An Aptamer-Array-Based Sample-to-Answer Biosensor for Ochratoxin A Detection via Fluorescence Resonance Energy Transfer

Abstract

Food toxins are a hidden threat that can cause cancer and tremendously impact human health. Therefore, the detection of food toxins in a timely manner with high sensitivity is of paramount importance for public health and food safety. However, the current detection methods are relatively time-consuming and not practical for field tests. In the present work, we developed a novel aptamer-chip-based sample-to-answer biosensor (ACSB) for ochratoxin A (OTA) detection via fluorescence resonance energy transfer (FRET). In this system, a cyanine 3 (Cy3)-labeled OTA-specific biotinylated aptamer was immobilized on an epoxy-coated chip via streptavidin-biotin binding. A complementary DNA strand to OTA aptamer at the 3′-end was labeled with a black hole quencher 2 (BHQ2) to quench Cy3 fluorescence when in proximity. In the presence of OTA, the Cy3-labeled OTA aptamer bound specifically to OTA and led to the physical separation of Cy3 and BHQ2, which resulted in an increase of fluorescence signal. The limit of detection (LOD) of this ACSB for OTA was 0.005 ng/mL with a linearity range of 0.01–10 ng/mL. The cross-reactivity of ACSB against other mycotoxins, ochratoxin B (OTB), aflatoxin B1 (AFB1), zearalenone (ZEA), or deoxynilvalenol (DON), was less than 0.01%. In addition, this system could accurately detect OTA in rice samples spiked with OTA, and the mean recovery rate of the spiked-in OTA reached 91%, with a coefficient of variation (CV) of 8.57–9.89%. Collectively, the ACSB may represent a rapid, accurate, and easy-to-use platform for OTA detection with high sensitivity and specificity.

Aptamer based detection and separation platforms for ochratoxin A: A systematic review

“…In the presence of OTA, the Cy3-labeled OTA aptamer bound specifically to OTA and led to the physical separation of Cy3 and BHQ2, which resulted in an increase in fluorescence signal. This aptasensor was tested in rice samples spiked with OTA with a mean recovery rate of 91% (Li et al, 2021).…”

Design of a Quencher-Free Fluorescent Aptasensor for Ochratoxin A Detection in Red Wine Based on the Guanine-Quenching Ability

“…The application of aptamers has brought hope to this expectation. In 2008, Cruz-Aguado selected the first OTA aptamer for OTA detection [21]; since then, this aptamer has been widely used to construct many biosensors [11,[22][23][24]. Among these, fluorescence aptasensors are the simplest, easiest to operate, and are extremely sensitive.…”

Smartphone-Based Quantitative Analysis of Protein Array Signals for Biomarker Detection in Lupus

“…However, for complicated chronic diseases such as lupus, lupus nephritis, or other CKDs, multiple disease markers are needed for an accurate diagnosis. Microarray technology provides a new opportunity to develop low-cost, high-throughput, multiplexing, and minimal-volume detection assays for clinical and point-of-care diagnostics [ 7 - 12 ], a laboratory-based, computerized microarray scanner is large, expensive, and not suitable for point-of-care testing.…”