Photonic Label-Free Biosensors for Fast and Multiplex Detection of Swine Viral Diseases

Abstract

In this paper we present the development of photonic integrated circuit (PIC) biosensors for the label-free detection of six emerging and endemic swine viruses, namely: African Swine Fever Virus (ASFV), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PPRSV), Porcine Parvovirus (PPV), Porcine Circovirus 2 (PCV2), and Swine Influenza Virus A (SIV). The optical biosensors are based on evanescent wave technology and, in particular, on Resonant Rings (RRs) fabricated in silicon nitride. The novel biosensors were packaged in an integrated sensing cartridge that included a microfluidic channel for buffer/sample delivery and an optical fiber array for the optical operation of the PICs. Antibodies were used as molecular recognition elements (MREs) and were selected based on western blotting and ELISA experiments to ensure the high sensitivity and specificity of the novel sensors. MREs were immobilized on RR surfaces to capture viral antigens. Antibody-antigen interactions were transduced via the RRs to a measurable resonant shift. Cell culture supernatants for all of the targeted viruses were used to validate the biosensors. Resonant shift responses were dose-dependent. The results were obtained within the framework of the SWINOSTICS project, contributing to cover the need of the novel diagnostic tools to tackle swine viral diseases.

Keywords: antibody; biosensor; label-free; photonic integrated circuit (PIC); photonics; ring resonator; swine disease.

Figure 1

Optical pictures of (a) the fabricated biosensor PIC and (b) of the PIC close to the region of the RRs, where the window is opened to allow buffer/sample access.

Figure 2

(a) 3D sketch of the microfluidic system together with the input and output fluidic ports, and the PIC assembled. (b) Bottom view of the silicon-based PIC assembled into the microfluidic system. (c) Representative picture of the optofluidic system; for the sake of clarity the required tubing to connect the microfluidic channel with the pump and the analyte container to handle the fluidics was not connected.

Figure 3

Panel (1A): WB results of the mAb anti-PCV2 against virus sample (line S), recombinant ORF2 capsid protein (line P), and purified mAb and pAb anti-PCV2 (lines mAb and pAb). Panel (1B): WB results of the pAb anti-PCV2 against virus sample (line S), recombinant ORF2 capsid protein (line P), and purified mAb and pAb anti-PCV2 (lines mAb and pAb). Panel (2A): WB results of the mAb anti-PRRSV against virus sample (line S), recombinant NP nucleocapsid protein (line P), and purified mAb and pAb anti-PRRSV (lines mAb and pAb). Panel (2B): WB results of the pAb anti-PRRSV against virus sample (line S), recombinant NP nucleocapsid protein (line P), and purified mAb and pAb anti-PRRSV (lines mAb and pAb). Panel (3A): WB results of the mAb anti-PPV against ST and SK-6 virus samples (lines S1 an S2), recombinant _VP2 protein (line P), and purified mAb and pAb anti-PPV (lines mAb and pAb). Panel (3B): WB results of the pAb anti-PPV against ST and SK-6 virus samples (lines S1 an S2), recombinant VP2 protein (line P), and purified mAb and pAb anti-PPV (lines mAb and pAb). Panel (4A): WB results of the mAb anti-ASFV against virus sample (line S), recombinant p30 capsid protein (line P), and purified mAb and pAb anti-ASFV (lines mAb and pAb). Panel (4B): WB results of the pAb anti-ASFV against virus sample (line S), recombinant p30 capsid protein (line P), and purified mAb and pAb anti-ASFV (lines mAb and pAb). Panel (5A): WB results of the mAb anti-CSFV against virus sample (line S), recombinant E2 envelope protein (line P), and purified mAb and pAb anti-CSFV (lines mAb and pAb). Panel (5B): WB results of the pAb anti-CSFV against virus sample (line S), recombinant E2 envelope protein (line P), and purified mAb and pAb anti-CSFV (lines mAb and pAb).

Figure 4

Differences between resonant notch shift obtained from the RRs with MREs immobilized on top of the sensors (black line) and from the references (dotted line) for the detection of PPV at dilutions of PPV at 1/100 (a) and at 1/5000 (b).

Figure 5

(a) Dose dependence signal shift (measured in pm) for pAb (alpha diagnostic) for each PPV dilution tested. The detection level for each dilution was obtained as the average shift of the data acquired after the rinse step and depicted as a horizontal line. (b) The detection levels for each dilution tested are summarized in a bar chart.

Figure 6

(a) Dose dependence signal shift (measured in pm) for pAb (alpha diagnostic) for each CSFV dilution tested. The detection level for each dilution was obtained as the average shift of the data acquired after the rinsing step and depicted as a horizontal line. (b) The detection levels for each dilution tested are summarized in a bar chart. For the highest dilution, quantification of the detection level it is not feasible, and this is marked with an (*).

Figure 7

(a) Dose dependence signal shift (measured in pm) for pAb (alpha diagnostic) for each PRRSV dilution factor. The peak observed at minute 20 for the (1/1000) dilution involves a RI increase. It is ascribed to fluidic instabilities in the fluidic chamber, where the two solutions are being exchanged, giving rise to an increase of the refractive index [17]. (b) The detection levels for each dilution tested are summarized in a bar chart.

Figure 8

(a) Signal shift (measured in pm) obtained for the detection of ASFV and SIV viruses at 1/20 and 1/100 dilutions in PBS-T + BSA 0.5%, respectively. (b) The detection levels for each virus tested are summarized in a bar chart.