Enzyme-free fluorometric assay for chloramphenicol based on double stirring bar-assisted dual signal amplification
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An enzyme-free fluorometric assay is described that accomplishes dual signal amplification by making use of a two stirring bars. Two Y-shaped DNA probes were designed and placed on the bars. When the target (with chloramphenicol as model analyte) is added, it triggers target recycling and simultaneously catalyzes hairpin assembly (CHA). A large fraction of DNA primers is released by the analyte from the bar to the supernatant and open hairpins with G-quadruplex DNA sequence. The G-quadruplex can specifically bind thioflavin T (ThT) to emit fluorescence (with excitation/emission maxima at 445 and 485 nm) for quantification of chloramphenicol. An enzyme is not needed. ThT is added to the system as a fluorescent DNA probe. All this strongly reduces the cost for sensor construction and usage. The dual signal amplification steps occur simultaneously which reduces the detection time. The assay was successfully employed to the determination of CAP in spiked milk and fish samples within 60 min and with a 16 pM limit of detection (at S/N = 3).
KeywordsCatalyzed hairpin assembly Thioflavin T Target recycling Food safety Antibiotics detection
This work was supported by Natural Science Foundation of Zhejiang (LY19B050001,Y18B070008), Zhejiang Province Welfare Technology Applied Research Project (LGN18H300001), the Natural Science and Huiming Foundation of Ningbo (2017C50035), Natural Science Foundation of Zhejiang (LY17C200007), Zhejiang Province Welfare Technology Applied Research Project (2017C37023), Open Fund of Key Laboratory of Marine New Materials and Applied Technology in Chinese Academy of Sciences(2018K08) and K. C. Wong Magna Fund in Ningbo University.
Compliance with ethical standards
The author(s) declare that they have no competing interests.
- 1.Versporten A, Bolokhovets G, Ghazaryan L, Abilova V, Pyshnik G, Spasojevic T, Korinteli I, Raka L, Kambaralieva B, Cizmovic L, Carp A, Radonjic V, Maqsudova N, Celik HD, Payerl-Pal M, Pedersen HB, Sautenkova N, Goossens H, Grp WE-EP (2014) Antibiotic use in eastern Europe: a cross-national database study in coordination with the WHO regional Office for Europe. Lancet Infect Dis 14(5):381–387. https://doi.org/10.1016/S1473-3099(14)70071-4 CrossRefPubMedGoogle Scholar
- 3.Chafer-Pericas C, Maquieira A, Puchades R, Miralles J, Moreno A (2011) Multiresidue determination of antibiotics in feed and fish samples for food safety evaluation. Comparison of immunoassay vs LC-MS-MS. Food Control 22(6):993–999. https://doi.org/10.1016/j.foodcont.2010.12.008 CrossRefGoogle Scholar
- 6.Yang SJ, Zhu XD, Wang JS, Jin X, Liu YC, Qian F, Zhang SC, Chen JM (2015) Combustion of hazardous biological waste derived from the fermentation of antibiotics using TG-FTIR and Py-GC/MS techniques. Bioresour Technol 193:156–163. https://doi.org/10.1016/j.biortech.2015.06.083 CrossRefPubMedGoogle Scholar
- 14.Wang YS, Ma TC, Ma SY, Liu YJ, Tian YP, Wang RN, Jiang YB, Hou DJ, Wang JL (2017) Fluorometric determination of the antibiotic kanamycin by aptamer-induced FRET quenching and recovery between MoS2 nanosheets and carbon dots. Microchim Acta 184(1):203–210. https://doi.org/10.1007/s00604-016-2011-4 CrossRefGoogle Scholar
- 15.Ramezani M, Danesh NM, Lavaee P, Abnous K, Taghdisi SM (2016) A selective and sensitive fluorescent aptasensor for detection of kanamycin based on catalytic recycling activity of exonuclease III and gold nanoparticles. Sensor Actuat B-Chem 222:1–7. https://doi.org/10.1016/j.snb.2015.08.024 CrossRefGoogle Scholar
- 19.Hou T, Li W, Liu XJ, Li F (2015) Label-free and enzyme-free homogeneous electrochemical biosensing strategy based on hybridization chain reaction: a facile, sensitive, and highly specific MicroRNA assay. Anal Chem 87(22):11368–11374. https://doi.org/10.1021/acs.analchem.5b02790 CrossRefPubMedGoogle Scholar
- 20.Zhang K, Gan N, Hu FT, Chen XX, Li TH, Cao JX (2018) Microfluidic electrophoretic non-enzymatic kanamycin assay making use of a stirring bar functionalized with gold-labeled aptamer, of a fluorescent DNA probe, and of signal amplification via hybridization chain reaction. Microchim Acta 185(3):181. https://doi.org/10.1007/S00604-017-2635-Z CrossRefGoogle Scholar
- 26.Hun X, Liu BR, Meng Y (2017) Ultrasensitive chemiluminescence assay for the lung cancer biomarker cytokeratin 21-1 via a dual amplification scheme based on the use of encoded gold nanoparticles and a toehold-mediated strand displacement reaction. Microchim Acta 184(10):3953–3959. https://doi.org/10.1007/s00604-017-2430-x CrossRefGoogle Scholar
- 27.Hong F, Chen XX, Cao YT, Dong YR, Wu DZ, Hu FT, Gan N (2018) Enzyme- and label-free electrochemical aptasensor for kanamycin detection based on double stir bar-assisted toehold-mediated strand displacement reaction for dual-signal amplification. Biosens Bioelectron 112:202–208. https://doi.org/10.1016/j.bios.2018.04.017 CrossRefPubMedGoogle Scholar
- 29.Pilehvar S, Dierckx T, Blust R, Breugelmans T, De Wael K (2014) An electrochemical Impedimetric Aptasensing platform for sensitive and selective detection of small molecules such as chloramphenicol. Sensors-Basel 14(7):12059–12069. https://doi.org/10.3390/s140712059 CrossRefPubMedPubMedCentralGoogle Scholar
- 33.Chen M, Gan N, Zhang HR, Yan ZD, Li TH, Chen YJ, Xu Q, Jiang QL (2016) Electrochemical simultaneous assay of chloramphenicol and PCB72 using magnetic and aptamer-modified quantum dot-encoded dendritic nanotracers for signal amplification. Microchim Acta 183(3):1099–1106. https://doi.org/10.1007/s00604-015-1695-1 CrossRefGoogle Scholar