A novel DNA-based colorimetric biosensor enables the ultra-sensitive detection of Ochratoxin A via starch-iodine complexation
Colorimetric detection methods are particularly well-suited for point-of-care (POC) biosensing due to their simple readout and operation. In this context, Akkapol Suea-Ngam from the deMello group has developed a novel and simple colorimetric assay for the highly sensitive detection of a fungal toxin on a paper platform.
Paper-based analytical devices (PADs) offer an ideal platform for detecting biological analytes at the point-of-need, primarily due to the lightweight, affordable, and wide availability of paper. In particular, PADs combined with a colorimetric detection scheme can easily meet many requirements for POC testing, such as being affordable, user-friendly, robust, and equipment-free. Despite their advantages, paper devices with colorimetric detection have often suffered from poor sensitivity. A wide range of colorimetric assays has been developed that rely on noble metal nanoparticles. However, these costly materials can limit the affordability of the device.
In this work, Akkapol has designed a novel colorimetric assay using an aptamer-antibody pair that uses starch-iodine complexation to produce a colorimetric signal. The target analyte is Ochratoxin A (OTA), a common food-contaminating mycotoxin produced by fungi, which potentially causes cancer in animals and humans. The method employs a common strategy termed a sandwich assay in which consecutive addition of reagents leads to the formation of a sensing structure. Briefly, anti-OTA antibodies are immobilized on the paper surface and serve to specifically capture OTA, then, aptamer-enzyme conjugates will bind to the immobilized OTA, forming the so-called sandwich complex. Aptamers are short DNA sequences that are designed to bind specifically to particular species, in this case OTA. Therefore, after washing, the enzyme (glucose-oxidase) attached to the aptamer will only be present if OTA had been captured in the first place. The last step involves the addition of glucose, starch, and iodine, all easy to use and affordable reagents. The reaction of glucose with the enzyme releases hydrogen peroxide, which reacts with iodine and starch to produce a strong blue color. In the absence of OTA, the PAD remains colorless. The assay shows a high sensitivity with a limit of detection of 20 pg/mL for OTA using only microliters of reagents. Such a low limit of detection compares well with other reported detection schemes for OTA, and shows that the assay performs particularly well for a paper-based format. To assess the utility of the device, the authors successfully performed OTA detection in real-world samples, including beer, urine, and human serum.
The high sensitivity of this assay, along with its non-toxic and affordable reagents, makes it ideally suited for in-the-field use, with the devices being safely and easily operated and disposed of. The authors expect that this sensing approach could be applied to a wide range of point-of-use testing devices for biological analyte detection, as well as testing of contaminated food samples.