Fluorescence detection for enrofloxacin by using Aptamer and fluorophore SYBR Green I

doi:10.21203/rs.3.rs-4911437/v1

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Fluorescence detection for enrofloxacin by using Aptamer and fluorophore SYBR Green I

https://doi.org/10.21203/rs.3.rs-4911437/v1

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Enrofloxacin (ENR), as a type of antimicrobial, is commonly used in animal sterilization. However, excessive ENR residues in food will seriously harm human health. Therefore, in this work, we developed a quantitative fluorescence method for ENR based on aptamer and SYBR Green I (SG-I). DNA aptamers with G-quadruplex space structure can be recognized and inserted by SG-I to emit intense green fluorescence. In the presence of ENR, the aptamer preferentially recognized and bound specifically to ENR resulting in the change of aptamer conformation, which prevented the SG-I from inserting into the aptamer to cause fluorescence quenching. The aptamer sensor can quantitatively detect ENR with linear concentration range of 0-600 nM, and the limit of detection was 0.158 nM. The aptamer sensor was used detecting ENR in chicken, fish and pork samples with recovery rate from 99.74–104.02%, and relative standard deviation (RSD) of ENR of less than 6%. Therefore, this aptamer-based sensor provided the fluorescence detection method for ENR in food safety.

Enrofloxacin

Aptamer

SYBR Green Ⅰ

G-quadruplex

Fluorescence detection

Enrofloxacin (ENR), as the third-generation antibiotics, can inhibit bacterial DNA deconjugase and topoisomerase IV by binding to the cycloheximide subunit, blocking the replication and transcription process of bacterial DNA to realize bacteriostatic and bacteriophageal effects (Grabowski, et al. 2022; Xi 2022) ENR has been widely used in the fields of aquaculture (Huang, et al. 2019 )and animal husbandry (Li, et al. 2019; Du and Liu 2021). However, the excessive use of ENR has result in bacterial resistance and symptoms such as immune system damage, physiological abnormalities, and intestinal flora dysbiosis in aquatic animals (Wei, et al. 2023). In addition, the enrichment effect in organisms affects human health and environmental ecology (Bao, et al. 2024; Liu, et al. 2024). Therefore, the use of ENR and maximum residue limits (MRLs) in different animal products were strictly regulated (Zhang, et al. 2020). Therefore, the rapid and precise analytical method is urgent need to be developed for monitoring ENR to ensure the sustainable development of the farming industry, ecological balance and human food safety.

Various methods have been reported for analyzing ENR residues including high performance liquid chromatography (HPLC) (Aslam, et al. 2016), electrochemical methods (Guo, et al. 2024; Huang, et al. 2022), immunochromatographic analyses (ICAs) (Hu, et al. 2023), enzyme-linked immunosorbent assay (ELISA) (El Tahir, et al. 2021; Zhang, et al. 2011), and colorimetric methods (Wang, et al. 2023; Zhu, et al. 2022). These methods are highly sensitive, but the high cost of equipment, complexity of operation and long detection time increase the difficulty of detection (Shen, et al. 2019). Therefore, it is particularly important to develop a faster and simple method for quantitative detection of ENR.

The aptamer, as a single-stranded DNA or RNA, can be rapidly screened and synthesized by in vitro screening SELEEX (Syste matic Evolution of Ligand Exponential Enrichment) technique to be prepared in the laboratory (Hou, et al. 2023; Zhang, et al. 2024; Teng, et al. 2018). The aptamers can form the specific secondary structures (e.g., stem-loop structures, three-way junctions, etc.) and tertiary folds (e.g., G -tetramers), which make the aptamers can bind to a variety of target molecules with non-covalent interaction, such as protein, nucleic acid, small-molecule drugs, and so on (Guo, et al. [2021]; Lv, et al. [2022]; Li, et al. [2020]; Shang, et al. [2023]; Zhang, et al. [2023]). Therefore, the aptamers have been widely used in biosensor detection, target recognition and delivery (Wan, et al. [2023]). SYBR Green I (SG-I), as a fluorescence dye frequently utilized for the identification of double-stranded DNA (dsDNA), is able to embed itself to the base pairs of double-stranded DNA to significantly emit fluorescence (Kaczmarek, et al. [2021]; Chen, et al. [2022]; Wang, et al [2024]). Based on above feature, SG-I is widely used in electrophoresis, real-time PCR, and other forms of DNA qualitative and quantitative analysis (Xia, et al. [2021]). Aptamer with G-quadruplex structure can be identified and inserted by SG-I to turn on fluorescence (Zhang, et al. [2024]; Yi, et al. [2019]). Therefore, we envisioned that designed an aptamer-based label-free fluorescence with SG-I for detection of ENR.

In this work, we established a rapid detection method for ENR based on aptamer with G-quadruplex structure and SG-I. As shown in Scheme 1, in the absence of ENR, the ENR aptamer with G-quadruplex structure can be recognized and inserted SG-I with forming fluorescence. After the addition of ENR, the high affinity ability made the prioritized combination ENR and the aptamer to cause release of SG-I, which led to decrease of fluorescence signal. Therefore, the ENR was quantitatively analyzed based on change of SG-I fluorescent signal. The results showed the aptamer sensor was capable of detecting ENR with concentration from0 to 600 nM (R² = 0.996). The limit of detection was 0.158 nM. In addition, this method can be used for detecting ENR on fish, chicken and pork samples with the relative standard deviations (RSD) below 6% and the average recoveries of these samples ranged from 99.74–104.02%. Therefore, this strategy provided a convenient assay for detecting ENR.

2.1. Chemicals and devices

The nucleic acid aptamer sequence 5’-TCT CTG AGC CCG GGG GTT ATT TCA GGG GGA-3’ was synthesized at Anhui Amptomai Biotechnology Co. ltd (Anhui, China). Tris was purchased from Biotopped Life Science Co. Sodium chloride (NaCI), hydrochloric acid (HCl), sodium dihydrogen phosphate (NaH₂PO₄) and disodium hydrogen phosphate (Na₂HPO₄) were purchased from Sinopharm Chemical Reagent Co. Enrofloxacin (ENR) and 3-(N-morpholino) propanesulfonic acid (MOPS) were purchased from Energy Chemical Co. TYL were purchased from Aladdin Biotechnology Company Limited (Shanghai, China). SYBR Green l (10,000 x) was purchased from Solarbio Life Sciences Co. ltd (Beijing, China).

The fluorescence intensity was measured by using the microplate reader (Molecular Devices, China). A microplate thermal oscillator device (Allsheng, China) was used to mix the solution. The aptamer stock solution was centrifuged by using a high-speed centrifuge (Sigma, Germany).

2.2. Fluorescence detection of ENR

The protocol for the quantitative determination of ENR was as follows: The solution of ENR aptamer (30 nM) and ultrapure water were introduced into a black 96-well plate and incubated for 30 min. Then, SG-I (1 x) was added and incubated for 50 min at the constant temperature. Finally, ENR (1 µM) was added to the mixture with final volume reach 200 µL, and the fluorescence signal was immediately measured. The fluorescence intensity was marked as F with addition of ENR and F₀ without ENR. The influence of ENR on the fluorescence intensity of SG-I in the system can be reflected by calculating ΔF (ΔF = F₀-F).

2.3. Characterization of the detection system

In order to verify the feasibility of the principle, the four samples were established, and the fluorescence intensity of was measured: (a) SG-I (1 x); (b) SG-I (1 x) + ENR (1 µM); (c) SG-I (1 x) + aptamer (30 nM); (d) SG-I (1 x) + ENR (1 µM) + aptamer (30 nM). All the experiments consisted of at least three parallel experiments.

2.4. Optimization of experimental conditions

To obtain the reliable detection results of ENR, a series of experiments of optimal condition were conducted, such as the reaction buffer solution (MOPS, PBS, Tris-HCl, ddH₂O), pH (5.0, 6.0, 7.0, and 8.0), reaction time of aptamer and SG-I (0, 10, 20, 30, 40, 50, 60, and 70 min), concentration of SG-I (0.2 x, 0.4 x, 0.6 x, 0.6 x, 0.8 x, 0.8 x, and 2 x), and concentration of aptamer (130, 140, 150, 160, 170, 180, 190, and 200 nM). The specificity and sensitivity of the system were tested under the optimized conditions. All the experiments consisted of at least three parallel experiments.

2.5 Real sample detection

Three actual samples were used in the experiment, namely pork, chicken and fish, all purchased from local supermarkets. First, the pork was homogenized. Then the sample (1 g) and HCl (0.01 M, 0.49 mL) were mixed, followed by ultrasonic treatment for 2 min and centrifugation at 12000 rpm for 10 min. The supernatant was aspirated and mixed with NaOH (0.01 M, 0.49 mL), and centrifuged at 12000 rpm for 10 min (Xiao, et al. [2022]). Finally, the supernatant was aspirated with a syringe and filtered through 0.22 µm membrane filter and collected for backup.

Chopped chicken (0.5 g) and fish (0.5 g) were mixed with acetonitrile (5 mL), and an appropriate amount of ethanol was added to remove proteins. The mixture was then sonicated for 10 min at room temperature and centrifuged for 10 min at 10,000 rpm (Shen, et al. [2024]). The supernatant was collected and filtered through 0.22 µm organic filter membrane to obtain the final solution for subsequent measurement. The final filtrate collected was mixed with different concentrations of ENR for fluorescence measurement.

3.1 detection mechanism

Firstly, the secondary structure of the 28-nt nucleic acid aptamer was predicted and analyzed by using the NUPACK program. As shown in Figure. 1a, the aptamer in its natural state had partial base complementation and presented a relatively stable state. Subsequently, we verified the feasibility of detection system by measuring the fluorescence intensity of SG-I under different conditions. As shown in Fig. 1b, the fluorescence intensity of SG-I at 534 nm was weak and not nearly affected with addition of ENR. However, when the aptamer was added to the SG-1 solution, which specifically recognized and inserted the aptamer with a G-quadruplex structure to emit the strong fluorescence signal. Subsequently, with further addition of ENR, the aptamer can be combined preferentially with ENR to cause the conformational change of aptamer, which prevented the SG-I from being inserted into the aptamer to result in decrease of SG-I fluorescence intensity. Therefore, this sensor strategy provided feasibility for quantitative analysis of ENR.

3.2 Optimization of experimental conditions

The fluorescence signal change of the detection system depended importantly on the experimental conditions. Therefore, to obtain the best performance in detecting ENR, we optimized the detection conditions, such as the type of buffer, pH, reaction time between SG-I and aptamer, concentration of aptamer and SG-I.

Firstly, ultrapure water, PBS (10 mM pH 7.4), Tris-HCl (50 mM pH 7.4) and MOPS (10 mM pH 7.0) buffers were selected for this experiment to compare the difference in fluorescence intensity between the blank group and the experimental group. As shown in Fig. 2, the most significant change in fluorescence intensity was observed in the ultrapure water system. Therefore, ultrapure water was chosen as the buffer solution for subsequent experiments.

Secondly, the fluorescence properties of SG-I may be altered by the pH of the environment. Usually, pH affected the protonation state of the dye, which in turn led to change in fluorescence intensity (Xing, et al. [2022]). Therefore, we examined the fluorescence intensity with and without the addition of ENR under the condition of pH 5.0, 6.0, 7.0, and 8.0, respectively. As shown in Fig. 3, ΔF was the largest at pH 6.0. Therefore, ultrapure water with pH 6.0 was chosen for subsequent experiments.

In addition, we studied the reaction time of aptamer and SG-I. As shown in Figure. 4a, the fluorescence intensity of the system increased with the reaction time, and the fluorescence intensity tended to level off after 50 min. Consequently, the optimal reaction time of aptamer and SG-I was 50 min.

Meanwhile, the concentrations of SG-I was screened. As showed in Figure. 4b, the fluorescence intensity was increased with SG-I concentration and reached the maximum with SG-I concentration of 0.6x. The fluorescence intensity remained basically stable with the increase of concentration from 0.8x to 1.0x, but the 0.6x was chosen as the optimum concentration of SG-I based on economy.

Finally, it was necessary to optimize the concentration of the solution of aptamer in the system, because it affected the sensitivity and feasibility of the detection system. As shown in Fig. 4c, with the increase in the concentration of aptamer (130–200 nM), ΔF first increased and then started to decrease gradually. The fluorescence intensity was enhanced with the increase of aptamer concentration and reached the maximum with concentration of 160 nM. Therefore, 160 nM was chosen as the optimal concentration of aptamer.

3.3 Selectivity and sensitivity of the detection system

Under optimized conditions, the selectivity of the assay was analyzed. We evaluated the response of the sensor to varieties of antibiotics under the same experimental conditions, including ENR, LOM, FLU, DIF, DAN, TYL, 7-ACA, KAN and Mix. As shown in Fig. 5a, the fluorescence intensity change (ΔF) caused by ENR was significantly stronger than that of other antibiotics. And when these antibiotics coexisted, the difference in fluorescence intensity of the system was almost identical to that of alone ENR. Therefore, this detection system had excellent selectivity for detecting ENR without interference of simultaneous other analogs.

Meanwhile, under optimized conditions, we studied the relationship between the fluorescence intensity change and the concentration of ENR (0, 20, 40, 80, 120, 160, 200, 400, 600, 800, 1000, 2000 nM). As shown in Fig. 5b, the fluorescence intensity of the system gradually increased with ENR concentration (0-600 nM), which displayed a good linear relationship. The linear equation was ΔF = 48961.3 + 8697.51 [ENR] (R² = 0.996), and the limit of detection was 0.158 nM. The aptasensor displayed wider linear detection range, high sensitivity and low detection limit for detecting ENR compared with related reports (Table 1). Therefore, this sensor system provided the potential for detection of ENR residues in meat products.

Table 1

Comparison with the reported methods for ENR determination.
Method applied	Linear range	LOD	Application	Reference
HPLC	0.50–53 µM	0.33 µM	plasma	(Aslam, et al. 2016)
Electrochemical assay	0.03-27.8nM	13.6 pM	chicken	(Guo, et al. 2024)
Electrochemical assay	0.03–278 nM	0.01 nM	fish	(Huang, et al. 2022)
ICAs	0.70–27.8 nM	79.4 pM	milk	(Hu, et al. [13])
Colorimetric assay	1.4–1400 nM	321.1 pM	seafood	(Wang, et al. 2023)
Fluorescent assay	0.03–278 nM	22.3 pM	milk	(Zhang, et al. [2024])
Electrochemical assay	0.28–27.8 nM	0.14 nM	pond water	(Van, et al. [2022])
Fluorescent assay	0-600 nM	0.158 nM	pork, fish, chicken	This work

3.4 Detection of ENR in real samples

In this experiment, the practical application was conducted on fish, chicken and pork samples spiked with ENR. As shown in Table 2, the relative standard deviations (RSD) values were all below 6%, and the average recoveries of these samples was from 99.74–104.02%. These results indicated that the detection system had potential for the detection of ENR levels in real samples.

Table 2

Levels of ENR in pork sample, fish sample and chicken sample were measured.
Sample	Spiked concentration (nM)	Mean found Concentration (nM)	Recovery (%)	RSD (%)
	100	100.51	100.51	2.63
pork	200	199.67	99.84	0.67
	300	301.56	100.52	1.73
	100	101.76	101.76	5.21
fish	200	205.63	102.82	2.88
	300	303.94	101.31	2.09
chicken	100 200 300	104.02 201.16 299.22	104.02 100.58 99.74	4.50 2.02 0.93

In summary, we developed a quantitative fluorescence method for the determination of ENR based on SG-I and aptamer. Under the optimized conditions, the aptasensor showed good linearity between fluorescence intensity and ENR concentration from 0 to 600 nM, with the limit of detection of 0.158 nM. In addition, the method was used for the detection of ENR residues in chicken, fish and pork, and showed good performance with average recoveries in the range of 99.74%-104.02%, and RSD of the ENR determination were less than 6%. Therefore, the method was low cost and easy to operate, and showed a wide application potential in the safety testing of meat products.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author Contribution

C.-C. Li and W.-C. Kong wrote the main manuscript text.J.-L. Cui and X.-L. Li prepared all figures.Y.-L. Meng and X.-J. Jia supervised the work.X.-L. Zhang and Y.-F. Kang reviewed the manuscript.

Acknowledgements

We thank for the support of the Key Project of Hebei Science and Technology Department (21326612D).

Data Availability

The data that support the findings of this study are available on request from the corresponding author upon reasonable request.

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No competing interests reported.

floatimage1.png
Scheme 1. Schematic illustration for determination of ENR based on the aptamer with G-quadruplex structure and SG-I.

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Fluorescence detection for enrofloxacin by using Aptamer and fluorophore SYBR Green I

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Chemicals and devices

2.2. Fluorescence detection of ENR

2.3. Characterization of the detection system

2.4. Optimization of experimental conditions

2.5 Real sample detection

3. Results and discussion

3.1 detection mechanism

3.2 Optimization of experimental conditions

3.3 Selectivity and sensitivity of the detection system

3.4 Detection of ENR in real samples

4. Conclusions

Declarations

Declaration of Competing Interest

Author Contribution

Acknowledgements

Data Availability

References

Additional Declarations

Supplementary Files

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