A Wild-Type Nanopore Sensor for Protein Kinase Activity | Analytical

Jun 26, 2019 - Protein kinases play a critical role in regulating virtually all cellular processes. Here, we developed a novel one-step method based o...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/ac

Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

A Wild-Type Nanopore Sensor for Protein Kinase Activity Fu-Na Meng,§,† Yi-Lun Ying,*,†,‡ Jie Yang,† and Yi-Tao Long*,†,‡ †

School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, 200237, P. R. China State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, P. R. China

‡

Downloaded via GUILFORD COLG on July 23, 2019 at 01:02:39 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: Protein kinases play a critical role in regulating virtually all cellular processes. Here, we developed a novel onestep method based on a wild-type aerolysin nanopore, which enables kinase activity detection without labeling/modification, immobilization, cooperative enzymes and complicated procedures. By virtual of the positively charged confinement of the aerolysin nanopore, the kinase-induced phosphopeptides are specially captured while the positively charged substrate peptides might move away from the pore by the electric field. Combining with internal standard method, the event frequency of the phosphopeptides exhibited a dose-dependent response with kinases. The detection limit of 0.005 U/μL has been achieved with protein kinase A as a model target. This method also allowed kinase inhibitor screening, kinase activity sensing in cell lysates and the real-time monitoring of kinase-catalyzed phosphorylation at singe molecule level, which could further benefit fundamental biochemical research, clinical diagnosis and kinase-targeted drug discovery. Moreover, this nanopore sensor shows strong capacity for the other enzymes that altered substrate charge (e.g., sulfonation, carboxylation, or amidation).

P

Nanopore sensor provides a single-molecule platform for sensitive detection.14,15 When a certain voltage is applied across a nanoscale pore, target molecules will be driven into the pore and interact with it, resulting in the typical blockages of the pore’s ionic current. Both the target identity and quantity can be determined by analyzing the current signatures. The nanopore technique has been extensively employed to detect DNA/RNA,16−20 peptides,21,22 proteins,23,24 enzymes25−28 and host−guest molecules.29,30 Aerolysin,31−33 a promising protein sensor, has been applied to investigate the peptides with different charges,21,34,35 discriminate very short oligonucleotides with single base resolution36 and monitor enzymatic degradation kinetics,37 et al. Although the protein kinase inhibitors and the binding constants between protein kinases and the substrate could be evaluated by nanopore via genetic engineering and chemical modifying,38,39 the direct detection of kinase activity has not been achieved by nanopore up to now. Besides, the chemical modification of nanopores complicated the analysis procedures and increased the cost. Here we utilize the positively charged confinement of the wild-type aerolysin nanopore to achieve one-step method for the kinase activity sensing including

rotein kinases are a critical family of enzymes that regulate virtually all cellular processes, including differentiation, proliferation, motility, and apoptosis, and thus cell function.1−3 Modulation of cell function by kinases is the result of the phosphorylation of target protein substrates that are involved in intracellular signaling pathways.4,5 The phosphorylation of protein substrates is evidenced to activate or deactivate target proteins at the molecular level. The resulting activation or deactivation of target proteins can in turn lead to aberrant signal transduction if levels of kinase activity are altered, as is the case in many disease states.6,7 Therefore, monitoring protein kinase activity is significant for fundamental biochemical research, clinical diagnosis, and kinase-targeted drug discovery. A variety of methods have been developed to assess protein kinase activities, including radiometric assays,8 fluorescence,9,10 electrochemistry,11 surface plasmon resonance12 and mass spectrometry.13 These methods mentioned above have their own unique advantages, such as high sensitivity and high specificity, etc. However, most of them suffer from certain drawbacks such as harmful radioactive labels, sophisticated and costly fluorescence-labeling peptide, multistep detection procedures, inadequate contact in reactants and expensive recognition proteins. Thus, it is still challenging but highly desirable to develop new principle for simple, convenient, label-free detection for protein kinase. © XXXX American Chemical Society

Received: March 29, 2019 Accepted: June 26, 2019 Published: June 26, 2019 A

DOI: 10.1021/acs.analchem.9b01570 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Figure 1. (a) Schematic representation of PKA activity detection via a wild-type aerolysin nanopore. Positive-charged S-peptide (LRRASLG) hardly induced any blockage events while the product P-peptide produced large amounts of current blockage events after catalyzed by PKA. The red circle on the P-peptide represents the phosphate group with two negative charges. The raw current traces were recorded with the reaction solution containing 100 μM S-peptide before and after catalyzed by PKA (0.1 U/μL). The detailed information on this PKA catalysis experiment is shown in the Experimental Section. The detection buffer was composed of 1.0 M KCl, 20 mM MgCl2 and 50 mM Tris (pH 7.5). The potential was set at +100 mV, which was applied from stem side and the cap side was connected to the ground. (b) Correlation curve between the relative event frequency of P-peptide and [PKA]. Based on the curve, the PKA can be quantified via the nanopore. (c) The phosphorylation evolution of Speptide in the course of the enzyme catalysis reaction. The aerolysin nanopore achieved to real-time monitor the kinase catalysis at single molecule level.

(Shanghai, China). cAMP-dependnet protein kinase (PKA) catalytic subunit was purchased from New England Biolabs (Beverly, MA, USA). Forskolin (Fsk) was purchased from GL Biochem Ltd. (Shanghai, China). 3-Isobutyl-1-methylxanthine (IBMX), H-89 and decane were obtained from SigmaAldrich (St Louis, MO, USA). ATP and ADP were purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). MCF-7 cells were from MoXi Biotech. Co. Ltd. (Shanghai, China). All other reagents were of analytical grade and were used as received without further purification. Nanopore Formation and Data Collection. The details of the nanopore formation are described in our previous research.29 The two chambers beside the nanopore were filled with the buffer solution (1 M KCl, 20 mM MgCl2 and 50 mM Tris at pH 7.5). The experiments were conducted using eONE amplifier (Elements SRL, Cesena, Italy). The sampling rate was set to 100 kHz and the final bandwidth 5 kHz. The data were acquired by EDR 3 software (Elements SRL, Cesena, Italy). Data analysis was performed using MOSAIC software44 and Origin 8.0 (OriginLab Corporation, Northampton, MA). The event frequencies were obtained by plotting the relation curves between cumulative event counts versus recording time. PKA Activity Detection and Inhibition Study. In the presence of ATP and Mg2+, PKA can transfer γ-phosphate group of ATP to the serine residue of S-peptide at pH 7.5. Then, the assays for assessing kinase activity were performed as follows. Typically, the PKA-catalyzed phosphorylation reaction was carried out in a 100 μL volume of reaction solution (1 M KCl, 20 mM MgCl2, 50 mM Tris, pH 7.5) containing 100 μM S-peptide, 100 μM ATP and a certain concentration of PKA at 30 °C for 12 h. Afterward, the reaction system was immediately put into water bath with the constant temperature 65 °C for 20 min to denature the enzyme. Then, the reaction system was added into the cap side chamber of the aerolysin nanopore, which contained 900 μL buffer solution, after it restored to the room temperature (27 ± 1 °C). Then, the current traces of aerolysin nanopore were recorded for analyzing the reaction system. The experimental procedures for the inhibition assay were similar to those stated above for detection PKA activity, except for the preincubation of a fixed

ultrasensitive kinase activity detection in cell lysate, the kinase inhibitor screening and the real-time monitoring of the kinase kinetics. A previous research utilized the α-hemolysin nanopore to detect the protein phosphorylation by tagging the protein on the C terminus with a 30-mer oligodeoxycytidine.40 In this paper, the peptide phosphorylation without any labeling and chemical modifications can be determined via the aerolysin nanopore, aiming to obtain the target kinase activity. The principle that underlies the sensing platform is shown in Figure 1. Here, we selected protein kinase A (PKA) as a model target and its recognition consensus sequence LRRASLG41 as the substrate peptide (S-peptide) to examine the kinase activity. Under the catalysis of PKA, the serine of S-peptide will be phosphorylated, resulting in phosphorylated peptide (Ppeptide) (Supporting Information (SI) Figure S1). The Ppeptide induced the evident current blockages while the positively charged molecule, S-peptide, might move away from the pore and hardly caused any blockages under the bias voltage. Combining with internal standard method, the frequencies of the blockage events induced by P-peptide can be used to quantify the kinase activity. This one-step method realizes facile quantitative analysis of kinase without peptide labeling/modification and other cooperative enzymes, representing significant advantages over traditional kinase assays.9,42 This strategy is also successfully applied to evaluate the kinase inhibitor and monitor drug-triggered PKA activation in cell lysates. Moreover, this nanopore system can realize a real-time monitoring of the kinase-catalyzed phosphorylation, which provides an approach to obtain the kinase kinetics at single molecule level.

■

EXPERIMENTAL SECTION Materials. All aqueous solutions for analytical studies were prepared with ultrapure water (reaching a resistivity of 18.2 MΩ·cm at 25 °C) from the Milli-Q System (EMD Millipore, Billerica, MA, USA). We obtained the 1,2-Diphytanoly snglycero-3-phosphocholine from Avanti Polar Lipids Inc., Alabaster, AL,USA. Proaerolysin was prepared according to our previous work and activated by digestion with trypsin for 4 h at room temperature.43 S-peptide (LRRASLG) and Ppeptide (LRRApSLG) were synthesized by GL Biochem Ltd. B

DOI: 10.1021/acs.analchem.9b01570 Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

■

Article

RESULTS AND DISCUSSION Measurement of PKA Activity via One-Step Method. As shown in Figure 1, the reaction solution containing Speptide without PKA could not induce any current blockages of aerolysin nanopore while the blockage events emerged evidently in the presence of 0.1 U/μL PKA. The enlargement of the typical blockage events was shown in Figure 2a. From

PKA concentration 0.5 U/μL with varying concentrations of inhibitors in the reaction system. MCF-7 Cell Culture and Cell Lysate Preparation. MCF-7 cells (1 × 105 cells) were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS, Sigma), streptomycin (100 μg/mL) and penicillin (100 μg/mL) in a humid atmosphere with 5% CO2 at 37 °C. Then the cultured medium was replaced by serum-free RPMI-1640 (1 mL) and the cells were incubated for 4 h before drug stimulation. Subsequently, the cultured cells were treated with different concentrations of Fsk and IBMX in dimethyl sulfoxide (DMSO) to activate intracellular PKA. DMSO (equal volume) without Fsk/IBMX solution was added to the medium for no drug unstimulated sample as the control. After 45 min of stimulation, the cultured cells were removed by scraping and lysed in Dulbecco’s phosphate-buffered saline (DPBS) by sonication (200 W) for 5 s × 6 times at an interval of 5 s for each time. The cell lysate were clarified by centrifugation at 14 000 rpm for 30 min at 4 °C. To eliminate the interference of other small molecules in the clarified lysates, including short DNA/RNA, sugars, peptides and small proteins etc., the lysates were continuously centrifuged by Amicon 10 K Ultra Centrifugal Filter Device (Merck KGaA, Darmstadt, Germany). Therefore, the molecules whose molecular weight less than 10 kDa were almost excluded from the lysate. Then the lysates were ready for phosphorylation reactions. The total protein concentration of cell lysate was assessed by BCA protein assay kit with BSA as the standard. Briefly, standard solutions with different concentrations of BSA (25− 2000 μg/mL) were incubated with BCA reagent for 30 min at 37 °C. The absorbance of the resulting solutions was recorded at 562 nm using a UV−vis spectrophotometer and the calibration curve of the standard concentration versus the absorbance was obtained by using a linear-regression program. The correlation coefficient of the absorbance with respect to the concentration was >0.99. Finally, an aliquot of the cell extract was mixed with BCA reagent and detected as described above. Its total protein concentration was then calculated by reference to the calibration curve. PKA Assay in MCF-7 Cell Lysates. The experimental procedures were similar to those stated above for detecting PKA activity, except that the incubation of the cell lysates instead of a certain concentration of PKA. The total protein concentrations of the cell lysate in the reaction system were kept at 12 μg/mL. In Situ Monitoring of the Kinase-Catalyzed Phosphorylation. In an attempt to perform a real-time reaction monitoring, the enzymatic catalysis was directly carried out in the cap side compartment. Five U/μL PKA, 100 μM ATP, and 10 μM S-peptide were added into the cap side compartment filled with the detection buffer composed of 1.0 M KCl, 20 mM MgCl2 and 50 mM Tris (pH 7.5). After a certain incubation time, the number of blockage events was counted for a time-interval of 1 min and the events were recorded for 9 min. The slopes of “recording time-cumulative number” fitting lines represent the event frequencies. We considered the average event frequency within the 9 min as the true frequency after a certain incubation time. With the same procedure, we statistics the event frequencies after different incubation times and obtained the phosphorylation evolution curve of S-peptide in the course of the enzyme reaction. The temperature was controlled at 27 ± 1 °C.

Figure 2. (a) The typical blockage events induced by the product Ppeptide in the PKA-catalyzed reaction system. (b) The corresponding 2D scatter plot induced by the catalysis product P-peptide. The plot was obtained via collecting the events for 15 min. The data were recorded after S-peptide was catalyzed by PKA (0.1 U/μL). The recording potential was +100 mV. The detailed information on this PKA catalysis experiment is shown in the Experimental Section.

the 2D contour plot (Figure 2b), we can find the blockage events mainly distributed in the region with duration time (t) ranging from 0.3 to 10 ms and I/I0 falling between 0.4 and 0.7. Here, I is the residue blockage current and I0 is the open pore current. Since S-peptide, ATP, PKA and the catalysis byproduct ADP could not induce any blockages (Figure 1 and SI Figure S2), the observable current blockages were derived from the catalyzed product P-peptide. Its identity signature was further confirmed by direct measurement of the pure sample of P-peptide via aerolysin nanopore. As shown in Figure 3 and SI Figure S3, P-peptide resulted in the current blockages clearly, which were also located in the same region with duration time ranging from 0.3 to 10 ms and I/I0 lying between 0.4 and 0.7. The event frequency grew as the concentration of P-peptide increased (Figure 3d and SI Figure S4). It was found that the relative event frequency compared to 1 μM P-peptide (f/f1 μM) increased linearly as [P-peptide] grew (Figure 3e). The relative frequency will help reduce the frequency errors among different experiments. Thus, the blockage events of the reaction solution were truly ascribed to the catalyzed product P-peptide. To probe the relationship between the PKA concentrations and its catalysis induced event frequencies, different concentrations of PKA ranging from 0.005 to 0.5 U/μL were investigated by the aerolysin nanopore (Figure 4 and SI Figure S5−S7). Then, the resulting event frequencies were analyzed. Since the relative frequency could help reduce the frequency errors among different experiments, we utilized the relative frequency to quantify the kinase-induced P-peptide in the reaction solution. As the event frequencies of standard Ppeptide increased linearly with its concentration ranging from 0 to 11 μM (Figure 3e), we likewise select 1 μM P-peptide as interior label for detecting [P-peptide] produced in the reaction solution which ranged from 0 to 10 μM. The interior label, 1 μM P-peptide, was added into the cap side chamber after recording for the reaction solution (the frequency named as f) and the enhanced frequency resulting from 1 μM Ppeptide was regarded as f1 μM (Figure 4 and SI Figure S5−S7). C

DOI: 10.1021/acs.analchem.9b01570 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Figure 4. (a−c) The raw current traces of the reaction solution without (left) and with (right) adding 1 μM P-peptide as interior label. The reaction solution contained 100 μM S-peptide, which was catalyzed by varying concentrations of PKA at 0.01 U/μL (a), 0.05 U/μL (b) and 0.2 U/μL (c), respectively. The detailed information is described in the Experimental Section. The width of the light yellow band in the current trace was calculated according to the current Gaussian peak width of the reaction solution (SI Figure S8), which was almost the same with that from pure sample experiments of Ppeptide (SI Figure S4). (d) The cumulative blockage number versus recording time (min) for the reaction solution catalyzed by 0.01 U/ μL PKA without (black) and with (red) adding 1 μM P-peptide. Results for more reaction solution catalyzed by different [PKA] was shown in SI Figure S7. The slopes of fitted lines represent the event frequencies. (e) Correlation between the f/f1 μM of blockage events and [PKA] in the reaction solution. All the experiments were performed at +100 mV.

Figure 3. (a−c) The raw current traces and the 2D contour plots of different concentrations of P-peptide: 1 μM (a), 5 μM (b), 10 μM (c). The width of the light yellow band in the current trace was calculated according to the current Gaussian peak width for standard P-peptide (SI Figure S4). All the 2D plots were based on the blockage events within 10 min. (d) The cumulative blockage number versus recording time (min) for different concentrations of P-peptide: 0.25, 1, 3, 5, 7, 10, 11 μM. The slopes of fitted lines represent the event frequencies (f). The frequencies for more concentrations of P-peptide were shown in SI Figure S4. (e) The linear relationship between the relative event frequency and the concentrations of P-peptide. The different concentrations were achieved via continuously adding Ppeptide into the cap side chamber during the experiment. All the experiments were performed at +100 mV.

preincubation of a fixed PKA concentration 0.5 U/μL with varying concentrations of H-89 in the reaction system. The inhibitor H-89 could not induce any interfere (SI Figure S2). As the concentration of H-89 increased from 0.1 to 105 nM, the f/f1 μM gradually decreased from 3.69 ± 0.32 to 0.29 ± 0.07, which is attributed to PKA activity decreasing (Figure 5a, SI Figure S9 and Table S2). Notable, the half-maximal inhibitory concentration (IC50) value of H-89 was determined to be 104 nM, which is in agreement with previous literature values.47 Evaluation of PKA activity in cell lysates. Protein kinase plays crucial role in intracellular signaling pathways and their activities are highly regulated in cells, so a practical kinase assay should be applicable for kinase detection in real biological samples. Thus, we testify whether the nanopore-based PKA assay could work in MCF-7 cell lysates. It is well-known that stimulation of MCF-7 cells by the combination of Fsk and IBMX can greatly increase the intracellular level of cAMP, leading to the activation of cAMP-dependent PKA.48 Therefore, in this study, MCF-7 cells were stimulated with different doses of Fsk/IBMX for 45 min and then the cell lysates were extracted. The PKA activities in these cell lysates were explored by the nanopore assay. The experimental protocols were similar to those stated above for detecting PKA activity, except for the incubation of the cell lysates instead of PKA. The total

Then, we utilized the relative frequency f/f1 μM to quantify PKA activity. When the concentration of PKA increased from 0.005 to 0.5 U/μL, the f/f1 μM increased from 0.05 ± 0.01 to 4.14 ± 0.47 (SI Table S1). We identified a positive correlation between PKA concentration and its corresponding f/f1 μM, which could be linearly fitted on a log−log plot (Figure 4e). The detection limit for PKA in the nanopore system was 0.005 U/μL. Such a detection limit is comparable with those of other various sensitive PKA detection methods10,45 in spite of our extremely simple detection strategy. Screening of PKA Inhibitor. To demonstrate the ability of the nanopore sensor to screen kinase inhibitor, the inhibition of the small molecule kinase inhibitor H-89 to PKA activity was measured (Figure 5a). The inhibitor H-89 competitively inhibits PKA by binding to the ATP binding cleft.46 The experimental procedures were similar to those stated above for PKA activity detection, except for the D

DOI: 10.1021/acs.analchem.9b01570 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

time recording of the PKA-catalyzed phosphorylation, which serves as a method for monitoring phosphorylation kinetics at the single molecule level.

■

CONCLUSIONS In conclusion, the wild-type aerolysin nanopore provided a novel one-step method for PKA activity without exogenous labels, immobilization and cooperative enzymes. The detection was based on the special response of aerolysin nanopore to the phosphorylated peptide in the reaction system. This nanopore method can be used as a sensing platform to identify kinase inhibitor as well as assaying kinase activity in cell lysate. More importantly, the aerolysin nanopore provides a label-free approach for real-time monitoring the kinase kinetics. The nanopore strategy may be used to evaluate the activity and kinetics of other enzymes that catalyze post-translational modifications that alter substrate charge (e.g., sulfonation, carboxylation, or amidation), thus providing a platform to screen a broad spectrum of protein or biomolecule modifications.

Figure 5. (a) Relationship between the relative event frequency (f/ f1 μM) and [H-89] added in the reaction solution. The IC50 was obtained from this figure. (b) The f/f1 μM of the reaction solution with the unstimulated cell lysate, 10 μM Fsk/20 μM IBMX-stimulated cell lysate and 50 μM Fsk/100 μM IBMX-stimulated cell lysate. All the experiments were performed at +100 mV. (c) The cumulative blockage number versus recording time (min) for the phosphorylation process after different incubation times. The slopes of fitted lines represent the event frequencies after different incubation times. (d) The phosphorylation evolution of S-peptide in the course of the enzyme reaction. The reaction system included 10 μM S-peptide, 100 μM ATP, 20 mM MgCl2, and 5 U/μL PKA. The data were recorded at +100 mV. The reaction temperature was 27 ± 1 °C.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b01570. Additional nanopore data as noted in text PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*(Y.L.Y.) E-mail: [email protected]. *(Y.T.L.) E-mail: [email protected].

protein concentrations of the cell lysates incubated in the reaction solution were kept at 12 μg/mL. The nanopore results demonstrated that the Fsk/IBMX-stimulated cell lysates aroused obvious blockage events with 0.3 ms < t < 10 ms and 0.4 < I/I0 < 0.7 (Figure S10 and S11). The event frequency ascended with increasing concentration of stimulants (Figure 5b and SI Figure S12). Thus, this clearly manifested that the cell lysate-actuated nanopore response truly resulted from the successful activation of PKA by drug stimulation. These results conclusively show that the proposed nanopore strategy can measure changes in cellular kinase activities in response to drug stimuli. Real-Time Monitoring of the Kinase-Catalyzed Phosphorylation. In the real-time monitoring of enzymatic catalysis, any blockage events were hardly observed before the addition of PKA to the cap side chamber containing 100 μM ATP, 10 μM S-peptide and 20 mM MgCl2 (SI Figure S13, t = 0 h). After addition of 5 U/μL PKA, the blockage events were clearly observed and the event frequency increased as incubation time grew (SI Figure S13). Via statistical analysis, the blockage event also distributed in the target region (SI Figure S14). Since the active PKA could not induce any interfere (SI Figure S2), the variation of the event frequency here resulted from the increase of the catalyzed product Ppeptide in the compartment. We considered the average event frequency within a short period relatively constant. Therefore, the event frequencies fitted linearly (Figure 5c), which were used to quantify phosphorylation level of S-peptide catalyzed by PKA. Then, a classic progress curve for an enzyme reaction was obtained (Figure 5d). It is worth noting that all the Speptide in the chamber was almost phosphorylated after 9 h reaction. Thus, this result evidenced the feasibility of a real-

ORCID

Yi-Lun Ying: 0000-0001-6217-256X Yi-Tao Long: 0000-0003-2571-7457 Present Address

§ The author now works in Heze University, Heze City 274015, Shandong Province, P. R. China.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (21834001 and 61871183), and Excellent Research Program of Nanjing University (ZYJH004). Dr. Yi-Lun Ying is sponsored by National Ten Thousand Talent Program for Young Top-Notch Talent, Shanghai Rising-Star Program (19QA1402300) and “ChenGuang” Project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (17CG27).

■

REFERENCES

(1) Adams, J. A. Chem. Rev. 2001, 101 (8), 2271−2290. (2) Johnson, L. N.; Lewis, R. J. Chem. Rev. 2001, 101 (8), 2209− 2242. (3) Manning, G. Science 2002, 298 (5600), 1912−1934. (4) Su, S. C.; Tsai, L.-H. Annu. Rev. Cell Dev. Biol. 2011, 27 (1), 465−491. (5) Liu, X.; Winey, M. Annu. Rev. Biochem. 2012, 81 (1), 561−585. (6) Geraldes, P.; King, G. L. Circ. Res. 2010, 106 (8), 1319−1331. (7) Zaha, V. G.; Young, L. H. Circ. Res. 2012, 111 (6), 800−814. E

DOI: 10.1021/acs.analchem.9b01570 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry (8) Altai, M.; Orlova, A.; Tolmachev, V. Curr. Pharm. Des. 2014, 20 (14), 2275−2292. (9) Zhang, X.; Liu, C.; Wang, H.; Wang, H.; Li, Z. Angew. Chem., Int. Ed. 2015, 54 (50), 15186−15190. (10) Shen, C.; Xia, X.; Hu, S.; Yang, M.; Wang, J. Anal. Chem. 2015, 87 (1), 693−698. (11) Wang, Z.; Sun, N.; He, Y.; Liu, Y.; Li, J. Anal. Chem. 2014, 86 (12), 6153−6159. (12) Yan, Z.; Wang, Z.; Miao, Z.; Liu, Y. Anal. Chem. 2016, 88 (1), 922−929. (13) Deng, Z.; Ye, M.; Bian, Y.; Liu, Z.; Liu, F.; Wang, C.; Qin, H.; Zou, H. Chem. Commun. 2014, 50 (90), 13960−13962. (14) Cao, C.; Long, Y.-T. Acc. Chem. Res. 2018, 51 (2), 331−341. (15) Gooding, J. J.; Gaus, K. Angew. Chem., Int. Ed. 2016, 55 (38), 11354−11366. (16) Zhang, X.; Zhang, D.; Zhao, C.; Tian, K.; Shi, R.; Du, X.; Burcke, A. J.; Wang, J.; Chen, S.-J.; Gu, L.-Q. Nat. Commun. 2017, 8 (1), 1458. (17) Larkin, J.; Henley, R. Y.; Jadhav, V.; Korlach, J.; Wanunu, M. Nat. Nanotechnol. 2017, 12 (12), 1169−1175. (18) Clarke, J.; Wu, H.-C.; Jayasinghe, L.; Patel, A.; Reid, S.; Bayley, H. Nat. Nanotechnol. 2009, 4 (4), 265−270. (19) Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. Proc. Natl. Acad. Sci. U. S. A. 1996, 93 (24), 13770−13773. (20) Olasagasti, F.; Lieberman, K. R.; Benner, S.; Cherf, G. M.; Dahl, J. M.; Deamer, D. W.; Akeson, M. Nat. Nanotechnol. 2010, 5 (11), 798−806. (21) Piguet, F.; Ouldali, H.; Pastoriza-Gallego, M.; Manivet, P.; Pelta, J.; Oukhaled, A. Nat. Commun. 2018, 9 (1), 966. (22) Asandei, A.; Chinappi, M.; Kang, H.-K.; Seo, C. H.; Mereuta, L.; Park, Y.; Luchian, T. ACS Appl. Mater. Interfaces 2015, 7 (30), 16706−16714. (23) Waduge, P.; Hu, R.; Bandarkar, P.; Yamazaki, H.; Cressiot, B.; Zhao, Q.; Whitford, P. C.; Wanunu, M. ACS Nano 2017, 11 (6), 5706−5716. (24) Li, T.; Liu, L.; Li, Y.; Xie, J.; Wu, H. C. Angew. Chem., Int. Ed. 2015, 54 (26), 7568−7571. (25) Zhou, S.; Wang, L.; Chen, X.; Guan, X. ACS Sensors 2016, 1 (5), 607−613. (26) Lin, L.; Liu, Y.; Yan, J.; Wang, X.; Li, J. Anal. Chem. 2013, 85 (1), 334−340. (27) Macrae, M. X.; Blake, S.; Jiang, X.; Capone, R.; Estes, D. J.; Mayer, M.; Yang, J. ACS Nano 2009, 3 (11), 3567−3580. (28) Zhao, Q.; de Zoysa, R. S. S.; Wang, D.; Jayawardhana, D. A.; Guan, X. J. Am. Chem. Soc. 2009, 131 (18), 6324−6325. (29) Meng, F.-N.; Yao, X.; Ying, Y.-L.; Zhang, J.; Tian, H.; Long, Y.T. Chem. Commun. 2015, 51 (7), 1202−1205. (30) Astier, Y.; Braha, O.; Bayley, H. J. Am. Chem. Soc. 2006, 128 (5), 1705−1710. (31) Parker, M. W.; Buckley, J. T.; Postma, J. P. M.; Tucker, A. D.; Leonard, K.; Pattus, F.; Tsernoglou, D. Nature 1994, 367 (6460), 292−295. (32) Wang, Y.; Gu, L.-Q.; Tian, K. Nanoscale 2018, 10 (29), 13857− 13866. (33) Cressiot, B.; Ouldali, H.; Pastoriza-Gallego, M.; Bacri, L.; Van der Goot, F. G.; Pelta, J. ACS Sensors 2019, 4 (3), 530−548. (34) Stefureac, R.; Long, Y.; Kraatz, H.-B.; Howard, P.; Lee, J. S. Biochemistry 2006, 45 (30), 9172−9179. (35) Li, S.; Cao, C.; Yang, J.; Long, Y.-T. Chem. Electro Chem. 2019, 6 (1), 126−129. (36) Cao, C.; Ying, Y.-L.; Hu, Z.-L.; Liao, D.-F.; Tian, H.; Long, Y.T. Nat. Nanotechnol. 2016, 11 (8), 713−718. (37) Fennouri, A.; Daniel, R.; Pastoriza-Gallego, M.; Auvray, L.; Pelta, J.; Bacri, L. Anal. Chem. 2013, 85 (18), 8488−8492. (38) Harrington, L.; Alexander, L. T.; Knapp, S.; Bayley, H. Angew. Chem., Int. Ed. 2015, 54 (28), 8154−8159. (39) Harrington, L.; Cheley, S.; Alexander, L. T.; Knapp, S.; Bayley, H. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (47), E4417−E4426.

(40) Rosen, C. B.; Rodriguez-Larrea, D.; Bayley, H. Nat. Biotechnol. 2014, 32 (2), 179−181. (41) Shabb, J. B. Chem. Rev. 2001, 101 (8), 2381−2412. (42) Li, X.; Zhu, L.; Zhou, Y.; Yin, H.; Ai, S. Anal. Chem. 2017, 89 (4), 2369−2376. (43) Cao, C.; Liao, D.-F.; Yu, J.; Tian, H.; Long, Y.-T. Nat. Protoc. 2017, 12 (9), 1901−1911. (44) Balijepalli, A.; Ettedgui, J.; Cornio, A. T.; Robertson, J. W. F.; Cheung, K. P.; Kasianowicz, J. J.; Vaz, C. ACS Nano 2014, 8 (2), 1547−1553. (45) MacConaghy, K. I.; Chadly, D. M.; Stoykovich, M. P.; Kaar, J. L. Anal. Chem. 2015, 87 (6), 3467−3475. (46) Murray, A. J. Sci. Signaling 2008, 1 (22), No. re4-re4. (47) MacConaghy, K. I.; Geary, C. I.; Kaar, J. L.; Stoykovich, M. P. J. Am. Chem. Soc. 2014, 136 (19), 6896−6899. (48) Dodge-Kafka, K. L.; Soughayer, J.; Pare, G. C.; Carlisle Michel, J. J.; Langeberg, L. K.; Kapiloff, M. S.; Scott, J. D. Nature 2005, 437 (7058), 574−578.

F

DOI: 10.1021/acs.analchem.9b01570 Anal. Chem. XXXX, XXX, XXX−XXX