shanghai, China). Human MMP-2, MMP-3, MMP-9 and Trypsin-3 protein were purchased from Sino Biological Inc. (Beijing, China), and recombinant human MMP-7 and MMP-14 were obtained from R&D Systems Inc. (Minneapolis, USA). 4-Aminophenyl mercuric acetate (APMA) was acquired from GenMed Medical Science and Technology Ltd. (Shanghai, China). The FRET peptide substrate 2 and 9 were synthesized by Synpeptide Co., Ltd. (Shanghai, China), substrate 3, 7 and 14 were synthesized by Lifetein (Beijing, China), and the details about the peptides were shown in Table S1 in Supporting Information. Gelatinsepharose 4B was supplied by Solarbio Science & Technology Co., Ltd. (Beijing, China). The microscope slides and aldehyde group-modified glass slides were purchased from CapitalBio Ltd. (Beijing, China). Other chemicals were all of analytical grade, and Milli-Q water (18.2 MΩ cm) was used in all experiments. Characterization The scanning electron microscope (SEM) micrographs of as-prepared substrates were obtained by a XL30 ESEM FEG system (FEI, USA) at an accelerating voltage of 10 kV. Transmission electron microscopy (TEM) micrographs, fast-Fourier transform (FFT) and energy-dispersive X-ray spectroscopy (EDX) analysis of as-prepared nanostructures were performed on a JEM 2000FX (JEOL Ltd, Japan) microscope operated at an accelerating voltage of 120 kV. The as-prepared nanostructures were peeled off from the glass slide by ultrasonication. X-ray diffraction (XRD) patterns of as-prepared substrates were conducted on a D8 ADVANCE X-ray diffractometer (Bruker, Germany). Extinction spectra of the as-prepared substrates were obtained by a V-570 UV/VIS/NIR spectrophotometer (JASCO, Japan). X-ray pho-

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toelectron spectra (XPS) were conducted with a VG ESCALAB MKII spectrometer (VG Scientific Ltd., UK). Preparation of Au/Ag@SiO2 substrate 13 nm GNPs were synthesized by the traditional Turkevich–Frens method.25,26 The plain microscope glass slides were treated as previously described.27 In order to introduce amino groups on the slides surface, the hydroxylated slides were immersed in 10% (v/v) APTES ethanol solution for 4 h, washed by 30 mL ethanol (3 times) and dried by centrifugation (300 g). Subsequently, the aminated slides were incubated with 1 nM GNPs at 30 °C for 6 h, rinsed with 30 mL water (3 times) and dried by centrifugation (300 g). Then, silver enhancer solution A and B were mixed well with the volume ratio of 1:1, and applied to the GNPs modified slides for 5 min followed by washing with 30 mL water (3 times) and drying by centrifugation (300 g). The SiO2 shells were grown on the GNP@Ag core@shell nanoparticles (termed as, GNP@Ag NPs) according to reported strategy with slight modification.28 Briefly, the GNP@Ag NPs modified slides (named as Au/Ag slides) were immersed into a homogeneous mixture containing 30 mL ethanol, 500 µL TEOS and 4 mL water under shaking for 20 min. 560 µL ammonium hydroxide (28%) was then added into the mixture and reacted for another 15 min, washed with 30 mL water (3 times) and dried by centrifugation (300 g). The slides were silanized with 5% (v/v) APTES ethanol solution for 10 h, washed with 30 mL ethanol (3 times) and 30 mL water (3 times). Finally, the aminated slides were incubated with 5% (w/v) GA in PBS (pH 7.5, 50 mM PB, 0.15 M NaCl) for 4 h, washed with 30 mL water (5 times), dried by centrifugation (300 g) and stored at desiccator. The final product was termed as Au/Ag@SiO2 substrate. Fabrication of peptide microarray and detection of MMPs activities Peptides were spotted on the Au/Ag@SiO2 substrates, commercial aldehyde group modified glass slides and SiO2 substrates by a SmartArrayer 136 system using a standard contact printing procedure according to our previous report with slight modifications (see the Supporting Information for details).27 The various concentrations of activated pure MMPs and cell-secreted MMPs were subjected to the asfabricated peptide microarrays for evaluating the activities of MMPs (see the Supporting Information for details). Extracting MMPs from clinical thyroid tissue samples All the clinical thyroid tissue samples were obtained from First Hospital of Jilin University (Changchun, China). Approval was obtained from the Local Research Ethics Committee and informed consents for tissue sampling were obtained from all subjects. 19 thyroid tissues of papillary thyroid carcinoma (PTC) patients and 6 thyroid tissues of thyroid nodule (TN) patients were collected by standard surgical procedures (see Table S2 in Supporting Information for details). The extraction and purification of MMPs from the samples were performed according to the Zhang’s work.29 The tissues were cut into tiny pieces, and homogenized by a 1 mL glass homogenizer with 400 µL working buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl2, 0.05% (w/v) Brij-35 and 0.02% (w/v) NaN3) supplied with 1% (v/v) Triton X-100. Total protein content of 10 µL homogenate was measured by the bicinchoninic acid (BCA) method according to the manufacturer’s instructions (Bioteke, Beijing, China). The homogenate was centrifuged at 12000 rpm at 4 °C for 15 min, and the supernatant was reserved. Then, the supernatant was divided into 2 equal parts. One part of the supernatant was treated by gelatin-

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Analytical Chemistry

sepharose 4B to purify MMPs. (see the Supporting Information for details). The activities of MMPs in crude supernatant and in gelatin-sepharose 4B treated supernatant were profiled by the FRET peptide microarray-based assay as previously described. Fluorescence measurement and analysis The fluorescence images of microarrays were acquired by a LuxScan-10K fluorescence microarray scanner (CapitalBio Ltd., Beijing, China) with the green channel. Six replicate spots per sample were used to calculate the mean values and standard deviations. The Z-factor (Z') and coefficient of variation (CV) were calculated according to the following equations: Z'=1−3×(σs+σb)/|µs−µb| and CV=σs/µs, where µs, µb and σs, σb are the mean values and standard deviations of sample and background, respectively. The relative fluorescence recovery (F/F0-1) is used to quantitatively analyze the MMP activities, where F and F0 are the mean fluorescence intensities of 6 spots on the same microarray treated with and without MMPs, respectively.

RESULTS AND DISCUSSION Preparation and characterization of Au/Ag@SiO2 substrate The overall procedure of Au/Ag@SiO2 substrate preparation is shown in Scheme 1. The 13 nm GNPs were firstly self-assembled on the APTES modified glass surface through the interaction of Au atom with amino group (i.e., formation of strong Au-N covalent bond). The silvers were then electroless deposited on the 13 nm GNP seeds and formed GNP@Ag NPs. Compared with conventional colloid-based method, this strategy can produce GNP@Ag NPs with more uniform size, better spatial homogeneity and higher surface coverage.19,23,30 A SiO2 thin layer was coated on the GNP@Ag NP to form GNP@Ag@SiO2 core@shell@shell nanostructure (named as, Au/Ag@SiO2 NP) on the slide surface by the hydrolysis of TEOS. Subsequently, APTES was used to functionalize the slide surface with amino groups. Finally, the amino group modified slide was activated by GA, an amine-reactive homobifunctional crosslinker. After GA activation, the slide (named as, Au/Ag@SiO2 substrate) is ready for microarray fabrication. Scheme 1. Preparation of Au/Ag@SiO2 substrate and the principle of FRET-peptide microarray-based MEF assay for multiple profiling MMPs activities.

MEF is closely related with the extinction properties of metal nanoparticles which depend on the type, size and morphology of the metal nanoparticle and the internanoparticle distance.21,30,31 In this case, the size and morphology of GNP@Ag NPs are directly affected by silver disposition time and GNP dispersity. The internanoparticle distance can be easily adjusted by the self-assembly time and the concentration of GNP seeds in the assembly solution. The 13 nm GNPs were arbitrarily selected as seeds because 13 nm GNPs pos-

sess uniform shape and monodispersity (as shown in the Figure S1). We found that the relative high MEF was achieved with 6 h of self-assembly time, 5 min of silver disposition time and 1 nM GNPs in solution (as shown in Figure S2). The successful stepwise surface reaction on the glass slide was confirmed by XPS analysis. The appearance of a nitrogen signal at 400.0 eV demonstrates that APTES has been attached on the glass slide (as shown in Figure S3a). After the selfassembly of GNPs seeds, Au 4f7/2 (83.5 eV) and Au 4f5/2 (87.2 eV) peaks are clearly observed (as shown in Figure S3b).32 An obvious Ag 3d core level spectrum is observed in Figure S3c. The two peaks at the binding energy of 368.1 and 374.1 eV are corresponded to Ag 3d5/2 and Ag 3d3/2, respectively, which are in good agreement with metallic silver (Ag0).33,34 As shown in Figure S3d, the surface contents of Si, C and O are distinctly increased while the surface content of Ag is decreased. The results indicate that SiO2 is coated on GNP@Ag NPs. The deconvolution of C1s spectrum of APTES functionalized slide shows three types of carbon bonds: C−C at 284.5 eV, C−O at 286.1 eV and C−N at 287.0 eV (as shown in Figure S3e). After activated by GA, the high-resolution C1s signal can be fitted with four peaks at 284.5 eV, 285.9 eV, 287.2 eV and 288.5 eV attributable to C−C, C=N, C−N and C=O, respectively (as shown in Figure S3f), demonstrating the successful modification of aldehyde group. The SEM micrographs of as-prepared Au/Ag@SiO2 substrate are shown in Figure 1a. The slide surface is covered by uniform nanoleaves (about 270 nm in long axis) which are composed of Ag nanospheres. The abundant nanogaps between the nanoleaves are correlated with local electrical field enhancement which contributes to the MEF.21 The typical TEM micrographs of GNP@Ag NPs before and after hydrolysis of TEOS are shown in Figure 1b and Figure 1c, a thin layer of SiO2 shell is conformally coated on the surface of GNP@Ag NPs. The thickness (ca., 8 nm) of the SiO2 shell is very close to the optimal distance (ca., 9 nm) between the fluorophore and nanoparticle surface for MEF.35,36 The thin SiO2 shell can provide three distinct advantages in the following experiments, (1) it can provide a precise distance control between the fluorophore and Ag nanoparticles surface; (2) it acts as a protective layer to prevent the oxidization of Ag, resulting in improved stability of GNP@Ag NPs; and (3) it serves as a platform to produce versatile surfaces for easily conjugating biomolecules through chemical reaction with various available silane coupling agents. The GNP@Ag NPs have a lattice spacing value of 0.240 nm which is attributed to (111) plane of the Ag (as shown in Figure 1d). The result is also confirmed by the well-aligned hexagonal spots in the corresponding FFT of HRTEM micrograph of GNP@Ag NPs (as shown in inset of Figure 1d). The elemental mapping and EDX analysis indicate that Ag, Au, Si, C and N elements are homogeneously distributed throughout the GNP@Ag NPs (as shown in Figure S4). Both GNP@Ag NPs and Au/Ag@SiO2 NPs have same characteristic peaks at 2θ degrees of 38.17, 44.47, 64.68, 77.42 and 81.6° (as shown in Figure 1e), which are assigned to (111), (200), (220), (311) and (222) planes of the face-centered cubic Ag.37 The strongest diffraction peak of (111) face is in good agreement with the result of HRTEM study. The extinction spectrum of Au/Ag@SiO2 substrate shows two distinct peaks at 441 nm and 548 nm which are corresponded to the absorption and scattering peaks of the GNP@Ag NPs, respectively. (as shown in Figure 1f). 30,38

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Figure 1. (a) SEM micrographs of the Au/Ag@SiO2 substrate, the inset is a higher magnification of the substrate; TEM micrographs of the GNP@Ag NPs (b) and Au/Ag@SiO2 NPs (c); (d) HRTEM micrograph of as-prepared Au/Ag@SiO2 NPs, the inset is the corresponding FFT image; (e) XRD patterns of the GNP@Ag NPs modified slide before and after SiO2 shell coating; (f) extinction spectrum of the asprepared Au/Ag@SiO2 substrate.

Performance of the as-prepared Au/Ag@SiO2 substrate To evaluate its MEF performance, Cy3 or FITC modified peptide microarrays were fabricated on the Au/Ag@SiO2 substrate and commercial aldehyde group-modified glass slide, respectively. As shown in Figure 2, because the Au/Ag@SiO2 substrate has relatively rough surface, the spots of Au/Ag@SiO2 substrate-based peptide microarray are much larger in diameter than the spots of glass slide-based microarray.39 The normalized excitation and emission spectra of FITC and Cy3 are well overlapped with the extinction spectrum of the Au/Ag@SiO2 substrate (as shown in Figure S5a). The phenomenon can generate highly efficient MEF since MEF is determined by the spectral overlap between the fluorophore excitation/emission and plasmonic resonance of the nanoparticle.40 As expected, the fluorescence intensities of Cy3 and FITC immobilized on the Au/Ag@SiO2 substrate are as 7-fold and 5-fold high as those on the glass slide. However, the observed enhancement of fluorescence intensities may also be due to the increased surface area. We estimated the surface area of the Au/Ag@SiO2 substrate by literature reported method.22 The surface area of the Au/Ag@SiO2 substrate is about 2.04-fold larger than that of glass slide, which is consistent with literature reported value (1.9-fold).30 We also prepared a SiO2 substrate with silica nanoparticles of 250 nm in diameter (as shown in Figure S6). Compared with glass slide, the SiO2 substrate exhibits 62% increase in surface area. After the same spotting and washing steps, the fluorescence intensities of Cy3 and FITC on the SiO2 substrate are about 1.95-fold and 1.86fold higher than those on the glass slide, respectively. In addition, as shown in Figure S7, the fluorescence intensities of Cy3 and FITC on the Au/Ag@SiO2 substrate are about 5.6-fold and 3.5-fold higher than those on the glass slide, respectively, while the microarrays were scanned just after spotting without further washing treatment. These results suggest that the ob-

served increased fluorescence intensity on Au/Ag@SiO2 substrate is the combination of increased surface area and MEF. In particular, MEF plays a main role (c.a., 80%) in the enhancement of fluorescence signal. Furthermore, for large metal nanoparticles, the contribution of scattering component in the fluorescence enhancement is much more than that of absorption.30 The excitation/emission spectra of Cy3 are better overlapped with scattering peak of the GNP@Ag NPs than those of FITC, which may result in higher fluorescence enhancement of Cy3 than that of FITC. In addition, 500 identical spots were analyzed on a single Au/Ag@SiO2 substrate-based peptide microarray, and Z-factor (Z') and coefficient of variation (CV) were calculated (as shown in Figure S5b). The relative low CV (4.92%) and high Z' (0.75) suggest uniform nanoparticle coverage at the macroscopic scale and good quality of the Au/Ag@SiO2 substrate-based peptide microarray.

Figure 2. The fluorescence intensities and microarray images of Cy3 (a) and FITC (b) modified peptide on the Au/Ag@SiO2 substrate and aldehyde modified glass slide after washing.

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FRET-based MEF assay of MMPs activities A FRETpeptide microarray-based MEF assay for determining MMPs activities is developed on the Au/Ag@SiO2 substrate. Five MMPs are studied by the assay, including secreted extracellular MMPs (MMP-2, MMP-9, MMP-3 and MMP-7) and membrane-tethered MMP (MMP-14), which are widely expressed in the tumor tissues. For instance, the roles of MMP-2, MMP9, MMP-7 and MMP-3 in tumor invasive and metastasis have been well demonstrated, while MMP-3 is also involved in the pathogenesis and progression of the osteoarthritis (OA) and rheumatoid arthritis (RA).41,42 MMP-14 not only participates in extracellular MMPs (EC-MMPs) activations and multiple signaling pathways, but also plays crucial role in cancer cell 3D invasion.43 Therefore, it is important to develop a method for simultaneous detection of these five MMPs with high selectivity and sensitivity. Five FRET-peptide substrates containing the specific recognition sequences of MMPs with FAM as fluorophore and Dabcyl as quencher group were carefully designed (as shown in Table S1). Considering the synthetic difficulty and cost of FRET peptides, the fluorophore/quencher pair, FAM/Dabcyl is selected to prepare the FRET peptide substrates since the FAM/Dabcyl pair is extensively employed in the peptide synthesis, and the characteristic excitation/emission peaks of FAM (Ex/Em=495/517 nm) are almost the same as those of FITC (Ex/Em=495/519 nm). The FRET peptide substrates consist of 15 or 16 amino acid residues with specific recognition sequences of MMPs flanked by the fluorophore on the N terminus and quencher on the C terminus. The three arginines at the N terminus are used for increasing the hydrophilicity of peptides. The cysteine serves as an anchor site for covalent immobilization of FRET peptides on the Au/Ag@SiO2 substrate. Upon cleavage by MMPs, the peptide fragments carrying Dabcyl moieties are released from the slide surface, and the fluorescence of FAM is recovered and further enhanced by Au/Ag@SiO2. In order to obtain good assay performance, the reaction conditions are optimized including the concentration of FRET-peptide in the spotting solution, the activation time of MMPs, and the cleavage time of MMPs with peptides. Considering time and cost of assay, MMP-3, MMP-7 and MMP14 are activated for 2 h (as shown in Figure S8). The activation times for MMP-2 and MMP-9 are 1 h which have been optimized in our previous study.27 The cleavage times are 2 h for four EC-MMPs and 4 h for MMP-14, respectively. The concentrations of FRET-peptide in spotting buffer are 0.4 mg mL-1 for MMP-9, 1.0 mg mL-1 for MMP-2, 0.5 mg mL-1 for MMP-3, and 0.3 mg mL-1 for MMP-7 and MMP-14, respectively. As expected, the relative fluorescence recoveries (F/F01) are proportional to the logarithm of MMPs concentrations on the Au/Ag@SiO2 substrate-based peptide microarray (as shown in Figure 3a-e). Under the optimal conditions, the assay performances including detection limits and dynamic ranges are summarized in Table 1, which are much better than those of literature reports.44-47 In addition, the assays were also conducted on commercial aldehyde slides. As shown in Figure S9, under the optimal conditions, the relative fluorescence recoveries (F/F0-1) on Au/Ag@SiO2 substrate are up to 5 times higher than those on commercial aldehyde slide. Furthermore, as displayed in Table S3, the assays on Au/Ag@SiO2 substrates exhibit lower detection limits and broader linear ranges than on commercial aldehyde slides. Taking MMP-2 as an example, the assay was also performed on the SiO2 substrates. As shown in Figure S10, the

Figure 3. (a-e) Calibration curves of the relative fluorescence recovery (F/F0-1) as a function of the logarithm of MMPs concentration, the insets are corresponding fluorescence images of microarrays. (f) Individual pure MMP cleavage signatures against the five FRET-peptide substrates.

detection limit is 1 pg mL-1, which is much higher than that on Au/Ag@SiO2 substrate. Besides, the relative fluorescence recoveries on Au/Ag@SiO2 substrate are also about 5-fold of that on glass slide under same experimental conditions, further demonstrating the MEF of the Au/Ag@SiO2 substrate. Table 1. Summary of the limits of detection (LOD) and linear ranges of the proposed FRET-peptide microarraybased MEF assay. Limit of detection -1

MMP-2

12.2 fg mL

MMP-3

60 pg mL-1

Linear range 0.1 pg mL-1−100 ng mL-1 0.1 ng mL-1−500 ng mL-1

-1

10 pg mL-1−100 ng mL-1

MMP-7

0.22 pg mL

MMP-9

-1

102 fg mL

1 pg mL-1−100 ng mL-1

MMP-14

0.68 ng mL-1

1 ng mL-1−250 ng mL-1

Specificity is critical for accurate MMPs detection in complex sample since MMPs exhibit close homology and have overlapping cleavage preferences for substrates. The five FRET-peptide substrates for specific MMPs were carefully selected (as shown in Table S1), and the individual pure MMP cleavage signatures against the five FRET-peptide substrates are depicted in Figure 3f. The set of selected FRET-peptide substrates display a trend toward diagonal dominance with much lower off-diagonal signal, indicating fairly high specificities of the substrates. Therefore, the proposed FRET-peptide microarray-based MEF assay can be employed to determine the activities of MMPs in complex samples. Profiling cell-secreted MMPs activities For testing its capability, the FRET-peptide microarray-based MEF assay is employed to determine the activities of cancer cell-secreted EC-MMPs in conditioned medium. As a membrane-tethered MMP, MMP-14 can’t be secreted into culture media, so only

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four EC-MMPs were detected by four FRET peptide substrates. Seven different cell lines including three high invasive cells (HT-1080, SW620 and MDA-MB-231) and four low or non-invasive cells (SW480, HT-29, MCF-7 and HeLa) are selected. All the cells were seeded in 48-well plate with various cell densities, and the MMPs activities in culture medium were directly detected. As shown in Figure 4, although the MMPs activities exhibit significant diversity among the cell lines, strong correlation between proteolytic activity and cell density is observed. The activities of MMPs secreted by sparsely plated cells are higher than the densely cultured ones. The activities of EC-MMPs reach the maximum when cells are plated at about 5000 cells/well, and gradually decreased with the decreasing cell density and/or increasing cell density. The results are well consistent with previous reports, i.e., the expression levels of major MMPs are higher in the less densely growing cells.48,49 In addition, the activities of EC-MMPs can be detected from cells seeded as low as 50 cells/well, further indicating that the FRET-peptide microarray-based MEF assay has high sensitivity.

Figure 4. (a-g) Proteolytic activity profiles of MMPs secreted by different cell lines plated at various densities: (a) SW620, (b) HT29, (c) SW480, (d) MDA-MB-231, (e) MCF-7, (f) HT-1080 and (g) HeLa. The cell densities are (1) 3×104, (2) 1×104, (3) 5000, (4) 1000, (5) 500, (6) 100 and (7) 50 cells/well. (h) Hierarchical clustering analysis (HCA) of activities of MMPs secreted by different cell lines plated at 5000 cells/well.

The levels of MMPs activities are closely correlate with the biological behaviours of cancer cell lines, i.e. the high malignant cells, SW620, MDA-MB-231 and HT-1080 (Figure 4a, d and f) exhibit higher MMPs activities than those of low malignant cells, HT-29, SW480, MCF-7 and HeLa (Figure 4b, c, e and g). The cell-secreted EC-MMPs activities of three colorectal cancer cell lines are followed in the order, SW620 > HT-29 > SW480, which is agree with their metastasis abilities.50 With regard to breast cancer cell lines, higher invasive MDA-MB231 cells show higher activities of four EC-MMPs, especially MMP-2, MMP-3 and MMP-7, which is in good agreement with the previous study (Figure 4d and Figure 4e).51 In addition, the MMPs activities patterns vary with the different cell types. As shown in Figure 4a, b and c, the colorectal cancer cell lines show relative high MMP-7 activity, which is consistent with that MMP-7 is expressed abundantly in colon cancer cells.52 The colorectal cancer cells also have co-expression of MMP-3 and MMP-9, since activated MMP-3 is activator of proMMP-9.53 The secreted MMPs (especially MMP-2 and MMP-9) activities of HT-1080 cell, a typical MMPsoverexpressing cell line, are higher than those of other cells (as shown in Figure 4f). For HeLa cells, the activities of MMP-2, 3 and 7 are lower than activity of MMP-9 (Figure

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4g). The results are consistent with the report in literature.54 The result demonstrates that the FRET-peptide microarraybased MEF assay can be used to detect MMPs activities in complex samples. With the ability to determine multiplexed proteolytic activities, different types of cancer cells could be classified into new cell populations by the similarity of their MMPs expression characteristics. Hierarchical clustering analysis (HCA) is performed based on the dataset of four EC-MMPs activities of all the cell lines seeded at 5000 cells/well. It was performed by SPSS Statistics 22 software using Euclidian distances and Between-Groups Linkage, and a dendrogram with welldefined cluster groups is produced (Figure 4h). The HCA diagram obviously categorizes the cells into two distinct groups of cells with high MMPs expression and the relatively lower expressed ones. The proteolytic activities of MMPs are closely related with tumor migration and invasion, so the metastasis potential of the cells can be reflected by the dendrogram. For instance, the high invasive cell lines, SW620, MDA-MB-231 and HT-1080 exhibit high MMPs activities, while HeLa, HT29, MCF-7 and SW480, the cell lines with relatively low metastasis potential, show low MMPs activities.48,50 MMPs activities analysis from thyroid tissue For addressing its practicability, the FRET-peptide microarray-based MEF assay is further explored to detect the MMPs activities of clinical thyroid tissues. In this case, the MMPs activities of both crude samples and gelatin-sepharose 4B treated samples (termed as, purified samples) are evaluated. The MMPs activities can be detected as low as 1 mg mL-1 total proteins in the MMP assay buffer. Given a reaction solution volume of 30 µL, this means that only 0.5 mg thyroid tissues can meet the requirement of single test, which is beneficial for the clinical examination. As shown in Figure 5, the crude samples and purified samples have same patterns of MMPs activities. The phenomenon suggests that the FRET-peptide microarraybased MEF assay is a robust method. The MMPs activities of purified samples are much higher than those of crude samples since gelatin-sepharose 4B can effectively separate and concentrate mammalian MMPs from other tissue proteins including the inhibitors of MMPs.55 The MMPs expression patterns of PTC samples are strikingly different from those of TN samples. Generally, the MMP-2, MMP-3, MMP-9 and MMP-14 activities of PTC samples are higher than those of TN samples. It has demonstrated that overexpression and activation of MMP-2 and MMP-9 in thyroid carcinomas is strongly associated with malignant behavior and lymph node metastasis of thyroid carcinomas.55,56 The activities of MMP-3 and MMP-14 are also increased in PTC samples since activated MMP-3 and MMP-14 are involved in the activation of pro-MMP-9 and pro-MMP-2, respectively.7,53 The patterns of MMPs activities are consistent with the pathologic analysis of PTC stages. For example, the samples (sample 1-5, 10, 14 and 19) from PTC patients with vessel/neural invasion and lymph node metastasis (LNM) exhibit much higher MMPs activities than the samples from patients with carcinoma in situ. In addition, the sample (sample 15) from paracancerous tissue of patient with advanced-stage PTC (i.e., PTC patient with severe LNM and vessel invasion, sample 14) exhibits high MMPs activities, while the pericarcinous tissue (sample 18) of patient with carcinoma in situ (sample 17) shows low MMPs activities. It further confirms that the microarray-based assay could be used as an effective approach for high-throughput profile of MMPs activities in clinical samples.

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ACKNOWLEDGMENT The authors would like to thank the National Natural Science Foundation of China (Grant no. 21475126 and YZ201561) for financial support.

REFERENCES

Figure 5. MMPs activities profiling of thyroid tissues from patients with PTC (1-19) and TN (20-25). MMPs activities measured from crude samples (a) and gelatin-sepharose 4B treated samples (b).

CONCLUSION In summary, we have developed a robust FRET-peptide microarray-based MEF assay for profiling the activities of MMPs in native biological samples through immobilization of FRETpeptide on a novel Au/Ag@SiO2 substrate. The results of multiplexed MMPs (MMP-2, 3, 7, 9, 14) activities detection indicate that our method has higher sensitivity and wider dynamic range than traditional/commercial MMP assays or previously reported approaches. The utility of the FRET-peptide microarray-based MEF assay is demonstrated by profiling MMPs activities patterns of different cell lines and thyroid tissues. Using PTC as a typical model, the relationship between MMPs activities pattern and progression of PTC is evaluated, and the obtained result is comparable with the result of pathologic analysis. In the light of its throughput, simplicity and sensitivity, the FRET-peptide microarray-based MEF assay provides an accessible tool for the simultaneous evaluation of multiplexed MMPs activities in clinical samples for human tumor diagnosis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional experimental section, details of the FRET peptide substrate sequences and clinical thyroid tissue samples, comparison of the assay performance, characterization figures including TEM and UV-visible spectrum of GNPs, XPS of stepwise modified glass slides, TEM mapping and EDX analysis of the as-prepared Au/Ag@SiO2 nanostructures, normalized spectrum and fluorescence intensities of Cy3 of 500 spots, the assay performance on the SiO2 substrate, condition optimization during preparation of Au/Ag@SiO2 substrate and MMPs detection, comparison between the Au/Ag@SiO2 substrate, commercial aldehyde groupmodified glass slide and SiO2 substrate. (PDF)

AUTHOR INFORMATION Corresponding Author *E-mails: [email protected]. Tel.: +86 431 85262243.

ORCID Zhenxin Wang: 0000-0002-1908-9848

Author Contributions All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

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