A chimeric aptamers-based and MoS2 nanosheet-enhanced label

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A chimeric aptamers-based and MoS2 nanosheet-enhanced label-free fluorescence polarization strategy for ATP detection Yao-Yao Fan, Zhao-Li Mou, Man Wang, Jun Li, Jing Zhang, Fuquan Dang, and Zhi-Qi Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04107 • Publication Date (Web): 23 Oct 2018 Downloaded from http://pubs.acs.org on October 25, 2018

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Analytical Chemistry

A chimeric aptamers-based and MoS2 nanosheet-enhanced label-free fluorescence polarization strategy for ATP detection Yao-Yao Fan,†, ‡ Zhao-Li Mou,†,‡ Man Wang,† Jun Li,† Jing Zhang,† Fu-Quan Dang† and Zhi-Qi Zhang†

, ‡*

†

Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China ‡ Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry (Shaanxi Normal University), Ministry of Education, Xi’an 710062, China *Corresponding Author. [email protected] ABSTRACT: Adenosine triphosphate (ATP) as a primary energy source plays an unique role in the regulation of all cellular events. The necessity to detect ATP requires sensitive and accurate quantitative analytical strategies. Herein, we present our study of developing a MoS2 nanosheet-enhanced aptasensor for fluorescence polarization-based ATP detection. A bifunctional DNA strand was designed to consist of chimeric aptamers that recognize and capture ATP and berberine, a fluorescence enhancer. In the absence of ATP, the DNA strand bound to berberine will be hydrolyzed when Exonuclease I (Exo I) is introduced, releasing berberine as a result. On the contrary, when ATP is present, ATP aptamer folds into a G-quadruplex structure, thus the complex can resist degradation by Exo I to maintain berberine for fluorescent detection purpose. In addition, to magnify the fluorescence polarization (FP) signal, MoS2 nanosheets were also adopted in the system. This nanosheets-enhanced FP strategy is simple and facile which does not require traditional dye-labeled DNA strands and complex operation steps. The developed fluorescence polarization aptasensor showed high sensitivity for the quantification of ATP with a detection limit of 34.4 nM, performing well both in buffer solution and in biological samples.

Adenosine triphosphate (ATP) is the ubiquitous energy cur-

cently, Zhao et al. designed a novel multifunctional adenosine

rency which plays a critical role in a lot of biochemical reac-

aptamer holding the ability of recognizing adenosine and cap-

tions such as regulating cellular metabolism processes and

turing the malachite green.19 The aptamer-based methods have

biochemical pathways.1,2 Therefore, it is of great significance

been applied in ATP monitoring using detection methods of

to establish an accurate and sensitive method for specific ATP

fluorescence resonance energy transfer (FRET) and electro-

monitoring. Numerous ATP analytical methods have been

chemistry luminescence.20-23 Nevertheless, fluorescent meth-

reported,3-6 particularly focusing on developing small molecule

ods usually requires tedious labeling process, which gives rise

or aptamer probes combined with florescence and nanotech-

to efficiency and accuracy concerns 24. A label-free fluorescent

nologies.

aptasensor is generally more attractive due to its simplicity, high sensitivity and fast response time.25,26

Aptamers are functional oligonucleotides generated from an in vitro selection process, with the ability to recognize plenti-

The fluorescence polarization (FP) method is promising in

ful targets such as small molecules, biomacromolecules, and

analyzing a wide range of targets.27-29 At constant temperature

organisms.7-11 Their superior binding specificity and affinity

and solution viscosity, the FP value is sensitive to changes in

was determined by their unique nucleotide sequences as well

the rotational motions of fluorophore-labeled molecules de-

as three-dimensional folded conformations.

12-14

Currently,

pending upon the molecular weight (or molecular volume) of

and the aptamer-

fluorescent molecules.30,31 FP, as an intrinsic parameter, is

based biosensor has become attractive in analytical applica-

less sensitive to confounding elements such as photobleaching

tions because of its advantages of cheap and easy synthesis,

and instrumental parameters.27 However, it is challenging to

chemical and thermal stability, and in situ monitoring capabil-

develop a general method of mass amplification to deal with

hundreds of aptamers have been reported

ity.

16-18

15

To improve the detection ability, designing a novel

the fact that most of molecular weights are too small to make

multipurpose aptamer probe can be a promising strategy. Re-

detectable FP changes.32,33 To obtain an enhanced FP signal,

1 ACS Paragon Plus Environment

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nanomaterials were usually adopted in the system to improve

Human serum samples were obtained from the Shaanxi Nor-

the detection sensitivity. These include but not limited to gold

mal University Hospital, and all other chemical reagents were

32-

of analytical grade without further purification and commer-

and carbon nano-

cially available. Ultrapure water used throughout all experi-

2D transition metal dichalcogenide nanosheets,

ments was obtained from a Millipore water purification system

nanoparticles, 35

31

SiO2 nanoparticles,

28

graphene oxide (GO),

metal-organic frameworks (MOFs)

materials.

29

36

(Z18 MΩ, Milli-Q, Millipore).

like MoS2, for example, with single- and few-layers became attractive worldwide in recent years. Despite the ability

Preparation of the MoS2 Nanosheets. A previously report-

MoS2 nanosheet exhibits in quenching the fluorescence of

ed hydrothermal method for the synthesis of MoS 2 nanosheets

organic dyes and the finding of its better adsorption affinity to ssDNA over dsDNA,

37-39

40

was used with some modification. In brief, Na2MoO4·2H2O

effort of exploration of the MoS2

(0.25 g) and Lcysteine (0.50 g) were dissolved in 25 mL water

nanosheets in biosensing application remains in early stages

and stirred for 10 min at 40 °C, respectively. After the two

today.

solutions were mixed, the pH of mixture was approximately

In present work, employing berberine as the fluorescence

adjusted to 5 with HCl (0.1 M). The mixture was put into a

reporter, along with the chimeric berberine binding-aptamer

100-mL Teflon-lined autoclave and was kept at 220 °C for 30

(BBA) and ATP binding-aptamer (ABA), and the MoS2

hours. After the autoclave cooling down, the black product

nanosheets as the fluorescence polarization signal magnifier,

was taken out, washed successively with deionized water and

we built a label-free system for ATP detection. The proposed

ethanol thrice, and finally dried at 60 °C for 12 hours in open

sensing strategy for ATP detection was displayed in Scheme 1.

air.

Scheme 1. Illustration of a chimeric aptamers -based and

ATP Fluorescence Signal Measurement. DNA stock solu-

MoS2 nanosheet-enhanced label-free fluorescence polariza-

tion (10 μM) of 6 μL and berberine solution (1 mM) of 6 μL

tion strategy for ATP detection

were added into 138 μL of Tris-HCl buffer (20 mM, pH 7.6, 60 μM MgCl2,75 mM NaCl) containing various concentration ATP to react for 70 min at 20 °C. Thereafter, Exo I (12U) was added and reacted again at 37 °C for 45 min. The fluorescence signal of 523 nm was then measured at an excitation wavelength of 364 nm using an LS55 fluorescence spectrometer (PerkinElmer Inc., USA). ATP Fluorescence Polarization (FP) Measurement. After 150 μL reaction system containing 400 nM DNA, 40 μM berberine and various concentrations of target ATP were preincubated in Tris-HCl buffer for 70 min at 20 °C and reacted with Exo I (12U) at 37 °C for 45 min, MoS2 nanosheets solu-

EXPERIMENTAL SECTION

tion (100 μg mL-1 ) of 8 μL was added, and then the fluores-

Chemicals and Solvents. Adenosine 5′-triphosphate (ATP),

cence polarization (FP) values were then collected using an

thymidine 5′-triphosphate (TTP), uridine 5′-triphosphate

LS55 fluorescence spectrometer with the excitation and emis-

(UTP), cytosine 5′-triphosphate (CTP) and guanosine 5′-

sion wavelengths of 364 nm and 523 nm respectively and cal-

triphosphate (GTP) were obtained from Shanghai Sangon Bio-

culated using the following equation (1):

technology (Shanghai, China). Exo I solution (20000 U/mL) was provided by New England Biolabs (Beijing, China) and stored at -20 °C prior to use. All DNA were synthesized and

Ivv and Ivh are the fluorescence intensities parallel and perpen-

HPLC-purified by Shanghai Sangon Biotechnology (Shanghai,

dicular to the excitation plane respectively. G is the instrumen-

China) (Table S1) and the stock solution of DNA was pre-

tal correction factor, correcting for the different detection effi-

pared by dissolving the lyophilized powder in distilled water.

ciencies of vertical and horizontal emission pathways, and

2

G=Ihv/Ihh.41

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Analytical Chemistry Characterization for MoS2 Nanosheets. Power X-ray dif-

inserted in the strand with or without the addition of ATP.

fraction (PXRD) characterization was performed using a

Accordingly, the fluorescence intensity value difference be-

Bruker D8 Advance Powder X-ray Diffractometer (Germany).

fore and after the addition of ATP, ΔFI, of the sensing system

The Cu Kα radiation was at a wavelength of 1.542 Å and the

gradually increased with the T base number extended from 0

data were collected from 10° to 70°. Electron microscopy

to 21 while declined with further extension, indicating that the

characterization was performed using a JEOL JEM-2100

insert of poly T strand with number of 21 mer could maxi-

Transmission Electron Microscope (TEM) at 200 kV. The

mumly improve the sensitivity of ATP detection. Compared

samples were prepared on carbon-film-covered copper grids

with ABA,26 BBA-T21-ABA enhanced the ΔFI over 5.5

and images were obtained.

times (Figure S2). Thus, it was chosen for the following experiments.

RESULTS AND DISCUSSION Design and Optimization of Chimeric Aptamers. Berberine oligomer-specific DNA aptamer was identified by the Pei et al. with an improved affinity chromatography-based SELEX, which has great potential in a variety of fluorescent biosensor applications.41 Wei et al. developed a label-free fluorescent aptasensor for detecting ATP using berberine as the fluorescence probe.26 Inspired by these reports, a bifunctional DNA strand that combines BBA and ABA was designed to detect ATP. Taking berberine as the fluorescence reporter, the experiment results showed that the conjugated chimeric DNA, BBA-ABA, has the maximum intensity of emission compared with ABA and BBA (Figure S1), indicating feasibility of our fluorescent strategy. Exo I preferentially hydrolyze ssDNA from its 3′ in a specific manner. The designed BBA-ABA ssDNA has a random coil structure and it maintains in this structure when berberine is introduced and captured, with intensified fluorescence emission of berberine at 523 nm. In the absence of ATP, the BBAABA was digested by Exo I, making the fluorescence intensity weakened. While when ATP is present, the ABA aptamer on 3′ prime of BBA-ABA bound specifically to its target, switching its secondary structure from random coil to G-quadruplex, which can resist attack from Exo I. This makes the fluorescence probe berberine remain bound to BBA, keeping its en-

Figure 1. The affection of number of poly T strand on ATP detec-

hanced fluorescence intensity.

tion. (A) Fluorescence spectra and (B) Fluorescence intensity. Berberine, 40 μM; number of poly T in BBA-ABA-derived DNA

According to the previously reported assay, extending the

(400 nM), 0, 3, 9, 15, 21, and 27, respectively; ATP, 106.7 μM.

poly T at the 5′-end of ABA had little influence on the interaction between ATP and ABA, which makes it an applicable

Optimization of Nanomaterials. It has been widely report-

spacer when designing chimeric aptamers.24 To avoid the in-

ed that nanosheet materials can enhance FP signal and im-

terfere between the functions of BBA and ABA, a series of

prove the sensitivity of detection.31-36 Comparing the FP en-

BBA-ABA chimeric aptamers with different number of poly T

hancement effects, the ΔFP with MoS2 nanosheets was higher

in the middle (Table S1) were tested in terms of their fluores-

than that with GO (Figure S3), which indicated that the MoS2

cent performance. It was seen in Figure 1A that the fluores-

nanosheets as FP enhancer was better than GO in this system.

cence intensity gradually increased with the number of T base



Further study showed that the difference of enhancement ef-

in the presence of ATP because of the complex of berberine

fects between MoS2 and GO probably lies in the different ef-

and G-quadruplex away from nanosheet surface, as a result of

fect of Exo I (Figure S4). Exo I as a protein having amino

diminished π-π stacking interactions between the honeycomb

groups could interact with GO through hydrogen bonding,

surface of nanomaterials and the aromatic rings of the nucleo-

causing the more complex of berberine and DNA desorption

tide bases of G-quadruplex.

from GO than MoS2.

Next, the amount of the MoS2 nanosheets was optimized to maximize the assay sensitivity with FP method. Too low concentration was insufficient to adsorb the berberine-DNA complex on the MoS2 surface, while excess amount of MoS2 nanosheets would result in the attachment of berberine-Gquadruplex complex on redundant nanomaterials. The ΔFP value of this sensor platform enhanced with gradually increasing the concentration of MoS2 nanosheets from 0 to 5.33 μg

Figure 2. Characterization for MoS2 Nanosheets. (A) PXRD pat-

mL-1 while declined with further MoS2 addition (Figure S5).

tern, (B) TEM image.

Hence, 5.33 μg mL-1 MoS2 was chosen as the optimal amount for next experiments. Optimization of the Assay Conditions. To further achieve the highest sensitivity of ATP detection, a series of other experimental conditions was optimized for fluorescence assay and FP assay. It was found that Mg2+ concentration and NaCl concentration could affect signals of this aptasensor. The maximum ΔFI and ΔFP were both obtained following the addition

Figure 3. The affection of MoS2 nanosheets (5.33 μg mL-1) on

concentration of Mg2+ at 60 μM (Figure S6A and S7A) and

ATP (106.7 μM) detection. (A) Fluorescence intensity method

NaCl at 75 mM (Figure S6B and S7B), respectively.

and (B) FP method.

The emission at 523 nm of the complex of BBA-T21-ABA

The synthesized MoS2 nanosheets were first characterized.

and berberine quickly decreased with the Exo I dosage in-

The significant intense peaks in Powder X-ray Diffraction

creasing from 4 to 12 U and no further decrease was observed

(PXRD) pattern were at 13.76°, 33.64°, 39.77°, and 59.41°

as dosage went higher (Figure S6C), and the FP signal reached

(Figure 2A) corresponding to the planes of MoS2 (0 0 2), (1 0

platform (Figure S7C), suggesting that the BBA-T21-ABA

43

0), (1 0 3) and (1 1 0) (JCPDS Card No. 37-1492). The la-

was digested sufficiently at Exo I dosage of 12 U. Subsequent-

mellar structure of the MoS2 nanosheet was shown in Figure

ly, the enzymatic kinetic behaviors were further explored

2B via a TEM image. Furthermore, affections of MoS2

(Figure S6D and S7D) and the complete reaction could be

nanosheets on fluorescence intensity and fluorescence polari-

finished within 45 min.

zation were investigated. Figure 3A showed that MoS2

Based on the above results, 60 μM of Mg2+ and 75 mM of

nanosheets elicited a little bit decrease on ΔFI instead of caus-

NaCl, 12 U of Exo I and 45 min of enzymatic hydrolysis time

ing enhancement. Whereas the addition of MoS2 nanosheets

were taken for further ATP assay.

(5.33 μg mL-1) dramatically enhanced the ΔFP over 2.7 times higher than that without MoS2 nanosheets (Figure 3B). Although comparison of ΔFPs showed promising results, it is worth noting that absolute value of FP decreases with addition of ATP, with or without MoS2 nanosheets presence. When absence of ATP, the rotation of berberine probe was depended upon its entirety through the van der Waals force and π-π stacking interactions. On the contrary, lower FP was obtained


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Figure 4. (A) Fluorescence spectra of system in the presence of various concentration of ATP (0, 0.3, 0.8, 5.33, 13.3, 26.7, 40, 53.3, 66.7, 80, 106.7, 133.3, 160 μM). (B) Fluorescence intensities at different concentrations of ATP (inset: linear relationship between (FI-FI0) / FI0 and ATP concentration, where FI0 repreFigure 5. FP values in the presence of various concentration of

sents the fluorescence intensity without ATP, and FI represent the

ATP (inset shows the linear relationship between (FP 0-FP) / FP0

fluorescence intensities at different ATP concentrations).

and logarithm of ATP concentration, where FP0 represents the

Sensitivity for ATP Detection. As illustrated in Figure 4A,

fluorescence polarization without ATP, and FP represents the

a gradual increase of the solution fluorescence intensity at 523

fluorescence polarization at different ATP concentrations).

nm with the concentration of ATP from 0 to 160 μM was In order to investigate the storage stability of the FP assay

clearly seen. By plotting fluorescence intensity against the

components (aptamers, berberine, Exo I, MoS2 nanosheets),

concentration of ATP, a calibration curve for ATP was ob-

the repeatability (inter-day) of the FP and ΔFP values were

tained (Figure 4B). The linear correlation to the ATP concen-

evaluated (Figure S8) for this aptasenor during seven day in

tration was at a range of 0.3-40 μM with a correlation coeffi-

the absence and presence of ATP (26.7 μM), respectively. The

cient of 0.999. The detection limit (3σ) of this method for ATP

RSDs of FP were both less than 1.0% and RSD of ΔFP was

was estimated to be 123 nM and lower than that of previously

1.4%. These results indicated that the assay components have

reported platform based on ATP aptamer and berberine

of very good storage stability and repeatability.

probe.26 It suggests that higher sensitive ATP detection is real-

Selectivity of the Proposed Aptasensor. The selectivity of

ized with this chimeric aptamers.

this proposed strategy was evaluated under the same condi-

As the Figure 5 shows, the FP values of the interaction sys-

tions by challenging this system against other ATP analogs

tem gradually decreased as the concentration of ATP from 0 to

including UTP, TTP, CTP and GTP. It was apparent that only

133.3 μM. The calibration plot of (FP0-FP) / FP0 versus the

ATP can induce significant fluorescence intensity and FP val-

logarithm of ATP concentration showed a high-quality linear

ue change. Neither significant changes in fluorescence intensi-

relationship (R = 0.996) from 0.067-26.7 μM (FP and FP0 are

ty nor in FP value were observed following the addition of

the fluorescence polarization values in the presence and ab-

other ATP analogs, indicating that this method is highly selec-

sence of ATP, respectively), and the detection limit (3σ) of

tive for ATP (Figure 6).

this FP strategy for ATP was as low as 34.4 nM. This detection limit obtained with this strategy is lower than most of ATP aptasenors previously reported.24, previous labeled-aptasenor,

23

3-6

Compared to our

although the detection limit is

slightly worse, much wider linear range was obtained and the tedious labeling process was avoided.

Figure 6. Selectivity of ATP detection. (A) fluorescence intensity method, (B) fluorescence polarization method. ATP, 100 μM; ATP analogs (UTP, CTP, GTP, and TTP), 100 μM, respectively.



Application in Real Sample Detection. To further evaluate

Corresponding Author

the feasibility of the proposed method in real biological sam-

*E-mail: [email protected]

ples, the ATP spiked in human serum was detected. The aver-

Notes

age recoveries obtained for the 5% diluted human serum sam-

The authors declare no competing financial interest.

ples were in the range of 97.1-109.3% for the fluorescence intensity method and 93.1-102.8% for the FP method (Table

ACKNOWLEDGMENT

S2 and Table S3). And relative standard deviations (RSDs)

This work is financially supported by National Natural Science

were in the range of 2.2-4.9%, revealing that the ability of this

Foundation of China (No. 21775100).

strategy is reliable and practical for detection of ATP in com-

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A chimeric aptamers-based and MoS2 nanosheet-enhanced label

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