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Nanochannel-Ionchannel Hybrid Device for Ultrasensitive Monitoring of Biomolecular Recognition Events Xiao-Ping Zhao, Yue Zhou, Qian-Wen Zhang, Dong-Rui Yang, Chen Wang, and Xing-Hua Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05162 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 11, 2018

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Analytical Chemistry

Nanochannel-Ionchannel Hybrid Device for Ultrasensitive Monitoring of Biomolecular Recognition Events Xiao-Ping Zhao,†‡ Yue Zhou,† Qian-Wen Zhang,† Dong-Rui Yang,† Chen Wang,*‡ Xing-Hua Xia*†

†State

Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and

Chemical Engineering, Nanjing University, Nanjing 210023, China ‡Key

Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education; Key

Laboratory of Biomedical Functional Materials, School of Science, China Pharmaceutical University, Nanjing, 211198, China *E-mail

address: [email protected]; [email protected]

ABSTRACT ： We propose an in-situ and label-free method for detection of biomolecular recognition event using nanochannel-ionchannel hybrid device integrated with an electrochemical detector. The aptamer is first immobilized on the outer surface of the nanochannel-ionchannel hybrid. Its binding with target thrombin in solution considerably regulates the mass transfer behavior of the device owing to the varied surface charge density and effective channel size. Using the electrochemical detector, the changed mass transport property can be in real-time monitored, which enables in-situ and label-free detection of thrombin-aptamer recognition. The solution pH has a significant influence on the detection sensitivity. Under optimal pH condition, the detection limit as low as 0.22 fM thrombin can be achieved, which is much lower than most reported work. The present nanofluidic device provides a simple, ultrasensitive and label-free platform for monitoring the biomolecular recognition events, which would hold great potential in exploring the biomolecules functions and reaction mechanism in the living systems. KEYWORDS: biomolecular recognition, nanochannel-ionchannel hybrid, electrochemical detector, nanofluidic, thrombin

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INTRODUCTION Biomolecular recognition between receptors and their cognate ligands participates in virtually every process within organisms.1 Typically biomolecular recognition processes include enzyme catalysis, cellular signalling, protein-protein interaction, DNA-DNA hybridization, and even targets transport in living system. Therefore, detection of these biomolecular recognition events is of critical importance in exploring the biomolecule function and reaction mechanism. Up to now, many methods have been developed for the detection of biomolecular recognition, including colorimetry,2 fluorescence,3 electrochemical assay,4-6 surface plasmon resonance (SPR),7-9 and surface-enhanced Raman spectroscopy (SERS).10 In recent years, some novel and sensitive methods have been developed for biomolecular recognition study. As an example, a photoelectrochemical (PEC) strategy was proposed for antibody-antigen recognition assay.4 Using the near-infrared light-excited upconversion nanospheres, ultrasensitive detection of antibody-antigen binding was successfully achieved with a detection limit of 5.3 pg/mL target protein. In another report, an electrochemiluminescence (ECL) assay was used for studying the tryptase-DNA recognition event.6 The detection limit for target protein as low as 0.81 pg/mL could be achieved. Although these reported approaches are of vital importance in pushing forward the study of biomolecular recognition events, they need prominent quantities of samples, hampering their real application in practical diagnosis, especially when the target amount is rare and limited. As an alternative platform, nanofluidics based on nanopore/channel has found extensive applications in chemical and biomedical fields.11-14 Among the particular attributes, a unique asymmetric mass transfer property can be achieved by symmetry breaking in the charge distribution or channel geometry of the nanofluidic devices,15-18 which we call as ionic current rectification (ICR). Based on the asymmetric mass transfer, Jiang’s group developed a series of biomimetic smart nanofluidic devices which could be activated by temperature,19 light,20 ion concentration,21 and pH.22 Due to the sensitive response to the nanochannel surface property, the mass transfer characteristics can be used in many fields, such as biomolecular recognition events,23-26 ion detection,27 energy storage and conversion.13 As a classic example, the change in the ICR properties of an asymmetric nanopore has been used for precisely quantification of protein.23 By investigating ICR change through the synthetic conical


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nanopore, the DNA-peptide nucleic acid recognition was systematically invsetigated.24 These proposed methods open new avenues in developing highly sensitive biosensors via the ICR property. Especially, in 2013, Szleifer’s group observed that ICR with high current rectification factor could occur in cylindrical nanochannels with chemically modified outer membrane and neutral inner wall.28 It has been confirmed by a recent work that described the importance of the outer surface of nanochannels in regulating the ion gating.29 These interesting observations would bring new opportunities for application of nanofluidic platform in biosensing and analysis. Porous anodic alumina (PAA) membrane fabricated by electrochemical method has attracted tremendous scientific interest owing to the perfect chemical or mechanical stability and high pore densities, which has been widely used in construction of (bio)chemical assays.30-36 In the applications, the barrier layers formed at the end of PAA membrane need to be removed, and thus the formed nanochannels are symmetric. As a matter of fact, the barrier layer is composed of numerous ionchannels which allow ions transfer during the electrochemical fabrication of PAA.37-39 We pioneered to utilize the ionchannels integrated with the nanochannels forming novel nanochannel-ionchannel hybrid membrane device for Cu2+ detection.40 However, in this previous work, the probe for Cu2+ recognition was immobilized on the inner walls of the nanochannels. The phenomenon of channel blockage induced by biomolecules probably takes place during experiment process. In comparison, if the immobilization of probers and affinity recognition events can be carried out on the outer surface of PAA, the blockage and damage of channel can be effectively avoided, and the experimental operation will also be significantly simplified. More importantly, such unique nanochannel-ionchannel hybrid membrane device possesses high detection sensitivity benefited from high rectification ratio owing to the existence of extremely asymmetric geometry and asymmetric charge distribution. The varied charge property by biomolecular recognition on the outer surface of the hybrid will also considerately influence the mass transfer characteristics,28,29 which allows for real-time monitoring the biomolecular reaction kinetics. Herein, we use the nanochannel-ionchannel hybrid membrane device for sensitive and label-free studying the biomolecular recognition event using thrombin (Thr)-aptamer reaction



as the demonstration. Thrombin aptamer (TBA) is immobilized on the outer surface of the ionchannel through covalent bonding. In the presence of thrombin ion solution, the biomolecular recognition reaction occurs (Scheme 1A). The biomolecular binding process leads to varied charge density on outer surface, resulting in changed mass transfer property of the device. In addition, the bounded thrombin will block the ions flowing through the nanochannel-ionchannel hybrid device. Both the effects of surface charge and steric hindrance caused by molecular recognition event influence the mass transfer property, which is monitored in real-time using an electrochemical detector (Scheme 1B, C). Especially, we find that the detection sensitivity is considerably regulated by solution pH. Under the optimal conditions, ultralow concentration of thrombin as low as 0.22 fM can be successfully detected using the present device. The present strategy provides an ultrasensitive, label-free, and in-situ detection technique for monitoring the biomolecular recognition events, which would hold great potential in exploring the biomolecule function and corresponding reaction kinetics.

Scheme 1. (A) Illustration of the biomolecular recognition detection principle. (B) Schematic diagram of the setup for I-V measurement. (C) I-V curves of PAA under different conditions


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with a scan rate of 100 mV/s. Pink curve: pure PAA; Red curve: amino-group modified PAA; Green curve: aptamer-modified PAA; Blue curve: thrombin-recognized PAA.

EXPERIMENTAL SECTION Materials and Reagents. Human thrombin (Thr, ≥2000NIH), bovine serum albumin (BSA, ≥98%), ascorbic acid (AA), 6-mercapto-1-hexanol (MCH) and L-cysteine (L-Cys, ≥97%) were purchased from Sigma-Aldrich (Shanghai, China). Thrombin aptamer (TBA: COOH-5’ -GGTTGGTGTGGTTGG-3’; NH2-5’-GGTTGGTGTGGTTGG-3’) was from Shanghai Sangon

Biotechnology

company

(Shanghai,

China).

Glucose

(Glu),

3-aminopropyltrimethoxy-silane (APTMS), phosphocromic acid and tin (П) chloride were from

Sinopharm

chemical

reagent

company

(Shanghai,

1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC,

China).

≥ 98%) and

N-Hydroxy succinimide (NHS, 98%) were from Sigma Aldrich (Shanghai, China). Oxalic acid, chromic acid and potassium chloride were purchased from Shanghai Lingfeng chemical reagents company. Hydrogen peroxide (30%) was from Nanjing chemical reagent company. All reagents were of analytical grade and used as received. All aqueous solutions were prepared using deionized water (18 MΩ·cm, PURELAB Classic, PALL, USA), and were filtered with a 0.22 µm syringe filter before use. Instrumentation. The morphology of the fabricated nanochannel-ionchannel hybrid device was imaged using a scanning electron microscope (SEM) (S-4800, Japan). X-ray photo-electron spectroscopy (XPS) (K-Alpha, Thermo Fisher Scientific, USA) was used to characterize the APTMS and TBA modified ionchannel-nanochannel hybrid device. Fourier-transform infrared (FTIR) spectroscopy was performed on a Nicolet 6700 model 912A0637. The electrochemical detection was conducted on an electrochemical workstation (CHI660E, Chenhua, China) with two Ag/AgCL electrodes as the anode and cathode, respectively. Fabrication of the Nanochannel-Ionchannel Hybrid Device. The nanochannel-ionchannel hybrid device was fabricated using a two-step aluminum oxidation process.40 First, a clean



aluminum sheet was anodically oxidized in 0.3 M oxalic acid for 30 min at 25 °C and 40 V voltage, forming an alumina membrane. This preformed alumina membrane was then removed by a mixture of phosphocromic acid (5% wt) and chromic acid (1.5% wt) solution for 40 min at 60 °C, and subsequently formed a textured pattern of concave substrate. In the following step, this concave substrate was used for second anodic oxidation process, during which, a PAA with well-ordered pores formed in 0.3 M oxalic acid for 4 h with the same temperature and voltage as the first oxidized step. Afterwards, the aluminum substrate was removed by Tin (П) chloride. Modification of the Nanochannel-Ionchannel Hybrid Device. The fabricated hybrid device was functionalized with TBA as illustrated below. First, the hybrid device was hydroxylated in boiled hydrogen peroxide (30% H2O2) for 0.5 h at 98-100 °C to generate abundant -OH groups. Then, 10 % of APTMS (180 μL APTMS diluted in 1620 μL acetone) was added into the ionchannel side overnight and then rinsed with deionized water three times. After that the APTMS-grafted hybrid device was heated at 120 °C for 1h to crosslink the silane layer. Finally, 800 μL of 0.9 mg/mL EDC and 200 μL of 1 μM TBA was added to the ionchannel side, incubating for 10 min. After that 800 μL of 0.8 mg/mL NHS was added into the mixed solution for 6 h at 4 °C and followed by complete rinsing by 10 mM PBS buffer solution. The functionalized hybrid device with aptamer immobilized on the side of ionchannels was thus obtained. Biomolecular Recognition Assay. The hybrid device was clamped between two silica gel films and then placed between two 2 mL home-made half cells for electrochemical detection. First, 1800 μL 1 mM different pH KCL solution was filled into the two half cells instead of thrombin solution. Then, 20 μL different concentrations of thrombin solutions (1 fM-11.111 nM thrombin final concentration) were added into the ionchannel side for 50 min incubating, allowing diffusion of free thrombin binding to TBA on the ionchannel side at room temperature. The recognition kinetics were investigated using 11.111 nM thrombin. Linear sweep voltammetric experiments from -0.8 V to +0.8 V with a scan rate 100 mV/s were carried out using CHI 660E (Chenhua, China) with two Ag/AgCL electrodes as the anode and cathode respectively. Electrochemical Impedance Spectroscopy (EIS) Analysis. To achieve the EIS analysis, a


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gold electrode was used for simplicity. First, the electrode was immersed in H2SO4 and 30% H2O2 mixture solution with the volume ratio 3:1 for 5 min to remove the adsorbed organics, and then was rinsed with deionized water. After that, the electrode was polished by 0.3 micron alumina powder, followed by sonicated three times in ethanol and water, and then dried with nitrogen gas. For electrode modification, 100 μL NH2-TBA (1.0 μM) was dropped on the gold electrode for 2 h, then 20 μL 1.0 nM of MCH was added on the electrode surface for 1 h to block the uncovered gold surface. After the modified electrode was washed with 10 mM PBS buffer solution, 100 μL of thrombin (100 nM) was added to the TBA-modified electrode surface for protein-aptamer interaction at room temperature for 50 min. After washing with 10 mM PBS buffer solution, the electrode was ready for measurement. EIS was performed in 0.1 M different pH KCL solution containing 5.0 mM [Fe(CN)6]3−/4− and the frequency range from 1 to 106 Hz at open circuit potential (0.242V in the present work). Analysis of Standard Spiked Human Plasma Sample. Human serum was diluted 1000 times by HBS-EP and then different concentrations of thrombin (0.111 nM, 1.111 nM, 11.111 nM) were added into the diluted serum. Before assay, the diluted human serum samples were added onto the TBA-modified nanochannel-ionchannel hybrid device for 50 min. The electrochemical measurement was then conducted after biomolecular recognition.

RESULTS AND DISCUSSION Characterization of the Nanochannel-Ionchannel Hybrid Membrane Device. The morphology of the prepared nanochannel-ionchannel hybrid membrane device was characterized using a SEM (Figure 1). As shown in Figure 1A and B, the barrier layer attached to the PAA shows the regular hexagonal structure with a thickness of ~100 nm. Figure 1C and D respectively shows the images of cross section and bottom of PAA. The average diameter of the array nanochannels is ~40 nm. The ionchannels existing in the hexagonal units could not be observed by SEM image owing to its super small size. XPS was used to characterize the PAA modification process. The intrinsic nanochannel-ionchannel hybrid membrane (red curves in Figure 1E and F) does not display the signals for Si 2p and P 2p. Mobilization of APTMS and TBA onto the outer surface of ionchannel results in appearance of an obvious Si 2p (103.5 eV) peak (Figure 1E) and P 2p



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peak (134.0 eV, Figure 1F). To ensure the successful chemical bonding between APTMS and the aptamer probe, FTIR was also used. The result was shown in Figure S1. The absorption of amide I (1690 cm-1) and amide II (1547 cm-1) respectively corresponding to the C=O and the C-N stretching were clearly observed, confirming the successful bonding between carboxylic groups in aptamer and amine groups in APTMS. The above results indicate the successful immobilization of APTMS and aptamer on the outer surface of the nanochannel-ionchannel hybrid device.

Figure 1. SEM images of the nanochannel-ionchannel hybrid membrane device. A: the barrier layer. B: the cross section of nanochannel-ionchannel hybrid. C: the cross section of the nanochannel layer. D: the porous layer. (E) XPS spectra of the pure PAA (red curve) and APTMS modified PAA (black curve). (F) XPS spectra of the pure PAA (red curve) and TBA modified PAA (black curve).

The current-potential (I-V) properties of the nanochannel-ionchannel hybrid before and after TBA modification were measured in the absence or presence of thrombin. Since the isoelectric point of pure PAA is ~6.5,37 the nanochannel-ionchannel hybrid membrane charges negatively

at

pH

7.4.

As

shown

in

Figure

2A

(pink

curve),

the

intrinsic

nanochannel-ionchannel hybrid device presents a fine rectified I-V curve in 1 mM KCL at pH 7.4 owing to its negative charge and extreme asymmetric structure similar to the cone-shaped structure.41 With APTMS immobilized on the ionchannel side, the charge on the outer surface


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of hybrid turns from negative to positive owing to the positively charged amino groups in APTMS, while the inner walls in the nanochannel walls keep negatively charged as before. The asymmetric surface charge distribution along with the extremely asymmetric geometry makes the ionchannel-nanochannel hybrid device acting as a bipolar nanofluidic diode, in which the asymmetric charge distribution dominates the direction of ICR.23 The different ICR directions in the pink curve and red curve further confirm the successful immobilization of APTMS. After covalently bonding with TBA, the ionchannel side is covered by a layer of negatively charged DNA probe (TBA). The direction of ICR turns reversed again (green curve in Figure 2A). Notably, the current decreases considerably compared to the pure PAA (pink curve), which could be explained by the blocking effect of the immobilized TBA on the ionic flow through the hybrid device. For the same reason, the ionic current decreases further upon binding with thrombin (blue curve in Figure 2A). The results in Figure 2A well confirm the successfully immobilization of TBA probe and the thrombin-aptamer affinity recognition on the outer surface of hybrid device. The biomolecular recognition can also be reflected by the changed ICR ratio (fR=I+0.8/I-0.8, where I+0.8 is the magnitude of ionic current recorded at +0.8 V, I-0.8 is the magnitude of ionic current recorded at -0.8 V). As displayed in Figure 2B, the fR values (calculated from Figure 2A) are respectively 11.4, 0.14, 15.5, 5.7 for the pure, APTMS-modified, TBA-immobilized and thrombin-recognized hybrid devices. A super small fR value is observed for the APTMS-modified device due to the reversed charge property after APTMS modification as that have been described above. Since the isoelectric point (pI) of thrombin is 7.0-7.4,42 the outer surface of hybrid membrane carries almost zero charge at pH 7.4. The uncharged thrombin layer plays dominant role in determining the ionic current, thus the ICR ratio decreases in the case of thrombin-recognized hybrid device compared to the TBA-immobilized device. The results in Figure 2 verify the successful modification processes as well as the thrombin-aptamer recognition.



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Figure 2. (A) I-V profiles of pure (pink curve) and modified hybrid device (different modification processes. APTMS: red curve, aptamer: green curve, thrombin: blue curve). (B) ICR ratio fR of the nanochannel-ionchannel hybrid device corresponding to the different modification processes (data adapted from Figure 2A).

Thrombin-TBA Recognition. The biomolecular recognition temperature was fixed at room temperature. The kinetics of biomolecular recognition was first investigated at pH 7.4. The measured I-V profiles are shown in Figure 3A and the corresponding current values at + 0.8 V was displayed in Figure 3B. When thrombin (11.111 nM) interacts with the aptamer-modified device, the ionic current decreases continuously with the reaction time, then reaches a stable state after 50 min. Therefore, in the following experiments, the optimal recognition time is chosen as 50 min. To investigate the applicability of the proposed method for quantifying biomolecule, the responses of the aptamer modified device to different concentrations of thrombin were studied using the electrochemical detection technique. As shown in Figure 3C, with increasing the thrombin concentration (e.g., from 10-6 nM to 11.111 nM), the current at +0.8 V obviously decreases with the increase of the concentration ranging from 1.0 pM to 11.111 nM. However, the change becomes relatively slight when further decreasing the thrombin concentration (form 1.0 pM to 1.0 fM). By plotting the currents (y) at +0.8 V vs. the logarithm of thrombin concentration (Log C), a calibration curve is obtained (Figure 3D). The linear part is y = 6.97 x 10-9-4.2 x 10-9 Log C (R=0.999). The detection limit is estimated to be 20 fM based on a 3 SD/L method (SD is the standard deviation of the blank, L is the slope of the calibration curve). To clearly show the ICR change during the biomolecular recognition, the


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corresponding rectification ratio (fR) was shown in the inset in Figure 3D. The fR nearly remains the same in the range of 1 fM-100 fM thrombin. From 100 fM to the same in the range of 1 fM-100 fM thrombin. From 100 fM to 11.111 nM, the fR decreases with the concentration due to the obvious amount of thrombin bound onto the hybrid device.

Figure 3. (A) I-V profiles monitoring the reaction kinetics of thrombin-TBA recognition from within 5-60 min for 5 min interval. The concentration of thrombin was 11.111 nM. (B) Current at +0.8 V from Panel A versus as a function of reaction time. (C) I-V profiles of the TBA modified nanochannel-ionchannel hybrid device for different concentrations of thrombin from 10-6 nM to 11.111 nM at pH 7.4. (D) Calibration plot (the current at + 0.8 V from Panel C versus Log C, C denotes the thrombin concentration) of the proposed hybrid device. The inset is the fR value of the nanochannel-ionchannel hybrid device with different thrombin concentrations at pH 7.4.

Effect of pH on Biomolecular Detection. Solution pH is always related to the protonation and activity of thrombin and exerts significant impact on the surface charge density. To test



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the pH-tunable performance of the present nanodevice, the experiments were conducted at pH 9.0 and pH 6.3 in addition to pH 7.4 (the stability of the thrombin-aptamer complex at different pH was verified by the electrochemical impedance spectroscopy (EIS) measurement (Figure S2 in the supporting information). The reaction kinetics was investigated (Figure S3 and Figure S4), showing that the reaction can be completed within 50 min in both pH environments. Then, the I-V curves of the nanochannel-ionchannel hybrid device were recorded after addition of a gradient concentration of thrombin from 10-6 nM to 11.111 nM at different pH values for 50 min. Figure 4A shows the I-V curves of thrombin-aptamer recognition at pH 9.0. The corresponding curve of ionic current versus Log C is displayed in Figure 4B. Figure 4C and D show the result at pH 6.3. The detection limits are respectively calculated as 5.0 fM, 20 fM, 0.22 fM at pH 6.3, 7.4, 9.0 using the method of 3 SD/L described above. A comparison of the performance for thrombin sensing using various methods is listed in Table S1. Compared with other methods, the sensitivity of the nanochannel-ionchannel hybrid device can be improved by adjusting pH environment without any signal amplification strategy. The biomolecular recognition between thrombin and aptamer has been studied within nanochannels of PAA in our previous reports.32 The sensitivity of 1.0 pM was achieved. Compared to the method within nanochannels, the detection sensitivity could be considerably increased using the present method.


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Figrue 4. (A) I-V profiles of the TBA modified nanochannel-ionchannel hybrid device for different concentrations of thrombin from 10-6 nM to 11.111 nM at pH 9.0. (B) The calibration plot (the current at +0.8 V from Panel A versus the Log C) of the proposed nanodevices. (C) I-V profiles of the TBA modified nanochannel-ionchannel hybrid device for different concentrations of thrombin from 10-6 nM to 11.111 nM at pH 6.3. (D) Calibration plot (the current at -0.8 V from Panel C versus Log C) of the proposed nanodevices.

From Figure 3 and Figure 4, we can see that the detection sensitivity varies with solution pH. The highest sensitivity is achieved at pH 9.0, while in pH 7.4 the detection sensitivity is lowest. This phenomenon can be explained by the different mass transfer properties under different pH conditions. The mass transfer behaviour in nanofluidics is dependent of two factors: charge effect and size effect.17 It has been reported that the change in charge density may have more sensitive response toward the same quantity of biomolecules detection as compared to the size effect.23 In the present case, the isoelectric point of thrombin is ~7.0-7.4. One could expect that different pH could induce different outer surface charge density on the device surface, and thus pH-dependent ionic current responses are varied accordingly. When



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the solution is equal to the pI, thrombin is neutral, efficiently shielding the outer surface charge of the ionchannels. Under this circumstance, the mass transfer property is mainly determined by the size effect. After binding with the thrombin, the formed thrombin-aptamer considerably covers the ionchannel side, blocking the mass transfer through the nanochnnel-ionchannel hybrid device. Therefore, a decreased current and ICR could be observed (Figure 2). When pH > pI, thrombin will be negatively charged. The thrombin-aptamer binding enables the further increase in the charge density on the device surface, resulting in more obvious asymmetric charge distribution along the channels. This will lead to an increase in the migration of cations flowing preferentially from the outer surface to the inner channels. Again, the charge effect predominates the mass transfer behaviour of the device as compared to the size effect.23 Thus, increased changes in ionic current and ICR are observed upon thrombin-aptamer recognition in alkaline medium as compared to the neutral condition (Figure 4A and Figure S5). The enhanced current response results in an increased detection sensitivity at pH 9.0 as compared to pH 7.4. When pH < pI, the charge of thrombin is switched from negative to positive, the system changes from cation to anion-selective. Consequently, the rectification of the ionic current becomes reverted as well. The corresponding I-V curve has characteristics opposite to that measured at pH 9.0 (Figure 4C). However, the thrombin-aptamer complex is relative less stable in acid medium than in alkaline medium,43 leading to slight decrease in detection sensitivity at pH 6.3. Therefore, the highest detection sensitivity for thrombin-aptamer recognition can be achieved at pH 9.0. The mechanism of the pH-tunable detection sensitivity is schematically illustrated in Figure 5A. The corresponding current changes after recognition with thrombin from 10-6 nM to 11.111 nM are shown in Figure 5B (18.7 nA at pH 7.4; 22.0 nA at pH 6.3; 253.0 nA at pH 9.0), clearly showing the pH-tunable detection sensitivity on the present nanofluidic device.


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Figure 5. (A) Mechanism of the thrombin-functionalized nanochaneel-ionchannel hybrid device with breaking symmetry under different solution pH (pink spheres represent the anions; green spheres represent the cations). (B) I-V profiles depending on pH of the solution. Different surface charge patterns are established on the outer surface of the hybrid (the schematics in the upper part of the figure), which leads to the different I-V curves with thrombin from 10-6 nM to 11.111 nM (Black curves: thrombin concentration at 10-6 nM; Red curves: thrombin concentration at 11.111 nM).

Specificity and Reproducibility of the Present Biosensor. A vital issue is the specificity of the constructed strategy. It is well known that the aptamers are artificial single-strand DNA or RNA sequences with extremely high affinity and specificity towards their target.44 Thus the aptamer was used for special binding with thrombin. For comparison, the I-V curve of the aptamer-modified nanochannel-ionchannel hybrid was used as the blank (black curve in Figure 6A). The same experiments were conducted using ascorbic acid (AA: 100 μM), bovine serum albumin (BSA, 100 μM), L-cysteine (L-Cys, 10 mM) and glucose (Glu, 10 mM) instead of thrombin (10 fM) at pH 9. The different biomolecular recognitions occurring on the ionchannel side was monitored by ionic current (Figure 6A). Upon exposure of the modified nanochannel-ionchannel hybrid device to AA, BSA, L-Cys, Glu solution, only slight changes in the ionic current are observed, which nearly overlapped with each other (the enlarged part



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refers to the inset of Figure 6A). Instead, in the case of thrombin, the current increases remarkably. Figure 6B presents the current at + 0.8 V of the device after addition of AA, BSA, L-Cys, Glu and thrombin. For thrombin, the ionic current shows the largest over the other species. The data indicate that the aptamer-functionalized nanochannel-ionchannel hybrid device can well distinguish thrombin from other species, exhibiting a remarkable specificity. To further evaluate the reproducibility of modified nanochannel-ionchannel hybrid, the experiments were performed on four different hybrid devices. The relative ion current (at + 0.8 V) in the presence of 10 fM thrombin were obtained (Figure S6), which did not change remarkably for the four parallel experiments. The result indicates the good reproducibility of the present method. Thrombin Recognition in Real Sample. In order to further investigate the practical application of the nanochannel-ionchannel hybrid device in real sample detection, we challenged the thrombin-aptamer recognition detection in human serum samples. During the experiment, the target thrombin is spiked into the serum with varied concentrations (0.111 nM, 1.111 nM, 11.111 nM). The results are shown in Figure 6C. It is found that in a range of thrombin concentrations from 0.111 nM to 11.111 nM, this nanochannel-ionchannel hybrid sensor shows good response to thrombin. The ionic current increases generally with the spiked thrombin concentration. The ionic current at + 0.8 V versus thrombin concentration in serum sample and PBS buffer (from Figure 4A) is displayed in Figure 6D. The similar change trends in serum sample and PBS buffer demonstrate that the proposed device performs well in serum samples, showing its potential in the practical applications.


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Figure 6. (A) I-V profiles of selective detection of thrombin (10 fM) in the present method against other control substances of AA, BSA, Glu, L-Cys. The blank is aptamer-modified hybrid. The inset is the enlarged part of the curves from +0.55 to +0.8 V. (B) The current at + 0.8 V of different substances. (C) I-V profiles of different concentrations of thrombin in real human plasma (0.111 nM, 1.111 nM, 11.111 nM). (D) The ionic current at + 0.8 V versus different concentration thrombin in PBS buffer solution and human plasma samples.

CONCLUSION In conclusion, we have presented an ultrasensitive and label-free approach for the detection of biomolecular recognition using nanochannel-ionchannel hybrid device integrated with an electrochemical detector. By monitoring the ionic current and rectification properties of the hybrid device during the recognition processes, the thrombin-aptamer recognition kinetics can be obtained in real-time. More importantly, the detection sensitivity of thrombin-aptamer recognition can be finely-tuned by modulating the surface charge of the nanochannel-ionchannel hybrid using solution pH. Under optimum pH condition, the detection limit can be achieved as low as 0.22 fM, which is much lower than the ones



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reported. Furthermore, this strategy exhibits not only high specificity and reproducibility but also excellent performance in real human plasma samples. Due to the unique mass transfer property of this nanochannl-ionchannel hybrid as well as the super small size of ionchannels, the established hybrid device has great potential in sensitive, label-free and in-situ mornitoring the biomolecular recognition events.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program of China (2017YFA0206500) and the National Natural Science Foundation of China (21874155, 21635004, 21575163). Supporting Information Available: Supporting Information is available free of charge from the Analytical Chemistry home page (http://pubs.acs.org/journal/ancham). REFERENCE (1) Renaud, J. P.; Chung, C.; Danielson, U. H.; Egner, U.; Hennig, M.; Roderick, E. H.; Nar, H. Nat. Rev. Drug. Discov. 2016, 15, 679. (2) Wang, Z. Y.; Zhao, J.; Bao, J. C.; Dai, Z. H. ACS Appl. Mater. Interfaces. 2016, 8, 827-833. (3) Liu, G.; Li, J.; Feng, D. Q.; Zhu, J. J.; Wang, W. Anal. Chem. 2017, 89, 1002-1008. (4) Luo, Z. B.; Zhang, L. J.; Zeng, R. J.; Su, L. S.; Tang, D. P. Anal. Chem. 2018, 90, 9568-9575. (5) Luna, P. D.; Mahshid, S. S.; Das, J.; Luan, B.; Sargent, E. H.; Kelley, S. O.; Zhou, R. Nano. Lett. 2017, 17, 1289-1295. (6) Wu, F. F.; Zhou, Y.; Zhang, H.; Yuan, R.; Chai, Y. Q. Anal. Chem. 2018, 90, 2263-2270. (7) Cohen, L.; Walt, R. D. Chem. Rev. 2018, DOI: 10.1021/acs.chemrev.8b00257


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For TOC Only Illustration of the biomolecular recognition detection principle 271x218mm (150 x 150 DPI)


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