Microfluidic paper device for rapid detection of aflatoxin B1 using an aptamer based colorimetric assay

Abstract

Contamination of milk by mycotoxins is a serious problem worldwide. Herein, we focused on the detection of aflatoxin B1 (AflB1) using a paper microfluidic device fabricated with specific aptamers as biorecognition elements. The fabrication process resulted in the generation of a leak proof microfluidic device where two zones were designed: control and test. Detection is achieved by color change when aflatoxin reacts with an aptamer followed by salt induced aggregation of gold nanoparticles. Specific aptamers for aflatoxin B1 were immobilized successfully onto the surface of gold nanoparticles. Biophysical characterization of the conjugated AuNPs-aptamer was done by UV-vis spectroscopy, DLS (dynamic light scattering), TEM (transmission electron microscopy). Under optimal conditions, the microfluidic device showed an excellent response for aflatoxin B1 detection in the range of 1 pM to 1 μM with a detection limit of up to 10 nM in spiked samples. Disadvantages associated with conventional techniques of ELISA were overcome by this one step detection technique with low operation cost, simple instrumentation, and user-friendly format with no interference due to chromatographic separation. The developed microfluidic paper-based analytical device (μPAD) can provide a tool for on-site detection of food toxins in less than a minute which is the main requirement for both qualitative and quantitative analysis in food safety and environmental monitoring.

Scheme 1. Colorimetric assay for the detection of aflatoxin B1 (Afl-B1) using salt induced AuNPs aggregation. In the absence of aflatoxin B1, aptamer adsorbed on the surface of via physical adsorption and enhanced the stability against salt (NaCl) induced aggregation by formation of G-quadruplex. In presence of aflatoxin B1, aptamer bound with AuNPs displaced and form a complex with aflatoxin B1 leaving AuNPs to react with NaCl that resulted in aggregation (change in color from wine red to blue).

Fig. 1. Optimization of NaCl concentration for the detection of aflatoxin B1. (a) TEM showed the (i) average AuNPs size of 19 ± 5 nM (ii) AuNPs coated with AflB1 aptamer; (b) DLS spectra showed hydrodynamic diameters of 19 nm; (c) UV-vis spectroscopy at different concentration of NaCl (20, 40, 80, 160, 340, 640 mM) showed the red shift from 520 nm (AuNPs) to 525 nm (AuNPs–NaCl complex), as the concentration of NaCl increased, the spectra became flattened; (d) calibration curve and corresponding color change w.r.t. optimum concentration of NaCl; (e) zeta potential of similar concentration of NaCl reacted with AuNPs.

Fig. 2. a) Diagrammatic representation for optimization of aptamer concentration for the detection of aflatoxin B1; (b) absorption spectra of different concentration of aptamer (800, 400, 200, 100, 50, 25 nM); (c) calibration curve for detection of optimum concentration of aptamer (25, 50, 100, 200, 400, 800 nM) and correspondence color change in the presence of different concentration of aptamer; (d) zeta potential of different concentration of aflatoxin B1 aptamer; (e) hydrodynamic diameter of AuNPs, AuNPs + AflB1 Apt, AuNPs + NaCl, and AuNPs–AflB1 + NaCl nanocomplex.

Fig. 3. a) Scheme showed the step by step displacement assay; (b) (i) UV-vis spectroscopy of AuNPs–Apt complex in the presence and absence of aflatoxin B1 at different concentrations (1 μM to 1 pM) wit fix concentration of NaCl and (ii and iii) corresponding change in color at various concentration of aflatoxin B1 and ochratoxin (1 μM-blue to 1pM-wine red); (c) (i) calibration curve at A630/520 and (ii) fluorescence spectra of Afl B1 and ochratoxin; (d) (i) Hydrodynamic diameter and (ii) zeta potential of AuNPs, AuNPs + Apt, AuNPs + Apt + AflB1 nanocomplex.

Fig. 4. Schematic representation of μPAD paper microfluidic device. (a) (i) Concept of μPAD-positive control (PC), negative control (NC), analyte pad (AP), (ii) dimensions and (iii) fabrication of the paper device; (b) (i) the color development after spiking Afl B1 in water and (ii) color development in presence of AflB1/no change in color in presence of ochratoxin in water.