Atomic Layer Deposition Modified Track-Etched Conical

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Analytical Chemistry

Atomic Layer Deposition Modified Track-Etched Conical Nanochannels for Protein Sensing Ceming Wang,a ‡Qibin Fu,a ‡ Xinwei Wang,b * Delin Kong,d Qian Sheng,a Yugang Wang,a Qiang Chen,d and Jianming Xue,a c * a

State Key Laboratory of Nuclear Physics and Technology, School of Physics, Peking University, Beijing 100871, P. R. China

b

Peking University Shenzhen Graduate School, Shenzhen 518055, P. R. China

c

Center for Applied Physics and Technology, Peking University, Beijing 100871, P. R. China

d

Laboratory of Plasma Physics and Materials, Beijing Institute of Graphic Communication, Beijing 102600, P. R. China

ABSTRACT: Nanopore-based devices have recently become popular tools to detect biomolecules at the single-molecule level. Unlike the long-chain nucleic acids, protein molecules are still quite challenging to detect, since the protein molecules are much smaller in size and usually travel too fast through the nanopore with poor signal-to-noise ratio of the induced transport signals. In this work, we demonstrate a new type of nanopore devices based on atomic layer deposition (ALD) Al2O3 modified track-etched conical nanochannels for protein sensing. These devices show very promising properties of high protein (bovine serum albumin) capture rate with well time-resolved transport signals and excellent signal-tonoise ratio for the transport events. Also, a special mechanism involving transient process of ion redistribution inside the nanochannel is proposed to explain the unusual biphasic waveshapes of the current change induced by the protein transport.

good detection sensitivity.9,17 In addition, the surface inside the nanopore should also be carefully prepared, since the surface charge, roughness, and/or functional groups could significantly influence the signal-to-noise ratio17-20 and the capture rate of the analytes,8,17 and they could even lead to unwanted permanent adsorption of the analytes21 or clogging,9 if not enough attention was paid.

In the past decade, nanopores have become a powerful tool to study biomolecules at the single–molecule level. Besides many works focusing on the sequencing of nucleic acids,1,2 protein molecules have also become another important target for investigation.3-13 However, proteins are generally much more difficult to detect than the nucleic acids. Unlike the nucleic acids which typically have one dimension much larger than the length of the pore, protein molecules normally with well-defined globular structures are small in three dimensions, so if the length of the nanopore was only a few tens of nanometers,1 the transport time for a protein molecule to pass through the nanopore would be only a few tens of microseconds, which could result in a high-percentage loss of the transport events due to the limited temporal resolution of the measurement instruments.12 Therefore, a critical challenge for detecting proteins is to prolong their dwell time inside the nanopore, so that time-resolvable signals of their translocation events can be collected.7,9,10,14-16 Precise control of the pore size in nanometer level is another important challenge, since the pore size needs to be just a little larger than the protein size in order to achieve a

Track-etched polymeric nanochannels have recently emerged as a cost-effective and versatile source of nanopores for biosensing,22-24 and ion-channel mimicking.2528 Both ‘‘nanopore’’ and ‘‘nanochannel’’ are in common mutual use.28 If the pore depth is much larger than the diameter, the structure is generally referred to as ‘‘nanochannel’’. Conically-shaped polymeric nanochannels, which can be reproducibly fabricated by an asymmetric etching method,29 are of great interest, as they show many unique and interesting properties, such as the ability to rectify the ion current.30,31 The tip size of these conical nanochannels could be made with only a few nanometers, which is much smaller than the pore base size, so a strong geometrical field focusing effect30 could occur at the tip.

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This focusing effect could strongly converge the electrical field as well as the ion current flow at the pore tip, and lead to the measured current very sensitive to the tip conditions. Thus, if an analyte molecule travels through the sensitive tip region, a significant change of the current could occur. We call this sensitive tip region as the detection zone of the analytes. The detection zone for this type of the track-etched polymeric nanochannels is usually the first ~1 μm zone from the tip,7 which is much longer than ~10s nm for the Si3N4-based solid-state nanopores,1 and thus a longer signal dwell time is expected for the protein transport process. Therefore, these track-etched conical nanochannels have been considered very promising for protein sensing. 32

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in the buffered neutral solution (pH = 7.4) and therefore could weakly attract the negatively charged BSA molecules. Due to this weak electrostatic attraction, very welldefined time-resolved transport signals were obtained. In addition, it was found that the current change for the BSA translocation process did not follow the common physical blocking mechanism, but rather involved a transient ion redistribution process inside the nanochannel. This process could induce a biphasic current waveshape with fairly large current change in magnitude, and therefore an excellent signal-to-noise ratio was achieved.

EXPERIMENTAL SECTION

However, precise control of the pore size is still a challenge for these polymeric nanochannels, and also the inner surface of these nanochannels is still somewhat uncertain at this stage.21 Their inner surface is considered to contain many negatively charged dangling polymeric chains, and these chains may become problematic, as they may repel the transport of proteins when the nominal pore size is small.30 There are already many approaches for surface modification of track-etched nanochannels, including electroless deposition7,22, ion sputtering33,34, plasma treatment35, and solution chemical modification36; however, it is still a challenge for these methods to finetune the diameters of nanochannels in a highly controllable and precise way. Moreover, these methods are usually time-consuming or hard to control the quality (e.g., density, stability and uniformity) of surface modification. The development of surface modification of single/multinanochannels has been described in detail by Hou et al.37 Instead, we use a simple and straightforward method of atomic layer deposition (ALD) to modify the nanochannel inner surface in this work. ALD is an important vaporphase thin-film deposition technology. 38 During the deposition, the substrate is alternately exposed to two reactants, and due to the self-limiting nature of the surface chemistry reactions, only one layer of material is allowed to grow at a time. Therefore, ALD is particularly advantageous for preparing highly conformal coatings with great reproducibility and digital control of the film thickness. Recently, ALD of Al2O320,39and HfO216 have been applied to modify solid-state Si3N417 and silica nanopores,15 and enhanced performance for sensing DNA was achieved.

Fabrication of Single Conical Polymeric Nanochannels by the track-etching technique The 12-μm-thick polyethylene terephthalate (PET) foils were irradiated with single swift heavy ions (Au) with energy of 11.4 MeV per nucleon at the GSI in Darmstadt, Germany. A irradiated foil was subsequently etched at room temperature (295 K) by an asymmetric etching method, 29 where the foil was mounted in between of two isolated containers that contained an etchant solution of 2.5 M NaOH in 1:1 MeOH/H2O and an etch-stopping solution of 1 M HCOOH and 1 M KCl aqueous solution, respectively. The etching process started from one side of the PET foil, but was immediately stopped when etched through (Figure S1, Supporting Information), and, as a result, a single conical nanochannel was formed on each irradiated PET foil. The addition of methanol in the etching solution could enlarge the cone angle of the conical nanochannels. It is noteworthy that the asymmetric nanochannels created by etching high-LET ion tracks (such as Au tracks) are actually funnel-like and not ideal conical41,42. The approximate tip diameter can be obtained by conductance measurement in the framework of conical model. Precise consideration of the sensing process should take into account the reconstruction of nanochannel profile suggested by Apel et al..41 Detailed fabrication information can be found in our previous publications.43 Atomic Layer Deposition (ALD) of Al2O3 Atomic layer deposition (ALD) was used to coat a conformal Al2O3 film on the conical nanochannels. The deposition was performed in a home-made flow-through ALD system (Figure S2, Supporting Information) with trimethylaluminum (TMA) and H2O as the precursors. A low deposition temperature of 120 oC was chosen to prevent thermal damage to the polymer PET. Detailed ALD process parameters can be found in Supporting Information (Figure S2).

In this work, we demonstrate a new type of devices with ALD-modified single conical polymeric nanochannels to detect single protein molecules. The nanochannels were made by the track-etching method and then followed by an ALD process for conformal thin-film coating to shrink the pore size to be close to the size of the analyte molecules. Bovine serum albumin (BSA), a major multifunctional protein present in the circulatory system, was used as the protein analyte. ALD Al2O3 was chosen for the coating, since Al2O3 is insulating and chemically stable and it also has a nominal isoelectric point at pH 9.0,40 which should lead to the surface positively charged

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Figure 1. (a) Schematic illustration of uniform ALD coating on a single conical nanochannel (not to scale). Insets show SEM images of the tip side (top) and the base side (bottom) of an ALD Al2O3 coated polymeric conical nanochannel. Scale bar = 100 nm. (b) SEM images of conical Al2O3 replicas obtained by coating the conical nanochannels by 150 cycles of ALD Al2O3 and then treated with RIE to partially remove the PET polymer matrix. A broken part of the Al2O3 replica (blue arrow) revealed that the ALD coating was only on the pore wall, indicating excellent step coverage inside the nanochannels.

Figure 2. (a) SEM images of three PET foils that contain multi-nanochannels before (top row) and after (bottom row) the ALD coatings of Al2O3 with 80, 120 and 200 cycles, respectively. Scale bar = 100 nm. (b) Comparisons of the average pore-opening diameters before and after the ALD Al2O3 coatings with different cycles. (c) I-V characteristics of a single conical nanochannel measured in 1 M KCl before and after 75 cycles of Al2O3 ALD. After the ALD, the tip diameter of the nanochannel shrank from 37 nm to 19 nm, as determined by the change of conductance. The initial pore base diameter was 950 nm. (d) The measured tip diameter as a function of the ALD cycle number. The good linear relation suggested a good controllability of the pore tip size by ALD.

Experiment of BSA transport A PET foil with a single conical nanochannel on it was mounted in between of two isolated containers that were both filled with buffered 0.5 M KCl aqueous solution (0.1x PBS, pH = 7.4). A patch clamp amplifier (Axopatch 200B, Molecular Devices Inc.) with Ag/AgCl electrodes were used to measure the current trace and the current-voltage response across the nanochannel. The polarity of the applied voltage was referenced to the tip side electrode. The current data were collected at 100 kHz with a low pass Bessel filter of 10 kHz. For the protein transport experiment, the buffered 100 nM BSA (Sigma, A2058) solution (in 0.5 M KCl, 0.1x PBS, pH = 7.4) was always freshly made prior to each experiment, and was injected to the tip side of the nanochannel. Unless otherwise specified, a positive voltage of 500 mV was used in the transport experiment to drive the negatively charged protein to pass through the nanochannel from the tip to base.

containing PET foil was coated with 150 cycles of ALD Al2O3, and then treated with reactive ion etching (RIE) to partially remove the PET polymer matrix and expose the solid component of the deposited Al2O3, which served as a replica of the nanochannel inner surface. As shown in Figure 1b, well-defined conical Al2O3 nanotubes were observed under the scanning electron microscope (SEM), which clearly demonstrated that a complete coating of Al2O3 was uniformly formed on the inner surface of the nanochannel. Due to the self-limiting nature of the ALD surface chemistry reactions, the film thickness can be precisely controlled by setting a certain number of the ALD cycles. Figure 2a shows the comparisons of the PET foils that contain multi-nanochannels before and after the ALD coatings of Al2O3 with 80, 120, and 200 cycles, respectively. Clear shrinkage of the nanochannel openings was observed after the ALD. As plotted in Figure 2b, the average diameters of the pore openings decreased by 17.3, 30.0, and 48.2 nm for 80, 120, and 200 cycles, respectively. These decreased values agreed quite well with the values expected from the growth rate (i.e. twice the thickness of the deposited Al2O3 film), showing that the diameters of polymeric nanochannels can be finely tuned by ALD in a highly controllable way.

RESULTS AND DISCUSSION The conical nanochannels fabricated by tracketching method was coated with a thin layer of Al2O3 by ALD as illustrated in Figure 1a. Under our experimental condition, the film growth rate was around 0.12 nm per cycle, as measured by ellipsometry. To examine the conformality of the coated Al2O3, a nanochannel-

Then we used this ALD process to modify single conical nanochannels. A conical nanochannel typically has a tip

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Figure 3. (a) Schematic illustration of the transport of BSA molecules through an Al2O3 coated conical nanochannel. (b) Example of current trace seen before and after adding buffered 100 nM BSA solution to the tip side of the conical nanochannel. Before adding BSA, the current trace was relatively steady (2201 ± 23 pA). But after BSA was added, many spikes corresponding to the BSA translocation events appeared in the current trace. (c) Magnified views of typical current pulses (see the text for details). The current trace was collected at +500 mV in 0.5 M KCl solution. The single conical nanochannel used in this experiment had a 25-cycle ALD coating of Al2O3, and the final diameters were 13 nm and 700 nm for the tip and the base, respectively.

end much smaller than the base end (tens of nanometers versus sub-micrometer in diameter), and the tip end is very critical for biosensing,7,44 since the diameter of the tip is usually comparable with the size of biomolecules. The tip diameter (dtip) was estimated by a conductance method29, in which the channel conductance (G) was extracted from the linear current-voltage (I-V) relation and a larger conductance value corresponded to a larger tip diameter. As the I-V curves shown in Figure 2c, a pronounced current drop was found after 75 cycles of Al2O3 ALD. The extracted conductance values were 27.4 and 14.8 nS for the nanochannel before and after the ALD, respectively, corresponding to a decrease of the tip diameter from 37 to 19 nm by the ALD. The capability of finetuning the tip size was further demonstrated by performing step-wise ALD on one single conical nanochannel and monitoring the shrinkage of the tip size during the process. As shown in Figure 2d, the tip diameter continuously shrank from initially ~67 nm down to only 3 nm, with a very good linear relation between the tip size and the ALD cycle number. The linear fit revealed that the growth rate was 0.128 ± 0.006 nm per cycle, which was reasonably close to the previously obtained value. This again demonstrated the unique advantage of ALD for precisely tuning the nanochannel size in nanometer level.

Information). Using the theoretical model developed by Yan et al.,46 the charge density of the ALD Al2O3 surface inside the nanochannel was estimated to be σs = +0.039 C/m2 (Figure S11, Supporting Information). We also investigated the stability of the ALD-Al2O3-coated nanochannels, which is an important feature for application to sensors. Due to the stable properties of Al2O3 coating layer, these nanochannels show very stable current response under different constant voltages (Figure S4a, Supporting Information). They can be stabilized for more than six months (Figure S4b, Supporting Information). The ALD-Al2O3-coated single conical nanochannels were used for protein transport experiments. Bovine serum albumin (BSA, 66 kDa), a negatively charged protein, was used as the analyte molecule. The measurement scheme is illustrated in Figure 3a. An Al2O3 coated nanochannel with a final tip diameter of 13 nm was mounted in between of two isolated cells that both contained 0.5 M KCl solution (buffered at pH = 7.4). A positively biased voltage of 500 mV was applied on the base end of the nanochannel, and a relatively steady current of 2201 pA with a small fluctuation of ± 23 pA was observed (Figure 3b). Then we added the BSA solution (100 nM) to the tip side of the conical nanochannel, and the BSA molecules were expected to translocate through the nanochannel by electrophoresis and electroosmosis. These translocation events resulted in a series of clear pulses in the current trace, as shown in Figure 3b. By a close examination of each pulse, we found that the waveshapes of the translocation pulses were quite

Surface charge is known to have significant influence on the translocation behavior of biomolecules, since the biomolecules are often charged and thus can interact strongly with a charged surface through electrostatic and/or electrokinetic forces.8,17 After the ALD Al2O3 coating, surface charge of nanochannel wall was reversed from negative (carboxylate groups45) to positive, as evidenced by the reversed rectification polarities of the I- V curves before and after the coating (Figure S3, Supporting

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Analytical Chemistry baseline current. On the other hand, if imagining that the time interval between the edges of the sudden current rise and fall (i.e. t1 in Figure 4a) was significantly shortened (e.g. to ~100 μs), the waveshape should be a superposition of the two opposite decaying processes, and the resulted superposition would be just a simple single-pulse waveshape as (vi) in Figure 3c. Therefore, there should be a fundamental connection between these two types of superficially different waveshapes (i.e. simple single-pulse waveshapes and the biphasic waveshapes). In fact, the observed transition waveshapes, such as (v) in Figure 3c, further reinforced this point of view. Therefore, for simplicity, we first chose to analyze a representative current waveshape with a distinct biphasic feature, and then extend our analysis to a statistical point of view to study the mechanism of the BSA translocation. The biphasic waveshape of (i) in Figure 3c was first chosen for analysis, and was again plotted in Figure 4a. The waveshape was divided into five featured segments, as labelled as I – V in Figure 4a. Before the translocation event, the background current (I0) was quite steady, as shown in Segment I. When a BSA molecule entered the nanochannel, the current suddenly rose to a higher value (I1) and then gradually decayed to approach a new steady value (Is), as shown in Segment II. Then the current stayed roughly at Is (Segment III) until a sudden current fall occurred. The current fell to a value (I2) even lower than the background and again gradually decayed back to approach the background current, as shown in Segment IV. Finally, the current was recovered back to the background steady state, as shown in Segment V. Note that the current changes of I1, Is, and I2 were all referenced to the background current of I0, so I2 was negative in our discussion. We noticed that, in Segments II and IV, the two decay processes could be both well fitted with exponential decay functions (Figure S7, Supporting Information) with fitted characteristic times of τ1 and τ2, respectively. This exponentially decaying behavior suggested that these two segments should be associated with some transient relaxation processes (vide infra). On the other hand, we also noticed that the noise level in Segments II and III was appreciably larger than that of the background (Segment I). As the detailed statistical histograms shown in Figure 4b, the standard deviations (σ) of the current fluctuations in Segment II and III were relatively close (i.e. σ = 55 pA and 48 pA, respectively), but they were considerably larger than that of the background (i.e. σ = 26 pA). However, surprisingly, after the current suddenly fell below the background current in Segment IV, the noise level was much reduced to nearly the background level (i.e. σ = 29 pA). This difference in noise level was quite pronounced and was consistently seen in all other biphasic waveshapes (e.g. (ii) and (iii) in Figure 3c), which intrigued us to find out the underlying mechanism that led to the difference. A well-known fact6 is that when a BSA molecule is adsorbed on a nanopore wall, its adhering conformation can rapidly fluctuate and thus result in

Figure 4. (a) Re-plotting of the biphasic waveshape (i) in Figure 3c for analysis. The waveshape was divided into five segments, as labelled as I – V. (b) Histogram analysis of the current fluctuation in each segment. For Segments I, III, and V, the means of the current were 2202, 2267, and 2206 pA, respectively, and the standard deviations were 26, 48, and 26 pA, respectively. For Segments II and IV, the plotted values of the current fluctuation were the differences between the original current data and the fitted data using exponential decay functions, respectively. The standard deviations for the current fluctuations in Segments II and IV were 55 and 29 pA, respectively.

interesting. About one third of the pulses showed a single-pulse waveshape with a short pulse duration less than 200 μs (e.g. Figure 3c (vi)). Another third of the pulses showed a biphasic waveshape, and the time interval between the edges of pulse rise and fall was typically greater than 1 ms (e.g. Figure 3c (i)-(iv)). The remaining one third of the pulse waveshapes looked like the transition states between the above two cases: as the waveshape (v) shown in Figure 3c, the pulse duration was between 200 μs and 1 ms, and a slight dip could be seen on the falling edge of the pulse. The biphasic waveshapes were of particular interest. This type of the pulses was found to have a few common features: the current pulse started with a sudden positive rise, and then gradually decayed for a certain period. If the decay period was long enough, a steady state current could be reached as the waveshapes of (i) and (ii) shown in Figure 3c. Then, suddenly, the current fell below the baseline, followed by a gradual decay/increase back to the

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the current to fluctuate as well. Thus, very likely, the increased noise observed in Segments II and III was due to the conformation fluctuation of the adsorbed BSA, and afterwards, at the moment of the falling edge of the current (i.e. the boundary between Segments III/IV), the adsorbed BSA molecule suddenly desorbed from the wall and immediately left the nanochannel detection zone, leading to the noise level in the later Segment IV much reduced to nearly the same level as the background. In addition, after the decaying/recovering period in Segment IV, the current trace was completely recovered back to the steady state before the translocation event, as the current histogram of Segment V was almost identical as that of Segment I.

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Figure 5. Statistical analysis of the BSA translocation pulses: (a) the histogram distribution of t1; (b) the plot of the values of the current rises (I1) and falls (I2) with respect to t1; (c) the scatter plot of the characteristic decay times (τ1 and τ2); and the plot of I2τ2 versus (I1 - Is)τ1 relation, where same notations as in Figure 4 were used.

From the above analysis, the difference in the noise level was able to provide the fingerprint to reveal that a BSA molecule was adsorbed on the nanochannel wall in Segments II and III. But, increased current was seen in these two segments, suggesting that the adsorbed BSA increased the nanochannel conductivity. This was rather opposite to the expectation from the simple physical blockage mechanism,47 where a decrease in conductivity should be seen. Nevertheless, increased conductivity was indeed experimentally observed in a few DNA transport experiments,48,49 where this phenomenon was explained by the high concentration mobile ions around the DNA that contributed to the increased conductivity. Similar explanation could also be applied in this case, since the highly charged BSA molecule was also surrounded by high-concentration mobile counter ions which formed as a screening ion cloud50,51. When the BSA molecule entered the nanochannel and adsorbed on the wall, it could bring in the surrounding ion cloud, increase the local ion concentration, and therefore increase the ionic conductivity inside the nanochannel. Consequently, an increased current of 2267 ± 48 pA, which was roughly 65 ± 55 pA (Is) higher than the background, was seen in the steady-state segment of III. Before reaching this steady state, the transient decaying process in Segment II could be, from the view of its characteristic time, ascribed to the redistribution process of the extra ions induced by the adsorption of BSA inside the nanochannel. The characteristic time (τ1) associated with this exponential decay was 1.0 ms. Assuming that the ion diffusivity (D) was 2×10-9 m2/s for both K+ and Cl-, the diffusion length for 1.0 ms was roughly Dτ 1.4 μm, which was quite consistent with the length of the detection zone inside the nanochannel.7 In addition, similar microsecond-scale characteristic time was also observed for the ion redistribution process in other types of conical nanochannels (e.g. glass nanochannels52). On the other hand, when the adsorbed BSA molecule suddenly desorbed from the wall and immediately left the detection zone, we expected the current trace to show a reversed pattern of the adsorption, i.e. a sudden current fall followed by a recovering process with exponential

decay, which was exactly what we observed in Segment IV of the current trace. In addition, this decay showed a similar characteristic time (τ2) of 1.8 ms, implying that a similar but reversed process of ion redistribution occurred at this stage. To further look into the sudden current rise/fall in Segments II/IV, we estimated the translocation time of a free BSA molecule passing through the detection zone of the nanochannel. The translocation time for free BSA should be no longer than thirty microseconds (Figure S12, Supporting Information), which was too fast for the measurement instruments. Consequently, the detailed transient processes before the adsorption (or after the desorption) that involved the motion of free BSA could hardly be captured by the measurement instruments, and therefore, only sudden current rises/falls appeared in the current trace. Next, we extended our analysis to a statistical point of view. A typical section of 30-second current trace including 516 successive translocation pulses was chosen for the statistical analysis. The critical characteristic parameters of the translocation waveshapes, including t1, I1, I2, τ1, and τ2 (same notations as in Figure 4), were extracted, whenever possible, to study their correlations. The histogram of t1 distribution was plotted in Figure 5a, where a wide range of the adsorption duration was observed, suggesting that the desorption of BSA was a random process. Figure 5b shows the values of the current rises (I1) and the current falls (I2) with respect to t1. Notice that I2 was referenced to the background current (I0), and thus, if the current fell very shortly after the current rise (i.e. very short t1), I2 would be very small in magnitude. As an extreme case, I2 could be almost zero as for the waveshape of (vi) in Figure 3c. Thus, as shown in Figure 5b, an increasing trend of I2 in magnitude with respect to t1 until a transition point was observed. The transition point in this case was t1 ≈ 2.5 ms; beyond this point (i.e. t1 > 2.5 ms),

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Analytical Chemistry static force to draw the BSA molecule into the nanochannel; the high electroosmotic flow induced by the focused electrical field that could carry the BSA into the nanochannel; and the positively charged Al2O3 wall that could further electrostatically attract the negative BSA molecule into the nanochannel. Secondly, the pulse duration of the BSA translocation was comparatively long.37,11,12,53 Thanks to the electrostatic attraction between the BSA and the wall, the dwell time of the BSA at the detection zone was significantly prolonged, which allowed an easy capturing of the translocation events by the measurement instruments. Meanwhile, the magnitude of the current change associated to the translocation event was fairly large (i.e. on the order of 1 nA). Though it was due to a different mechanism that involved transient ion redistribution, as a result, the signal-to-noise ratio was greatly improved. Thirdly, the weak electrostatic attraction between BSA and the wall was able to prolong the transport time of BSA, but not too strong to cause permanent adsorption or clogging. Therefore, the problem of fouling is not obvious in our measurement period (typically less than 24 h). One of the biggest challenges of the biosensing application is to develop antifouling sensor system. Long-term antifouling behavior in nanopore can be achieved through gating a pore by liquid reconfiguration. This approach reported by Hou et al.54 uses a capillary-stabilized liquid as reversible, reconfigurable gate that fills and seals pores in the closed state, and creates a non-fouling, liquid-lined pore in the open state. In addition, the sensitivity of the nanochannel should be at its best when the size of the nanochannel is just a little larger the protein size; otherwise, the signal-to-noise ratio would be much deteriorated if the tip size was considerably larger than the analyte protein (Figure S10, Supporting Information). Therefore, the capability of fine-tuning the pore size adds another important advantage for the ALDmodified nanochannels.

well-separated biphasic waveshapes appeared (e.g. waveshapes of (i-iii) in Figure 3c), and therefore I2 should not depend on t1 anymore. Moreover, we extracted the characteristic decay times of τ1 and τ2 for the waveshapes with t1 > 2.5 ms, and found that τ1 and τ2 were two independent parameters for all of these translocation events; they did not depend on I1 or I2 (Figure S8, Supporting Information), nor on each other (Figure 5c). This observation was quite consistent with the above-proposed mechanism, where τ1 and τ2 corresponded to the relaxation processes of the ion redistribution inside the nanochannel, and therefore they should only depend on the features of the nanochannel, but not on the details of the adsorption process. However, on the other hand, the magnitudes of the current rises (I1) and falls (I2) might be affected by the details of the BSA adsorption. For instance, the adsorption could occur at different locations inside the nanochannel, and thus lead to different magnitudes of the current change. But, nevertheless, for a certain biphasic waveshape, the corresponding I1 and I2 should associate with the same BSA molecule and the same location that adsorption/desorption occurred. Therefore, the change of the charge induced by the adsorption/desorption inside the nanochannel should be equal for each translocation event. This change could be obtained by integrating the transient exponential decay function ( ∆Ie / dt ∆Iτ), and therefore I − I τ −I τ was expected to be valid for each waveshape with t1 > 2.5 ms (notice that I2 is negative in our notation). Figure 5d shows the scatter plot of the corresponding data extracted from the experimental current trace. A linear fitting with fixed zero intercept was performed, and an expected unity slope of -0.99±0.02 was obtained, which further reinforced the proposed transient mechanism. To sum up the entire translocation process of a BSA molecule through the ALD-Al2O3-coated nanochannel, a negatively charged free BSA molecule was drawn into the nanochannel from the tip side by both electrophoresis and electroosmosis, then the BSA was weakly attracted by the positively charged Al2O3 pore wall and adsorbed on the wall for a certain period (from ~10s μs to ~10s ms), during which a transient redistribution of the ions inside the nanochannel occurred as a result of the extra charges the adsorbed BSA brought in, afterwards the BSA suddenly desorbed from the wall and immediately left the pore detection zone, and then the ion distribution inside the nanochannel gradually recovered back to the original state.

CONCLUSIONS In this work, we demonstrated a simple and straightforward approach of using ALD to modify single conical polymeric nanochannels for protein single-molecule sensing. The nanochannels were prepared by the tracketching method followed by a highly conformal coating of Al2O3 via ALD. As a distinct advantage of ALD, the thickness of the Al2O3 coating layer could be precisely controlled to make the tip size of the nanochannel to be just a little larger than the size of the analyte protein (BSA), in order to achieve a good sensitivity. The deposited positively charged Al2O3 layer could weakly attract the negatively charged BSA molecules, which could appreciably prolong the transport time of BSA through the nanochannel. Therefore, very good time-resolved signals could be captured for the translocation events. Moreover, the translocation process in the ALD-modified nanochannels was found to follow a special mechanism, which involved transient processes of ion redistribution as the BSA molecules adsorb on (or desorb from) the pore

It should be pointed out that there were quite a few special beneficial features of using this type of ALD-Al2O3coated conical PET nanochannels for the BSA transport experiment. Firstly, compared with other types of nanopores,4-7,11,12,53 the BSA capture rate was very high (i.e. ~17 s-1), and could be further increased if a higher bias voltage was applied (Figure S9, Supporting Information). The reasons might from three aspects: the highly focused electrical field at the tip that could exert a strong electro-

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wall. These ion redistribution processes could result in very large current variation with interesting biphasic waveshapes in the current trace. Consequently, excellent signal-to-noise ratio was obtained for the translocation events. In addition, these nanochannels showed very high analyte capture rate, which was also beneficial for biosensing. Therefore, we believe that these ALD-modified track-etched conical nanochannels are very promising for biosensing at the single-molecule level.

AUTHOR INFORMATION Corresponding Author [email protected] [email protected]

Author Contributions ‡ Ceming Wang and Qibin Fu contributed equally.

ACKNOWLEDGMENT This work is financially supported by NSFC (Grant Nos. 51302007, 11375031, and 11335003), and Shenzhen Science and Technology Innovation Committee (Grant No. JCYJ20130329181509637). We acknowledge Prof. C. Trautmann for providing the irradiated samples.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.

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Atomic Layer Deposition Modified Track-Etched Conical

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