Photoinduced Regeneration of an Aptamer-Based Electrochemical

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Photo-Induced Regeneration of An Aptamer-based Electrochemical Sensor for Sensitively Detecting Adenosine Triphosphate Xiaoyu Zhang, Chunxia Song, Ke Yang, Wenwen Hong, Ying Lu, Ping Yu, and Lanqun Mao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05442 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 25, 2018

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Analytical Chemistry

Photo-Induced Regeneration of An Aptamer-based Electrochemical Sensor for Sensitively Detecting Adenosine Triphosphate Xiaoyu Zhang,†,§ Chunxia Song,†,§ Ke Yang,† Wenwen Hong,† Ying Lu,*,† Ping Yu,‡ Lanqun Mao*,‡ †

Department of Applied Chemistry, Anhui Agricultural University, Hefei 230036, China

‡

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for

Living Biosystems, Institute of Chemistry, The Chinese Academy of Sciences (CAS), Beijing 100190, China

*

Corresponding Authors: Fax: + 86-551- 65785833. E-mails: [email protected], [email protected]

1

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Analytical Chemistry 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

ABSTRACT: Electrochemical aptasensors generally include three elements, i.e., recognition element, signal-transformation element, and regeneration element. In this study, a new ATP-aptasensor is developed by combining three elements into one DNA oligonucleotide chain. In the DNA oligonucleotide chain, DNA aptamer is used as the recognition element, ferrocene group attached at the 3’-end of the aptamer as the signal-transformation element, and azobenzene moiety embedded into the DNA chain as the regeneration element. In addition to the similar analytical properties with the traditional ones, the aptasensor developed here is easily regenerated with UV light irradiating. The current response recorded on the aptasensor increases with increasing the concentration of ATP in the incubation solution and is linear with the logarithm of ATP concentration in a range from 1 nM to 100 µM. The limit of detection is 0.5 nM (S/N = 3). The basal level of ATP in the rat brain cortex microdialysate is determined to be 21.33 ± 4.1 nM (n = 3). After being challenged with ATP, the aptasensor could be readily regenerated by UV light irradiation for more than seven cycles. The regeneration of the aptasensor is proposed to be regulated by conversing azobenzene from its trans- to cis-form under UV irradiation.

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Aptamers represent oligonucleotide- or peptide-molecules that bind a specific target molecule with high affinity.1-3 This intermolecular interaction features a high sensitivity, fast kinetics, and simplicity, which have intrigued considerable attention over the last decade in developing aptamer-based electrochemical sensors (i.e., aptasensors) for practical applications.4,5 Typically, the preparation of such sensors includes terminating an aptamer with redox moieties while linking the other end on a substrate electrode.6-8 Aptamers binding to target molecules can cause their conformation change to re-position the terminal redox center closer to or further away the surface of electrodes. This significant change in configuration of aptamer ligands is often used to generate electrochemical signals that are used as analytical signals for electroanalysis. Aptasensors of this category, known as the electrochemical aptamer switches, have proven their performance in selectivity and sensitivity.9 Furthermore, the fabrication is usually reagentless and cost-effective. These unique properties, however, are fairly compromised by their poor renewability because aptamers bind a target molecule strongly to re-activate the electrochemical active center for repeatable measurements. As such, regenerating aptasensors in a simple and reproducible way remains very essential to applying the aptasensors for real applications. Photo-regulation is an attractive stimuli mode since it operates remotely and accurately and well minimizes contamination for the reaction systems.10 Azobenzene is an ideal photoresponsive molecule that is characteristic of trans-to-cis conversion upon irradiation with particular wavelengths of light (i.e., photo-isomerization). Ultraviolet light, whose energy is comparable to the energy gap of the π-π* transition, can evoke trans-to-cis conversion of azobenzene while blue light with energy equivalent to the n-π* transition is able to help its reverse cis-to-trans isomerization.11-13 As azobenzene is introduced into the paired bases of a DNA chain and thus the DNA duplex becomes photo-responsive. Very interestingly, visible light irradiation has insignificant impact on the chemical structure of DNA duplexes if azobenzene is in a trans-form (a planar molecule), while the UV irradiation on trans-azobenzene can cause trans-to-cis conversion then the duplex would dissociate. 3



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This photo-stimulation paves a straightforward way to make transition between the hybridization and dissociation of azobenzene-tethered DNA duplexes. On the other hand, the identification and determination of adenosine triphosphate (ATP) has been an important but difficult issue, and this is particularly true for the measurements in rat brain due to its extreme low concentration.14,15 Electrochemical ATP biosensors with aptamers are demonstrated to be very useful for sensitive detection of ATP.16-18 In ATP aptasensors, the aptamer binds with two ATP units to form an ATP-aptamer adduct that would fold into a hairpin conformation. In the folding structure of the aptamer, there are two G-quartets to form a loop with four pairs of based duplex on its two ends. The ATP units would inset into the grooves in the loop area.19 For this reason, if the aptamer is immobilized on electrode surface to form an aptasensor, the regeneration of the aptasensor by loosening the folding aptamer to release ATP units from the grooves becomes an important issue. Here, we for the first time demonstrate a photo-induced method for the regeneration of the aptasensors by tailor-making a photo-switchable aptamer that has two azobenzene molecules inserted in the vicinity of its 5’-end. As reported previously, two azobenzene residues should be separated by two

natural

nucleotides.

The

sequence

of

the

aptamer

5’-SH-(CH2)6-AXCCXTGGGGGAGTATTGCGGAGGAAGGT-Fc-3’

is

rationally (X

residue

designed

as

denotes

an

azobenzene moiety, and Fc is short for ferrocene group). As shown in Scheme 1, when the aptamer-modified electrode is incubated in an ATP solution, one aptamer chain would combine two ATP molecules and then fold itself into a hairpin conformation with four pairs of bases positioning on its two ends. Due to the shortened distance between the redox moiety and electrode, the electron transfer of the redox moiety is facilitated. Upon UV light irradiation, the azobenzene moieties experience trans-to-cis isomerization. Therefore, the hairpin structure of the aptamer would be destructured by dissociation of the four pairs of bases. By this way, the electrode would be readily regenerated for the next detection of ATP. This study offers a new method for regeneration of 4


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electrochemical aptasensors, which would be of great importance in promoting aptasensors for practical applications.

EXPERIMENTAL SECTION Chemicals and Reagents. The 27-mer azobenzene-tethered ATP-binding aptamer, i.e., 5’-SH-(CH2)6-AXCCXTGGGGGAGTATTGCGGAGGAAGGT-NH2-3’, and the control aptamer containing no azobenzene, i.e., 5’-SH-(CH2)6-ACCTGGGGGAGTATTGCGGAGGAAGGT-NH2-3’ were synthesized and purified by Sangon Biotech Co. Ltd. (Shanghai, China). The aptamers were concentrated by distillation to 100 µM with 20 mM Tris buffer. The distilled aptamers were labeled with the ferrocene moiety (Fc) by adding 100 µM ferrocene carboxylic acid, 5 mM 1-ethyl-3-[(3-dimethylamino) propyl] carbodiimide, and 25 mM N-hydroxysuccinimide into the DNA solution; and the resulting mixture was incubated at room temperature for 2 h. Then, the Fc-labeled aptamer was purified by ultrafiltration. All aqueous solutions were prepared with ultrapure water (Millipore-D 24uv, Millipore Instruments, France). Preparation of Electrochemical Aptasensor. The electrochemical aptasensor was prepared on Au electrode (1.6 mm in diameter, CHI Instruments, Shanghai, China). Au electrode was polished with alumina powder (0.3 and 0.05 µm) and sonicated in acetone and ultrapure water (each for 3-5 min). The electrode was then electrochemically pretreated by consecutively cycling the potential between -0.2 and +1.6 V at 0.5 V s-1 in 0.5 M H2SO4 solution until a cyclic voltammogram characteristic of a clean Au electrode was obtained. An amount of 50 µL of azobenzene-tethered Fc-labeled ATP-binding aptamer was dropped onto the electrode, and the electrode was covered with a plastic cap to avoid the solution evaporation. After being kept at room temperature for 2 h, the electrode was immersed into 20 mM Tris buffer containing 0.10 M 3-mercaptopropionic acid for 10 min to further form a submonolayer at the unoccupied Au surface. Apparatus and Measurements. Electrochemical measurements were performed with a 5



computer-controlled electrochemical analyzer (Model CHI 660E, CHI Instruments, Shanghai, China) in a conventional three-electrode electrochemical cell with modified Au electrode as working electrode, a platinum wire as counter electrode, and Ag/AgCl electrode (KCl-saturated) as reference electrode. Differential pulse voltammetry (DPV) was performed with an initial potential of 0.1 V, amplitude of 0.05 V, and scan rate of 0.025 V s-1. The visible and UV light sources were a 60 W table lamp and a 6 W UV lamp (365 nm), respectively. To regenerate the aptasensors, the aptasensors previously being challenged were covered by a PE tube, in which 100 µL of Tris buffer (20 mM) was filled (Figure 1). The end of PE tube was cut off to permit irradiation of UV light. The UV lamp was placed on the above of the aptasensors. The regeneration was carried out by irradiating UV light for 1 h.

RESULTS AND DISCUSSION Figure 2 displays typical DPV responses at Au electrode modified with Fc-labeled and photoswitchable aptamer before and after the electrode was incubated in the Tris buffer containing ATP for 1 h. The peak current at +0.30 V for the oxidation of the Fc group clearly increases after the electrode was incubated in the aqueous solution of ATP. The peak current (Ip) increases with increasing the concentration of ATP in the incubation solution (inset in Figure 2) and is linear with the logarithm of ATP concentration in the range from 1 nM to 100 µM (Ip /µA = 0.15log CATP / nM + 0.082, R2 = 0.996) with a limit of detection of (0.5 nM (S/N = 3). To investigate the selectivity of the aptasensor, we treated the aptasensor with 100 µM of cytidine-5’-trophosphate (CTP), guanosine-5’-triphosphate (GTP), and uridine-5’-triphosphate (UTP) and we did not found significant current response when compared with that of ATP (Figure 3). This result show the aptasensor is very selective for the detection of ATP. With the apatasensor developed here, the basal level of ATP in the rat brain cortex microdialysate was determined to be 21.33 ± 4.1 nM (n = 3), which was consistent with the reported value.14 6


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The electrochemical aptasensor with the azobenzene-tethered aptamer was ready to be regenerated by using UV light irradiation. As shown in Figure 4, after being challenged with 100 µM of ATP and then irradiated by UV light for 1 h, the aptasensor was regenerated for more than seven cycles. The regeneration of the aptasensor was proposed to be regulated by the conversion of azobenzene from its trans- to cis-form under UV irradiation. To verify this mechanism, we carried out control experiments using ATP aptamer without being tethered with azobenzene and we did not find obvious changes in current response signal changes upon UV light irradiation under the same experimental conditions employed in Figure 4. This result suggests that the photo-induced isomerization of azobenzene functions as a chemical switcher in the on-and-off cycling of the aptasensor. The light-induced signal change of the aptasensors versus irradiation time was shown in Figure 5. When irradiated by UV light, the Ip values recorded with the azobenzene-tethered aptamer modified electrode decreases largely and reaches equilibrium after 1 h with continuous irradiation of UV light (black line). This decrease of Ip values was attributed to the UV light-induced trans-to-cis isomerization of azobenzene and the subsequent unfolding of the aptamer. As the aptamer was converted from a hairpin structure into a loosen structure, the distance between Fc moiety and the electrode surface becomes larger and the signal of Fc would decrease. As shown in Figure 5 (red and blue lines), when the aptamer was changed to azobenzene-free (i.e., without being tethered with azobenzene), or the irradiation light source was changed to be visible light, the current responses of the aptasensors did not change by the light irradiation. These results show that the UV light-induced change in the form of the azobenzene moiety plays a key role in the regeneration of the aptasensor.

CONCLUSION In summary, we have demonstrated a new approach to preparation of easily regenerated electrochemical aptasensors by using azobenzene-tethered aptamers as a probe. Under different light 7



sources, the conversion of azobenzene between trans- and cis-forms can change the conformation of the aptamer. In this case, azobenzene acts as a hinge inserted into the DNA chain of the aptamer. When the hinge is opened or closed, the DNA chain would fold or unfold consequently. This study may pave a new approach to the development of electrochemical aptasensors with an easily regenerated property, which is believed to be particularly useful to advance the aptasensors for real practical applications.

AUTHOR INFORMATION Corresponding Authors *

E-mail: [email protected]

*

E-mail: [email protected]

Author Contributions §

X.Z. and C.S. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is financially supported by NSFC of China (Grant Nos. 21305002 and 21705002).

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(18) Shen, L.; Chen, Z.; Li, Y.; Jing, P.; Xie, S.; He, S.; He, P.; Shao, Y. Chem. Commun. 2007, 2169-2171. (19) Huizenga, D. E.; Szostak, J. W. Biochemistry 1995, 34, 656-665.

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vis UV

Trans

Cis

：

：

N

N N

N

：Ferrocene (Fc)

：

ATP

Scheme 1 Schematic Illustration of the Mechanism of Photo-Renewable Electrochemical Aptasensor with Azobenzene-Tethered and Ferrocene-Labeled Aptamer as the Probe.

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UV

UV light

Tris buffer PE tube

Au electrode

Figure 1. Schematic illustration of the experimental setup for photo-regeneration of the electrochemical aptasensor.

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Figure 2. DPV responses recorded with the Fc-labeled photoresponsive aptamer-modified Au electrodes in 20 mM Tris buffer containing 140 mM NaCl before and after the electrodes were incubated with ATP solution in 20 mM Tris buffer with different concentrations of (from bottom to top) 0, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM and 100 µM. Inset, plot of current responses versus logarithm of ATP concentration.

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0.8

Ip / µA

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

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0.6

0.4

0.2

0.0 blank

CTP

GTP

UTP

ATP

Figure 3. The selectivity of the photo-regulated electrochemical aptasensor. The Ip values were recorded with the aptasensor after the separate incubation in 100 µM of CTP, GTP, UTP or ATP solution for 1 h.

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0.8

0.6

Ip / µA

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


0.4

0.2

0.0 0

1

2

3

4

5

6

7

N Figure 4. Graphics for the regeneration of the aptasensor toward 100 µM of ATP and after the aptasensor was irradiated by UV light for different cycles. N represents the times of regeneration cycles. Black symbols, the initial and regenerated signals of the aptasensor. Red symbols: current responses recorded with the aptasensor when the aptasensor was challenged with 100 µM of ATP.

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1.0

0.8

Ip / µA

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

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0.6

0.4

0.2

0.0 0

20

40

60

80

100

120

t / min Figure 5. Light induced change of the regenerated signals on the aptasensors as a function of irradiation time. Black symbol, azobenzene modified-aptamer irradiated by UV light. Blue symbol, azobenzene modified-aptamer irradiated by visible light. Red symbol: control aptamer (i.e., aptamer without being tethered with azobenzene) irradiated by UV light.

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Photoinduced Regeneration of an Aptamer-Based Electrochemical

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