Mechanism of sensing detection
Figure 1 illustrates the sensing strategy for the DA detection based on the fluorescence resonance energy transfer (FRET) between aptamer and GO. In the absence of target molecule (DA), FAM-modified aptamer is adsorbed onto the surface of GO via hydrophobic and π-π stacking interaction between the nucleotide bases and the sp2 honeycomb network of carbon [59–61]. Therefore, these interactions induce the formation of a stable complex that will lead to quench the fluorescence of FAM easily through energy transfer from the FAM to GO [62]. However, in the presence of target molecule (DA), The conformation of DA-aptamer is altered (target-induced allosteric effect), and switched from a random coil to rigid stem–loop structure-DA that have a weak affinity to GO and keep the dyes away from graphene surface [63]. Consequently, the FRET process will be hindered, and the FAM fluorescence is recovered and measured as a function of DA. In the experimental mixture, the fluorescence recovery increases by increasing DA-concentrations and the fluorescence will be proportional to the concentration of dopamine [64, 65]. On the other hand, the addition of interferents couldn’t change the conformation of DA-aptamer, so as it can’t be released from GO surface, resulting in non-emission of fluorescence due to the quenching effect of GO.
Based on ssDNA-GO interactions, Ye and collaborators [65] demonstrated the generation of a versatile molecular beacon-like probe as a multiplex sensing platform. This probe has been effectively applied as an example of a fluorescent biosensor-based FRET method to detect a specific target, for instance: specific sequence of DNA, as well as thrombin, metal ions such as Ag+ and Hg2+, and amino acids such as cysteine, with detection limits of 5 nM, 20 nM, 5.7 nM, and 60 nM, respectively.
Optimization of assay conditions
Aptamer and quencher concentrations, and incubation time are experimental variables that play an important role in the fabrication of sensitive and selective aptasensing platforms based on FRET principle. In this context, different experiments have been realized to optimize aptamer and quencher concentrations, as well as incubation time.
GO concentration
The choice of GO concentration is a critical factor because it may affect the performance of the sensing system. Optimizations were carried out by varying GO concentration between 0 and 5 µg/ml where the aptamer concentration was fixed at 400 nM. The quenching efficiency was defined by 100% × (F–F0)/F, where F0 and F are the fluorescence intensities of the aptamer solution with and without GO, respectively. As shown in Fig. 2, the quenching of fluorescence increased dramatically with the increasing amounts of GO, indicating that aptamers are still free in the solution. Then, the fluorescence quenching efficiency (~ 75 %) stabilized when the GO concentration was higher than 3 µg/ml, indicating that the FAM-labeled aptamer was almost completely adsorbed onto the surface of GO. Since higher concentrations of GO haven’t shown an enhanced quenching efficiency, a concentration of 3 µg/ml was selected as the GO amount generating the optimum quenching. Thus, it has been used in the next experiments.
GO quenching time
The effect of reaction time between the aptamer and GO on the fluorescence quenching of FAM probe was also investigated. In this context, different incubation times have been tested ranging from 0 to 30 min. As we can see from Fig. 3, the quenching efficiency increased by increasing the reaction time indicating that the FAM-aptamer was adsorbed onto the surface of GO. After 10 min of incubation, the signal reached the maximum of 75% of quenching and stabilized. Therefore, 10 min was chosen to be the optimal incubation time for GO and FAM-aptamer.
Aptamer concentration
To obtain the maximum sensitivity of biosensing, four different concentrations of aptamer (200, 400, 600 and 800 nM) were tested. According to the results presented in Fig. 4, the change of fluorescence intensity kept increasing gradually with increasing aptamer concentrations. The optimal concentration is determined according to the sensitivity of the proposed aptasensor. It should be noted that a high concentration of FAM-aptamer provides a better fluorescence signal. However, a high concentration of FAM-aptamer may influence the assay sensitivity leading to an erroneous results [66]. Therefore, the aptamer concentration of 400 nM was adopted to perform the next experiments.
Detection of DA
The sensitivity of any detection method is considered as a key factor to determine its applicability. It was investigated by monitoring the fluorescence intensity of increasing concentrations of DA at the emission wavelength of 538 nm. The fluorescence intensity was quantified by calculating the percentage of fluorescence recovery [(F-F0)/F0 × 100], where F is the fluorescence intensity in different dopamine concentrations and F0 is the fluorescence intensity in the absence of dopamine. The calibration curve presenting the fluorescent recovery percentages as function of DA concentrations ranging from 3 nM to 1680 nM was illustrated in Fig. 5. It was revealed that with increasing the concentration of DA, the fluorescence recovery increased gradually with a good linear relationship (Regression coefficient R2 = 0.997), indicating the growth of the number of FAM-aptamer attached to the DA target even at low concentrations (3 nM). The detection limit was estimated to be 0.031 nM based on 3δ/S calculation (δ is the standard deviation for the blank solution, and S is the slope of the calibration curve).
The performance of our technique was compared to other electrochemical and optical aptasensors, previously reported in the literature for dopamine detection. It has been noted that the LOD achieved in this new approach is much lower than those of these techniques (Table 1). The high sensitivity of our aptasensor could be attributed to the high affinity of the aptamer toward DA, and the ultra-high fluorescence quenching ability of GO. The change of the conformation of the aptamer upon the addition of target molecules enhanced the distance between FAM-labeled signal probe and the surface of GO, inducing a higher fluorescence restoration. Moreover, since this assay was performed in a homogenous solution, it avoided the need for time-consuming immobilization, coating and washing steps usually required in common heterogeneous assays [67–69]. Indeed, our biosensor provides a simple and rapid detection of DA since it is based on monitoring the fluorescence change due to the target binding. These features, as well as its other merits, such as low cost and fluorescence background, make it a promising tool for a rapid label free detection of DA.
Table 1
Comparison between this method and other reported techniques for detection of dopamine
Detection method
|
Detection range
nM
|
LOD
nM
|
Principle
|
Refs
|
Electrochemical
|
5×103- 75×103
|
3.36 × 103
|
DNA aptamer/AuNP/rGO/ modified glassy carbon electrode.
|
[70]
|
5–300
|
2.1
|
Au-electrode/Au-NPs/PEI/CNTs.
|
[67]
|
5–150
|
1
|
MB/MCH/DNA/Au electrode.
|
[71]
|
1–1000
|
0.75
|
Aptamer/GCSC-GO/GCE.
|
[72]
|
5 ×103-50×103
|
1000
|
Split aptamer1/split aptamer 2 / Au-electrode.
|
[73]
|
5–5×102
|
1.8
|
aptamer/AuNPs/GCE.
|
[74]
|
Colorimetric
|
540–5400
|
360
|
Aptamer/ unmodified AuNPs.
|
[75]
|
200–1100
|
70
|
AHMT/ AuNPs.
|
[76]
|
Fluorescence
|
26 − 2.90 × 103
|
2
|
DNA/ rhodamine B/ AuNPs.
|
[68]
|
0.1–10.0
|
0.08
|
tDNA/cDNA/aptamer/dopamine/hDNA/ Exo III.
|
[69]
|
50 − 2.5×105
|
10
|
CQDs/AuNPs/aptamer.
|
[77]
|
5× 104 − 106
|
104
|
CB/ Fe2+/ DNA
|
[29]
|
30–210
|
19
|
Ru/ QDs/ DNA
|
[78]
|
3–1680
|
0.031
|
GO/DNA
|
This work
|
Selectivity of the sensing system
Selectivity is another important feature of a good sensing system as though the presence of many interferents in the real sample would affect the accuracy of the detection mechanism. In this regard, the selectivity of the aptamer-GO sensing platform was studied by monitoring the fluorescence recovery percentage for different interferential substances including glycine, cysteine, glucose, and lactic acid. For that, we set the concentration of DA and the other interferents at 1120 nM, and then each one was incubated with 25 µl of FAM-aptamer. After 25 min, 25 µl of GO was added to the mixture, and the fluorescence recovery was measured after 30 min of incubation. As shown in Fig. 6, under the same experimental conditions, the fluorescence recoveries of the sensing system in the presence of DA are highly significant, whereas a minor fluorescence restoration was observed in the presence of the tested interferents. Furthermore, it can be seen that the fluorescence enhancement in the presence of the target was more than 4-fold as compared to the other interferents. These results indicates that glycine, cysteine, glucose, and acid lactic are not recognized by DA-aptamer. Consequently, the aptamer remains adsorbed on the GO surface inducing the fluorescence quenching. In particular, the aptamer was strongly bound to dopamine, thus inducing its release from the surface of GO and the fluorescence recovery. Therefore, this confirms that the label-free fluorescent aptasensor based on GO exhibited an excellent specificity for the DA detection, thus indicating its potential application in complex matrices.
Determination of DA in human serum
Table 2
Recoveries of DA from human serum samples (n = 3)
Sample
|
Standard value of DA (nM)
|
Found (nM)
|
Recovery (%)
|
RSD (%, n = 3)
|
Human serum
|
3
|
3.02
|
100.53
|
0.76
|
7
|
7.01
|
100.21
|
2.09
|
280
|
288.8
|
103.16
|
0.61
|
1200
|
1999.83
|
99.99
|
1.89
|
In order to investigate the applicability of the method, our biosensor was tested in human serum samples. For that, the samples free of DA were collected from a local biomedical analysis laboratory. Then, they were diluted 10 times and spiked with 3, 7, 280, and 1200 nM of DA. After incubation with the aptamer, 25 µl of GO was added and the fluorescence recovery was measured. As it is shown in Fig. 7, the fluorescent recovery percentages of human serum and tris-buffer were almost the same. This latter conclusion is confirmed by the analytical results for the samples spiked with DA presented in Table 2. As illustrated in this table, the developed aptasensor exhibited good recoveries ranging from 89–103%, with RSD between 0.61 and 2.09 %. These experimental results confirm the good reliability and applicability of the proposed method for DA detection in complex biological samples.