An Origami Paper-Based Biosensor for Allergen Detection by Chemiluminescence Immunoassay on Magnetic Microbeads

Abstract

Food allergies are adverse health effects that arise from specific immune responses, occurring upon exposure to given foods, even if present in traces. Egg allergy is one of the most common food allergies, mainly caused by egg white proteins, with ovalbumin being the most abundant. As allergens can also be present in foodstuff due to unintended contamination, there is a need for analytical tools that are able to rapidly detect allergens in food products at the point-of-use. Herein, we report an origami paper-based device for detecting ovalbumin in food samples, based on a competitive immunoassay with chemiluminescence detection. In this biosensor, magnetic microbeads have been employed for easy and efficient immobilization of ovalbumin on paper. Immobilized ovalbumin competes with the ovalbumin present in the sample for a limited amount of enzyme-labelled anti-ovalbumin antibody. By exploiting the origami approach, a multistep analytical procedure could be performed using reagents preloaded on paper layers, thus providing a ready-to-use immunosensing platform. The assay provided a limit of detection (LOD) of about 1 ng mL^-1 for ovalbumin and, when tested on ovalbumin-spiked food matrices (chocolate chip cookies), demonstrated good assay specificity and accuracy, as compared with a commercial immunoassay kit.

Keywords: chemiluminescence; immunoassay; magnetic beads; origami; ovalbumin; paper-based biosensors.

Figure 1

(a) Design of the hydrophobic areas of the origami µPAD. Black and red dashed lines represent folding lines (created by a manual rotary perforating blade) and cutting lines, respectively. A: base layer; B: anti-leaching layer; C: immunoreaction layer; D: washing layers; E: CL detection layers. (b) Images of the origami µPAD just after cutting of excess paper (left) and upon loading and air-drying of reagents and OVA-MBs (right). (c) Images of the spring-loaded 3D-printed holding clips one equipped with magnets and used for loading of OVA-MBs in the µPAD (left) and one without magnets and employed in the assay procedure (right). The scale bars represent 1 cm.

Figure 2

Scheme of the analytical procedure for the determination of OVA using the origami µPAD. In each assay step, upon folding, the origami µPAD was inserted into the 3D-printed holding clip (not shown).

Figure 3

(a) CL signals obtained by employing different EDC/sulfo-NHS concentrations for the activation of the surface carboxyl groups of the MBs. Activated carboxyl groups were quantified by reaction with an excess of HRP, followed by CL detection of the bound enzyme. (b) CL signals obtained by employing different OVA concentrations in the coating of activated MBs to obtain OVA-MBs. Ovalbumin bound to MBs was quantified by reaction with an excess of anti-OVA-HRP, followed by CL detection of the conjugate. The optimal experimental conditions are highlighted in red. Each of the data are the mean ± SD of three measurements.

Figure 4

Simulation of 3D trajectory of fluid across the washing layers D1–D3 at various times (t₀ > t₁ > t₂ > t₃ > t₄). The fluid trajectory was described according to a system of parametric equations in polar coordinates (Equation (1)), considering only the direction of the contours of the fluid in each layer. A bell-shaped trajectory is obtained for the fluid front moving across the D layers, characterized by the increasing diameter of the hydrophilic area. This can be ascribed to the combination of a radial movement towards the boundary of the hydrophilic zone of a given D layer and a vertical movement between the adjacent D layers.

Figure 5

CL signals obtained by analyzing OVA-free solutions (PBS) in origami µPADs prepared using anti-OVA-HRP solutions at different concentrations. The optimal anti-OVA-HRP concentration is highlighted in red. Each of the data are the mean ± SD of three measurements.

Figure 6

Calibration curve generated by combining the results obtained by analyzing OVA standard solutions with different biosensors. A four-parameter logistic equation was used to fit the experimental data and the equation of the resulting calibration curve was Y = 0.925/(1 + 10(0.932(1.257 + X))) + 0.057 (R2 = 0.994), where Y and X were the CL signal ratio and the logarithm of concentration of OVA standard solutions. The dashed lines show the assay range (see text). Each of the data are the mean ± SD of three measurements.

Figure 7

(a) CL image of the origami µPAD acquired during the assay. The scale bar represents 1 cm. (b) CL emission intensity kinetic profiles obtained by the analysis of the CL images acquired during the assay. (c) Application of the two-standard calibration approach to the calibration data of Figure 6. The readings obtained for concentrations that correspond to the upper and lower limits of the assay working range (dark points) were used to obtain the two-point calibration curve, while the other readings (light points) were simply plotted on the graph.

Figure 8

CL signals measured in the origami µPAD for an OVA-free standard solution (PBS) and 10 µg mL⁻¹ standard solutions of the potentially interfering proteins RSA, BSA, and Lys. For comparison, the CL signal measured for a 10 µg mL⁻¹ OVA standard solution is reported. Each of the data are the mean ± SD of three measurements.

Figure 9

Most probable 3D structures obtained by molecular docking simulations for the complexes of (a) OVA, (b) Lys, and (c) BSA with anti-OVA. The scale bars represent 50 Å.

Figure 10

Comparison between OVA concentrations measured in real samples by using the origami µPAD and the colorimetric ELISA kit reference method. The equation of the linear regression curve is Y = 1.087 X − 0.018 (R² = 0.992), where Y and X are the OVA concentrations measured with the origami µPAD and the colorimetric ELISA kit reference method, respectively. Each of the data are the mean ± SD of three measurements.

Figure 11

Decrease in the CL signal measured upon storage at 4 °C for origami µPADs containing the reagents (a) OVA-MBs, (b) anti-OVA-HRP, and (c) luminol/enhancer and sodium perborate. The remaining reagents were loaded in the µPADs just before the measurement. Each of the data are the mean ± SD of three measurements.