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An Aptamer/Protein Proximity Binding-Triggered Molecular Machine for Amplified Electrochemical Sensing of Thrombin Jianmei Yang, Baoting Dou, Ruo Yuan, and Yun Xiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00827 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 10, 2017

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Analytical Chemistry

An Aptamer/Protein Proximity Binding-Triggered Molecular Machine for Amplified Electrochemical Sensing of Thrombin Jianmei Yang, Baoting Dou, Ruo Yuan, Yun Xiang* Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China E-mail: [email protected] (Y. Xiang). * Corresponding author. Tel.: +86-23-68252277 (Y. Xiang).

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ABSTRACT The development of convenient and sensitive methods without involving any enzymes or complex nanomaterials for the monitoring of proteins is of great significance in disease diagnostics. In this work, we describe the validation of a new aptamer/protein proximity binding-triggered molecular machinery amplification strategy for sensitive electrochemical assay of thrombin in complex serum samples. The sensing interface is prepared by self-assembly of three-stranded DNA complexes on the gold electrode. The association of two distinct functional aptamers with different sites of thrombin triggers proximity binding-induced displacement one of the short ssDNA from the surface-immobilized three-stranded DNA complexes, exposing a pre-locked toehold domain to hybridize with a methylene blue (MB)-tagged fuel ssDNA strand (MB-DNA). Subsequent toehold-mediated strand displacement by the MB-DNA leads to the release and recycling of the aptamer/protein complexes and the function of the molecular machine. Eventually, a large number of MB-DNA strands are captured by the sensor surface, generating drastically amplified electrochemical responses from the MB tags for sensitive detection of thrombin. Our signal amplified sensor is completely enzyme-free and shows a dynamic range from 5 pM to 1 nM with a detection limit of 1.7 pM. Such sensor also has a high specificity for thrombin assay in serum samples. By changing the affinity probe pairs, the developed sensor can be readily expanded as a more general platform for sensitive detection of different types of proteins.


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INTRODUCTION Simultaneous binding of two DNA linked-affinity ligands to one target molecule at distinct domains can bring the corresponding complementary oligonucleotides into close proximity, enabling dramatic increase of their local concentrations.1,2 The increased local DNA concentrations thus favor the hybridization of the separate DNA components that are otherwise unable to hybridize spontaneously.3 Such proximity binding effects have inspired the development of various assays for highly sensitive detection of proteins and other molecules with the first demonstration of the proximity ligation assay (PLA) of proteins.4 In a typical PLA approach, simultaneous recognition of one target molecule via a pair of proximity probes places the free two oligonucleotide termini into close position, which facilitates downstream DNA ligation of the two probes by cooperative hybridization to an external connector oligonucleotides. The ligated products are then amplified and detected by polymerase chain reaction (PCR) to achieve highly sensitive and indirect detection of proteins. With this approach, protein detections can be transformed into the assay of specific oligonucleotides that are formed by proximity ligation.5 Inspired by the successful establishment of the PLA mechanism, two new methods, the binding-induced DNA annealing6,7 and DNA assembly assays,8 have been developed by taking advantages of such proximity effects. Moreover, on the basis of this principle, Le and co-workers9 have reasoned that the increased local concentrations of DNA induced by the protein binding event should be able to accelerate the rate of a strand displacement reaction and then to initiate the strand displacement event. This new approach, termed binding-induced DNA strand displacement, could be applied to detect a variety of protein targets. The binding-induced DNA strand displacement is an interesting process that can convert specific protein recognition to DNA amplification or subsequent DNA assembly for signal generation. Several exquisite sensing platforms for protein assays have been fabricated based on this strategy, in which proximity probes were generally prepared by the conjugation of antibodies to the tails of the DNA strands.10,11


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The toehold-mediated strand displacement reaction (TSDR), first pioneered by Yurke et al.12 for the construction of a tweezer-like dynamic molecular machine, occurs when a shorter single-stranded DNA (ssDNA) of a dsDNA complex is displaced by an exterior intruding ssDNA with its binding to an overhang region (the toehold domain) to initiate the strand migration process.13 The TSDR is an enzyme-free and entropy-driven process that can take place at room temperature. These unique properties of TSDR have shown to be useful in the development of a number of non-enzymatic DNA-fueled molecular machines14,15 and programmable self-assembly of DNA tiles.16 Of particular interest is that TSDR can be used as an enzyme-free signal enhancement approach for amplified detection of various nucleic acid targets.17,18 In these systems, the target oligonucleotide sequences act as catalysts to trigger the operation of DNA-fueled molecular machines through a series of TSDRs to recycle and reuse the target sequences, thus leading to significant amplification of the signal outputs.19 Inspired by the wide application of TSDR in amplified detection of different nucleic acid targets, the integration of such mechanism in protein assays is expected to facilitate the establishment of new protein detection strategies without involving any enzymes. In this regard, we report here the fabrication of an enzyme-free electrochemical sensor for sensitive determination of thrombin in complex serums by coupling aptamer/protein proximity binding with DNA-fueled TSDR recycling signal amplification. Aptamers are synthetic short oligonucleotides obtained through a process of in vitro selection for specific, high-affinity binding to a broad range of target molecules.20,21 Interestingly, aptamers have long shelf-life, can be easily synthesized, manipulated and modified.22-24 Considering the nucleic acid nature of aptamers, they are particularly suitable for proximity-binding assay of proteins because the common DNA-antibody conjugation steps in this type of assays can be avoided to simplify the protocol. Importantly, different DNA-based amplification strategies can be incorporated into the assay approaches to achieve sensitive detection of more general targets. In our design, simultaneous recognition of the target thrombin by two aptamers triggers the binding-induced DNA strand displacement reaction to displace the blocker DNAs from the surface-immobilized, three-stranded DNA complexes, causing the exposure of the hidden toeholds. ACS Paragon Plus Environment

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Subsequently, the methylene blue (MB)-tagged fuel ssDNA strands (MB-DNA) hybridize with the exposed toehold regions to initiate the TSDR and lead to the release of the aptamer/protein proximity complexes and the attachment of the MB-DNA onto the electrode. The liberated aptamer/protein proximity complexes can thus be recycled to enable continuous operation of this molecular machine, resulting in the introduction of many MB-DNA onto the electrode with notably amplified signals to achieve sensitive detection of thrombin. This protein proximity binding-triggered molecular machine allows a simple and enzyme-free assay toward the target thrombin with high sensitivity and selectivity. With a high specificity, the fabricated sensor can be applied to determine thrombin in complex serum samples. EXPERIMENTAL SECTION Chemicals and materials: Tris-HCl, tris(2-carboxyethyl) phosphine hydrochloride (TCEP), 6-mercapto-1-hexanol (MCH), bovine serum albumin (BSA), mouse immunoglobin G (IgG) and thrombin were supplied by Sigma-Aldrich (St, Louis, MO). α-fetoprotein (AFP) was ordered from Abcam Co., Ltd. (Shanghai, China). The HPLC-purified synthetic oligonucleotides (Table 1) were provided by Sangon Biotechnology Inc. (Shanghai, China). Ultrapure water (specific resistance of 18.2 MΩ cm) was adopted in the whole experimental processes. Other reagents (analytical grade) involved in this work were used as received. Table 1. The designed oligonucleotide sequences involved in this work Oligonucleotides

Sequences

Substrate sequence (ST)

5’-SH-(CH2)6-TTTTTTCCGTAAGTTAGTTGGAGACGTAGTA GGGCGAACTAC-3’

Assistant DNA (AS)

5’-CTACGTCTCCAACTAACTTACGG-3’

Blocker DNA (BK)

5’-GTTCGCCCTA-3’

Proximity probe 1 (P1)

5’-GGTTGGTGTGGTTGGTTTTTTTTTTTTTTGTAGTTCG-3’

Proximity probe 2 (P2)

5’-AGTCCGTGGTAGGGCAGGTTGGGGTGACTTTTTTTTTT TTTTTGTAGTTCG-3’ ACS Paragon Plus Environment

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MB-tagged fuel ssDNA strand (MB-DNA)

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5’-GTTCGCCCTACTACGTCTCCAACTAACTTACGG-MB-3’

Fabrication of the sensing electrode: The gold electrode (AuE, 3 mm in diameter) was first pretreated using previously reported protocols.25 Briefly, the AuE was immersed into a fresh piranha solution (concentrated 98% H2SO4/30% H2O2, 3:1 in volume) for 30 min. This was followed by rinsing the electrode with water. Next, the AuE was polished with 0.3 and 0.05 m aluminum oxide slurries for 5 min, respectively, and washed with water. After that, the AuE was sonicated in ethanol and ultrapure water, and further electrochemically treated in 0.5 M H2SO4 by scanning the potential in the -0.3 V to 1.55 V range until a stable cyclic voltammogram was observed. Finally, the pretreated AuE was rinsed with water and dried in a nitrogen stream. The three-stranded DNA complexes were obtained by annealing the mixture of the thiolated substrate sequence (ST, 10 M), assistant DNA (AS, 12 M) and blocker DNA (BK, 12 M) in 20 mM Tris-HCl buffer (5 mM KCl, 100 mM NaCl, 2 mM MgCl 2, pH 7.4) at 95 °C for 10 min, followed by cooling down to 25 °C at 1 °C min-1. Prior to the immobilization of the three-stranded DNA complexes on the pretreated AuE, the complexes were treated with 10 mM TCEP for 1 h and then diluted with Tris-HCl to a final concentration of 0.8 M. Then, 10 L of the above complexes (0.8 M) was added onto the electrode and incubated for 2 h at room temperature. After rinsing with Tris-HCl buffer and drying with nitrogen, the resulting AuE was allowed to incubate with 10 L of 1 mM MCH for 2 h to obtain the sensing interface. Amplified electrochemical detection of thrombin: Firstly, the Tris-HCl buffer supplemented with proximity probe 1 (P1, 50 nM), proximity probe 2 (P2, 50 nM) and MB-DNA fuel strand (MB-DNA, 0.5 M) was incubated with thrombin at various concentrations for 90 min at room temperature. After that, the above mixture was transferred to the sensing AuE to incubate for 80 min. Finally, the electrode was rinsed with Tris-HCl buffer and electrochemical measurement was accomplished according to our


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previously reported procedure26 by using a conventional three-electrode system (Ag/AgCl as the reference electrode, platinum wire as counter electrode and the modified AuE as working electrode). Cyclic voltammetry (CV) from -0.1 to 0.6 V was performed in 0.1 M KCl solution containing 1 mM [Fe(CN)6]3-/4- at a scan rate of 50 mV s-1. Square wave voltammetry (SWV) from -0.5 V to -0.1 V was carried out in Tris-HCl buffer with a 25 mV amplitude signal at a step potential of 4 mV and a frequency of 25 Hz. RESULTS AND DISCUSSION

Scheme 1. Schematic diagram of the aptamer/protein proximity binding-triggered molecular machine for amplified electrochemical determination of thrombin. The graphical representation of our proposed signal amplification strategy based on aptamer/protein proximity binding-triggered molecular machine for sensitive electrochemical detection of thrombin is displayed in Scheme 1. The thermally annealed three-stranded DNA complexes (ST/AS-BK) are first self-assembled on AuE through the thiol groups on ST to prepare the sensing interface. During the thermal annealing process, AS and BK hybridize with ST to block the toehold domain (5 nt, the red region in Scheme 1) in the middle of ST and to expose a 3-nt tail at the 3’-terminus. Meanwhile, the two aptamer proximity probes (Apt15 in P1 and Apt29 in P2) are designed to have three functional regions: the thrombin binding aptamer region, the T14 poly(T) spacer to avoid any steric hindrance when binding to the thrombin target and the 8-nt tail sequence to trigger the strand displacement from the 3’-terminus ACS Paragon Plus Environment

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in ST. The complementary sequence between ST and BK is 2-nt longer than the counterpart of ST and the tail sequences of the proximity probes. Therefore, in the absence of thrombin, the proximity probes are unable to displace BK from the three-stranded DNA complexes because the hybrids of ST/BK have a higher stability than that of ST and the tail sequences of the proximity probes. In this case, the toehold domain in the middle region of ST is blocked and unavailable for binding with the MB-DNA. Subsequent TSDRs are therefore inhibited to capture the MB-DNA on AuE and the background signal can be suppressed as expected. However, after the introduction of thrombin, synchronous binding of the pair of proximity probes (P1 and P2) to one thrombin target molecule brings the proximity probes into close proximity with their local concentration increased substantially to allow for intramolecular binding-induced displacement reaction on the electrode surface.27,28 The intramolecular interaction leads to a significantly increased stability between ST and the tail sequences of the proximity probes than that of the ST/BK duplex, driving the formation of a more stable hybrid between ST and the tail sequences of the proximity probes by displacing BK from the three-stranded DNA complexes. The binding-induced displacement enables the formation of a highly stable closed-loop architecture29 and exposes the initially blocked toehold domain in the middle of ST. Consequently, the MB-DNA fuel strand (MB-DNA) hybridizes with this toehold region and triggers TSDR, resulting in the release of AS and the tail sequences of the proximity probe. The dissociated proximity probes thus quickly explore the neighboring sites to trigger another intramolecular binding-induced displacement reaction to expose more toeholds for binding with MB-DNA, leading to autonomous movement of the proximity probes on the electrode surface. As a result, many MB-DNA are captured by the electrode and significantly amplified signal output can be obtained for the detection of thrombin at low levels.


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Figure 1. CV responses corresponding to: (a) unmodified AuE, (b) (ST/AS-BK)/AuE and (c) MCH/(ST/AS-BK)/AuE. CV of [Fe(CN)6]3-/4- in 0.1 M KCl was applied to monitor the stepwise modification process of the sensing interface. As displayed in Figure 1, the unmodified AuE gives a pair of well-defined and reversible redox peaks (curve a) because of the excellent electrical conductivity of AuE. When the AuE was modified with the three-stranded DNA complexes (ST/AS-BK), dramatic decrease in redox currents and a significant peak separation can be observed (curve b), owing to the negative charges on the DNA phosphate skeletons that prevent the access and redox of [Fe(CN)6]3-/4- on the electrode surface. Upon being blocked with MCH, the redox currents show slight increases (curve c) due to the enhanced accessibility of the redox probes to the electrode interface. This is assumably ascribed to the fact that MCH could displace the physically adsorbed three-stranded DNA complexes (ST/AS-BK) from the electrode surface and force the ST/AS-BK complexes to orientate towards the electrolyte solution.30 These results reveal the successful construction of the sensing interface.


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Figure 2. SWV responses of the MCH/(ST/AS-BK)/AuE sensing electrodes incubated with: (a) Tris-HCl buffer, (b) MB-DNA fuel strand (0.5 M), (c) mixture of P1 (50.0 nM), P2 (50.0 nM) and MB-DNA (0.5

M) in the absence and (d) the presence of thrombin (100 pM). The feasibility of the aptamer/protein proximity binding-triggered molecular machine for amplified electrochemical assay of thrombin was verified by interrogating the sensor electrodes with different solutions. As depicted in Figure 2, No SWV peak is observed on the MCH/(ST/AS-BK)/AuE electrode (curve a) in Tris-HCl buffer, owing to the lack of electroactive species in this case. After incubating the sensor electrode with the MB-DNA alone (curve b) or the mixture of P1, P2 and MB-DNA without the addition of thrombin (curve c), the SWV peak currents exhibit slight increases because of the relatively low rate of the strand displacement reaction without the target molecules. Nevertheless, when the sensor electrode was incubated with the mixture of P1, P2 and MB-DNA in the presence of thrombin (100 pM), the aptamer pairs bind to the same target to trigger the displacement of BK by P1 and P2, resulting in the function of the molecular machine and the attachment of many MB-DNA on the electrode. Thus, a significant SWV peak (curve d) can be observed. The proof-of-concept results here suggest the potential of our sensor for sensitive determination of thrombin.

Figure 3. Experimental optimizations for amplified electrochemical detection of thrombin. (A) the immobilization concentration of ST/AS-BK and (B) the reaction time. Error bars: SD, n = 3. To achieve the optimal assay performance, the experimental conditions for thrombin assay with the developed method were optimized. We first employed different immobilization concentrations of ST/AS-BK to optimize the performance of the fabricated sensor with the reaction time of 120 min. As shown in Figure 3A, the SWV peak current increases with increasing concentration of the ST/AS-BK from 0.3 M to 0.8 M and tends to reach a maximum value at 0.8 M. After that, it decreases with ACS Paragon Plus Environment

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further increment of the ST/AS-BK concentration beyond 0.8 M. Such a phenomenon can be ascribed to the fact that the detection efficiency of the sensor is suppressed at a lower concentrations (0.3 and 0.5 M), while higher concentrations (1.0 and 1.2 M) could induce steric hindrance to inhibit the TSDR, preventing the capture of more MB-DNA on electrode. Therefore, the immobilization concentration of ST/AS-BK was fixed at 0.8 M for all subsequent experiments. Furthermore, the reaction time was evaluated. Figure 3B shows that the signal response increases along with the extension of the reaction time and tends to level off after 80 min. Therefore, the optimal reaction time for thrombin assay is 80 min.

Figure 4. (A) SWV current responses of the sensors to 0, 5, 10, 20, 50, 100, 200, 500 and 1000 pM of thrombin (from a to i). (B) Calibration plot of SWV peak current versus the logarithm of thrombin concentration. Error bars: SD, n = 3.

To examine the analytical performance of the assay method, the sensors were challenged with various concentrations of thrombin under optimal experimental conditions. According to Figure 4A, one can observe that the SWV peak current elevates with increasing thrombin concentration. In addition, according to Figure 4B, the value of the peak current linearly depends on the logarithm of thrombin concentration in the range from 5 pM to 1000 pM. The linear equation for thrombin detection is Y = -0.0170 + 0.2260 log c (R2 = 0.9943), where Y and c represent the SWV peak current and thrombin concentration, respectively. The detection limit is found to be 1.7 pM based on the 3σ calculation. The analytical performance comparison between the proposed thrombin assay method and other reported approaches with complex amplification strategies are shown in Table 2. Furthermore, the reproducibility of the assay strategy was investigated and a relative standard deviation of 4.5% corresponding to six


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repetitive measurements for 100 pM thrombin was obtained, indicating an acceptable reproducibility of the method. Table 2. Analytical performances of different methods for thrombin detection Detection technology

Signal amplification

Linear range

Detection limit

Fluorescence

Nanomaterialbased amplification

0.6 nM-100 nM

0.20 nM

31

Colorimetry

Enzymatic amplification

50 pM-5 nM

20 pM

32

Electrochemilumi nescence

Nanomaterial-base d amplification

0.01 nM-10 nM

6.3 pM

33

Electrochemistry

Enzymatic amplification

1 nM-200 nM

0.1 nM

34

Electrochemistry

Nanomaterial-base d amplification

0.3 nM-50 nM

10 pM

35

Electrochemistry

DNAzyme assisted amplification

10 pM-50 nM

5.6 pM

36

Electrochemistry

TSDR-mediated amplification

5 pM-1 nM

1.7 pM

Reference

This work

Figure 5. Selectivity assessment of our method for thrombin (100 pM) against other interference proteins (1000 pM of AFP, BSA, IgG) by comparing the SWV peak current responses. Error bars: SD, n = 3.

We further examined the selectivity of the fabricated molecular machine by measuring its response to thrombin against three other interference proteins, AFP, BSA, and IgG (Figure 5). As expected, under


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the experimental conditions, the developed sensor shows significant current response to the target thrombin (100 pM), while the current responses of the interference proteins (with their concentrations 10 folds higher than thrombin) are close to that of the blank (in the absence of the thrombin). These results suggest that our sensor is specifically responsive to the target thrombin and the specificity of the assay is satisfactory. The high specificity of the sensor largely arises from its distinct feature that the activation of the molecular machine necessitates simultaneous recognition of the same target molecule by the aptamer pairs.

Figure 6. SWV responses of the sensing platform to diluted serum samples spiked with thrombin at 0, 20, 100 and 500 pM (from a to d). Table 3. Results for thrombin determination in diluted serum samples (n = 3) Samples Added Founda Recovery (%) RSD (%)

a

1

20 pM

20.4 pM

102

2.7

2

100 pM

103.4 pM

103.4

3.2

3

500 pM

488.2 pM

97.6

3.6

The mean of three measurements.

To demonstrate the applicability of the fabricated sensor for real samples, 10-fold diluted healthy serum samples (from the 9th people’s hospital of Chongqing) with different concentrations of spiked thrombin (20, 100 and 500 pM) were measured. The typical SWV responses corresponding to the three different measurements were displayed in Figure 6. The recoveries for thrombin are from 97.6% to 103.4% with relative standard deviation (RSD) between 2.7% and 3.6% (Table 3), which is acceptable for quantitative assays performed in complex real samples.


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CONCLUSIONS In conclusion, this work has demonstrated a simple, sensitive and selective aptamer/protein proximity binding-triggered molecular machine approach for electrochemical detection of thrombin without involving any enzymes. Synchronous binding of the proximity probe pairs to one target thrombin molecule triggers the displacement of the blocker DNA from the three-stranded DNA complexes and the exposure of the pre-locked toehold region in the middle of the substrate sequence. Subsequent TSDRs lead to the capture of many MB-DNA onto the sensor interface with significantly amplified signal response for thrombin assay. The developed electrochemical sensor shows three attractive features. Firstly, the signal amplification of the sensor is realized without the participation of any enzymes or nanomaterials. Secondly, the method has high sensitivity because the aptamer/protein complexes are recycled in the detection protocol. Thirdly, the sensor offers great potential for the detection of other proteins by using the corresponding affinity pairs. With these advantages, this strategy can be used to develop more universal sensing platform for simple, selective and sensitive determination of proteins. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (Nos. 21275004 and 21675128). REFERENCES (1) Zhang, H. Q.; Li, F.; Dever, B.; Wang, C.; Li, X. F.; Le, X. C. Angew. Chem. Int. Ed. 2013, 52, 10698-10705. (2) Li, F.; Tang, Y. N.; Traynor, S. M.; Li, X. F.; Le, X. C. Anal. Chem. 2016, 88, 8152-8157. (3) Zong, C.; Wu, J.; Liu, M. M.; Yang, L. L.; Liu, L.; Yan, F.; Ju, H. X. Anal. Chem. 2014, 86, 5573-5578. (4) Fredriksson, S.; Gullberg, M.; Jarvius, J.; Olsson, C.; Pietras, K.; Gústafsdóttir, S. M.; Östman, A.; Landegren, U. Nat. Biotechnol. 2002, 20, 473-477. (5) Zhang, L.; Zhang, K. X.; Liu, G. C.; Liu, M. J.; Liu, Y.; Li, J. H. Anal. Chem. 2015, 87, 5677-5682. ACS Paragon Plus Environment

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For TOC only:


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Protein Proximity Binding-Triggered Molecular Machine for

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