Nandi Zhou

Selection and identification of streptomycin-specific single-stranded DNA aptamers and the application in the detection of streptomycin in honey

Single-stranded DNA (ssDNA) aptamers specific to streptomycin were screened and identified from a random oligonucleotides library by affinity magnetic beads-based SELEX. After eight rounds of selection, 16 ssDNA with different sequences were identified. Then the dissociation constants (Kd) of these ssDNA were determined and an aptamer (STR1) with highest affinity for streptomycin was identified. Further study showed that aptamer STR1 exhibits very low affinity for other aminoglycoside antibiotics, indicating high specificity. With this aptamer, detection of streptomycin was achieved by using gold nanoparticles (AuNPs)-based colorimetric method. In the presence of streptomycin, the competitive binding of the target and the aptamer decreases the stability of AuNPs in NaCl solution, triggers the aggregation, and exhibits visible color change of AuNPs solution. Through UV-visible spectroscopic quantitative analysis, streptomycin can be detected in the range of 0.2-1.2 μM. The presence of other aminoglycoside antibiotics shows neglectable disturbance. Furthermore, the established method was utilized to detect streptomycin in honey, and the same low detection limit and linear detection range were achieved.

Aptamer-based spectrophotometric detection of kanamycin in milk

Aminoglycoside antibiotics are widely used drugs. The residual antibiotics in animal-derived food are proven to be harmful to human health and therefore should be strictly controlled in the food industry. A spectrophotometric method based on a kanamycin-specific aptamer and gold nanoparticles (AuNPs) was developed for the quantitative detection of kanamycin. Two types of functionalized AuNPs were synthesized by self-assembly of thiol-modified single-stranded DNAs (ssDNAs), which were complementary to the 5′ terminal and 3′ terminal sequences of the kanamycin aptamer. As the kanamycin aptamer is mixed with these AuNPs, the AuNPs aggregate due to the hybridization of the aptamer with the complementary ssDNAs on the surface of AuNPs. However, when different concentrations of kanamycin are present, it can bind with the aptamer specifically and competitively, which leads to the disaggregation of the AuNPs aggregate, and a change in the UV-visible absorption spectrum of AuNPs. The absorbance at 527 nm is utilized to detect the concentration of kanamycin. The detection range of this method is 1–500 nM, with a detection limit of 1 nM. The developed method was then successfully employed to detect kanamycin in milk samples, and a similar response range and detection limit were obtained.

A label-free electrochemical aptasensor for the detection of kanamycin in milk

Kanamycin is a widely used aminoglycoside antibiotic, and residual kanamycin in animal-derived food causes serious side effects. Herein, a simple aptamer-based assay is proposed for the label-free electrochemical detection of kanamycin. A 5′-SH-modified kanamycin-specific aptamer was self-assembled on the surface of a gold electrode through Au–S chemistry. Upon binding to kanamycin, the conformation of the aptamer changes and its coverage of the electrode surface increases, owing to the formation of a stem-loop structure in the complex. As a result, the electron transfer resistance between the solution species and the electrode is increased. Using [Fe(CN)6]3−/4− as a probe, the square wave voltammetry (SWV) current response was utilised to determine the concentration of kanamycin. The detection range of the aptasensor was found to be 10–2000 nM, indicating a high sensitivity and a broad detection range. The specificity of detection was also determined. Furthermore, the assay was successfully employed in the detection of kanamycin in milk samples, with a similar response range and detection limit.

Simultaneous electrochemical detection of multiple antibiotic residues in milk based on aptamers and quantum dots

A sensitive simultaneous detection of streptomycin (STR), chloramphenicol (CHL) and tetracycline (TET) residues was achieved based on aptamers and quantum dot (QD) tags. Three complementary DNA1 sequences (cDNA1s) were first designed, which are not only complementary to corresponding aptamer DNAs (Ap-DNAs) specific for STR, CHL and TET, but also complementary to part of capture DNAs (Cap-DNAs) and part of complementary DNA2s (cDNA2s), respectively. cDNA1s initially hybridize with corresponding Ap-DNAs to form duplex DNAs. However, when the target antibiotics are present, STR, CHL and TET can bind with their Ap-DNAs specifically with higher affinity, which leads to the release of cDNA1s. Then the liberated cDNA1s hybridize with corresponding Cap-DNAs which are immobilized on the surface of a gold electrode by self-assembly. After that, the prepared PbS, CdS and ZnS QDs-tagged cDNA2s further hybridize with the other ends of corresponding cDNA1s on the electrode surface. The captured QDs yield distinct electrochemical signals after acid dissolution, which reflect the type and concentration of target antibiotics. Due to the inherent amplification feature of QD tags, the low detection limits for STR, CHL and TET reached 10 nM, 5 nM and 20 nM, respectively. In addition, the established assay was applied in the detection of STR, CHL and TET residues in milk samples and showed similar response ranges.

Spectrophotometric determination of ethyl carbamate through bi-enzymatic cascade reactions

A highly sensitive spectrophotometric method for ethyl carbamate (EC) determination was established through glutamate dehydrogenase/urethanase cascade reactions and the corresponding change in NADH concentration. The absorbance at 340 nm is linearly related to the EC concentration within the range of 0.3–50 μM, with a low detection limit of 0.00928 μM. The assay was further applied to analyse EC in mimic Chinese rice wine samples.

Graphene oxide-based selection and identification of ofloxacin-specific single-stranded DNA aptamers

Graphene oxide (GO) was introduced in the process of systematic evolution of ligands by exponential enrichment (SELEX) to screen ofloxacin-specific single-stranded DNA aptamers. Four sequences with high homology, ap1, ap3, ap4 and ap5 were picked out. Their secondary structures were simulated, and the dissociation constants (Kd) were measured, which were 251.3, 130.1, 159.1 and 304.4 nM, respectively. The specificity of the aptamers was also evaluated.

Electrochemical detection of tobramycin based on enzymes-assisted dual signal amplification by using a novel truncated aptamer with high affinity

An aptamer with the length of only 15 nucleotides specific for tobramycin was obtained through rationally designed truncation from a previously reported long sequence. The structural and binding properties of the aptamer were characterized. The dissociation constant (Kd) was determined to be 42.12 nM, indicating high affinity of the aptamer for tobramycin. Then an electrochemical sensor based on this aptamer was developed, which employed an enzymes-assisted dual signal amplification cycle through target recycling and strand-displacement DNA polymerization. A hairpin probe containing the aptamer sequence was designed and used to start the production cycle of a short ssDNA fragment in the presence of tobramycin, with the help of phi29 DNA polymerase and nicking endonuclease Nt.AlwI. The ssDNA fragment was captured by a signal transduction probe modified on gold electrode to form a triple-helix structure. With the help of [Ru(NH3)6]3+, a significant electrochemical signal was observed in differential pulse voltammetry (DPV). Under the optimal conditions, the current in DPV is linearly related with the concentration of tobramycin in the range of 10-200 nM, and the detection limit is 5.13 nM. The electrochemical sensor showed high specificity for tobramycin when it was challenged by other antibiotics. In addition, the constructed sensor was used to detect tobramycin in milk and water samples, and showed satisfactory performance. Therefore, the screened aptamer as well as the developed sensor has great application prospects in the fields of food safety control, medical test and environment monitoring.

UV-visible spectroscopic detection of kanamycin based on target-induced growth of gold nanoparticles

A UV-visible spectroscopic detection method of kanamycin was successfully developed based on target-induced growth of gold nanoparticles (AuNPs), using AuNPs as the probe and a kanamycin-specific aptamer as the recognition element. Firstly, the aptamer was incubated with AuNPs, which blocked the surface of the AuNPs, and inhibited the further growth of AuNPs. However, kanamycin bound with the aptamer on the surface of AuNPs with high affinity, competitively desorbed the aptamer and exposed the surface of AuNPs. Then, the growth of AuNPs proceeded by addition of NH2OH and HAuCl4. After the growth of AuNPs, the color and the maximum absorption wavelength of the AuNP solution changed, which was utilized to determine the concentration of kanamycin. Under the optimized conditions, the proposed UV-visible spectroscopic assay showed high sensitivity and high specificity for kanamycin, with a linear detection range from 20 to 100 nM, and the detection limit of 9.21 nM. Moreover, the assay has been successfully applied to detect kanamycin in honey samples without pretreatment. Therefore, it has great application prospects to detect residual kanamycin in food, medicine and water environments.