A label-free fluorescence aptasensor was studied for adenosine triphosphate (ATP) detection that combines low biosensor toxicity with a simplified preparation process. In this study, the aptamer of the gold nanoparticle@aptamer@carbon quantum dot nanostructure could specifically identify ATP, resulting in the change of the fluorescence signal. In order to analyze the performance of the sensor, the effects of the carbon quantum dot (CQD) concentration and centrifugal rate on the stability of the probe were investigated. The results show that the sensor was superior under the 220 µl CQD volume and 2000 rpm centrifugal rate. Furthermore, the linear relationship between the change of the fluorescence signal and ATP concentration is Y = 359.747 + 0.226X within the volume range of 20 µM–280 µM. The correlation coefficient is 0.98, and the detection limit is 20 µM. No obvious fluorescence change was observed in solutions containing other common ions. On the basis of no pollution and simplicity, this sensor demonstrates great potential as a low-cost diagnostic tool for the detection of various targets, particularly for use in the fields of food safety and biomedical diagnostics.

Adenosine triphosphate (ATP) provides a direct source of energy for a broad range of cell metabolisms including muscle contraction, transport of molecules and ions, and synthesis of important biomolecules.1 The ATP concentration will sharply drop in the cell when the cell has been dead.2 Therefore, the abnormal ATP concentration can lead to a number of diseases, such as malignant tumors, hypoglycemia, and Parkinson's disease.3,4 Current methods to detect ATP include mass spectrometry and high performance liquid chromatography.5,6 However, these standard technologies generally involve expensive laboratory equipment, highly trained technicians, and timely sample processing.7–9 Thus, it is necessary to develop an ATP sensor that is low cost and easy to use.

Recently, aptasensors for ATP detection have gained much attention due to their excellent biocompatibility, high specificity, and easy synthesis.10–12 Although an aptamer can recognize and capture the targets, it needs to be modified by a transduction element to achieve detection.13 Therefore, fluorophores are usually modified on the aptamer to complete the signal transformation, for example, Deng’s team constructed a sensor of molecular beacons for detecting kanamycin, which was required to mark Cy3 and Cy5 at both ends of the aptamer.14 Wang’s team established a sensor for the detection of aflatoxin, which was required to mark 6-carboxy-fluorescein (FAM) and fluorescence quencher at both ends of the aptamer.15 The above sensors can realize the detection of different targets. However, since they have a certain extent of toxicity and complex marking process, these fluorophores are not beneficial for detecting biomolecules in food.16 Compared with traditional fluorophores, CQDs are a new type of fluorescent material, which has the advantages of low toxicity and low cost.17,18 Thus, a label-free fluorescence aptasensor combines the superiority of the aptamer and CQDs to have a broad application in biomolecule and heavy metal ion detection.

In this study, a label-free fluorescence aptasensor was researched that was conjugated with CQDs for detecting ATP in aqueous solution. The sensor platform used in this research reduces the complicated experimental procedure and biomaterial toxicity. The technology of label-free detection provides valuable information for a range of applications including food safety, water analysis, and clinical diagnosis.

Tris(2-carboxyethyl) phosphine (TCEP) and adenosine triphosphate aptamer were purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). ATP aptamer: “5-SH-ACC TGG GGG AGT ATT GCG GAG GAA GGT-3” and adenosine triphosphate (ATP) were purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Phosphate buffer solution (PBS, 10 mM, pH 7.4) was used. All chemical reagents were of analytical grade, and the ultrapure water used in this study was prepared by a PURELAB Option-R (ELGA Lab Water, UK).

Fluorescence measurements were performed by using an F-7000 fluorescence spectrophotometer (Hitachi, Japan). An Eppendorf centrifuge 5418 (Hamburg, Germany) was used to centrifuge solutions. All pH values were acquired by using an FE-20K pH-meter (METTLER TOLEDO, Switzerland). An HZQ-F200 constant temperature shaker (Beijing Donglian haer Instrument Co., Beijing, China) was used to promote the hybridization.

Above all, 10 ml 1 mol/l glucose solution was mixed with 10 ml NaOH solution. Second, the mixed solution was placed in an ultrasonic box for 4 h to obtain carbon element. Subsequently, the pH of the mixed solution was adjusted to 7 after the ultrasonic treatment. 100 ml absolute ethanol was added into the solution by dropping and stirred together. Finally, after adding 12% (wt. %) magnesium sulfate, the solution was stored for 24 h to obtain the CQDs.19 

Gold nanoparticles (AuNPs) were prepared by reducing chloroauric acid with sodium citrate according to the method in Ref. 20. In this preparation process, 250 ml 0.1 mmol HAuCl4 solution was placed in a clean beaker and heated to boiling with vigorous stirring. After adding 5 ml 38.8 mmol sodium citrate into the beaker, the color of the solution changed from pale yellow to wine red. The AuNPs were obtained after the solution was cooled to room temperature. As shown in the supplementary material the size of the synthesized AuNPs was about 20 nm according to Ref. 20.

The S–H bond of the ATP aptamer (5 µM) was activated by TCEP (pH = 5.4), and then, the AuNPs were added to the aptamer solution. The aptamer could be immediately modified on the AuNPs after adding a little citric acid–hydrochloric acid (500 mm, pH = 3.0). The mixed solution was placed into the shaking box to cultivate for 3 h. Then, the precipitate was obtained by centrifugation (15000 rpm) for 25 min. The centrifugation process was repeated three times. Finally, the probe (Au/Aptamer/CQDs) was obtained after adding the CQDs.

After a fast reaction of a different ATP concentration (20 µM–280 µM) and the probe, the supernatant and the precipitate were separated by centrifugation (2000 rpm). Then, the fluorescence intensity was measured from the supernatant. The fluorescence spectra were recorded, and a standard curve was drawn.

The principle of ATP detection is shown in Fig. 1(a). First, the AuNPs and aptamer are conjugated to one another by an Au–S bond, constructing the Au@Aptamer nanostructure in the sample solution [as shown in Fig. 1(b)]. Subsequently, CQDs were added to the solution to form the Au@Aptamer@CQD nanostructure by the electrostatic force because the CQDs and aptamer carry opposite charges.21 Specifically, the CQD fluorescence was quenched based on FRET due to the close proximity of the CQDs and AuNPs.22 

FIG. 1.

(a) Based on AuNPs and CQDs for the detection principle of ATP. (b) The binding process of AuNPs and aptamer.

FIG. 1.

(a) Based on AuNPs and CQDs for the detection principle of ATP. (b) The binding process of AuNPs and aptamer.

Close modal

The aptamer can be bound preferentially to ATP. Therefore, when ATP was present, CQDs would separate from the aptamer. Finally, the content of detached CQDs can be measured from the supernatant by centrifugation. Using this design strategy, the concentration of ATP can be determined by detecting the fluorescence changes of the supernatant [as shown in Fig. 2, curves (b) and (c) are between curve (a) and (d), and curve (b) is higher than (c)].

FIG. 2.

The fluorescence spectra of (a) pure CQDs, (b) probe@ATP (280 µM), (c) probe@ATP (20 µM), and (d) probe at the same CQD concentration.

FIG. 2.

The fluorescence spectra of (a) pure CQDs, (b) probe@ATP (280 µM), (c) probe@ATP (20 µM), and (d) probe at the same CQD concentration.

Close modal

The change in the CQD concentration has a significant effect on the sensor sensitivity and measuring range. If there are too many CQDs, the solution background noise will be high, affecting the sensor detection sensitivity and detection limit. On the other hand, if the CQD concentration is too small, there will be fewer CQDs that can be replaced by ATP, resulting in a narrower detection range for the sensor.

Because CQDs are in the unlabeled state, they are susceptible to react with various substances in solution. Study was performed to analyze the influence of the concentration change of CQDs in different structures. Figures 3(a)–3(e) present the fluorescence spectra of pure CQDs, Aptamer@CQDs, Probe@ATP, Au@CQDs, and probe at different CQD volumes. Figure 4 shows the corresponding state of the above structure in solution, in particular, for comparing the changes of fluorescence intensity in different structures at the same CQD volume. The peak of fluorescence spectra at 500 nm is taken as the y axis, and the CQD volume is taken as the x axis, plotting Fig. 3(f).

FIG. 3.

Fluorescence spectra of (a) pure CQDs, (b) Aptamer@CQDs, (c) Probe@ATP, (d) Au@CQDs, and (e) probe at different CQD volumes (140 μl–300 µl). (f) presents the comparison of peak values for different nanostructures at 500 nm wavelength.

FIG. 3.

Fluorescence spectra of (a) pure CQDs, (b) Aptamer@CQDs, (c) Probe@ATP, (d) Au@CQDs, and (e) probe at different CQD volumes (140 μl–300 µl). (f) presents the comparison of peak values for different nanostructures at 500 nm wavelength.

Close modal
FIG. 4.

The mechanism of (a) pure CQDs, (b) Aptamer@CQDs, (c) Probe@ATP, (d) Au@CQDs, and (e) probe.

FIG. 4.

The mechanism of (a) pure CQDs, (b) Aptamer@CQDs, (c) Probe@ATP, (d) Au@CQDs, and (e) probe.

Close modal

As shown in Fig. 3(f), with the increase of the CQD volume, the fluorescence intensity of CQDs, Aptamer@CQDs, Probe@ATP (20 µM), and Au@CQDs also increased, in addition to the probe. Because the CQD fluorescence intensity increases, the volume increases before CQDs reach their own quenching concentration. Analyzing the curve relationship in Fig. 3(f), first, the aptamer has little effect on the CQD fluorescence intensity22 [structure diagram is shown in Fig. 4(b)], resulting in the fact that curve b is close to curve a. When there is the presence of only AuNPs and CQDs in the solution [Fig. 4(d)], FRET more easily occurs between the AuNPs and CQDs, causing curve d to stay away from curve a as the CQD volume is increased.

After adding the same ATP volume [Fig. 4(c)], the supernatant fluorescence intensity increases with the increase of the CQD volume compared to the probe structure. In order to obtain greater detection sensitivity and avoid the interference of background noise as small as possible, comparing the effect of adding ATP on the fluorescence signal of the probe, the CQD volume aptamer was selected to be 220 µl for subsequent experiments.

The centrifugal rate during the experiment has a significant effect on the sensor detection performance. Therefore, as shown in Fig. 5, at the same concentration of the target, the change in the CQD fluorescence was measured in the supernatant at different centrifugation rates. If the centrifugal rate was lower than 1500, a part of Au/Aptamer/ATP, probe, and CQDs existed in the supernatant. Contrastingly, if the centrifugal rate was higher than 2000, the AuNPs@Aptamer@CQD nanostructure will be disintegrated, leading to the existence of more CQDs in the supernatant. In this experiment, 2000 rpm was selected as the optimal speed of centrifugation. In this condition, the nanostructure could be better centrifuged to the bottom and the state of the solution was more stable.

FIG. 5.

Fluorescence spectra and peak of the supernatant at different centrifugal speeds.

FIG. 5.

Fluorescence spectra and peak of the supernatant at different centrifugal speeds.

Close modal

Different concentrations of ATP were added to the solution that contained the probe for 10 min. After centrifugation, the supernatant fluorescence intensity was detected. Fluorescence spectra of different ATP concentrations are shown in Fig. 6(a). As shown in Fig. 6(b), there is a good linear relationship between the fluorescence intensity of the CQDs in the supernatant and the added ATP concentration. The fluorescence intensity increased with the increase in the ATP concentration (20 µM–280 µM). The linear formula is Y = 359.747 + 0.226X. The correlation coefficient is 0.98. The detection limit is 20 µM.

FIG. 6.

(a) Fluorescence spectra of different ATP concentrations. (b) Linear detection range of ATP.

FIG. 6.

(a) Fluorescence spectra of different ATP concentrations. (b) Linear detection range of ATP.

Close modal

The specificity is one of the important parameters for evaluating the sensor performance. Therefore, at the same concentration (20 µM) of the target, this experiment prepared different small molecules that include the DA, biotin, sucrose, urea, and epinephrine to test the specificity of the sensor. As shown in Fig. 7, the sensor was able to distinguish the ATP target with high selectivity in the presence of interfering targets. It verifies that the sensor has a good specificity for ATP.

FIG. 7.

Fluorescence intensity of different targets at the same concentration (20 µM).

FIG. 7.

Fluorescence intensity of different targets at the same concentration (20 µM).

Close modal

In this study, a label-free fluorescence aptasensor for ATP detection based on a low-cost and simple production process was constructed. Additionally, the influence of the CQD volume and centrifugal rate on the sensor fluorescence signal was investigated, which improves the sensor detection performance. The optimized CQD concentration is 220 µl, and the centrifugal rate is 2000 rpm. Subsequently, analysis of the ATP detection performance showed that the sensor has a good linear relationship for ATP detection (20 µM–280 µM), and it also has obvious specificity for ATP. The aptasensor detection limit is 20 µM. Consequently, this sensor has great potential for applications in many areas, including food safety, biomedicine, and environmental management.

As shown in the figure of the supplementary material, the AuNPs of 20 nm size were prepared by reducing chloroauric acid with sodium citrate. The distribution of AuNPs was uniform and round, which was beneficial to the construction of label-free fluorescence aptasensors. TEM images of AuNPs (a) 20 nm scale and (b) 50 nm scale.

This research was funded by the National Natural Science Foundation of China (Grant Nos. 61801436 and 62073299), Program for Innovative Research Team (in Science and Technology) in the University of Henan Province (Grant No. 20IRTSTHN017), Henan Province Science and Technology Plan Project (Grant No. 202102210186), and Henan Key Laboratory of Biomolecular Recognition and Sensing (Grant No. HKLBRSK1801).

The data that support the findings of this study are available within this article.

1.
S.
Li
,
X. T.
Zhao
,
X. X.
Yu
,
Y. Q.
Wan
,
M. Y.
Yin
,
W. W.
Zhang
,
B. Q.
Cao
, and
H.
Wang
, “
Fe3O4 nanozymes with aptamer-tuned catalysis for selective colorimetric analysis of ATP in blood
,”
Anal. Chem.
91
,
14737
14742
(
2019
).
2.
Y.
Liu
,
D.
Lee
,
D.
Wu
,
K. M. K.
Swamy
, and
J.
Yoon
, “
A new kind of rhodamine-based fluorescence turn-on probe for monitoring ATP in mitochondria
,”
Sens. Actuators, B
265
,
429
434
(
2018
).
3.
J.
Wang
,
Y.
Wang
,
S.
Liu
,
H.
Wang
,
X.
Zhang
,
X.
Song
, and
J.
Huang
, “
Duplex featured polymerase-driven concurrent strategy for detecting of ATP based on endonuclease-fueled feedback amplification
,”
Anal. Chim. Acta
1060
,
79
87
(
2019
).
4.
F.
Li
,
X.
Hu
,
F.
Wang
,
B.
Zheng
,
J.
Du
, and
D.
Xiao
, “
A fluorescent ‘on-off-on’ probe for sensitive detection of ATP based on ATP displacing DNA from nanoceria
,”
Talanta
179
,
285
291
(
2018
).
5.
X. Z.
Jiang
,
L.
Zhen
,
S. G.
Mingqin
,
S. L.
Yi
,
X. Y.
Zeng
,
Y. L.
Zhang
, and
L. X.
Hou
, “
A fluorescence ‘turn-on’ sensor for detecting hydrazine in environment
,”
Microchem. J.
152
,
5
(
2020
).
6.
H.
Li
,
Z. J.
Guo
,
W. C.
Xie
,
W. Y.
Sun
,
S.
Ji
,
J.
Tian
, and
L.
Lv
, “
A label-free fluorometric aptasensor for adenosine triphosphate (ATP) detection based on aggregation-induced emission probe
,”
Anal. Biochem.
578
,
60
65
(
2019
).
7.
Y.
Dai
,
Y.
Zhang
,
W.
Liao
,
W.
Wang
, and
L.
Wu
, “
G-quadruplex specific thioflavin T-based label-free fluorescence aptasensor for rapid detection of tetracycline
,”
Spectrochim. Acta Part A
238
,
118406
(
2020
).
8.
D. A.
Raja
,
S. G.
Musharraf
,
M. R.
Shah
,
A.
Jabbar
,
M. I.
Bhanger
, and
M. I.
Malik
, “
Poly(propylene glycol) stabilized gold nanoparticles: An efficient colorimetric assay for ceftriaxone
,”
J. Ind. Eng. Chem.
87
,
180
186
(
2020
).
9.
G.
Ren
,
X.
Hou
,
Y.
Kang
,
R.
Zhang
,
M.
Zhang
,
W.
Liu
,
L.
Li
,
S.
Wei
,
H.
Wang
,
B.
Wang
, and
H.
Diao
, “
Efficient preparation of nitrogen-doped fluorescent carbon dots for highly sensitive detection of metronidazole and live cell imaging
,”
Spectrochim. Acta Part A
234
,
118251
(
2020
).
10.
A.
Ahmadi
,
N. M.
Danesh
,
M.
Ramezani
,
M.
Alibolandi
,
P.
Lavaee
,
A. S.
Emrani
,
K.
Abnous
, and
S. M.
Taghdisi
, “
A rapid and simple ratiometric fluorescent sensor for patulin detection based on a stabilized DNA duplex probe containing less amount of aptamer-involved base pairs
,”
Talanta
204
,
641
646
(
2019
).
11.
D.
Song
,
R.
Yang
,
S.
Fang
,
Y.
Liu
, and
F.
Long
, “
A FRET-based dual-color evanescent wave optical fiberaptasensor for simultaneous fluorometric determination of aflatoxin M1 and ochratoxin A
,”
Microchim. Acta
185
,
508
(
2018
).
12.
S. M.
Taghdisi
,
N. M.
Danesh
,
M.
Ramezani
, and
K.
Abnous
, “
A new amplified fluorescent aptasensor based on hairpin structure of G-quadruplex oligonucleotide-Aptamer chimera and silica nanoparticles for sensitive detection of aflatoxin B-1 in the grape juice
,”
Food Chem.
268
,
342
346
(
2018
).
13.
F.
Zhang
,
F.
Deng
,
G.-J.
Liu
,
R.
Middleton
,
D. W.
Inglis
,
A.
Anwer
,
S.
Wang
, and
G.
Liu
, “
IFN-gamma-induced signal-on fluorescence aptasensors: From hybridization chain reaction amplification to 3D optical fiber sensing interface towards a deployable device for cytokine sensing
,”
Mol. Syst. Des. Eng.
4
,
872
881
(
2019
).
14.
J.
Deng
,
Y.
Liu
,
X.
Lin
,
Y.
Lyu
,
P.
Qian
, and
S.
Wang
, “
A ratiometric fluorescent biosensor based on cascaded amplification strategy for ultrasensitive detection of kanamycin
,”
Sens. Actuators, B
273
,
1495
1500
(
2018
).
15.
C.
Wang
,
L.
Sun
, and
Q.
Zhao
, “
A simple aptamer molecular beacon assay for rapid detection of aflatoxin B1
,”
Chin. Chem. Lett.
30
,
1017
1020
(
2019
).
16.
X.
Xu
,
M. H.
Zhang
,
L. L.
Wang
,
S. M.
Zhang
,
M. Y.
Liu
,
N.
Long
,
X. Y.
Qi
,
Z. Z.
Cui
, and
L.
Zhang
, “
Determination of rhodamine B in food using ionic liquid-coated multiwalled carbon nanotube-based ultrasound-assisted dispersive solid-phase microextraction followed by high-performance liquid chromatography
,”
Food Anal. Methods
9
,
1696
1705
(
2016
).
17.
Y.
Ma
,
A. Y.
Chen
,
Y. Y.
Huang
,
X.
He
,
X. F.
Xie
,
B.
He
,
J. H.
Yang
, and
X. Y.
Wang
, “
Off-on fluorescent switching of boron-doped carbon quantum dots for ultrasensitive sensing of catechol and glutathione
,”
Carbon
162
,
234
244
(
2020
).
18.
Z.
Cui
,
Z.
Li
,
Y.
Jin
,
T.
Ren
,
J.
Chen
,
X.
Wang
,
K.
Zhong
,
L.
Tang
,
Y.
Tang
, and
M.
Cao
, “
Novel magnetic fluorescence probe based on carbon quantum dots-doped molecularly imprinted polymer for AHLs signaling molecules sensing in fish juice and milk
,”
Food Chem.
328
,
127063
(
2020
).
19.
L.-j.
Ren
,
P.
Zhang
,
R.-b.
Qi
,
J.
Yin
,
S.
Liu
,
J.-t.
Zhang
,
Q.-h.
Chen
, and
L.-y.
Jiang
, “
Influencing factors of luminescence properties of carbon dots prepared by ultrasonic
,”
Spectrosc. Spect. Anal.
37
,
3354
3359
(
2017
).
20.
X.
Ji
,
X.
Song
,
J.
Li
,
Y.
Bai
,
W.
Yang
, and
X.
Peng
, “
Size control of gold nanocrystals in citrate reduction: The third role of citrate
,”
J. Am. Chem. Soc.
129
,
13939
13948
(
2007
).
21.
B.
Wang
,
Y.
Chen
,
Y.
Wu
,
B.
Weng
,
Y.
Liu
,
Z.
Lu
,
C. M.
Li
, and
C.
Yu
, “
Aptamer induced assembly of fluorescent nitrogen-doped carbon dots on gold nanoparticles for sensitive detection of AFB(1)
,”
Biosens. Bioelectron.
78
,
23
30
(
2016
).
22.
S.
Ghayyem
and
F.
Faridbod
, “
A fluorescent aptamer/carbon dots based assay for Cytochrome c protein detection as a biomarker of cell apoptosis
,”
Methods Appl. Fluoresc.
7
,
015005
(
2019
).

Supplementary Material