Rational Engineering a Multichannel Upconversion Sensor for

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Rational Engineering a Multichannel Upconversion Sensor for Multiplex Detection of Matrix Metalloproteinases Activities Sunan Cao, Zhi Li, Jinyuan Zhao, Mi Chen, and Nan Ma ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00320 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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Rational Engineering a Multichannel Upconversion Sensor for Multiplex Detection of Matrix Metalloproteinases Activities Sunan Cao, Zhi Li, Jinyuan Zhao, Mi Chen, Nan Ma*

The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, P. R. China

Abstract Optical sensing of cancer-relevant protease is of great value for cancer diagnostics, prognosis, and drug discovery. Multiplex sensing is known to improve predicative accuracy, yet remains challenging because of severe fluorescence signal crosstalk in a single assay. Herein, we developed a multichannel optical sensor based on upconversion nanoparticles (UCNPs) for multiplex ratiometric sensing of proteolytic activities of two matrix metalloproteinases (MMP-2 and MMP-7). To this end, we rationally designed a NaYF4:Gd3+/Yb3+@NaYF4:Yb3+/Tm3+/Er3+ core-shell structure that favors multicolor narrow-band emission of both Tm3+ and Er3+ dopants and efficient luminescence resonance energy transfer (LRET) between the dopants in the shell and the fluorophores on particle surface. The sensor was constructed via a facile phase transfer protocol using two polyhistidine-containing peptides conjugated with different fluorophores (FITC and TAMRA) as co-ligands. The blue emission and green emission could be specifically activated by MMP-7 and MMP-2 respectively upon peptide cleavage, and the red emission could serve as an internal reference for ratiometric sensing. The sensor exhibits high specificity and sensitivity towards both targets with little signal crosstalk and crossreactivity. It could potentially serve as a general platform for multiplex detection of various types of proteases.

Keywords: matrix metalloproteinases, multiplex detection, upconversion, sensor, peptide, phase transfer

Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases that participate in extracellular matrix (ECM) degradation, play important roles in vital biological process such as cell migration, embryogenesis, and tissue remodeling.1-3 Upregulated expression levels and activities of

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MMPs are closely associated with cancer progression including angiogenesis, invasion, and metastasis.4-6 Sensitive measurement of MMPs activities is of great value for cancer diagnostics, prognosis, and MMP inhibitor screening.7,8 Members of MMPs exert different functions in cancer development and in many cases act cooperatively to determine a disease state.7 Therefore, it is necessary to measure the activities of different MMPs simultaneously in order to improve the diagnostics and prognosis accuracy. Resonance energy transfer (RET)-based detection is a valuable approach for direct measurement of enzymatic activity via fluorescence readout.9-15 Great efforts have been made to detect single protease using organic fluorophores or inorganic nanoparticles as reporters.9 However, multiplex detection of proteases activities remains less accomplished. The relatively broad emission spectra of conventional fluorescent reporters usually lead to spectra crosstalk that requires further deconvolution for data analysis.16,17 Integrating different optical channels in a single sensor for multiplex ratiometric sensing remains undeveloped.

Lanthanide-doped upconversion nanoparticles (UCNPs) hold great promise for biomedical application because of their unique optical properties including sharp emission bandwidths (FWHM = 5-15 nm), dopant-dependent multicolor emission, and minimal autofluorescence background under near-infrared (NIR) excitation.18-21 UCNPs have been utilized for luminescence resonance energy transfer (LRET)-based detection of single bioanalyte.22-27 However, multiplex detection of proteases using multicolor UCNPs has not been previously demonstrated. It has been suggested that co-doping of Tm3+ and Er3+ in a homogenous structure leads to either substantial quenching of the blue emission of Tm3+ due to cross-relaxation between Er3+ and Tm3+ at elevated dopant concentrations or insufficient luminescence at low dopant concentrations.28 This issue has been partially resolved by spatially isolating Tm3+ and Er3+ in core@shell structures (e.g. NaYF4:Yb3+/Er3+@NaYF4:Yb3+/Tm3+ and NaYF4:Yb3+/Tm3+@NaYF4:Yb3+/Er3+).29 However, these structures are incompetent for LRET-based multiplex detection because LRET efficiency significantly drops above a separation distance of 10 nm for inner core dopants.22

Results and discussions Herein, we present a strategy for rationally engineering a LRET-based multichannel UCNP sensor for simultaneous detection of MMP-2 and MMP-7 activities. To resolve the above challenges, we propose a NaYF4:Gd3+/Yb3+@NaYF4:Yb3+/Tm3+/Er3+ core-shell UCNP structure to achieve efficient


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Scheme 1. Schematic illustration of the UCNP sensor for multiplex detection of MMPs activities. (a) Schematic illustration of the NaYF4:Gd3+/Yb3+@NaYF4:Yb3+/Tm3+/Er3+ core-shell structure; (b) proposed energy transfer mechanisms in the core-shell UCNPs; (c) schematic illustration of LRET-based sensing of MMP-2 and MMP-7 activities.

multicolor (blue, green, red) emission of Tm3+ and Er3+ dopants while minimizing the separation distance between the dopants and the surface-functionalized energy acceptors. The NaYF4:Gd3+/Yb3+ core contains a high concentration of Yb3+ sensitizer (60%) to enhance light harvesting and suppress Yb3+-defect energy transfer, thereby significantly improve the upconversion luminescence efficiency.30 Also, a certain percentage of Gd3+ is incorporated into the core structure to control the core size and maintain its crystal phase.31 Tm3+ and Er3+ are co-doped into the shell layer at low concentrations (0.5% and 0.2%) to minimize cross-relaxation, and the core activator promotes efficient luminescence of both dopants (Scheme 1a and 1b). Two peptides, which contain the substrate sequences of MMP-7 and MMP-2, are conjugated to FITC and TAMRA respectively and self-assembled on the surface of the UCNPs through polyhistidine tails via a facile phase transfer protocol.33 In this context the blue emission of Tm3+ and the green emission of Er3+ are efficiently quenched by FITC and TAMRA


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Figure 1. Characterization of NaYF4:Gd3+/Yb3+ core and NaYF4:Gd3+/Yb3+@NaYF4:Yb3+/Tm3+/Er3+ core-shell nanoparticles. (a) TEM images of NaYF4:Gd3+/Yb3+ core and NaYF4:Gd3+/Yb3+@NaYF4:Yb3+/Tm3+/Er3+ coreshell nanoparticles prepared with different percentages of Gd3+ (0-30%). Scale bars in all the images are 100 nm; (b) luminescence spectra of NaYF4:Gd3+/Yb3+@NaYF4:Yb3+/Tm3+/Er3+ core-shell nanoparticles prepared with different percentages of Gd3+ (10-30%).

respectively through LRET (Scheme 1c). Upon MMP treatment, the peptide substrate is specifically cleaved, leading to dissociation of the quencher and recovery of the corresponding emission peak. Meanwhile, the red emission of Er3+ remains unchanged and serves as an internal reference for ratiometric sensing. The multicolor-emitting core-shell UCNPs were prepared via two steps. The NaYF4:Yb3+/Gd3+ core containing 60% Yb3+ and various percentages of Gd3+ (0%-30%) was synthesized via a solvothermal method. Incorporation of Gd3+ into the core has a pronounced effect on the size of nanoparticles. As


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shown in transmission electron microscopy (TEM) images (Figure 1a, top panel), hexagonal-shaped near-monodisperse nanoparticles with different sizes were produced under various Gd3+ concentrations. The nanoparticle size gradually decreases from 90 nm to 18 nm as the percentage of Gd3+ increases from 0% to 30%. It is known that the Gd3+ ion in the crystal could increase the electron charge density on the surface and thereby slow the diffusion of F- ions to the surface, leading to decrease of NaYF4 nanocrystal

size.31

Next,

successive

epitaxial

growth

of

a

layer

of

NaYF4:0.2%Er3+/0.5%Tm3+/30%Yb3+ on the core was conducted. TEM images show that the large core (0%-20% Gd3+) yields heterogeneous products including a population of small particles whereas the medium and small core (21.5%-30% Gd3+) leads to homogeneous core-shell nanoparticles. The upconversion luminescence of each product was further characterized. Strong emission of both Tm3+ (1D2 – 3F4 450 nm, 1G4 – 3H6 475 nm) and Er3+ (4S3/2 – 4I15/2 541 nm, 4F9/2 – 4I15/2 655 nm) were detected for larger particles (10% Gd3+, 20% Gd3+, 21.5% Gd3+). The upconversion luminescence gradually diminishes for small particles when Gd3+ concentration exceeds 22.5% (Figure 1b). This is

Figure 2. Characterization of NaYF4:Gd3+/Yb3+@NaYF4:Yb3+/Tm3+/Er3+ core-shell UCNPs (21.5% Gd3+). (a) TEM images of NaYF4:Gd3+/Yb3+ core (left) and NaYF4:Gd3+/Yb3+@NaYF4:Yb3+/Tm3+/Er3+ core-shell nanoparticles (right). Scale bars in all the images are 50 nm; (b) size statistics of NaYF4:Gd3+/Yb3+ core (left) and NaYF4:Gd3+/Yb3+@NaYF4:Yb3+/Tm3+/Er3+ core-shell nanoparticles (right); (c) X-ray powder diffraction pattern of the core-shell UCNPs; (d) EDX spectrum of the core-shell UCNPs.


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likely because the smaller nanoparticles have larger surface-to-volume ratio and higher surface detects density, leading to more pronounced PL suppression for smaller UCNPs.32 This is attributed to Therefore, we choose core-shell particles with 21.5% Gd3+ for subsequent sensing experiments because of their homogeneity and strong luminescence. The mean size of these particles (21.5% Gd3+) increases from 44.5 nm to 52.5 nm after shell growth (Figure 2a and 2b). X-ray powder diffraction (XRD) pattern reveals hexagonal phase of these particles (Figure 2c). Energy dispersive X-ray spectroscopy (EDX) confirms their elemental composition including Gd, Yb, Tm, Er et al. (Figure 2d).

Next, phase transfer was performed on the as-prepared UCNPs through ligand exchange using polyhistidine-containing peptides (HHHHHHVPLSLTMGK) without fluorophore labeling. As shown in Figure 3, UCNPs transferred to the aqueous layer retains the characteristic emission peaks of Tm3+ and Er3+ (Figure 3a). 150 µM peptides were sufficient to produce water-soluble UCNPs with efficient luminescence (Figure 3b). To construct LRET pairs for MMP sensing, FITC and TAMRA are used to selectively quench the emission peak of Tm3+ (1D2 – 3F4, 1G4 – 3H6) and Er3+ (4S3/2 – 4I15/2) respectively because of the good overlap between the emission peak of UCNPs and the absorption peak of each fluorophore (Figure 4). Also, Tm3+ and Er3+ localized in the shell layer have minimal separation distance with the fluorophores for efficient LRET.

Figure 3. Peptide-mediated phase transfer of the core-shell UCNPs. (a) Luminescence spectra and (b) integrated emission peak area (500-600 nm) of UCNPs transferred with different concentrations of peptides (50-300 µM).


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Figure 4. Spectra overlay of UCNP luminescence, FITC absorption and emission, TAMRA absorption and emission.

Figure 5. Preparation of (H)6VPLSLTMGK−FITC-capped UCNPs for MMP-7 detection. (a) Photographs of core-shell UCNPs pre- and post-phase transfer under NIR excitation; (b) luminescence spectra of UCNPs preand post-phase transfer; (c) luminescence spectra of UCNPs treated with various concentrations of MMP-7; (d) calibration curve for MMP-7 quantification.


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We subsequently evaluated the responsivity of each LRET pair to MMP-7 and MMP-2-induced peptide cleavage. Firstly, we used a FITC-labeled peptide with MMP-7 substrate sequence to transfer UCNPs to the aqueous layer. Efficient phase transfer was revealed by strong luminescence in aqueous layer and disappearance of luminescence in organic layer (Figure 5a). The nanoparticles remained monodispersed in aqueous solution and the mean hydrodynamic size increased after phase transfer (SI Figure S1-S3). Fourier-transform infrared spectroscopy (FTIR) confirmed ligand exchange of oleic acid with peptides (SI Figure S4). Notably, the two blue emission peaks of Tm3+ are selectively quenched by FITC after phase transfer (Figure 5b). The partial decrease of other emission peaks is likely due to the water quenching effect.34 Next, a titration experiment was conducted to detect MMP-7 activities at different concentrations. Gradual recovery of blue emission was observed with increasing MMP-7 concentration (Figure 5c), and a linear dynamic range was achieved between 0.005 and 0.35

Figure 6. Preparation of (H)6GPLGVRGK−TAMRA-capped UCNPs for MMP-2 detection. (a) Photographs of core-shell UCNPs pre- and post-phase transfer under NIR excitation; (b) luminescence spectra of UCNPs preand post-phase transfer; (c) luminescence spectra of UCNPs treated with various concentrations of MMP-2; (d) calibration curve for MMP-2 quantification.


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µg/mL MMP-7 (Figure 5d). Next, a TAMRA-labeled peptide with MMP-2 substrate sequence was used for efficient phase transfer of UCNPs (Figure 6a). In this context the green emission peak of Er3+ was selectively quenched by TAMRA (Figure 6b). Upon MMP-2 treatment, the green emission was gradually recovered with increasing MMP-2 concentration (Figure 6c). A linear dynamic range for MMP-2 detection was obtained between 0.001 and 0.12 µg/mL MMP-2 (Figure 6d). These results indicate that the peptide linker could be specifically cleaved by the MMP, leading to destruction of LRET pair and luminescence enhancement.

On the basis of the above results, we proceeded to construct a dual-channel UCNP sensor for multiplex detection of MMP-2 and MMP-7 activities. The two peptides labeled with FITC and TAMRA respectively were used as co-ligands for phase transfer of UCNPs. The average numbers of FITC- and TAMRA-labeled peptides per particle were calculated to be 1267 and 1048 respectively (see SI for calculation details). Both the blue emission of Tm3+ and the green emission of Er3+ were efficiently quenched after phase transfer (Figure 7a and 7b). The as-prepared UCNP sensor was titrated against a mixture of various concentrations of MMP-2 (0.001-0.2 µg/mL) and MMP-7 (0.01-1 µg/mL). Gradual enhancement of blue and green luminescence was detected with increasing MMP concentrations (Figure 7c and 7d). The limit of detection (LOD) for MMP-2 and MMP-7 were determined to be 2.2 ng/mL and 13.9 ng/mL respectively, which is comparable or even lower than the other reported methods (SI Table S1). Meanwhile, little cross-reactivity of MMP-2 and MMP-7 with each other’s substrate was observed (Figure 8a), revealing high specificity of proteolytic activity of both MMPs. Furthermore, metal ions (K+, Mg2+) or other non-specific biomolecules (glucose, BSA) did not induce obvious signal changes (Figure 8b), confirming the robustness of the UCNP sensor for MMPs detection. Also, a non-specific protease – trypsin induced minimal luminescence enhancement, indicating that the peptide cleavage is highly specific to MMP-2 and MMP-7 (Figure 8b). MMP-2 and MMP-7 pretreated with MMP inhibitor (BB-94) did not induce luminescence enhancement, confirming that the detection signal is caused by proteolytic activities of MMPs (Figure 8c). Standard addition experiments were conducted for quantification of MMPs in human serum. As shown in Table 1, concentrations of MMP-7 and MMP-2 in human serum determined using the UCNP sensor are consistent with the standard concentration by weighting. The relative standard deviation (RSD) for MMP-7 and MMP-2 concentrations is typically below 7% and 3% respectively.


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Figure 7. Preparation of (H)6VPLSLTMGK−FITC- and (H)6GPLGVRGK−TAMRA-capped UCNPs for multiplex MMP-2 and MMP-7 detection. (a) Photographs of core-shell UCNPs pre- and post-phase transfer under NIR excitation; (b) luminescence spectra of UCNPs pre- and post-phase transfer; (c) luminescence spectra of UCNPs treated with various concentrations of MMP-2 and MMP-7; (d) calibration curves for MMP-2 and MMP-7 quantification.

In addition, we monitored the activity of MMP-2 and MMP-7 secreted by two cancer cell lines – HT1080 and K562 cells respectively. Expression of MMP-2 and MMP-7 was validated by SDS PAGE (SI Figrue S7). As shown in Figure 9, the MMP-2 and MMP-7 secreted by cancer cells lead to enhancement of Er3+ and Tm3+ emissions respectively. The concentrations of active MMP-2 and MMP7 in cell medium were determined to be 11.5 ng/mL and 1.8 ng/mL.


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Figure 8. Specificity evaluation of the UCNP sensor for MMP-2 and MMP-7 detection. (a) Luminescence (450 nm and 541 nm) enhancement of UCNPs treated with MMP-2 or MMP-7 alone; (b) Luminescence (450 nm and 541 nm) enhancement of UCNPs treated with KCl (100 mM), MgCl2 (2.5 mM), glucose (10 mM), BSA (1 mM), trypsin (1 µM), and MMP-7 (0.5 µg/mL) and MMP-2 (0.15 µg/mL); (c) luminescence spectra of UCNPs incubated with MMP-2 (0.15 µg/mL)/MMP-7 (0.5 µg/mL) with or without MMP inhibitor inactivation. Sample

Added (µg/mL) MMP-7, MMP-2

Measured (µg/mL) MMP-7, MMP-2

RSD (%) MMP-7, MMP-2

1

0.030, 0.030

0.0278, 0.0310

4.7, 2.4

2

0.050, 0.050

0.0496, 0.0467

4.3, 2.6

3

0.080, 0.080

0.0762, 0.0791

6.9, 1.9

4

0.100, 0.100

0.105, 0.101

5.4, 1.1

Table 1. Determination of the concentrations of MMP-7 and MMP-2 in human serum using the UCNP sensor.


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Figure 9. Detection of active MMP-2 and MMP-7 secreted by tumor cells. (a) Luminescence spectra of UCNPs incubated with cell medium of HT1080 cells; (b) incubation time dependence of I541/I655 emission intensity ratio for MMP-2 detection; (c) luminescence spectra of UCNPs incubated with cell medium of K562 cells; (d) incubation time dependence of I450/I655 emission intensity ratio for MMP-7 detection.

Conclusions Taken together, we devised a multichannel UCNP sensor for multiplex ratiometric sensing of MMP2 and MMP-7 activities. We rationally designed a core-shell UCNP structure that is favorable for multicolor emission and high LRET efficiencies. This allows integration of multiple optical sensing channels in a single sensor for multiplex detection. The UCNP sensor exhibits high specificity and sensitivity toward MMP-2 and MMP-7 with little cross-reactivity. The ratiometric sensing mode provides higher accuracy than single signal-on mode because it is less susceptible to sample volume changes. The number of sensing channels in the UCNP sensor could be potentially expanded using the


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additional emission peaks in the ultraviolet or near-infrared region, or alternatively by incorporating a third dopant with different emission wavelength. We expect that the reported sensor is potentially generalizable for multiplex detection of various types of proteases in a single assay.

Experimental Section Materials. YCl3·6H2O (99.99%), YbCl3·6H2O (99.9%), GdCl3·xH2O (99.99%), TmCl3 (99.9%), ErCl3·6H2O (99.9%), NaOH (98%), NH4F (98%), oleic acid (OA, 90%), 1-octadecene (ODE, 90%), trypsin, and bovine serum albumin (BSA, 98%) were purchased from Sigma-Aldrich. Matrix metalloproteinase inhibitor (BB-94) was purchased from Selleck Chemicals. MMP-2 (Recombinant Human Matrix Metalloproteinase-2) and MMP-7 (Recombinant Human Matrix Metalloproteinase-7) were purchased from ProSpec. MMP activator (4-aminophenyl)mercuric acetate (APMA) was purchased from Genmed Scientifics. Healthy human serum samples were provided by Blood Transfusion Department, No. 97 Hospital of PLA. HT1080 Cells and K562 Cells were purchased form Procell Life Science & Technology Co,. Ltd. The following peptides were synthesized and purified by Ningbo Karebay Biochem, Co., Ltd. (left: N-terminal; right: C-terminal) HHHHHHVPLSLTMGK HHHHHHGPLGVRGK−TAMRA HHHHHHVPLSLTMGK−FITC

Synthesis of NaYF4:60%Yb/ X %Gd (X = 0, 10, 20, 21.5, 25, 30) nanoparticles. In a typical experiment, X mmol GdCl3·xH2O, (0.4-X) mmol YCl3·6H2O, and 0.6 mmol YbCl3·6H2O were mixed with 6 mL of OA and 15 mL of ODE in a three-neck round-bottom flask. The resulting mixture was heated at 150 °C under argon flow for 30 min to form a clear light yellow solution. After cooling to 50 °C, 10 mL of methanol solution containing 0.1482 g of NH4F and 0.10 g of NaOH was dropwise added and the solution was vigorously stirred for 2 hours. Then, the slurry was slowly heated and kept at 110 °C for 10 min to remove methanol. Next, the reaction mixture was protected with an argon atmosphere, quickly heated to 305 °C (~15 °C min−1), and kept for 65 min. The products were extracted with ethanol by centrifugation at 6000 rpm for 10 min. The materials were washed with ethanol and cyclohexane for several times, and finally redispersed in 6 mL chloroform.


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Synthesis of NaYF4:60%Yb/ X %Gd (X = 0, 10, 20, 21.5, 25, 30) @NaYF4 :30%Yb/0.5%Tm /0.2%Er core-shell nanoparticles. For the synthesis of core-shell structure, YCl3·6H2O (0.693 mmol), YbCl3·6H2O (0.3 mmol), TmCl3·6H2O (0.005 mmol) and ErCl3·6H2O (0.002 mmol) were mixed with 6 mL of OA and 15 mL of ODE in a three-neck round-bottom flask at room temperature. Then, the slurry was heated to 150 °C under argon flow for 30 min to form a clear light yellow solution. After cooling to 50 °C, the asprepared NaYF4:Yb/Gd nanoparticles in 6 mL chloroform were added into the above solution and kept at 65 °C for 30 min. After the removal of chloroform, the mixture was cooled down to 50 °C and 10 mL methanol solution containing 0.1482 g of NH4F and 0.10 g of NaOH was dropwise added and the solution was vigorously stirred for 2 hours. Then, the slurry was slowly heated and kept at 110 °C for 10 min to remove methanol. Next, the reaction mixture was protected with an argon atmosphere, quickly heated to 305 °C (~15 °C min−1), and kept for 100 min. The products were extracted with ethanol by centrifugation at 6000 rpm for 10 min, washed with ethanol and cyclohexane for several times, and finally redispersed in chloroform with a 2 mg mL-1 concentration.

Peptide-mediated phase transfer of UCNPs. A total of 300 µL OA-capped UCNPs (2 mg/mL) and 300 µL peptide in H2O (peptide concentration = 50, 100, 150, 200, 300 µM; peptide sequence: HHHHHHVPLSLTMGK) were added to a glass vial and then vigorously stirred for 6−8 hours. The UCNPs were efficiently transferred to the top water layer from the chloroform layer. The UCNP solution (top layer) was then collected and stored at 4 °C for further use.

TEM and EDX characterization. Each UCNP sample (1 mg/mL) was dispersed onto a 3 mm copper grid covered with a continuous carbon film and dried at room temperature. TEM was performed using a Tecnai G2 20 (FEI, Hillsboro, OR) transmission electron microscope operating at 185 kV. HRTEM and EDX characterization were performed using a Tecnai F20 (FEI) transmission electron microscope operating at 200 kV.

AFM characterization. OA-capped UCNP solution (0.2 mg/mL) was dropped onto fleshly prepared mica and dried at room


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temperature. The UCNPs were imaged with AFM (Bruker Multi-Mode 8) under ScanAsyst-Air mode using a ScanAsyst-Air probe (Bruker).

Optical characterization. The photoluminescence spectra were recorded using a fiber fluorescence spectrophotometer (AvaSpec2048) equipped with a 980 nm laser (1 W, Shanghai Dream Lasers Technology Co., Ltd., power density of 6.6 W/cm2) as the excitation light source. The integration time was set to 300 ms. The absorption spectra were recorded using a UV−Vis spectrophotometer (Agilent 8453).

DLS and zeta potential. Hydrodynamic sizes and zeta potential of UCNPs were measured on a Zetasizer Nano ZS90 (Malvern).

XRD characterization. UCNP samples were prepared by evaporating chloroform solutions of OA-capped UCNPs in vacuum. Powder XRD patterns were recorded on a Bruker D8-Venture diffractometer with a Turbo X-ray Source (Mo−Kα radiation, λ = 0.71073 Å) adopting the direct drive rotating anode technique and a CMOS detector at room temperature.

FTIR characterization. Samples were prepared by evaporating chloroform solutions of OA-capped UCNPs and water solutions of peptide-capped UCNPs in vacuum. FTIR spectra were recorded on a Bruker VERTEX 70 V FTIR spectrometer.

Peptide-coated UCNPs for MMP-7 detection. To prepare peptide-coated UCNPs, 500 µL of OA-capped UCNPs (2 mg/mL) in chloroform and 500 µL of (H)6VPLSLTMGK−FITC (150 µM) in H2O were added to a glass vial and then vigorously stirred for 8 hours. The UCNPs were efficiently transferred to the upper water layer from the chloroform layer. The UCNP solution was transferred to a microtube and sonicated for 3 min. Afterwards, peptide-coated UCNPs were centrifuged at 4500 rpm for 10 min to remove excess peptides in the supernatant and resuspended in 500 µL of TCNB buffer (50 mM Tris, pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij) containing 150 µM (H)6VPLSLTMGK−FITC. The resulting UCNP solution was left at room


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temperature for 1 day, and the free peptides were removed as described above. The purified nanoparticles were redispersed in TCNB buffer and stored at 4 °C for further use. UCNP−(H)6VPLSLTMGK−FITC (120 µL, UCNP concentration =2.0 mg/mL) and various amounts of MMP-7 (final MMP-7 concentration for the reaction = 0, 0.005, 0.01, 0.02, 0.05, 0.10, 0.15, 0.25, 0.35,0.50, and 1 µg/mL) were added to TCNB buffer to a total volume of 200 µL. The enzyme cleavage reaction was conducted in a water bath at 37 °C for 2 hours.

Peptide-coated UCNPs for MMP-2 detection. The probe for MMP-2 detection was prepared as described above using (H)6GPLGVRGK−TAMRA peptide for phase transfer. MMP-2 was activated with 1 mM of APMA by incubation in a water bath at 37 °C for 1 hour prior to use. UCNP−(H)6GPLGVRGK−TAMRA (120 µL, UCNP concentration =2.0 mg/mL) and various amounts of MMP-2 (final MMP-2 concentration for the reaction = 0, 0.001, 0.015, 0.03, 0.06, 0.08, 0.10, 0.12, 0.15, and 0.20 µg/mL) were added to TCNB buffer to a total volume of 200 µL. The enzyme cleavage reaction was conducted in a water bath at 37 °C for 2 hours.

Peptide-coated UCNPs for MMP-2 and MMP-7 multiplex detection. The probe for MMP-2 and MMP-7 duplex detection was prepared as described above using both (H)6GPLGVRGK−TAMRA (150 µM) and (H)6VPLSLTMGK−FITC (150 µM) peptides for phase transfer. UCNP−(H)6GPLGVRGK−TAMRA/(H)6VPLSLTMGK−FITC (120 µL, UCNP concentration =2.0 mg/mL) and various amounts of MMP-7 and MMP-2 (final MMP-7 concentration for the reaction = 0, 0.01, 0.05, 0.01, 0.15, 0.25, 0.35, 0.5, and 1 µg/mL, final MMP-2 concentration for the reaction = 0, 0.001, 0.015, 0.03, 0.06, 0.08, 0.12, 0.15, and 0.20 µg/mL) were added to TCNB buffer to a total volume of 200 µL. The enzyme cleavage reaction was conducted in a water bath at 37 °C for 2 hours.

Specificity test. UCNP−(H)6GPLGVRGK−TAMRA/−(H)6VPLSLTMGK−FITC (120 µL, UCNP concentration =2.0 mg/mL) and KCl (100 mM), MgCl2 (2.5 mM), glucose (10 mM), trypsin (1 µM), or BSA (1 mM) were added to TCNB buffer to a total volume of 200 µL, respectively. The reaction was conducted in a water bath at 37 °C for 2 hours.

MMP inhibition assay.


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2 µL MMP inhibitor BB-94 (1 mM) in DMSO was added to 200 µL TCNB buffer containing 2 µg/mL MMP-7 and 200 µL TCNB buffer containing 1 µg/mL MMP-2 respectively. The reaction was conducted in a water bath at 37 °C for 30 min. Then the above MMPI-treated 50 µL TCNB buffer containing 2 µg/mL MMP-7 and 30 µL TCNB buffer containing 1 µg/mL MMP-2 were added to UCNP−(H)6GPLGVRGK−TAMRA/(H)6VPLSLTMGK−FITC solution (120 µL, UCNP concentration =2.0 mg/mL). The reaction was conducted in a water bath at 37 °C for 2 hours. MMP-7 and MMP-2 without MMPI treatment were used as controls.

Human serum sample analysis. 40 µL of diluted (1:10) healthy human serum samples containing different concentrations of MMP-2 or MMP-7 were prepared and used for sensing experiments.

Detection of active MMP-2 and MMP-7 secreted by tumor cells. HT1080 cell line (human fibrosarcoma, adhesion cell line) were cultured on 25 cm2 cell culture plates with vent caps (Corning) in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% pen/strep (100 units mL−1 penicillin and 100 mg mL−1 streptomycin) in a humidified atmosphere containing 5% CO2 at 37 °C. When cell density reached 70%, the cells were washed three times with serum-free DMEM, and then cultured at 37 °C for 24 h with serum-free DMEM. The cell culture media was collected and centrifuged at 1,000 rpm for 10 min to remove cell debris and then concentrated (10×) by Centricon (Millipore, molecular weight cutoff 30 K). MMP-2 activity was detected by incubation of UCNP−(H)6GPLGVRGK−TAMRA in the media. K562 cell line (human fibrosarcoma, suspension cell line) were cultured on 25 cm2 cell culture plates with vent caps (Corning) in IMDM medium supplemented with 10% fetal bovine serum (FBS) and 1% pen/strep (100 units mL−1 penicillin and 100 mg mL−1 streptomycin) in a humidified atmosphere containing 5% CO2 at 37 °C. When cell density reached 70%, the medium was removed by centrifugation, and the cells were cultured at 37 °C for 24 h with serum-free IMDM. The cell culture media was collected and centrifuged at 1,000 rpm for 10 min to remove cell debris and then concentrated (40×) by Centricon (Millipore, molecular weight cutoff 10 K). MMP-7 activity was detected by incubation of UCNP-(H)6VPLSLTMGK−FITC in the media. MMP-2 and MMP-7 expression in cell culture media was examined by SDS-PAGE. The gel was stained with Coomassie blue and destained with 10% glacial acetic acid and 10% methanol solution.


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ASSOCIATED CONTENT Supporting Information. TEM images and DLS measurements of UCNPs before and after phase transfer using different peptides as ligands (Figure S1-S3); FTIR spectra of UCNPs before and after ligand exchange (Figure S4); detailed calculations of the average number of peptides on UCNPs (Figure S5 and S6). AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ACKNOWLEDGMENT This work was supported in part by the NSFC (21475093, 21522506), the National High-Tech R&D Program (2014AA020518), 1000-Young Talents Plan, PAPD, and startup funds from Soochow University. References 1. Visse, R.; Nagase, H. Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases: Structure, Function, and Biochemistry. Circ. Res. 2003, 92, 827-839 2. Shapiro, S. D. Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Curr. Opin. Cell Biol. 1998, 10, 602-608. 3. Nagase, H.; Visse, R.; Murphy, G. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc. Res. 2006, 69, 562-573. 4. Egeblad, M.; Werb, Z. New functions for the matrix metalloproteinases in cancer progression. Nat. Rev. Cancer 2002, 2, 161-174. 5. Ii, M.; Yamamoto, H.; Adachi, Y.; Maruyama, Y.; Shinomura, Y. Role of Matrix Metalloproteinase-7 (Matrilysin) in Human Cancer Invasion, Apoptosis, Growth, and Angiogenesis. Exp. Biol. Med. 2006, 231, 20-27 6. Gialeli, C.; Theocharis, A. D.; Karamanos, N. K. Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting. FEBS J. 2011, 278, 16-27. 7. Roy, R.; Yang, J.; Moses, M. A. Matrix Metalloproteinases As Novel Biomarkers and Potential Therapeutic Targets in Human Cancer. J. Clin. Oncol. 2009, 27, 5287-5297.


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For TOC only


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Rational Engineering a Multichannel Upconversion Sensor for

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