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Ultrasensitive and label-free detection of ATP by using gold nanorods coupled with enzyme assisted target recycling amplification
Journal Pre-proof Ultrasensitive and label-free detection of ATP by using gold nanorods coupled with enzyme assisted target recycling amplification Ning Xue, Shujie Wu, Zongbing Li, Xiangmin Miao PII:
S0003-2670(19)31559-4
DOI:
https://doi.org/10.1016/j.aca.2019.12.073
Reference:
ACA 237356
To appear in:
Analytica Chimica Acta
Received Date: 14 October 2019 Revised Date:
2 December 2019
Accepted Date: 28 December 2019
Please cite this article as: N. Xue, S. Wu, Z. Li, X. Miao, Ultrasensitive and label-free detection of ATP by using gold nanorods coupled with enzyme assisted target recycling amplification, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.073. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.
*Graphical Abstract
Ultrasensitive and label-free detection of ATP by using gold nanorods coupled with enzyme assisted target recycling amplification Ning Xue, Shujie Wu, Zongbing Li, Xiangmin Miao* School of Life Science, Jiangsu Normal University, Xuzhou 221116, China
An ultrasensitive and label-free sensor was designed to detect ATP coupling the fluorescence quenching of positively charged gold nanorods ((+)AuNRs) with exonuclease
(Exo
) assisted target recycling amplification.
Ultrasensitive and label-free detection of ATP by using gold nanorods coupled with enzyme assisted target recycling amplification Ning Xue, Shujie Wu, Zongbing Li, Xiangmin Miao* School of Life Science, Jiangsu Normal University, Xuzhou 221116, China
Corresponding Author *Tel.: +86-516-83403170 E-mail address: [email protected]
ABSTRACT Abnormal concentration of adenosine triphosphate (ATP) is directly asscociate with several diseases. Thus, sensitive detection of ATP is essential to early diagnosis of disease. Herein, we described an ultrasensitive strategy for ATP detection by using positively charged gold nanorods ((+)AuNPs) as an efficient fluorescence quenching platform, coupled with exonuclease
(Exo
) assisted target recycling amplification.
To construct the sensor, DNA template that contained ATP aptamer was used for the formation of AgNCs signal probe (DNA/AgNCs), the structure of it could change to duplex after the interaction of it with ATP. Such DNA template or duplex DNA product could electrostatically adsorb onto (+)AuNRs surface, resulting in the quenching of the fluorescence signal due to the vicinity of AgNCs to (+)AuNRs. With the addition of Exo
, DNA duplex could be hydrolyzed and released from (+)AuNRs
surface, leading to the recovery of a strong fluorescent signal, while ATP could be regenerated for next target recycling. Combing the good fluorescence quenching ability of (+)AuNRs and the Exo
assisted signal amplification, a low detection limit
of 26 pM was achieved for ATP detection. Notably, the proposed method can be successfully applied for detecting ATP in serum samples, indicating a potential application value in early cancer diagnosis. Keywords: Fluorescent sensor, (+)AuNRs, DNA/AgNCs, ATP detection, Exo Target recycling amplification
,
1. Introduction Adenosine triphosphate (ATP) plays an important role in the regulation of cellular metabolism and can supply energy for the regulating of various cell life activities [1]. In addition, the variation of ATP concentration is directly associated with several diseases including angiocardiopathy, Parkinson’s disease and malignant tumors [2]. Thus, developing efficient methods for ATP detection is crucial for the disease diagnose at an early stage. Currently, a series of biosensing methods have been developed for ATP detection [3-9]. Thereinto, fluorescence method as one type of promising analytical techniques have been widely used for sensors preparation due to the advantages of it including selectivity, ease of operation and fast analysis [10-14]. To enhance the detection sensitivity of ATP, various signal amplification strategies, including enzyme-assisted target recycling amplification [15-19], hybridization chain reaction
(HCR)
[20,21],
rolling
circle
amplification
(RCA)
[22],
nanomaterial-mediated signal amplification [23,24] and target-catalyzed hairpin assembly [25-27] have been developed. Thereinto, enzyme-assisted signal amplification, especially Exo
assisted target recycling amplification, has attracted
tremendous attention due to the advantages of it such as high selectivity, low immunogenicity, and can be realized at isothermal condition. Based on of these, numbers of labeled [28-34] or label-free [35,36] biosensors have been reported for ATP detection coupled with the Exo
assisted signal amplification.
Gold nanorods (AuNRs), as an anisotropic nanomaterial, possess the advantages of
easy preparation and unique optical properties [37], which shows more predominant performance in optics and electrics in the longitudinal plasmon band than gold nanoparticles (AuNPs) [38]; The surface plasmon resonance band of AuNRs endow with the higher adsorption cross section than AuNPs, and the extinction coefficient of it is much larger than AuNPs [39]. Moreover, AuNRs present prominent properties of super-quenching efficiency to a fluorophore when fluorescent dyes are located in their vicinity [40]. Thus, AuNRs have been suggested as an efficient nanomaterial for the preparation of fluorescent sensors. For example, Wang’s group developed the fluorescent sensors for spermine and heparin detection using gold nanorods [41,42]; Yan et al realized the miRNA detection based on the electrostatically binding of gold nanorods with nucleic acid probe [43]; Du’s group reported the visualization of endogenous hydrogen sulfide based on Au nanorods@silica enhanced fluorescence [44]; et al [45-49]. However, the main limitation of these methods are their low sensitivity (nanomolar level, nM), which limiting the widely application of them for biomolecular analysis. To the best of our knowledge, no analytical platform has yet been developed for ATP detection coupling the fluorescence quenching of AuNRs with Exo
assisted target recycling amplification.
Inspired by the high fluorescence quenching ability of (+)AuNPs and the effective signal amplification of Exo
, in this work, a fluorescent sensor was designed for
ultrasensitive ATP detection by subtle combining AuNRs with Exo
assisted target
recycling amplification. AgNCs were utilized as a stable fluorescence signal probe, which was synthetized by using the unmodified DNA template, avoiding the labelling
of DNA with fluorophore. Notably, taking advantages of the unique fluorescence quenching ability of (+)AuNRs coupled with Exo
assisted signal recycling
amplification, sensitive detection of ATP was realized with a detection limit of 26 pM. In addition, such strategy possess good selectivity for ATP detection because of the high affinity of the aptamer-target interaction. 2. Experimental section 2.1. Reagents and chemicals Cetyltrimethylammonium bromide (CTAB), sodium oleate (NaOL), hydrochloric acid (HCl, 37 wt%), ascorbic acid (AA) and sodium borohydride (NaBH4) were purchased from Aladdin Biotech CO. Ltd. (Beijing, China). Exonuclease III (Exo III) was obtained from Thermo scientific Reagent Co. Ltd. (USA). Chloroauric acid (HAuCl4), ATP, cytidine triphosphate (CTP), gytidine Triphosphate (GTP), uridine Triphosphate (UTP), and thymidine triphosphate (TTP) were obtained from Sigma-Aldrich Co. Ltd. (Shanghai, China). Tris-HCl was purchased from X-Y Biotech CO. Ltd. (1.0 M, pH 8.0, Shanghai, China), and was diluted to 20 mM before using. DNA/AgNCs were prepared according to our previous method with a concentration of 100 µM [50], which were formed on the 13C bases of the DNA template. The fluorescence intensity of DNA/AgNCs were determined at 560 nm with an excitation wavelength of 430 nm. DNA template was synthesized by Sangon Biotechnology
(Shanghai,
China)
with
the
sequence
5'-CCCCCCCCCCCCCACCTGGGGGAGTATTGCGGAGGAAGGT-3',
of which
containing two parts: the AgNCs formation template (italic section) and the ATP
aptamer (underlined section). 2.2. Apparatus The fluorescence spectra were recorded using the Agilent Cary Eclipse G9800A fluorescence spectrophotometer (Agilent, America). All UV-vis absorption spectra were obtained with a UV-260 spectrometer (Thermo Fisher Corporation, USA). TEM images of (+)AuNRs and AgNCs were recorded by transmission electron microscope (TEM, JEM-2010HR, Japan). The zeta potential of the proposed AuNRs were recorded using the particle size analyzer (MS2000, England). 2.3. Synthesis of (+)AuNRs (+)AuNRs were synthesized according to the literature method by seed-mediated growth containing two steps [51]. Gold seed formation: Firstly, all glass instruments were immersed in aqua regia for more than 24 h. After that, 60 µL of freshly prepared ice-cold NaBH4 (0.01 M) was added to the mixed solution containing 5.0 mL of CTAB (0.2 M) and 5.0 mL of HAuCl4 (0.5 mM), following by stirring rapidly for 2 min at 30 °C. At this time, the color of the mixed solution changed from transparent to yellowish brown, indicating the formation of gold seed solution. Then, the seed solution was stirred vigorously for 2.0 min, and maintained at 30 °C for 2.0 h. Preparation of the growth solution: Briefly, 0.35 g CTAB and 0.0617 g NaOL were dissolved in 12.5 mL of warm water (50 °C). After that, the solution was cooled down to 30 °C and mixed with 4.0 mM of AgNO3 solution. Subsequently, 12.5 mL of HAuCl4 (1.0 mM) was added into above solution and kept undisturbed at 30 °C for 15 min, following by stirring for 90 min (700 rpm). Then, 0.05 mL of HCl (1.0 M) was
introduced to adjust the pH. After another slowly stirring (400 rpm) for 15 min, 0.0625 mL of ascorbic acid (AA, 0.064 M) was added and vigorously stirred for 30 s. For AuNRs formation, 0.02 mL of prepared seed solution was injected into the growth solution, and the solution was allowed to sit overnight at 30 °C. At last, the product was isolated by centrifugation (7,000 rpm) for 30 min to remove excess CTAB. 2.4. Detection procedure of ATP Detection of ATP was constructed in Tris-HCl buffer (20 mM). In the typical process, 100 µL of (+)AuNRs solution was mixed with 80 µL of prepared DNA/AgNCs (1.0 µM) and incubated at room temperature for 30 min. Subsequently, different concentrations of ATP were added into above mixture, respectively, and followed by incubating for 60 min at room temperature. After that, 35 U of Exo III was added and incubated at 37 °C for 45 min, and then incubated at 75 °C for 10 min to inactivate. Before detecting, the mixture was diluted to 300 µL with Tris-HCl buffer solution. For fluorescence measurements, the excitation wavelength and the emission wavelength were set at 430 and 560 nm, the slit width for both excitation and emission were set at 10 nm, and the samples were scanned from 500 to 700 nm. 3. Results and discussion 3.1. Detection principle of the sensing system for ATP The working principle of the sensing platform for ATP detection was illustrated in Fig. 1, DNA/AgNCs were employed to produce a strong fluorescent signal. After the incubation of such DNA/AgNCs with (+)AuNRs, the fluorescence signals of them were effectively quenched due to the electrostatic absorption of them with (+)AuNRs
and the vicinity of AgNCs to (+)AuNRs. In the presence of ATP, DNA duplex that contained a recessed end was formed based on the specific interaction of ATP with DNA template, such DNA duplex presented stronger electrostatic adsorption with (+)AuNRs, and the fluorescence signal of AgNCs decreased further. Upon addition of Exo III, such DNA duplex could be hydrolyzed and released from (+)AuNPs surface, which directly resulted in the recovery of a strong fluorescence signal. Meantime, ATP will returned to the solution and reacted with another DNA/AgNCs to realize enzyme-assisted target recycling amplification. And the recovery of the fluorescent intensity was proportional with the concentrations of ATP, which could be detected by using a fluorescence spectrophotometer with a scanning range from 500 to 700 nm. Fig. 1 3.2. Characteristics of the nanomaterials TEM image showed the size and disperse states of (+)AuNRs. As shown in Fig. 2A, an obvious rod-like structure of (+)AuNRs was emerged with an average length of 50 nm and an average width of 20 nm. The transverse and longitudinal surface plasmon absorptions of such (+)AuNRs observed from UV-vis results were 508 nm and 766 nm (Fig. 2C, curve a). In addition, the zeta-potential of such AuNRs monitored from the particle size analyzer was +65.3 mV (Fig. 2B). Meantime, the stability of such (+)AuNRs was investigated by using UV-vis spectra (Fig. 2C). Compared with (+)AuNRs (curve a), the absorption spectra and solution colors of (+)AuNRs did not change obviously while adding 50 mM of BSA (curve b), NaCl (curve c), metal ions
(including K+, Ca2+, Mg2+, Al3+, curve d), and ATP (curve e), respectively, indicating a good stability of (+)AuNRs in complex biological samples. From TEM result it could be seen that AgNCs were spherical with uniform distribution and particle size of about 2.0 nm (Fig. 2D). To further prove the formation of DNA/AgNCs, UV-vis adsorption spectra were applied and two characteristic absorption peaks at 265 nm and 445 nm were appeared for DNA/AgNCs (curve b), while only one peak at 265 nm obtained for DNA template (curve a), which directly indicating the formation of DNA/AgNCs (Fig. 2E). Fig. 2 3.3. Characterization of the sensor The stability of DNA/AgNCs can affected the characteristics of the sensor. Thus, DNA/AgNCs were stored and measured every 0.5 h to monitor the stability of them. As shown in Fig. 3A, the fluorescence signal of DNA/AgNCs still maintain 98.6% of the corresponding original response after 4.0 h, demonstrating a good stability of DNA/AgNCs. Moreover, the feasibility of the sensor for ATP detection was investigated in Fig. 3B, and a strong fluorescence signal appeared for DNA/AgNCs (curve a). Then, an obvious fluorescence quenching happened after the mixing of DNA/AgNCs with (+)AuNRs (curve b), such result mainly due to the electrostatic interaction of DNA/AgNCs with (+)AuNRs, directly resulted in the vicinity of AgNCs to (+)AuNRs. In the presence of 5.0 nM of ATP, DNA duplex formed based on the specific interaction between ATP and DNA template, inducing the fluorescence signal decreased further (curve c), because of the strong electrostatic interaction of
negatively charged DNA duplex onto (+)AuNRs surface. After adding 35 U of Exo III, the fluorescence intensity increased greatly (curve d), indicating the release of AgNCs from (+)AuNRs and the recovery of the fluorescence signal, which obviously illustrated that the DNA duplex could be effectively hydrolyzed by Exo III from the 3’ terminus. 3.4. Circular dichroism spectroscopy of DNA probe Circular dichroism (CD) spectroscopy was used to investigate the formation of dsDNA. As shown in Fig. 3C, DNA probe showed a weak positive Cotton effect peak around 278 nm and a weak negative Cotton effect peak around 244 nm (a). After the addition of 5.0 nM of ATP, the intensity at the peak of 278 nm and 244 nm increased obviously (b), indicating the formation of dsDNA structure. However, when such dsDNA product was incubated with 35 U of Exo III, the peak is substantially disappeared, these results might be attributed to the cleavage of dsDNA product (c). Fig. 3 3.5. Optimization of the experimental conditions To ensure the ATP detection was constructed at optimal conditions, the reaction time between (+)AuNRs and DNA/AgNCs, the concentration of (+)AuNRs, the reaction time of ATP with DNA template, the amount of Exo III, and the incubating time of Exo III with DNA duplex were optimized. From Fig. 4A, we can see that the fluorescence signal decreased with increasing reaction time of (+)AuNRs with DNA/AgNCs from 0 to 30 min, and then reached a plateau after 30 min. Thus, the optimal time of 30 min was selected in our work. Fig. 4B displayed the effect of the
concentration of (+)AuNRs. It could be seen that the change of the fluorescence signal (∆F, ∆F=Fthe
value of curve b-Fthe value of curve a)
increased along with the increase of
(+)AuNRs concentration in the range of 0-15 nM, and reached a maximum value at 15 nM. Therefore, 15 nM of (+)AuNRs was chosen for the following study. Just as shown in shown in Fig. 4C, a maximum fluorescence signal was obtained while the reaction time between ATP and DNA template reached 60 min, illustrating that the DNA template could effectively interact with ATP within 60 min. Meanwhile, the fluorescence intensity increased along with the increase of Exo III amount from 5.0 to 35 U, and reached a plateau after that (Fig. 4D). So, 35 U of Exo III was selected in the experiments. In addition, it could be seen from Fig. 4E that the fluorescence signal increased while the incubating time between Exo III and DNA duplex increased from 5 to 45 min. Thus, 45 min was used as an optimal incubating time of Exo III with DNA duplex. Fig. 4 3.6. Performance of the sensor for ATP detection Under optimal conditions, the quantitative detection of ATP was shown in Fig. 5. It was found that the fluorescence intensity increased dramatically along with the increase of ATP concentration from 50 pM to 30 nM (Fig. 5A), and a linear range was obtained between 50 pM to 1.0 nM with a detection limit of 26 pM (S/N = 3) (Fig. 5B, insert). The linear regression equation for ATP was F= 54.40+122.50 c (where F is the fluorescent intensity and c is the concentration of ATP), with a correlation coefficient of R2 =0.9968. The detection performance of the present work was compared with
literatures, and the results were listed in Table 1. Taking advantage of the good fluorescence quenching ability of (+)AuNRs coupled with Exo III assisted target recycling amplification, the detection limit of our proposed method was much lower than most of other recently reported fluorescence based methods. Furthermore, we also investigated the selectivity of the sensor for ATP detection by using the other analogous molecules including GTP, CTP, UTP and TTP as the interfering substances (Fig. 5C), due to the fact that the similar molecule structures of them may interfere the practical detection of ATP. It could be seen that 5.0 nM of ATP induced a strong fluorescence enhancement, while the fluorescence change was weak and even can be ignored for 50 nM of GTP, CTP and UTP, respectively. Such results clearly indicated that the fluorescence sensor was highly specific for ATP detection. Meanwhile, five different batches of the system were prepared under the same conditions to detect 1.0 nM of ATP for evaluating the reproducibility of the sensor. As shown in Fig. 5D, the five batches performed similar fluorescence responses, with the RSD that less than 10%, indicating acceptable reproducibility of the sensor. Fig. 5 Table 1 3.7. Application of the fluorescent sensor To construct the addition and recovery experiments of ATP in real samples, the clinical serum sample was collected from healthy donors in Xuzhou central hospital in Jiangsu Province, China. As shown in Table 2, a good recovery that in the range of 95.3–103.5% was obtained, with the RSD that less than 10%, indicating that the
proposed system provided an effective platform for the assay of ATP in real biological samples. Table 2 4. Conclusions In conclusion, an ultrasensitive and label-free detection of ATP was realized based on the fluorescence quenching of (+)AuNRs coupled with Exo recycling amplification. The utilization of (+)AuNRs and Exo
associated target
realized the sensitive
detection of ATP detection with a detection limit of 26 pM, which were comparable with many of other fluorescent methods. Moreover, the detection of ATP was realized mainly based on the electrostatic adsorption of the negatively charged DNA template with (+)AuNRs, which greatly simplifying the detection process of ATP. Notably, the proposed method possess high selectivity due to the utilization of ATP aptamer, indicating a potential application value in early cancer diagnosis. Acknowledgements This work was supported by the Natural Science Foundation of Xuzhou City (KC18140), the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18-2141).
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Figure captions Fig. 1 Schematic illustration of ATP detection by using (+)AuNRs as the fluorescence quenching platform coupled with Exo
assisted target recycling amplification.
Fig. 2 (A) TEM image of (+)AuNRs; (B) UV–vis absorption spectrum of (+)AuNRs; (C) UV-vis absorption spectra of (+)AuNRs (curve a) upon addition with 50 mM of BSA (curve b), NaCl (curve c), metal ions (including K+, Ca2+, Mg2+, Al3+, curve d), and ATP (curve e), respectively, Inset showed the corresponding color of (+)AuNRs in different solutions; (D) TEM image of DNA/AgNCs; (E) UV–vis absorption spectra of the DNA template (curve a) and the DNA/AgNCs (curve b). Fig. 3 (A) The stability of DNA/AgNCs after kept for different times (a to i: 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 h); (B) The fluorescence spectra of DNA/AgNCs (a), (b) after the incubation of DNA/AgNCs with (+)AuNRs, (c) “(b)” in the presence of 5.0 nM of ATP, (d) “c” in the presence of 35 U of Exo
; (C) CD spectra of 8.0 µM of DNA probe (a), dsDNA product (b) and DNA
fragments in the presence of 35 U of Exo
(c).
Fig. 4 (A) Effect of the reaction time of AuNRs with DNA, (B) the concentration of AuNRs in the absence (a), and presence of 1.0 nM of ATP and 35 U of Exo DNA template and 1.0 nM of ATP, (D) the amount of Exo Exo
(b); (C) the reaction time between ; (E) the incubation time of 35 U of
with DNA duplex on the fluorescent responses of the system. The error bars indicated
the standard deviations of three replicates.
Fig. 5 (A) Fluorescence spectra of the system in the presence of ATP with concentrations of 0.05, 0.1, 0.3, 0.5, 0.7, 1, 3, 5, 10, 20, and 30 nM, respectively; (B) The relationship between the fluorescence intensity and the concentration of ATP (insert: calibration curve); (C) Selectivity of the sensor for 5.0 nM of ATP compared with the interfering substances containing 50 nM of UTP, CTP, GTP and TTP; (D) The reproducibility of five different batches of the system for the detection of 1.0 nM of ATP.
Table 1 Comparison of our method with other fluorescence-based works Materials
Linear range
Detection limit
Selectivity
Reference
Ag@SiO2 nanoparticles
100 nM – 5 mM
14.2 nM
UTP, GTP, CTP
6
Water-soluble carbon nanotubes
0. 1 – 1 mM
24 µM
ADP, AMP, UTP, GTP, CTP
52
G-quadruplex DNAs
0.1 – 60 µM
33 µM
UTP, GTP, CTP
53
Polydopamine nanospheres
20 – 600 nM
8.32 nM
UTP, GTP, CTP
54
PDANTs
0.35 – 800 µM
150 nM
UTP, GTP, CTP
10
Copper nanoparticles
0.2 – 50 µM
93 nM
UTP, GTP, CTP
55
G-quadruplex
0.05 – 1 µM
18 nM
UTP, GTP, CTP
56
Graphene oxide
0.1 – 5 nM
80 pM
UTP, GTP, CTP
57
DNA/Ag nanoclusters
0.5 – 8.0 µM
91.6 nM
UTP, GTP, CTP
5
Berberine and Exo I
0.5 – 50 µM
140 nM
UTP, GTP, CTP
3
0.3 – 2.0 mM,
0.27 mM
UTP, GTP, CTP
7
Gold nanoparticles
0.5-10 µM
100 nM
UTP, GTP, CTP
58
AuNP@SiO2
100 pM-50 uM
43 pM
UTP, GTP, CTP
59
Gold nanorods
50 pM-1.0 nM
26 pM
UTP, GTP, CTP, TTP
Our method
a
AuNCs-Gr
a
b
PDANTs: polydopamine nanotubes AuNCs-Gr: gold nanocrosses and graphene quantum dots
b
Table 2 Detection results of ATP in serum samples (n=5) Samples
Added (pM)
Detected (pM)
Recovery (%)
RSD, n=3(%)
1
50
48.6
97.2
4.3
2
100
103.5
103.5
3.6
3
300
285.9
95.3
4.7
4
700
713
101.8
5.1
5
1000
1006.8
100.7
6.2
The authors declared that they have no conflicts of interest to this work.
*Highlights An ultrasensitive and label-free sensor was designed to detect ATP coupling the fluorescence quenching of positively charged gold nanorods ((+)AuNRs) with exonuclease
assisted target recycling amplification, and several merits contained in
the system: 1. (+)AuNRs with individual fluorescence quenching ability were utilized for the detection of ATP; 2. A low detection limit of 26 pM was obtained for ATP detection due to the combination of (+)AuNRs with Exo
assisted target recycling amplification;
3. Highly stable DNA/AgNCs were utilized as good fluorescence signal probes to detect ATP. 4. The sensing system possess high selectivity for ATP detection due to the employment of ATP aptamer.
S0003-2670(19)31559-4
DOI:
https://doi.org/10.1016/j.aca.2019.12.073
Reference:
ACA 237356
To appear in:
Analytica Chimica Acta
Received Date: 14 October 2019 Revised Date:
2 December 2019
Accepted Date: 28 December 2019
Please cite this article as: N. Xue, S. Wu, Z. Li, X. Miao, Ultrasensitive and label-free detection of ATP by using gold nanorods coupled with enzyme assisted target recycling amplification, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.073. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.
*Graphical Abstract
Ultrasensitive and label-free detection of ATP by using gold nanorods coupled with enzyme assisted target recycling amplification Ning Xue, Shujie Wu, Zongbing Li, Xiangmin Miao* School of Life Science, Jiangsu Normal University, Xuzhou 221116, China
An ultrasensitive and label-free sensor was designed to detect ATP coupling the fluorescence quenching of positively charged gold nanorods ((+)AuNRs) with exonuclease
(Exo
) assisted target recycling amplification.
Ultrasensitive and label-free detection of ATP by using gold nanorods coupled with enzyme assisted target recycling amplification Ning Xue, Shujie Wu, Zongbing Li, Xiangmin Miao* School of Life Science, Jiangsu Normal University, Xuzhou 221116, China
Corresponding Author *Tel.: +86-516-83403170 E-mail address: [email protected]
ABSTRACT Abnormal concentration of adenosine triphosphate (ATP) is directly asscociate with several diseases. Thus, sensitive detection of ATP is essential to early diagnosis of disease. Herein, we described an ultrasensitive strategy for ATP detection by using positively charged gold nanorods ((+)AuNPs) as an efficient fluorescence quenching platform, coupled with exonuclease
(Exo
) assisted target recycling amplification.
To construct the sensor, DNA template that contained ATP aptamer was used for the formation of AgNCs signal probe (DNA/AgNCs), the structure of it could change to duplex after the interaction of it with ATP. Such DNA template or duplex DNA product could electrostatically adsorb onto (+)AuNRs surface, resulting in the quenching of the fluorescence signal due to the vicinity of AgNCs to (+)AuNRs. With the addition of Exo
, DNA duplex could be hydrolyzed and released from (+)AuNRs
surface, leading to the recovery of a strong fluorescent signal, while ATP could be regenerated for next target recycling. Combing the good fluorescence quenching ability of (+)AuNRs and the Exo
assisted signal amplification, a low detection limit
of 26 pM was achieved for ATP detection. Notably, the proposed method can be successfully applied for detecting ATP in serum samples, indicating a potential application value in early cancer diagnosis. Keywords: Fluorescent sensor, (+)AuNRs, DNA/AgNCs, ATP detection, Exo Target recycling amplification
,
1. Introduction Adenosine triphosphate (ATP) plays an important role in the regulation of cellular metabolism and can supply energy for the regulating of various cell life activities [1]. In addition, the variation of ATP concentration is directly associated with several diseases including angiocardiopathy, Parkinson’s disease and malignant tumors [2]. Thus, developing efficient methods for ATP detection is crucial for the disease diagnose at an early stage. Currently, a series of biosensing methods have been developed for ATP detection [3-9]. Thereinto, fluorescence method as one type of promising analytical techniques have been widely used for sensors preparation due to the advantages of it including selectivity, ease of operation and fast analysis [10-14]. To enhance the detection sensitivity of ATP, various signal amplification strategies, including enzyme-assisted target recycling amplification [15-19], hybridization chain reaction
(HCR)
[20,21],
rolling
circle
amplification
(RCA)
[22],
nanomaterial-mediated signal amplification [23,24] and target-catalyzed hairpin assembly [25-27] have been developed. Thereinto, enzyme-assisted signal amplification, especially Exo
assisted target recycling amplification, has attracted
tremendous attention due to the advantages of it such as high selectivity, low immunogenicity, and can be realized at isothermal condition. Based on of these, numbers of labeled [28-34] or label-free [35,36] biosensors have been reported for ATP detection coupled with the Exo
assisted signal amplification.
Gold nanorods (AuNRs), as an anisotropic nanomaterial, possess the advantages of
easy preparation and unique optical properties [37], which shows more predominant performance in optics and electrics in the longitudinal plasmon band than gold nanoparticles (AuNPs) [38]; The surface plasmon resonance band of AuNRs endow with the higher adsorption cross section than AuNPs, and the extinction coefficient of it is much larger than AuNPs [39]. Moreover, AuNRs present prominent properties of super-quenching efficiency to a fluorophore when fluorescent dyes are located in their vicinity [40]. Thus, AuNRs have been suggested as an efficient nanomaterial for the preparation of fluorescent sensors. For example, Wang’s group developed the fluorescent sensors for spermine and heparin detection using gold nanorods [41,42]; Yan et al realized the miRNA detection based on the electrostatically binding of gold nanorods with nucleic acid probe [43]; Du’s group reported the visualization of endogenous hydrogen sulfide based on Au nanorods@silica enhanced fluorescence [44]; et al [45-49]. However, the main limitation of these methods are their low sensitivity (nanomolar level, nM), which limiting the widely application of them for biomolecular analysis. To the best of our knowledge, no analytical platform has yet been developed for ATP detection coupling the fluorescence quenching of AuNRs with Exo
assisted target recycling amplification.
Inspired by the high fluorescence quenching ability of (+)AuNPs and the effective signal amplification of Exo
, in this work, a fluorescent sensor was designed for
ultrasensitive ATP detection by subtle combining AuNRs with Exo
assisted target
recycling amplification. AgNCs were utilized as a stable fluorescence signal probe, which was synthetized by using the unmodified DNA template, avoiding the labelling
of DNA with fluorophore. Notably, taking advantages of the unique fluorescence quenching ability of (+)AuNRs coupled with Exo
assisted signal recycling
amplification, sensitive detection of ATP was realized with a detection limit of 26 pM. In addition, such strategy possess good selectivity for ATP detection because of the high affinity of the aptamer-target interaction. 2. Experimental section 2.1. Reagents and chemicals Cetyltrimethylammonium bromide (CTAB), sodium oleate (NaOL), hydrochloric acid (HCl, 37 wt%), ascorbic acid (AA) and sodium borohydride (NaBH4) were purchased from Aladdin Biotech CO. Ltd. (Beijing, China). Exonuclease III (Exo III) was obtained from Thermo scientific Reagent Co. Ltd. (USA). Chloroauric acid (HAuCl4), ATP, cytidine triphosphate (CTP), gytidine Triphosphate (GTP), uridine Triphosphate (UTP), and thymidine triphosphate (TTP) were obtained from Sigma-Aldrich Co. Ltd. (Shanghai, China). Tris-HCl was purchased from X-Y Biotech CO. Ltd. (1.0 M, pH 8.0, Shanghai, China), and was diluted to 20 mM before using. DNA/AgNCs were prepared according to our previous method with a concentration of 100 µM [50], which were formed on the 13C bases of the DNA template. The fluorescence intensity of DNA/AgNCs were determined at 560 nm with an excitation wavelength of 430 nm. DNA template was synthesized by Sangon Biotechnology
(Shanghai,
China)
with
the
sequence
5'-CCCCCCCCCCCCCACCTGGGGGAGTATTGCGGAGGAAGGT-3',
of which
containing two parts: the AgNCs formation template (italic section) and the ATP
aptamer (underlined section). 2.2. Apparatus The fluorescence spectra were recorded using the Agilent Cary Eclipse G9800A fluorescence spectrophotometer (Agilent, America). All UV-vis absorption spectra were obtained with a UV-260 spectrometer (Thermo Fisher Corporation, USA). TEM images of (+)AuNRs and AgNCs were recorded by transmission electron microscope (TEM, JEM-2010HR, Japan). The zeta potential of the proposed AuNRs were recorded using the particle size analyzer (MS2000, England). 2.3. Synthesis of (+)AuNRs (+)AuNRs were synthesized according to the literature method by seed-mediated growth containing two steps [51]. Gold seed formation: Firstly, all glass instruments were immersed in aqua regia for more than 24 h. After that, 60 µL of freshly prepared ice-cold NaBH4 (0.01 M) was added to the mixed solution containing 5.0 mL of CTAB (0.2 M) and 5.0 mL of HAuCl4 (0.5 mM), following by stirring rapidly for 2 min at 30 °C. At this time, the color of the mixed solution changed from transparent to yellowish brown, indicating the formation of gold seed solution. Then, the seed solution was stirred vigorously for 2.0 min, and maintained at 30 °C for 2.0 h. Preparation of the growth solution: Briefly, 0.35 g CTAB and 0.0617 g NaOL were dissolved in 12.5 mL of warm water (50 °C). After that, the solution was cooled down to 30 °C and mixed with 4.0 mM of AgNO3 solution. Subsequently, 12.5 mL of HAuCl4 (1.0 mM) was added into above solution and kept undisturbed at 30 °C for 15 min, following by stirring for 90 min (700 rpm). Then, 0.05 mL of HCl (1.0 M) was
introduced to adjust the pH. After another slowly stirring (400 rpm) for 15 min, 0.0625 mL of ascorbic acid (AA, 0.064 M) was added and vigorously stirred for 30 s. For AuNRs formation, 0.02 mL of prepared seed solution was injected into the growth solution, and the solution was allowed to sit overnight at 30 °C. At last, the product was isolated by centrifugation (7,000 rpm) for 30 min to remove excess CTAB. 2.4. Detection procedure of ATP Detection of ATP was constructed in Tris-HCl buffer (20 mM). In the typical process, 100 µL of (+)AuNRs solution was mixed with 80 µL of prepared DNA/AgNCs (1.0 µM) and incubated at room temperature for 30 min. Subsequently, different concentrations of ATP were added into above mixture, respectively, and followed by incubating for 60 min at room temperature. After that, 35 U of Exo III was added and incubated at 37 °C for 45 min, and then incubated at 75 °C for 10 min to inactivate. Before detecting, the mixture was diluted to 300 µL with Tris-HCl buffer solution. For fluorescence measurements, the excitation wavelength and the emission wavelength were set at 430 and 560 nm, the slit width for both excitation and emission were set at 10 nm, and the samples were scanned from 500 to 700 nm. 3. Results and discussion 3.1. Detection principle of the sensing system for ATP The working principle of the sensing platform for ATP detection was illustrated in Fig. 1, DNA/AgNCs were employed to produce a strong fluorescent signal. After the incubation of such DNA/AgNCs with (+)AuNRs, the fluorescence signals of them were effectively quenched due to the electrostatic absorption of them with (+)AuNRs
and the vicinity of AgNCs to (+)AuNRs. In the presence of ATP, DNA duplex that contained a recessed end was formed based on the specific interaction of ATP with DNA template, such DNA duplex presented stronger electrostatic adsorption with (+)AuNRs, and the fluorescence signal of AgNCs decreased further. Upon addition of Exo III, such DNA duplex could be hydrolyzed and released from (+)AuNPs surface, which directly resulted in the recovery of a strong fluorescence signal. Meantime, ATP will returned to the solution and reacted with another DNA/AgNCs to realize enzyme-assisted target recycling amplification. And the recovery of the fluorescent intensity was proportional with the concentrations of ATP, which could be detected by using a fluorescence spectrophotometer with a scanning range from 500 to 700 nm. Fig. 1 3.2. Characteristics of the nanomaterials TEM image showed the size and disperse states of (+)AuNRs. As shown in Fig. 2A, an obvious rod-like structure of (+)AuNRs was emerged with an average length of 50 nm and an average width of 20 nm. The transverse and longitudinal surface plasmon absorptions of such (+)AuNRs observed from UV-vis results were 508 nm and 766 nm (Fig. 2C, curve a). In addition, the zeta-potential of such AuNRs monitored from the particle size analyzer was +65.3 mV (Fig. 2B). Meantime, the stability of such (+)AuNRs was investigated by using UV-vis spectra (Fig. 2C). Compared with (+)AuNRs (curve a), the absorption spectra and solution colors of (+)AuNRs did not change obviously while adding 50 mM of BSA (curve b), NaCl (curve c), metal ions
(including K+, Ca2+, Mg2+, Al3+, curve d), and ATP (curve e), respectively, indicating a good stability of (+)AuNRs in complex biological samples. From TEM result it could be seen that AgNCs were spherical with uniform distribution and particle size of about 2.0 nm (Fig. 2D). To further prove the formation of DNA/AgNCs, UV-vis adsorption spectra were applied and two characteristic absorption peaks at 265 nm and 445 nm were appeared for DNA/AgNCs (curve b), while only one peak at 265 nm obtained for DNA template (curve a), which directly indicating the formation of DNA/AgNCs (Fig. 2E). Fig. 2 3.3. Characterization of the sensor The stability of DNA/AgNCs can affected the characteristics of the sensor. Thus, DNA/AgNCs were stored and measured every 0.5 h to monitor the stability of them. As shown in Fig. 3A, the fluorescence signal of DNA/AgNCs still maintain 98.6% of the corresponding original response after 4.0 h, demonstrating a good stability of DNA/AgNCs. Moreover, the feasibility of the sensor for ATP detection was investigated in Fig. 3B, and a strong fluorescence signal appeared for DNA/AgNCs (curve a). Then, an obvious fluorescence quenching happened after the mixing of DNA/AgNCs with (+)AuNRs (curve b), such result mainly due to the electrostatic interaction of DNA/AgNCs with (+)AuNRs, directly resulted in the vicinity of AgNCs to (+)AuNRs. In the presence of 5.0 nM of ATP, DNA duplex formed based on the specific interaction between ATP and DNA template, inducing the fluorescence signal decreased further (curve c), because of the strong electrostatic interaction of
negatively charged DNA duplex onto (+)AuNRs surface. After adding 35 U of Exo III, the fluorescence intensity increased greatly (curve d), indicating the release of AgNCs from (+)AuNRs and the recovery of the fluorescence signal, which obviously illustrated that the DNA duplex could be effectively hydrolyzed by Exo III from the 3’ terminus. 3.4. Circular dichroism spectroscopy of DNA probe Circular dichroism (CD) spectroscopy was used to investigate the formation of dsDNA. As shown in Fig. 3C, DNA probe showed a weak positive Cotton effect peak around 278 nm and a weak negative Cotton effect peak around 244 nm (a). After the addition of 5.0 nM of ATP, the intensity at the peak of 278 nm and 244 nm increased obviously (b), indicating the formation of dsDNA structure. However, when such dsDNA product was incubated with 35 U of Exo III, the peak is substantially disappeared, these results might be attributed to the cleavage of dsDNA product (c). Fig. 3 3.5. Optimization of the experimental conditions To ensure the ATP detection was constructed at optimal conditions, the reaction time between (+)AuNRs and DNA/AgNCs, the concentration of (+)AuNRs, the reaction time of ATP with DNA template, the amount of Exo III, and the incubating time of Exo III with DNA duplex were optimized. From Fig. 4A, we can see that the fluorescence signal decreased with increasing reaction time of (+)AuNRs with DNA/AgNCs from 0 to 30 min, and then reached a plateau after 30 min. Thus, the optimal time of 30 min was selected in our work. Fig. 4B displayed the effect of the
concentration of (+)AuNRs. It could be seen that the change of the fluorescence signal (∆F, ∆F=Fthe
value of curve b-Fthe value of curve a)
increased along with the increase of
(+)AuNRs concentration in the range of 0-15 nM, and reached a maximum value at 15 nM. Therefore, 15 nM of (+)AuNRs was chosen for the following study. Just as shown in shown in Fig. 4C, a maximum fluorescence signal was obtained while the reaction time between ATP and DNA template reached 60 min, illustrating that the DNA template could effectively interact with ATP within 60 min. Meanwhile, the fluorescence intensity increased along with the increase of Exo III amount from 5.0 to 35 U, and reached a plateau after that (Fig. 4D). So, 35 U of Exo III was selected in the experiments. In addition, it could be seen from Fig. 4E that the fluorescence signal increased while the incubating time between Exo III and DNA duplex increased from 5 to 45 min. Thus, 45 min was used as an optimal incubating time of Exo III with DNA duplex. Fig. 4 3.6. Performance of the sensor for ATP detection Under optimal conditions, the quantitative detection of ATP was shown in Fig. 5. It was found that the fluorescence intensity increased dramatically along with the increase of ATP concentration from 50 pM to 30 nM (Fig. 5A), and a linear range was obtained between 50 pM to 1.0 nM with a detection limit of 26 pM (S/N = 3) (Fig. 5B, insert). The linear regression equation for ATP was F= 54.40+122.50 c (where F is the fluorescent intensity and c is the concentration of ATP), with a correlation coefficient of R2 =0.9968. The detection performance of the present work was compared with
literatures, and the results were listed in Table 1. Taking advantage of the good fluorescence quenching ability of (+)AuNRs coupled with Exo III assisted target recycling amplification, the detection limit of our proposed method was much lower than most of other recently reported fluorescence based methods. Furthermore, we also investigated the selectivity of the sensor for ATP detection by using the other analogous molecules including GTP, CTP, UTP and TTP as the interfering substances (Fig. 5C), due to the fact that the similar molecule structures of them may interfere the practical detection of ATP. It could be seen that 5.0 nM of ATP induced a strong fluorescence enhancement, while the fluorescence change was weak and even can be ignored for 50 nM of GTP, CTP and UTP, respectively. Such results clearly indicated that the fluorescence sensor was highly specific for ATP detection. Meanwhile, five different batches of the system were prepared under the same conditions to detect 1.0 nM of ATP for evaluating the reproducibility of the sensor. As shown in Fig. 5D, the five batches performed similar fluorescence responses, with the RSD that less than 10%, indicating acceptable reproducibility of the sensor. Fig. 5 Table 1 3.7. Application of the fluorescent sensor To construct the addition and recovery experiments of ATP in real samples, the clinical serum sample was collected from healthy donors in Xuzhou central hospital in Jiangsu Province, China. As shown in Table 2, a good recovery that in the range of 95.3–103.5% was obtained, with the RSD that less than 10%, indicating that the
proposed system provided an effective platform for the assay of ATP in real biological samples. Table 2 4. Conclusions In conclusion, an ultrasensitive and label-free detection of ATP was realized based on the fluorescence quenching of (+)AuNRs coupled with Exo recycling amplification. The utilization of (+)AuNRs and Exo
associated target
realized the sensitive
detection of ATP detection with a detection limit of 26 pM, which were comparable with many of other fluorescent methods. Moreover, the detection of ATP was realized mainly based on the electrostatic adsorption of the negatively charged DNA template with (+)AuNRs, which greatly simplifying the detection process of ATP. Notably, the proposed method possess high selectivity due to the utilization of ATP aptamer, indicating a potential application value in early cancer diagnosis. Acknowledgements This work was supported by the Natural Science Foundation of Xuzhou City (KC18140), the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18-2141).
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Figure captions Fig. 1 Schematic illustration of ATP detection by using (+)AuNRs as the fluorescence quenching platform coupled with Exo
assisted target recycling amplification.
Fig. 2 (A) TEM image of (+)AuNRs; (B) UV–vis absorption spectrum of (+)AuNRs; (C) UV-vis absorption spectra of (+)AuNRs (curve a) upon addition with 50 mM of BSA (curve b), NaCl (curve c), metal ions (including K+, Ca2+, Mg2+, Al3+, curve d), and ATP (curve e), respectively, Inset showed the corresponding color of (+)AuNRs in different solutions; (D) TEM image of DNA/AgNCs; (E) UV–vis absorption spectra of the DNA template (curve a) and the DNA/AgNCs (curve b). Fig. 3 (A) The stability of DNA/AgNCs after kept for different times (a to i: 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 h); (B) The fluorescence spectra of DNA/AgNCs (a), (b) after the incubation of DNA/AgNCs with (+)AuNRs, (c) “(b)” in the presence of 5.0 nM of ATP, (d) “c” in the presence of 35 U of Exo
; (C) CD spectra of 8.0 µM of DNA probe (a), dsDNA product (b) and DNA
fragments in the presence of 35 U of Exo
(c).
Fig. 4 (A) Effect of the reaction time of AuNRs with DNA, (B) the concentration of AuNRs in the absence (a), and presence of 1.0 nM of ATP and 35 U of Exo DNA template and 1.0 nM of ATP, (D) the amount of Exo Exo
(b); (C) the reaction time between ; (E) the incubation time of 35 U of
with DNA duplex on the fluorescent responses of the system. The error bars indicated
the standard deviations of three replicates.
Fig. 5 (A) Fluorescence spectra of the system in the presence of ATP with concentrations of 0.05, 0.1, 0.3, 0.5, 0.7, 1, 3, 5, 10, 20, and 30 nM, respectively; (B) The relationship between the fluorescence intensity and the concentration of ATP (insert: calibration curve); (C) Selectivity of the sensor for 5.0 nM of ATP compared with the interfering substances containing 50 nM of UTP, CTP, GTP and TTP; (D) The reproducibility of five different batches of the system for the detection of 1.0 nM of ATP.
Table 1 Comparison of our method with other fluorescence-based works Materials
Linear range
Detection limit
Selectivity
Reference
Ag@SiO2 nanoparticles
100 nM – 5 mM
14.2 nM
UTP, GTP, CTP
6
Water-soluble carbon nanotubes
0. 1 – 1 mM
24 µM
ADP, AMP, UTP, GTP, CTP
52
G-quadruplex DNAs
0.1 – 60 µM
33 µM
UTP, GTP, CTP
53
Polydopamine nanospheres
20 – 600 nM
8.32 nM
UTP, GTP, CTP
54
PDANTs
0.35 – 800 µM
150 nM
UTP, GTP, CTP
10
Copper nanoparticles
0.2 – 50 µM
93 nM
UTP, GTP, CTP
55
G-quadruplex
0.05 – 1 µM
18 nM
UTP, GTP, CTP
56
Graphene oxide
0.1 – 5 nM
80 pM
UTP, GTP, CTP
57
DNA/Ag nanoclusters
0.5 – 8.0 µM
91.6 nM
UTP, GTP, CTP
5
Berberine and Exo I
0.5 – 50 µM
140 nM
UTP, GTP, CTP
3
0.3 – 2.0 mM,
0.27 mM
UTP, GTP, CTP
7
Gold nanoparticles
0.5-10 µM
100 nM
UTP, GTP, CTP
58
AuNP@SiO2
100 pM-50 uM
43 pM
UTP, GTP, CTP
59
Gold nanorods
50 pM-1.0 nM
26 pM
UTP, GTP, CTP, TTP
Our method
a
AuNCs-Gr
a
b
PDANTs: polydopamine nanotubes AuNCs-Gr: gold nanocrosses and graphene quantum dots
b
Table 2 Detection results of ATP in serum samples (n=5) Samples
Added (pM)
Detected (pM)
Recovery (%)
RSD, n=3(%)
1
50
48.6
97.2
4.3
2
100
103.5
103.5
3.6
3
300
285.9
95.3
4.7
4
700
713
101.8
5.1
5
1000
1006.8
100.7
6.2
The authors declared that they have no conflicts of interest to this work.
*Highlights An ultrasensitive and label-free sensor was designed to detect ATP coupling the fluorescence quenching of positively charged gold nanorods ((+)AuNRs) with exonuclease
assisted target recycling amplification, and several merits contained in
the system: 1. (+)AuNRs with individual fluorescence quenching ability were utilized for the detection of ATP; 2. A low detection limit of 26 pM was obtained for ATP detection due to the combination of (+)AuNRs with Exo
assisted target recycling amplification;
3. Highly stable DNA/AgNCs were utilized as good fluorescence signal probes to detect ATP. 4. The sensing system possess high selectivity for ATP detection due to the employment of ATP aptamer.