Voltage-driven translocation behaviors of IgG molecule through nanopore arrays
© Liu et al.; licensee Springer. 2013
Received: 17 November 2012
Accepted: 12 January 2013
Published: 15 May 2013
Nanopore-based biosensing has attracted more and more interests in the past years, which is also regarded as an emerging field with major impact on bio-analysis and fundamental understanding of nanoscale interactions down to single-molecule level. In this work, the voltage-driven translocation properties of goat antibody to human immunoglobulin G (IgG) are investigated using nanopore arrays in polycarbonate membranes. Obviously, the background ionic currents are modulated by IgG molecules for their physical place-holding effect. However, the detected ionic currents do ‘not’ continuously decrease as conceived; the currents first decrease, then increase, and finally stabilize with increasing IgG concentration. To understand this phenomenon, a simplified model is suggested, and the calculated results contribute to the understanding of the abnormal phenomenon in the actual ionic current changing tendency.
In recent years, the new generation of analytical technology based on nanopores or nanochannels provides possibilities for low-cost and rapid biosensing and DNA sequencing. It is regarded as an emerging field which is expected to have major impact on bio-analysis and fundamental understanding of nanoscale interactions down to single-molecule level. Nowadays, more and more theoretical and experimental work aiming to understand and design nanopore-based devices have been done, which is at the forefront of life science, chemistry, material science, and (bio) physics. Among these studies, nanopore fabrication and nanopore-based nanofluidic device design are two key issues [1–4]. Generally speaking, there are two major types of nanopores which have been used for DNA sequencing and biosensing applications (e.g., using nanopores as analytical sensors for molecular or biomolecular analytes): protein nanopores [5–8] (such as the α-hemolysin pore) and artificial pores in solid-state films (such as silicon nitride nanopores [9–13], graphene nanopores [14–16], and silicon oxide nanopores [17, 18]).
Although much progress has been achieved in nanopore techniques, it is still difficult to sense nucleotides at single-base resolution in DNA. That is mainly because the thickness of nanopores (about several nanometers) can permit 10 to 15 nucleotides occupying them at one time. On the other hand, the momentary change in ionic currents is at only nano-ampere or pico-ampere level, and the duration of this change is at millisecond or so, which is hard to detect and analyzed. To improve the intensity of signals is an important issue in this area. Nanopore arrays, which can be regarded as the integration of multiple nanochannels in the same direction, can improve the intensity of signals in ionic current changes compared to single pore. Now, nanopore arrays are widely used in biomolecular separation, detection and analysis, although it seems difficult for DNA sequencing at present. In this work, the single molecule translocation properties through polycarbonate nanopore arrays are studied and discussed.
Experimental device and reagent
Polycarbonate membranes containing nanopore arrays (nanopore diameter 50 nm, nanopore distribution density 6 pores/μm2, thickness of polycarbonate membranes 6 to 11 μm) are purchased from the branch in China of Whatman, Inc. (Shanghai, China), and hydrophilic treatments are carried out before its usage. Goat antibody to human immunoglobulin G (IgG) is imported from America Basic Gene Associate Bioscience, Inc. through Nanjing Boquan Technology Co., Ltd. (Nanjing, China). KCl is commercially available, and it is of analytical grade. Ultra-pure water (resistivity 18.25 MΩ·cm) is used for the preparation of all solutions and rinsing. Keithley 2000 61/2-digital multimeter (Keithley Instruments Inc., Beijing, China) is used for ionic current recording. The applied voltage used in the experiments is varied 0.5 to 2V. AFM image in tapping mode is obtained from MFP-3D-SA atomic force microscope produced by Asylum Research (Santa Barbara, USA), and the scanning rate is 1.0 Hz.
A test device (Figure 1) integrated by two separated liquid cells linked by PC membrane containing nanopore arrays (sealed by PDMS) is used for measuring ionic currents. At room temperature, KCl solution is added to the feed cell and permeation cell, and IgG is dissolved in the reservoir. After that, the electric field is applied to the two sides of the membrane, and the trans-membrane ionic current can be measured by Keithley 2000 61/2-digital multimeter and recorded simultaneously by computer.
Parameters for simulation
Relative molecular mass 140 kDa, surface charge density σ = 2.0 × 1,017/m2, concentration 10 ng/mL
Nanopore arrays in PC membrane
Pore diameter 50 nm, pore density 6 pores/μm2, membrane thickness 6 to 11 μm; its effective contact area contacting the solution is around 7 mm
The applied electric field E = 0.1 V/nm, 0.1 M KCl solution
Results and discussions
The experimental approach
Generally, the change in the ionic current will be mainly affected by two factors: (1) physical place-holding effect. Once IgG molecules enter the nanopores, the volumes in the nanopores are partially occupied, which will prevent certain amounts of K+ and Cl− from passing through PC membrane. It is so-called physical place-holding effect, and it will decrease the background ionic current. (2) Surface charge density of IgG molecule: as we know, the surface charge of IgG molecule will also contribute to the increase of total ionic current when it passes through the nanopore. The final current changes will be determined by the combined effects of the above two factors. When the concentration of electrolyte is quite higher, the density of anions and cations in the solution is also higher, and the lost number in anions and cations due to the physical place-holding effect is quite bigger. At the same time, the surface charge density of IgG molecules does not change if the pH of the solution remains at 7.48. In this condition, the decrease in ionic current generated by physical place-holding effect is bigger than the increase due to the contribution of IgG surface charge; so, there will be a decrease blockade in the background ionic current. When the concentration of electrolyte is quite lower, so that the decrease in current generated by physical place-holding effect is smaller than the increase in current due to the contribution of IgG surface charge, there will be an increase blockade in the background ionic current.
Based on the above analysis, the physical place-holding effect will be enhanced with the increasing concentration of IgG molecules in the solution within certain ranges; on the other hand, the volume of IgG molecule (IgG is one kind of molecule with “Y”-type structure and its size is about 20 nm) is much larger than K+ and Cl−, so the bulk charge density is much lower in the occupied nanopore arrays, which results in the decrease of ionic current. Of course, the modulated ionic current is affected not only by the physical place-holding effect but also by many other factors (such as electric double layer effect), so the decrease will nonlinearly change with the concentration of KCl. The differences between the background currents and the recorded currents at 40 ng/mL of IgG are plotted versus the concentration of KCl (insets of Figures 4 and 5), from which it can be found that the difference of current increase does ‘not’ linearly rise with the concentration of electrolyte.
According the above analysis and common sense, the current should continue to decrease along with the increasing concentration of IgG, but abnormal phenomenon appears when the concentration of IgG is higher than 40 ng/mL: the ionic currents do not decrease but increase with increasing IgG concentration. Undoubtedly, the physical place-holding effect also exists at these concentrations. The experimental results show that when IgG concentration is high enough, the translocation probability will not always increase with increasing IgG concentration. This is just like the following case: imagine a stadium with limited doors, the maximum allowed flux of people in unit time is N. When the number of people who need to enter the stadium is lower than N, the number of entering people will increase with the number of people who need to enter. If the number of people who need to enter the stadium in unit time is larger than N, the actual number of entering people will equal to or less than N (especially for disordered case). When IgG concentration is higher than a certain value (threshold value), the number of passing molecules will remain or be decreased. The physical place-holding effect is weakened, which will result in the ‘abnormal’ increase in the ionic current. The further explanation from the view of simulation is suggested in the following part.
The simulation approach
The calculated results based on the suggested model are the outputs of the program after running 10,000 steps, which correspond to the number of IgG molecules passing through the nanopores in 10 ps. These obtained numbers in each step are discrete, but the numbers of passing IgG molecules in unit time can be regarded as the IgG moving velocity in the nanopores if the thickness of the nanopores is ignored. To simplify the calculation, we suppose that the nanopores move only in single row direction; the biomolecules passing through the nanopores can be investigated from a quasi two-dimensional perspective. In this slide cell, the acceleration of biomolecules is determined by total force, and then the velocity and position are determined. In one limited cell, the periodic boundary conditions are applied to guarantee the number of biomolecules in the cell being constant. The starting status is that IgG molecules are distributed homogeneously in the solution; as time goes on, the molecules in the solution become increasingly chaotic states, which are more close to the actual situation of the molecule movements and distributions. Therefore, only the last 5,000 steps are adopted and averaged of molecules in order to understand the change tendency of the number of molecules passing through the nanopores in unit time.
In summary, the transporting properties of IgG molecules are investigated using nanopore arrays. The experimental results indicate that the ionic currents do not decrease continuously with increasing IgG concentration, as general consideration; the current decrease at first, then increase, and stabilize with the increasing concentration. The calculated passing velocity of IgG molecules based on a simplified model will first increase, then decrease, and finally stabilize with the increasing IgG concentration, which can provide support for our experimental results.
This work is supported by the National Basic Research Program of China (2011CB707601 and 2011CB707605), the Natural Science Foundation of China (51003015, 51005047), the Fundamental Research Funds for the Central Universities (3202001103), the Qing Lan Project and the International Foundation for Science, Stockholm, Sweden, the Organization for the Prohibition of Chemical Weapons, The Hague, Netherlands, through a grant to Lei Liu (F/4736-1), and the Student Research Training Programme in Southeast University.
- Fologea D, Gershow M, Ledden B, McNabb DS, Golovchenko JA, Li J: Detecting single stranded DNA with a solid state nanopore. Nano Lett 2005, 5: 1905–1909. 10.1021/nl051199mView ArticleGoogle Scholar
- Chen Z, Jiang YB, Dunphy DR, Adams DP, Hodges C, Liu N, Zhang N, Xomeritakis G, Jin X, Aluru NR, Gaik SJ, Hillhouse HW, Brinker CJ: DNA translocation through an array of kinked nanopores. Nature Mater 2010, 9: 667–675. 10.1038/nmat2805View ArticleGoogle Scholar
- Mulero R, Prabhu AS, Freedman KJ, Kim MJ: Nanopore-based devices for bioanalytical applications. JALA 2010, 15: 243–252.Google Scholar
- Liang KZ, Qi JS, Mu WJ, Chen ZG: Biomolecules/gold nanowires-doped sol–gel film for label-free electrochemical immunoassay of testosterone. J Biochem Biophy Methods 2008, 70: 1156–1162. 10.1016/j.jprot.2007.11.007View ArticleGoogle Scholar
- Jin Q, Fleming AM, Burrows CJ: Unzipping kinetics of duplex DNA containing oxidized lesions in an alpha-hemolysin nanopore. J Am Chem Soc 2012, 134: 11006–11011. 10.1021/ja304169nView ArticleGoogle Scholar
- Wen S, Zeng T, Liu L: Highly sensitive and selective DNA-based detection of mercury(II) with alpha-hemolysin nanopore. J Am Chem Soc 2011, 133: 18312–18317. 10.1021/ja206983zView ArticleGoogle Scholar
- de Zoysa RSS, Krishantha DMM, Zhao Q: Translocation of single-stranded DNA through the alpha-hemolysin protein nanopore in acidic solutions. Electrophoresis 2011, 32: 3034–3041. 10.1002/elps.201100216View ArticleGoogle Scholar
- Shen JW, Shi YY: Current status on single molecular sequencing based on protein nanopores. Nano Biomed Eng 2012, 4: 1–5.View ArticleGoogle Scholar
- Lu B, Hoogerheide DP, Zhao Q: Effective driving force applied on DNA inside a solid-state nanopore. Phy Rev E 2012, 86: 011921.View ArticleGoogle Scholar
- Rosenstein JK, Wanunu M, Merchant CA, Drndic M, Shepard KL: Integrated nanopore sensing platform with sub-microsecond temporal resolution. Nat Methods 2012, 9: 487-U112. 10.1038/nmeth.1932View ArticleGoogle Scholar
- Wei RS, Gatterdam V, Wieneke R: Stochastic sensing of proteins with receptor-modified solid-state nanopores. Nature Nanotechnol 2012, 7: 257–263. 10.1038/nnano.2012.24View ArticleGoogle Scholar
- Spinney PS, Howitt DG, Smith RL: Nanopore formation by low-energy focused electron beam machining. Nanotechnology 2010, 21: 375301. 10.1088/0957-4484/21/37/375301View ArticleGoogle Scholar
- Edmonds CM, Hudiono YC, Ahmadi AG: Polymer translocation in solid-state nanopores: dependence of scaling behavior on pore dimensions and applied voltage. J Chem Phy 2012, 136: 065105. 10.1063/1.3682777View ArticleGoogle Scholar
- Zhao Q, Wang Y, Dong JJ, Zhao L, Rui XF, Yu D: Nanopore-based DNA analysis via graphene electrodes. J Nanomater 2012, 2012: 318950.Google Scholar
- Venkatesan BM, Estrada D, Banerjee S: Stacked graphene-Al2O3 nanopore sensors for sensitive detection of DNA and DNA-protein complexes. ACS Nano 2012, 6: 441–450. 10.1021/nn203769eView ArticleGoogle Scholar
- Saha KK, Drndic M, Nikolic BK: DNA base-specific modulation of microampere transverse edge currents through a metallic graphene nanoribbon with a nanopore. Nano Lett 2012, 12: 50–55. 10.1021/nl202870yView ArticleGoogle Scholar
- Storm AJ, Chen JH, Zandbergen HW: Translocation of double-strand DNA through a silicon oxide nanopore. Phy Rev E 2005, 71: 051903.View ArticleGoogle Scholar
- Chang H, Kosari F, Andreadakis G: DNA-mediated fluctuations in ionic current through silicon oxide nanopore channels. Nano Lett 2004, 4: 1551–1556. 10.1021/nl049267cView ArticleGoogle Scholar
- Vlassarev DM, Golovchenko JA: Trapping DNA near a solid-state nanopore. Biophy J 2012, 103: 352–356. 10.1016/j.bpj.2012.06.008View ArticleGoogle Scholar
- Melnikov DV, Leburton JP, Gracheva ME: Slowing down and stretching DNA with an electrically tunable nanopore in a p-n semiconductor membrane. Nanotechnology 2012, 23: 255501. 10.1088/0957-4484/23/25/255501View ArticleGoogle Scholar
- Timp W, Comer J, Aksimentiev A: DNA base-calling from a nanopore using a Viterbi algorithm. Biophy J 2012, 102: L37-L39. 10.1016/j.bpj.2012.04.009View ArticleGoogle Scholar
- Liu J, Pham P, Haguet V: Polarization-induced local pore-wall functionalization for biosensing: from micropore to nanopore. Anal Chem 2012, 84: 3254–3261. 10.1021/ac2033744View ArticleGoogle Scholar
- Bessonov A, Takemoto JY, Simmel FC: Probing DNA-Lipid membrane interactions with a lipopeptide nanopore. ACS Nano 2012, 6: 3356–3363. 10.1021/nn3003696View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.