Toehold Mediated One-Step Conformation-Switchable âSignal-On

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Toehold Mediated One-Step Conformation-Switchable "Signal-on" Electrochemical DNA Sensing Enhanced with Homogeneous Enzymatic Amplification Siqi Wang, Fan Yang, Dan Jin, Qi Dai, Jiyuan Tu, Yanju Liu, Yong Ning, and Guo-Jun Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on April 28, 2017

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 free 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 accessible to all readers and 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.

Analytical Chemistry 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 26

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

Analytical Chemistry

Toehold

Mediated

One-Step

Conformation-Switchable

"Signal-on" Electrochemical DNA Sensing Enhanced with Homogeneous Enzymatic Amplification

Siqi Wanga, Fan Yang a,*, Dan Jina, Qi Daib, Jiyuan Tuc, Yanju Liuc, Yong Ninga, Guo-Jun Zhanga,* a

School of Laboratory Medicine, Hubei University of Chinese Medicine, 1 Huangjia Lake West Road, Wuhan 430065, China.

b

Huangjia Lake Hospital, Hubei University of Chinese Medicine, 1 Huangjia Lake West Road, Wuhan 430065, China.

c

School of Pharmacy, Hubei University of Chinese Medicine, 1 Huangjia Lake West Road, Wuhan 430065, China.

*Corresponding author: Tel: +86-27-68890259, Fax: +86-27-68890259 E-mail: [email protected]; [email protected]

ACS Paragon Plus Environment


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

Page 2 of 26

ABSTRACT: The development of highly sensitive and sequence-specific electrochemical DNA (E-DNA) sensors featuring flexible, one-step and "signal-on", is a long-lasting goal. Here we present a single-step, toehold-triggered structure-switchable signaling design that is "signal-on" and compatible with homogeneous enzyme-assisted target recycling (EATR). In this design, a partially hybridized duplex is bifunctional, which consists of a signal probe having foldable hairpin sequence and a target recognition probe with exposed toehold domain. In the presence of both target and exonuclease, the toehold sequence rapidly fuels the strand displacement reaction, liberating the surface-confined toehold-target duplex into homogeneous solution for target recycling, and meanwhile leaving the dehybridized signal probe to form a stem-loop structure for signaling. Through such an 1: N enzymatic catalysis, more and more unfolded probes self-hybridize to their original folded configuration, giving a remarkable signal gain. This enzyme and toehold-assisted E-DNA (etE-DNA) sensor achieves a satisfactory detection limit down to 42 fM, which is lower than that of the routine switchable E-DNA sensor by several orders of magnitude. In addition, the strategy shows high selectivity against a single-base mismatch, and is capable of probing low abundant target DNA directly in human serum with minimal interference. By synergizing

the

toehold-based

high

selectivity,

EATR

and

one-step

conformation-switchable signaling, this functional etE-DNA sensor appears to be a promising bioassay approach for clinical diagnostics.

KEYWORDS: Electrochemistry; Conformation-switchable sensor; Toehold; Strand displacement; Exonuclease Ⅲ


Page 3 of 26

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 Highly specific and predictable Watson-Crick base-pairing allows DNA molecules not only to encode the mystery of life, but also to serve as building blocks to engineer DNA nanostructures of various sizes, shapes and geometries via dynamical self-assembly.1,2 Up to now, a series of dynamic DNA nanostructures,3 such as computation logic gates,4 catalytic circuits,5 and artificial molecular machines,6-8 have been successfully constructed, most of them are driven by toehold-mediated strand displacement reaction (SDR). Of note, the toehold-mediated SDR functions based on an overhanging single-stranded domain-toehold (typical 5-8 nucleotides) in a partially hybridized duplex, which enables itself to displace from the original duplex and hybridize with the stimulative (target) sequence in a programmable and fast "base-by-base" fashion.9-11 Such a highly specific self-powered dynamic self-assembly mechanism has inspired the design of multiple nucleic acid biosensors, including homogeneous fluorescence detection and atomic force microscope imaging,11,12 heterogeneous quartz crystal microbalance,13 field-effect

transistor and

electrochemistry

biosensing.7,14-16

In

comparison,

surface-confined SDR-based electrochemical DNA (E-DNA) sensor is more competent due to its low cost, small size, simple operation as well as the multiplexed chip-like

readout

with

high

addressability.

Nevertheless,

many

reported

toehold-triggered E-DNA sensors are "signal-off" because of the intrinsic displacing and releasing of signal probes, resulting in a limitation of the maximal signal suppression (100%) under any experimental conditions.15,16 To circumvent this hurdle, several "signal-on" SDR-based E-DNA sensors have been constructed. They rely, however, on either the uncontrollable flexibility of the post-SDR single-stranded DNA to approach the electrode surface for electron transport,17 or the addition of multiple exogenous assisted probes.7 To better control and facilitate the surface-confined "signal-on" E-DNA signaling, the reagentless conformation-switchable sensor design is appealing. Since the first structurally switchable E-DNA sensor was reported in 2003,18 such dynamic sensing design evolved rapidly from original stem-loop structured probe ("signal-off") to pseudoknot probe ("signal-on").19-21 Note that this target-responsive switchable signaling features reagentless and one-step readout, signifying great potential to be deployed at the point of care settings. Despite these advantages, one limitation is the 1:1 response that



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

Page 4 of 26

means each target sequence could only induce a single signaling event by hybridizing with one copy of capture probe, thus limiting its sensitivity and application in real world.22 To address this issue, a signal amplification strategy that is simple and can make full use of surface-confined SDR is becoming highly necessary. Although a variety of nucleic acid amplification methods have been reported,23 such as polymerase chain reaction,24 hybridization chain reaction,25-29 rolling circle amplification,30,31

and

nanomaterials-based

DNA

amplification,32,33

these

methodologies either require multistep thermal cycling, complex conjugation or elaborate probe design for target extension, which are incompatible with the heterogeneous surface-attached DNA switches. In contrast, enzyme-assisted target recycling (EATR) appears effective and competent, wherein the nuclease can repeatedly recognize and selectively cleave the probe-target complex to release the nucleic acid target, thereby amplifying the signal produced by the target without altering its copy number.34 To further enhance the functionality of the E-DNA sensor, we here present a toehold-mediated structurally switchable E-DNA sensor integrated with EATR strategy for single-step, “signal-on” readout. The partially hybridized probe complex with a toehold domain is first self-assembled on the electrode via thiol-gold chemistry. The duplex maintains the signal probe with a rigid linear structure, keeping the electroactive reporter (methylene blue, MB) far away from the interrogating electrode. In the presence of target sequence, this signal probe can return to its original stem-loop structure by liberating the newly formed toehold-target duplex, and can simultaneously bring the redox molecules in proximity to the interrogating electrode surface for "signal-on" sensing. Moreover, the released toehold-target complex with a blunt 3' terminus could be specifically recognized and digested by Exonuclease Ⅲ (Exo Ⅲ).35-40 In comparison to the surface-confined enzymatic digestion,41-43 such a homogeneous, highly efficient 1:N target-responsive recycling mechanism allows more and more signal probes to form their intrinsic stem-loop conformation, yielding a significantly enhanced faradaic current. This enzyme-assisted toehold E-DNA (etE-DNA) sensor achieves a high sensitivity (42 fM). More importantly, this simple design can combine multiple advantages, such as the toehold-based high selectivity, enzyme-triggered target recycling and one-step conformation-switchable signaling. Such a powerful sensor is also capable of reading out the trace amounts of target


Page 5 of 26

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


DNA directly in human serum. Experimental section Materials and Chemicals. All oligonucleotides (Table S1) were synthesized and purified by Sangon Inc. (Shanghai, China). A stem-looped DNA probe thiolated with a -(CH2)6-spacer at the 3' end and modified with MB at the other end (5') was used as signal probe. A 24-base DNA probe was employed as a toehold probe. Other oligonucleotides including target DNA, mismatched target sequences (i.e., 1 M, 2 M, 3 M) and the random non-complementary sequence were listed in Table S1. 6-Mercapto-1-hexanol (MCH), hexaammineruthenium(III) chloride ([Ru(NH3)6]3+, RuHex) and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were all purchased from Sigma-Aldrich (St. Louis, MO). Tris-(hydroxymethyl) aminomethane (Tris) was obtained from Solarbio Science Co. Ltd. (Beijing, China). Exonuclease III (Exo Ⅲ) was purchased from New England Biolabs Co. Ltd. (Beijing, China). DEME medium (Hyclone, USA) is a mixture of 10% fetal calf serum with a variety of amino acids, glucose and antibiotics. Healthy human serum was provided by the affiliated hospital of Hubei University of Chinese Medicine (Wuhan, China). All other chemicals were of analytical grade. Ultrapure water (Millipore Corp., Bedford, MA) with 18.2 MΩ resistivity was used throughout the research. Preparation of the sensor. The detailed experimental procedure could be found in a previous literature.44 Briefly, prior to assembling sensing molecular structures, Au electrodes (2-mm in diameter, CH Instruments, Inc., Austin, TX) were polished with 0.05-µm γ-alumina powder (CH Instruments, Inc.) to obtain a mirror surface, followed by sonicating them in water and ethanol for 5 min, respectively, to remove residual particulates. After electrochemical cleaning in 0.5 M H2SO4, the well-prepared electrodes were thoroughly rinsed with Milli-Q water and dried with nitrogen, and should be immediately used for probe immobilization.



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

Page 6 of 26

The E-DNA sensor fabrication involved several steps. At first, the duplex probe was prepared by mixing 10 µM signal probe (with 1 mM TCEP) and 15 µM toehold probe in equal volumes in hybridization buffer (10 mM Tris-Hcl, 50 mM NaCl, 10 mM MgCl2, pH 7.4). Then, the mixed sequences were heated to 85oC for 10 min and cooled down to room temperature for at least 2 h. The as-prepared duplex probe (5 µM) was diluted to the required concentration before use. For probe immobilization, 6 µL of droplets of the duplex probes were dropped on the well-prepared electrodes for self-assembly overnight at room temperature. After immobilization, the functional electrode was thoroughly rinsed with wash buffer (10 mM Tris-HCl, 100 mM NaCl, pH 7.4) and gently dried under a stream of nitrogen. The modified electrodes were subsequently immersed in 2 mM MCH solution for 1 h to remove the nonspecific DNA adsorption. Next, this sensor was challenged in displacement buffer (10 mM Tris-HCl, 100 mM NaCl, 20 mM MgCl2, pH 7.4) containing Exo III (2 U/µL) and diverse concentrations of the target DNA at 37oC for 60 min to accomplish SDR and target recycling. Electrochemical measurements. All electrochemical measurements

were

performed

with

a

CHI660D

electrochemical workstation (CH Instruments Inc., Austin, TX). A conventional three-electrode configuration was employed all through the experiment, which comprised a gold working electrode, a platinum wire auxiliary electrode, and an Ag/AgCl reference electrode. Cyclic voltammetry (CV) was carried out in 0.1 M KCl solution containing 5 mM [Fe(CN)6]3-/4- with a potential window from -0.2 to 0.6 V under a scan rate of 0.1 V/s. CV was employed to characterize the interfacial assembly process of DNA molecules. Chronocoulometry at a pulse period of 250 ms and pulse width of 700 mV, was conducted in 10 mM Tris-HCl buffer (50 µM [Ru(NH3)6]3+) to determine the surface probe coverage. Square wave voltammetry (SWV) measurements were performed by scanning the potential from -0.4 V to -0.1 V in 10 mM Tris-HCl buffer (0.1 M NaCl and 20 mM MgCl2, pH 7.4) or in 10% serum with a step potential of 4 mV, a frequency of 25 Hz, and an amplitude of 25 mV. The


Page 7 of 26

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


signal enhancement percentage (%) is calculated as the ratio between the MB peak current in the presence and absence of target molecules. The equation is as follows: signal gain (%) = [(IT-I0)/I0] × 100, in which IT is the baseline-subtracted peak current obtained in the presence of the target, and I0 is the baseline-subtracted peak current in the absence of target. Results and discussions Design of the etE-DNA sensor and signaling mechanism. The architecture of the etE-DNA sensor and its signaling mechanism are illustrated in Figure 1. A 32-base stem-loop signal DNA (stem, 9-nt; loop, 14-nt) dually labeled with 3'-SH and 5'-MB, respectively, was first partially hybridized with a toehold probe sequence (24-nt) through a 17 base-pair (bp) duplex structure by rapid one-step annealing. The formation of this partially complementary duplex (-24.35 kcal/mol, free energy of secondary structure) is thermodynamically more favorable as compared to the hairpin-like structure (-10.55 kcal/mol) (Figure S1). Importantly, this signal-toehold probe complex allows a 7-nt toehold domain exposed to be flanked by target molecules as the complex has been self-assembled onto the interrogating gold electrode followed by passivation with MCH. Without target DNA, the rigid duplex forces the distal end redox reporter, MB, away from the electrode surface, generating a low background current ("turn-off"). In the presence of target molecules, the toehold sequence can trigger a highly specific SDR by forming a toehold-target duplex that is then released into the bulk solution. Concurrently, the dehybridized signal probe can, in theory, spontaneously return to its original hairpin-like conformation, and thus brings MB in proximity to the interrogating electrode surface for high-efficiency electron transfer, a "signal-on" readout ("turn-on"). More importantly, the liberated duplex with blunt 3' terminus in the homogeneous solution would be specifically recognized and digested by Exo Ⅲ that is preferential to cleave the blunt or recessed 3' end of double-stranded DNA. After hydrolysis of toehold probe, the intact target sequence continues to flank next toehold-target complex. Such an 1:N enzyme-assisted target recycling strategy could induce an increasing number of signal



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

probes to form surface-confined stem-loop constructs, and significantly improve the target-responsive faradaic current. That is to say, in the coexistence of target DNA and Exo Ⅲ, an enhanced electrical signal would be attained in a single step because of the inherent conformation-switchable signaling mechanism. Beyond that, our etE-DNA sensor also features (i) high selectivity based on toehold structure, which cooperates with (ii) target recycling-triggered high sensitivity and (iii) one-step conformation-switchable signaling, forming a functional "signal-on" sensor. Toehold and Exo III-assisted synergistic signal amplification The feasibility of our etE-DNA sensor design was verified by square wave voltammetry (SWV). Cyclic voltammetry (CV) was also employed to characterize the stepwise construction of the sensing interface aided by a redox couple of [Fe(CN)6]3-/4-. Prior to immobilization of signal-toehold duplex, a pair of quasi-reversible, well-defined redox peaks of [Fe(CN)6]3−/4− with the maximum current intensity (curve a in Figure S2) were observed, reflecting the barrier-free diffusion of [Fe(CN)6]3−/4− to the bare gold electrode. After self-assembling the duplex probe on the interrogating electrode followed by MCH passivation, the peak current reduced significantly and an increase in peak separation emerged (Figure S2b), due mainly to the electrostatic repulsion between the negatively charged [Fe(CN)6]3−/4− and phosphate backbones of the DNA probes, resulting in the reduction of electrons transfer. We also observed a typical and relatively low SWV peak originated from MB centered at around -0.2 V (Figure 2a). This is presumably attributed to the rigid 17-bp domain that forces the terminal electroactive MB separating from the electrode surface with a distance of ~6 nm, disrupting the efficient electron transfer. In the presence of target DNA (1 µM), the SWV response attained an obvious increase (60%) (Figure 2b). We speculate that the target sequence aiding toehold domain triggers a SDR along the 17-bp rigid duplex, thereby enabling the surface-confined signal probe folding back to form its original stem-loop conformation. In this case, the MB reporter is held in close proximity to the interrogation electrode surface, generating a large electrochemical signal. Importantly, a sharp increase (~120%) in the SWV signal occurred in the coexistence of target DNA and Exo Ⅲ (Figure 2c), as compared to the case without target. This may be due to the enzymatic hydrolysis of the toehold oligonucleotide from toehold-target duplex, liberating target for the next round of


Page 8 of 26

Page 9 of 26

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


SDR and enabling more signal probes folding back to their stem-loop structures. This hypothesis was further confirmed by the partially restored redox currents and narrowed peak separation (Figure S2c), because the cooperative effect between the toehold-mediated SDR and enzymatic target recycling drastically reduced the surface-confined nucleic acid probes. Therefore, the surface negative charge decreased responsively, thereby enhancing the electrode interfacial electron transfer. The polyacrylamide gel electrophoresis analysis also verified this hypothesis (Figure S3). Upon introduction of only Exo III, however, the signal almost retained the same as control with minimal change (Figure 2d). This signifies that Exo III can't trigger SDR or digest the signal-toehold complex without target assistance. All the characterization results reveal that such a synergic dynamic folding mechanism renders the sensor highly sensitive via homogeneous signal amplification. Effect of measurement frequency and probe density on the sensor To achieve the optimal performance of etE-DNA sensor, we emphatically investigated two critical parameters, such as measurement frequency, probe density on the electrode. The signaling behavior of our conformation-switch E-DNA sensor is directly linked to the formation of stem-loop structure, which forces the distal MB reporter (slow electron transfer rate) in close to the electrode surface, and shows a much faster electron transfer rate. This difference in rate allows us to optimize the SWV frequency to maximize signal gain. On the basis of previously reported critical frequencies in folding triplex (100 Hz),45 or supersandwich (60 Hz) E-DNA sensors,46 the measurement frequency ranging from 10 to 100 Hz was studied (Figure 3A). The percentage signal gain obtained upon target binding got gradually higher with increasing frequency (< 60 Hz). The highest signal gain (~120%) reached at 60 Hz, and then the attenuated signal was instead observed. This is presumably due to the fact that the ratio of signal between linear and stem-looped states is variable by varying the SWV frequency, enabling a highly tunable signaling. In addition, the signal of the sensor strongly depends on surface-attached probe density. As shown in Figure 3B, the maximal current response (~120% signal change) occurred at 2 µM signal probe assembly concentrations. The higher or lower assembly



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

density would degrade the sensor's performance because of the inherent surface effect, such as molecular crowding and lying down of probes. For example, the low surface density (0.2 µM signal probe) was estimated to be 1.3×1011 molecules/cm2 via chronocoulometry (Figure 3C, Figure S4).47-49 It means that the intermolecular distance is 27.8 nm, much larger than the probe size (~10 nm), thus making many DNA probes falling down on the electrode surface and degrading the subsequent hybridization efficiency. Comparatively, the relatively high assembly concentration (5.0 µM signal probe) has a surface density of ~2.0×1012 molecules/cm2 (Figure 3C), equal to 7.1 nm separation between neighboring molecules (＜10 nm probe length). Such a densely packed probe monolayer is detrimental to surface-based hybridization due to the improved steric hindrance and electrostatic repulsion. Besides, the highly crowded unfolding regime may decrease the stability of the surface-tethered folding molecules,50 which would belie the "real" target-responsive structure-switch events and further attenuate the signal change. In contrast, 2 µM signal probe assembly concentration generates a surface density of ~1.2×1012 molecules/cm2 (i.e., 9.3 nm intermolecular separation) that may allow DNA probes to adopt a relatively upright orientation. This would enhance molecular recognition and hybridization efficiency with reduced steric hindrance. Therefore, the optimal assembly concentration of signal probe is 2 µM, which guarantees rational probe separation to enhance the hybridization and folding efficiency for maximizing the signal change. Synergistic response kinetics of etE-DNA sensor We further explored the kinetic response of our sensor to the perfectly matched target DNA and exonuclease. In our design, the signal gain is governed by the numbers of target-responsive folding molecule and the separation between MB reporter and the interrogating electrode surface. Of note, MB molecule is stable and able to real-time reflect the surface-confined dynamical molecular configurations. On the basis of these unique properties, we perform a real-time monitoring of signal change versus the time of synergic enzyme digestion and SDR in the presence both of target DNA (1 µM) and Exo Ⅲ (2 U/µL). A signal gain saturation at ~120% within 50


Page 10 of 26

Page 11 of 26

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


min was clearly observed. However, upon addition of only target DNA, the sensor achieved saturation after 30 min at a much lower signal gain (~60%). This time delay (20 min) of signal saturation in the coexistence of target and exonuclease indicates that the synergic reactions involving multiple cycles of SDR, enzymatic digestion and conformation-switch, are taking place indeed. We also found a rapid hybridization kinetics during the first 30 min, in which the enzyme-amplified synergic reaction generated an ~3% signal gain per minute. Nevertheless, a slightly lower rate of signal gain (~2% per minute) was exhibited in the toehold-mediated SDR with target only (Figure 4). In contrast, the exonuclease-assisted synergic reaction responded with a larger amplitude of faradaic current change probably due to the cooperative stimuli by the added target and the enzyme-released target. After the first 30 min of reaction, the velocity of SDR began to slow with the depletion of the original target DNA and saturate the signal gain after 50 min of enzymatic digestion. This signifies that the most, if not all, potential signal probes can fold back to their intrinsic stem-loop constructs by means of synergic SDR and signal amplification within ~50 min. Sensitive detection of target DNA Sensor sensitivity and dynamic response range are two other critical indexes for evaluating sensor performance in the real-world nucleic acid analysis. We next use the optimized etE-DNA sensor to challenge with a series of target DNA with concentrations ranging from 100 fM to 1 µM (Figure 5). It was noticed that the SWV peak current gradually increased along with the enhancement of target DNA concentration, suggesting a highly concentration-dependent signal response for quantitative assay. A dose-response calibration curve was fitted (inset in Figure 5), where the signal gain (%) was logarithmically related to the concentration of target DNA, spanning a linear dynamic range of 7 orders of magnitude (100 fM - 1 µM). The regression equation is expressed as s=14.13 lgc-14.97, (s is the signal change ratio and c is the concentration of the target DNA), with a correlation coefficient value (R2) of 0.994. A limit of detection of 42 fM could be estimated using 3σ/S (σ is standard deviation of the blank signal and S is the slope of the fit line in Figure 5



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

inset). Such a low detection limit for a 1:N input-output sensor is comparable to or even more sensitive than that of other structural responsive E-DNA sensors.51 This is probably attributed to the synergic effect of highly efficient SDR and homogeneous enzymatic catalysis, where the rigid duplex structure renders the toehold an “upright” orientation for enhanced molecular recognition, and the EATR can further accelerate the SDR. To better show the advantages of this etE-DNA sensor, the associated performance indexes (e.g., sensing strategy, detection limit, linear range) have been compared with those of other structure-switchable E-DNA sensors, and the result was outlined in Table 1. In comparison, our etE-DNA sensor has higher sensitivity and wider dynamic range, presumably due to the cooperative target recycling and molecular folding, as above-mentioned. Selectivity of the sensor Besides sensitivity, selectivity is another significant performance indicator for sensors. We next evaluated the discrimination capability of our eatE-DNA sensor by using multiple control oligonucleotides, such as single-base (1M), two-base (2M), three-base (3M) mismatched sequences and noncognate DNA sequence (NC) as targets. It was found that all the mismatched sequences could be readily distinguished from the fully complementary DNA, in which the concentration of the perfectly matched sequence (1 µM) was equal to that of the mismatched counterparts (1 µM) (Figure 6A). It was noticed that only the fully matched DNA exhibited prominent signal change (~120%), while the alteration caused by all the other mismatched oligonucleotides was negligible. Such an excellent selectivity of the sensor might benefit from the toehold design, in which a protruded functional domain (7-nt in length), serving as a fueling probe to trigger SDR, possesses an outstanding ability capable of differentiating single-base mismatch sequence.13 It should be noted that our novel sensor can easily distinguish a single-base mismatch DNA isothermally. This high selectivity of toehold mediated SDR was further verified by polyacrylamide gel electrophoresis analysis (Figure S5). It demonstrated that 1M DNA could displace the signal-toehold probe complex in part, which leaded to a relative weak duplex stripe


Page 12 of 26

Page 13 of 26

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


(the top) as compared to that in lane 3, where the 2M DNA is more difficult to displace the signal-toehold probe complex. In contrast, both of 3M DNA and the random sequence failed to trigger the SDR, which yielded two strong stripes in the lane 4 and 5. It is anticipated that the selectivity might be further improved by using highly specific locked nucleic acid or peptide nucleic acid as capture probes, and such an excellent selectivity combined with the femtomolar sensitivity will make our novel sensor more suitable for single nucleotide polymorphism analysis.15 Stability and anti-interference The stability and anti-interference of this solid DNA sensor were also investigated as they are two important indicators of biosensor for use in practical multicomponent biofluids. We found that the E-DNA sensor showed high stability (RSD = 3.3%) in the repeated SWV readout of the target-responsive signal, which was measured every 24 h for a week storage at 4oC (Figure S6). Impressively, it still retained its original displacement efficiency even after storage of half a month, and the signal change was less than 10%, indicating that our sensor has a good stability. To further demonstrate the superiority of the etE-DNA sensor, the sensor was challenged directly in real samples by spiking target DNA (1 µM) into 10% human serum and undiluted DEME cell-culture medium, respectively. From Figure 6B, we observed a negligible signal change (＜15%) as the sensor was incubated either in buffered saline or in the interfering substances (10% human serum and 100% DEME), despite their extremely complex and multicomponent nature that might degrade the performance of the sensor. This prominent anti-interference originates mainly from the outstanding sequence-specific hybridization, enzymatic catalysis and the toehold-dependent high-efficiency SDR. Conclusions In brief, we have tailored an etE-DNA sensor for single-step, highly sensitive and sequence-specific electrochemical readout of target sequence in complex matrices using a toehold and enzyme-mediated target-stimulative structurally switchable



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

Page 14 of 26

sensing design. This unique design features (i) the toehold-based high selectivity, (ii) enzymatic target recycling and (iii) one-step conformation-switchable signaling. This functional sensor appears to be more efficient than its traditional counterparts, and competes well with them in sensitivity and selectivity. Besides, this sensor has several impressive merits. For example, 1) the partially hybridized duplex design guarantees a protruded toehold domain that facilitates the probe-target binding; 2) surface-confined target-responsive SDR combined with homogeneous enzyme-assisted target recycling produces a synergic signaling mechanism; 3) our etE-DNA sensor is stable, and can be deployed directly in human serum (10%) with minimal signal degradation. We believe that, in combination with other emerging technologies, such as electric field enhanced

hybridization

and

single-step

microfluidic

electrochemical

array

sensing,52,53 this sensor will be more competent for use in practical clinical diagnostics. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 21305034, 21475034 and 21675041) and Hubei Province health and family planning scientific research project (Grant No. WJ2017Q032). Supporting Information Sequences and involved oligonucleotides, secondary structure of the DNA probes by NUPACK analysis, CV characterization of DNA probe assembly, polyacrylamide gel electrophoresis (PAGE) analysis and calculation of probe density based on chronocoulometric curves. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) (2) (3) (4) (5)

Seeman, N. C. Nature 2003, 421, 427-431. Jones, M. R.; Seeman, N. C.; Mirkin, C. A. Science 2015, 347. Zhang, D. Y.; Seelig, G. Nat Chem 2011, 3, 103-113. Zhu, J.; Zhang, L.; Li, T.; Dong, S.; Wang, E. Adv. Mater. 2013, 25, 2440-2444. Seelig, G.; Soloveichik, D.; Zhang, D. Y.; Winfree, E. Science 2006, 314, 1585-1588. (6) Song, T.; Xiao, S.; Yao, D.; Huang, F.; Hu, M.; Liang, H. Adv. Mater. 2014, 26, 6181-6185.


Page 15 of 26

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


(7) Shi, K.; Dou, B.; Yang, C.; Chai, Y.; Yuan, R.; Xiang, Y. Anal. Chem. 2015, 87, 8578-8583. (8) Dunn, K. E.; Trefzer, M. A.; Johnson, S.; Tyrrell, A. M. Sci. Rep. 2016, 6, 29581. (9) Zhang, D. Y.; Winfree, E. J. Am. Chem. Soc. 2009, 131, 17303-17314. (10) Xing, Y.; Yang, Z.; Liu, D. Angew. Chem. Int. Ed. 2011, 50, 11934-11936. (11) Khodakov, D. A.; Khodakova, A. S.; Linacre, A.; Ellis, A. V. J. Am. Chem. Soc. 2013, 135, 5612-5619. (12) Zhang, Z.; Wang, Y.; Fan, C.; Li, C.; Li, Y.; Qian, L.; Fu, Y.; Shi, Y.; Hu, J.; He, L. Adv. Mater. 2010, 22, 2672-2675. (13) Wang, D.; Tang, W.; Wu, X.; Wang, X.; Chen, G.; Chen, Q.; Li, N.; Liu, F. Anal. Chem. 2012, 84, 7008-7014. (14) Hwang, M. T.; Landon, P. B.; Lee, J.; Choi, D.; Mo, A. H.; Glinsky, G.; Lal, R. Proc. Natl. Acad. Sci. USA 2016, 113, 7088-7093. (15) Gao, Z. F.; Ling, Y.; Lu, L.; Chen, N. Y.; Luo, H. Q.; Li, N. B. Anal. Chem. 2014, 86, 2543-2548. (16) Yang, F.; Wang, S.; Zhang, Y.; Tang, L.; Jin, D.; Ning, Y.; Zhang, G.-J. Biosens. Bioelectron. 2016, 82, 32-39. (17) Xiao, Y.; Lubin, A. A.; Baker, B. R.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. USA 2006, 103, 16677-16680. (18) Fan, C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. USA 2003, 100, 9134-9137. (19) Jin, Y.; Yao, X.; Liu, Q.; Li, J. Biosens. Bioelectron. 2007, 22, 1126-1130. (20) Yao, W.; Wang, L.; Wang, H.; Zhang, X.; Li, L. Microchim. Acta. 2009, 165, 407-413. (21) Xiao, Y.; Qu, X.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2007, 129, 11896-11897. (22) Duan, R.; Lou, X.; Xia, F. Chem. Soc. Rev. 2016, 45, 1738-1749. (23) Zhao, Y.; Chen, F.; Li, Q.; Wang, L.; Fan, C. Chem. Rev. 2015, 115, 12491-12545. (24) Yeung, S. S.; Lee, T. M.; Hsing, I.-M. J. Am. Chem. Soc. 2006, 128, 13374-13375. (25) Dirks, R. M.; Pierce, N. A. Proc. Natl. Acad. Sci. USA 2004, 101, 15275-15278. (26) Chen, X.; Hong, C.-Y.; Lin, Y.-H.; Chen, J.-H.; Chen, G.-N.; Yang, H.-H. Anal. Chem. 2012, 84, 8277-8283. (27) Chen, Y.; Xu, J.; Su, J.; Xiang, Y.; Yuan, R.; Chai, Y. Anal. Chem. 2012, 84, 7750-7755. (28) Liu, S.; Wang, Y.; Ming, J.; Lin, Y.; Cheng, C.; Li, F. Biosens. Bioelectron. 2013, 49, 472-477. (29) Yang, H.; Gao, Y.; Wang, S.; Qin, Y.; Xu, L.; Jin, D.; Yang, F.; Zhang, G.-J. Biosens. Bioelectron. 2016, 80, 450-455. (30) Ji, H.; Yan, F.; Lei, J.; Ju, H. Anal. Chem. 2012, 84, 7166-7171. (31) Zhang, S.; Wu, Z.; Shen, G.; Yu, R. Biosens. Bioelectron. 2009, 24, 3201-3207. (32) Wang, J. Analyst 2005, 130, 421-426. (33) Wu, L.; Xiong, E.; Zhang, X.; Zhang, X.; Chen, J. Nano Today 2014, 9, 197-211. (34) Gerasimova, Y. V.; Kolpashchikov, D. M. Chem. Soc. Rev. 2014, 43, 6405-6438. (35) Xuan, F.; Luo, X.; Hsing, I. M. Anal. Chem. 2012, 84, 5216-5220. (36) Liu, S.; Wang, C.; Zhang, C.; Wang, Y.; Tang, B. Anal. Chem. 2013, 85, 2282-2288. (37) Wu, D.; Yin, B.-C.; Ye, B.-C. Biosens. Bioelectron. 2011, 28, 232-238. (38) Lee, H. J.; Li, Y.; Wark, A. W.; Corn, R. M. Anal. Chem. 2005, 77, 5096-5100.



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

(39) Tian, Y.; Wang, Y.; Xu, Y.; Liu, Y.; Li, D.; Fan, C. Sci. China Chem. 2015, 58, 514-518. (40) Zuo, X.; Xia, F.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2010, 132, 1816-1818. (41) Biagiotti, V.; Porchetta, A.; Desiderati, S.; Plaxco, K. W.; Palleschi, G.; Ricci, F. Anal. Bioanal. Chem. 2012, 402, 413-421. (42) Hsieh, K.; Xiao, Y.; Tom Soh, H. Langmuir 2010, 26, 10392-10396. (43) Chen, J.; Zhang, J.; Guo, Y.; Li, J.; Fu, F.; Yang, H.-H.; Chen, G. Chem. Commun. 2011, 47, 8004-8006. (44) Zhang, J.; Song, S.; Wang, L.; Pan, D.; Fan, C. Nat. Protoc. 2007, 2, 2888-2895. (45) Idili, A.; Amodio, A.; Vidonis, M.; Feinberg-Somerson, J.; Castronovo, M.; Ricci, F. Anal. Chem. 2014, 86, 9013-9019. (46) Xia, F.; White, R. J.; Zuo, X.; Patterson, A.; Xiao, Y.; Kang, D.; Gong, X.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2010, 132, 14346-14348. (47) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670-4677. (48) Shen, L.; Chen, Z.; Li, Y.; Jing, P.; Xie, S.; He, S.; He, P.; Shao, Y. Chem. Commun. 2007, 2169-2171. (49) Yao, B.; Liu, Y.; Tabata, M.; Zhu, H.; Miyahara, Y. Chem. Commun. 2014, 50, 9704-9706. (50) Watkins, H. M.; Simon, A. J.; Ricci, F.; Plaxco, K. W. J. Am. Chem. Soc. 2014, 136, 8923-8927. (51) Yu, Z.-g.; Lai, R. Y. Anal. Chem. 2013, 85, 3340-3346. (52) Kaiser, W.; Rant, U. J. Am. Chem. Soc. 2010, 132, 7935-7945. (53) Yang, F.; Zuo, X.; Li, Z.; Deng, W.; Shi, J.; Zhang, G.; Huang, Q.; Song, S.; Fan, C. Adv. Mater. 2014, 26, 4671-4676.


Page 16 of 26

Page 17 of 26

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


Table 1. Comparison between the proposed etE-DNA sensor and other conformation-switchable E-DNA sensors reported for nucleic acid assay. signaling mechanisma SL switch SL switch SDR triplex SL switch+AP pseudoknot switch SL switch+EXO SL switch+SDR+EXO

signal on/off

tagb and readout protocolc

off off on on on on on on

Fc, CV Ru, ECL MB, ACV MB, SWV MB, ACV MB, ACV MB, ACV MB, SWV

linear range 30 pM - 5 μM 1 pM - 0.1 µM 0.4pM - 20 nM 1 pM - 3 μM 2 nM - 100 nM 0.1 pM - 1 μM

a

LOD

ref

10 pM 0.5 pM 0.4 pM 1 pM 2 nM 2 nM 42 fM

18 20 17 45 51 21 42 this work

SL, stem-loop; AP, assisted probe; EXO, exonuclease. bFc, ferrocene; Ru, Ru(bpy)2(cbpy). c ECL, electrochemiluminescence; ACV, alternating current voltammetry.



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

Page 18 of 26

Figure Caption: Figure 1. Design and signaling mechanism of both exonuclease and toehold-assisted switchable etE-DNA sensor with single-step, "signal-on" readout. In the presence of both target DNA and Exo Ш, the toehold domain hybridizes with target molecule and triggers a rapid SDR by releasing toehold-target duplex into enzyme solution for selectively

digestion

and

target recycling.

Concurrently,

the

dehybridized

single-stranded signal probe self-folds into the original stem-loop structure, thus bringing the redox MB in proximity to the interrogating electrode surface for accelerated electron transfer and "signal-on" readout. This process synergizing with Exo-assisted

target

recycling

(N

cycles)

can

significantly

enhance

the

target-responsive electric signal. Figure 2. Typical SWV responses of Au/ signal-toehold duplex probe/MCH (a) in 20 mM Tris-HCl buffer (100 mM NaCl, 20 mM MgCl2), in the presence of (b) target DNA (1 µM), (c) both target DNA (1 µM) and Exo Ш (2 U/µL), (d) Exo Ш (2 U/µL) only. SWV measurements were performed by scanning the potential from -0.4 V to 0 V with a step potential of 1 mV, a frequency of 60 Hz, and an amplitude of 50 mV. Figure 3. Optimization of etE-DNA sensor. Effects of (A) the measurement frequency and (B) assembly concentration of the signal-toehold duplex probe on the sensor response to the perfectly matched target (1 µM) and Exo Ш (2 U/µL). (C) The intermolecular distance between surface-confined duplex probes depends on the probe immobilization concentration that can tune the hybridization efficiency at nanoscale. Error bars represent standard deviations of measurements (n = 3). Figure 4. The kinetic response of the etE-DNA sensor to (top curve) both the fully matched target (1 µM) and exonuclease (Exo Ш, 2 U/µL), (middle curve) the perfectly complementary target DNA (1 µM) only, and (middle curve) buffer without target and exonuclease. In the presence of only target DNA, sensors reached saturation in approximately 30 min (light green bar). In contrast, delayed signal saturation (~60 min, light red bar) occurred as both target DNA (1 µM) and Exo Ш (2 U/µL) are co-present, probably due to the multiple cycling of synergic reactions. Error bars represent standard deviations of measurements (n = 3). Figure 5. Typical SWV responses of the sensors to different concentrations of the target DNA, from bottom to top (arrow): 0 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM,


Page 19 of 26

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


10 nM, 100 nM and 1 µM in buffer (10 mM Tris-HCl, 100 mM NaCl, 20 mM MgCl2, pH 7.4). Logarithmic plot (inset) of signal gain (%) versus target sequence concentrations (from 0.1 pM to 1 µM). Error bars represent standard deviations of measurements (n = 3). Figure 6. (A) Selective response of the sensor to different DNA sequences, including target (T), 1M, 2M, 3M, R (random sequence) and Control (blank). All the sequences were in equal concentration at 1 µM. (B) Signal change (%) for the etE-DNA sensor challenged with target (1 µM) in buffer (20 mM Tris-HCl, 100 mM NaCl, 20 mM MgCl2) and complex matrices, such as 10% human serum and DMEM. Error bars represent standard deviations of measurements (n = 3).



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 157x126mm (300 x 300 DPI)


Page 20 of 26

Page 21 of 26

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 2 81x63mm (300 x 300 DPI)



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 3 139x58mm (300 x 300 DPI)


Page 22 of 26

Page 23 of 26

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





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



Page 24 of 26

Page 25 of 26

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 6 163x57mm (300 x 300 DPI)



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

For TOC only 86x69mm (300 x 300 DPI)


Page 26 of 26

Toehold Mediated One-Step Conformation-Switchable âSignal-On

Recommend Documents

Toehold Mediated One-Step Conformation-Switchable âSignal-On