Selection and characterization of a novel DNA aptamer for label-free fluorescence biosensing of ochratoxin A - PubMed (original) (raw)

Selection and characterization of a novel DNA aptamer for label-free fluorescence biosensing of ochratoxin A

Maureen McKeague et al. Toxins (Basel). 2014.

Abstract

Nucleic acid aptamers are emerging as useful molecular recognition tools for food safety monitoring. However, practical and technical challenges limit the number and diversity of available aptamer probes that can be incorporated into novel sensing schemes. This work describes the selection of novel DNA aptamers that bind to the important food contaminant ochratoxin A (OTA). Following 15 rounds of in vitro selection, sequences were analyzed for OTA binding. Two of the isolated aptamers demonstrated high affinity binding and selectivity to this mycotoxin compared to similar food adulterants. These sequences, as well as a truncated aptamer (minimal sequence required for binding), were incorporated into a SYBR® Green I fluorescence-based OTA biosensing scheme. This label-free detection platform is capable of rapid, selective, and sensitive OTA quantification with a limit of detection of 9 nM and linear quantification up to 100 nM.

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Figures

Figure 1

Figure 1

Secondary structure of OTA aptamers predicted by RNAstructure folding software [51] and rendered using VARNA software [55].

Figure 2

Figure 2

SYBR Green I fluorescence-based OTA aptasensing scheme. In the presence of the aptamer, SG fluoresces at 520 nM, due to intercalation and other hydrophobic interactions with the DNA aptamer. Increasing concentrations of the aptamer target (OTA in this assay) causes a displacement of SG, and a concentration-dependent loss of emitted fluorescence.

Figure 3

Figure 3

The SG-based label-free assay (A) Aptamer A08, OTA, SG, and SG with OTA show no fluorescence from 500–600 nm; (B) Combining DNA and SG results in significant emission at 520 nm. A non-specific DNA control does not respond to the addition of OTA; (C–E) The A08, B08 and A08 minimer aptamers selected in this experiment display enhanced fluorescence when incubated with SG. Upon the addition of OTA, a concentration dependent loss of fluorescence is observed; (F) The A08 minimer-based sensor has a linear response between 9 and 100 nM (y = 4.89 × 106_x_; _R_2 = 0.991).

Figure 4

Figure 4

Selectivity of the SG aptamer biosensor at 30 and 100 nM concentrations of targets. The sensor is more sensitive to the target OTA compared to similar compounds. All the fluorescence spectra for the aptamers with these three targets are available as Supplementary Information (Figures S5–S7).

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