FULL PAPER DOI: 10.1002/asia.201402221
A Mix-and-Read Fluorescence Strategy for the Switch-On Probing of Kinase Activity Based on an Aptameric-Peptide/Graphene-Oxide Platform Chunyang Lei,[a] Xiahong Xu,[a, b] Jiang Zhou,[a] Xin Liu,[a] Zhou Nie,*[a] Meng Qing,[a] Pei Li,[a] Yan Huang,*[a] and Shouzhuo Yao[a] Abstract: Protein kinase plays a vital role in regulating signal-transduction pathways and its simple and quick detection is highly desirable because traditional kinase assays typically rely on a time-consuming kinase-phosphorylation process (ca. 1 h). Herein, we report a new and rapid fluorescencebased sensing platform for probing the activity of protein kinase that is based on the super-quenching capacity of gra-
phene oxide (GO) nanosheets and specific recognition of the aptameric peptide (FITC-IP20). On the GO/peptide platform, the fluorescence quenching of FITC-IP20 that is adsorbed onto GO can be restored by selective binding of active protein kinase to the aptameric Keywords: biosensors · fluorescence · graphene · kinase · peptides
Introduction
avoid these shortcomings, great effort has been devoted to the design of alternative methods and a number of methods based on fluorescence,[5] electrochemistry,[6] surface plasmon resonance spectroscopy,[7] and mass spectroscopy[8] have been developed. Conventionally, these assays involve kinase-catalyzed phosphorylation reactions, which typically require about 1 h reaction time to accumulate enough phosphorylated product for effective detection. Moreover, multistep detection processes have been required in some methods, including labeling, incubation, washing, generation, and amplification of the detection signal, which are labor-intensive and time-consuming. Therefore, convenient, rapid, and cost-effective assays for detecting protein kinase are in demand but highly challenging. Recently, we found that the specific inhibitory peptide of protein kinase could be exploited as a potent aptameric peptide for recognizing kinase and we developed a one-step approach for the rapid detection (< 10 min) of protein kinase by using the quartz crystal microbalance (QCM).[9] This aptameric peptide is the natural protein kinase A (PKA)-inhibitory peptide, IP20, which contains a RRNAI sequence that replaces the RRXS recognition motif of the PKA substrate.[10] The aptameric peptide has specific affinity with the free catalytic subunit of PKA—the activated form of PKA inside the cell—and shows a much higher affinity for PKA than the substrate peptide, in accordance with their relative K values (IP20 : Ki = 2.3 nm, substrate kemptide: Km = 5 mm).[11] By using this peptide, the stimulated activation of PKA in the cell lysate has been successfully detected by QCM. Because the feasibility of this aptameric peptide for kinase recognition has been demonstrated, the application of this aptameric peptide in various detection methods is anticipated, in particular in homogenous fluorescent assays,
The protein-phosphorylation reaction catalyzed by protein kinases plays a vital role in regulating the signal-transduction pathways in the majority of crucial cellular processes, such as cell growth, metabolism, differentiation, proliferation, and apoptosis.[1] Indeed, the over-expression and aberrant activity of protein kinase, which causes abnormal cellular signaling, is associated with a range of diseases, including cancer, diabetes, inflammation, cardiac diseases, and Alzheimer’s disease.[2] Therefore, the detection of protein kinase is valuable for understanding the diseases related to fundamental biological processes, as well as for clinical diagnosis and drug-discovery applications.[1b, 2b, 3] The standard method that is used to detect kinase activity relies on radiolabeled g-32/33P-ATP; however, this method may pose a threat to human health and to the environment, owing to the use of hazardous radioactive reagents.[1a, 4] To
[a] C. Lei,+ Dr. X. Xu,+ J. Zhou, X. Liu, Prof. Dr. Z. Nie, M. Qing, P. Li, Dr. Y. Huang, Prof. S. Yao State Key laboratory of Chemo/Biosensing and Chemometrics College of Chemistry and Chemical Engineering Hunan University, Changsha, 410082 (P. R. China) Fax: (+ 86) 731-8882-1848 E-mail: [email protected] [email protected] [b] Dr. X. Xu+ College of Biosystem Engineering and Food Science Zhejiang University Hangzhou, Zhejiang, 310058 (P. R. China) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201402221.
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peptide, thereby resulting in the fast switch-on detection of kinase activity (ca. 15 min). The feasibility of this method has been demonstrated by the sensitive measurement of the activity of cAMP-dependent protein kinase (PKA), with a detection limit of 0.053 mU mL 1. This assay technique was also successfully applied to the detection of kinase activation in cell lysate.
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which are highly sensitive, convenient, and ready for adaption to high-throughput screening. Graphene oxide (GO)—a 2D carbon material with singleatom thickness—has attracted considerable attention, owing to its remarkable mechanical, thermal, optical, and electronic properties.[12] Owing to its intrinsic properties, including large surface area, water solubility, good biocompatibility, and facile surface modification, GO represents a promising material in biosensing, bioengineering, and drug delivery.[13] Moreover, GO has been used as a fluorescence superquencher of various fluorophores over a wide wavelength range, owing to its long-range nanoscale energy transfer. GO/DNA-based sensing platforms have been widely applied in DNA and RNA analysis.[14] By comparison, the application of GO/peptide nanocomposites in fluorescence-based biosensors has been much less explored and only a few reports have appeared so far. Qu’s group reported the selective detection of Cyclin A2 by using a graphene-oxide/peptide platform,[15] whilst Leblanc’s group developed a fluorescence assay for peptides and proteins by using the quenching effect of GO on the intrinsic fluorescence of the peptide.[16] Further application of GO/peptide systems in protease detection has also been reported;[17] however, the application of GO/peptide sensing systems for post-translation modification-related enzymes assay remains scarce. Recently, we developed a GO-based fluorescence biosensing method for determining the activity of protein kinase that employed phosphorylation protection against protease cleavage.[18] This method was efficient and versatile, but it was still hampered by the relatively long reaction time of the phosphorylation and subsequent protease-hydrolysis steps, as well as by its “turn-off” detection mode.
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Herein, we report a new and rapid fluorescence-sensing platform for the “turn-on” probing of the activity of protein kinase that is based on the super-quenching capability of graphene-oxide nanosheets and the specific recognition of an aptameric peptide (FITC-IP20). In this GO/peptide platform, the fluorescence quenching of an FITC-labeled aptameric peptide (FITC-IP20) that is adsorbed onto GO can be restored by selective binding of the aptameric peptide to active protein kinase (PKA), thereby resulting in “turn-on” kinase detection. This GO-based platform is simple and feasible to construct, with small background interference, thereby providing superior sensitivity and rapid response without the need to perform phosphorylation processes. Moreover, our approach does not require multi-step washing, prolonged incubation, or complicated sample preparation. Because a variety of specific inhibitor peptides for their cognate kinases are naturally occurring, this method has potential to be a versatile technique for rapid kinase detection.
Results and Discussion Detection Mechanism The principle of the GO/peptide kinase assay is shown in Scheme 1. It is well-known that GO is an amphiphile that contains a hydrophobic basal plane and ionizable hydrophilic edges (e.g., COOH groups).[19] As calculated by using ProtParam, the aptameric peptide IP20 is a 20-amino-acid-
Abstract in Chinese:
Scheme 1. Principles of the fluorescence biosensor for PKA, based on FITC-IP20 and GO.
long peptide that contains two aromatic (Phe and Tyr) residues, with a pI value of 8.43 and moderate hydrophobicity (aliphatic index: 54.00). Hence, mixing FITC-IP20 with GO at pH 7.5 allows the FITC-IP20 to be non-covalently adsorbed onto the surface of GO through p–p, hydrophobic, and electrostatic interactions and the proximity of the GO and FITC moieties effectively quench the fluorescence of FITC (Scheme 1 a). In the presence of PKA, the strong affinity between FITC-IP20 and PKA causes the formation of
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without the presence of GO (40 mg mL 1) at various concentrations of FITC-IP20 (0–30 mm) and the corresponding bar plots are shown in the Supporting Information, Figure S2. The highest QE value was obtained at 10 mm FITC-IP20. Thus, 40 mg mL 1 GO and 10 mm FITC-IP20 in 10 mm TrisHCl buffer were used in the subsequent experiments. The adsorption of aptameric peptide FITC-IP20 onto GO was further investigated by investigating the thickness and height profile of GO and the FITC-IP20/GO complex by atomic force microscopy (AFM; see the Supporting Information, Figure S3). AFM analysis revealed that the thickness of GO was 1.0 nm, consistent with the previously reported apparent thickness of GO.[14a] The thickness of the FITC-IP20/GO complex was 2.1 nm, higher than that of pristine GO, thus indicating that FITC-IP20 was absorbed onto the GO surface. The adsorption of FITC-IP20 onto GO was further investigated by CD spectroscopy. The FITC-IP20/GO complex was prepared by centrifugation to remove any unbound FITC-IP20. Peaks were observed at 203 and 220 nm in the FITC-IP20 sample, which were characteristic of an a-helical structure (see the Supporting Information, Figure S4). The CD spectrum of FITC-IP20 that had been incubated with GO alone showed minimal changes compared with that with FITC-IP20 alone, in good agreement with a previous report.[20] This result indicated that FITC-IP20 was successfully adsorbed onto the GO surface without remarkable conformation changes.
a FITC-IP20/PKA complex, thereby resulting in the tagged FITC moving away from the GO surface and subsequent recovery of the fluorescence (Scheme 1 b). Therefore, specific recognition of the aptameric peptide by PKA readily transduces to afford a measurable signal enhancement and provides a facile “turn-on” assay for PKA detection. Investigation of Fluorescence Quenching Effect of Graphene Oxide (GO) on FITC-IP20 First, we studied the interactions between FITC-IP20 and GO. The non-covalent adsorption of FITC-IP20 onto GO was studied by evaluating the change in fluorescence intensity of FITC-IP20 (10 mm) on mixing with different concentrations of GO. Fluorescence emission spectra and the corresponding changes in fluorescence intensity at 520 nm are shown in Figure 1. On the increasing concentration of GO,
Detection of PKA by the GO/Aptameric-Peptide System The detection of PKA by the proposed GO/aptameric-peptide sensor was performed under the optimized conditions. As shown in Figure 2 a, the addition of GO induced 95.8 % quenching of the fluorescence of FITC-IP20, which indicated the strong adsorption of FITC-IP20 onto GO and a high fluorescence-quenching efficiency of GO towards FITC-IP20. Moreover, in the presence of 250 mU mL 1 PKA, the fluorescence intensity increased 7.1-fold compared with that of the FITC-IP20/GO sample, thus implying that the binding of aptameric peptide by PKA caused a remarkable restoration of the fluorescence and provided a “turn-on” detection of PKA. Furthermore, the kinetic behavior of the interactions between FITC-IP20 and GO with or without PKA was studied by monitoring the fluorescence intensity as a function of time (30 min). As shown in Figure 2 b, the change in fluorescence intensity of FITC-IP20 (10 mm) was negligible during the measurements (Figure 2 b, curve 1). The addition of GO caused a rapid decrease in the fluorescence intensity of FITC-IP20 (within 5 min; Figure 2 b, curve 2). In the presence of PKA, upon the addition of GO, the change in fluorescence intensity followed the same pattern as that with FITC-IP20 alone, but was much smaller, probably because the formation of the FITC-IP20/PKA complex distanced FITC from GO and the unbound FITC-IP20 adsorbed onto the GO surface. This result indicated that an incubation time of 5 min was sufficient for the complete adsorption of the aptameric peptide on GO. Therefore, the feasibility of
Figure 1. Fluorescence emission spectra of FITC-IP20 on the addition of 0–50 mg mL 1 GO in TB buffer (10 mm Tris-HCl at pH 7.5, 25 8C). Inset shows a plot of the corresponding fluorescence intensity of FITC-IP20 (10 mm) at 520 nm versus the concentration of GO.
the fluorescence intensity of FITC-IP20 gradually decreased and plateaued at 40 mg mL 1 GO. Quenching efficiency (QE) is defined as (F0 F)/F0, where F0 and F are the fluorescence intensity of FITC-IP20 before and after the addition of GO, respectively. The QE of FITC-IP20 with 40 mg mL 1 GO was 95.8 %. The superior fluorescence-quenching ability of GO enables this method to have a low background. Furthermore, the effect of buffer on the fluorescencequenching efficiency of GO on FITC-IP20 was also investigated. Buffers were compared with or without the presence of magnesium ions. As shown in the Supporting Information, Figure S1, the fluorescence quenching of mixtures of FITC-IP20 with equal amounts of GO in TB buffer (10 mm Tris-HCl at pH 7.5) was stronger than in TBS buffer (10 mm Tris-HCl, 10 mm MgCl2 at pH 7.5). As a result, we concluded that increasing ionic strength decreased the quenching efficiency of GO, thus indicating that electrostatic interactions contributed to the fluorescence quenching between positively charged FITC-IP20 and negative charged GO. Next, we compared the fluorescence intensity of FITC-IP20 with and
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Figure 3. Fluorescence anisotropy values of (1) FITC-IP20 ; (2) FITC-IP20/ GO; (3) FITC-IP20/PKA; and (4) FITC-IP20/PKA/GO. The concentrations of FITC-IP20, PKA, and GO were 10 mm, 250 mU mL 1, and 40 mg mL 1, respectively.
suggesting that fluorescence detection was preferable for this sensor. Figure 2. a) Fluorescence emission spectra of FITC-IP20 (10 mm, curve 1), FTC-IP20/GO (40 mg mL 1, curve 2), and FITC-IP20 incubated with PKA (250 mU mL 1) for 10 min upon the addition of GO (40 mg mL 1, curve 3); the excitation wavelength was at 480 nm. b) Plot of fluorescence intensity changes of FITC-IP20 (curve 1), FTC-IP20/GO (curve 2), and FITC-IP20/ PKA/GO (curve 3) as a function of incubation time (0–30 min).
Specific Recognition and Detection of PKA First, various incubation times (0–30 min) of FITC-IP20 and PKA were investigated to optimize the reaction (see the Supporting Information, Figure S5). On increasing the reaction time, the corresponding fluorescence intensity gradually increased and then reached a plateau after 10 min, thus indicating that the binding of PKA to FITC-IP20 was fast. Therefore, a binding time of 10 min was employed in the subsequent assays. To investigate the sensitivity of our proposed biosensor for the detection of PKA, various concentrations of PKA (0–625 mU mL 1) were tested (Figure 4). The results clearly revealed that the fluorescence intensity continuously increased with increasing PKA concentration and that the fluorescence intensity reached a plateau at a PKA concentration of 250 mU mL 1. Thus, the fluorescence-intensity change was successfully used to assess the degree of binding of the aptameric peptide to PKA. Figure 4, inset, shows a calibration curve of the fluorescence intensity as function of the logarithm of PKA concentration. The EC50 value (the amount of kinase needed to achieve 50 % of the maximum signal) using this approach was 30.28 mU mL 1, similar to that for our previously reported QCM sensor for PKA based on the aptameric peptide (33.64 mU mL 1). The assay allowed for the detection of PKA at concentrations as low as 0.053 mU mL 1, lower than with many previously reported assays.[4c, 6c, 22] To compare our aptameric-peptide-based approach with the traditional kinase-activity assay, the commercial fluorescence kinase-activity assay kit was employed as a reference.
this biosensing platform for the quick detection of PKA has been demonstrated. Fluorescence Anisotropy Detection of PKA The fluorescence anisotropy (FA) of a fluorophore, which is defined as the ability of a molecule to rotate in its microenvironment, is sensitive to the molecular weight of the fluorophore.[21] Therefore, anisotropy measurements were used to further investigate the aptameric-peptide/PKA and peptide/ GO interactions. As shown in Figure 3, the original FA value of FITC-IP20 in Tris-HCl buffer was 0.075, and its FA value significantly increased 4.7-fold to 0.354 upon the addition of GO, thus indicating the formation of large-mass complex FITC-IP20/GO. The introduction of PKA into a solution of FITC-IP20 caused a 2-fold increase in the FA value compared with FITC-IP20 alone, thus confirming the specific binding of FITC-IP20 to the target PKA. The FA value of the FITC-IP20/PKA complex in the presence of GO (0.227) was smaller than that of FITC-IP20/GO, thus indicating that PKA liberated FITC-IP20 from the capture of GO. These results provided further support for the mechanism of this peptide/GO system. Notably, the PKA-induced change in FA was not as significant as the change in fluorescence, thus
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Figure 4. Changes in the fluorescence spectra with increasing concentration of PKA (0–625 mU mL 1). Inset shows the calibration curve for fluorescence intensity change at 520 nm as a function of the logarithm of PKA concentration.
Figure 5. Fluorescence intensity responses of the sensor to different proteins: (1) 250 mU mL 1 PKA; (2) 0.25 mg mL 1 glucose oxidase; (3) 0.25 mm catalase; (4) 0.25 mm lysozyme; (5) 0.25 mm BSA; (6) 250 mU mL 1 thrombin; (7) 250 mU mL 1 CKII instead of PKA; (8) FITC-CP20 instead of FITC-IP20 peptide; and (9) PKA substrate peptide FITC-kemptide instead of FITC-IP20 peptide, respectively. The excitation wavelength was 480 nm.
Plots of the fluorescence-intensity response of both assays as a function of the logarithm of PKA concentration are shown in the Supporting Information, Figure S6. These results indicated that the fluorescence signal from our directrecognition approach perfectly corresponded with that from the commercial PKA assay kit. The EC50 value for our approach (30.28 mU mL 1) was quite close to that with the commercial kit (31.36 mU mL 1). In addition, the fluorescence-intensity response of a sample of FITC-IP20 that had been incubated with thermally inactivated PKA was negligible compared with that with the activated PKA (see the Supporting Information, Figure S7 A), in accordance with that for the commercial PKA assay kit (see the Supporting Information, Figure S7 B). These results successfully demonstrate that the aptameric-peptide-based method can also effectively reflect the level of kinase activity through specific binding between the aptameric peptide (IP20) and the active form of PKA, without the need for a phosphorylation process. Furthermore, the robustness and reproducibility of this aptameric-peptide-based approach for kinase assay was evaluated. As shown in the Supporting Information, Figure S8, the Z factor of this assay was 0.75, which indicated that this method was a solid assay with good reproducibility.[23] The specificity of our proposed sensor towards PKA was examined in a series of control experiments. Six other control proteins, including non-target kinase CKII, and other enzymes, such as glucose oxidase, catalase, lysozyme, BSA, thrombin, and FITC-CP20 (two mutated amino acids compared with FITC-IP20), as well as PKA substrate peptide FITC-kemptide as a control peptide, were investigated. None of the non-specific proteins gave the same remarkable fluorescence response in the assay as PKA did (Figure 5). Similarly, FITC-CP20 and FITC-kemptide caused negligible fluorescence responses compared with the aptameric peptide (IP20), because their affinities towards PKA were significantly weaker than that of IP20. This result clearly demonstrated that this platform based on GO and FITC-IP20 could
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be used as a selective probe for the targeted detection of the PKA protein. Active Protein Kinase Detection in Cell Lysate It is well-known that the activation of kinases in cells by extracellular stimulation can trigger a range of important cellular processes, including transcription, differentiation, and apoptosis.[24] Thus, whether an assay is available for detecting kinase activity in cell lysate is important for the study of kinase regulation in cell systems. As shown in Scheme 2, PKA in human cells could be activated through extracellular stimulation by forskolin (an adenylyl-cyclase activator) and IBMX (a phosphodiesterase inhibitor). The activation of PKA induced the separation of the catalytic subunits—the active form of PKA—from the regulation units and these catalytic subunits could be captured by the aptameric peptide. As shown in Figure 6, the fluorescence response of
Scheme 2. Schematic representation of the activation of PKA in MCF-7 cell lysate by forskolin and IBMX. The active catalytic subunits of PKA were recognized by the FITC-IP20/GO platform.
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detection within 15 min. The super-quenching capacity of GO and the high affinity of the aptameric peptide for kinase endow this method with high sensitivity and good selectivity. The rapid detection of active PKA in stimulated cell lysate revealed the feasibility of our method in “real” samples of cell lysate and the potential biochemical investigation of signal transduction. Furthermore, this GO-based assay could serve as a universal platform for detecting different protein kinases by simply changing their cognate aptameric peptides. Therefore, this method not only represents a promising technique for protein-kinase biosensing, but is also an intriguing example for expanding the application of GO to the research of post-translation modification-related enzymes.
Experimental Section Materials and Measurements A dispersion of graphite oxide in double-distilled water (0.5 mg mL 1) was purchased from XF Nano (Nanjing, China). Cyclic adenosine-3’,5’monophosphate-dependent protein kinase (PKA; catalytic subunit; Mw = 38 kDa; 2500 U mL 1) and Casein kinase II (CKII) were purchased from New England Biolabs Inc. (Beverly, MA). In general, PKA was diluted in the stock solutions (50 mm NaCl; 1 mm EDTA; 2 mm DTT; 50 % glycerol in 20 mm Tris-HCl buffer, pH 7.5, 25 8C) and stored at 80 8C. Fluorescein isothiocyanate (FITC)-labeled aptameric peptide (IP20) for PKA (FITC-TTYADFIASGRTGRRNAIHD), control peptides (FITC-CP20, FITC-TTYADAIASGRTGRRNAGHD; FITC-kemptide, FITCLRRASLG), and TARMA-IP20 (TARMA-TTYADFIASGRTGRRNAIHD) were synthesized by GL Biochem Ltd (Shanghai, China). Forskolin and 3-isobutyl-1-methylxantine (IBMX) were purchased from Sigma–Aldrich (St. Louis, MO). Protease inhibitor and the improved Bradford protein assay dye-reagent kit were purchased from Sangon (Shanghai, China). The commercial kinase-activity assay kit (ProFluor PKA Assay) was purchased from Promega (Madison, WI, USA). All other chemical reagents, including bovine serum albumin (BSA), glucose oxidase, thrombin, lysozyme, thrombin, catalase, and 2-amino-2-(hydroxymethy)-1, 3-propanediol (Tris), were supplied by Bio Basic (Ontario, Canada). Human breast-cancer cells (MCF-7) were obtained from the Cell Bank of Xiangya Central Experiment Laboratory of Central South University (Changsha, China). The MCF-7 breast-cancer cell supplement contained 10 % fetal bovine serum, 0.1 mm Mimimum Eagle’s essential medium (MEM) nonessential amino-acid solution, 1 % insulin-transferrin-selenium A supplement, penicillin (100 U mL 1), streptomycin (100 mg mL 1), and amphotericin B (0.25 mg mL 1). The solution was prepared by using ultrapure water (18.3 MW·cm) from the Millipore Milli-Q system in all experiments.
Figure 6. a) Fluorescence emission spectra of the GO/aptameric peptide sensor in response to various samples of cell lysate. b) Bar chart of the fluorescence intensity responses of cell lysate samples 1–7; a blank sample, which was treated with unstimulated cell lysate, was also recorded. The concentrations of the activator (forskolin/IBMX) are shown in the table.
FITC-IP20 towards cell lysate increased with increasing concentration of the stimulators and the fluorescence intensity almost reached a plateau when the concentrations of forskolin and IBMX reached 50 and 100 mm, respectively. This stimulator-dependent fluorescence-intensity profile was very similar to that with the commercial PKA activity assay kit for the same samples of cell lysate (see the Supporting Information, Figure S9). Therefore, the stimulator-triggered activation of PKA in cells was successfully detected by using the aptameric-peptide-based GO sensor, thus indicating that this approach holds great potential for cellular kinase assays.
Fluorescence emission spectra were recorded from 500 to 700 nm at an excitation wavelength of 480 nm on a Synergy Mx multimode microplate reader (BioTek Instruments, Inc.) at 30 8C. The fluorescence emission intensity was monitored at the emission maximum (520 nm). Atomic force microscopy (AFM) measurements were recorded on a MultiMode AFM (Veeco Instruments Inc., U.S.). Fluorescence anisotropy measurements were performed on a PTI QM40 fluorescence spectrometer. Cell-breaking was performed on a JY92-IIN ultrasound cell-disruption system (Scientz, Ningbo, China). Circular dichroism (CD) spectra were recorded at RT on a JASCO J-815 spectropolarimeter (Tokyo, Japan) in a rectangular quartz cuvette (path length: 1 mm). The spectra were collected from 190 to 300 nm with 0.1 nm intervals.
Conclusion We have developed a new peptide/GO fluorescence biosensing platform for the “turn-on” detection of PKA that employs protein-kinase recognition by an aptameric peptide. Compared with conventional kinase assays, which normally require time-consuming phosphorylation processes (about 1 h) and multiple detection steps, this method is rapid and facile, as confirmed by its “mix-and-read” property and fast
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Investigation of Fluorescence Quenching of FITC-IP20 by GO Samples of FITC-IP20 (60 mL, 10 mm) in 10 mm Tris-HCl buffer (pH 7.5, 25 8C) were added into a black 96-well microplate, followed by aliquots of GO dispersions with various concentrations of seriatim (0–
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50 mg mL 1). The samples were shaken for 1 min and then measured immediately afterwards at an excitation wavelength of 480 nm by using a multimode microplate reader.
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Detection of PKA by Using the GO/Peptide Sensor FITC-IP20 (60 mL) was treated with various concentrations of PKA (0– 625 mU mL 1) in 10 mm Tris-HCl buffer (pH 7.5) for 10 min at 30 8C. Then, 60 mL of the resulting solution (FITC-IP20/PKA complex) was added into a black 96-well microplate, followed by GO (5 mL, 40 mg mL 1), and the PKA detection was performed under the same conditions as described in the previous paragraph. A range of incubation times (0–30 min) was employed to optimize the reaction time between FITC-IP20 and PKA. Preparation of the MCF-7 Cell Culture and Lysate MCF-7 cells (1 106 cells) were incubated under a humidified atmosphere with 5 % CO2 at 37 8C. After incubation for 4 h in serum-free culture medium, a mixture of forskolin and IBMX in DMSO was added into the medium at various concentrations (for the concentrations of forskolin and IBMX, see Figure 6a) to stimulate intracellular PKA activity. An equal volume of DMSO was also added into the medium as in the control sample. After stimulation for 30 min, the cultured cells were removed by scraping and lysed in Dulbecco’s phosphate-buffered saline (D-PBS) that contained protease inhibitor by using sonication (200 W) for 2 s for 60 times with 3 s intervals for protein extraction. The samples were centrifuged at 22 000 rpm for 60 min at 4 8C and the extracted supernatants were stored at 20 8C. Then, the Bradford method was employed to determine the total protein content in the cell lysate supernatants. Subsequent kinase assays in MCF-7 cell lysate were performed by using the above-mentioned standard procedure, except for the involvement of pre-prepared cell lysate samples instead of PKA. Measurement of Kinase Activity with the Fluorescent PKA Activity Assay Kit
The ProFluor PKA assay was used as a reference method for evaluating the proposed kinase assay. The assay was performed according to the manufacturer’s instructions. A solution (25 mL) of the PKA R110 substrate (10 mm) and various concentrations of PKA (0–625 mU mL 1) in 1 buffer solution were added into a black 96-well microplate, followed by a 0.1 mm solution of ATP (25 mL) in the buffer. The resulting solution was shaken for 15 s and incubated for 20 min at 30 8C. Then, the protease solution (25 mL) was added into each well and the microplate was mixed and incubated for 30 min at RT. Finally, the stabilizer solution (25 mL) was added into each well. The fluorescence signal was recorded at an emission wavelength of 530 nm with an excitation wavelength of 485 nm. For the kinase-activity assay with cell lysate, the experiments were performed by using the above-mentioned procedures with the prepared cell lysate samples instead of solutions of PKA.
Acknowledgements This work was financially supported by the National Basic Research Program of China (973 Program; 2009CB421601 and 2011CB911002), the Foundation for Innovative Research Groups of NSFC (21221003), the National Natural Science Foundation of China (21222507, 21175036, 21190044, 21075031, and 21205106), and the Program for New Century Excellent Talents in University (NCET-10-0366). X. H. Xu acknowledges the financial support from a Project Supported by Scientific Research Fund of Zhejiang Provincial Education Department (Y201225536).
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Received: March 12, 2014 Published online: July 22, 2014
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A Mix-and-Read Fluorescence Strategy for the Switch-On Probing of Kinase Activity Based on an Aptameric-Peptide/Graphene-Oxide Platform Chunyang Lei,[a] Xiahong Xu,[a, b] Jiang Zhou,[a] Xin Liu,[a] Zhou Nie,*[a] Meng Qing,[a] Pei Li,[a] Yan Huang,*[a] and Shouzhuo Yao[a] Abstract: Protein kinase plays a vital role in regulating signal-transduction pathways and its simple and quick detection is highly desirable because traditional kinase assays typically rely on a time-consuming kinase-phosphorylation process (ca. 1 h). Herein, we report a new and rapid fluorescencebased sensing platform for probing the activity of protein kinase that is based on the super-quenching capacity of gra-
phene oxide (GO) nanosheets and specific recognition of the aptameric peptide (FITC-IP20). On the GO/peptide platform, the fluorescence quenching of FITC-IP20 that is adsorbed onto GO can be restored by selective binding of active protein kinase to the aptameric Keywords: biosensors · fluorescence · graphene · kinase · peptides
Introduction
avoid these shortcomings, great effort has been devoted to the design of alternative methods and a number of methods based on fluorescence,[5] electrochemistry,[6] surface plasmon resonance spectroscopy,[7] and mass spectroscopy[8] have been developed. Conventionally, these assays involve kinase-catalyzed phosphorylation reactions, which typically require about 1 h reaction time to accumulate enough phosphorylated product for effective detection. Moreover, multistep detection processes have been required in some methods, including labeling, incubation, washing, generation, and amplification of the detection signal, which are labor-intensive and time-consuming. Therefore, convenient, rapid, and cost-effective assays for detecting protein kinase are in demand but highly challenging. Recently, we found that the specific inhibitory peptide of protein kinase could be exploited as a potent aptameric peptide for recognizing kinase and we developed a one-step approach for the rapid detection (< 10 min) of protein kinase by using the quartz crystal microbalance (QCM).[9] This aptameric peptide is the natural protein kinase A (PKA)-inhibitory peptide, IP20, which contains a RRNAI sequence that replaces the RRXS recognition motif of the PKA substrate.[10] The aptameric peptide has specific affinity with the free catalytic subunit of PKA—the activated form of PKA inside the cell—and shows a much higher affinity for PKA than the substrate peptide, in accordance with their relative K values (IP20 : Ki = 2.3 nm, substrate kemptide: Km = 5 mm).[11] By using this peptide, the stimulated activation of PKA in the cell lysate has been successfully detected by QCM. Because the feasibility of this aptameric peptide for kinase recognition has been demonstrated, the application of this aptameric peptide in various detection methods is anticipated, in particular in homogenous fluorescent assays,
The protein-phosphorylation reaction catalyzed by protein kinases plays a vital role in regulating the signal-transduction pathways in the majority of crucial cellular processes, such as cell growth, metabolism, differentiation, proliferation, and apoptosis.[1] Indeed, the over-expression and aberrant activity of protein kinase, which causes abnormal cellular signaling, is associated with a range of diseases, including cancer, diabetes, inflammation, cardiac diseases, and Alzheimer’s disease.[2] Therefore, the detection of protein kinase is valuable for understanding the diseases related to fundamental biological processes, as well as for clinical diagnosis and drug-discovery applications.[1b, 2b, 3] The standard method that is used to detect kinase activity relies on radiolabeled g-32/33P-ATP; however, this method may pose a threat to human health and to the environment, owing to the use of hazardous radioactive reagents.[1a, 4] To
[a] C. Lei,+ Dr. X. Xu,+ J. Zhou, X. Liu, Prof. Dr. Z. Nie, M. Qing, P. Li, Dr. Y. Huang, Prof. S. Yao State Key laboratory of Chemo/Biosensing and Chemometrics College of Chemistry and Chemical Engineering Hunan University, Changsha, 410082 (P. R. China) Fax: (+ 86) 731-8882-1848 E-mail: [email protected] [email protected] [b] Dr. X. Xu+ College of Biosystem Engineering and Food Science Zhejiang University Hangzhou, Zhejiang, 310058 (P. R. China) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201402221.
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peptide, thereby resulting in the fast switch-on detection of kinase activity (ca. 15 min). The feasibility of this method has been demonstrated by the sensitive measurement of the activity of cAMP-dependent protein kinase (PKA), with a detection limit of 0.053 mU mL 1. This assay technique was also successfully applied to the detection of kinase activation in cell lysate.
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which are highly sensitive, convenient, and ready for adaption to high-throughput screening. Graphene oxide (GO)—a 2D carbon material with singleatom thickness—has attracted considerable attention, owing to its remarkable mechanical, thermal, optical, and electronic properties.[12] Owing to its intrinsic properties, including large surface area, water solubility, good biocompatibility, and facile surface modification, GO represents a promising material in biosensing, bioengineering, and drug delivery.[13] Moreover, GO has been used as a fluorescence superquencher of various fluorophores over a wide wavelength range, owing to its long-range nanoscale energy transfer. GO/DNA-based sensing platforms have been widely applied in DNA and RNA analysis.[14] By comparison, the application of GO/peptide nanocomposites in fluorescence-based biosensors has been much less explored and only a few reports have appeared so far. Qu’s group reported the selective detection of Cyclin A2 by using a graphene-oxide/peptide platform,[15] whilst Leblanc’s group developed a fluorescence assay for peptides and proteins by using the quenching effect of GO on the intrinsic fluorescence of the peptide.[16] Further application of GO/peptide systems in protease detection has also been reported;[17] however, the application of GO/peptide sensing systems for post-translation modification-related enzymes assay remains scarce. Recently, we developed a GO-based fluorescence biosensing method for determining the activity of protein kinase that employed phosphorylation protection against protease cleavage.[18] This method was efficient and versatile, but it was still hampered by the relatively long reaction time of the phosphorylation and subsequent protease-hydrolysis steps, as well as by its “turn-off” detection mode.
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Herein, we report a new and rapid fluorescence-sensing platform for the “turn-on” probing of the activity of protein kinase that is based on the super-quenching capability of graphene-oxide nanosheets and the specific recognition of an aptameric peptide (FITC-IP20). In this GO/peptide platform, the fluorescence quenching of an FITC-labeled aptameric peptide (FITC-IP20) that is adsorbed onto GO can be restored by selective binding of the aptameric peptide to active protein kinase (PKA), thereby resulting in “turn-on” kinase detection. This GO-based platform is simple and feasible to construct, with small background interference, thereby providing superior sensitivity and rapid response without the need to perform phosphorylation processes. Moreover, our approach does not require multi-step washing, prolonged incubation, or complicated sample preparation. Because a variety of specific inhibitor peptides for their cognate kinases are naturally occurring, this method has potential to be a versatile technique for rapid kinase detection.
Results and Discussion Detection Mechanism The principle of the GO/peptide kinase assay is shown in Scheme 1. It is well-known that GO is an amphiphile that contains a hydrophobic basal plane and ionizable hydrophilic edges (e.g., COOH groups).[19] As calculated by using ProtParam, the aptameric peptide IP20 is a 20-amino-acid-
Abstract in Chinese:
Scheme 1. Principles of the fluorescence biosensor for PKA, based on FITC-IP20 and GO.
long peptide that contains two aromatic (Phe and Tyr) residues, with a pI value of 8.43 and moderate hydrophobicity (aliphatic index: 54.00). Hence, mixing FITC-IP20 with GO at pH 7.5 allows the FITC-IP20 to be non-covalently adsorbed onto the surface of GO through p–p, hydrophobic, and electrostatic interactions and the proximity of the GO and FITC moieties effectively quench the fluorescence of FITC (Scheme 1 a). In the presence of PKA, the strong affinity between FITC-IP20 and PKA causes the formation of
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without the presence of GO (40 mg mL 1) at various concentrations of FITC-IP20 (0–30 mm) and the corresponding bar plots are shown in the Supporting Information, Figure S2. The highest QE value was obtained at 10 mm FITC-IP20. Thus, 40 mg mL 1 GO and 10 mm FITC-IP20 in 10 mm TrisHCl buffer were used in the subsequent experiments. The adsorption of aptameric peptide FITC-IP20 onto GO was further investigated by investigating the thickness and height profile of GO and the FITC-IP20/GO complex by atomic force microscopy (AFM; see the Supporting Information, Figure S3). AFM analysis revealed that the thickness of GO was 1.0 nm, consistent with the previously reported apparent thickness of GO.[14a] The thickness of the FITC-IP20/GO complex was 2.1 nm, higher than that of pristine GO, thus indicating that FITC-IP20 was absorbed onto the GO surface. The adsorption of FITC-IP20 onto GO was further investigated by CD spectroscopy. The FITC-IP20/GO complex was prepared by centrifugation to remove any unbound FITC-IP20. Peaks were observed at 203 and 220 nm in the FITC-IP20 sample, which were characteristic of an a-helical structure (see the Supporting Information, Figure S4). The CD spectrum of FITC-IP20 that had been incubated with GO alone showed minimal changes compared with that with FITC-IP20 alone, in good agreement with a previous report.[20] This result indicated that FITC-IP20 was successfully adsorbed onto the GO surface without remarkable conformation changes.
a FITC-IP20/PKA complex, thereby resulting in the tagged FITC moving away from the GO surface and subsequent recovery of the fluorescence (Scheme 1 b). Therefore, specific recognition of the aptameric peptide by PKA readily transduces to afford a measurable signal enhancement and provides a facile “turn-on” assay for PKA detection. Investigation of Fluorescence Quenching Effect of Graphene Oxide (GO) on FITC-IP20 First, we studied the interactions between FITC-IP20 and GO. The non-covalent adsorption of FITC-IP20 onto GO was studied by evaluating the change in fluorescence intensity of FITC-IP20 (10 mm) on mixing with different concentrations of GO. Fluorescence emission spectra and the corresponding changes in fluorescence intensity at 520 nm are shown in Figure 1. On the increasing concentration of GO,
Detection of PKA by the GO/Aptameric-Peptide System The detection of PKA by the proposed GO/aptameric-peptide sensor was performed under the optimized conditions. As shown in Figure 2 a, the addition of GO induced 95.8 % quenching of the fluorescence of FITC-IP20, which indicated the strong adsorption of FITC-IP20 onto GO and a high fluorescence-quenching efficiency of GO towards FITC-IP20. Moreover, in the presence of 250 mU mL 1 PKA, the fluorescence intensity increased 7.1-fold compared with that of the FITC-IP20/GO sample, thus implying that the binding of aptameric peptide by PKA caused a remarkable restoration of the fluorescence and provided a “turn-on” detection of PKA. Furthermore, the kinetic behavior of the interactions between FITC-IP20 and GO with or without PKA was studied by monitoring the fluorescence intensity as a function of time (30 min). As shown in Figure 2 b, the change in fluorescence intensity of FITC-IP20 (10 mm) was negligible during the measurements (Figure 2 b, curve 1). The addition of GO caused a rapid decrease in the fluorescence intensity of FITC-IP20 (within 5 min; Figure 2 b, curve 2). In the presence of PKA, upon the addition of GO, the change in fluorescence intensity followed the same pattern as that with FITC-IP20 alone, but was much smaller, probably because the formation of the FITC-IP20/PKA complex distanced FITC from GO and the unbound FITC-IP20 adsorbed onto the GO surface. This result indicated that an incubation time of 5 min was sufficient for the complete adsorption of the aptameric peptide on GO. Therefore, the feasibility of
Figure 1. Fluorescence emission spectra of FITC-IP20 on the addition of 0–50 mg mL 1 GO in TB buffer (10 mm Tris-HCl at pH 7.5, 25 8C). Inset shows a plot of the corresponding fluorescence intensity of FITC-IP20 (10 mm) at 520 nm versus the concentration of GO.
the fluorescence intensity of FITC-IP20 gradually decreased and plateaued at 40 mg mL 1 GO. Quenching efficiency (QE) is defined as (F0 F)/F0, where F0 and F are the fluorescence intensity of FITC-IP20 before and after the addition of GO, respectively. The QE of FITC-IP20 with 40 mg mL 1 GO was 95.8 %. The superior fluorescence-quenching ability of GO enables this method to have a low background. Furthermore, the effect of buffer on the fluorescencequenching efficiency of GO on FITC-IP20 was also investigated. Buffers were compared with or without the presence of magnesium ions. As shown in the Supporting Information, Figure S1, the fluorescence quenching of mixtures of FITC-IP20 with equal amounts of GO in TB buffer (10 mm Tris-HCl at pH 7.5) was stronger than in TBS buffer (10 mm Tris-HCl, 10 mm MgCl2 at pH 7.5). As a result, we concluded that increasing ionic strength decreased the quenching efficiency of GO, thus indicating that electrostatic interactions contributed to the fluorescence quenching between positively charged FITC-IP20 and negative charged GO. Next, we compared the fluorescence intensity of FITC-IP20 with and
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Figure 3. Fluorescence anisotropy values of (1) FITC-IP20 ; (2) FITC-IP20/ GO; (3) FITC-IP20/PKA; and (4) FITC-IP20/PKA/GO. The concentrations of FITC-IP20, PKA, and GO were 10 mm, 250 mU mL 1, and 40 mg mL 1, respectively.
suggesting that fluorescence detection was preferable for this sensor. Figure 2. a) Fluorescence emission spectra of FITC-IP20 (10 mm, curve 1), FTC-IP20/GO (40 mg mL 1, curve 2), and FITC-IP20 incubated with PKA (250 mU mL 1) for 10 min upon the addition of GO (40 mg mL 1, curve 3); the excitation wavelength was at 480 nm. b) Plot of fluorescence intensity changes of FITC-IP20 (curve 1), FTC-IP20/GO (curve 2), and FITC-IP20/ PKA/GO (curve 3) as a function of incubation time (0–30 min).
Specific Recognition and Detection of PKA First, various incubation times (0–30 min) of FITC-IP20 and PKA were investigated to optimize the reaction (see the Supporting Information, Figure S5). On increasing the reaction time, the corresponding fluorescence intensity gradually increased and then reached a plateau after 10 min, thus indicating that the binding of PKA to FITC-IP20 was fast. Therefore, a binding time of 10 min was employed in the subsequent assays. To investigate the sensitivity of our proposed biosensor for the detection of PKA, various concentrations of PKA (0–625 mU mL 1) were tested (Figure 4). The results clearly revealed that the fluorescence intensity continuously increased with increasing PKA concentration and that the fluorescence intensity reached a plateau at a PKA concentration of 250 mU mL 1. Thus, the fluorescence-intensity change was successfully used to assess the degree of binding of the aptameric peptide to PKA. Figure 4, inset, shows a calibration curve of the fluorescence intensity as function of the logarithm of PKA concentration. The EC50 value (the amount of kinase needed to achieve 50 % of the maximum signal) using this approach was 30.28 mU mL 1, similar to that for our previously reported QCM sensor for PKA based on the aptameric peptide (33.64 mU mL 1). The assay allowed for the detection of PKA at concentrations as low as 0.053 mU mL 1, lower than with many previously reported assays.[4c, 6c, 22] To compare our aptameric-peptide-based approach with the traditional kinase-activity assay, the commercial fluorescence kinase-activity assay kit was employed as a reference.
this biosensing platform for the quick detection of PKA has been demonstrated. Fluorescence Anisotropy Detection of PKA The fluorescence anisotropy (FA) of a fluorophore, which is defined as the ability of a molecule to rotate in its microenvironment, is sensitive to the molecular weight of the fluorophore.[21] Therefore, anisotropy measurements were used to further investigate the aptameric-peptide/PKA and peptide/ GO interactions. As shown in Figure 3, the original FA value of FITC-IP20 in Tris-HCl buffer was 0.075, and its FA value significantly increased 4.7-fold to 0.354 upon the addition of GO, thus indicating the formation of large-mass complex FITC-IP20/GO. The introduction of PKA into a solution of FITC-IP20 caused a 2-fold increase in the FA value compared with FITC-IP20 alone, thus confirming the specific binding of FITC-IP20 to the target PKA. The FA value of the FITC-IP20/PKA complex in the presence of GO (0.227) was smaller than that of FITC-IP20/GO, thus indicating that PKA liberated FITC-IP20 from the capture of GO. These results provided further support for the mechanism of this peptide/GO system. Notably, the PKA-induced change in FA was not as significant as the change in fluorescence, thus
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Figure 4. Changes in the fluorescence spectra with increasing concentration of PKA (0–625 mU mL 1). Inset shows the calibration curve for fluorescence intensity change at 520 nm as a function of the logarithm of PKA concentration.
Figure 5. Fluorescence intensity responses of the sensor to different proteins: (1) 250 mU mL 1 PKA; (2) 0.25 mg mL 1 glucose oxidase; (3) 0.25 mm catalase; (4) 0.25 mm lysozyme; (5) 0.25 mm BSA; (6) 250 mU mL 1 thrombin; (7) 250 mU mL 1 CKII instead of PKA; (8) FITC-CP20 instead of FITC-IP20 peptide; and (9) PKA substrate peptide FITC-kemptide instead of FITC-IP20 peptide, respectively. The excitation wavelength was 480 nm.
Plots of the fluorescence-intensity response of both assays as a function of the logarithm of PKA concentration are shown in the Supporting Information, Figure S6. These results indicated that the fluorescence signal from our directrecognition approach perfectly corresponded with that from the commercial PKA assay kit. The EC50 value for our approach (30.28 mU mL 1) was quite close to that with the commercial kit (31.36 mU mL 1). In addition, the fluorescence-intensity response of a sample of FITC-IP20 that had been incubated with thermally inactivated PKA was negligible compared with that with the activated PKA (see the Supporting Information, Figure S7 A), in accordance with that for the commercial PKA assay kit (see the Supporting Information, Figure S7 B). These results successfully demonstrate that the aptameric-peptide-based method can also effectively reflect the level of kinase activity through specific binding between the aptameric peptide (IP20) and the active form of PKA, without the need for a phosphorylation process. Furthermore, the robustness and reproducibility of this aptameric-peptide-based approach for kinase assay was evaluated. As shown in the Supporting Information, Figure S8, the Z factor of this assay was 0.75, which indicated that this method was a solid assay with good reproducibility.[23] The specificity of our proposed sensor towards PKA was examined in a series of control experiments. Six other control proteins, including non-target kinase CKII, and other enzymes, such as glucose oxidase, catalase, lysozyme, BSA, thrombin, and FITC-CP20 (two mutated amino acids compared with FITC-IP20), as well as PKA substrate peptide FITC-kemptide as a control peptide, were investigated. None of the non-specific proteins gave the same remarkable fluorescence response in the assay as PKA did (Figure 5). Similarly, FITC-CP20 and FITC-kemptide caused negligible fluorescence responses compared with the aptameric peptide (IP20), because their affinities towards PKA were significantly weaker than that of IP20. This result clearly demonstrated that this platform based on GO and FITC-IP20 could
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be used as a selective probe for the targeted detection of the PKA protein. Active Protein Kinase Detection in Cell Lysate It is well-known that the activation of kinases in cells by extracellular stimulation can trigger a range of important cellular processes, including transcription, differentiation, and apoptosis.[24] Thus, whether an assay is available for detecting kinase activity in cell lysate is important for the study of kinase regulation in cell systems. As shown in Scheme 2, PKA in human cells could be activated through extracellular stimulation by forskolin (an adenylyl-cyclase activator) and IBMX (a phosphodiesterase inhibitor). The activation of PKA induced the separation of the catalytic subunits—the active form of PKA—from the regulation units and these catalytic subunits could be captured by the aptameric peptide. As shown in Figure 6, the fluorescence response of
Scheme 2. Schematic representation of the activation of PKA in MCF-7 cell lysate by forskolin and IBMX. The active catalytic subunits of PKA were recognized by the FITC-IP20/GO platform.
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detection within 15 min. The super-quenching capacity of GO and the high affinity of the aptameric peptide for kinase endow this method with high sensitivity and good selectivity. The rapid detection of active PKA in stimulated cell lysate revealed the feasibility of our method in “real” samples of cell lysate and the potential biochemical investigation of signal transduction. Furthermore, this GO-based assay could serve as a universal platform for detecting different protein kinases by simply changing their cognate aptameric peptides. Therefore, this method not only represents a promising technique for protein-kinase biosensing, but is also an intriguing example for expanding the application of GO to the research of post-translation modification-related enzymes.
Experimental Section Materials and Measurements A dispersion of graphite oxide in double-distilled water (0.5 mg mL 1) was purchased from XF Nano (Nanjing, China). Cyclic adenosine-3’,5’monophosphate-dependent protein kinase (PKA; catalytic subunit; Mw = 38 kDa; 2500 U mL 1) and Casein kinase II (CKII) were purchased from New England Biolabs Inc. (Beverly, MA). In general, PKA was diluted in the stock solutions (50 mm NaCl; 1 mm EDTA; 2 mm DTT; 50 % glycerol in 20 mm Tris-HCl buffer, pH 7.5, 25 8C) and stored at 80 8C. Fluorescein isothiocyanate (FITC)-labeled aptameric peptide (IP20) for PKA (FITC-TTYADFIASGRTGRRNAIHD), control peptides (FITC-CP20, FITC-TTYADAIASGRTGRRNAGHD; FITC-kemptide, FITCLRRASLG), and TARMA-IP20 (TARMA-TTYADFIASGRTGRRNAIHD) were synthesized by GL Biochem Ltd (Shanghai, China). Forskolin and 3-isobutyl-1-methylxantine (IBMX) were purchased from Sigma–Aldrich (St. Louis, MO). Protease inhibitor and the improved Bradford protein assay dye-reagent kit were purchased from Sangon (Shanghai, China). The commercial kinase-activity assay kit (ProFluor PKA Assay) was purchased from Promega (Madison, WI, USA). All other chemical reagents, including bovine serum albumin (BSA), glucose oxidase, thrombin, lysozyme, thrombin, catalase, and 2-amino-2-(hydroxymethy)-1, 3-propanediol (Tris), were supplied by Bio Basic (Ontario, Canada). Human breast-cancer cells (MCF-7) were obtained from the Cell Bank of Xiangya Central Experiment Laboratory of Central South University (Changsha, China). The MCF-7 breast-cancer cell supplement contained 10 % fetal bovine serum, 0.1 mm Mimimum Eagle’s essential medium (MEM) nonessential amino-acid solution, 1 % insulin-transferrin-selenium A supplement, penicillin (100 U mL 1), streptomycin (100 mg mL 1), and amphotericin B (0.25 mg mL 1). The solution was prepared by using ultrapure water (18.3 MW·cm) from the Millipore Milli-Q system in all experiments.
Figure 6. a) Fluorescence emission spectra of the GO/aptameric peptide sensor in response to various samples of cell lysate. b) Bar chart of the fluorescence intensity responses of cell lysate samples 1–7; a blank sample, which was treated with unstimulated cell lysate, was also recorded. The concentrations of the activator (forskolin/IBMX) are shown in the table.
FITC-IP20 towards cell lysate increased with increasing concentration of the stimulators and the fluorescence intensity almost reached a plateau when the concentrations of forskolin and IBMX reached 50 and 100 mm, respectively. This stimulator-dependent fluorescence-intensity profile was very similar to that with the commercial PKA activity assay kit for the same samples of cell lysate (see the Supporting Information, Figure S9). Therefore, the stimulator-triggered activation of PKA in cells was successfully detected by using the aptameric-peptide-based GO sensor, thus indicating that this approach holds great potential for cellular kinase assays.
Fluorescence emission spectra were recorded from 500 to 700 nm at an excitation wavelength of 480 nm on a Synergy Mx multimode microplate reader (BioTek Instruments, Inc.) at 30 8C. The fluorescence emission intensity was monitored at the emission maximum (520 nm). Atomic force microscopy (AFM) measurements were recorded on a MultiMode AFM (Veeco Instruments Inc., U.S.). Fluorescence anisotropy measurements were performed on a PTI QM40 fluorescence spectrometer. Cell-breaking was performed on a JY92-IIN ultrasound cell-disruption system (Scientz, Ningbo, China). Circular dichroism (CD) spectra were recorded at RT on a JASCO J-815 spectropolarimeter (Tokyo, Japan) in a rectangular quartz cuvette (path length: 1 mm). The spectra were collected from 190 to 300 nm with 0.1 nm intervals.
Conclusion We have developed a new peptide/GO fluorescence biosensing platform for the “turn-on” detection of PKA that employs protein-kinase recognition by an aptameric peptide. Compared with conventional kinase assays, which normally require time-consuming phosphorylation processes (about 1 h) and multiple detection steps, this method is rapid and facile, as confirmed by its “mix-and-read” property and fast
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Investigation of Fluorescence Quenching of FITC-IP20 by GO Samples of FITC-IP20 (60 mL, 10 mm) in 10 mm Tris-HCl buffer (pH 7.5, 25 8C) were added into a black 96-well microplate, followed by aliquots of GO dispersions with various concentrations of seriatim (0–
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50 mg mL 1). The samples were shaken for 1 min and then measured immediately afterwards at an excitation wavelength of 480 nm by using a multimode microplate reader.
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Detection of PKA by Using the GO/Peptide Sensor FITC-IP20 (60 mL) was treated with various concentrations of PKA (0– 625 mU mL 1) in 10 mm Tris-HCl buffer (pH 7.5) for 10 min at 30 8C. Then, 60 mL of the resulting solution (FITC-IP20/PKA complex) was added into a black 96-well microplate, followed by GO (5 mL, 40 mg mL 1), and the PKA detection was performed under the same conditions as described in the previous paragraph. A range of incubation times (0–30 min) was employed to optimize the reaction time between FITC-IP20 and PKA. Preparation of the MCF-7 Cell Culture and Lysate MCF-7 cells (1 106 cells) were incubated under a humidified atmosphere with 5 % CO2 at 37 8C. After incubation for 4 h in serum-free culture medium, a mixture of forskolin and IBMX in DMSO was added into the medium at various concentrations (for the concentrations of forskolin and IBMX, see Figure 6a) to stimulate intracellular PKA activity. An equal volume of DMSO was also added into the medium as in the control sample. After stimulation for 30 min, the cultured cells were removed by scraping and lysed in Dulbecco’s phosphate-buffered saline (D-PBS) that contained protease inhibitor by using sonication (200 W) for 2 s for 60 times with 3 s intervals for protein extraction. The samples were centrifuged at 22 000 rpm for 60 min at 4 8C and the extracted supernatants were stored at 20 8C. Then, the Bradford method was employed to determine the total protein content in the cell lysate supernatants. Subsequent kinase assays in MCF-7 cell lysate were performed by using the above-mentioned standard procedure, except for the involvement of pre-prepared cell lysate samples instead of PKA. Measurement of Kinase Activity with the Fluorescent PKA Activity Assay Kit
The ProFluor PKA assay was used as a reference method for evaluating the proposed kinase assay. The assay was performed according to the manufacturer’s instructions. A solution (25 mL) of the PKA R110 substrate (10 mm) and various concentrations of PKA (0–625 mU mL 1) in 1 buffer solution were added into a black 96-well microplate, followed by a 0.1 mm solution of ATP (25 mL) in the buffer. The resulting solution was shaken for 15 s and incubated for 20 min at 30 8C. Then, the protease solution (25 mL) was added into each well and the microplate was mixed and incubated for 30 min at RT. Finally, the stabilizer solution (25 mL) was added into each well. The fluorescence signal was recorded at an emission wavelength of 530 nm with an excitation wavelength of 485 nm. For the kinase-activity assay with cell lysate, the experiments were performed by using the above-mentioned procedures with the prepared cell lysate samples instead of solutions of PKA.
Acknowledgements This work was financially supported by the National Basic Research Program of China (973 Program; 2009CB421601 and 2011CB911002), the Foundation for Innovative Research Groups of NSFC (21221003), the National Natural Science Foundation of China (21222507, 21175036, 21190044, 21075031, and 21205106), and the Program for New Century Excellent Talents in University (NCET-10-0366). X. H. Xu acknowledges the financial support from a Project Supported by Scientific Research Fund of Zhejiang Provincial Education Department (Y201225536).
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Received: March 12, 2014 Published online: July 22, 2014
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