Article pubs.acs.org/ac
Poly(m‑phenylenediamine)-Based Fluorescent Nanoprobe for Ultrasensitive Detection of Matrix Metalloproteinase 2 Zhe Wang, Xiaohua Li,* Duan Feng, Lihong Li, Wen Shi, and Huimin Ma* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *
ABSTRACT: A novel fluorescence nanoprobe for the detection of matrix metalloproteinase 2 (MMP2) has been developed by engineering the fluorescein isothiocyanate-labeled peptide onto the surface of poly(m-phenylenediamine) (PMPD) nanoparticles through covalent linkage. The nanoprobe itself displays a low background signal due to the effective fluorescence quenching by electronrich PMPD, but its reaction with MMP2 causes 11-fold fluorescence enhancement. Compared with similar fluorescence nanosystems for MMP2 assembled through physical adsorption, the as-prepared nanoprobe is significantly more stable and displays a strikingly higher signal-to-background ratio, which leads to a high sensitivity for MMP2 assay, with a detection limit of 32 pM. Most notably, the nanoprobe has been successfully applied to determine MMP2 in human serum samples, demonstrating that the MMP2 level in serum from colorectal cancer (CRC) patients is 2 times higher than that from healthy people. Moreover, the nanoprobe has also been used to monitor MMP2 secreted by CRC cells that were grown under normoxic and hypoxic conditions, respectively, and the results show that the cells under hypoxic conditions produce higher level of MMP2 than those under normoxic conditions. Our method is simple and can offer a highly sensitive detection of MMP2 in relevant clinical samples. fluorophore is quenched by the nanomaterial and reaction with MMPs results in the cleavage of the peptide bond as a consequence of the specific substrate recognition, accompanying the release of the fluorophore-labeled peptide segment and thereby the recovery of fluorescence. Nevertheless, most of these nanoprobes are constructed through electrostatic adsorption or π−π stacking interaction between the peptides and the nanomaterials, instead of through covalent linkage; such nanoprobes usually exhibit insufficient fluorescence quenching and thus poor sensitivity for analyte detection. Moreover, some of the aforementioned nanomaterials themselves suffer from instability. For instance, gold nanoparticles are apt to precipitate in biological high-salt environments.23−26 Thus, new fluorescent nanoprobes with high sensitivity and stability are still demanded for a more effective MMPs detection in biosystems. Poly(m-phenylenediamine) (PMPD), an electron-rich aromatic polymer, has attracted considerable attention in recent years due to its good water solubility and fluorescence quenching capability, which has led to the development of a few fluorescence off−on nanoprobes for different biomolecules by using the physical adsorption of nucleic acids on PMPD.27−29 However, to the best of our knowledge, PMPD
M
atrix metalloproteinases (MMPs), with Zn2+ as cofactor, are a class of endopeptidases capable of degrading virtually all kinds of extracellular matrix proteins,1,2 and their overexpression has been found to be closely related with tumor invasion, metastasis, and angiogenesis.3 Matrix metalloproteinase 2 (MMP2) is one of the most vital MMPs and has been reported to be abnormal in many types of cancer, such as breast cancer,4 bladder cancer,5 and colorectal cancer.6 Because of the crucial role of MMP2 as a tumor marker, its sensitive detection is of great importance for clinical diagnosis and therapy of cancer at its early stage. The traditional methods for MMPs detection mainly include immunoassay7 and zymography.8,9 Although immunoassay is sensitive and specific for MMPs, it requires costly antibody proteins and also complex washing steps; zymography, instead, is suitable for qualitative but not for quantitative determination. In addition, the gas chromatography/mass spectrometry method has also been reported for quantifying MMPs,10 which, however, is time-consuming and involves complicated procedures for pretreatment of the sample. Alternatively, fluorescence methods have received continually growing interest due to their high sensitivity,11−17 and a number of fluorescence off−on nanoprobes have been proposed to detect MMPs based on the fluorescence quenching property of a variety of nanomaterials, such as metal nanoparticles,18−20 carbon nanoparticles,21 and graphene oxide.22 In these approaches, the fluorophore-labeled peptides and the nanoquenchers are assembled to form a complex as a nanoprobe, in which the fluorescence of the organic © 2014 American Chemical Society
Received: May 5, 2014 Accepted: July 16, 2014 Published: July 16, 2014 7719
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Scheme 1. Schematic Diagram to Illustrate the Fluorescence Response of the Nanoprobe to MMP2
purchased from Sigma-Aldrich and were used as received without further purification. TCNB buffer (50 mM Tris with 10 mM CaCl2, 150 mM NaCl, and 0.05% Brij 35; pH 7.4) was employed in the experiments. MMP2 commercial enzymelinked immunosorbent assay (ELISA) kit was purchased from Shanghai Yanxin Biological Technology Co., Ltd. Ultrapure water (over 18 MΩ·cm) from a Milli-Q Reference system (Millipore) was used throughout. Human sera from five healthy individuals and five CRC patients were provided by Xijing Hospital, and an informed consent was obtained from each donor. Apparatus. Fluorescence measurements were performed on a Hitachi F-4600 spectrophotometer in 10 mm × 10 mm quartz cells (Tokyo, Japan), with a 400 V PMT voltage. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses were made on a JEM-1011 instrument and Hitachi S-4800 field emission scanning electron microscope, respectively. ζ-Potential measurements were performed on a Nano-ZS Zetasizer ZEN3600 (Malvern Instruments Ltd., U.K.). A model HI-98128 pH meter (Hama Instruments Inc., U.S.A.) was employed for pH measurements. UV−vis absorption spectra were recorded in 1 cm quartz cells with a TU-1900 spectrophotometer (Beijing, China). Fourier transform infrared (FT-IR) spectra were taken in KBr disks on a Tensor 27 spectrometer (Bruker, Germany). The absorbance for ELISA analysis was recorded on a microplate reader (BIOTEK Synergy HT, U.S.A.). The density of cells was determined by cell counting chamber [Biosystem Medical Technology (Shanghai) Co., Ltd.]. The incubation was carried out in Shaker incubator (SKY-100C, Shanghai Sukun Industry & Commerce Co., Ltd.). Preparation of PMPD Nanoparticles. PMPD nanoparticles were synthesized by room-temperature chemical oxidation polymerization of m-phenylenediamine monomer with (NH4)2S2O8.27 Typically, 0.6 mL of aqueous solution (0.5 M) of (NH 4 ) 2 S 2 O 8 was mixed with 8.4 mL of Nmethylpyrrolidone at room temperature. To this mixture, 1 mL of aqueous solution (0.1 M) of m-phenylenediamine was then injected under stirring. During the reaction, a large amount of black precipitate was gradually produced. After reaction for different periods of time (6, 12, 24, and 36 h), the resulting precipitate was washed three times with ethanol and water by centrifugation, respectively. Finally, the obtained
has never been used as a component to construct a nanoprobe via covalent linkage. Herein, we report such an attempt by designing a fluorescent off−on nanoprobe for MMP2 assay, in which the fluorescein isothiocyanate (FITC)-labeled peptide containing the core substrate sequence (PLGVR) of MMP22 was directly linked to the amine groups on the surface of the PMPD nanoparticles through covalent bond formation (PMPD−peptide−FITC, Scheme 1). In this nanoprobe, the FITC-labeled peptide serves as a fluorophore, and its fluorescence is efficiently quenched by electron-rich PMPD; upon reaction with MMP2, however, the quenched fluorescence is turned-on through the selective cleavage of the FITClabeled peptide and thus the release of the FITC-containing peptide segment. Most notably, because of electrostatic repulsion between identical charges, the feature of the positive charge of both PMPD and the core peptide (its pI was 9.7522) in the near-neutral media during the nanoprobe preparation effectively eliminates the nonspecific adsorption of the fluorophore on the surface of PMPD, which is also rather favorable for achieving a low background signal. With this nanoprobe the concentrations of MMP2 in the serum samples from colorectal cancer patients as well as those from healthy people have been determined and compared. In addition, the concentrations of MMP2 secreted by colorectal cancer (CRC) cells grown under normoxic and hypoxic conditions have also been monitored, which reveals that hypoxia can elevate the secretory level of MMP2. This method is simple and useful for the detection of MMP2 in clinical samples for the diagnosis of MMP2-related diseases.
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EXPERIMENTAL SECTION Reagents. 4-Aminophenylmercuric acetate, 1,10-phenanthroline, 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), bovine serum albumin, human serum albumin, and MMP2 were obtained from Sigma-Aldrich. Matrix metalloproteinase 1 (MMP1) was obtained from Sino Biological Inc. The FITC-labeled peptide (FITC−GPLGVRG) was purchased from Beijing SBS Genetech Co., Ltd. N-Hydroxysuccinimide (NHS) and tris(hydroxymethyl)-aminomethane (Tris) were purchased from J&K Chemical. CaCl2, NaCl, KCl, MgCl2, glucose, vitamin B1, and glutamine were obtained from Beijing Chemicals, Ltd. All the other reagents employed were 7720
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Figure 1. TEM image (A), FT-IR spectrum (B), and UV−vis absorption spectrum (C) of the PMPD nanoparticles (0.025 mg/mL) prepared with the reaction time of 12 h.
precipitate product was dispersed in water and stored at 4 °C for characterization and further use. Preparation of the PMPD−Peptide−FITC Nanoprobe. EDC (0.8 mg) and NHS (1.4 mg) were first mixed in 1.8 mL of borate buffer (10 mM, pH 7.2) containing 0.23 mg of the FITC-labeled peptide to activate the carboxylic acid groups of the peptide. The mixture was incubated by shaking at room temperature for 2 h in dark. After the pH value of the mixture was adjusted to 7.5 with NaOH (1 mM), an appropriate amount of PMPD (0.25 mg/mL) was added. After reaction at 35 °C for 90 min, 10 mg of bovine serum albumin was added to block the unreacted NHS. Finally, the PMPD−peptide−FITC nanoprobe (referred to the nanoprobe) was centrifuged, washed with water, and then dispersed in 2 mL of TCNB buffer (pH 7.4) for further applications. General Procedure for MMP2 Detection. Prior to use, MMP2 (20 μL, 1.4 μM) was activated by incubating with an equal volume of 4-aminophenylmercuric acetate (2.5 mM) in TCNB buffer (pH 7.4) at 37 °C for 1 h following the known procedure.30 Then, different concentrations of the activated MMP2 were incubated with the nanoprobe at 37 °C for 90 min, followed by measuring the fluorescence of the samples at room temperature with λex/em = 480/518 nm. Determination of MMP2 in Human Serum Samples by Nanoprobe. Human serum samples were diluted 10 times before analysis. Then appropriate aliquots of the serum samples were transferred to the nanoprobe solution, and MMP2 in the diluted serum samples was quantified following the general procedure as described above. Determination of MMP2 in Human Serum Samples by ELISA. The concentrations of MMP2 in human serum samples were also determined by measuring the absorbance values at 450 nm using a commercial ELISA kit. In this detection, the standard curve (OD = 0.43[MMP2] (nM) + 0.84, R = 0.987) was first obtained following the direction of the kit in the MMP2 concentration range from 0 to 2 nM. Then 50 μL of the human serum sample was added to the ELISA kit wells, and 100 μL of horseradish peroxidase conjugate reagent was introduced into each well, followed by incubation at 37 °C for 60 min. After all of the samples were washed five times with 400 μL of wash solution, 50 μL of chromogen solution A and 50 μL of chromogen solution B were added to each well, followed by incubation at 37 °C for 15 min in dark. Finally, 50 μL of the stop solution was added to each well to stop the reaction, and the optical density was read immediately on a microplate reader at 450 nm. Determination of MMP2 Secreted by CRC Cells Grown under Different Oxygen Levels. CRC cells, with a density of about 1.0 × 106 cells/mL, were grown at 37 °C for 12 h on
glass-bottom culture dishes (MatTek Co.) in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin−streptomycin in a humidified incubator under normoxic (95% air and 5% CO2, i.e., 20% O2) and different hypoxic (80% N2, 15% O2, and 5% CO2; 85% N2, 10% O2, and 5% CO2; 90% N2, 5% O2, and 5% CO2; or 94% N2, 1% O2, and 5% CO2) conditions,31,32 respectively; the cell densities in the culture media were measured by the cell counting chamber. Then, an appropriate volume (typically 50 μL) of the culture media was collected and transferred to the nanoprobe solution. The MMP2 concentration in the solution was quantified following the general procedure as described above.
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RESULTS AND DISCUSSION Preparation and Characterization of PMPD Nanoparticles. The PMPD nanoparticles were synthesized following the procedure as described above and characterized by SEM analysis. As shown in Figure S1 in the Supporting Information, the size of the PMPD nanoparticles prepared with the reaction time of 12 h becomes smaller than that of 6 h, which may result from the fact that PMPD nanoparticles with a reaction time of 6 h are loosely assembled, and then they can form tightly organized structure until 12 h; further increase of the reaction time to 24 or 36 h does not lead to uniform nanoparticles (Supporting Information Figure S1, parts C and D), which indicates that the PMPD nanoparticles can polymerize together with longer reaction time, generating irregular blocks. Because small size of nanoparticles is beneficial for uniform distribution in homogeneous bioassays,33 PMPD prepared with the reaction time of 12 h was used for the nanoprobe construction. Figure 1A shows the TEM image of such PMPD. As is seen, the nanoparticles have a rather uniform spherical shape, with an average diameter of ca. 20 nm. Figure 1B depicts their FT-IR spectrum, which indicates the existence of Ar−NH2 moiety on the surface of PMPD, as evidenced by N−H stretching (3138 cm−1), N−H bending (1624 cm−1), and C−N stretching (1401 and 1117 cm−1). In addition, the UV− vis absorption spectrum (Figure 1C) shows that PMPD exhibits a wide range of absorption wavelengths from 200 to 800 nm,29 which can overlap the fluorescence spectra of most of fluorochromes, suggesting that the electron-rich PMPD is a good fluorescence quencher. The PMPD nanoparticles prepared with the reaction time of 12 h possess excellent dispersibility in water, whereas the others are easier to precipitate, though PMPD prepared with the reaction time of 6 h displays a relatively slow precipitation (Figure S2A in the Supporting Information). Furthermore, by 7721
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revealing that the PMPD nanoparticles linked to the FITClabeled peptide can quench the fluorescence very efficiently as expected. However, addition of MMP2 (10 nM) to the nanoprobe solution causes 11-fold fluorescence enhancement (curve c in Figure 2), which is ascribed to the specific cleavage of the substrate (the peptide) by MMP2 and the release of the FITC-containing peptide segment. On the other hand, the quenching efficiency of the nanoprobe was compared with that of the simple mixture of PMPD and the FITC−peptide. As shown in Figure 2 (curve d), about 29% of the fluorescence is quenched in the simple mixture, which is much less than that of the nanoprobe, clearly indicating that covalent linkage of the peptide to PMPD results in more efficient fluorescence quenching than physical adsorption. It is also important to note that the fluorescence of the free FITC-labeled peptide is scarcely influenced by the addition of MMP2 in the absence of PMPD (curve e in Figure 2), and PMPD itself exhibits nearly no fluorescence (curve f in Figure 2). All the above observations indicate that this fluorescent nanoprobe is wellsuitable for MMP2 detection. The amount of PMPD as a nanoquencher was optimized for preparing the nanoprobe. As shown in Figure 3 (curve a), 94%
dispersing PMPD prepared with 12 h in water, the resulting colloidal solution is stable at room temperature at least for 4 days because no obvious precipitation is observed in such solution (Figure S2B, Supporting Information). In addition, the ζ-potential values of the PMPD nanomaterials prepared with the reaction time of 6, 12, 24, and 36 h were determined to be +19.7, +16.6, +12.8, and +7.5 mV (Figure S3 in the Supporting Information), respectively, indicating that these nanoparticles are positively charged in water, and their ζ-potential values decrease with increasing the reaction time. This may result from the fact that the PMPD nanoparticles polymerize together with increasing time and the amine group takes part in the polymerization, consistent with the SEM and dispersibility experimental results. Preparation of Nanoprobe and Its Fluorescence Response to MMP2. As mentioned above, the nanoprobe was synthesized by linking the FITC-labeled peptide to the PMPD nanoparticles prepared with the reaction time of 12 h. To verify the successful preparation of the nanoprobe, a comparative study was made by FT-IR spectroscopy with the following three samples: the first one was PMPD itself; the second one was PMPD separated from the simple mixture of PMPD and FITC-labeled peptide by thoroughly washing with water; the third one was the nanoprobe. As shown in Figure S4 in Supporting Information, the FT-IR spectrum of PMPD separated from the simple mixture is rather similar to that of PMPD itself, indicating that no covalent reaction occurs between PMPD and the peptide, and thus, the peptide can be washed away. However, the FT-IR spectrum of the nanoprobe shows the typical amide I band (1686 cm−1) and amide II band (1529 cm−1) as well as stronger N−H stretching vibration (3415 cm−1), and is quite different from that of PMPD itself or PMPD separated from the simple mixture, clearly demonstrating the preparation of the nanoprobe with the covalent linkage. Then, the analytical performance of the nanoprobe was characterized in detail. Figure 2 shows the fluorescence spectra of the FITC-labeled peptide itself before and after reaction with PMPD. As can be seen, without PMPD the FITC-labeled peptide exhibits strong fluorescence emission at 518 nm characteristic of FITC (curve a in Figure 2). In contrast, reaction of the peptide with 0.03 mg/mL of PMPD leads to about 94% quenching of the fluorescence (curve b in Figure 2),
Figure 3. Effect of the amount of PMPD as a nanoquencher on the fluorescence of different systems: (a) the nanoprobe prepared through covalent bond in the presence of FITC-labeled peptide (100 nM) and different concentrations (0−0.05 mg/mL) of PMPD; (b) simple mixture of FITC-labeled peptide (100 nM) with different concentrations (0−0.05 mg/mL) of PMPD.
of the nanoprobe fluorescence can be quenched when 0.03 mg/ mL of PMPD is used, but in the simple mixture of PMPD and the FITC-labeled peptide, only 37% of the fluorescence is quenched even in the presence of 0.05 mg/mL of PMPD, also showing that much lower background signal can be obtained when the two components are combined through covalent linkage than through physical adsorption. Furthermore, the change of fluorescence intensity of the nanoprobe during its preparation was monitored as a function of time. As shown in Figure S5 (Supporting Information), after PMPD (0.025 mg/mL) is added to the FITC-labeled peptide solution (100 nM), the fluorescence intensity decreases rapidly in the beginning and reaches to a plateau after 90 min. Thus, the reaction time of 90 min was selected for preparing the nanoprobe. The effect of pH on the nanoprobe fluorescence was also studied (Figure S6, Supporting Information). With the change of pH value from 2 to 11, the fluorescence intensities of both the nanoprobe and the simple mixture of the FITC-labeled peptide with PMPD increase. However, the fluorescence of the nanoprobe increases much less than that of the simple mixture,
Figure 2. Fluorescence emission spectra (λex = 480 nm) of different reaction systems: (a) the free FITC-labeled peptide (100 nM); (b) the nanoprobe prepared through the covalent reaction of the FITClabeled peptide (100 nM) with PMPD (0.03 mg/mL); (c) the nanoprobe solution (100 nM) + MMP2 (10 nM); (d) the simple mixture of the FITC-labeled peptide (100 nM) and PMPD (0.03 mg/ mL); (e) the FITC-labeled peptide (100 nM) + MMP2 (10 nM); (f) PMPD (0.03 mg/mL). 7722
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indicating that the nanoprobe is more stable against pH change, including the physiological pH around 7. Fluorescence kinetic curves of the nanoprobe reacting with MMP2 at varied concentrations are depicted in Figure S7 (Supporting Information), which reveals that higher concentrations of MMP2 result in faster cleavage reaction and larger fluorescence enhancement. For MMP2 of no more than 10 nM, the fluorescence intensity increases to a plateau in about 90 min. In contrast, the fluorescence of the nanoprobe without MMP2 (control) hardly changes during the same period of time, also suggesting that the nanoprobe is stable under the experimental conditions. On the basis of the above data, an incubation time of 90 min was employed for the nanoprobe reacting with MMP2 in analytical application. To test the selectivity of the nanoprobe for MMP2, various potentially interfering substances, such as inorganic salts (KCl and MgCl2), glucose, glutamine, vitamin B1, bovine serum albumin, human serum albumin, and some proteases (esterase, glutamine dehydrogenase, MMP1, and thrombin), were examined under the same conditions. As shown in Figure 4,
Figure 5. Fluorescence intensities of the nanoprobe in the presence of varied concentrations of MMP2 (from bottom to top: 0, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 5.0, 10, and 20 nM) in TCNB buffer of pH 7.4 at 37 °C. λex = 480 nm.
which is much lower than those of most of the other reported detection systems for MMP217,21,22,36 To confirm that the fluorescence enhancement was caused solely by MMP2, the effect of a common inhibitor of MMP2, 1,10-phenanthroline, on the activity of the enzyme was investigated. As shown in Figure S9 in the Supporting Information, when 1,10-phenanthroline was mixed with the nanoprobe, there is no obvious change in fluorescence intensity (compare curve a with curve c), indicating that the inhibitor has no effect on the nanoprobe. However, the fluorescence intensity in the presence of both MMP2 and 1,10-phenanthroline (curve d) is much less than that without 1,10-phenanthroline (curve b), suggesting that the activity of MMP2 can be effectively suppressed by 1,10-phenanthroline. Hence, it can be concluded that the fluorescence off−on response indeed arises from the action of MMP2, and such fluorescence suppression may be used to screen new anticancer drugs for MMP2. Determination of MMP2 in Human Serum Samples. With the nanoprobe described here the concentrations of MMP2 in human serum samples were determined. In these experiments, 10 individual serum samples from five healthy people and five CRC patients were analyzed, and the results are shown in Table 1. As is seen, the average concentration of MMP2 in the serum samples of the CRC patients is 3.92 nM, which is roughly 2 times higher than that (2.17 nM) of the healthy people. However, it should be noted that one test result of the serum samples from the CRC patients (CRC 1) shows unexpectedly the same level as those from the healthy people, namely, a false-negative result occurs. This is a quite normal
Figure 4. Fluorescence responses of the nanoprobe (100 nM) to various species: KCl (150 mM), MgCl2 (2.5 mM), glucose (10 mM), glutamine (1 mM), vitamin B1 (1 mM), bovine serum albumin (100 nM), human serum albumin (100 nM), esterase (5 nM), glutamine dehydrogenase (5 nM), MMP1 (5 nM), thrombin (5 nM), and MMP2 (5 nM). ΔF is the difference of the fluorescence intensity of the nanoprobe in the presence and absence of a species. The results are the mean ± standard deviation of three separate measurements. λex/em = 480/518 nm.
the fluorescence intensity enhanced by MMP2 is 6−30 times higher than those by the other species, indicating that the nanoprobe has a pronounced selectivity for MMP2 over the other species tested, even including MMP1 (another member of MMPs). Under the optimized conditions (incubation at 37 °C for 90 min in TCNB buffer of pH 7.4), the fluorescence response of the nanoprobe to MMP2 at varied concentrations is shown in Figure 5. As can be seen, the fluorescence intensity increases with increasing the concentration of MMP2 from 0 to 20 nM, and a linear equation of ΔF = 12.04[MMP2] (nM) + 1.71 (R = 0.995) is obtained in the range of 0.1−2.0 nM MMP2 (Figure S8 in the Supporting Information), where ΔF is the difference of fluorescence intensity of the nanoprobe in the presence and absence of MMP2. The detection limit (3S/m, in which S is the standard deviation of blank measurements, n = 11, and m is the slope of the linear equation)34,35 is determined to be 32 pM,
Table 1. Determination of the MMP2 Level in Human Serum Samples serum samples healthy healthy healthy healthy healthy CRC 1 CRC 2 CRC 3 CRC 4 CRC 5
1 2 3 4 5
current method [nM]a
av value [nM]b
± ± ± ± ± ± ± ± ± ±
2.17
2.74 1.99 1.80 2.51 1.81 2.00 4.91 4.15 3.93 4.60
0.13 0.14 0.09 0.22 0.12 0.16 0.11 0.03 0.17 0.16
3.92
ELISA [nM]a
av value [ nM]b
± ± ± ± ± ± ± ± ± ±
1.99
2.56 1.77 1.61 2.35 1.61 2.11 4.71 3.93 3.73 4.37
0.12 0.12 0.14 0.62 0.11 0.05 0.13 0.19 0.08 0.15
3.77
Mean of three determinations ± standard deviation. bThe average value from five individuals for different groups. a
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observation because a large number of clinical markers show a noticeable percentage of false-positive or false-negative results, which may be caused by individual differences.37−39 In addition, the obtained results by our method were compared with those obtained by a commercial ELISA kit using a Student’s t test.40 It is seen that the agreement between both independent methods was remarkably good, and no significant difference between the two methods was statistically found at the 90% confidence level. Thus, the above results show that the proposed method can be satisfactorily applied to the quantitative determination of MMP2 in the complicated biological samples such as human serum. Determination of MMP2 Secreted by CRC Cells Grown under Different O2 Levels. As is known, hypoxia is an important feature of solid tumor cells and has been considered to be an indicator of an adverse prognosis.41−43 In order to explore the effect of hypoxia on the MMP2 level in CRC cells, we also used the nanoprobe to determine the concentration of MMP2 secreted by CRC cells grown for 12 h under normoxic and different hypoxic conditions, respectively. As shown in Table 2, the concentration of MMP2 increases considerably
20 15 10 5 1
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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a
± ± ± ± ±
AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. *E-mail: [email protected]. Phone: +86-10-62554673. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to the financial support from the NSF of China (Nos. 21275146 and 21321003), the Ministry of Science and Technology (2011CB935800), and the Chinese Academy of Sciences (XDB14030102, KJCX2-EW-N06-01, and CMS-PY201301).
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concn of MMP2 found (nM)a 13.80 20.27 29.00 30.13 37.53
ASSOCIATED CONTENT
S Supporting Information *
Table 2. Determination of MMP2 Secreted by CRC cells Grown under Different O2 Levels O2 level (%)
Article
1.58 1.72 1.25 0.42 2.02
Mean of three determinations ± standard deviation.
(ca. 3-fold) with the decrease of O2 concentration from 20% to 1%, suggesting that hypoxia influences the MMP2 level secreted by CRC cells. This is understandable because the extent of hypoxia and the level of MMP2 are both associated with the progress of tumor cells. Furthermore, this is also another proof that MMP2 is overexpressed in tumors.
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CONCLUSIONS In summary, we have developed a highly sensitive and selective nanoprobe for the determination of MMP2, which is designed through covalent linkage of an FITC-labeled peptide to the surface of electron-rich PMPD. Compared with similar fluorescent MMP2 nanoprobes assembled through physical adsorption, the present nanoprobe produced through covalent bond is significantly more stable and displays a much lower background signal, which enables MMP2 to be determined at a lower detection limit of 32 pM. The nanoprobe has been used to quantify MMP2 in human serum, revealing that the MMP2 level in the serum samples from CRC patients is about 2 times higher than that from healthy people. Moreover, with this nanoprobe MMP2 secreted by CRC cells grown under normoxic and hypoxic conditions has also been monitored, which shows that the cells under hypoxic conditions secrete a higher level of MMP2 than those under normoxic conditions. Compared with ELISA, the most significant advantage of the present method is its simplicity and cost-effectiveness, which makes it being of great potential for the detection of MMP2 in relevant clinical samples. 7724
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(24) Ai, K. L.; Liu, Y. L.; Lu, L. H. J. Am. Chem. Soc. 2009, 131, 9496−9497. (25) Xue, J. P.; Shan, L. L.; Chen, H. Y.; Li, Y.; Zhu, H. Y.; Deng, D. W.; Qian, Z. Y.; Achilefu, S.; Gu, Y. Q. Biosens. Bioelectron. 2013, 41, 71−77. (26) Song, Y. C.; Liu, J. X.; Zhang, Y. Y.; Shi, W.; Ma, H. M. Acta Chim. Sin. 2013, 71, 1607−1610. (27) Zhang, Y. W.; Sun, X. P. Chem. Commun. 2011, 47, 3927−3929. (28) Zhang, Y. W.; Li, H. L.; Luo, Y. L.; Shi, X.; Tian, J. Q.; Sun, X. P. PLoS One 2011, 6, e20569. (29) Wang, Y. H.; Wu, Z. J.; Liu, Z. H. Anal. Chem. 2013, 85, 258− 264. (30) Lee, S.; Cha, E. J.; Park, K.; Lee, S. Y.; Hong, J. K.; Sun, I. C.; Kim, S. Y.; Choi, K.; Kwon, I. C.; Kim, K.; Ahn, C. H. Angew. Chem., Int. Ed. 2008, 47, 2804−2807. (31) Li, Z.; Li, X. H.; Gao, X. H.; Zhang, Y. Y.; Shi, W.; Ma, H. M. Anal. Chem. 2013, 85, 3926−3932. (32) Yuan, L.; Lin, W.; Yang, Y.; Chen, H. J. Am. Chem. Soc. 2012, 134, 1200−1211. (33) Shi, W.; Li, X. H.; Ma, H. M. Angew. Chem., Int. Ed. 2012, 51, 6432−6435. (34) Chen, S. M.; Lu, J. X.; Sun, C. D.; Ma, H. M. Analyst 2010, 135, 577−582. (35) Sun, C. D.; Shi, W.; Song, Y. C.; Chen, W.; Ma, H. M. Chem. Commun. 2011, 47, 8638−8640. (36) Zheng, T. T.; Zhang, R.; Zhang, Q. F.; Tan, T. T.; Zhang, K.; Zhu, J. J.; Wang, H. Chem. Commun. 2013, 49, 7881−7883. (37) Handy, B. LabMedicine 2009, 40, 99−103. (38) Lewandrowski, K.; Chen, A.; Januzzi, J. Am. J. Clin. Pathol. 2002, 118, s93−s99. (39) EL-Ebiary, M.; Soler, N.; Monton, C.; Torres, A. Clin. Intensive Care 1995, 6, 121−126. (40) Lu, J. X.; Sun, C. D.; Chen, W.; Ma, H. M.; Shi, W.; Li, X. H. Talanta 2011, 83, 1050−1056. (41) Kizaka-Kondoh, S.; Inoue, M.; Harada, H.; Hiraoka, M. Cancer Sci. 2003, 94, 1021−1028. (42) Shinohara, E. T.; Maity, A. Curr. Mol. Med. 2009, 9, 1034−1045. (43) He, F. Q.; Deng, X. L.; Wen, B. X.; Liu, Y. P.; Sun, X. R.; Xing, L. G.; Minami, A.; Huang, Y. H.; Chen, Q.; Zanzonico, P. B.; Ling, C. C.; Li, G. C. Cancer Res. 2008, 68, 8597−8606.
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Poly(m‑phenylenediamine)-Based Fluorescent Nanoprobe for Ultrasensitive Detection of Matrix Metalloproteinase 2 Zhe Wang, Xiaohua Li,* Duan Feng, Lihong Li, Wen Shi, and Huimin Ma* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *
ABSTRACT: A novel fluorescence nanoprobe for the detection of matrix metalloproteinase 2 (MMP2) has been developed by engineering the fluorescein isothiocyanate-labeled peptide onto the surface of poly(m-phenylenediamine) (PMPD) nanoparticles through covalent linkage. The nanoprobe itself displays a low background signal due to the effective fluorescence quenching by electronrich PMPD, but its reaction with MMP2 causes 11-fold fluorescence enhancement. Compared with similar fluorescence nanosystems for MMP2 assembled through physical adsorption, the as-prepared nanoprobe is significantly more stable and displays a strikingly higher signal-to-background ratio, which leads to a high sensitivity for MMP2 assay, with a detection limit of 32 pM. Most notably, the nanoprobe has been successfully applied to determine MMP2 in human serum samples, demonstrating that the MMP2 level in serum from colorectal cancer (CRC) patients is 2 times higher than that from healthy people. Moreover, the nanoprobe has also been used to monitor MMP2 secreted by CRC cells that were grown under normoxic and hypoxic conditions, respectively, and the results show that the cells under hypoxic conditions produce higher level of MMP2 than those under normoxic conditions. Our method is simple and can offer a highly sensitive detection of MMP2 in relevant clinical samples. fluorophore is quenched by the nanomaterial and reaction with MMPs results in the cleavage of the peptide bond as a consequence of the specific substrate recognition, accompanying the release of the fluorophore-labeled peptide segment and thereby the recovery of fluorescence. Nevertheless, most of these nanoprobes are constructed through electrostatic adsorption or π−π stacking interaction between the peptides and the nanomaterials, instead of through covalent linkage; such nanoprobes usually exhibit insufficient fluorescence quenching and thus poor sensitivity for analyte detection. Moreover, some of the aforementioned nanomaterials themselves suffer from instability. For instance, gold nanoparticles are apt to precipitate in biological high-salt environments.23−26 Thus, new fluorescent nanoprobes with high sensitivity and stability are still demanded for a more effective MMPs detection in biosystems. Poly(m-phenylenediamine) (PMPD), an electron-rich aromatic polymer, has attracted considerable attention in recent years due to its good water solubility and fluorescence quenching capability, which has led to the development of a few fluorescence off−on nanoprobes for different biomolecules by using the physical adsorption of nucleic acids on PMPD.27−29 However, to the best of our knowledge, PMPD
M
atrix metalloproteinases (MMPs), with Zn2+ as cofactor, are a class of endopeptidases capable of degrading virtually all kinds of extracellular matrix proteins,1,2 and their overexpression has been found to be closely related with tumor invasion, metastasis, and angiogenesis.3 Matrix metalloproteinase 2 (MMP2) is one of the most vital MMPs and has been reported to be abnormal in many types of cancer, such as breast cancer,4 bladder cancer,5 and colorectal cancer.6 Because of the crucial role of MMP2 as a tumor marker, its sensitive detection is of great importance for clinical diagnosis and therapy of cancer at its early stage. The traditional methods for MMPs detection mainly include immunoassay7 and zymography.8,9 Although immunoassay is sensitive and specific for MMPs, it requires costly antibody proteins and also complex washing steps; zymography, instead, is suitable for qualitative but not for quantitative determination. In addition, the gas chromatography/mass spectrometry method has also been reported for quantifying MMPs,10 which, however, is time-consuming and involves complicated procedures for pretreatment of the sample. Alternatively, fluorescence methods have received continually growing interest due to their high sensitivity,11−17 and a number of fluorescence off−on nanoprobes have been proposed to detect MMPs based on the fluorescence quenching property of a variety of nanomaterials, such as metal nanoparticles,18−20 carbon nanoparticles,21 and graphene oxide.22 In these approaches, the fluorophore-labeled peptides and the nanoquenchers are assembled to form a complex as a nanoprobe, in which the fluorescence of the organic © 2014 American Chemical Society
Received: May 5, 2014 Accepted: July 16, 2014 Published: July 16, 2014 7719
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Scheme 1. Schematic Diagram to Illustrate the Fluorescence Response of the Nanoprobe to MMP2
purchased from Sigma-Aldrich and were used as received without further purification. TCNB buffer (50 mM Tris with 10 mM CaCl2, 150 mM NaCl, and 0.05% Brij 35; pH 7.4) was employed in the experiments. MMP2 commercial enzymelinked immunosorbent assay (ELISA) kit was purchased from Shanghai Yanxin Biological Technology Co., Ltd. Ultrapure water (over 18 MΩ·cm) from a Milli-Q Reference system (Millipore) was used throughout. Human sera from five healthy individuals and five CRC patients were provided by Xijing Hospital, and an informed consent was obtained from each donor. Apparatus. Fluorescence measurements were performed on a Hitachi F-4600 spectrophotometer in 10 mm × 10 mm quartz cells (Tokyo, Japan), with a 400 V PMT voltage. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses were made on a JEM-1011 instrument and Hitachi S-4800 field emission scanning electron microscope, respectively. ζ-Potential measurements were performed on a Nano-ZS Zetasizer ZEN3600 (Malvern Instruments Ltd., U.K.). A model HI-98128 pH meter (Hama Instruments Inc., U.S.A.) was employed for pH measurements. UV−vis absorption spectra were recorded in 1 cm quartz cells with a TU-1900 spectrophotometer (Beijing, China). Fourier transform infrared (FT-IR) spectra were taken in KBr disks on a Tensor 27 spectrometer (Bruker, Germany). The absorbance for ELISA analysis was recorded on a microplate reader (BIOTEK Synergy HT, U.S.A.). The density of cells was determined by cell counting chamber [Biosystem Medical Technology (Shanghai) Co., Ltd.]. The incubation was carried out in Shaker incubator (SKY-100C, Shanghai Sukun Industry & Commerce Co., Ltd.). Preparation of PMPD Nanoparticles. PMPD nanoparticles were synthesized by room-temperature chemical oxidation polymerization of m-phenylenediamine monomer with (NH4)2S2O8.27 Typically, 0.6 mL of aqueous solution (0.5 M) of (NH 4 ) 2 S 2 O 8 was mixed with 8.4 mL of Nmethylpyrrolidone at room temperature. To this mixture, 1 mL of aqueous solution (0.1 M) of m-phenylenediamine was then injected under stirring. During the reaction, a large amount of black precipitate was gradually produced. After reaction for different periods of time (6, 12, 24, and 36 h), the resulting precipitate was washed three times with ethanol and water by centrifugation, respectively. Finally, the obtained
has never been used as a component to construct a nanoprobe via covalent linkage. Herein, we report such an attempt by designing a fluorescent off−on nanoprobe for MMP2 assay, in which the fluorescein isothiocyanate (FITC)-labeled peptide containing the core substrate sequence (PLGVR) of MMP22 was directly linked to the amine groups on the surface of the PMPD nanoparticles through covalent bond formation (PMPD−peptide−FITC, Scheme 1). In this nanoprobe, the FITC-labeled peptide serves as a fluorophore, and its fluorescence is efficiently quenched by electron-rich PMPD; upon reaction with MMP2, however, the quenched fluorescence is turned-on through the selective cleavage of the FITClabeled peptide and thus the release of the FITC-containing peptide segment. Most notably, because of electrostatic repulsion between identical charges, the feature of the positive charge of both PMPD and the core peptide (its pI was 9.7522) in the near-neutral media during the nanoprobe preparation effectively eliminates the nonspecific adsorption of the fluorophore on the surface of PMPD, which is also rather favorable for achieving a low background signal. With this nanoprobe the concentrations of MMP2 in the serum samples from colorectal cancer patients as well as those from healthy people have been determined and compared. In addition, the concentrations of MMP2 secreted by colorectal cancer (CRC) cells grown under normoxic and hypoxic conditions have also been monitored, which reveals that hypoxia can elevate the secretory level of MMP2. This method is simple and useful for the detection of MMP2 in clinical samples for the diagnosis of MMP2-related diseases.
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EXPERIMENTAL SECTION Reagents. 4-Aminophenylmercuric acetate, 1,10-phenanthroline, 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), bovine serum albumin, human serum albumin, and MMP2 were obtained from Sigma-Aldrich. Matrix metalloproteinase 1 (MMP1) was obtained from Sino Biological Inc. The FITC-labeled peptide (FITC−GPLGVRG) was purchased from Beijing SBS Genetech Co., Ltd. N-Hydroxysuccinimide (NHS) and tris(hydroxymethyl)-aminomethane (Tris) were purchased from J&K Chemical. CaCl2, NaCl, KCl, MgCl2, glucose, vitamin B1, and glutamine were obtained from Beijing Chemicals, Ltd. All the other reagents employed were 7720
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Figure 1. TEM image (A), FT-IR spectrum (B), and UV−vis absorption spectrum (C) of the PMPD nanoparticles (0.025 mg/mL) prepared with the reaction time of 12 h.
precipitate product was dispersed in water and stored at 4 °C for characterization and further use. Preparation of the PMPD−Peptide−FITC Nanoprobe. EDC (0.8 mg) and NHS (1.4 mg) were first mixed in 1.8 mL of borate buffer (10 mM, pH 7.2) containing 0.23 mg of the FITC-labeled peptide to activate the carboxylic acid groups of the peptide. The mixture was incubated by shaking at room temperature for 2 h in dark. After the pH value of the mixture was adjusted to 7.5 with NaOH (1 mM), an appropriate amount of PMPD (0.25 mg/mL) was added. After reaction at 35 °C for 90 min, 10 mg of bovine serum albumin was added to block the unreacted NHS. Finally, the PMPD−peptide−FITC nanoprobe (referred to the nanoprobe) was centrifuged, washed with water, and then dispersed in 2 mL of TCNB buffer (pH 7.4) for further applications. General Procedure for MMP2 Detection. Prior to use, MMP2 (20 μL, 1.4 μM) was activated by incubating with an equal volume of 4-aminophenylmercuric acetate (2.5 mM) in TCNB buffer (pH 7.4) at 37 °C for 1 h following the known procedure.30 Then, different concentrations of the activated MMP2 were incubated with the nanoprobe at 37 °C for 90 min, followed by measuring the fluorescence of the samples at room temperature with λex/em = 480/518 nm. Determination of MMP2 in Human Serum Samples by Nanoprobe. Human serum samples were diluted 10 times before analysis. Then appropriate aliquots of the serum samples were transferred to the nanoprobe solution, and MMP2 in the diluted serum samples was quantified following the general procedure as described above. Determination of MMP2 in Human Serum Samples by ELISA. The concentrations of MMP2 in human serum samples were also determined by measuring the absorbance values at 450 nm using a commercial ELISA kit. In this detection, the standard curve (OD = 0.43[MMP2] (nM) + 0.84, R = 0.987) was first obtained following the direction of the kit in the MMP2 concentration range from 0 to 2 nM. Then 50 μL of the human serum sample was added to the ELISA kit wells, and 100 μL of horseradish peroxidase conjugate reagent was introduced into each well, followed by incubation at 37 °C for 60 min. After all of the samples were washed five times with 400 μL of wash solution, 50 μL of chromogen solution A and 50 μL of chromogen solution B were added to each well, followed by incubation at 37 °C for 15 min in dark. Finally, 50 μL of the stop solution was added to each well to stop the reaction, and the optical density was read immediately on a microplate reader at 450 nm. Determination of MMP2 Secreted by CRC Cells Grown under Different Oxygen Levels. CRC cells, with a density of about 1.0 × 106 cells/mL, were grown at 37 °C for 12 h on
glass-bottom culture dishes (MatTek Co.) in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin−streptomycin in a humidified incubator under normoxic (95% air and 5% CO2, i.e., 20% O2) and different hypoxic (80% N2, 15% O2, and 5% CO2; 85% N2, 10% O2, and 5% CO2; 90% N2, 5% O2, and 5% CO2; or 94% N2, 1% O2, and 5% CO2) conditions,31,32 respectively; the cell densities in the culture media were measured by the cell counting chamber. Then, an appropriate volume (typically 50 μL) of the culture media was collected and transferred to the nanoprobe solution. The MMP2 concentration in the solution was quantified following the general procedure as described above.
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RESULTS AND DISCUSSION Preparation and Characterization of PMPD Nanoparticles. The PMPD nanoparticles were synthesized following the procedure as described above and characterized by SEM analysis. As shown in Figure S1 in the Supporting Information, the size of the PMPD nanoparticles prepared with the reaction time of 12 h becomes smaller than that of 6 h, which may result from the fact that PMPD nanoparticles with a reaction time of 6 h are loosely assembled, and then they can form tightly organized structure until 12 h; further increase of the reaction time to 24 or 36 h does not lead to uniform nanoparticles (Supporting Information Figure S1, parts C and D), which indicates that the PMPD nanoparticles can polymerize together with longer reaction time, generating irregular blocks. Because small size of nanoparticles is beneficial for uniform distribution in homogeneous bioassays,33 PMPD prepared with the reaction time of 12 h was used for the nanoprobe construction. Figure 1A shows the TEM image of such PMPD. As is seen, the nanoparticles have a rather uniform spherical shape, with an average diameter of ca. 20 nm. Figure 1B depicts their FT-IR spectrum, which indicates the existence of Ar−NH2 moiety on the surface of PMPD, as evidenced by N−H stretching (3138 cm−1), N−H bending (1624 cm−1), and C−N stretching (1401 and 1117 cm−1). In addition, the UV− vis absorption spectrum (Figure 1C) shows that PMPD exhibits a wide range of absorption wavelengths from 200 to 800 nm,29 which can overlap the fluorescence spectra of most of fluorochromes, suggesting that the electron-rich PMPD is a good fluorescence quencher. The PMPD nanoparticles prepared with the reaction time of 12 h possess excellent dispersibility in water, whereas the others are easier to precipitate, though PMPD prepared with the reaction time of 6 h displays a relatively slow precipitation (Figure S2A in the Supporting Information). Furthermore, by 7721
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revealing that the PMPD nanoparticles linked to the FITClabeled peptide can quench the fluorescence very efficiently as expected. However, addition of MMP2 (10 nM) to the nanoprobe solution causes 11-fold fluorescence enhancement (curve c in Figure 2), which is ascribed to the specific cleavage of the substrate (the peptide) by MMP2 and the release of the FITC-containing peptide segment. On the other hand, the quenching efficiency of the nanoprobe was compared with that of the simple mixture of PMPD and the FITC−peptide. As shown in Figure 2 (curve d), about 29% of the fluorescence is quenched in the simple mixture, which is much less than that of the nanoprobe, clearly indicating that covalent linkage of the peptide to PMPD results in more efficient fluorescence quenching than physical adsorption. It is also important to note that the fluorescence of the free FITC-labeled peptide is scarcely influenced by the addition of MMP2 in the absence of PMPD (curve e in Figure 2), and PMPD itself exhibits nearly no fluorescence (curve f in Figure 2). All the above observations indicate that this fluorescent nanoprobe is wellsuitable for MMP2 detection. The amount of PMPD as a nanoquencher was optimized for preparing the nanoprobe. As shown in Figure 3 (curve a), 94%
dispersing PMPD prepared with 12 h in water, the resulting colloidal solution is stable at room temperature at least for 4 days because no obvious precipitation is observed in such solution (Figure S2B, Supporting Information). In addition, the ζ-potential values of the PMPD nanomaterials prepared with the reaction time of 6, 12, 24, and 36 h were determined to be +19.7, +16.6, +12.8, and +7.5 mV (Figure S3 in the Supporting Information), respectively, indicating that these nanoparticles are positively charged in water, and their ζ-potential values decrease with increasing the reaction time. This may result from the fact that the PMPD nanoparticles polymerize together with increasing time and the amine group takes part in the polymerization, consistent with the SEM and dispersibility experimental results. Preparation of Nanoprobe and Its Fluorescence Response to MMP2. As mentioned above, the nanoprobe was synthesized by linking the FITC-labeled peptide to the PMPD nanoparticles prepared with the reaction time of 12 h. To verify the successful preparation of the nanoprobe, a comparative study was made by FT-IR spectroscopy with the following three samples: the first one was PMPD itself; the second one was PMPD separated from the simple mixture of PMPD and FITC-labeled peptide by thoroughly washing with water; the third one was the nanoprobe. As shown in Figure S4 in Supporting Information, the FT-IR spectrum of PMPD separated from the simple mixture is rather similar to that of PMPD itself, indicating that no covalent reaction occurs between PMPD and the peptide, and thus, the peptide can be washed away. However, the FT-IR spectrum of the nanoprobe shows the typical amide I band (1686 cm−1) and amide II band (1529 cm−1) as well as stronger N−H stretching vibration (3415 cm−1), and is quite different from that of PMPD itself or PMPD separated from the simple mixture, clearly demonstrating the preparation of the nanoprobe with the covalent linkage. Then, the analytical performance of the nanoprobe was characterized in detail. Figure 2 shows the fluorescence spectra of the FITC-labeled peptide itself before and after reaction with PMPD. As can be seen, without PMPD the FITC-labeled peptide exhibits strong fluorescence emission at 518 nm characteristic of FITC (curve a in Figure 2). In contrast, reaction of the peptide with 0.03 mg/mL of PMPD leads to about 94% quenching of the fluorescence (curve b in Figure 2),
Figure 3. Effect of the amount of PMPD as a nanoquencher on the fluorescence of different systems: (a) the nanoprobe prepared through covalent bond in the presence of FITC-labeled peptide (100 nM) and different concentrations (0−0.05 mg/mL) of PMPD; (b) simple mixture of FITC-labeled peptide (100 nM) with different concentrations (0−0.05 mg/mL) of PMPD.
of the nanoprobe fluorescence can be quenched when 0.03 mg/ mL of PMPD is used, but in the simple mixture of PMPD and the FITC-labeled peptide, only 37% of the fluorescence is quenched even in the presence of 0.05 mg/mL of PMPD, also showing that much lower background signal can be obtained when the two components are combined through covalent linkage than through physical adsorption. Furthermore, the change of fluorescence intensity of the nanoprobe during its preparation was monitored as a function of time. As shown in Figure S5 (Supporting Information), after PMPD (0.025 mg/mL) is added to the FITC-labeled peptide solution (100 nM), the fluorescence intensity decreases rapidly in the beginning and reaches to a plateau after 90 min. Thus, the reaction time of 90 min was selected for preparing the nanoprobe. The effect of pH on the nanoprobe fluorescence was also studied (Figure S6, Supporting Information). With the change of pH value from 2 to 11, the fluorescence intensities of both the nanoprobe and the simple mixture of the FITC-labeled peptide with PMPD increase. However, the fluorescence of the nanoprobe increases much less than that of the simple mixture,
Figure 2. Fluorescence emission spectra (λex = 480 nm) of different reaction systems: (a) the free FITC-labeled peptide (100 nM); (b) the nanoprobe prepared through the covalent reaction of the FITClabeled peptide (100 nM) with PMPD (0.03 mg/mL); (c) the nanoprobe solution (100 nM) + MMP2 (10 nM); (d) the simple mixture of the FITC-labeled peptide (100 nM) and PMPD (0.03 mg/ mL); (e) the FITC-labeled peptide (100 nM) + MMP2 (10 nM); (f) PMPD (0.03 mg/mL). 7722
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indicating that the nanoprobe is more stable against pH change, including the physiological pH around 7. Fluorescence kinetic curves of the nanoprobe reacting with MMP2 at varied concentrations are depicted in Figure S7 (Supporting Information), which reveals that higher concentrations of MMP2 result in faster cleavage reaction and larger fluorescence enhancement. For MMP2 of no more than 10 nM, the fluorescence intensity increases to a plateau in about 90 min. In contrast, the fluorescence of the nanoprobe without MMP2 (control) hardly changes during the same period of time, also suggesting that the nanoprobe is stable under the experimental conditions. On the basis of the above data, an incubation time of 90 min was employed for the nanoprobe reacting with MMP2 in analytical application. To test the selectivity of the nanoprobe for MMP2, various potentially interfering substances, such as inorganic salts (KCl and MgCl2), glucose, glutamine, vitamin B1, bovine serum albumin, human serum albumin, and some proteases (esterase, glutamine dehydrogenase, MMP1, and thrombin), were examined under the same conditions. As shown in Figure 4,
Figure 5. Fluorescence intensities of the nanoprobe in the presence of varied concentrations of MMP2 (from bottom to top: 0, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 5.0, 10, and 20 nM) in TCNB buffer of pH 7.4 at 37 °C. λex = 480 nm.
which is much lower than those of most of the other reported detection systems for MMP217,21,22,36 To confirm that the fluorescence enhancement was caused solely by MMP2, the effect of a common inhibitor of MMP2, 1,10-phenanthroline, on the activity of the enzyme was investigated. As shown in Figure S9 in the Supporting Information, when 1,10-phenanthroline was mixed with the nanoprobe, there is no obvious change in fluorescence intensity (compare curve a with curve c), indicating that the inhibitor has no effect on the nanoprobe. However, the fluorescence intensity in the presence of both MMP2 and 1,10-phenanthroline (curve d) is much less than that without 1,10-phenanthroline (curve b), suggesting that the activity of MMP2 can be effectively suppressed by 1,10-phenanthroline. Hence, it can be concluded that the fluorescence off−on response indeed arises from the action of MMP2, and such fluorescence suppression may be used to screen new anticancer drugs for MMP2. Determination of MMP2 in Human Serum Samples. With the nanoprobe described here the concentrations of MMP2 in human serum samples were determined. In these experiments, 10 individual serum samples from five healthy people and five CRC patients were analyzed, and the results are shown in Table 1. As is seen, the average concentration of MMP2 in the serum samples of the CRC patients is 3.92 nM, which is roughly 2 times higher than that (2.17 nM) of the healthy people. However, it should be noted that one test result of the serum samples from the CRC patients (CRC 1) shows unexpectedly the same level as those from the healthy people, namely, a false-negative result occurs. This is a quite normal
Figure 4. Fluorescence responses of the nanoprobe (100 nM) to various species: KCl (150 mM), MgCl2 (2.5 mM), glucose (10 mM), glutamine (1 mM), vitamin B1 (1 mM), bovine serum albumin (100 nM), human serum albumin (100 nM), esterase (5 nM), glutamine dehydrogenase (5 nM), MMP1 (5 nM), thrombin (5 nM), and MMP2 (5 nM). ΔF is the difference of the fluorescence intensity of the nanoprobe in the presence and absence of a species. The results are the mean ± standard deviation of three separate measurements. λex/em = 480/518 nm.
the fluorescence intensity enhanced by MMP2 is 6−30 times higher than those by the other species, indicating that the nanoprobe has a pronounced selectivity for MMP2 over the other species tested, even including MMP1 (another member of MMPs). Under the optimized conditions (incubation at 37 °C for 90 min in TCNB buffer of pH 7.4), the fluorescence response of the nanoprobe to MMP2 at varied concentrations is shown in Figure 5. As can be seen, the fluorescence intensity increases with increasing the concentration of MMP2 from 0 to 20 nM, and a linear equation of ΔF = 12.04[MMP2] (nM) + 1.71 (R = 0.995) is obtained in the range of 0.1−2.0 nM MMP2 (Figure S8 in the Supporting Information), where ΔF is the difference of fluorescence intensity of the nanoprobe in the presence and absence of MMP2. The detection limit (3S/m, in which S is the standard deviation of blank measurements, n = 11, and m is the slope of the linear equation)34,35 is determined to be 32 pM,
Table 1. Determination of the MMP2 Level in Human Serum Samples serum samples healthy healthy healthy healthy healthy CRC 1 CRC 2 CRC 3 CRC 4 CRC 5
1 2 3 4 5
current method [nM]a
av value [nM]b
± ± ± ± ± ± ± ± ± ±
2.17
2.74 1.99 1.80 2.51 1.81 2.00 4.91 4.15 3.93 4.60
0.13 0.14 0.09 0.22 0.12 0.16 0.11 0.03 0.17 0.16
3.92
ELISA [nM]a
av value [ nM]b
± ± ± ± ± ± ± ± ± ±
1.99
2.56 1.77 1.61 2.35 1.61 2.11 4.71 3.93 3.73 4.37
0.12 0.12 0.14 0.62 0.11 0.05 0.13 0.19 0.08 0.15
3.77
Mean of three determinations ± standard deviation. bThe average value from five individuals for different groups. a
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observation because a large number of clinical markers show a noticeable percentage of false-positive or false-negative results, which may be caused by individual differences.37−39 In addition, the obtained results by our method were compared with those obtained by a commercial ELISA kit using a Student’s t test.40 It is seen that the agreement between both independent methods was remarkably good, and no significant difference between the two methods was statistically found at the 90% confidence level. Thus, the above results show that the proposed method can be satisfactorily applied to the quantitative determination of MMP2 in the complicated biological samples such as human serum. Determination of MMP2 Secreted by CRC Cells Grown under Different O2 Levels. As is known, hypoxia is an important feature of solid tumor cells and has been considered to be an indicator of an adverse prognosis.41−43 In order to explore the effect of hypoxia on the MMP2 level in CRC cells, we also used the nanoprobe to determine the concentration of MMP2 secreted by CRC cells grown for 12 h under normoxic and different hypoxic conditions, respectively. As shown in Table 2, the concentration of MMP2 increases considerably
20 15 10 5 1
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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a
± ± ± ± ±
AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. *E-mail: [email protected]. Phone: +86-10-62554673. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to the financial support from the NSF of China (Nos. 21275146 and 21321003), the Ministry of Science and Technology (2011CB935800), and the Chinese Academy of Sciences (XDB14030102, KJCX2-EW-N06-01, and CMS-PY201301).
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concn of MMP2 found (nM)a 13.80 20.27 29.00 30.13 37.53
ASSOCIATED CONTENT
S Supporting Information *
Table 2. Determination of MMP2 Secreted by CRC cells Grown under Different O2 Levels O2 level (%)
Article
1.58 1.72 1.25 0.42 2.02
Mean of three determinations ± standard deviation.
(ca. 3-fold) with the decrease of O2 concentration from 20% to 1%, suggesting that hypoxia influences the MMP2 level secreted by CRC cells. This is understandable because the extent of hypoxia and the level of MMP2 are both associated with the progress of tumor cells. Furthermore, this is also another proof that MMP2 is overexpressed in tumors.
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CONCLUSIONS In summary, we have developed a highly sensitive and selective nanoprobe for the determination of MMP2, which is designed through covalent linkage of an FITC-labeled peptide to the surface of electron-rich PMPD. Compared with similar fluorescent MMP2 nanoprobes assembled through physical adsorption, the present nanoprobe produced through covalent bond is significantly more stable and displays a much lower background signal, which enables MMP2 to be determined at a lower detection limit of 32 pM. The nanoprobe has been used to quantify MMP2 in human serum, revealing that the MMP2 level in the serum samples from CRC patients is about 2 times higher than that from healthy people. Moreover, with this nanoprobe MMP2 secreted by CRC cells grown under normoxic and hypoxic conditions has also been monitored, which shows that the cells under hypoxic conditions secrete a higher level of MMP2 than those under normoxic conditions. Compared with ELISA, the most significant advantage of the present method is its simplicity and cost-effectiveness, which makes it being of great potential for the detection of MMP2 in relevant clinical samples. 7724
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