Integrated Solid-State Nanopore Electrochemistry Array for Sensitive

Subscriber access provided by READING UNIV

Interfaces: Adsorption, Reactions, Films, Forces, Measurement Techniques, Charge Transfer, Electrochemistry, Electrocatalysis, Energy Production and Storage

Integrated Solid-state Nanopore Electrochemistry Array for Sensitive, Specific, and Label-free Biodetection Xinchun Li, Tianchi Zhang, Pengcheng Gao, Benmei Wei, Yongmei Jia, Yong Cheng, Xiaoding Lou, and Fan Xia Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02010 • Publication Date (Web): 21 Aug 2018 Downloaded from http://pubs.acs.org on August 22, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Integrated Solid-state Nanopore Electrochemistry Array for Sensitive, Specific, and Label-free Biodetection Xinchun Li,1,2 Tianchi Zhang,1 Pengcheng Gao,3 Benmei Wei,1 Yongmei Jia,1 Yong Cheng,1 Xiaoding Lou,*,1,3 Fan Xia*,1,3 1

Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and

Chemical Engineering, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, P. R. China. 2

Pharmacuetical Analysis Division, School of Pharmacy, Guangxi Medical University, 22

Shuangyong Road, Nanning 530021, P. R. China. 3

Engineering Research Center of Nano-Geomaterials of Ministry of Education, Faculty of Materials

Science and Chemistry, China University of Geosciences, 388 Lumo Road, Wuhan 430074, P. R. China.

Abstract: Nanopore ionic current measurement is currently a prevailing readout and offers considerable opportunities for bioassays. Extending conventional electrochemistry to nanoscale space, albeit noteworthy, remains challenging. Here, we report a versatile electrochemistry array established on a nanofluidic platform by controllably depositing gold layers on the two outer sides of anodic aluminum oxide (AAO) nanopores, leading to form an electrochemical microdevice capable of performing amperometry in a label-free manner. Electroactive species ferricyanide ions passing through gold decorated nanopores act as electrochemical indicator to generate electrolytic current signal. The electroactive species flux that dominates current signal response is closely related to the nanopore permeability. Such well-characteristic electrolytic current-species flux correlation lays a premise for quantitative electrochemical analysis. As a proof-of-concept demonstration, we preliminarily verify the analytical utility by detection of nucleic acid and protein at picomolar concentration levels. Universal surface modification and molecule assembly, specific target recognition and reliable signal output in nanopore enable direct electrochemical detection of biomolecules without the need of cumbersome probe labeling and signal amplification. Keywords: Nanopores; Electrochemistry array; Anodic aluminum oxide; Nanofluidic; Biodetection *

Corresponding authors. E-mail: [email protected], [email protected]; [email protected], [email protected]

1

ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Introduction Nanopore has emerged as a useful tool in chemistry, biology, and material science territory in the past decades,1 and has been an ardent topic of energy storage, protein characterization, gene mutation identification, nucleic acid sequencing, as well as chemical analysis and biosensing applications.2-7 Native protein porins are widely used for biosensors.8-11 In contrast, biomimetic nanopore is also a topic of nanopore technology, which offers considerable possibilities to simulate ion transport process as biological ion channels.12,13 Functionalized solid-state nanopore is commonly fabricated using silicon nitride,14 glass,15 quartz,16 anodic aluminum oxide (AAO),17 and graphene.18 Besides, organic polymers such as polyethylene terephthalate19 and polyimide20 have also been widely used. Among them, porous AAO membrane has gained particular interest due to uniform aperture, tunable pores density, and favorable stability.21 It is well known that signal recording is critical to information conversion and takes responsibility in delineating the sensing event occurring in nanopore. At present, ionic current dominates the readout strategy in nanopores-associated researches.22-29 In this mode, trans-membrane current of charged ions driven by electrophoretic force is recorded, and the amplitude of current variation correlative to channel-blockage state can reveal molecule binding, dissociation and motion. However, such an electrical recording is observed to suffer from practical challenge due to background noise,30,31 which is particularly a case in the solid-state nanopore.32 Therefore, development of novel and reliable signal modes shall appreciably advance this field. For instance, to improve nanopore technology, a variety of signal recordings have been proposed, such as optical methods,33-39 field effect/tunneling modes,40,41 mass spectrometry,42 resistive pulse sensing,43 and electrochemiluminescence detection.44 Electrochemical technique (here referring to voltammetric mode) is very attractive because of its unambiguous merits that include high sensitivity, low cost and miniaturized instrumentation.45 The alliance of electrochemistry with nanopore would thus be feasible to construction of miniaturized analytical devices. Single-nanopore electrochemistry has proven to offer procedural simplification and fueled extensive research enthusiasm;46-48 by contrast, integrated nanopore electrochemistry array, although intriguing, is yet far from its destination. Of paramount importance is that this research platform can offer new insights into nanoscale mass transport and electrochemistry mechanisms49. Muniz et al50 reported a nanochannel-based electrochemical assay for detection of carcinoembryonic antigen; in this work, a screen printed carbon electrode was in roughly contacted with the solid-state nanochannel. Yu and coworkers employed the similar strategy to investigation of antigen-antibody reaction kinetics,51 and detection of potassium ion and ATP molecule,52 in which an extrinsic metal disc electrode (gold or platinum) was simply laid underneath the nanochannel to acquire the current

2


Page 2 of 18

Page 3 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

signal. These nanopore-based electrochemistry platforms showed to some extent the feasibility for analytical purpose; however, the electrochemical unit (e.g., electrode system) was actually discrete from the nanopores, thus falling short of flexibility. An elegant work associated with nanopore array-based electrochemical device was presented that adopted in-situ preparation of working electrode at nanopore, while auxiliary electrode and reference electrode were independently placed in an electrochemical measurement cell.53 Later, this method was used for detection of single nucleotide polymorphisms.54 Surely, ongoing endeavors are still needed to move forward the integration and miniaturization for nanopore research. Recently, Liu et al55 designed an all-in-one nanopore battery array through successively sputtering metal and metal oxide layers onto AAO nanopores to function as anode and cathode using atomic layer deposition technique, thereby creating a fully integrated microscale battery array and experimentally verifying the significant improvement of the battery performance. Inspired by this, we present an integrated nanopores-based electrochemistry array using porous AAO membrane. We used electron beam evaporation technique to controllably deposit gold layers onto the both outer surfaces of the AAO nanopores. As a result, the porous AAO nanopores evolved to be nanoelectrochemistry integrity. The developed nanopore electrochemistry array was coupled with a nanofluidic platform to quantitatively detect sequence-specific oligonucleotide through assembly of DNA complex structure and amperometric monitoring of trans-membrane transported electroactive species. Such a label-free electroanalytical strategy was also examined for sensitive and specific insulin assay via aptamer-mediated biorecognition event.

Experimental Chemicals and Reagents All of the reagents were of analytical grade and used as received otherwise specified notification. 3-aminopropyltrimethyloxysilane (APTMS), glutaraldehyde (GA, 50 wt%), and tris(hydroxymethyl) aminomethane (Tris) were obtained from Sinopharm reagent (Shanghai, China). Hydroquinone and potassium ferricyanide were from Aladdin reagent (Shanghai, China). [Ru(NH3)6]Cl3, human insulin, human serum albumin (HSA), hemoglobin，thrombin, immunoglobulin G (IgG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Single-stranded DNA (ssDNA) sequences were commercially obtained from Sangon Biotech (Shanghai, China).

3


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1.Overview of the nanopore electrochemistry setting. (a) Block diagram of the nanofluidic-based AAO nanopore electrochemical measurement device. A, reservoir; B, magnetic stirrer; C, communicating holes; D, electrode pads; E, conducting strip for auxiliary electrode; F, AAO nanopore membrane; G, conducting strip for working electrode; H, Ag/AgCl reference electrode. The horizontal dashed arrow indicated the fluid direction. (b) Schematic representation of the AAO nanopore electrochemistry array. WE, working electrode; AE, auxiliary electrode; RE, reference electrode. (c) Photo of the AAO nanopore membrane deposited with gold. (d) SEM image of the as-prepared gold decorated AAO nanopores. Also shown inset was the pristine AAO nanopores morphology. Accelerating voltage, 10 kV; scale bar, 200 nm. (e) Energy dispersive spectrum (EDS) of gold decorated AAO nanopores surface. (f) EDS revealing the signal variation of the relevant constituents inside the AAO nanopores. The colored arrow indicated gold signal decay from the outer surface to inner of the nanopores. Condition: deposition rate, 0.05 nm s-1; deposition depth, 10 nm. 4


Page 4 of 18

Page 5 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Preparation of Gold Decorated AAO Nanopore Membrane AAO nanopore membrane (60 µm thick and 25 mm in diameter) was obtained commercially (Puyuan Nano, China); the density was about 109-1011 pores/cm-2. An electron beam evaporation system (Angstrom engineering, Canada) was used to prepare AAO nanopore electrochemistry array. Briefly, AAO nanopore membrane was thoroughly rinsed with ethanol and water in order, and baked o

for 1 h at 110 C. Then, a 10 nm-thick Au film was sputtered onto one side, followed by 30 nm-thick Au film deposition on the other side of the AAO membrane at deposition rate of 0.05 nm s-1 to form an electrochemical ensemble containing both working electrode and auxiliary electrode. Note that this process can be readily manipulated by controlling deposition rate and time, giving rise to well-defined nanopores-based electrochemistry array.

Nanopore Characterization and Electrochemical Measurement The morphology and elements characteristics of gold decorated AAO nanopores were characterized by field emission scanning electron microscopy (FESEM, Nova NanoSEM 450, Netherlands) and energy dispersive X-ray spectroscopy (EDS, Oxford, UK); respectively. Prior to use, gold decorated AAO nanopore membranes were immersed in buffer solution for at least 30 min to guarantee complete wetting. Then, the membrane was clamped between a laboratory-built measuring device (Figure 1a). The side of AAO membrane deposited with 10-nm-thick gold layer was exposed to the receiving cell to serve as working electrode while the other side exposed to the feeding cell was used for auxiliary electrode. Electrochemical measurements were carried out on a CHI 630D (Chenhua Instrument, China) analyzer. As for the quantitative analyses, current-time (i-t) mode was employed. Typically, a 1.5 mL of electrolyte solution was loaded to the feeding cell meanwhile the receiving cell was filled with the same buffer. The buffer solution was driven to permeate the AAO nanopores by magnetic stirring. An electrolytic potential was applied to monitor the current response. After the current trace was stable, an appropriate volume of K3[Fe(CN)6] solution was added to the feeding cell; electrochemical reduction would occur when [Fe(CN)6]3- ions pass through the gold decorated nanopores, producing characteristic electrolytic current signal.

Results and Discussion Configuration of the Nanopore Electrochemistry Array As shown in Figure 1a, the nanofluidic electrochemical setting was composed of two chambers (feeding cell and receiving cell) isolated by the AAO nanopore membrane which was fixed and electrically contacted with the conducting pads (Supporting Information). Under magnetic stirring, the electroactive species (ferricyanide ions, [Fe(CN)6]3-) can transport through the AAO nanopores.

5


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Electrochemical reduction of [Fe(CN)6]3- would occur at the gold interface to produce electrolytic current signal at given operating potential. Figure 1b depicted the configuration of the integrated AAO nanopore electrochemistry array. Porous AAO membrane was covered by gold layers on its both sides; meanwhile, gold layer extended to the inner walls of the AAO nanopores, but the inner gold layers were isolated by the AAO base. In this viewpoint, the gold decorated AAO nanopores turned to be an electrochemical device that contained both working electrode and auxiliary electrode, thus improving the functionality of the integrated nanosystem. A picture of gold coating AAO membrane was shown in Figure 1c. Additionally, the SEM image revealed that relatively uniform gold layers covered the membrane surface, forming a well-characteristic electrochemical sensing interface network (Figure 1d). It is worth mentioning that the pores remain to be open after gold deposition, which is necessary for the transport of electroactive species and electrolyte ions. We further used energy dispersive spectroscopy (EDS) to delve the surface constituents of the gold decorated AAO nanopore membrane. As expected, oxygen, aluminum and gold signals can be observed (Figure 1e). Moreover, gold distribution at the inner walls of the nanopores was also revealed (Figure 1f). From the inner to outer surface of the nanopores, the increase of gold signal was accompanied by the decrease of oxygen and aluminum signals, implying that an amount of gold layer (ca. 0.3 µm in depth) was extended to the inner of the nanopores, which constituted the working electrode. Meanwhile, relatively uniform gold layer covered the outer surface of the reverse side of the AAO nanopore membrane and about 1.2 µm-deep gold layer was extended to the inner wall (Figure S1), which jointly served as the auxiliary electrode. Thus, it can be perceived that the total electrochemical sensing domain included two parts, one was from the nanopore outer surface gold layer and the other was contributed by gold coating inner wall.

Electrochemical Characterization Cyclic voltammetry (CV) was initially used to profile the electrochemical characteristics of the established nanopore analytical device by using K3[Fe(CN)6] as electrochemical indicator (Figure S2-S3). It was found that both the anodic and cathodic peak currents were linearly dependent on the square root of scan rate, suggesting a diffusion-controlled process in the nanopores electrochemistry array.56 Then, we investigated the electrochemical response on the nanofluidic platform (Figure 1a). In this experiment, current-time (i-t) mode was adopted. Current response of [Fe(CN)6]3- ions was directly proportional to the concentration of electroactive species. The steady-state reduction current (Iss) can be achieved in less than 30 s (Figure 2), indicating a rapid electrochemical response behavior. This may substantially benefit from the favorable permeability and electron transfer ability of the nanopore electrochemistry array. Additionally, a serial of electrochemical investigations demonstrated

6


Page 6 of 18

Page 7 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the favorable analytical performance of the developed electrochemistry platform (Figure S4).

Figure 2. Electrochemical response of serial concentrations (0.01, 0.05, 0.2, 1.0 and 5 mM) of K3[Fe(CN)6] at the integrated nanopore electrochemistry setting. The dashed arrow indicated the addition of K3[Fe(CN)6] solution. Inset: (left) an amplified view of i-t traces of 0.01mM of K3[Fe(CN)6] and blank electrolyte; (right) the steady-state current (Iss) as a function of electroactive species concentration. Conditions: background electrolyte, 10 mM phosphate buffer (pH 6.0); electrolytic potential, 0 V (vs. Ag/AgCl wire); stirring speed, 1500 rpm.

Detection of Biomolecules The present nanopore electrochemistry platform was then used for quantitative bioanalysis. The surface modification and molecular assembly process was schematically presented in Figure 3a. The nanopores were initially modified using silane chemistry and Schiff’s base reaction,57 and then were functionalized by nucleic acid capture probe (CP). For the oligonucleotide assay, another capture probe (CP2) was introduced to facilitate the formation of a multiplexed DNA hybridization structure at the present of target DNA (Supporting Information, Table S1). This strategy was successfully used for electrochemical signal amplification toward sensitive detection of sequence-specific DNA on planar gold electrode,58 as well as polymer nanopore-based ionic current analysis19 in our previous researches. Such a cascade assembly of DNA architecture has proven to effectively block the nanopores, causing drastic ionic current change and thus enhancing the detection sensitivity. Therefore, it was an ideal model analyte to interrogate the performance of the present nanopore electrochemistry system. As usual, ionic current signal was used a reference to trace the molecular assembly process. The immobilization of CP molecules resulted in slightly reduced transmembrane ionic current signal, whereas the concatenation of CP2 and the target DNA led to remarkable ionic current drop due to channel blockage effect, indicating the formation of DNA hybridization structure (Supporting Information, Figure S5). Equally, this strategy would also be feasible to the present nanopores-based electrochemical assays, because the electro-reduction current of [Fe(CN)6]3- ions

7


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

generated on the nanopore array electrode was directly correlated to the flux of the electroactive species (Figure 3b). In addition, we can readily detect protein in this fashion. Also as shown in Figure 3a, CP molecules were immobilized in the nanopores and then hybridized with insulin binding aptamer.59 At the presence of the target, the bioaffinity interaction between the aptamer with insulin would induce the conformational change, thus affecting the flux of electroactive species and producing a distinct electrolytic current signal.

Figure 3. (a) Molecular assembly for the detection of DNA (i) and insulin (ii) in the nanopores. (b) Cartoon presentation of the working principle. Electrochemical reduction of [Fe(CN)6]3- ions takes place at the gold film electrode after transporting the biomolecules functionalized nanopores, thereby producing flux-dependent electrolytic current signal. AE, auxiliary electrode; WE, working electrode.

Figure 4a illustrated the representative current-time traces obtained from DNA detection. Clearly, the decrease of steady-state current after binding of the target DNA can be observed, as compared to the background current signal (without the target molecules). Due to the multiplexed DNA configuration, our nanopore-based electrochemistry array exhibited sensitive signal response to the sequence-specific DNA; for instance, 1 nM target brought about more than 70% current signal decrease, and 1 µM target resulted in approximately 90% current drop. Moreover, the current change amplitude was found to be highly dose-dependent. This can be interpreted that the target triggered the assembly of a multiplexed DNA configuration in the nanopores and reduced the effective aperture, which consequently restrained the transport of electroactive ions, thus declining the electrochemical current. For comparison, ionic current signal was employed to investigate the biosensing events in the nanopores. As can be seen, the I-V recordings also showed typical concentration-dependent response feature (Figure 4b); however, the signal variation amplitude roughly appeared to be less sensitive compared to electrolytic current readout. In order to quantitative estimation, we defined the signal change ratio (%) by using an expression of (I0-It)/I0, in which I0 was the steady-current in the absence of target and It was the steady-current obtained after binding of target in the nanopores. Plotting the

8


Page 8 of 18

Page 9 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

signal change ratio versus the target concentration yielded a well-characteristic dose-effect relationship, which spanned a concentration region from 1 pM to 1 µM for the sequence-specific DNA. In comparison, a linear response range of 10 pM ~ 100 nM was obtained by ionic current signal (Figure 4c), declaring more appealing performance of the developed electroanalytical strategy.

Figure 4. DNA detection. (a) Typical i-t curves obtained from the nanopore electrochemistry array. In this experiment, 0.1 mM K3[Fe(CN)6] was used as signal reporter. Supporting electrolyte: 5 mM Tris buffer (pH 7.4). (b) I-V recordings from ionic current mode using uncoated AAO nanopores. Ionic current was measured with a Keithley 6487 picoammeter through two pieces of 0.5 mm Ag/AgCl electrodes. Running buffer: 10 mM Tris +

9


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

500 mM KCl (pH 7.4). (c) The corresponding quantitative calibration curves achieved by electrolytic current (EC) and ionic current (IC) measurements; respectively. The shaded region defined the threshold for quantitative limits and the error bars represented the standard deviations of independent triple measurements.

To further validate the utility, we applied the present electrochemistry platform to protein analysis. Taking insulin as an example, the recognition of the target molecules in the gold decorated nanopores, likewise resulted in decreased steady-state current signatures as compared to the CP-aptamer duplex status. As shown in Figure 5a, even 10 pM insulin could produce a comparatively evident current variation, manifesting a rather sensitive response at the nanopore electrochemistry array, which can also be elucidated that the CP-aptamer-target complex certainly diminished the aperture of the AAO nanopores that dominated the transport of electroactive ions. The electrostatic interaction was also possibly response for this issue, because the DNA moieties (CP and aptamer) were negatively charged and insulin (with pI value of 5.35-5.45) would also bear negative charge in our experiment conditions.60 The enhanced negative charge capacity upon the binding of target molecules in the nanopores strengthened the electrostatic repulsion toward the electroactive ions. In addition, steric hindrance effect stemming from the multiplexed bioconjunction entity in the nanopores was reasonably involved with this phenomenon.61 As to the quantitative analysis, one can see that a definite concentration response region from 10 pM to 10 nM has been achieved (Figure 5b). Therefore, our nanopore-based electrochemistry protocol showed a wide range of signal response to insulin detection. It should be noted that because of the potential nonspecific adsorption of protein molecules on gold-decorated nanopore surface,62 the reliable quantification limit for insulin was defined to be 50 pM (see discussion below). As regards the present functionalized nanopores pertaining to ion permittivity and electrochemical response, we compared different electroactive species including [Fe(CN)6]3-, hydroquinone and [Ru(NH3)6]3+ representing negatively charged, electroneutral and positively charged substances; respectively. For both DNA and insulin detection, [Fe(CN)6]3- ion was experimentally verified to offer much higher detection sensitivity (Figure S6). Generally, transmembrane transport of ions or molecules in confined space is governed by pore size, charge and hydrophilicity.63 In our experiments, the flux of electroactive ions was envisioned to be primarily affected by the aperture and electrostatic effect in the biomolecules-functionalized nanopores.64 AAO is an amphoteric substance; the inner wall of the nanopores would bear negative charge after the surface modification by DNA probes and the conjugated targets. Actually, more significant current change observed at the use of [Fe(CN)6]3- ion confirmed that electrostatic interaction had considerable influence on the current signal response. Comparatively, positively charged [Ru(NH3)6]3+ ion brought about fairly insensitive current change;

10


Page 10 of 18

Page 11 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the (I0-It)/I0 values were 25.6% and 13.9% for the detection of DNA and insulin, respectively, inferior to 75.8% and 63.4% current change when use of [Fe(CN)6]3- ion as signal reporter, at the equal condition. This can be attributed that the hybridized probe-target complex preferentially allowed positively charged species to transport to maintain electroneutral property in the nanopores.53 Also note that [Ru(NH3)6]3+ has been widely used for label-free electrochemical detection of nucleic acids;65-67 this electroactive specie, however, was less amenable to the present electrochemical assays.

Figure 5. Protein assay. (a) Representative i-t curves from sensing of insulin using the nanopore electrochemistry array. Analytical condition was as in Figure 4. (b) The corresponding quantitative calibration curves. The shaded region defined the threshold for quantitative limits and the error bars represented the standard deviations of independent triple measurements.

Furthermore, we inspected the selectivity of the established nanopores-based electrochemical protocols. A couple of base mismatched DNA analogues were used (Table S2). Surely, our present method displayed excellent selectivity to the target DNA, and can discriminate even single-base mismatched DNA sequence, primarily due to the exquisitely designed DNA configuration that improved the differentiating capacity (Figure 6a). Additionally, various proteins that were 100-fold

11


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

concentration higher than insulin, including human serum albumin (HSA), hemoglobin, thrombin, immunoglobulin G (IgG) were interrogated to challenge the selectivity. It was found that IgG induced considerable turbulence while the others were largely agreeable to insulin detection (Figure 6b). Apart from the non-specific adsorption, such interfering was possibly ascribed to relatively bulky molecular size of IgG (150 kDa) producing undesirable channel blockage effect.68 Nonetheless, this analytical strategy would, in principle, be suitable for detection of other proteins having clinical significance by virtue of the analytical flexibility and maneuverability of our present nanopore-based electrochemistry array platform (Table S3).

Figure 6. The selectivity. The present nanopore-based electrochemical protocols were challenged by examination of the specificity using a variety of mismatched DNA sequences (1 nM each) and potential interfering proteins including human serum albumin (HSA), hemoglobin, thrombin, immunoglobulin G (IgG) with respect to DNA (a) and insulin (b) assays. Concentration: 0.5 nM for insulin and 50 nM for other proteins.

Conclusions We have reported on the development of a novel electrochemistry array that integrates the sensing electrodes at porous AAO nanopore membrane. By facile preparation of gold decorated AAO nanopores, in combination with a nanofluidic device, we can readily perform electrochemical

12


Page 12 of 18

Page 13 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

measurements. Electroactive ions are modulated to transport the functionally modified nanopores and are electrochemically reduced at the array electrode interface to produce electrolytic current. The electrochemical signal relates closely to the status of the nanopores and could reflect the biosensing events. In the herein proof-of-concept demonstrations, DNA and protein biomolecules can be detected at picomolar concentration levels in a label-free manner. Although current success is reaped, barriers to in situ biodetection still exist. This ought to be overcome by using far more compatible and ingenious nanofluidic setting capable of implementing the assays closer to reality. To sum up, the present nanopores-based electrochemistry platform and the pertinent methodology can be expanded to a wide range of bioanalytical applications provided that high-efficiency biorecognition probes (e.g., nucleic acid aptamer or antibody) as well as competent molecular assembly methods are rationally introduced to the nanopores.

Supporting Information Fabrication of measurement cell and electrode pads, nanopore modification and molecular assembly, nanopore characterization and electrochemical test. This material is available free of charge via the Internet at http://pubs.acs.org.

Notes The authors declare no competing financial interest.

Acknowledgments This work was supported by the National Basic Research Program of China (973 Program, 2015CB932600), the National Key R&D Program of China (2017YFA0208000, 2016YFF0100800), the National Natural Science Foundation of China (21525523, 21722507, 21574048, 81302743, 21665004, and 21605053), Guangxi Natural Science Foundation (2018GXNSFAA138022), the Fok Ying-Tong Education Foundation, China (151011). X. C. Li is also thankful to the financial support from China Postdoctoral Science Foundation (2015M570637) and to Professor Junshan Liu, Key Laboratory for Micro/Nano Technology and System of Liaoning Province, Dalian University of Technology, for providing technical support of electrode pads fabrication.

References (1)

Dekker, C. Solid-state nanopores. Nat.Nanotechnol. 2007, 2, 209-215.

(2)

Banerjee, P.; Perez, I.; Henn-Lecordier, L.; Lee, S. B.; Rubloff, G. W. Nanotubular Metal-Insulator-Metal

Capacitor Arrays for Energy Storage. Nat. Nanotechnol. 2009, 4, 292-296. (3)

Biesemans, A.; Soskine, M.; Maglia, G. A Protein Rotaxane Controls the Translocation of Proteins Across

a ClyA Nanopore. Nano. Lett. 2015, 15, 6076-6081.

13


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(4)

Shim, J.; Kim, Y.; Humphreys, G. I.; Nardulli, A. M.; Kosari, F.; Vasmatzis, G.; Taylor, W. R.; Ahlquist, D.

A.; Myong, S.; Bashir, R. Nanopore-Based Assay for Detection of Methylation in Double-Stranded DNA Fragments. ACS Nano 2015, 9, 290-300. (5)

Manrao, E. A.; Derrington, I. M.; Laszlo, A. H.; Langford, K. W.; Hopper, M. K.; Gillgren, N.; Pavlenok,

M.; Niederweis, M.; Gundlach, J. H. Reading DNA at Single-Nucleotide Resolution with a Mutant MspA Nanopore and Phi29 DNA Polymerase. Nat. Biotechnol. 2012, 30, 349-353. (6)

Kawano, R.; Osaki, T.; Sasaki, H.; Takinoue, M.; Yoshizawa, S.; Takeuchi, S. Rapid Detection of a

Cocaine-Binding Aptamer Using Biological Nanopores on a Chip. J. Am. Chem. Soc. 2011, 133, 8474-8477. (7) Zhang, Y. Q.; Rana, A.; Stratton, Y.; Czyzyk-Krzeska, M. F.; Esfandiari, L. Sequence-Specific Detection of MicroRNAs Related to Clear Cell Renal Cell Carcinoma at fM Concentration by an Electroosmotically Driven Nanopore-Based Device. Anal. Chem. 2017, 89, 9201-9208. (8) Zhang, L.; Zhang, K.; Rauf, S.; Dong, D.; Liu, Y.; Li, J. H. Single-Molecule Analysis of Human Telomere Sequence Interactions with G-quadruplex Ligand. Anal. Chem. 2016, 88, 4533-4540. (9) Liu, G.; Zhang, L.; Dong, D.; Liu, Y.; Li, J. H. A label-free DNAzyme-based Nanopore Biosensor for Highly Sensitive and Selective Lead Ion Detection. Anal. Methods 2016, 8, 7040-7046. (10) Rauf, S.; Zhang, L.; Ali, A.; Ahmad, J.; Liu, Y.; Li, J. H. Nanopore-Based, Label-Free, and Real-Time Monitoring Assay for DNA Methyltransferase Activity and Inhibition. Anal. Chem. 2017, 89, 13252-13260. (11) Rauf, S.; Zhang, L.; Ali, A.; Liu, Y.; Li, J. H. Label-Free Nanopore Biosensor for Rapid and Highly Sensitive Cocaine Detection in Complex Biological Fluids. ACS Sens. 2016, 2, 227-234. (12) Zhang, Z.; Wen, L. P.; Jiang, L. Bioinspired Smart Asymmetric Nanochannel Membranes. Chem. Soc. Rev. 2018, 47, 322-356. (13)

Li, P.; Kong, X. Y.; Xie, G.; Xiao, K.; Zhang, Z.; Wen, L. P.; Jiang, L. Adenosine-Activated Nanochannels

Inspired by G-Protein-Coupled Receptors. Small 2016, 12, 1854-1858. (14)

Lin, Y.; Shi, X.; Liu, S. C.; Ying, Y. L.; Li, Q.; Gao, R.; Fathi, F.; Long, Y. T.; Tian, H. Characterization of

DNA Duplex Unzipping through a sub-2 nm Solid-state Nanopore. Chem. Commun. 2017, 53, 3539-3542. (15)

White, R. J.; Ervin, E. N.; Yang, T.; Chen, X.; Daniel, S.; Cremer, P. S.; White, H. S. Single Ion-Channel

Recordings Using Glass Nanopore Membranes. J. Am. Chem. Soc. 2007, 129, 11766-11775. (16) Sze, J. Y.; Ivanov, A. P.; Cass, A. E. G.; Edel, J. B. Single Molecule Multiplexed Nanopore Protein Screening in Human Serum using Aptamer Modified DNA Carriers. Nat. Commun. 2017, DOI: 10.1038/s41467-017-01584-3. (17) Li, X. C.; Zhai, T. Y.; Gao, P. C.; Cheng, H. L.; Hou, R. Z.; Lou, X. D.; Xia, F. Role of Outer Surface Probes for Regulating Ion Gating of Nanochannels. Nat. Commun. 2018, doi: 10.1038/s41467-017-02447-7. (18) Garaj, S.; Hubbard, W.; Reina, A.; Kong, J.; Branton, D.; Golovchenko, J. A. Graphene as a Subnanometre Trans-electrode Membrane. Nature 2010, 467, 190-193. (19) Liu, N. N.; Jiang, Y. N.; Zhou, Y. H.; Xia, F.; Guo, W.; Jiang, L. Two-Way Nanopore Sensing of Sequence-Specific Oligonucleotides and Small-Molecule Targets in Complex Matrices Using Integrated DNA Supersandwich Structures. Angew. Chem. Int. Ed. 2013, 52, 2007-2011. (20) Liu, Q.; Wen, L. P.; Xiao, K.; Lu, H.; Zhang, Z.; Xie, G. H.; Kong, X. Y.; Bo, Z. S.; Jiang, L. A Biomimetic Voltage-Gated Chloride Nanochannel. Adv. Mater. 2016, 28, 3181-3186. (21) MutalibMd Jani, A.; Kempson, I. M.; Losic, D.; Voelcker, N. H. Dressing in Layers: Layering Surface

14


Page 14 of 18

Page 15 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Functionalities in Nanoporous Aluminum Oxide Membranes. Angew. Chem. Int. Ed. 2010, 49, 7933-7937. (22) Haque, F.; Li, J.; Wu, H. C.; Liang, X. J.; Guo, P. X. Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA. Nano Today 2013, 8, 56-74. (23)

Wang, Y.; Zheng, D.; Tan, Q.; Wang, M. X.; Gu, L. Q. Nanopore-Based Detection of Circulating

MicroRNAs in Lung Cancer Patients. Nat. Nanotechnol. 2011, 6, 668-674. (24) Jiang, Y.; Liu, N.; Guo, W.; Xia, F.; Jiang, L. Highly-Efficient Gating of Solid-State Nanochannels by DNA Supersandwich Structure Containing ATP Aptamers: A Nanofluidic IMPLICATION Logic Device. J. Am. Chem. Soc. 2012, 134, 15395-15401. (25)

Rosen, C. B.; Rodriguez, L. D.; Bayley, H. Single-Molecule Site-Specific Detection of Protein

Phosphorylation with a Nanopore. Nat. Biotechnol. 2014, 32, 179-181. (26) Lin, L.; Yan, J.; Li, J. H. Small-Molecule Triggered Cascade Enzymatic Catalysis in Hour-Glass Shaped Nanochannel Reactor for Glucose Monitoring. Anal. Chem. 2014, 86, 10546-10551. (27) Zhang, L.; Zhang, K.; Liu, G.; Liu, M.; Liu, Y.; Li, J. H. Label-Free Nanopore Proximity Bioassay for Platelet-Derived Growth Factor Detection. Anal. Chem. 2015, 87, 5677-5682. (28) Wang, L. L.; Yao, F. J.; Kang, X. F. Nanopore Single-Molecule Analysis of Metal Ion-Chelator Chemical Reaction. Anal. Chem. 2017, 89, 7958-7965. (29) Zhao, Y. X.; Janot, J. M.; Balanzat, E.; Balme, S. Mimicking pH-Gated Ionic Channels by Polyelectrolyte Complex Confinement Inside a Single Nanopore. Langmuir 2017, 33, 3484-3490. (30) Gu, Z.; Ying, Y.-L.; Cao, C.; He, P.; Long, Y. T. Accurate Data Process for Nanopore Analysis. Anal. Chem. 2015, 87, 907-913. (31) Zhang, J. H.; Liu, X. L.; Ying, Y. L.; Gu, Z.; Meng, F. N.; Long, Y. T. High-bandwidth Nanopore Data Analysis by Using a Modified Hidden Markov Model. Nanoscale 2017, 9, 3458-3465. (32) Smeets, R. M.; Keyser, U. F.; Dekker, N. H.; Dekker, C. Noise in Solid-state Nanopores. Proc. Natl. Acad. Sci. 2008, 105, 417-421. (33) Shi, X.; Gao, R.; Ying, Y. L.; Si, W.; Chen, Y. F.; Long, Y. T. A Scattering Nanopore for Single Nanoentity Sensing. ACS Sens. 2016, 1, 1086-1090. (34) McNally, B.; Singer, A.; Yu, Z.; Sun, Y.; Weng, Z.; Meller, A. Optical Recognition of Converted DNA Nucleotides for Single-Molecule DNA Sequencing Using Nanopore Arrays. Nano Lett. 2010, 10, 2237-2244. (35) Kurz, V.; Nelson, E. M.; Shim, J.; Timp, G. Direct Visualization of Single-Molecule Translocations through Synthetic Nanopores Comparable in Size to a Molecule. ACS Nano 2013, 7, 4057-4069. (36) Zhao, Y.; Ashcroft, B.; Zhang, P.; Liu, H.; Sen, S.; Song, W.; Im, J.; Gyarfas, B.; Manna, S.; Biswas, S.; Borges, C.; Lindsay, S. Single-molecule Spectroscopy of Amino Acids and Peptides by Recognition Tunnelling. Nat. Nanotechnol. 2014, 9, 466-473. (37) Belkin, M.; Chao, S. H.; Jonsson, M. P.; Dekker, C.; Aksimentiev, A. Plasmonic Nanopores for Trapping, Controlling Displacement, and Sequencing of DNA. ACS Nano 2015, 9, 10598-10611. (38)

Huang, S.; Romero, R. M.; Castell, O. K.; Bayley, H.; Wallace, M. I. High-throughput Optical Sensing of

Nucleic Acids in a Nanopore Array. Nat. Nanotechnol. 2015, 10, 986-991. (39) Assad, O. N.; Gilboa, T.; Spitzberg, J.; Juhasz, M.; Weinhold, E.; Meller, A. Light-Enhancing Plasmonic-Nanopore Biosensor for Superior Single-Molecule Detection. Adv. Mater. 2017, DOI: 10.1002/adma.201605442. (40) Xie, P.; Xiong, Q.; Fang, Y.; Qing, Q.; Lieber, C. M. Local Electrical Potential Detection of DNA by 15


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nanowire-Nanopore Sensors. Nat. Nanotechnol. 2012, 7, 119-125. (41) Ivanov, A. P.; Instuli, E.; McGilvery, C. M.; Baldwin, G.; McComb, D. W.; Albrecht, T.; Edel, J. B. DNA Tunneling Detector Embedded in a Nanopore. Nano Lett. 2011, 11 (1), 279-285. (42) Baaken, G.; Ankri, N.; Schuler, A.-K.; Rühe, J.; Behrends, J. C. Nanopore-Based Single-Molecule Mass Spectrometry on a Lipid Membrane Microarray. ACS Nano 2011, 5, 8080-8088. (43) Sexton, L. T.; Mukaibo, H.; Katira, P.; Hess, H.; Horne, L. P.; Martin, C. R. An Adsorption-Based Model for Pulse Duration in Resistive-Pulse Protein Sensing. J. Am. Chem. Soc. 2010, 132, 6755-6763. (44) Huo, X. L.; Yang, H.; Zhao, W.; Xu, J. J.; Chen, H. Y. Nanopore-Based Electrochemiluminescence for Detection of MicroRNAs via Duplex-Specific Nuclease-Assisted Target Recycling. ACS Appl. Mater. Interfaces 2017, 9, 33360-33367. (45)

Murray, R. W. Nanoelectrochemistry: Metal Nanoparticles, Nanoelectrodes, and Nanopores. Chem. Rev.

2008, 108, 2688-2720. (46) Wang, G. L.; Zhang, B.; Wayment, J. R.; Harris, J. M.; White, H. S. Electrostatic-Gated Transport in Chemically Modified Glass Nanopore Electrodes. J. Am. Chem. Soc. 2006, 128, 7679-7686. (47) Sun, P.; Mirkin, M. V. Electrochemistry of Individual Molecules in Zeptoliter Volumes. J. Am. Chem. Soc. 2008, 130, 8241-8250. (48) Jena, B. K.; Percival, S. J.; Zhang, B. Au Disk Nanoelectrode by Electrochemical Deposition in a Nanopore. Anal. Chem. 2010, 82, 6737-6743. (49) Branagan, S. P.; Contento, N. M.; Bohn, P. W. Enhanced Mass Transport of Electroactive Species to Annular Nanoband Electrodes Embedded in Nanocapillary Array Membranes. J. Am. Chem. Soc. 2012, 134, 8617-8624. (50) de la Escosura-Muniz, A.; Merkoci, A. A Nanochannel/Nanoparticle-Based Filtering and Sensing Platform for Direct Detection of a Cancer Biomarker in Blood. Small 2011, 7, 675-682. (51) Yu, J.; Luo, P.; Xin, C.; Cao, X.; Zhang, Y.; Liu, S. Quantitative Evaluation of Biological Reaction Kinetics in Confined Nanospaces. Anal. Chem. 2014, 86, 8129-8135. (52) Yu, J.; Zhang, L.; Xu, X.; Liu, S. Quantitative Detection of Potassium Ions and Adenosine Triphosphate via a Nanochannel-Based Electrochemical Platform Coupled with G-quadruplex Aptamers. Anal. Chem. 2014, 86, 10741-10748. (53) Li, S. J.; Li, J.; Wang, K.; Xu, J. J.; Chen, H. Y.; Xia, X. H.; Huo, Q. A Nanochannel Array-Based Electrochemical Device for Quantitative Label-Free DNA Analysis. ACS Nano 2010, 4, 6417-6424. (54) Gao, H. L.; Wang, M.; Wu, Z. Q.; Wang, C.; Wang, K.; Xia, X. H. Morpholino-Functionalized Nanochannel Array for Label-Free Single Nucleotide Polymorphisms Detection. Anal. Chem. 2015, 87, 3936-3941. (55) Liu, C.; Gillette, E. I.; Chen, X.; Pearse, A. J.; Kozen, A. C.; Schroeder, M. A.; Gregorczyk, K. E.; Lee, S. B.; Rubloff, G. W. An All-in-One Nanopore Battery Array. Nat. Nanotechnol. 2014, 9, 1031-1039. (56) Luo, X. L.; Pan, J. B.; Pan, K. M; Yu, Y. Y.; Zhong, A. N.; Wei, S. S.; Li, J.; Shi, J. Y.; Li, X. C. An Electrochemical Sensor for Hydrazine and Nitrite Based on Graphene–Cobalt Hexacyanoferrate Nanocomposite: Toward Environment and Food Detection. J. Electroanal. Chem. 2015, 745, 80-87. (57) Gao, P.; Hu, L.; Liu, N.; Yang, Z.; Lou, X.; Zhai, T.; Li, H.; Xia, F. Functional “Janus” Annulus in Confined Channels. Adv. Mater. 2016, 28, 460-465. (58) Xia, F.; White, R. J.; Zuo, X. L.; Patterson, A.; Xiao, Y.; Kang, D.; Gong, X.; Plaxco, K. W.; Heeger, A. J. 16


Page 16 of 18

Page 17 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

An Electrochemical Supersandwich Assay for Sensitive and Selective DNA Detection in Complex Matrices. J. Am. Chem. Soc. 2010, 132, 14346-14348. (59) Wang, Y. H.; Gao, D. Y.; Zhang, P. F.; Gong, P.; Chen, C.; Gao, G. H.; Cai, L. T. A Near Infrared Fluorescence Resonance Energy Transfer Based Aptamer Biosensor for Insulin Detection in Human Plasma. Chem. Commun. 2014, 50, 811-813. (60) Li, C. Y.; Ma, F. X.; Wu, Z. Q.; Gao, H. L.; Shao, W. T.; Wang, K.; Xia, X. H. Solution-pH-Modulated Rectification of Ionic Current in Highly Ordered Nanochannel Arrays Patterned with Chemical Functional Groups at Designed Positions. Adv. Funct. Mater. 2013, 23, 3836-3844. (61) Guo, W.; Hong, F.; Liu, N.; Huang, J.; Wang, B.; Duan, R.; Lou, X.; Xia, F. Target-Specific 3D DNA Gatekeepers for Biomimetic Nanopores. Adv. Mater. 2015, 27, 2090-2095. (62) Yusko, E. C.; Johnson, J. M.; Majd, S.; Prangkio, P.; Rollings, R. C.; Li, J.; Yang, J.; Mayer, M. Controlling Protein Translocation through Nanopores with Bio-inspired Fluid Walls. Nat. Nanotechnol. 2011, 6, 253-260. (63) Savariar, E. N.; Krishnamoorthy, K.; Thayumanavan, S. Molecular Discrimination inside Polymer Nanotubules. Nat. Nanotechnol. 2008, 3, 112-117. (64) Chen , W.; Wu, Z. Q.; Xia, X. H.; Xu, J. J.; Chen, H. Y. Anomalous Diffusion of Electrically Neutral Molecules in Charged Nanochannels. Angew. Chem., Int. Ed. 2010, 49, 7943-7947. (65) Yang, F.; Yang, X.; Wang, Y.; Qin, Y.; Liu, X.; Yan, X.; Zou, K.; Ning, Y.; Zhang, G. J. Template-Independent, in Situ Grown DNA Nanotail Enabling Label-Free Femtomolar Chronocoulometric Detection of Nucleic Acids. Anal. Chem. 2014, 86, 11905-11912. (66) Zhang, J.; Song, S. P.; Wang, L. H.; Pan, D.; Fan, C. H. A Gold Nanoparticle-based Chronocoulometric DNA Sensor for Amplified Detection of DNA. Nat. Protocols 2007, 2, 2888-2895. (67) Zhang, J.; Song, S. P.; Zhang L. Y.; Wang, L. H.; Wu, H. P.; Pan, D.; Fan, C. H. Sequence-Specific Detection of Femtomolar DNA via a Chronocoulometric DNA Sensor (CDS): Effects of Nanoparticle-Mediated Amplification and Nanoscale Control of DNA Assembly at Electrodes. J. Am. Chem. Soc. 2006, 128, 8575-8580. (68) Wei, R.; Martin, T. G.; Rant, U.; Dietz, H. DNA Origami Gatekeepers for Solid-State Nanopores. Angew. Chem. Int. Ed. 2012, 51, 4864-4867.

17


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC

18


Page 18 of 18

Integrated Solid-State Nanopore Electrochemistry Array for Sensitive

Recommend Documents