Antibiotics have been widely used in human medicine, aquaculture and agriculture to treat serious infectious diseases. However, overuse and misuse of antibiotics in some areas have contributed to the residues of antibiotic contaminants in water bodies and food products such as meat and dairy. This misuse has resulted in generation of super bacteria with tolerance to normal antibiotics thereby reducing the efficiency of treatment. Therefore, there is a huge demand for highly efficient and practical methods for the detection and analysis of antibiotics in food and water samples.

Current methods for the detection of antibiotics typically involve the following methods such as:

  • ELISA
  • Chromatography
  • Mass-spectrometry
  • Surface-enhanced Raman scattering (SERS)
  • SPR and electrochemical biosensors.

The techniques however, are quite expensive, time consuming and operated by trained personnel. Moreover, development of biosensors platforms for the detection of antibiotic has been difficult too, due to the lack of suitable antibodies and their relatively small signals.

Aptamer based assays and biosensors on the other hand, have shown to offer faster, simpler, less expensive methods for detection of antibiotics. Aptamers are known to be:

  • More stable than antibodies
  • More cost effective
  • Easily modified chemically
  • Better stability than antibodies
  • Easily produced in bulk

Furthermore, the unique binding properties of aptamers have shown great potential for biosensors using optical, electrochemical, and mass- sensitive approaches.

Taking advantage of aptamers and enhanced sensitivity of colorimetric biosensors, the following researchers developed an easy point-of-care platform for sensitive detection of antibiotics in water and milk samples.

Aptamer based assay for detection of antibiotics in water samples.

Chloramphenicol (CAP) is a potent drug used to treat a variety of bacterial infections. However, due to its toxic effects on human health, use of CAP is banned in food-producing animals, emphasizing the necessity for its ultrasensitive detection. Li et al., 2019 developed an aptamer-based assay for the detection of CAP in water samples. The team utilized iron-based metal organic framework (MOF) that oxidized tetramethylbenzidine (TMB) in the presence of hydrogen peroxide for colorimetric detection (Figure 1). Binding of chloramphenicol to aptamer gold nanoparticles in the presence of MOF reduced oxidation of TMB and decreased absorbance. TEM images suggested that the CAP – aptamer – AuNP conjugates coat the outside of the MOF, restricting electron transfer.

Figure 1. Schematic representation of antibiotic detection based on the antibiotic aptamer/AuNP aptamer and the intrinsic capacity of iron-based MOF’s (top left). Zeta potential of Fe-MOF’s and CAP – conjugates indicated quenching catalytic activity in the presence of aptamer (top right). UV-vis absorbance spectra indicated decreased absorbance with the increase in CAP concentration (bottom left, a). UV-vis absorbance decreasing efficiency of the Fe-MIL-53 based sensing platform showed reduced absorbance of target CAP as compared to other interfering species (bottom right, b).

High selectivity and sensitivity were obtained for CAP detection with the limit of detection of 25 nM or 8 ng/ml (Figure 1a and 1b). The method was also demonstrated using aptamers selective for other interfering species such as oxytetracycline, tetracycline and ampicillin. The detection reliability and easy operation of aptamer-based platform render it as a promising candidate for antibiotic detection in water quality monitoring practices.

Aptamer based assay for detection of antibiotics in milk samples.

Xu et al., 2018 developed an ultrasensitive and selective colorimetric strategy using highly selective DNA aptamers for the determination of the antibiotics oxytetracycline (OTC) and kanamycin (KAN) in spiked milk samples. The team used magnetic beads (SDB) for separation, HRP for colorimetric detection and gold nanoparticles (AuNPs)- for signal amplification. The aptamers were hybridized with cDNA and immobilized on magnetic beads (SDB) (Figure 2A).

Figure 2. (A) Detailed working principle of the multiplex colorimetric aptasensor for the detection of OXY and KAN antibiotics. (B) Sensitive detection of antibiotics OTC and KAN using substrates TMB and OPD, respectively. The colour changed from transparent to deep blue or deep yellow upon addition of OTC or KAN respectively. (C) Selective detection of OTC and KAN in spiked milk samples with other interfering antibiotics.

In the presence of one target, the aptamer was released, and therefore, the cDNA was free to hybridize with the HRP-signal probe immobilized on AuNPs, which in turn catalyzed the conversion of substrates. Owing to the enzymatically catalyzed colorimetric signals (from transparent to blue or yellow), OTC and KAN were quantitatively detected in a wide linear range from 10-6 to 105pg/ml. The lower detection limit of 1 ag/ml for OTC and KAN demonstrated superiority of aptamer-based detection over currently available methods for antibiotic analysis in the applications of food monitoring and clinical diagnosis.

Overall, aptamers can be readily incorporated into existing colorimetric assays and ELISA platforms, showing target specific responses even in crude sample matrices enhancing its practical utility.

At Aptamer Group Ltd, we have developed and validated numerous antibiotics and other small molecule specific aptamers in many such biosensors and assay formats. These aptamers can be used in a rapid, gain of signal assay that can be used to discover, deduce and quantify sample compounds, which can reduce the heavy reliance on antibodies and mass spec. To know more about our small molecule aptamer detection methods and how they can benefit your research, please contact us using the form below.

References

Li, J. et al. Novel sensing platform based on gold nanoparticle-aptamer and Fe-metal-organic framework for multiple antibiotic detection and signal amplification. Environment International. 2019. 125: 135-141.

Xu, Y., Lu, C., Sun, Y. et al. A colorimetric aptasensor for the antibiotics oxytetracycline and kanamycin based on the use of magnetic beads and gold nanoparticles. Microchim Acta 185548 (2018). https://doi.org/10.1007/s00604-018-3077-y

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