Gold Nanoparticle Paper Immunoassays for Sensing the Presence of Vibrio parahaemolyticus in Oyster Hemolymph

Abstract

Seafood contamination with Vibrio bacteria is a problem for aquaculture, especially with oysters, which are often consumed raw. Current methods for diagnosing bacterial pathogens in seafood involve lab-based assays such as polymerase chain reaction or culturing, which are time consuming and must occur in a centralized location. Detection of Vibrio in a point-of-care assay would be a significant tool for food safety control measures. We report here a paper immunoassay that can detect the presence of Vibrio parahaemolyticus (Vp) in buffer and oyster hemolymph. The test uses gold nanoparticles conjugated to polyclonal anti-Vibrio antibodies in a paper-based sandwich immunoassay. A sample is added to the strip and wicked through by capillary action. If Vp is present, it results in a visible color at the test area that can be read out by eyes or a standard mobile phone camera. The assay has a limit of detection of 6.05 × 10⁵ cfu/mL and a cost estimate of $5 per test. Receiver operating characteristic curves with validated environmental samples showed a test sensitivity of 0.96 and a specificity of 1.00. Because the assay is inexpensive and can be used on Vp directly without the requirement for culturing, or sophisticated equipment, it has the potential to be used in fieldable settings.

Figure 1

Schematic of the vibrio paper sensor and its synthesis. (a) Presence of Vibrio results in the formation of a sandwich immunoassay at the test line, where anti-Vibrio antibodies are immobilized. NP-anti-Vibrio antibody conjugates bind to the Vibrio. (b) synthesis of the gold nanoparticles (NPs) and antibody conjugation.

Figure 2

NP synthesis and Ab conjugation. (a) TEM image of Au NPs, (b) optical absorption of bare NPs (red) and NPs conjugated to anti-Vibrio Abs (black), (c) agarose gel electrophoresis of bare (right) and NP–Ab conjugates (left), and (d) D_H (upper) and zeta potential (lower) of bare (right) and NP–Ab conjugates (right) measured by DLS. Error bars are averages of three independent measurements.

Figure 3

Paper sandwich immunoassay test with Vp spiked into 1× PBS. (a) Vp present (+) or absent (−). (b) Test line intensity as a function of Vp concentration in 1× PBS (circles). Error bars indicate averages of five independent strips. Fit to a modified Langmuir curve (line) to obtain an LOD.

Figure 4

Test run in hemolymph. (a) Extraction of hemolymph from oyster (b) test strips with increasing Vp concentration spiked into hemolymph and (c) intensity of test area as a function Vp concentration (red) andVibrio splendidus (blue). Error bars indicate averages of five and seven independent measurements, respectively. Fits to a modified Langmuir curve (lines) to obtain LODs.

Figure 5

Negative controls. (a) Different negative controls compared to Vp: hemolymph alone, Vs, E. coli, and Marinomonas. (b) Test strips run with Vs in hemolymph. (c) Line scans of strips in b for E. coli (blue), Marinomonas (green), hemolymph alone (gray), Vp (red), and Vs (orange).

Figure 6

Environmental samples from oysters collected from a Massachusetts harbor. (a) Test line intensities for different oysters that contained Vp. Optimal test intensity cutoff value (red dotted line) and (b) ROC curve analysis for environmental samples.