Detecting a single molecule using a micropore-nanopore hybrid chip
© Liu et al.; licensee Springer. 2013
Received: 4 September 2013
Accepted: 5 November 2013
Published: 21 November 2013
Nanopore-based DNA sequencing and biomolecule sensing have attracted more and more attention. In this work, novel sensing devices were built on the basis of the chips containing nanopore arrays in polycarbonate (PC) membranes and micropores in Si3N4 films. Using the integrated chips, the transmembrane ionic current induced by biomolecule's translocation was recorded and analyzed, which suggested that the detected current did not change linearly as commonly expected with increasing biomolecule concentration. On the other hand, detailed translocation information (such as translocation gesture) was also extracted from the discrete current blockages in basic current curves. These results indicated that the nanofluidic device based on the chips integrated by micropores and nanopores possessed comparative potentials in biomolecule sensing.
KeywordsNanopore Biosensing Biomolecule
Since voltage-driven biomolecule translocation through nanopores was first reported by Kasianowicz et al. in 1996 , nanopores in solid films have become an effective tool for bio-analysis [2–4]. Nowadays, more and more theoretical and experimental studies aiming to design nanopore-based sensing device have been carried out, and most of them are at the forefront of life sciences, chemistry, material sciences, and biophysics. For example, nanopore plays an important role in low-cost and rapid DNA sequencing, which is expected to have major impact on bio-analysis and to give fundamental understanding of nanoscale interactions down to single-molecule level. The mechanism of nanopore-based biomolecule sensing or DNA sequencing can be simply depicted as follows: analyte in electrolyte solution is driven through a nanopore by applied electric field, yielding a characteristic change in background ionic current due to its translocation. According to the existed work, analyte with its dimensions comparable to the size of nanopore is quite advantageous to obtain signals with better quality. The concentration information of analyte can be obtained by comparing the frequencies of translocation events, while the structural information of analyte can be acquired by analyzing the magnitude, duration, and shape of the current blockages. In addition, pore geometry, pore size, flow direction, and other factors also have influences on the detected current signals. On the other hand, theoretical studies in this area have attempted to give a fundamental understanding of the translocation, which are expected to obtain deeper comprehensions for the relevant existing experiments . Fabrication of smart nanopore-based device together with the sensitive collection and accurate analysis of current signals is regarded as a key issue in nanopore-based analysis and DNA sequencing.
Generally speaking, natural pores at nanometer scale (such as alpha-hemolysin) in biomembranes and artificial pores at nanometer scale in solid films are two major types of nanopores used in DNA sequencing and biomolecule sensing. In this area, Bayley and Cremer , and Bayley and Jayasinghe  have performed fundamental studies on alpha-hemolysin. On the basis of these pioneer efforts, other excellent research work on protein-based nanopore has been carried out [8, 9]. In recent years, the developments of artificial nanopores have become faster and faster with the rapid developments of nanoscience and nanotechnology. Novel fabricating methods, such as ion beams and electron beams [10–12], have been gradually used to manufacture artificial nanopore in thin solid materials (including silicon nitride [13–17], graphene [18–21], and silicon oxide [22, 23]) for sequencing or bio-analysis usage. These progresses are of great importance for nanopore-based sensing devices because of their great potentials in combination with developed MEMS technology. In addition, the group of Harrell et al. and other groups have utilized track etching method to prepare conically-shaped single nanopore in polymer membranes (such as polycarbonate, poly(ethylene terephthalate), polypropylene, poly-(vinylidene fluoride), and polyimide), which provides other possible choice for nanopore-based sensing device [24–27].
In this work, novel sensing devices were fabricated on the basis of nanopore arrays in polycarbonate (PC) membranes and micropores in Si-Si3N4 films, and related translocation properties of single molecule were investigated using these devices.
Experimental device and reagent
PC membranes containing nanopore (pore diameter 50 nm, pore density six pores per μm2, membrane thickness 6 to 11 μm) arrays were purchased from Whatman, Inc. (Shanghai, China), and hydrophilic treatments were carried out before usage. Ultrapure water (18.25 MΩ · cm) was used for the preparation and rinsing. Goat antibody to human immunoglobulin G (IgG) and λ-DNA (48 kB, 310 ng/mL) obtained from Nanjing Boquan Technology Co., Ltd. (Jiangsu, China) were used as analytes in the experiments. Potassium chloride (KCl) was commercially available and at analytical grade.
A test device containing separated liquid cells linked by nanopore chip (sealed by PDMS) was integrated to measure the ionic current. At room temperature (25°C ± 2°C), KCl solution (pH = 7.48) was added to both feed cell and permeation cell, and the analytes were dissolved in the reservoir. An electric field was applied to both sides of the nanopore chip, and the transmembrane current was recorded using a Keithley2000 61/2-digital multimeter (Cleveland, OH, USA) or HEKA EPC-10 patch clamp (Bellmore, NY, USA). The nanopores were characterized using a MFP-3D-SA atomic force microscope produced by Asylum Research (Goleta, CA, USA). The micropores in the Si3N4 film was fabricated and characterized using Helios NanoLab 600i dual beam (Hillsboro, OR, USA).
Fabrication of nanopore-based device
Results and discussion
Fabrication of nanopore-based device
Ionic currents induced by biomolecule translocation
The sensing device based on PC membranes containing nanopore arrays was used to detect the ionic currents modulated by the biomolecule's translocation. KCl solutions of 0.001, 0.01, and 0.1 mol/L were employed as electrolytes, and IgG was used as analyte.
Figure 5 shows the detected current changing, with IgG concentration increasing at the driven voltage of 1.0 V. The differences between the background currents and the modulated currents versus KCl concentrations (IgG concentration is 40 ng/mL) are plotted, as shown in the inset of Figure 5, which reflects the influence on the ionic current caused by the concentration of electrolyte solution. If KCl concentration continues to increase, the ion density in the solution becomes higher and higher. Then, the lost amounts in K+ and Cl− due to the physical place-holding effect are rather bigger. On the other hand, the obtained results about the current changing tendency with IgG concentration indicate that the detected ionic current decreases with IgG concentration increase when it is lower than 40 ng/mL. Obviously, the entry of the IgG molecules results in the partial occupations of nanopore arrays, which prevents K+ and Cl− from passing through the PC membrane. Within a certain concentration, the translocation probability of IgG increases with its increasing concentration. As we have known, the volume of IgG is much larger than that of K+ or Cl−, so the charge density is rather lower in the occupied channel space, which results in the decrease in the detected ionic current.
However, the ion current does not continue to drop with increasing IgG concentration as expected; on the contrary, the ionic current increases with the increasing IgG concentration when it is higher than 40 ng/mL, and then it tends to be stable with the concentration continuing to increase, as shown in Figure 5. An example will help us understand this phenomenon: imagine a big room with a door; the maximum allowable value of the entering people in unit time is N. When the number of people who need to enter the room in unit time is lower than N, the value of the entering people in unit time will increase with the increasing number of people who need to enter. When the number of people who need to enter the room in unit time is larger than N, the actual value of the entering people will equal to or even be lesser than N (especially for disordered conditions). Similarly, when IgG concentration is higher than the threshold value, the number of passing molecules will remain or decrease. The physical place-holding effect is weakened, which can result in the increase of ionic current.
Only an overall decline in the background current can be observed using PC membranes. In order to find the changes in the background current curve induced by a single biomolecule's translocation, the Si3N4 micropore is employed, and it is covered by the PC membrane containing nanopore arrays, which will significantly decrease the effective nanopore numbers. The effective areas of the two Si3N4 micropores used in our work are 1.77 μm2 (chip 1) and 3.14 μm2 (chip 2), which can decrease the effective nanopore number from 106 and 107 to 10 and 19, respectively. They are integrated into the nanofluidic device for DNA sensing, and the ionic current was recorded by patch clamp. In these cases, the probabilities of the simultaneous translocation events decreased dramatically. So, it is possible to obtain discrete ionic drops or blockades in the detected ionic curves during biomolecules' translocations, which can provide more information for the translocation.
On the other hand, the blockages in the base current curve can also provide detailed information of DNA translocation. The inset in the center of Figure 8 shows different magnified blockages, which stand for the three main types of DNA translocation gestures discriminated from varieties of translocation data, as following: (1) straight-line translocation: in this case, the decrease of ionic current at the blockages is smaller, while the duration time is rather bigger; (2) double-folded translocation: in this case, the decrease of the ionic current at the blockades is about twice that in case 1, but the duration time is only a half; (3) partly-folded translocation: in this case, the decrease of the ionic current at blockades and the duration time are both between case 1 and case 2, and the shapes and durations of the blockades change variously because of the different gestures in DNA translocation. Comparing the two curves in Figure 8, the amounts of the effective nanopore numbers can be modulated by adjusting the size of the Si3N4 micropore, which can change the frequency of the current drop signals in the ionic current curve.
In summary, the transporting properties and detailed translocation information of biomolecules are investigated using an integrated device based on nanopore arrays in PC membranes and micropore in silicon nitride films. The amounts of effective nanopore numbers can be modulated by adjusting the size of Si3N4 micropore, which can change the frequency of signals in ionic current curve. It is believed that the nanofluidic device based on integrated micropore-nanopore chips possessed comparative potentials in biosensing applications.
LL is an associate professor at the Southeast University, PR China. LZ is an undergraduate student at the same university. ZN and YC are professors at the Southeast University, PR China.
This work is financially supported by the Natural Science Foundation of China (51003015 and U1332134); the National Basic Research Program of China (2011CB707601 and 2011CB707605); the Natural Science Foundation of Suzhou (SYG201329); open fund offered by the State Key Laboratory of Fire Science (HZ2012-KF09), 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.
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