Ren Ren, Xiaoyi Wang, Shenglin Cai, Yanjun Zhang, Yuri Korchev, … · 2020. 11. 27. · processes...

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2000356 (1 of 8) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Selective Sensing of Proteins Using Aptamer Functionalized Nanopore Extended Field-Effect Transistors

Ren Ren, Xiaoyi Wang, Shenglin Cai, Yanjun Zhang, Yuri Korchev, Aleksandar P. Ivanov,* and Joshua B. Edel*

Dr. R. Ren, X. Wang, Dr. S. Cai, Dr. A. P. Ivanov, Prof. J. B. EdelDepartment of ChemistryMolecular Science Research HubImperial College LondonWhite City Campus80 Wood Lane, London W12 0BZ, UKE-mail: [email protected]; [email protected]. Y. Zhang, Prof. Y. KorchevDepartment of MedicineImperial College LondonHammersmith Campus, London W12 0NN, UKDr. Y. Zhang, Prof. Y. KorchevNano Life Science Institute (WPI-NanoLSI)Kanazawa UniversityKakuma-machi, Kanazawa 920-1192, JapanDr. Y. ZhangTianjin Neurological InstituteTianjin Medical University General HospitalTianjin 300052, China

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smtd.202000356.

DOI: 10.1002/smtd.202000356

within a complex mixture has been gaining in importance. Single-molecule techniques have emerged as powerful tools for the observation of individual molecules in real-time, which can provide rich infor-mation about molecular structures and interactions with other molecules.[4,5] By monitoring one molecule at a time and sta-tistically analyzing thousands of individual molecules, proteins can be identified by measuring their characteristic signals.[6,7]

Single molecules techniques, such as nanopore sensors are becoming popular due to their label-free operation, ease of fabrication, the ability of active transporta-tion, and in some cases, the ability to tailor the sensor chemistry via surface modifica-

tion.[8–14] Nanopores have been widely used for the detections of nucleic acids, DNA sequencing,[15] and protein sensing.[16–23] However, detection of proteins remains challenging due to fast analyte transport, low capture rates, and the need for relatively high protein concentration.[24,25] To date, a number of valuable approaches have emerged for nanopore based protein sensing. For example, the use of high salt conditions to slow down the translocation, or the use of high bandwidth instruments to resolve the fast transients.[26,27] However, selectivity is still something that needs to be optimized.

One promising strategy for addressing these challenges is to integrate nanopores with field-effect-transistors (FETs).[28–31] These combined sensors are capable of selectively detecting analytes at the single-molecule level and offer a degree of con-trol over the transport of individual analytes. Our group has also developed nanopore designs coupled with ionic-FETs, where a gate voltage was used to facilitate molecular transport at the single-molecule level.[32,33]

In one of these designs, a nanopore extended FET (nexFET) was functionalized with a polypyrrole (PPy) layer that incorporated protein receptors. By applying the gate voltage our group has dem-onstrated that molecular transport can be efficiently controlled at the single-molecule level.[32] nexFETs were able to selectively recognize the corresponding antibodies, with improved signal-to-noise and enhanced analyte capture. Despite these improve-ments, the generality of the detection strategy is somewhat limited as it requires optimization for each antibody–antigen pair.[34] An alternative and perhaps more general approach would be to use aptamers, as they simplify sensor functionalization, and typically can operate in a broader range of environments.[35] Aptamers are commonly selected from a random DNA/RNA sequence library using systematic evolution of ligands by exponential enrichment

The ability to sense proteins and protein-related interactions at the single-molecule level is becoming of increasing importance to understand biological processes and diseases better. Single-molecule sensors, such as nanopores have shown substantial promise for the label-free detection of proteins; however, challenges remain due to the lack of selectivity and the need for relatively high analyte concentrations. An aptamer-functionalized nanopore extended field-effect transistor (nexFET) sensor is reported here, where protein transport can be controlled via the gate voltage that in turn improves single-molecule sensitivity and analyte capture rates. Importantly, these sen-sors allow for selective detection, based on the choice of aptamer chemistry, and can provide a valuable addition to the existing methods for the analysis of proteins and biomarkers in biological fluids.

1. Introduction

Proteins play an essential role in biological function.[1] The accu-rate recognition and quantification of proteins are of particular significance in biochemical and biomedical applications, including clinical diagnostics and targeted therapeutics.[2,3] Developing tools to detect such targets at low concentrations

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(SELEX), which can be made applicable to a wide range of tar-gets from small molecules to cells and bacteria.[36] Unlike protein ligands/receptors, aptamers have a well-defined charge distribu-tion, that can be used in optimizing electrostatic interactions near the nanopore surface when using gating voltage.[37]

Here, we report on a nexFET sensor based on a double-barrel nanopipette, which was functionalized with aptamers (Figure 1). A method for the fabrication and the functionalization of these sensors is shown, whereby simple aptamer sequence is embedded in a polypyrrole gating layer. A thrombin binding aptamer (TBA) was used for the detection of human α-thrombin, a protein that is generally difficult to detect using more conven-tional nanopores.[38,39] In contrast, with the aptamer modified nexFET, the gate voltage facilitates single-molecule thrombin detection at low concentrations (pm) with improved signal-to-noise, higher capture rate and lower translocating speed.

2. Result and Discussion

2.1. Fabrication and Characterization of the Aptamer Modified nexFET

The aptamer modified nexFET is fabricated using a double-barrel nanopipette with one hollow barrel terminating with a

nanopore, and the other filled with carbon forming a nanoelec-trode. The carbon nanoelectrode is further functionalized by a two-step PPy/aptamer electrodeposition with real-time current feedback control.

First, double-barrel quartz capillaries were pulled using a laser-assisted pipette puller to achieve nanopipettes terminating with two 100 ± 20 nm (n = 5, number of repeats) nanopores (Figure 2a(ii); Figure S1, Supporting Informa-tion; Figure 2e; Figure S2, Supporting Information). Carbon was pyrolytically deposited in one barrel and at the end of the nanopore tip, as previously reported,[32,40] (Figure 2b; Figures S3 and S4, Supporting Information). The conduc-tive carbon at the nanopipette tip was electrically connected to carbon inside the barrel, and the electrochemically active carbon area was estimated via linear sweep voltammetry (LSVs) using 1 × 10−3 m ferrocenemethanol as the redox mediator (Figure 2f ). It should be noted that the uniformal carbon coating of the nanopipette tip is essential for the successful fabrication of functional nexFETs. Nonuniform deposition of carbon at the tip and the adjacent nanopore orifice results in an uneven coating of the PPy. This, in turn, would lead to a device that would lack gating control, as the drain–source channel would be dominated by the glass wall rather than the PPy gating layer (Figures S5, S6, and Note S1, Supporting Information).

Figure 1. Conceptual schematic of an aptamer functionalized nexFET. a) The sensor is based on a double-barrel quartz nanopipette. One of the bar-rels is hollow and terminates with a nanopore. The inside of the other barrel is filled with pyrolytic carbon, and the tip of the pipette is coated with a thin layer of carbon and a core PPy layer and a shell PPy–aptamer layer. b) A side view schematic of the nanopore, illustrating the sensor operation. When no gate voltage is applied, the inner layer is positively charged, and the outer layer is slightly negatively charged, resulting in limited binding and detection of the target protein. c) Positive gated device (VG = 400 mV) can catch and sense thrombin selectively, with higher throughput and signal-to-noise. Representative current–time traces showing translocation thrombin (concentration 50 × 10−12 m) in 100 × 10−3 m KCl, 1 × 10−3 m Tris–EDTA, pH 8, under a bias of VDS = 500 mV. Scale bar: horizontal: 1 s; Vertical: 20 pA).




The carbon nanoelectrode and carbon deposited at the nano-pipette tip served as a site for the electrochemical polymeriza-tion and formation of the PPy layers. The electropolymerization process was performed by applying a bias, VG, to the carbon working electrode to enable the oxidation of pyrrole while the second working electrode was used to monitor the change of the open pore current, IDS under a small bias across the nano-pore, VDS. Precise control of the nanopore dimension can be achieved by the real-time feedback, by monitoring the IDS (Figure 2c) similar to previous reports.[32,33] By using this con-trollable fabrication process, the deposition could be stopped at a defined threshold of the current passing through the nano-pore IDS. The process is reproducible and results in PPy-coated nexFET with very similar ionic I–V characteristics (Figure 2g).

Previously, we added antibody receptors to the PPy precursor prior to electrochemical polymerization.[32] These nexFET devices had the capability to selectively sense target antibodies as they bind to the receptors embedded in the PPy modified

nanopore surface. However, diminished FET performance was observed, due to the excess negative charge of the functional groups embedded in the PPy (Figure S8, Supporting Informa-tion). Herein, we improved on the FET performance by devel-oping a two-step electrodeposition protocol: In the first step, a PPy core layer was electropolymerized onto the carbon surface of the nanopipette tip, generating a PPy nanopore (Figure 2c). In the second step, a PPy shell embedded with aptamers was electropolymerized onto the existing PPy layer (Figure 2d). After electrochemical polymerization of the core layer, the ion transport through the nanopore showed a positive ionic cur-rent rectification (ICR), indicating the nanopore wall is posi-tively charged due to the protonated amino groups in PPy (Figure 2g). Upon addition of the shell, negative ICR (Note S2, Supporting Information) was observed, confirming the suc-cessful imprinting of negatively charged aptamers (Figure 2h). The FET performance of this double-layer system, compared to a single layer system, was improved significantly: the ICR

Figure 2. Fabrication and characterization of aptamer modified nexFETs. The aptamer modified nexFET was fabricated using a) double barrel quartz nanopipettes with b) one barrel filled with pyrolytically deposited carbon, followed by c) electropolymerization of a core PPy layer, and d) electrodeposi-tion of a shell PPy layer embedded with aptamers. i) shows the schematic representation and ii) the corresponding fabrication process. e) I–V charac-teristics of a double-barrel quartz nanopipette in 100 × 10−3 m KCl (prior to filling with carbon and electrodeposition). f) Linear sweep voltammograms recorded at the carbon surface area using 1 × 10−3 m ferrocenemethanol as a redox mediator, see Note S1 in the Supporting Information for additional discussion. g) The I–Vs of multiple devices with after the PPy deposition with real-time feedback control. h) The I–Vs for the multiple devices after aptamer/PPy deposition. The insets are the SEM images for the device at each stage. Scale bar: 100 nm. SEM images are shown in Figure S7 in the Supporting Information. In all cases, error bars indicate one standard deviation, n = 5.




changed from 0.17 to 0.5 within the gate voltage range of −400 to 400 mV in 100 × 10−3 m KCl (Figure S9, Supporting Informa-tion). Hence, the core PPy layer, which has high conductance, can be used to efficiently control the gate response of the FET, while the shell layer can be used for the selective sensing of target analytes.

Using this two-step PPy functionalization protocol and depo-sition feedback control, the final PPy coated nanopore diameter was 15.0 ± 0.7 nm (n = 5). This was achieved, by stopping the deposition when the nanopore current reaches a threshold of 2 nA at VDS = 100 mV. This resulted in an initial PPy coated nanopore ≈40 nm in diameter (Figure 2g; Figure S7, Supporting Information). Following deposition of the shell PPy layer, using a nanopore current threshold of 0.7 nA, the nanopore diameter decreased to ≈15 nm (Figure 2h; Figure S7, Supporting Infor-mation). Using this approach, the nanopore I–V characteristics can be easily tuned by changing the set point of the open cur-rent to match the size of the target molecules of interest.

2.2. Aptamer Modified nexFET with Different Functionalization Density

Sensor validation was performed using the well-established TBA–thrombin binding system. We used human α-thrombin (37.5 kDa; pI of 7.0–7.4), that is a multifunctional protease in the bloodstream and has a significant role in physiological and pathological processes, such as blood coagulation, thrombosis, and angiogenesis. It is therefore essential to detect thrombin at trace levels (pm–nm range) with high sensitivity. The TBA used in this study, is 15-mer (5′-GGTTGGTGTGGTTGG-3′) ssDNA (Kd between 35–100 nm) and folds into a stable intramolecular G-quadruplex conformation in the presence of K+ which is required for thrombin binding.[35,41,42] To ensure that the TBA is sufficiently extended from the PPy shell to facilitate thrombin binding, a 12 base random sequence was added to the aptamer (Figure S11, Supporting Information).

In control experiments, no translocations were observed using plain nanopipettes before PPy polymerization (Figure 3a). With smaller nanopore sizes (15–20 nm), transloca-tion events were rarely observed, even at thrombin concentra-tions of 1 × 10−9 m (Figure S10, Supporting Information). This was attributed to several factors including 1) The protein does not easily get transported electrophoretically through the nano-pore due to its heterogeneous charge of; 2) the translocation signal cannot be resolve from the noise due to the small analyte size and fast transport speed.

Quartz nanopores functionalized with PPy can slow down dsDNA translocation as a result of the interactions between the DNA and PPy. In this configuration, the translocation of thrombin is slowed by the electrostatic interaction with a more positively charged and smaller nanopore, resulting in sharp blockade events (Figure 3b). However, such a platform is not selective and cannot differentiate between molecules of similar size and charge.

With the addition of the aptamer/PPy shell layer, selectivity is improved, and the target protein can bind and unbind to the corresponding aptamer located on the shell. We studied the effect of TBA concentration, incorporated in the shell, at

a constant thrombin concentration of 1 × 10−9 m. Upon the addition of 100 × 10−9 m TBA into the PPy precursor prior to electrochemical polymerization, the ICR changed from 1.53 (Figure 3b(ii)) to 0.96 (Figure 3c(ii)). The addition of 1 × 10−6 m TBA results in a negatively charged nanopore with ICR = 0.38 (Figure 3d(ii)) and 10 × 10−6 m TBA results in an even more negatively charged nanopore with ICR = 0.15 (Figure 3e(ii)).

At higher TBA concentrations, the number of available binding sites increases, as is evident by the “closed” TBA–thrombin bound state becoming more dominant. The increase in the “closed” fraction is due to the higher capture rate (Figure S12, Supporting Information) and prolonged dwell time (Figure S13, Supporting Information). When the binding sites are more densely packed, the bound molecules can detach, move and reattach to nearby sites.[43,44] On one hand, the aptamers can act as a transport system: the proteins can be accumulated by the aptamers on the whole polymer surface and then attracted by the nanopore under the electrophoretic force, resulting in a higher capture rate (Figure S14a, Supporting Information). On the other hand, the protein can hop along the binding sites located in the orifice and keep occupying the space in the nano-pore, leading to prolonged dwell time (Figure S14b, Supporting Information).[29,31,32,44] A common problem when functional-izing nanopores is how to control the number of binding sites; if there are insufficient binding sites, the majority of the pro-teins will pass through the nanopore undetected (Figure 3c). With too many accessible binding sites (Figure 3e), the nano-pore could remain in the blocked state. In this study, by varying the aptamer concentration in the polymerization electrolyte, the number of these functional groups located on the nano-pore orifice can be controlled. It was found that a starting TBA concentration of 1 × 10−6 m was appropriate to ensure a reason-able capture rate (RC = 1.53 ± 0.25 event s−1, VDS = 500 mV) while avoiding spontaneous pore blocking (Figure 3d). The VDS dependence was similar to what would be expected with conven-tional transport through a nanopore, i.e., at higher bias voltage the capture rate increases, dwell time decreases, and peak ampli-tude increases (Figure S15, Supporting Information).[11,45,46] To further confirm the sensor is selective only to thrombin, we used an IgG antibody, which cannot bind to TBA, as a control. Compared to thrombin translocation (τD = 0.96 ± 0.61 ms, Ipeak = 32.32 ± 12.43 pA), the antibody unbound signal is sharp and short (τD = 0.40 ± 0.21 ms, Ipeak = 58.39 ± 19.09 pA), indicating there are no specific interactions between the TBA and the antibody (Figure S16, Supporting Information).

2.3. Protein Translocation Controlled by Gating

The rate of transport, along with peak amplitude and signal to noise could be tuned by biasing the gate electrode, VG For instance, a VG = −400 mV results in only sporadic events (RC = 0.04 ± 0.01 event s−1, VDS = 500 mV), Figure 4. A likely explanation is that a negatively charged pore prevents the negatively charged proteins from passing through; the protein passes through without binding to the aptamer, leading to fast translocation.[24] Thrombin has a slight negative charge under the experimental conditions used (pH 8) and was repelled from the nanopore surface under negative gate voltage.




A positive gate voltage helps attract the thrombin towards the TBA and facilitates binding resulting in overall higher cap-ture rate, Figure 4. A VG = 400 mV resulted in a 54-fold increase in capture rate, 4.1-fold increase in the mean peak current, and 3.9-fold increase in the signal-to-noise ratio, compared to VG = −400 mV. This was attributed to two contributions 1) the altered pore charge and hence, the increased likeliness of binding between aptamers and proteins, and 2) enhanced capture of the analyte due to electroosmotic flow. When the gate bias is sufficiently positive, the electroosmotic flow has the same direction as the electrophoretic force (from the bath to the inside of the pipette), facilitating analyte transport. Hence, it was possible to detect thrombin at concentrations as low as

50 × 10−12 m (Figure S17, Supporting Information). Importantly, it is possible to detect analytes at a concentration well below the dissociation constant and can distinguish between bound and unbound states with single-molecule resolution.

Apart from the capture rate, another key characteristic of the nexFET was an improvement is the signal-to-noise ratio, Figure 4f. This was in large part due to the PPy sur-face conductivity and its voltage-induced conformational change.[47,48] Under the experimental buffer conditions, adja-cent PPy polymer chains tend to repel each other as they carry a moderately positive charge. As a result, the structure tends to swell and expand, which has also been observed in other elastic polymers such as poly(N-isopropylacrylamide).[49]

Figure 3. Single-molecule detection of human α-thrombin using a) bare double-barrel nanopipette, b) unmodified nexFET, c) nexFET functionalized with 100 × 10−9 m TBA, d) nexFET functionalized with 1 × 10−6 m TBA, e) nexFET functionalized with 10 × 10−6 m TBA. i) Schematic of the protein trans-location process. The protein stays longer inside the nanopore when bound to the aptamer. ii) I–V characteristics of the nanopores in 100 × 10−3 m KCl, 1 × 10−3 m Tris–EDTA, pH 8. iii) 5 s representative translocation recordings of 1 × 10−9 m thrombin in 100 × 10−3 m KCl, 1 × 10−3 m Tris–EDTA, pH 8, under a bias of VDS = 500 mV. iv) Normalized all points histogram of the current–time traces shown in (iii), the gray peaks represent the distribu-tion for the open pore current while the green peaks show the distributions for the TBA–thrombin bound states (scale bar: horizontal: 0.5 s; vertical: 20 pA). Typical individual events are shown in Figure S13 in the Supporting Information.




Application of a negative gate voltage allows for the polymer to become less positively charged, thus decreasing repul-sion between polymer chains and leading to a larger effec-tive nanopore diameter and hence, lower signal-to-noise ratio (Figure S18a, Supporting Information). On the other hand, if a positive gate voltage is applied, then the struc-ture will swell, and the nanopore will have a smaller effec-tive diameter, resulting in a higher signal-to-noise ratio (Figure S18b, Supporting Information). This improvement is particularly useful as many of the proteins are too small to be detected. Interestingly, the dwell time of the protein translo-cation is not significantly affected by the gate voltage. This is attributed to interactions between the analyte and the nano-pore, that do not originate from electrostatic forces but from aptamer–protein specific interactions. Therefore, it is likely that the dwell times (Figure S19, Supporting Information) are

dependent on the number of binding sites rather than the gate bias as can be seen the current time traces in Figure 3.

3. Conclusion

In this work, we demonstrate that some of the challenges in nanopore protein sensing, that arise due to fast translocation speeds, low capture efficiency and recognition specificity[50] can be addressed when solid-state nanopores are integrated with a FET and functionalized with DNA aptamers. In par-ticular, we demonstrate that the aptamer modified nexFET can selectively detect thrombin at a low concentration with improved performance. This is due to a combination of factors including nonspecific electrostatic interactions between the nanopore sur-face and the analyte, specific aptamer–protein interactions, and

Figure 4. Gate voltage dependence on thrombin translocation through the TBA modified nexFET. a) Representative 60 s current–time traces of 1 × 10−9 m thrombin using a TBA functionalized nexFET (1 × 10−6 m) using a drain–source voltage VDS = 500 mV and a gate voltage VG ranging from −400 to 400 mV in 100 × 10−3 m KCl, 1 × 10−3 m Tris–EDTA, pH 8 (top to bottom), scale bar: horizontal: 5 s; vertical: 20 pA. b) The Nanopore I–V curves at different gate voltages. c) Scatter plots of dwell time versus peak current are shown and based on 180 s recordings. For a given voltage, the d) capture rate, e) mean peak current, and f) the signal-to-noise ratio were calculated. The histogram shows the change in percentile, compared to the value in VG = 0 mV. Error bars represent the standard deviation of three independent experimental repeats.




potential differences generated at the gating nanoelectrode. The platform has a universal design, and the sensing capability can be expanded further to selectively detect a broad range of ana-lytes for which aptamers are available.

4. Experimental SectionFabrication and Characterization of Double-Barrel Nanopipettes: The

double-barrel capillaries were plasma cleaned followed by careful loading into a laser-based pipette puller (Sutter Instrument, P-2000). Capillaries were heated and pulled to generate a pair of double barrel nanopipettes, as illustrated in Figure 2a(ii). The parameters of the one-step protocol are HEAT 700 FIL 3 VEL 45 DEL 130 PULL 93. It should be noted that the parameters of the pulling protocols are instrument specific and sensitive to both the temperature and humidity of the room.

Fabrication of Carbon-Coated Double-Barrel Nanopipettes with One Carbon Nanoelectrode: The carbon pyrolysis strategy was used, which was previously published.[32,40] Specifically, to deposit carbon only in one barrel, a plug was used to seal off the second barrel. Then, butane/propane was filled in the barrel and heated with a butane torch under a convective argon flow, which prevented the deposited carbon from being oxidized by the air under high temperatures. Within the inert atmosphere and high temperature, butane/propane can be pyrolyzed to form a conductive solid layer made up of carbon. To coat the whole cross-section of the nanopore rather than only one barrel, it is essential to heat location 1 for at least 15 s initially and then gradually move closer to the tip area (locations 2 and 3), as illustrated in Figure 2b(ii). The entire fabrication process took ≈60 s.

After fabrication, cyclic voltammetry (CV) was used to characterize the approximate pore size in 100 × 10−3 m of KCl (Sigma-Aldrich) before PPy polymerization. The area of the carbon nanoelectrode was examined by SLV using the redox mediator ferrocenemethanol (Sigma-Aldrich) in 100 × 10−3 m KCl.

PPy Predeposition with Real-Time Feedback Control: Pyrrole (Py, Sigma-Aldrich) was stored under an inert atmosphere, and the quality was always checked before use. The color of the liquid should be transparent otherwise, the pyrrole needs to be purified by column chromatography (using Al2O3). PPy was electropolymerized on the conductive surface of the carbon-coated dual-barrel nanopipettes by applying a 600 mV bias (this was determined by the voltage of the oxidation peak on the CV scan from −800 to 800 mV) on the carbon nanoelectrode using a solution of 500 × 10−3 m Py, 200 × 10−3 m of lithium perchlorate (LiClO4, Sigma-Aldrich), and 100 × 10−3 m of perchloric acid (HClO4, Sigma-Aldrich) in DI water. At the same time, the open barrel was filled with 100 × 10−3 m of KCl, and a small bias (100 mV) was applied to monitor the open pore current (Figure 2c(ii)). Real-time feedback control is crucial as the conductivity of the nanopore is tightly linked to its size. After PPy polymerization, the nexFET was stabilized in 100 × 10−3 m HCl. A CV was performed between −300 and 300 mV until the current stabilized.

To form the functional layer, a further deposition was conducted using a Py/aptamer solution. The same feedback control deposition conditions were used as described above.

Ionic Currents and Translocation Measurement: I–V characteristics, FET behavior, and single-molecule sensing were measured using a Multiclamp 700B amplifier and 1550b digitizer (Molecular Devices). Patch A Ag/AgCl electrode was used as the patch electrode to apply the drain-source voltage. The ground/reference electrode was also Ag/AgCl, and it was placed into the bath. A silver wire was incorporated into the carbon and was used to bias the PPy.

All characterization experiments were performed in Tris–EDTA pH-buffered 100 × 10−3 m KCl. There are two reasons for choosing this salt concentration: 1) The optimal condition of the nexFET performance was previously validated under different ionic strength conditions. It is clear that at 1 m salt, FET performance is reduced due to the shorter Debye screening length;[32] 2) Furthermore, the intention is to ultimately

use such a platform for practical use. One advantage of biosensors based on nanopipette is its geometry that can be used in vivo and living cell detection. Moreover, the highly demanding analytics and diagnosis require sensors to work well in real samples. Therefore, a condition (100 × 10−3 m KCl, pH 8.0) that was close to the physiological condition was chosen to perform measurements. In the future, using this sensor would be tried to detect targets in real samples and even living cells.

The data was recorded by pClamp software (Molecular Devices) and analyzed using the Nanopore App written by Joshua Edel.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

AcknowledgementsY.K., A.P.I., J.B.E. acknowledge support from EPSRC grant EP/P011985/1. A.P.I. and J.B.E acknowledge support from BBSRC grant BB/R022429/1 and Analytical Chemistry Trust Fund grant 600322/05. Y.Z. and Y.K. acknowledge support from the World Premier International Research Center Initiative (WPI), MEXT, Japan. Y.Z. acknowledges support from the National Natural Science Foundation of China (grant no. 31870990). J.B.E. received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 724300, NanoPD and 875525, NanoPD_P).

Conflict of InterestThe authors declare no conflict of interest.

Keywordsaptamers, ionic field-effect transistors, nanopores, single-molecule protein sensing

Received: May 12, 2020Revised: June 18, 2020

Published online: July 16, 2020

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