Article pubs.acs.org/ac
Analyte-Activable Probe for Protease Based on Cytochrome C‑Capped Mn: ZnS Quantum Dots Peng Wu,†,‡ Ting Zhao,‡ Jinyi Zhang,‡ Lan Wu,† and Xiandeng Hou*,†,‡ †
Analytical & Testing Center and ‡Key Laboratory of Green Chemistry and Technology of MOE in College of Chemistry, Sichuan University, Chengdu 610064, China S Supporting Information *
ABSTRACT: A new sensor format was proposed here by integrating conjugation of analyte-recognition sites and quenching the luminescence of quantum dots (QDs) in one pot during the synthesis of QDs, with protease as the model analyte. Inherently phosphorescence-attenuated Mn-doped ZnS QDs were prepared with electron transfer protein cytochrome C (Cyt C) as the ligand, which was capable of protease sensing in both label-free and activable format. This detection strategy eliminates the postsynthetic protein conjugation and responses to analyte in the turn-on mode, lowering the signal background. In the presence of protease, the initially “locked” phosphorescence of Mn-doped ZnS QDs could be activated, due to the enzymatic digestion of surface Cyt C ligand and removal of the electron-transfer quenching unit away from the close-proximity of QDs. The proposed probe exhibited good selectivity toward proteases over other proteins and enzymes. Besides, it was also capable of differentiating active and inactive serine proteases. Analytical performance of this probe was evaluated using trypsin as the model serine protease. Limits of detection (LOD) of 2 nM was obtained, which is well below the average urine trypsin level of patients. The analytical application of this probe was demonstrated in determination of trypsin in human pancreatic carcinoma (PANC-1 and 818.4) cells lysates, demonstrating the potential usefulness of this probe in future clinical diagnosis.
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into peptides in proteomics, in particular in mass spectroscopy based proteomics.13 In view of the vital importance of proteases, new convenient assays for proteases detection and their inhibitors screening are highly desired for the development of efficient diagnostic and therapeutic methods toward pancreatic diseases and applications in the proteomics area.14−17 However, these methods are often labeled protocols and often suffer from fluorescent biological background interferences. Herein, we proposed a new sensor format by integrating conjugation of analyte-recognition sites and quenching the luminescence of QDs in one-pot during the synthesis of QDs. Taking protease as a model analyte, a label-free and analyteactivable (turn-on) phosphorescent QD-based protease probe was developed (Scheme 1B). Through using cytochrome C (Cyt C) to direct the synthesis of Mn-doped ZnS QDs, we obtained protein-conjugated QDs that were capable of protease sensing in one-pot synthesis, which eliminated multiple QDlabeling and purification steps in conventional assays. Moreover, different from previous protein-directed synthesis of highly luminescent QDs,18−25 the phosphorescence of the asprepared Cyt C-capped Mn-doped ZnS QDs is “locked” due to Cyt C-induced quenching,26−29 which provides an inherent
emiconductor QDs are superior inorganic luminophores with large extinction coefficients, strong fluorescence, and robust photostability. The past 2 decades has witnessed increasing activity in using QDs as chromophores for bioanalysis.1−5 Particularly, the QD-biomolecule nanohybrids integrating the unique electronic and photonic properties of QDs and fascinating recognition and catalytic properties of biomolecules exhibit synergetic properties and functions for advanced biosensing applications.6 However, from the synthetic point of view, the fabrication of QD-based biosensors is often a laborious multistep process. Taking the typical QD-based protease assays7−10 as examples, the construction of the sensors often involves colloidal QDs synthesis and solubilization, biomolecule (peptides or proteins with protease cleavage site)-functionalization, and protease recognition (Scheme 1A). Besides, QDs after synthesis is in a “fluorescence-on” state. In order to effectively modulate the fluorescence of QDs, the biomolecules must be dually labeled with both fluorescent quencher and QDs. The synthetic complexity would probably prohibit the end-users from gaining direct access to customizable QD probes in an easy and efficient manner. Proteases are a major class of enzymes that catalyze the hydrolysis of peptide bounds to break down proteins into small pieces in a process known as proteolysis.11 They are key participants in many diseases such as cancer and stroke. In addition, many infectious microorganisms, including bacteria, viruses, and parasites use proteases as virulence factors.12 Proteases are also employed for the degradation of proteins © XXXX American Chemical Society
Received: April 7, 2014 Accepted: September 24, 2014
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with a model FLS-980 spectrofluorometer (Edinburgh Instruments, England). TEM together with EDX characterization was performed with a Tecnai G2 F20 S-TWIN transmission electron microscope at an accelerating voltage of 200 kV (FEI Co.). Synthesis of Cyt C-Capped Mn-Doped ZnS QDs. The synthetic strategy was based on our previous publication22 but with modifications. Briefly, to a three-necked flask, aqueous solutions of ZnSO4 (1.85 mL, 0.1 M), Mn(Ac)2 (0.15 mL, 0.1 M), and Cyt C (1.2 mL, 15 mg/mL) were added. The mixed solution was made up to 20 mL with ultrapure water and adjusted to pH 10 with 1 M NaOH. A volume of 2 mL of 0.1 M Na2S was quickly injected into the solution. The mixture was stirred mildly and then aged at 80 °C for 2 h to form Cyt Ccapped Mn-ZnS QDs. For control experiments, QDs were also synthesized at room temperature for investigation of the temperature effect. Protease Sensing with Cyt C-Capped Mn-Doped ZnS QDs. To a 10 mL-centrifuge tube, 250 μL of Cyt C-capped Mn-doped ZnS QDs, 1 mL of pH 8.0 PBS buffer (0.1 M), and various amounts of trypsin were mixed together and then diluted to volume (10 mL). The above solutions were incubated in a water bath (37 °C) for 1 h. Then, the mixture was taken out from the water bath and allowed to cool to room temperature for 5 min for phosphorescence measurements at an excitation wavelength of 280 nm. For evaluation of other proteases and proteins, the experimental procedures are generally the same as that for trypsin, except that trypsin was replaced with other proteins. Trypsin Inhibition Assay. Different amounts of the trypsin inhibitor were first mixed with 0.5 μM trypsin and incubated at 37 °C for 15 min. Then, the trypsin and trypsin inhibitor mixtures were mixed with Cyt C-capped Mn-doped ZnS QDs, with subsequent procedures the same as above. The inhibition efficiency was defined as follows:
Scheme 1. Schematic Illustration of the Principles of (A) Conventional QD-Based Fluorescent Protease Assays7−10 and (B) the Proposed Label-Free Turn-On Phosphorescent Assay. FRET: fluorescence resonance energy transfer, PIET: photo-induced electron transfer
turn-on sensing platform. In the presence of proteases, the surface Cyt C was digested away,30−32 resulting in mix-to-signal and turn-on detection of protease.
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EXPERIMENTAL SECTION Materials. All reagents used were of at least analytical grade. ZnAc2·7H2O, MnAc2·4H2O, and Na2S·9H2O were supplied by Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Trypsin, proteinase K, α-chymotrypsin, pepsin, papain, bromelain, alkaline phosphatase, ovalbumin, bovine serum albumin, human serum albumin, hemoglobin, horseradish peroxidase, and lysozyme were bought from Sigma-Aldrich (Shanghai, China). Trasylol, a known trypsin inhibitor isolated from Glycine max (soybean), was also obtained from SigmaAldrich (Shanghai, China). Ultrapure water (18.2 MΩ cm) was obtained from a water purification system (PCUJ-10, Chengdu Pure Technology Co., Ltd., Chengdu, China). Fetal calf serum and NEM (cell culture media) were obtained from Invitrogen Corporation. Apparatus. Fluorescence, phosphorescence, and phosphorescence lifetime measurements were performed on an F-7000 spectrofluorometer (Hitachi, Japan) equipped with a plotter unit and a quartz cell (1 cm × 1 cm). For measuring phosphorescence lifetime, the lighting source is still the xenon lamp (the same as that for steady-state measurments), but an excitation pulse is achieved via placing a chopper in front of the excitation gratings. It should be pointed out here that such equipment provides very rough lifetime in the scale of 0−20 ms. Absorption spectra were recorded on a UV-1700 UV−vis spectrophotometer (Shimadzu, Japan). The absolute quantum yield of the as-prepared Mn-doped ZnS QDs was evaluated
inhibition efficiency =
trypsin activity with inhibitor trypsin activity without inhibitor
The IC50 value is defined as the concentration of the inhibitor required to achieve 50% decrease of the enzyme activity. The IC50 values of the trypsin inhibitor (trasylol) could be evaluated from the plot of inhibition efficiency versus inhibitor concentration. Sample Analysis. All the cells were grown in cell culture media and incubated at 37 °C in a 5% CO2/95% air humidified incubator. After receiving the cells, they were centrifuged to remove the culture media as well as any trypsin used for resuspending the cells and washed with cold PBS buffer (pH 7.4) three times. In this manner, any residue trypsin in the cell solutions was removed. After redispersing in PBS buffer (1 mL), cells were sonicated for 10 min and then centrifuged again. The supernatant was collected and subjected to the above analytical procedure for trypsin directly. For fetal calf serum and NEM, they were diluted with PBS buffer for 20- and 10-fold before analysis, respectively.
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RESULTS AND DISCUSSION Characterization of the Cyt C-Capped Mn-Doped ZnS QDs. Inherently phosphorescence-attenuated Cyt C-capped Mn-doped ZnS QDs was synthesized in aqueous media (Supporting Information).22 We chose Mn-doped ZnS QDs as the sensor moiety because it can emit orange phosphorB
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escence due to the 4T1 → 6A1 triplet transition of Mn2+ dopant,33−40 which can efficiently eliminate autofluorescence background and light scattering of biological samples. Besides, it has been previously demonstrated that protein-capped Mndoped ZnS QDs showed good stability in media of high ionic strength as seen in biological media,21,22 which facilitates biological applications. As shown in Figure 1a, the absorption edge of the asprepared Mn-doped ZnS QDs located at about 325 nm.
Mn-doped ZnS QDs exhibited the characteristic Mn2+ dopant phosphorescent emission centered at 580 nm originated from the Mn2+ 4T1 → 6A1 transition (Figure 1b,c but relatively low with an absolute quantum yield of about 0.2−0.4%); but in the absence of Mn2+ dopant, no phosphorescent Mn2+ dopant emission was observed from Cyt C-capped ZnS QDs (Figure 1b). The decay time of the Mn2+ dopant emission is in the millisecond-scale (Figure 1d), due to spin and orbitally forbidden of the Mn2+ d−d transition (4T1 → 6A1). These spectral features clearly demonstrated successful synthesis of Mn-doped ZnS QDs. The transmission electron microscopy (TEM) image confirmed the presence of roughly spherical QDs, with an average diameter of 4.0 ± 0.6 nm (Figure 1e and Figure S3 in the Supporting Information). The high-resolution TEM image shows clear lattice fringes of Mn-doped ZnS QDs (inset in Figure 1e). The distinct fringe spacing and the corresponding selected-area electron diffraction (SAED) pattern (Figure 1f) reveal the reasonably good crystallinity of the as-prepared Mn-doped ZnS QDs. Selectivity of Using the Cyt C-Capped Mn-Doped ZnS QDs As an Activable Probe for Protease. The concept of protease sensing was demonstrated by incubating the asprepared Cyt C-capped Mn-doped ZnS QDs with trypsin (0.4 μM, a serine protease). As shown in Figure 1b, the phosphorescence emission of QDs was appreciably enhanced upon incubation with trypsin. The control experiment with deactivated trypsin (heated at 80 °C for 20 min) showed essentially little change to the phosphorescence intensity of Mn-doped ZnS QDs (Figure S4 in the Supporting Information), confirming that phosphorescence turn-on was caused by degradation of surface Cyt C by trypsin. Besides, it was also feasible to employ Cyt C-capped Mn-doped ZnS QDs as an activable probe for trypsin. The application of this assay is not solely limited to trypsin but also can be adapted for other proteases. As shown in Figure 2, both proteinase K (Pro-K) and α-chymotrypsin (α-Chy)
Figure 1. Characterization of the Cyt C-capped Mn-doped ZnS QDs: (a) UV−vis absorption spectra, (b) phosphorescence emission spectra, (c) schematic diagram of the energy levels of the conduction and valence bands of ZnS, the d states of Mn2+ dopant, and possible electron transfer pathways from Mn-doped ZnS QDs to Cyt C, (d) phosphorescence decay curve, (e) TEM image (Note: the inset is the high-resolution TEM image.), and (f) SAED pattern of the Mn-doped ZnS QDs.
Figure 2. Selectivity of using Cyt C-capped Mn-doped ZnS QDs as a phosphorescence turn-on probe for protease. Here Ph and Ph0 represent the phosphorescence intensity of QDs (at 580 nm) in the presence and absence of proteins, respectively. Before measurements, all proteins (0.4 μM each) were incubated with Cyt C-capped Mndoped ZnS QDs at 37 °C for 1 h.
According to the empirical relationship between the bandgap and the radius of nanocrystalline ZnS proposed by Suyver et al.,41 the size of the as-prepared Mn-doped ZnS QDs was calculated as 3.9 nm. Another absorption band at about 411 nm was also identified, which can be ascribed to the characteristic γband absorption of Cyt C (Figure S2 in the Supporting Information). Emission spectra revealed that the Cyt C-capped
exhibited similar phosphorescence turn-on effect to trypsin. However, another three proteases, namely, pepsin (Pep), papain (Pap), and bromelain (Bro), were not effective in turning on the phosphorescence of the Cyt C-capped Mndoped ZnS QDs. Although all these proteases are digestive enzymes for breaking down proteins, their origins and substrates are different. Trypsin, proteinase K, and α-Chy are C
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Figure 1c that the lifetime of Mn-doped ZnS QDs was prolonged appreciably in the presence of trypsin (1 μM), which indicates the removal of dynamic quencher (here heme in Cyt C) away from close proximity of QDs. Moreover, trypsindigestion of the ligand Cyt C would result in slight aggregation of QDs, which was confirmed by the light scattering spectra shown in Figure S6 in the Supporting Information.22 Analytical Performances of Using the Cyt C-Capped Mn-Doped ZnS QDs As an Activable Probe for Protease. The analytical performance of this sensor was demonstrated with trypsin as a model protease. Upon incubation with various concentrations of trypsin at 37 °C for 1 h, the phosphorescence of the Mn-doped ZnS QDs was gradually increased (Figure 4a).
all serine proteases, pepsin is a kind of aspartate protease, and papain and bromelain are cysteine proteases. Accordingly, the proposed Cyt C-capped Mn-doped ZnS QDs may be effective in differentiation of active and inactive serine proteases. Approximately 35 000 new cases of pancreatic cancer associated with misbalanced serine protease activity are reported annually,42 indicating potential application of this method for diagnosis of pancreatic cancer. Other common proteins and enzymes, including alkaline phosphatase (ALP), ovalbumin (Ob), human serum albumin (HSA), bovine serum albumin (BSA), hemoglobin (Hem), horseradish peroxidase (HRP), and lysozyme (Lys), did not show a similar appreciable phosphorescence turn-on effect as that of serine proteases, demonstrating good selectivity of the proposed assay. Cyt C is a well-known electron-transfer protein that can be entrapped into the mitochondrial membrane, with heme (an iron porphyrin, Figure 3) as the cofactor. Both Cyt C and heme
Figure 4. (a) Phosphorescence emission spectra of Cyt C-capped Mndoped ZnS QDs in the presence of various concentrations of trypsin and (b) the corresponding calibration plot for trypsin. Here Ph and Ph0 represent the phosphorescence intensity of QDs (at 580 nm) in the presence and absence of trypsin, respectively. Mn-doped ZnS QDs were synthesized with 0.9 mg/mL Cyt C as ligand. Before measurements, all trypsin solutions were incubated with Cyt Ccapped Mn-doped ZnS QDs at 37 °C for 1 h.
Figure 3. Mechanistic illustration of as-prepared phosphorescencequenched Cyt C-capped Mn-doped ZnS QDs. During the synthesis, the original Fe(II)-heme may be partially or totally converted to Fe(III)-heme. Both Fe(II)-heme and Fe(III)-heme can act as an electron-transfer quencher for Mn-doped ZnS QDs.
For different amounts of Cyt C used for the one-pot synthesis of Mn-doped ZnS QDs, the phosphorescence restoration caused by trypsin was investigated and the results were given in Figure S7 in the Supporting Information. When 0.9 mg/mL of Cyt C was used, the highest phosphorescence restoration is obtained. Here the optimal probe for protease should possess low signal background and high phosphorescence restoration. Accordingly, such optimal concentration of Cyt C is a compromise between the above two factors. With 0.9 mg/mL of Cyt C for synthesis of Mn-doped ZnS QDs, a linear relation between the phosphorescence intensity (at 580 nm) and the concentration of trypsin was observed in the range of 0.05−1 μM (Figure 4b). A calibration function of Ph/Ph0 = 1.42Ctrypsin + 1.49 (R = 0.9918) and a limit of detection (3σ) of 2 nM were obtained, which is comparable to those of the typical trypsin assays reported to date (Table S1 in the Supporting Information). Besides, control experiments with Cyt C postconjugated MPA-capped Mn-doped ZnS QDs for trypsin assay was also performed (Scheme 1A), with a calibration function of Ph/Ph0 = 3.56Ctrypsin + 1.18 (R = 0.9962) and a limit of detection of 2 nM (Figure S8 in the Supporting Information). Clearly, the direct conjugation-based trypsin detection possessed higher sensitivity (about 3-fold higher) than the proposed on-pot assay. It should be acknowledged that bioactivity loss of Cyt C occurred during the synthesis of Mndoped ZnS QDs (the one-pot assay, Figure S2 in the
have been identified as electron-transfer quencher for QDs,26−29,43−45 and the Cyt C-induced quenching can also be ascribed to the heme cofactor. In a control experiment, Cyt C-induced phosphorescence quenching to Mn-doped ZnS QDs was experimentally confirmed (Figure S5 in the Supporting Information). Accordingly, exploring Cyt C as the synthetic ligand yielded phosphorescence-quenched Mn-doped ZnS QDs (Figure 1b), presumably due to electron transfer from Mndoped ZnS QDs to heme (Figure S5 in the Supporting Information). On the other hand, Cyt C as a substrate for protease digestion has also been confirmed in the literature with either MALDI-TOF-MS30,31 or capillary electrophoresis,46 leading to the release of heme/peptide complex. In fact, the amino-acid sequence of Cyt C was determined with serine protease digestion.47 In this system, however, protease digestion of Cyt C would result in disturbance of the electron transfer quencher (heme) on the surface of QDs. Since electron transfer efficiency falls off exponentially upon distance variation of the quencher, the addition of protease activates the phosphorescence of Cyt C-capped Mn-doped ZnS QDs and gives rise to phosphorescence turn-on. Although our spectrofluorometer could not give very precise lifetime data (with a rough time scale of 0−20 ms), one can still see from D
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Supporting Information), which may result in lower sensitivity. Indeed, this is a drawback of the proposed one-pot assay when compared with the conventional conjugation-based protease assay in Scheme 1. However, the conventional assay did require more steps (including synthesis, conjugation, and purification) for preparation of the probe (Supporting Information). The original phosphorescence intensity of the Cyt C-cpped Mn-doped ZnS QDs could be lowered upon synthesis at room temperature, but in turn, the phosphorescence turn-on intensity of the probe was also significantly lower (Figure S9 in the Supporting Information). It should be noted that the average urine trypsin level of patients is as high as 84.4 μg/mL (3.6 μM).48 Accordingly, the proposed trypsin assay may be potentially useful in clinical diagnosis. Proteases are well-known for use in numerous biotechnological processes, particularly for the degradation of proteins into peptides in proteomics.13 Therefore, interference evaluation from coexisting proteins, amino acids, and metal ions are important for the proposed protease assay. An error of ±10.0% in the relative phosphorescence intensity was considered tolerable. As shown in Table S2 in the Supporting Information, 1000 μM of K+ and Na+, 200 μM of Ca2+ and Mg2+, 2 μM of Fe3+, and 1 μM of Zn2+ and Mn2+ have no appreciable effects on the restored phosphorescence caused by 0.4 μM of trypsin. For small biomolecules, such as amino acids, glucose, and ascorbic acid, the tolerant limits (for 0.4 μM of trypsin) are in the range of 10−200 μM. Since potential coexisting proteins can compete with the consumption of trypsin with Cyt C, the tolerance limits (below than 1 μM) are significantly lower. Determination of Trypsin in Pancreatic Carcinoma Cells Lysates. The potential application of the proposed method for trypsin detection was evaluated with several cellrelated biological samples. Pancreatic cancer is the fourth most common cause of cancer-related deaths in the United States and the eighth worldwide.49 Detection of trypsin in pancreatic cell lyastes is an important means for diagnosis of chronic pancreatic diseases. Accordingly, two human pancreatic carcinoma cells (PANC-1 and 818.4) were chosen as the sample matrix to demonstrate the potential application of this probe. Besides, to show the analytical specificity of this probe for pancreatic cells, two nonpancreatic cells (HeLa and HEK 293) and the “growth media” for cells (fetal calf serum and NEM, the cell culture media) were also included in the samples. As expected, severe fluorescence background was observed in these biological samples, but such background was eliminated in phosphorescence detection mode (Figure S10 in the Supporting Information), demonstrating the superiority of phosphorescence detection for biological samples. No trypsin was found in either the “growth media” of the cells or the lysates of nonpancreatic cells, but the phosphorescence of Cyt C-capped QDs was turned on when incubating with lysates of human pancreatic carcinoma cells (Figure 5), indicating trypsin was directly related to pancreatic carcinoma. Also, this probe was able to differentiate pancreatic and nonpancreatic cells. The accuracy of this method was further verified with a spikerecovery test; the results given in Table S3 in the Supporting Information indicated that the recoveries (89−108%) and feasibility of the method applied to determine trypsin in cell extracts were satisfactory. It should be pointed out that since the samples were diluted 20- and 10-fold prior to analysis, the LOD of this probe in real samples should be higher (20−40 nM) than that in pure buffer.
Figure 5. Determination of trypsin in biological samples. Here Ph and Ph0 represent the phosphorescence intensity of QDs (at 580 nm) in the presence and absence of samples, respectively. Before measurements, all samples were incubated with Cyt C-capped Mn-doped ZnS QDs at 37 °C for 1 h.
Protease Inhibitor Evaluation. Evaluation of protease inhibitors is of great importance for potential drug discovery assays. We tested this Cyt C-capped Mn-doped ZnS QDs against a known trypsin inhibitor, trasylol (isolated from Glycine max). The inhibition efficiency was evaluated under the same conditions used for trypsin detection described above. As shown in Figure 6, addition of the inhibitor did reduce the
Figure 6. Dependence of the trypsin inhibitor concentrations on inhibition efficiency of the trypsin (enzymatic digestion of Cyt C). Trypsin concentration, 0.5 μM; incubation time, 1 h.
activity of trypsin and thus reduce the degree of Cyt C digestion. The IC50 (defined as 50% inhibition efficiency) value was estimated to be 3 μg/mL. Since Cyt C is a substrate of serine proteases, these results demonstrated that the current method may be useful for screening potential drugs on the basis of the inhibition of the cleavage reactions catalyzed by serine proteases.
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CONCLUSION In summary, we have reported an activable protocol for labelfree phosphorescent sensing for protease based on Cyt Ccapped Mn-doped ZnS QDs. Inherently phosphorescencequenched but analyte-activable Mn-doped ZnS QDs were synthesized easily in one pot with the well-known electrontransfer protein Cyt C as the ligand. The sensing of protease was simply based on enzymatic digestion of surface Cyt C and removal of the quenching unit away from Mn-doped ZnS QDs. Besides, such Cyt C-capped Mn-doped ZnS QDs were capable of differential active and inactive serine proteases. This sensor was successfully exploited for detection of trypsin in pancreatic cancer lysates. However, it should also be noted that this assay suffers interferences from other proteins, which have arisen E
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(19) Zhao, L.; Gattas-Asfura, K. M.; Xu, J. M.; Patel, R. A.; Dadlani, A.; Sillero-Mahinay, M.; Cushmore, M.; Rastogi, V. K.; Shah, S. S.; Leblanc, R. M. Chem. Commun. 2011, 47, 7242−7244. (20) He, X.; Gao, L.; Ma, N. Sci. Rep. 2013, 3, 2825. (21) Zhou, W. B.; Baneyx, F. ACS Nano 2011, 5, 8013−8018. (22) Wu, P.; Zhao, T.; Tian, Y. F.; Wu, L.; Hou, X. D. Chem.Eur. J. 2013, 19, 7473−7479. (23) Makhal, A.; Sarkar, S.; Pal, S. K. Inorg. Chem. 2012, 51, 10203− 10210. (24) Goswami, N.; Giri, A.; Kar, S.; Bootharaju, M. S.; John, R.; Xavier, P. L.; Pradeep, T.; Pal, S. K. Small 2012, 8, 3175−3184. (25) Zhou, W.; Schwartz, D. T.; Baneyx, F. J. Am. Chem. Soc. 2010, 132, 4731−4738. (26) Ma, Y.; Bai, H. X.; Yang, C.; Yang, X. R. Analyst 2005, 130, 283−285. (27) Gerhards, C.; Schulz-Drost, C.; Sgobba, V.; Guldi, D. M. J. Phys. Chem. B 2008, 112, 14482−14491. (28) Li, D. W.; Qin, L. X.; Li, Y.; Nia, R. P.; Long, Y. T.; Chen, H. Y. Chem. Commun. 2011, 47, 8539−8541. (29) Wu, P.; Miao, L.-N.; Wang, H.-F.; Shao, X.-G.; Yan, X.-P. Angew. Chem., Int. Ed. 2011, 50, 8118−8121. (30) Wang, Y. Y.; Zhang, Y.; Liu, B. Anal. Chem. 2010, 82, 8604− 8610. (31) Zhang, Q. F.; Li, W. Y.; Chen, J.; Wang, F. Y.; Wang, Y.; Chen, Y.; Yu, C. Chem. Commun. 2013, 49, 3137−3139. (32) Li, X.; Zhu, S. J.; Xu, B.; Ma, K.; Zhang, J. H.; Yang, B.; Tian, W. J. Nanoscale 2013, 5, 7776−7779. (33) Wu, P.; Yan, X.-P. Chem. Soc. Rev. 2013, 42, 5489−5521. (34) Zhang, L.; Cui, P.; Zhang, B.; Gao, F. Chem.Eur. J. 2013, 19, 9242−9250. (35) Wang, H.-F.; Wu, Y.-Y.; Yan, X.-P. Anal. Chem. 2013, 85, 1920− 1925. (36) Sotelo-Gonzalez, E.; Fernandez-Argüelles, M. T.; CostaFernandez, J. M.; Sanz-Medel, A. Anal. Chim. Acta 2012, 712, 120− 126. (37) Zhao, Y. Y.; Ma, Y. X.; Li, H.; Wang, L. Y. Anal. Chem. 2012, 84, 386−395. (38) Zou, W.-S.; Sheng, D.; Ge, X.; Qiao, J.-Q.; Lian, H.-Z. Anal. Chem. 2011, 83, 30−37. (39) Wu, P.; Zhang, J. Y.; Wang, S. L.; Zhu, A. R.; Hou, X. D. Chem.Eur. J. 2014, 20, 952−956. (40) Tan, L.; Kang, C.; Xu, S.; Tang, Y. Biosens. Bioelectron. 2013, 48, 216−223. (41) Suyver, J. F.; Wuister, S. F.; Kelly, J. J.; Meijerink, A. Nano Lett. 2001, 1, 429−433. (42) Sarkar, F. H.; Banerjee, S.; Li, Y. W. Toxicol. Appl. Pharmacol. 2007, 224, 326−336. (43) Sharon, E.; Freeman, R.; Willner, I. Anal. Chem. 2010, 82, 7073−7077. (44) Zhang, L. B.; Zhu, J. B.; Guo, S. J.; Li, T.; Li, J.; Wang, E. K. J. Am. Chem. Soc. 2013, 135, 2403−2406. (45) Raichlin, S.; Sharon, E.; Freeman, R.; Tzfati, Y.; Willner, I. Biosens. Bioelectron. 2011, 26, 4681−4689. (46) Busnel, J. M.; Descroix, S.; Le Saux, T.; Terabe, S.; Hennion, M. C.; Peltre, G. Electrophoresis 2006, 27, 1481−1488. (47) Margoliash, E. J. Biol. Chem. 1962, 237, 2161−2174. (48) See, W. A.; Smith, J. L. Transplantation 1991, 52, 630−633. (49) Hariharan, D.; Saied, A.; Kocher, H. M. HPB 2008, 10, 58−62. (50) Yildiz, I.; Tomasulo, M.; Raymo, F. M. J. Mater. Chem. 2008, 18, 5577−5584.
from the competition consumption of trypsin by other proteins and demands future improvements. Currently, the knowledge for synthesis of various QDs is quite mature and the electrontransfer quenchers (typically redox-active species) for QDs have been well-demonstrated in the literature.50 Given that the ligands for synthesis of QDs can be elegantly designed by integrating the QD-anchoring group, analyte-recognition site, and electron-transfer quenching group for QDs, the proposed scheme for development of analyte-activable (turn-on) probe is expected to be extended for a number of other analytes.
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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 Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the Natural Science Foundation of China (Grant 21205084), Chengdu Bureau of Science and Technology (Grant 12DXYB303JH-002), and the Basic Research Program of Sichuan Province (Grant 2014JY0049).
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REFERENCES
(1) Petryayeva, E.; Algar, W. R.; Medintz, I. L. Appl. Spectrosc. 2013, 67, 215−252. (2) Algar, W. R.; Susumu, K.; Delehanty, J. B.; Medintz, I. L. Anal. Chem. 2011, 83, 8826−8837. (3) Algar, W. R.; Tavares, A. J.; Krull, U. J. Anal. Chim. Acta 2010, 673, 1−25. (4) Freeman, R.; Willner, I. Chem. Soc. Rev. 2012, 41, 4067−4085. (5) Wu, P.; Zhao, T.; Wang, S. L.; Hou, X. D. Nanoscale 2014, 6, 43− 64. (6) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042−6108. (7) Medintz, I. L.; Clapp, A. R.; Brunel, F. M.; Tiefenbrunn, T.; Uyeda, H. T.; Chang, E. L.; Deschamps, J. R.; Dawson, P. E.; Mattoussi, H. Nat. Mater. 2006, 5, 581−589. (8) Shi, L. F.; De Paoli, V.; Rosenzweig, N.; Rosenzweig, Z. J. Am. Chem. Soc. 2006, 128, 10378−10379. (9) Gao, X.; Tang, G.; Li, Y.; Su, X. Anal. Chim. Acta 2012, 743, 131−136. (10) Biswas, P.; Cella, L. N.; Kang, S. H.; Mulchandani, A.; Yates, M. V.; Chen, W. Chem. Commun. 2011, 47, 5259−5261. (11) Wolfe, M. S. Chem. Rev. 2009, 109, 1599−1612. (12) Puente, X. S.; Sanchez, L. M.; Overall, C. M.; Lopez-Otin, C. Nat. Rev. Genet. 2003, 4, 544−558. (13) Olsen, J. V.; Ong, S. E.; Mann, M. Mol. Cell. Proteomics 2004, 3, 608−614. (14) 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. (15) Kim, J. H.; Chung, B. H. Small 2010, 6, 126−131. (16) Orosco, M. M.; Pacholski, C.; Miskelly, G. M.; Sailor, M. J. Adv. Mater. 2006, 18, 1393−1396. (17) Mu, C. J.; LaVan, D. A.; Langer, R. S.; Zetter, B. R. ACS Nano 2010, 4, 1511−1520. (18) Ma, N.; Marshall, A. F.; Rao, J. H. J. Am. Chem. Soc. 2010, 132, 6884−6885. F
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