Sensitive and Selective Near-Infrared Fluorescent Off–On Probe and

Article pubs.acs.org/ac

Sensitive and Selective Near-Infrared Fluorescent Off−On Probe and Its Application to Imaging Different Levels of β‑Lactamase in Staphylococcus aureus Lihong Li, Zhao Li, Wen Shi,* Xiaohua Li, 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 highly sensitive and selective near-infrared (NIR) fluorescent probe, (E)-2-(2-(6-((2-carboxy-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl)methoxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-1-propyl-3H-indol-1ium (1), is developed for the determination of β-lactamase. The probe is designed and synthesized by incorporating the specific substrate (cephalosporin) of β-lactamase into a stable hemicyanine skeleton. The fluorescence of 1 itself is very weak due to the alkylation of the hydroxyl group of the hemicyanine fluorophore; however, β-lactamase can selectively react with its substrate (βlactam ring) in the probe, thereby causing a spontaneous fragmentation. This action leads to the release of the fluorophore and a large fluorescence enhancement at 707 nm (λex = 680 nm). Under the optimized conditions, the fluorescence response of probe 1 is directly proportional to the concentration of β-lactamase in the range of 0.05−2 nM, with a detection limit of 0.02 nM. The validity of the probe has been confirmed by determining βlactamase in human urine samples in comparison with that determined by iodimetry. Moreover, by taking advantage of its high sensitivity and NIR emission feature, the probe has also been utilized to image β-lactamase in three types of Staphylococcus aureus, including methicillin-resistant S. aureus ATCC BAA44, penicillin-resistant strain ATCC 11632, and penicillin-susceptible strain ATCC 29213, which clearly reveals the significantly different expression levels of β-lactamase in these S. aureus. knowledge, this probe is the first one for β-lactamase assay with an emission wavelength over 700 nm. The probe was designed by incorporating the specific substrate (cephalosporin) of βlactamase into a stable hemicyanine skeleton (2), which can be generated via the decomposition of its unstable precursor IR780 but still possesses an NIR feature (Scheme S1, Supporting Information). The probe itself is nearly nonfluorescent due to the hydroxyl alkylation of 2, which is favorable for achieving a low background signal; upon treatment with β-lactamase, however, the lactam ring of the cephalosporin moiety is opened, followed by a spontaneous cleavage of the ether bond and thus the release of free fluorophore 2 (Scheme 1). As a result, the fluorescence of the reaction system is turned on, which leads to the development of a sensitive and selective method for assaying β-lactamase. Moreover, the probe has been applied to both the determination of β-lactamase in human urine samples and the imaging of β-lactamase in different Staphylococcus aureus strains, revealing their significantly different expression levels of β-lactamase.

he β-lactam antibiotics have been used to treat bacterial infections clinically for a long time because of their high efficacy and broad-spectrum applicability. However, the bacterial resistance to β-lactam antibiotics is an unavoidable consequence of the usage of these drugs and has become a severe problem.1,2 Among the mechanisms that account for the antibiotic resistance, the production of β-lactamase by bacteria has been regarded as the most significant one.2−4 Thus, it is of great importance to develop analytical methods toward βlactamase for the diagnosis and treatment of bacterial infections. There are several methods for the detection of βlactamase,5−28 among which fluorescent probes have attracted a lot of attention due to their great temporal and spatial sampling capability as well as easy operation.29,30 In particular, fluorescent probes with a near-infrared (NIR) analytical wavelength are rather desirable for practical applications because of their minimal interference from biological samples.31−33 However, NIR fluorescent probes for β-lactamase are still rare.20,27,28 Herein we report (E)-2-(2-(6-((2-carboxy8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2en-3-yl)methoxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-1-propyl-3H-indol-1-ium (1) as a sensitive and selective NIR probe for β-lactamase (Scheme 1). To the best of our

T

© 2014 American Chemical Society

Received: April 9, 2014 Accepted: May 20, 2014 Published: May 20, 2014 6115

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incubator (SKY-100C, Shanghai Sukun Industry & Commerce Co., Ltd.). Synthesis of Probe 1. The synthetic route of probe 1 is shown in Scheme S1 in the Supporting Information. First, resorcinol (248 mg, 2.3 mmol) and K2CO3 (311 mg, 2.3 mmol) were placed in a flask containing CH3CN (5 mL), and the mixture was stirred for 10 min at ambient temperature under an argon atmosphere. Then IR-780 iodide (600 mg, 0.9 mmol) in CH3CN (5 mL) was introduced to the mixture, followed by heating at 50 °C for 2 h. After removal of the solvent from the reaction mixture under reduced pressure, the crude product was purified by silica gel chromatography with CH2Cl2/CH3OH (100/1 to 10/1, v/v) as the eluent, affording compound 2 as a blue solid (231 mg, yield 62%). 1H and 13C NMR spectra are shown in Figures S1 and S2 (Supporting Information), respectively. 1H NMR (400 MHz, 298 K, CDCl3): δ 8.03 (d, 1H, J = 14.2 Hz), 7.28−7.23 (m, 3H), 7.19 (d, 1H, J = 9.2 Hz), 7.04 (t, 1H, J = 7.6 Hz), 6.81 (d, 1H, J = 7.6 Hz), 6.74 (d, 1H, J = 8.8 Hz), 6.51 (s, 1H), 5.61 (d, 1H, J = 14.2 Hz), 3.76 (t, 2H, J = 7.2 Hz), 2.67 (t, 2H, J = 6.0 Hz), 2.61 (t, 2H, J = 6.0 Hz), 1.91−1.78 (m, 4H), 1.66 (s, 6H), 1.06 (t, 3H, J = 7.6 Hz). 13C NMR (400 MHz, 298 K, CD3OD): δ 174.5, 173.7, 163.8, 158.5, 144.0, 142.2, 141.6, 140.3, 131.1, 129.9, 126.1, 123.5, 123.2, 121.9, 116.2, 115.9, 112.0, 103.6, 100.1, 50.4, 46.6, 29.6, 29.0, 25.3, 22.1, 21.8, 11.9. HR-ESI-MS: m/z calcd for 2 (C28H30NO2+, M+), 412.2271; found, 412.2268. Second, a mixture of PACBE (666 mg, 1.3 mmol), sodium iodide (187 mg, 1.3 mmol), and K2CO3 (173 mg, 1.3 mmol) in 5 mL of CH3CN was stirred for 10 min at ambient temperature. Then the solution of 2 (206 mg, 0.5 mmol) in CH3CN (3 mL) was added dropwise. The mixture was stirred at ambient temperature for 2 h. After removal of the solvent under reduced pressure, the crude product was dissolved in a mixture of water (10 mL) and ethyl acetate (15 mL), followed by treatment of the water phase with ethyl acetate (15 mL × 2). Then the combined organic phase was washed with 10% Na2S2O3 aqueous solution (25 mL × 2) and brine (25 mL × 2) and dried over Na2SO4. The solvent was removed by evaporation under reduced pressure, and the residue was subjected to silica gel chromatography with CH2Cl2/CH3OH (100/1 to 25/1, v/v) as the eluent, affording compound 3 as a blue solid (147 mg, yield 32%). 1H and 13C NMR spectra are depicted in Figures S3 and S4 (Supporting Information), respectively. 1H NMR (300 MHz, 298 K, DMSO-d6): δ 9.25 (t, 1H, J = 7.8 Hz), 8.61 (d, 1H, J = 14.7 Hz), 7.74−6.80 (m, 24H), 6.64 (d, 1H, J = 15.0 Hz), 5.54 (q, 1H, J = 3.9 Hz), 5.23 (t, 1H, J = 3.9 Hz), 4.88−4.71 (m, 2H), 4.44 (t, 2H, J = 6.6 Hz), 3.78−3.50 (m, 4H), 2.73−2.71 (m, 4H), 1.86−1.76 (m, 10H), 1.02 (t, 3H, J = 7.2 Hz). 13C NMR (300 MHz, 298 K, DMSO-d6): δ 178.0, 171.5, 166.6, 165.8, 164.5, 161.3, 160.8, 154.1, 142.5, 142.0, 140.2, 140.1, 136.2, 129.6, 129.5, 129.0, 128.9, 128.7, 128.4, 127.5, 127.0, 126.6, 123.5, 123.2, 119.2, 116.2, 114.5, 113.9, 105.1, 102.2, 79.2, 78.8, 61.4, 53.6, 50.9, 46.7, 42.1, 29.0, 28.1, 24.2, 21.5, 11.6. HR-ESI-MS: m/z calcd for 3 (C57H54N3O6S+, M+), 908.3728; found, 908.3708. Finally, probe 1 was prepared as follows. Compound 3 (36 mg, 0.04 mmol), anisole (50 μL), and trifluoroacetic acid (200 μL) in 1 mL of dichloromethane was mixed at 0 °C and stirred for 1 h at room temperature. After removal of the solvent under reduced pressure, the crude product was purified by preparative HPLC, affording probe 1 as a blue solid (16 mg, 54%). 1H and 13 C NMR spectra are shown in Figures S5 and S6 (Supporting Information), respectively. 1H NMR (400 MHz, 298 K,

Scheme 1. Structure of Probe 1 and Its Mechanism of Response to β-Lactamase

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EXPERIMENTAL SECTION Reagents. IR-780 iodide, resorcinol, potassium clavulanate, and sulbactam were purchased from Sigma-Aldrich, and 7(phenylacetamido)-3-(chloromethyl)cephalosporanic acid benzhydryl ester (PACBE) was purchased from Struchem Co., Ltd. β-Lactamase, urea, uric acid, oxalic acid, and creatinine were obtained from Aladdin. KCl, CaCl2, MgCl2, FeCl3, ZnSO4, and CuSO4 were purchased from Beijing Chemicals, Ltd. Phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) solution was purchased from Invitrogen. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was purchased from Serva Electrophoresis GmbH. The stock solution (1 mM) of probe 1 was made by dissolving 1 in dimethyl sulfoxide (DMSO). The solution of β-lactamase was prepared in pure water and stored at 4 °C in a refrigerator. Ultrapure water (over 18 MΩ·cm) from a Milli-Q reference system (Millipore) was employed in all experiments. All the other reagents employed were of analytical grade. Apparatus. 1H and 13C NMR spectra were recorded on a Bruker DMX-300 or DMX-400 spectrometer. Electrospray ionization mass spectrometry (ESI-MS) was performed in positive mode with a Shimadzu LC−MS 2010A instrument (Kyoto, Japan). High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on an APEX IV FTMS instrument (Bruker, Daltonics). High-performance liquid chromatography (HPLC) analyses were performed as described previously.34 pH measurements were made on a model HI-98128 pH meter (Hanna Instruments Inc., Woonsocket, RI). Absorption spectra were measured with a TU-1900 spectrophotometer (Beijing, China) in 1 cm quartz cells. Fluorescence spectra were collected on a Hitachi F-4600 spectrofluorimeter in 1 × 1 cm quartz cells with both excitation and emission slit widths of 10 nm. Fluorescence imaging was done on an FV 1000-IX81 confocal laser scanning microscope (Olympus, Japan). The incubation was carried out in a shaker 6116

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for β-lactamase have a relatively short emission wavelength ( 700 nm) as a signal unit with cephalosporin as a recognition unit. In addition, a docking study was conducted to estimate the binding ability between βlactamase and the substrate (probe 1) by the Surflex-dock module (SYBYL version 1.1, Tripos Inc.), which indicates a strong binding between them as evidenced by the docking score of 7.44 (−(log Kd)). Moreover, the ribbon model created by Pymol shows six potential hydrogen bonds between the enzyme and probe 1 (Figure S8, Supporting Information), which also supports the strong binding. These results show that our design approach for the NIR probe is feasible, and its preparation can be achieved via the route depicted in Scheme S1 (Supporting Information). Fluorescent Response of Probe 1 to β-Lactamase. The fluorescent properties of probe 1 in the absence and presence of β-lactamase are shown in Figure 1. As is seen, probe 1 itself

DMSO-d6): δ 8.99 (d, 1H, J = 8.4 Hz), 8.67 (d, 1H, J = 14.8 Hz), 7.90−7.05 (m, 13H), 6.57 (d, 1H, J = 14.8 Hz), 5.48 (dd, 1H, J = 4.8, 3.6 Hz), 5.38 (d, 1H, J = 12.0 Hz), 4.95−4.90 (m, 2H), 4.39 (t, 2H, J = 7.2 Hz), 3.55−3.44 (m, 4H), 2.72−2.64 (m, 4H), 1.84−1.69 (m, 10H), 0.99 (t, 3H, J = 7.2 Hz). 13C NMR (300 MHz, 298 K, DMSO-d6): δ 178.1, 171.4, 169.2, 163.5, 161.9, 160.9, 154.2, 145.3, 142.6, 141.9, 136.3, 130.0, 128.7, 126.9, 123.2, 122.0, 120.0, 116.0, 114.4, 113.7, 104.9, 102.1, 70.3, 60.8, 53.5, 50.9, 46.5, 42.0, 29.5, 28.1, 24.1, 21.4, 11.5. HR-ESI-MS: m/z calcd for 1 (C44H44N3O6S+, M+), 742.2945; found, 742.2929. In addition, the purity of probe 1 was further confirmed by HPLC (Figure S7, Supporting Information). Modeling of the Binding Affinity between Probe 1 and β-Lactamase. The binding affinity between 1 and βlactamase was evaluated as described previously.34 The crystal structure of β-lactamase was obtained from the PDB under code 1CK3. General Procedure for β-Lactamase Assay. Unless otherwise noted, the fluorescence of 1 (10 μM) reacting with β-lactamase was measured in pH 7.4 PBS as follows. In a test tube, 4 mL of PBS and 50 μL of the stock solution of 1 were mixed, followed by addition of β-lactamase solution. The final volume was adjusted to 5 mL with PBS. After reaction at 37 °C for 15 min in a shaker incubator, a 3 mL portion of the reaction solution was transferred to a 1 cm quartz cell to measure fluorescence with λex/em = 680 nm/707 nm. At the same time, a solution containing no β-lactamase (control) was made and determined for comparison under the same conditions. Determination of β-Lactamase by Iodimetry. The determination of β-lactamase by iodimetry was done according to the known method.35,36 In brief, the solution of penicillin and β-lactamase was mixed and incubated at 30 °C for 20 min. Then a 3 mL portion of the reaction solution was added to 25 mL of iodine reagent (5 mM, pH 4.5), followed by the resulting solution standing at room temperature for 15 min. The solution was then titrated with Na2S2O3 solution (10 mM). When the color of the reaction solution turned light yellow, 2−3 drops of starch solution were introduced, and the titration was continued until the violet-blue color became colorless. Fluorescence Imaging of β-Lactamase in S. aureus. Different S. aureus strains, including methicillin-resistant S. aureus (MRSA) ATCC BAA44, penicillin-resistant strain ATCC 11632, and penicillin-susceptible strain ATCC 29213, were first grown at 37 °C in tryptone soya broth for 12 h. Then the bacteria were harvested by centrifugation (4000 rpm for 5 min) and washed three times with PBS. The supernatant was discarded, and the remaining bacteria were resuspended in PBS with an OD600 of 0.5. Then a 1 mL portion of the bacterial suspension was incubated with 10 μL of the stock solution of probe 1 for 30 min at 37 °C. After being washed with PBS, the bacterial cells were spotted on coverslips (Fisherbrand, 24 × 50 mm) and covered by smaller coverslips (20 × 20 mm). Then cell imaging was performed on an FV 1000-IX81 confocal laser scanning microscope with 635 nm excitation and 650−750 nm emission.

Figure 1. Fluorescence emission spectra of 1 (10 μM) before (a) and after (b) reaction with β-lactamase (200 nM) at 37 °C for 15 min in PBS (pH 7.4). λex = 680 nm.

has almost no fluorescence in the NIR region due to the hydroxyl alkylation of compound 2. However, addition of βlactamase produces a large fluorescence enhancement at 707 nm, which resembles the characteristic emission of 2 (Figure S9A, Supporting Information). Meanwhile, the maximum absorption peak of the reaction system is red-shifted from 600 to 665 nm (Figure S9B), which is also similar to the characteristic absorption spectrum of 2 (Figure S9A). These observations suggest that the reaction of 1 with β-lactamase causes the release of fluorophore 2, and the ESI-MS analysis further proves the generation of 2 (m/z 412.5 [M]+; Figure S10, Supporting Information). The fluorescence kinetic study of probe 1 reacting with βlactamase shows a fast enzymatic hydrolysis because the fluorescence signal at 707 nm reaches an approximate plateau in 15 min (Figure S11 in the Supporting Information). Moreover, as can be seen from Figure S11, higher concentrations of β-lactamase lead to faster reactions and larger fluorescence enhancement, but the fluorescence intensity of probe 1 barely changes during the same period of time in the absence of β-lactamase, suggesting the good stability of 1. Then the examination of the pH effect reveals that the maximum fluorescence enhancement is achieved in neutral media of about pH 7 (Figure S12, Supporting Information), which is rather suitable for biological applications. On the other hand, the fluorescence of probe 1 itself is scarcely affected by a temperature change from 20 to 40 °C, while that of the

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RESULTS AND DISCUSSION Design and Synthesis of the NIR Probe. Cephalosporin, a member of the β-lactam antibiotics, holds a lactam ring, which can be specifically hydrolyzed by β-lactamase. Therefore, cephalosporin could serve as an excellent recognition unit in the probe construction. Unfortunately, all the reported probes 6117

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reaction system can be turned on effectively. Therefore, the determination of β-lactamase by probe 1 can be performed under physiological conditions (37 °C and pH 7.4). Under the above-determined condition, the fluorescence response of probe 1 to β-lactamase exhibits a good linearity (Figure 2) in the concentration range of 0.05−2 nM with the

Figure 3. Fluorescence response of probe 1 (10 μM) to various substances in the absence (white) and presence (gray) of β-lactamase (5.0 nM): (a) blank; (b) KCl (150 mM); (c) CaCl2 (2.5 mM); (d) MgCl2 (2.5 mM); (e) FeCl3 (0.1 mM); (f) ZnSO4 (0.1 mM); (g) CuSO4 (0.1 mM); (h) glucose (10 mM); (i) vitamin C (1 mM); (j) alanine (1 mM); (k) aspartic acid (1 mM); (l) cysteine (1 mM); (m) glutathione (1 mM); (n) thrombin (1 U/mL); (o) esterase (1 U/ mL); (p) glutamate dehydrogenase (0.2 U/mL); (q) uric acid (0.3 mM); (r) oxalic acid (0.5 mM); (s) urea (20 mM); (t) creatinine (10 mM). The results are the mean ± standard deviation of three separate measurements. λex/em = 680 nm/707 nm.

Figure 2. (A) Fluorescence response of 1 (10 μM) to β-lactamase at varied concentrations (0, 0.05, 0.075, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0, and 7.5 nM). (B) Linear fitting curve of ΔF toward the concentration (C) of β-lactamase between 0.05 and 2 nM. λex = 680 nm.

infections.37 In the past decade, there have been alarming reports on the emergence and spread of antibiotic-resistant bacteria in UTIs, most of which can produce β-lactamase.38,39 Unfortunately, the current microbiological analysis for UTIs is time-consuming, which might lead to the missing of the best treatment time. Therefore, rapid determination of urinary βlactamase may provide crucial information for the precise diagnosis of UTIs. To assess the potential use of probe 1 in this aspect, different amounts of β-lactamase were spiked into urine to mimic the infected urine samples, and the β-lactamase contents were determined simultaneously by our method and iodimetry. As shown in Table 1, the results obtained from the

equation ΔF = 220.2[C (nM)] − 8.27 (R = 0.995), where ΔF is the fluorescence enhancement of probe 1 at 707 nm with and without β-lactamase. The detection limit (S/N = 3) is determined to be 0.02 nM β-lactamase. Next we analyzed the enzymatic kinetic parameters of probe 1 reacting with β-lactamase. As shown in Figure S13 (Supporting Information), the Michaelis constant (Km) and catalytic constant (kcat) for the enzyme-activated reaction were found to be 15.8 μM and 62 s−1, respectively. The catalytic efficiency (kcat/Km) is calculated to be 3.9 × 106 M−1 s−1, indicating a superior catalytic performance as compared to the previously reported results.17,23,24 The specificity of probe 1 was investigated by reaction with various substances, such as inorganic salts (KCl, CaCl2, MgCl2, FeCl3, ZnSO4, and CuSO4), organic molecules (glucose, vitamin C, alanine, aspartic acid, cysteine, glutathione, uric acid, oxalic acid, urea, and creatinine), and some enzymes (thrombin, esterase, and glutamate dehydrogenase). Moreover, the interferences from these potential coexisting substances were studied as well. The results (Figure 3) show that probe 1 exhibits excellent selectivity for β-lactamase and other substances do not significantly disturb the fluorescence intensity of the reaction solution, indicating the reliability of the detection system. To further confirm that the fluorescence enhancement was caused by the enzymatic hydrolysis of β-lactamase, the effects of two inhibitors, potassium clavulanate (PCA) and sulbactam (SUL), on the activity of the enzyme have been examined. As depicted in Figure S14 (Supporting Information), both of the inhibitors show significant inhibition effects on the reaction system. Specifically, 1 and 10 μM PCA can inhibit 63% and 99% of the activity of β-lactamase, respectively; 1 μM SUL hardly affects the fluorescence of the system, but 10 μM SUL can decrease the fluorescence intensity by 66%. The superior inhibition efficiency of PCA over SUL is consistent with the existing observation.14 These results not only prove the mechanism of fluorescence enhancement by the enzymatic reaction but also show the great potential of our probe in screening inhibitors of β-lactamase. Detection of β-Lactamase in Human Urine. The urinary tract infection (UTI) is one of the most common bacterial

Table 1. Determination of β-Lactamase Concentration (nM) in Urine Samplesa concn of β-lactamase added (nM)

proposed method

iodimetry

0 0.10 0.60 0.80 1.00

ATCC 11632 > ATCC 29213. This finding has not been reported to the best of our knowledge. Moreover, the highest expression level of β-lactamase in the ATCC BAA44 strain provides direct evidence for its strong resistance to most β-lactam antibiotics. From the above results, we may conclude that probe 1 is bacterium-permeable and has the ability to visualize the relative expression level of β-lactamase in bacteria.

Fluorescence Imaging of β-Lactamase in S. aureus. As is known, S. aureus is one of the most common causes of infections after injury or surgery,41 and especially the emergence of MRSA, which exhibits strong resistance to βlactam antibiotics, is a serious problem in clinical medicine.42,43 Developing a rapid and reliable imaging method to screen MRSA and other β-lactam-antibiotic-resistant S. aureus is thus beneficial to the clinical diagnosis as well as drug study. On the basis of the high sensitivity and NIR emission feature of probe 1, we investigated its applicability to image S. aureus strains. With the purpose of estimating the level of β-lactamase in different S. aureus strains, ATCC BAA44 (a clinical isolate of the MRSA strain), ATCC 11632 (a penicillin-resistant strain but not MRSA), and ATCC 29213 (a penicillin-susceptible strain) were subjected to confocal fluorescence imaging.24,44 As depicted in Figure 4A, after incubation with probe 1, the ATCC

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CONCLUSIONS In summary, we have developed 1 as a new sensitive and selective NIR fluorescent probe (λem = 707 nm) for β-lactamase assay. The probe displays an intense fluorescence off−on response to β-lactamase via the enzyme-catalyzed cleavage of the β-lactam ring, followed by the spontaneous fragmentation and the release of the NIR fluorophore. The applicability of 1 has been demonstrated by determining β-lactamase in urine in comparison with that determined by iodimetry. Most notably, by taking advantage of its high sensitivity and NIR emission, probe 1 has also been used to visualize the expression levels of β-lactamase in different S. aureus strains, revealing that the ATCC BAA44 strain has high-level expression of β-lactamase, which may be responsible for its strong resistance to most βlactam antibiotics. In addition, the probe is simple and may be of great potential for in vivo imaging of β-lactamase in some other complex biosamples.

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ASSOCIATED CONTENT

S Supporting Information *

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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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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Figure 4. (A) Confocal fluorescence images of different S. aureus strains: (a) ATCC BAA44; (b) ATCC 11632; (c) ATCC 29213. The strains were incubated with probe 1 (10 μM) at 37 °C for 30 min. The differential interference contrast images of the corresponding samples are shown at the bottom. Scale bar = 5 μm. The fluorescence images from the corresponding control experiments without 1 are given in Figure S16 (Supporting Information). (B) Relative pixel intensity of the corresponding fluorescence images in panel A (the pixel intensity from the ATCC BAA4 strain is defined as 1.0). The results are the mean ± standard deviation of three separate measurements.

ACKNOWLEDGMENTS We are grateful for the financial support from the National Natural Science Foundation (NSF) of China (Grants 21105104, 21275147, and 21321003), the Ministry of Science and Technology (Grant 2011CB935800), and the Chinese Academy of Sciences (Grants KJCX2-EW-N06-01, XDB14030102, and CMS-PY-201301).

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BAA44 strain shows strong NIR fluorescence, whereas the ATCC 11632 strain displays moderate fluorescence. In contrast, the ATCC 29213 strain gives the weakest fluorescence signal, which might be attributed to the very-low-level expression of β-lactamase in this strain. To quantitatively compare the β-lactamase levels in the three types of S. aureus, relative pixel intensity analysis45,46 was done by using ImageJ software (version 1.45 s, NIH). In doing so, the pixel intensity of at least 20 bacterial cells was averaged, and the results are given in Figure 4B. As is seen, the fluorescent intensity of ATCC BAA44 and ATCC 11632 is about 4.5 and 1.4 times higher than that of ATCC 29213, respectively, clearly revealing that the expression level of β-lactamase in these S. aureus strains

REFERENCES

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dx.doi.org/10.1021/ac501288e | Anal. Chem. 2014, 86, 6115−6120