Aptamer based point of care diagnostic for the detection of food allergens

Abstract

Aptamers, due to their small size, strong target affinity, and ease of chemical modification, are ideally suited for molecular detection technologies. Here, we describe successful use of aptamer technology in a consumer device for the detection of peanut antigen in food. The novel aptamer-based protein detection method is robust across a wide variety of food matrices and sensitive to peanut protein at concentrations as low as 12.5 ppm (37.5 µg peanut protein in the sample). Integration of the assay into a sensitive, stable, and consumer friendly portable device will empower users to easily and quickly assess the presence of peanut allergens in foods before eating. With many food reactions occurring outside the home, the type of technology described here has significant potential to improve lives for children and families.

Figure 1

Determination of dissociation constants (K_ds) for five peanut aptamers and targets. Five aptamers were incubated with increasing concentrations of target purified Ara h 1 protein to determine the K_d by fluorescence polarization. (a) Purified Ara h 1, (b) Peanut butter, (c) Peanut flour. Five independent replicates were tested and fitting of the binding isotherm yielded K_d values shown in SI Table 1. Error bars represent the standard deviation of the mean.

Figure 2

Principles of the assay. Fluorescently labeled aptamer is incubated with sample. If it binds its target, it cannot bind its complementary anchor on the solid surface, leading to low fluorescence.

Figure 3

Alexa Fluor 647-labeled P1-16 aptamer binds to Ara h 1 and Ara h 3. Binding specificity was assessed by incubating purified AraH proteins, Ara h 1, Ara h 2, Ara h 3, Ara h 6, and Ara h 8, with AF647-P1-16 aptamer and testing with the benchtop assay. Curve fitting was performed using non-linear regression analysis. Four replicates were tested for each concentration with error bars representing the standard deviation of the mean.

Figure 4

AF647-P1-16 aptamer binds to control anchor with or without peanut in the sample. (a) Comparison of AF647-P1-16 aptamer binding to two different anchors (test and control) spotted on the same surface in the presence of increasing concentrations of clarified peanut flour homogenate. Five replicates of each peanut protein concentration were tested. Error bars represent the standard error of the mean. (b) Representative image of AF647-P1-16 aptamer bound to both the test spots (top left and alternating) and the control spots. The brighter spots on the left and right sides represent alignment markers for optical performance.

Figure 5

Specificity against tree nuts. (a) AF647-P1-16 aptamer binds to peanut protein(s) preferentially to tree nuts, as indicated by the lower normalized difference of tree nut values versus peanut. (b) The addition of 0.1% milk added to the buffer as a non-specific food matrix changed the normalized differences of all assays and increased aptamer specificity for peanut versus the other tree nuts. (c) AF647-P1-16 aptamer was incubated with clarified tree nut homogenate at 50 ppm nut flour (or control buffer) and spiked with 0 or 12.5 ppm peanut protein. The percent decrease for each run was calculated as 1-(test intensity/control intensity)/100. Four or five replicates were tested for each concentration. Data are presented as a box and whisker plot, with the center line denoting the median value (50th percentile), while the box contains the 25th to 75th percentiles of dataset. The whiskers mark the 5th and 95th percentiles.

Figure 6

Assay Validation. (a) Peanut can be detected in major food components and common food additives. Assay was run using multiple food components and additives, both with and without 12.5 ppm of peanut protein. Four or five replicates of each peanut flour concentration were tested. Data are presented as a box and whisker plot, with the center line denoting the median value (50th percentile), while the box contains the 25th to 75th percentiles of dataset. The whiskers mark the 5th and 95th percentiles. (b) Food samples with and without peanut protein can be differentiated by comparing intensity of test spots to control spots. Fifty commercially available foods, spiked with 0 ppm or 12.5 ppm peanut protein, at least 5 replicates each, were tested with AF647-P1-16 aptamer. Using − 50% as an empirically determined cut off value, normalized differences (1 – test/control) are plotted as bars to show clear distinction between food samples containing peanut and those without; the error bars represent standard deviation. All foods containing peanut (at 12.5 ppm) show a normalized difference higher than the defined cut off of − 50% while all the foods that do not contain peanut (0 ppm) show a normalized difference lower than the defined cut off of − 50%.

Figure 7

Future work. (a) Cy5-GN5 aptamer binds to gluten in a concentration dependent manner and in a variety of food matrices. GN5 aptamer was incubated in buffer spiked with increasing concentrations of gluten. (b) Commercially available foods (gluten versus gluten-free) were homogenized, filtered, then incubated with Cy5-GN5. As described for the P1-16 aptamer, the samples were incubated with a chip spotted with a 10 oligonucleotide anchor complementary to sequence of GN5. Four replicates of each sample were tested. Error bars represent the standard deviation of the mean.