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Detection of Single Proteins with a General Nanopore Sensor Qiao Li, Yi-Lun Ying, Shao-Chuang Liu, Yao Lin, and Yi-Tao Long ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00228 • Publication Date (Web): 12 Mar 2019 Downloaded from http://pubs.acs.org on March 12, 2019
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ACS Sensors
Detection of Single Proteins with a General Nanopore Sensor Qiao Li1, Yi-Lun Ying1*, Shao-Chuang Liu1, Yao Lin1, Yi-Tao Long1, 2* 1
Key Laboratory for Advanced Materials, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China 2 School of Chemistry and Chemical Engineering Nanjing University, Nanjing, 210023, P. R. China.
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ABSTRACT: Single protein sensing based on solid-state 45 nanopore is promising but challenging because the fast 46 translocation velocity of a protein is beyond the bandwidth of 47 nanopore instruments. To decelerate the translocation speed,48 here, we employed a common protein crosslink interaction to 49 achieve a general and robust nanopore sensing platform for 50 single-molecule detection of protein. Benefiting from the 51 EDC/NHS coupling interaction between nanopore and 52 proteins, a ten-fold decrease in speed has been achieved. 53 The clearly distinguishable current signatures further reveal 54 the anisotropy of a protein produces three translocation 55 behaviors, which are horizontal, vertical and flipping transit 56 inside nanopore confinement. This strategy provides a 57 general platform for rapid detection of proteins as well as 58 exploring fundamental protein dynamics at the single- 59 60 molecule level.
61 62 63 KEYWORDS: solid-state nanopore, protein detection, cross 64 linker, dielectric breakdown, functionalization 65 66 67 68 Beyond the most promising application in DNA sequencing,1 69 nanopore sensors have shown their prominent ability in 70 protein sensing.2–10 Owing to the label-free and high 71 throughput merits, nanopore measurements yield rich 72 information about protein-protein interactions6, protein-DNA 73 interactions,11 protein size,12,13 conformations13,14 and protein 74 charges15. As a protein enters into nano-scaled confinement, 75 it would modulate the ionic current under applied potential 76 leading to a characteristic current signature. The 77 characteristics of the analytes, e.g. volume, charge and 78 geometry can be obtained by analyzing the blockage current 79 and duration time of current signatures caused by the 80 analyte.16 The solid-state nanopores, due to their tunable 81 geometry, have expected to sensor the large proteins in their 82 native state and adjustable surface properties.4,7,17–22 Unlike 83 DNA molecules which are uniformly charged, proteins are 84 anisotropically charged sphere molecules even with low 85 translocation probability through a nanopore in a linear 86 fashion in most cases. The sphere structure prejudices 87 protein recognition and sequencing with nanopore 88 technology compared with DNA molecules.23 Moreover, the 89 translocation speed of a protein through the solid-state 90 nanopore is too fast to be resolved in the present commercial 91 instruments.4 To improve protein detection sensitivity of 92 solid-state nanopore, one strategy is to enhance the 93 17 bandwidth of the measurement electronics.
Although the
high bandwidth instrument facilitates the nanopore sensing on the proteins with ultra-fast translocation speed, the desired nanopore blockages may be covered by high current noise at high bandwidth.17 The other solutions include slowing down the velocity of the protein by applying light control,24 adjusting pH near the isoelectric point (pI) of the interest protein,25 or introducing the pressure.26,27 But some of the harsh experimental conditions hold the possibility to damage the native structure of protein. Moreover, the velocity of the protein translocation through nanopore could be reduced by enhancing the interaction between the inner wall of nanopores and proteins.28 For example, by employing the specific interaction between molecules and nitrilotriacetic acid to solid-state nanopore, it could specifically detect Histagged proteins.29 Besides, the nanopore coating with fluid lipid bilayers and specific ligands is able to detect the specific protein.30 These approaches only are effective for detection of specific proteins. Therefore, a more generalized approach is necessary for a wider application of nanopore-based single protein sensing. In order to develop a generalized method for protein detection with solid-state nanopores, here we employ the non-specific interaction of amino and carboxyl rather than the specific interaction to enhance the detection sensitivity. To simplify the modification of the sensor interface, a 30 nm thick gold film was coated on the SiNX membrane. In this study, glucose oxidase (GOX) as a model protein was used to demonstrate that the non-specific interaction efficiently slows down the protein translocation speed (Figure 1). The common protein crosslinkers, N-(3-dimethylaminopropyl)-N’ethylcarbodimide (EDC)/N-hydroxysuccinimide (NHS), were introduced to activate the interaction between carboxylate on GOX surface and the amine groups on the inside wall of Aunanopore. Our results demonstrate that the translocation speed of proteins significantly decreases after introducing the amine and carboxyl interaction. The duration time of GOX through the functional Au-nanopore is approximately ten-fold longer than that of the bare Au-nanopore. This work provides a convenient and general method for single native protein detection that could greatly slow down the protein translocation speed. To achieve a simple modification of SiNX nanopore, here we coated a gold film on the SiNX chip via E-beam evaporation (Figure 2a-i). According to the previous study,31,32 the Aunanopores with the desired diameter were fabricated by controlled dielectric breakdown (CDB, Figure 2a-ii, and Figure S1). Comparing with ion-beam sculpting33 or transmission electron microscopy (TEM) drilling34 of solidstate nanopores under vacuum, the CDB fabrication is
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21 undergoes a fast translocation through the solid-state 22 nanopore. Note that the interaction between the native 23 protein and the bare Au-nanopore such as electrostatic 24 absorption may prolong the translocation time of the protein 25 comparing to the bare SiNx nanopore. 26 The statistical analysis of the current amplitudes (ΔI) from 27 each blockage shows two Gaussian distributions with the 28 peak value of ΔI = 1.44 nA and 2.41 nA, respectively. Here, 29 ΔI = I0 - Ib whereas I0 represents the baseline current and Ib 30 represents the residue current. Previous studies suggested 31 that the anisotropy of nanorods results in the different current 32 blockage levels.37–39 Nanopore sensing of protein detection 33 also demonstrates the anisotropy of proteins would distribute 34 the ion flow proximity to the proteins, resulting in the 35 characteristic current variations.14,40,41 In our study, the GOX 36 is a dimer protein (5 nm* 5nm *7nm) with an aspect ratio of 37 1.4.42 We speculate the spike-like signals in two current 38 distributions originate from the anisotropy induced proteins 39 translocation process. According to the conductance model Figure 1. Slowing the translocation of glucose oxidase (GOX) 40 and size exclusion effect (details see in Supplementary via a functionalized Au-nanopore. (a) Employment the non- 41 Information and Table S1), the deep current distribution with specific interaction between protein and nanopore inside a 42 Gaussian peak value of Δ I = 2.41 nA is caused by the cysteamine (Cys H) functionalized Au-nanopore. After 43 translocation of GOX with a horizon fashion while the shallow treating with EDC/NHS, the carboxyl (-COOH) on GOX 44 current distribution with Gaussian peak value of ΔI = 1.44 nA surface turns into quaternary amino (-N(CH3)2H+). The green 45 is induced by the vertically threading of the GOX. ellipse and purple ellipse present native and modified glucose oxidase, respectively. The dashed line represents 46 To reduce the translocation speed of GOX, we functionalized the direction of the force to which the protein is subjected. 47 a cysteamine hydrochloride (Cys H) monolayer on the
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The raw current trace (b) and statistical results (c) for driven GOX into bare Au-nanopore. The raw current trace (b) and statistical results (d) for driving a modified GOX into a functionalized Au-nanopore. The current distributions fit into two-peak Gaussian function and the duration histogram fit the single Gaussian function. The triangles in the raw current traces illustrate the magnified current blockages in (b) and (e). The scale bare in (d) and (e) is 1 ms and 3 nA. The difference in Δ I is mainly due to the difference in pore diameter. The current trace was obtained in 1 M KNO3 pH 8 solution in the presence of 0.1 mg/mL analyte with 5 kHz lowpass filter and 100 kHz sampling rate. To simplified the illustration, only one quaternary amino modification is presented in the cartoon. The modification should occur around the GOX. performed directly in the solution, which shows a simple, efficient and cheap manner for solid-state nanopores fabrication. The detailed CDB fabrication process can be found in Figure S1. The current-voltage (I-V) curve was used to characterize the diameter of Au-nanopores based on the previously reported conductance model (Figure S2 and Figure S3).35,36 Then, we carried out the control experiment by using the unmodified Au coated SiNX nanopore towards single GOX sensing. Since the native GOX owns a pI of 5.2, it carries a negative charge at pH 8. Under the applied positive voltage, the native GOX in the trans chamber solution was electrically driven through the nanopore to the cis chamber (Figure 1a, left, Figure S4), which produces the current blockade with a small current amplitude (Figure 1b). The Gaussian fitting provides the short duration of 0.17 ms as the native GOX transverses through the bare Au-nanopore at +500 mV. Moreover, the decreased duration with the increased potential suggests the native protein could translocated across the bare Au-nanopore (Figure S4). This value is similar to the previous reports4 that the protein often
Figure 2. Modification processes and characterizations of Cys H functionalized Au-nanopore. (a) The modification process: i) Employ E-beam evaporation to coat a 30 nm Au layer (details shown in Supplementary Information). ii) Fabricate a single nanopore with controlled diameter by CBD. iii) Perform single molecular layer self-assembly to form a Cys H layer inside the pore. (b) I-V characterization of Au-nanopore with a diameter about 14 nm before (blue) and after (red) modification of Cys H. (c) Current power spectral density characterization of Au-nanopore at +100 mV before (blue) and after (red) modification of Cys H. The current for I-V and PSD were obtained in 1 M KNO3 pH 8 solution with 5 kHz low-pass filter and 100 kHz sampling rate.
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20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 3. Illustration and statistical analysis of GOX 46 blockages from the Cys H modified Au-nanopore. (a) The 47 Type I blockage with deep blockage current represents the 48 horizontal presence of the GOX inside modified Au- 49 nanopore. This entrance fashion is denoted as State 1. (b) 50 The Type I blockage with small blockage current represents 51 the vertical confining of the GOX inside modified Au- 52 nanopore. This accommodation fashion is denoted as State 53 2. The statistical results of Type I blockage at -800 mV (c) 54 and -1000 mV (d). (e) The Type II blockage with two 55 blockage level illustrates the flip of GOX from the horizon 56 state (State 1) to the vertical state (State 2) inside nanopore 57 confinement. The initial deep current level is in blue color 58 while the following small current level is in red color. The 59 statistical results of the initial deep current level of Type II 60 blockages at -800 mV (f) and -1000 mV (g). The statistical 61 results of the following small current level of Type II blockage 62 at -800 mV (h) and -1000 mV (i). 63 64 surface of a 14 nm Au-coated nanopore via Au-S bond 65 (Figure 2a-iii). Then, the modified nanopore carries a 66 negative charge, which could decelerate the translocation of 67 positively charged proteins via electrostatic interaction and 68 the dynamics EDC/NHS coupling. The comparison of the I-V 69 curves and TOF-SIMS analysis before and after modification 70 immersing with of Cys H is able to certify the successful modification of Cys H (Figure 2b, Figure S5, S6). After 71 modification of Cys H, the rectification ratio, R, is decreased 72 to 1.7 compared to that of the bare Au-nanopore (R = 1.9, 73 Figure 2b). Here, the rectification ratio, R, is defined as the 74 ratio of the negative current value, 𝐼" , to the positive current 75 value, 𝐼# , at ±1.0 V. The polarized gold layer under applied 76 voltage causes the rectification of the bare Au-nanopore.43 77 After modifying with Cys H, the gold layer is covered with 78 amine groups, which dominates the surface charge 79 distribution on the Au-nanopore instead of Au polarization. 80 Since the positively charged amine groups on gold layer 81 would preferentially transport anions,44 the increased current 82
value suggests the successful modification of Cys H. The power spectral density (PSD) displays that the noise of the Au-nanopore hardly changed by the modification process (Figure 2c), which demonstrates the amine layer would not increase the noise of Au-coated nanopore system. And the PSD curves reflecting the noise of two experimental condition can be get in Figure S7. Moreover, the current root mean square (IRMS) remains stable before and after modification which are 0.32 nA to 0.28 nA, respectively. In order to develop a general process to slow the translocation speed of GOX, here, we treated GOX with EDC/NHS which is a popular procedure for adding the positive charge group onto the protein surface (Figure 1a). The carboxyl group (-COOH) which is widely presented in the protein surface is reacted with EDC/NHS to form quaternary amino (-N(CH3)2H+). This reaction ensures protein carrying positive charges. Therefore, under a voltage of -800 mV, the modified GOX was driving into the Cys H functionalized Aunanopore (Figure 1c). Surprisingly, the positively charged GOX generates the spike-like blockages (denoted as Type I) as well as the partial blockages with two current levels (denoted as Type II) as shown in Figure 3a, b, e. First, we evaluated the duration and current amplitude for all blockages both in Type I and Type II. In this analysis, we used the maximal current amplitude value and durations for the Type II blockages. Similar to the bare Au-nanopore, the negatively charged Au-nanopore sensor in the detection of positively charged GOX also exhibits two current distributions which could be fitted into two Gaussian peaks with the peak value of ΔI = 3.05 nA and ΔI = 4.64 nA, respectively (Figure 1e). However, the duration time is efficiently prolonged to 1.79 ms by employing the electrostatic interaction and dynamics coupling between protein and nanopore sensing interface. Compared to the bare Au-nanopore, the translocation speed is decreased ten-times inside functionalized Au-nanopore. Then, we further analyzed the Type I blockages in details as shown in Figure 3a-d. At both -800 mV and -1000 mV, twopeak Gaussian function could be fitted in the current distribution. Similar to the results from bare Au-nanopore, the higher current amplitude is ascribed to the horizontal presenting of GOX inside nanopore (State 1, Figure 3a) while the population with lower current amplitude could be assigned to the vertical translocation of GOX (State 2, Figure 3b). Note that State 1 shows the higher proportion than State 2 in total Type I blockages. These results suggest that the protein shows a preference for horizontally across the Aunanopore. The durations of Type I at -800 mV and -1000 mV are 0.73 ms and 0.82 ms, respectively, which suggests a high energy barrier presented in the functionalized Au-nanopore compared to the bare Au nanopore (0.17 ms). As shown in Figure 3f-i, we evaluated the duration and current amplitude from each level of Type II blockages. At the two applied voltage, the current amplitude of each level is voltage independent. This results further demonstrate that the volume exclusion effect dominates the current blockages. Comparing the current amplitude of two states in Type I with the two levels of Type II at -800 mV, the ΔI value of State 1 in Type I is comparable to the initial deep current level in Type II blockages which are 4.65 nA, while that of State 2 in Type I is consistent to the following low current level in Type II blockages ( ΔI = 2.23 nA). These results suggest that the Type II signatures are generated by the flip of the horizontal
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protein into the vertical direction after entering the nanopore. 60 As the negative voltage increased from -800 mV to -1000 mV,61 the State 1 duration of Type II decreases from 0.97 ms to 0.46 ms. However, State 2 durations remains relative 62 consistent which are 0.91 ms and 1.04 ms for -800 mV and 1000 mV, respectively. These results suggest that the State 63 1 is resulting from the electrophoretic driving entrance while 64 State 2 is prone to the electrostatic interaction between 65 nanopore and proteins. Considering the distribution of 66 carboxyl on the GOX surface, the horizontal presence of GOX 67 inside nanopore ensures the maximum quantity of EDC/NHS 68 activated -COOH groups to interact with the Cys H 69 functionalized Au-nanopore. Except for Type II event, we also observe the rare event shows the characteristic of 70 adjusting from State 2 to State 1 (Figure S8). These results 71 suggest that the GOX is prone to adopt horizon orientation 72 during the entering process but vertically transverse through 73 74 pore due to the less confined effect. 75 In summary, we elucidate the novel and general nanopore 76 sensor design approach by introducing the commonly used 77 protein coupling reaction. Due to the enhanced interaction 78 between proteins and nanopore, the protein translocation 79 velocity is greatly slowed down for about 10 times. Moreover, 80 owing to the anisotropic charge distribution at the protein 81 surface, once the active site is exposed to the sensing 82 interface, the interaction is much more pronounced, vice 83 84 versa. Since the sensitivity of a nanopore sensor is enhanced 85 by non-specific modification at the sensor interface, the 86 interaction between the sensor interface and the analytes 87 determines the sensitivity of the nanopore sensors. In the 88 future, it is vital to tune the non-specific interaction between 89 the nanopore sensor and the analyte for controlling the 90 orientation of the protein as well as modulate the 91 folding/unfolding of the protein. Our strategy just opens a 92 93 door for the nanopore-based protein sensor, which holds 94 great potential in disease diagnostics and fundamental 95 understanding of proteins. 96
97 98 99 100 ASSOCIATED CONTENT 101 Supporting Information 102 The Supporting Information is available free of charge on the103 104 ACS Publications website. 105 Reagents and chemicals; fabrication and characterization of106 Au-nanopore; modification of Au-nanopore and glucose107 oxidase; analysis of glucose oxidase translocation using a108 conductance model; I-V curves, ToF-SIMS and PSD109 characterization of modified Au-nanopore; typical Type III110 111 signal (PDF) 112 113 AUTHOR INFORMATION 114 115 Corresponding Author 116 *E-mail:
[email protected],
[email protected] 117 118 Author Contributions 119 Y.-T.L., Q.L., and Y.-L.Y conceived the idea; Q.L. and Y.-L.Y120 designed the experiments; Q.L. performed the nanopore121 experiments; Q.L. and S.-C.L analyze the experimental data;122 123 Q.L. and Y.-L.Y interpreted the data; Q.L., Y.-L.Y. and Y.L. 124 co-wrote the paper; Y.-T.L., Y.-L.Y. supervised the project.125 All authors have given approval to the final version of the126 manuscript.
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Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (61871183 and 21834001), Ten Thousand Talent Program for young top-notch talent, Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-02-E00023), and Shanghai Science and Technology Development Fund (19QA1402300).
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