Multiplexed aptasensing of food contaminants by using terminal deoxynucleotidyl transferase-produced primer-triggered rolling circle amplification: application to the colorimetric determination of enrofloxacin, lead (II), Escherichia coli O157:H7 and tropomyosin
- 39 Downloads
A colorimetric assay is described for simultaneous detection of multiple analytes related to food safety. It is based on the use of a sandwich aptasensor and terminal deoxynucleotidyl transferase (TdT) which produces a primer for subsequent rolling circle amplification (RCA). Two split aptamer fragments (Apt1 and Apt2) are firstly immobilized, Apt1 on gold nanoparticles (AuNPs), and Apt2 on magnetic beads (MBs). They are then used in a sandwich aptasensor. In the presence of analyte, two probes could specifically recognize target and form a ternary assembly, and the magnetic beads also act to separate rapidly and enrich the target. Then, the extension of template-free DNA is triggered by TdT at the exposed 3′-hydroxy terminals of Apt1. This produces polyA sequences that serve as primers for subsequent RCA. The product of RCA is hybridized with a complementary horse radish peroxidase (HRP) DNA probe. HRP catalyzes the H2O2-mediated oxidation of tetramethylbenzidine (TMB) and forms a blue chromogenic product. After magnetic separation, the absorption values of the blue product in the supernatant are measured at a wavelength of 600 nm. Based on this dual amplification mechanism, the assay was applied to multiplexed determination of enrofloxacin (ENR), lead(II), Escherichia coli O157:H7 and tropomyosin. Exemplarily, ENR is detectable at concentrations down to 2.5 pg mL−1 with a linear range that extends from 1 pg mL−1 to 1 μg·mL−1. The assay was validated by analysis of spiked fish samples. Recoveries range between 87.5 and 92.1%.
KeywordsSignal amplification method Isothermal nucleic acid amplification Food safety detection Biodetection Gold nanoparticles Magnetic beads Aptasensor Antibiotics
This work was financially supported by the National Natural Science Foundation of China (21775102, 21775104), National Key R&D Program of China (2017YFF0204603, 2018YFF0212803) and Shanghai Rising-Star Program (16QB1403100).
Compliance with ethical standards
Conflict of interest
This article does not contain any studies involving human participants performed by any of the authors.
- 9.Zhang K, Cao J, Wu Y, Hu F, Li T, Wang Y, Gan N (2019) A fluorometric aptamer method for kanamycin by applying a dual amplification strategy and using double Y-shaped DNA probes on a gold bar and on magnetite nanoparticles. Mikrochim Acta 186(2):120. https://doi.org/10.1007/s00604-018-3207-6 CrossRefPubMedGoogle Scholar
- 18.Yan P, Hao Y, Shu Z, Gu C, Zhou X, Liu X, Xiang H (2018) Double signal enhancement strategy based on rolling circle amplification and photoinduced electron transfer for ultrasensitive fluorometric detection of methylated DNA. Mikrochim Acta 185(6):299. https://doi.org/10.1007/s00604-018-2839-x CrossRefPubMedGoogle Scholar
- 22.Hu L (2017) Food safety-rapid detection and effective prevention of foodborne hazards. Apple Academic Press (T&F), FloridaGoogle Scholar
- 23.Gao F, Zhang T, Chu Y, Wang Q, Song J, Qiu W, Lin Z (2018) Ultrasensitive impedimetric mercury(II) sensor based on thymine-hg(II)-thymine interaction and subsequent disintegration of multiple sandwich-structured DNA chains. Mikrochim Acta 185(12):555. https://doi.org/10.1007/s00604-018-3097-7 CrossRefPubMedGoogle Scholar
- 27.Zhou Y, Fang W, Lai K, Zhu Y, Bian X, Shen J, Li Q, Wang L, Zhang W, Yan J (2019) Terminal deoxynucleotidyl transferase (TdT)-catalyzed homo-nucleotides-constituted ssDNA: inducing tunable-size nanogap for core-shell plasmonic metal nanostructure and acting as Raman reporters for detection of Escherichia coli O157:H7. Biosens Bioelectron 141:111419. https://doi.org/10.1016/j.bios.2019.111419 CrossRefPubMedGoogle Scholar
- 32.Cinquina AL, Roberti P, Giannetti L, Longo F, Draisci R, Fagiolo A, Brizioli NR (2003) Determination of enrofloxacin and its metabolite ciprofloxacin in goat milk by high-performance liquid chromatography with diode-array detection. Optimization and validation. J Chromatogr A 987(1–2):221–226CrossRefGoogle Scholar