A dichromatic label-free aptasensor for sulfadimethoxine detection in fish and water based on AuNPs color and fluorescent dyeing of double-stranded DNA with SYBR Green I
Introduction
Sulfonamides are commonly used as antibacterial agents for the treatment of bacterial infections in aquaculture (Chokejaroenrat et al., 2019). Sulfadimethoxine (SDM) is a sulfonamide that can be detected most frequently in water and fish samples (Gaffney et al., 2015, Yuan et al., 2015) and in fresh milk/dairy products (Beltrán et al., 2015, Chiesa et al., 2012, Furusawa, 2000). The intake of SDM through food chain may present toxic reactions, superinfection and drug resistance for human health (Białk-Bielińska et al., 2012). Therefore, European Commission (EC) and China have proposed a maximum residue limit (MRL) of SDM 100 ng mL−1 in foodstuffs (Wang et al., 2017). High performance liquid chromatography (HPLC) (Mahmoud et al., 2013), capillary electrophoresis (CE) (Castro-Puyana, Crego, & Marina, 2008), and enzyme-linked immunosorbent assay (ELISA) (Muldoon et al., 2000) and immunochromatographic lateral flow strip test (Chen et al., 2017) are the routine detection methods for SDM residue. These methods have low limits of detection (LODs) meeting the requirement of MRL, but have some shortcomings, including time-consuming, expensive instruments and complicate operation procedures (Ma et al., 2018, Yang et al., 2017). Therefore, it is urgent to develop an economical, portable, and high-throughput method to detect SDM residue sensitively and specifically.
Aptamers are single-stranded DNA/RNA (ssDNA/RNA) oligonucleotides that possess the advantages of simple synthesis, easy and controllable modification, and long-term stability (Chen, Li, Tu, & Luo, 2018). Aptamers have high affinity and specificity with a wide range of targets (Sun et al., 2016, Wang et al., 2017, Yang et al., 2017). Since DNA-aptamer against SDM was screened by Song, Jeong, Jeon, Jo, and Ban (2012), the aptamer has been employed to detect SDM in many strategies, such as colorimetric (Chen et al., 2013), electrochemical (You et al., 2018), fluorescence (Liu et al., 2014), and photoelectrochemical methods (Okoth, Yan, Liu, & Zhang, 2016). Among them, florescence method has received promising attention due to its fast response, simple operation and high sensitivity (Chen et al., 2018). However, the current fluorescence-based detection methods require laborious or expensive modification processes, which severely limit their practical applications (Cetin et al., 2014). SYBR Green I (SGI) is a popular commercial nucleic acid fluorescent dye that can specifically bind to double-stranded DNA (dsDNA) to produce strong fluorescent emission (Li, Tian, Kong, & Chu, 2015). Because SGI possesses temperature stability, good optical physical properties, and low fluorescent background in aqueous systems, florescence methods through SGI dyeing of dsDNA have been widely applied (Yang et al., 2018).
Gold nanoparticles (AuNPs) have been used to construct many fluorescent or colorimetric sensors due to their dimensional and distance-dependent optical properties, high absorption efficiencies, and large surface areas (Niu et al., 2014, Yang et al., 2017, Youssef et al., 2014). The dispersed AuNPs present red color, while a blue color can be observed when AuNPs aggregate in salt solutions (Mirkin, Letsinger, Mucic, & Storhoff, 1996). But ssDNA can prevent the salt-induced aggregation of AuNPs because of the protection of ssDNA by the coordination interaction between the exposed bases of ssDNA and the AuNPs (Li & Rothberg, 2004). As a non-luminescent energy acceptor, AuNPs have a high extinction coefficient and a broad absorption spectrum in visible light, which may be utilized as quenchers by fluorescence methods based on the resonance energy transfer (FRET) or internal filter effect (IFE) (Cao et al., 2013, Wang and Guo, 2009).
In the present study, a sensitive dichromatic aptasensor for SDM detection was constructed as shown in Fig. 1. The affinity of aptamer toward AuNPs results in a complex of aptamer-AuNPs which can be deposited in residue in red color after centrifugation. In supernatant where the solo cDNA can’t be dyed by SGI so that no fluorescence emission is observed. In the presence of SDM, the specific binding of aptamer to SDM leads to the separation of AuNPs and aptamer-SDM after centrifugation, in which AuNPs is deposited in blue color in residue while the aptamer-SDM is dispersed in supernatant. After the addition of cDNA and SGI in supernatant, the hybridization of cDNA and aptamer occurs because aptamer shows higher affinity toward cDNA than SDM, which results in a fluorescence emission at 530 nm due to the dyeing of dsDNA by SGI. Based on the dichromatic aptasensor, SDM in fish and water was detected with the fluorescence in solutions and the color changes in residues.
Section snippets
Reagents and materials
DNA-aptamer against SDM (5′-GGC AAC GAG TGT TTA-3′) and its complementary strand DNA (cDNA, 5′-TAA ACA CTC GTT GCC-3′), and SYBR Green I (SGI) were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). Sulfadimethoxine (SDM) and sulfathiazole (ST) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Sulfaguanidine (SG), sulfanilamide (SN), chloramphenicol (CAP), chloroauric acid tetrahydrate (AuCl3·HCl·4H2O), and trisodium citrate dihydrate (C6H5Na3O7·2H2O) were purchased
Construction of the aptamersensor
As shown in Fig. S1, the DNA aptamer against SDM consists of a loop and a stem with sequences of “GAG” and “GC-AT-AT”, by which SDM can be specifically recognized (Song et al., 2012). In the absence of target, the complexes of AuNPs-aptamer form by the coordination interaction between AuNPs and the bases of aptamer, and can be centrifuged and deposited in residue with a red color. In the presence of SDM, aptamer specifically binds to SDM by forming a hairpin structure which encapsulates the
Conclusions
A label-free aptasensor was constructed based on the different binding affinities of the aptamer toward to SDM and cDNA. By adding the AuNPs, the excessive aptamer which might hybride with cDNA was removed for eliminating the possible fluorescent interferences. And a dichromatic detection mode with the fluorescence at 530 nm and the color changes of AuNPs was achieved. Based on the subtle design of the aptasensor, SDM was detected with two linear ranges from 2 ng mL−1 to 300 ng mL−1 with
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.
Acknowledgement
This research was supported by the Foundation from the Science and Technology Planning Project of Fujian Province, China (2016Y0064), the Natural Science Foundation of Fujian Province of China (2018J01432, 2017J01633), National Key R and D Program of China (2018YFD0901003), the Science and Technology Planning Project of Xiamen, China (3502Z20183031), and the National Undergraduate Training Programs for Innovation and Entrepreneurship (201710390022, 201810390071, 20181xj008).
References (42)
- et al.
Performance of current microbial tests for screening antibiotics in sheep and goat milk
International Dairy Journal
(2015) - et al.
Sulfadimethoxine and sulfaguanidine: Their sorption potential on natural soils
Chemosphere
(2012) - et al.
Remediating sulfadimethoxine-contaminated aquaculture wastewater using ZVI-activated persulfate in a flow-through system
Aquacultural Engineering
(2019) - et al.
High sensitive rapid visual detection of sulfadimethoxine by label-freeaptasensor
Biosensors and Bioelectronics
(2013) - et al.
Label-free fluorescent aptasensor for thrombin detection based on exonuclease I assisted target recycling and SYBR Green I aided signal amplification
Sensors and Actuators B: Chemical
(2018) - et al.
Efficient inner filter effect of gold nanoparticles on the fluorescence of CdS quantum dots for sensitive detection of melamine in raw milk
Food Control
(2013) Simplified determining procedure for routine residue monitoring of sulphamethazine and sulphadimethoxine in milk
Journal of Chromatography A
(2000)- et al.
Target-induced DNA machine amplification strategy for high sensitive and selective detection of biotoxin
Sensors and Actuators B: Chemical
(2018) - et al.
Improving sensitivity of gold nanoparticle based fluorescence quenching and colorimetric aptasensor by using water resuspended gold nanoparticle
Biosensors and Bioelectronics
(2014) - et al.
Colorimetric aptasensors for determination of tobramycin in milk and chicken eggs based on DNA and gold nanoparticles
Food Chemistry
(2018)