Development of aptamer fluorescent switch assay for aflatoxin B1 by using fluorescein-labeled aptamer and black hole quencher 1-labeled complementary DNA. 2018

Yapiao Li, and Linlin Sun, and Qiang Zhao
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China.

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins and draws great concern in health and food safety. A DNA aptamer against AFB1 having a stem-loop structure shows high binding affinity to AFB1 and promise in assay development for AFB1 detection. Based on the structure-switching property of the aptamer, we report an aptamer fluorescence assay for AFB1 detection. Aptamer with fluorescein (FAM) label at 5' end was used as affinity ligand, while its short complementary DNA (cDNA) with BHQ1 (black hole quencher 1) label at 3' end was used as a quencher. In the absence of AFB1, FAM-labeled aptamer hybridized with BHQ1-labeled cDNA, forming a duplex of cDNA and aptamer, resulting in fluorescence quenching of FAM. When AFB1 bound with aptamer, the BHQ1-labeled cDNA was displaced from aptamer, causing fluorescence restoration of FAM. We tested a series of FAM-labeled aptamers and BHQ1-labeled cDNAs with different lengths. The lengths of the aptamer stem and the cDNA, Mg2+ in binding buffer, and temperature had significant influence on the performance of the assay. Under optimized conditions, we achieved sensitive detection of AFB1 by using a 29-mer FAM-labeled aptamer and a 14-mer BHQ1-labeled cDNA, and the detection limit of AFB1 reached 0.2 nM. The maximum fluorescence recovery rate of FAM-labeled aptamer caused by AFB1 was about 69-fold. This method enabled the detection of AFB1 in complex sample matrix, e.g., diluted wine samples and maize flour samples. This aptamer-based fluorescent assay for AFB1 determination shows potential for broad applications. Graphical abstract ᅟ.

UI MeSH Term Description Entries
D011042 Poisons Substances which, when ingested, inhaled, or absorbed, or when applied to, injected into, or developed within the body in relatively small amounts may, by their chemical action, cause damage to structure or disturbance of function. (From Dorland, 27th ed) Poison
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D000476 Alkanesulfonates Organic esters or salts of sulfonic acid derivatives containing an aliphatic hydrocarbon radical. Alkyl Sulfonates,Sulfonates, Alkyl
D001391 Azo Compounds Organic chemicals where aryl or alkyl groups are joined by two nitrogen atoms through a double bond (R-N Azo Dye,Azo Dyes,Compounds, Azo,Dye, Azo,Dyes, Azo
D016604 Aflatoxin B1 A potent hepatotoxic and hepatocarcinogenic mycotoxin produced by the Aspergillus flavus group of fungi. It is also mutagenic, teratogenic, and causes immunosuppression in animals. It is found as a contaminant in peanuts, cottonseed meal, corn, and other grains. The mycotoxin requires epoxidation to aflatoxin B1 2,3-oxide for activation. Microsomal monooxygenases biotransform the toxin to the less toxic metabolites aflatoxin M1 and Q1. Aflatoxin B(1),Aflatoxin B,Aflatoxin B1 Dihydrochloride, (6aR-cis)-Isomer,Aflatoxin B1, (6aR-cis)-Isomer, 14C-Labeled,Aflatoxin B1, (6aR-cis)-Isomer, 2H-Labeled,Aflatoxin B1, (6aR-cis)-Isomer, 3H-Labeled,Aflatoxin B1, cis(+,-)-Isomer,HSDB-3453,NSC-529592,HSDB 3453,HSDB3453,NSC 529592,NSC529592
D052157 Aptamers, Nucleotide Nucleotide sequences, generated by iterative rounds of SELEX APTAMER TECHNIQUE, that bind to a target molecule specifically and with high affinity. DNA Aptamer,DNA Aptamers,RNA Aptamers,Rna Aptamer,Nucleotide Aptamers,Oligonucleotide Ligands, DNA,Oligonucleotide Ligands, RNA,Aptamer, DNA,Aptamer, Rna,Aptamers, DNA,Aptamers, RNA,DNA Oligonucleotide Ligands,RNA Oligonucleotide Ligands
D057230 Limit of Detection Concentration or quantity that is derived from the smallest measure that can be detected with reasonable certainty for a given analytical procedure. Limits of Detection,Detection Limit,Detection Limits
D018076 DNA, Complementary Single-stranded complementary DNA synthesized from an RNA template by the action of RNA-dependent DNA polymerase. cDNA (i.e., complementary DNA, not circular DNA, not C-DNA) is used in a variety of molecular cloning experiments as well as serving as a specific hybridization probe. Complementary DNA,cDNA,cDNA Probes,Probes, cDNA
D019793 Fluorescein A phthalic indicator dye that appears yellow-green in normal tear film and bright green in a more alkaline medium such as the aqueous humor. Fluorescein Sodium,Sodium Fluorescein,C.I. 45350,Colircusi Fluoresceina,D & C Yellow No. 7,D & C Yellow No. 8,D and C Yellow No. 7,D and C Yellow No. 8,D&C Yellow No. 7,D&C Yellow No. 8,Diofluor,Disodium Fluorescein,Fluor-I-Strip A.T.,Fluorescein Dipotassium Salt,Fluorescein Disodium Salt,Fluorescein Monosodium Salt,Fluorescite,Fluorescéine sodique Faure,Fluorets,Ful-Glo,Funduscein,Minims Fluorescein Sodium,Minims Fluoresceine,Minims Stains,Optifluor Diba,Uranine,Dipotassium Salt, Fluorescein,Disodium Salt, Fluorescein,Fluor I Strip A.T.,Fluorescein Sodium, Minims,Fluorescein, Disodium,Fluorescein, Sodium,Fluoresceina, Colircusi,Fluoresceine, Minims,Ful Glo,Monosodium Salt, Fluorescein,Sodium, Fluorescein

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