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OPEN Rapid and accurate electrochemical

sensor for food allergen detection
in complex foods
Madanodaya Sundhoro, Srikanth R. Agnihotra, Nazir D. Khan, Abigail Barnes,
Joseph BelBruno & Lukasz Mendecki*

Food allergies are estimated to affect about 2–5% of adults and 6–8% of children, globally. Currently,
the most effective strategy for food allergy management is stringent avoidance of the offending
allergen. Unlike other major food allergens, soy is uniquely challenging to avoid due to its prevalence
and insidiousness in a wide variety of foods, such as infant formulas. Recently, we demonstrated a
simple, accurate, and consumer-friendly sensor using molecularly imprinted polymers (MIPs) for rapid
detection of soy allergenic tracers in complex food matrices at clinically relevant levels. In this work,
we build on these findings by subjecting MIP-based soy allergen sensors to test trials in 42 different
food products, representing over 300 ingredients. Foods were selected based on their compositional
complexity to capture a wide range of preparatory methods and processing conditions. In each case,
the Allergy Amulet correctly reported on the presence or absence of soy allergen tracer in investigated
samples and were subjected to immunoassay confirmatory analysis. The outcome of this research will
help resolve persistent difficulties with commercial technologies in detecting allergenic tracers with
minimal cross-interference in foods, and will give those with soy allergies the ability to easily, rapidly,
and accurately identify and avoid foods with soy allergens.

Soybean is one of the most common sources of dietary protein due to its reported health benefits, functional
properties, and high nutritional v­ alue1,2. Soy is typically introduced into the diet early in life, often in the form of
infant formula, as a substitute to human or cow milk for lactose intolerant i­ nfants3,4. Despite its reported nutri-
tional benefits, soy is also an important source of food allergens. Soy allergy is among the eight most common
forms of food allergy, and in severe cases it can trigger life-threatening a­ naphylaxis5. The estimated prevalence of
soy allergy ranges from 0.8 to 1.2% in children and from 0.3 to 0.4% in adults, currently affecting approximately
1.9 million Americans, including 0.4 million c­ hildren6,7. Since soy is nearly omnipresent in processed foods
today, consuming soy-based products is essentially unavoidable unless one makes a concerted effort to read
labels carefully, and effectively communicates the allergy to those preparing one’s food—even then, the risk of
inadvertent ingestion remains.
Currently, no preventative solutions are available for soy allergy sufferers other than strict avoidance. In
response, countries have developed legislations and allergen management strategies, requiring manufacturers to
identify allergen ingredients on labels to alert consumers on the presence or absence of allergen. In the US, the
Food Allergen Labeling and Consumer Protection Act (FALCPA) identifies eight food allergens, including soy,
milk, egg, peanut, shellfish, fish, wheat, and tree nuts, that must be identified on the food l­abel8. More recently,
sesame was added to this l­ist9. Even when assuming strict precautionary measures, consumers face a high risk
of accidental allergen exposure from adulterated products, undeclared substances, and cross-contamination. A
low-cost, accurate, rapid, and consumer-friendly solution for detecting allergens in foods would help account
for these risks through greater food transparency and would provide consumers with greater assurances that
their foods are safe.
To date, the most widely employed methods for food allergen detection include enzyme‐linked immunosorb-
ent assays (ELISA), lateral flow devices (LFDs), and polymerase chain reaction (PCR). ELISA and LFDs both use
monoclonal or polyclonal antibodies to recognize and capture targeted allergens, while PCR relies on detecting
DNA fragments of the allergenic s­ pecies10–12. Although many detection kits based on the ELISA, PCR, and
LFDs technologies have been successfully ­commercialized13, these methods present several practical limitations
undermining the credibility of test results. Among them is the denaturation and/or degradation of proteins and
DNA fragments during food processing, which can yield false negative r­ esponses11,14,15. In addition, antibodies
used in the production of immunological bioassays often demonstrate limited thermal s­ tability16, are expensive

Allergy Amulet, 600 Suffolk Street, Suite 268, Lowell, MA 01854A, USA. *email: [email protected]

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to manufacture, and can cross-react17 with other matrix components, which may produce false positive or false
negative ­responses11. While LFD strips are arguably the closest assay to a consumer device in terms of simplic-
ity and ease of use, these tests often demonstrate low detection a­ ccuracy18, requiring multiple samples to verify
accuracy. Additionally, changes in food viscosity and texture are known to strongly influence the accuracy of
LFD strips in food allergen a­ nalysis19. Existing commercial detection systems are accordingly ill-equipped for
consumer use, and underscore the need for a consumer device capable of rapidly and accurately detecting com-
mon allergenic ingredients on-site in food samples.
Recently, we demonstrated the first application of molecularly imprinted polymers (MIPs) to achieve electro-
chemical detection of soy in complex foods, through detecting a soy allergen marker: g­ enistein20. These sensors
correctly reported on the presence of soy in food samples subjected to both MIP and LFD measurements. Herein,
we carried out the first demonstration of imprinted polymer technology detecting allergens in a large number
of foods with varying levels of complexity and homogeneity (e.g., meats, sauces, confectionary, grains, curries,
liquids, etc.) prepared under a variety of processing conditions including heat, fermentation, and acidity. For this
purpose, we selected 42 food products representing store purchased foods and restaurant products. Together,
these foods consist of over 300 ingredients (Supplementary Table S1). In each case, our technology correctly
reported on the presence or absence of soy in food samples subjected to the standard LFD allergen detection
measurements, demonstrating the effectiveness of our sensors in a diverse range of chemical environments, and
the potential of MIP-based technology as a new benchmark for rapid and accurate allergen detection applications.

Materials and Methods

Genistein was purchased from BOC Sciences (Shirley, NY). Ortho-phenylenediamine (o-PD), catechin hydrate,
chrysin, acetic acid, and sodium acetate were purchased from Sigma-Aldrich (Milwaukee, WI). Amygdalin and
juglone (5-hydroxy-1,4-napthquinone) were purchased from Alfa Aesar (Tewksbury, MA). Denatured ethanol
(5% IPA, 5% n-propylacetate) was purchased from Oakwood Chemicals (Estill, SC). PBS 10X (pH = 7.4) was
sourced from Boston Bioproducts (Boston, MA). All reagents were of analytical grade and were used without
further purification. All aqueous solutions were prepared in ultra-pure water (resistance 18 MΩ ­cm−1) obtained
from Satorius arium mini plus Ultrapure Water System (Germany). 1× PBS solutions were prepared by perform-
ing a 1:10 dilution of 10× PBS in ultrapure water.
Electrochemical experiments were conducted with a PalmSens4 potentiostat (Palm Instruments BV, Neth-
erlands). Carbon ItalSens IS-C Screen Printed Electrodes (SPE) were purchased from PalmSens (Houten, Neth-
erlands) and were used during all electrochemical measurements. Our allergen sensors, which are part of the
Allergy Amulet platform detection system, were prepared according to the method previously developed in our
laboratory (Supplementary Experimental S1)20.

Food samples. Store purchased goods included soy curls (Butler), soybeans (Soymerica), tofu (House-
foods), soy sauce (Kim Ve Wong), vegetable oil (Hannaford), Roasted Garlic Parmesan Sauce (Ragu), Captain’s
Wafers Cream Cheese and Chives (Lance), Thousand Island dressing (Ken’s Steak House), soy protein isolate
(Now), granola protein (Nature Valley), soy lecithin (Modernist Pantry), Ritz Crackers with Cheese (Nabisco),
Lemon Flavor Crème Oreo (Nabisco), Toast Chee Peanut Butter Crackers (Lance), veggie burger (Morning Star
Farms), soy flour (Bob’s Red Mill), defatted soy flour (Scratch), Chicken (Not!) (Dixie Diner’s Club), Zante cur-
rant raisins (Sunmaid), tikka masala (Patak’s), sesame seeds (McCormick), rice milk (Rice Dream), red wine
vinegar (Market Basket), raisins (Sunmaid), Pure butter shortbread (Walkers), peanut oil (Hain), Moroccan
tomato sauce (Mina), mayonnaise (Hellman’s), Major Grey chutney (Patak’s), Original macadamia milk (Milka-
damia), Growing Years whole milk (Horizon Organic), green salsa (Mrs. Renfro’s), flax milk (Good Karma),
fish sauce (Thai Kitchen), Country French with Orange Blossom Honey dressing (Ken’s Steak House), Coffee
Mate creamer (Nestle), cashew milk (So Delicious), Breakfast Blend light roast coffee (Green Mountain), and
almond milk (Nature’s Promise) were sourced from local supermarkets. Restaurant dishes, including Ming’s
Bings Veggie-Filled Bing patty, duck fried rice and garlic ginger bok choy were sourced from Blue Dragon res-
taurant (Boston, MA). Confirmatory LFDs measurements were performed using a Soy Rapid Kit L25SOY LFD
kit purchased from 3 M.

Food testing method. For each solid food tested, 1 g of food was homogenized using a mortar and pestle
(5 min) until a fine powder was obtained. The resulting powder was then mixed with 10 mL of buffer solution
and stirred for 15 min. Liquid food samples were prepared by mixing 1 g of food directly with 10 mL of the buffer
solution. For both solid and liquid food tests, template-extracted MIP electrodes were inserted into a 10 mL of
the buffer solution and equilibrated for 5 min prior to the electrochemical measurements. The electrode was
then taken out of the solution and the liquid was removed from the surface. After 1 min incubation with 100 µL
of sample solution, the electrodes were subjected to differential pulse voltammetry (DPV) measurements. DPV
experimental parameters: scan rate: 50 mV/s; pulse width: 50 ms; and amplitude: 50 mV. All food measurements
were run at least in triplicate. Imprinting factor was calculated by dividing signal intensity of MIP with a non-
imprinted polymer (NIP) at their peak current maxima (peak position: 0.6 V vs Ag/AgCl reference electrode).
A positive response was noted when the sensor reported on the presence of an oxidation peak at approximately
0.60 V vs Ag/AgCl and an imprinting factor above 1.3, which corresponds to oxidative redox transformations of
genistein (Fig. 1A)20. This electrochemical behavior is consistent with the studies of Popa and ­Diculescu21 and
­ ork20.
our earlier w

LFD testing method. LFDs measurements were carried out according to the 3 M protocol (Fig. 1B)22.
Briefly, 100 µL of liquid food samples were mixed with 900 µL of 3 M extraction buffer and vortexed for 15 s to

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Figure 1.  Illustrative responses from: Allergy Amulet kit (A) and 3 M Soy Protein Rapid kit (B). The Allergy
Amulet sensor records a positive reading when a distinct oxidation peak for soy allergen tracer is present at
approximately 0.60 V vs Ag/AgCl reference electrode. For the LFD test kits a positive result is visualized by
the presence of three lines: a control line (C), a hook line (H) and a test line (T). Negative LFDs results were
indicated by the presence of a control and a hook line only. Concentrated LFDs results were indicated by the
presence of control line while the invalid results were indicated by the absences of all of three lines.

aid extraction. 100 µL of the resulting food mixture was then introduced to the 3 M Soy Protein LFD sample well
and left to incubate for 11 min. Solid food samples were prepared by homogenizing 1 g of food, using a mortar
and pestle, for 5 min. 1.8 mL of the 3 M extraction buffer was then added into a microcentrifuge tube contain-
ing 0.2 g of the homogenized samples. The resulting mixture was vortexed for 15 s until the sample was well
dispersed prior to 30 s centrifugation at 10,000 rpm using Bio Lion XC-10K. A suspension sample of 100 µL was
then introduced into the well of the 3 M Soy Protein LFD and left to incubate for 11 min.

Cross‑reactivity studies. For cross-reactivity measurements, a stock solution with concentration of 1 mg/
mL was prepared by dissolving 5 mg of the interferent molecule (amygdalin, juglone, chrysin, or catechin) in
5 mL of ethanol for 15 min. A 10 ppm solution of each interferent was prepared by adding 100 µL of stock solu-
tion (1 mg/mL) into a solution containing 900 µL of ethanol and 9 mL of PBS 1×. 10 ppm solutions of both the
soy allergen tracer and the analogous molecule were formed by adding 100 µL of soy allergen tracer stock solu-
tion and 100 µL of interference stock solution followed by adding 800 µL of ethanol and 9 mL of PBS 1× with
15 min of stirring. DPV measurements were conducted using the same parameters as those used for food testing.

Results and discussion

Processing and cooking can subject an allergen to denaturation and other conformational changes, which can
reduce—but will not necessarily remove completely—its potential to trigger an allergic response. Additionally,
allergens may be entrapped or physically constrained to their environment, inhibiting dissolution and/or binding
to the selective cavities of the polymer. For the sensor to be effective, it must detect the presence of the allergen
regardless of its chemical environment. In this study, we validated the effectiveness of the Allergy Amulet by
testing the sensor against 20 different foods known to contain soy and 22 different foods not containing soy. For
store-purchased products and restaurant dishes, information on the presence or absence of soy allergen was col-
lected directly from food ingredients used and allergen labels. The integrity of the results requires confirmation
of the presence of the allergen in the food by extant allergen detection technology. This was performed using
commercially available immunoassay methods (LFDs).
The detection of allergens in food products strongly depends on efficient extraction of soy allergen tracer from
complex food matrices. Food processing is known to cause allergen denaturation, conformational changes, aggre-
gation, or chemical ­modifications15. These changes have been reported to strongly influence allergen extractability
and antibody recognition of allergenic proteins or DNA fragments in immunoassay or PCR analysis. Conversely,
genistein (soy allergen tracer) has been shown to retain its structural stability after being subjected to extensive
food processing treatments, including heating and f­ ermentation23,24. This approach enables detection of soy in
foods even when the DNA and/or the allergenic protein was altered or degraded after food processing. This prop-
erty is important since it has been reported that soybean can retain its allergenicity even after food ­processing25.
To better understand the impact of food texture and composition on the extractability of soy allergen tracer
from foods, we have created a four-point rating scale for grouping different foods by their textural characteristics
including crispiness, tenderness, smoothness, toughness, chewiness, creaminess (Table 1). The following rat-
ings were assigned for all tested foods: (1) liquids (soy sauce, vegetable oil, rice milk, red wine vinegar, peanut
oil, Original macadamia milk, growing years whole milk, flax milk, fish sauce, Coffee Mate creamer, cashew
milk, Breakfast Blend light roast coffee, and almond milk); (2) viscous liquids and emulsions (Roasted Garlic
Parmesan Sauce, Thousand Island dressing, tikka masala, Moroccan tomato sauce, mayonnaise, Major Grey
chutney, green salsa, Country French with Orange Blossom and Honey dressing); (3) gelatinous substances

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Entry Food Brand Number of i­ ngredientsa Presence of s­ oya Source of ­soya Textureb MIP ­resultsc LFD results
Thousand island
1 Ken’s Steak House 36 Yes Soybean oil 2 Negative Negative
dressing
2 Almond milk Nature’s Promise 10 No – 1 Negative Negative
Breakfast blend light
3 Green Mountain 1 No – 1 Negative Negative
roast coffee
Captain’s wafers cream
4 Lance 30 Yes Soybean oil 4 Negative Negative
cheese and chives
5 Cashew milk So Delicious 14 No – 1 Negative Negative
6 Chicken (Not!) Dixie Diner’s Club 1 Yes Soy flour 4 Positive Concentrated
7 Coffee mate creamer Nestle 8 No – 1 Negative Negative
Country French with
8 orange blossom honey Ken’ Steak House 16 No – 2 Negative Negative
dressing
9 Defatted soy flour Scratch 1 Yes Soy flour 3 Positive Concentrated
10 Duck fried rice Blue Dragon Restaurant 15 Yes Soy sauce 3 Negative Negative
11 Fish sauce Thai Kitchen 3 No – 1 Negative Negative
12 Flax milk Good Karma 12 No – 1 Negative Negative
13 Garlic ginger bok choy Blue Dragon Restaurant 6 No – 3 Negative Negative
14 Granola protein Nature Valley 11 Yes Soy protein isolate 4 Positive Concentrated
15 Green salsa Mrs. Renfro’s 7 No – 2 Negative Negative
Growing years whole
16 Horizon Organic 7 No – 1 Negative Negative
milk
Lemon flavor crème
17 Nabisco 15 Yes Soy lecithin 4 Negative Negative
oreo
Original macadamia
18 Milkadamia 11 No – 1 Negative Negative
milk
19 Major grey chutney Patak’s 13 No – 2 Negative Negative
20 Mayonnaise Hellmann’s 8 No – 2 Negative Negative
21 Veggie filled Ming’s Bing Blue Dragon Restaurant 22 Yes Soy sauce 22 Positive Positive
22 Moroccan tomato sauce Mina 8 No – 2 Negative Negative
23 Peanut oil Hain 2 No – 1 Negative Negative
24 Pure butter shortbread Walkers 8 No – 4 Negative Negative
25 Raisin Sunmaid 1 No – 3 Negative Negative
26 Red wine vinegar Market Basket 1 No – 1 Negative Negative
27 Rice milk Rice Dream 8 No – 1 Negative Negative
Roasted garlic parmesan
28 Ragu 25 Yes Soybean oil 2 Negative Negative
sauce
29 Ritz crackers with cheese Nabisco 25 Yes Soy lecithin 4 Negative Negative
30 Sesame seeds McCormick 1 No – 4 Negative Negative
31 Soy curls Butler 1 Yes Soybeans 4 Positive Concentrated
32 Soy flour Bob’s Red Mill 1 Yes Soy flour 3 Positive Concentrated
33 Soy lecithin Modernist Pantry 1 Yes Soy lecithin 3 Negative Concentrated
34 Soy protein isolate Now 1 Yes Soy protein isolate 3 Positive Concentrated
35 Soy sauce Kim Ve Wong 5 Yes Soybeans 1 Positive Negative
36 Soybeans Soymerica 1 Yes Soybeans 4 Positive Positive
37 Tikka masala Patak’s 20 No – 2 Negative Negative
Toast chee peanut butter
38 Lance 25 Yes Soy lecithin, soybean oil 4 Negative Weak Positive
crackers
39 Tofu Housefoods 4 Yes Soybeans 3 Positive Positive
40 Vegetable oil Hannaford 1 Yes Soybean oil 1 Negative Negative
Soy flour, soy sauce
41 Veggie burger Morning star farms 27 Yes 3 Positive Positive
powder
42 Zante currant raisin Sunmaid 1 No – 3 Negative Negative

Table 1.  Detection responses recorded for MIP-coated electrodes and LFDs during food product
measurements. A positive test result for both MIP and LFD kit detection confirms that soy allergen is present
within a tested sample. a As reported in ingredients lists. b Based on scale 1–4 with 1 liquid, 2 viscous liquid and
emulsions, 3 gelatinous and soft solid, 4 hard solid. c Based on triplicated electrochemical readings.

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and soft-solids (tofu, duck fried rice, soy protein isolate, soy lecithin, veggie burger, soy flour, defatted soy flour,
Zante currant raisin, raisin, and garlic ginger bok choy); and (4) hard solids (soy curls, soybeans, Captain’s Wafers
Cream Cheese and Chives, granola protein, Ritz Cracker with Cheese, Lemon Flavor Crème Oreo, Toast Chee
peanut butter crackers, Chicken (Not!), sesame seeds, and Pure butter shortbread) (Table 1). For soy containing
foods, we further subcategorized these products based on the origin and source of the soy protein used for their
preparation, including soybeans, soy flour, soy protein isolate, soy sauce, tofu, soy lecithin, and soybean oil. We
then tested the electrochemical response of MIP-coated electrodes in 10% ethanol, 90% 1× PBS (v/v, pH 7.4)
solutions containing 10% by weight of each of the food product representing different textures and soy origins.
DPV measurements recorded for soy protein isolate, soy flour, defatted soy flour, tofu, and soybeans showed
distinct oxidation peak for soy allergen tracer at approximately 0.6 V (Supplementary Figs. S1–S4) which is
consistent with the analyte-centered redox activity of ­genistein20,21. Similarly, positive detection was recorded
for soy curls and Chicken (Not!) chunks (Table 1), which contain mainly soybeans or soy flour, respectively.
While LFD measurements also reported a positive response for the soybeans, the 3 M LFD test kit failed to
inform on the presence of soy allergens in soy protein isolate, soy curls, Chicken (Not!), and tofu (Supplementary
Fig. S5A–D), giving rise to a concentrated readout (invalid). The lack of response for LFDs could be caused by
protein oversaturation at the detection site, the area at which the biological recognition elements (antibodies,
proteins, enzymes, etc.) are immobilized. To overcome this problem, the sample solution—prepared according
to the manufacturer’s specifications—had to be repeatedly diluted to achieve the right concentration and enable
detection of a soy allergen using LFD. The differences in response characteristics of LFD strips between the soy
protein isolate, soy curls, Chicken (Not!) chunks, and the soybeans may be partially attributed to their textural
variability. When matured, soybeans can be characterized as a dry and hard solid, which can limit its grinding
efficiency using mortar and pestle. Indeed, the other soy-containing hard foods are easier to process using mor-
tar and pestle. As a result, manual processing of soybeans did not yield a well homogenized and homogenous
powder, contributing to inefficient extraction of allergenic proteins from soybean particles.
Further, LFD measurements did not register a positive response for soy flour samples (Supplementary Fig.
S5F). This inconclusive result may be attributed to the increased viscosity of the tested solution, inhibiting the
fluid flow across the LFD substrate even after hours of incubation. While diluting the soy flour solution four
times enabled the movement of the liquid to the detection area, the device still reported a concentrated result
(Supplementary Fig. S5G). The susceptibility of tested LFDs to produce positive responses in soy-rich foods
poses practical limitations if such products were to be used in consumer allergen testing applications, as the user
would in theory be required to undertake extensive sample processing steps, and run multiple measurements
to realize accurate detection.
We then tested the electrochemical response of MIP-coated electrodes for two soy-labelled liquid products:
soy sauce and soybean oil. The DPV measurements of soy sauce and soybean oil did not reveal a characteristic
response for soy tracer at 0.60 V, but instead generated a smaller signal at approximately 0.80 V for soy sauce
containing samples. The observed peak current at higher anodic potentials may be due to the presence of poly-
phenols in soy ­sauce26. Confirmatory LFDs tests also reported a negative responses for soy sauce (Supplementary
Fig. S5H) and soybean oil samples (Supplementary Fig. S5I). These experimental observations can most likely be
explained by very low content of allergenic soy protein, resulting from the industrial fermentation processes (e.g.,
microbial proteolytic enzymes) used for the manufacturing of many soy sauces. Indeed, food processing is known
to reduce the allergenicity of soy and wheat proteins in processed f­ oods27. Similarly, the industrial processes of
refining soybean oil typically involve multiple extraction steps using hot solvents, bleaching, and deodorization,
which serve to effectively eliminate the allergenic soy protein from the soybean oil-based products. Addition-
ally, it has been showed that soybean oil is generally safe to consume for soy allergenic ­individuals28. Unlike the
positive results, obtaining negative results is quite straightforward in LFDs. In most cases, clear hook and control
lines are easily observable. In most foods containing soy allergen, LFD results tend to give concentrated or faint
signals on both the hook line and the test line. A positive or invalid result in these cases would be subject to the
perception of the interpreter.
Lastly, we studied the response of our sensors when testing soy lecithin. Soy lecithin is a common soy-based
additive used in the food industry as an emulsifier, lubricant, antioxidant, and flavor protector. Because soy leci-
thin is produced from highly refined soy oils, it typically contains insufficient amounts of allergenic soy protein
to provoke allergic reactions in most soy-allergic individuals. For example, the Food & Drug Administration
(FDA) has granted exemptions regarding the labelling of soy lecithin as an allergen on food products. These
exemptions apply when soy lecithin is used directly as a release agent or a component of a release agent applied
to food contact ­surfaces29. DPV measurements with MIP-coated electrodes on soy lecithin reported a negative
response as evidenced by the lack of an anodic peak at 0.6 V vs Ag/AgCl reference electrode. Like soy flour, LFD
tests produced an inconclusive readout—the liquid could not readily travel from the loading well to the detection
site (Supplementary Fig. S5J). Although, LFDs have been widely regarded as rapid and portable food tests, these
are highly susceptible to the presence of matrix components and overall sample consistency which together can
cause pore obstruction, and thus limit the liquid flow. After carrying out additional sample dilutions (tenfold),
the resulting “slurry” was of the right consistency to produce a visible readout using immunoassay analysis. The
confirmatory LFD strips also reported a negative response, indicating the absence of soy allergenic protein at
clinically relevant levels in tested soy lecithin samples (Supplementary Fig. S5K).

Grocery store foods. After studying the influence of food texture and composition on detection perfor-
mance of the Allergy Amulet sensor, we focused our attention on expanding the range and number of use cases
in food allergen analysis applications. We accordingly selected 21 soy-free and 18 soy-containing store-bought
products, each prepared using a different manufacturing process and having a unique composition (Supplemen-

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Coffee
0.50

Sesame seeds
0.40

Current (µA)
0.30
Nut milk
Soy allergen tracer
0.20

0.10

Soy sauce
0.00
0.00 0.20 0.40 0.60 0.80 1.00
Potential (V)

Figure 2.  DPV responses for soy allergen tracer (red line), soy sauce (brown line), sesame seeds (purple line),
coffee (gold line), nut milk (blue line) 10% by weight in buffer solution containing 10% ethanol in 90% PBS 1×.

tary Table S1). Electrochemical measurements of Captain’s Wafers Cream Cheese & Chives sandwich crackers,
Toast Chee Peanut Butter Crackers, and Ritz Crackers with Cheese reported a negative response as evidenced
by the lack of a characteristic signal for soy allergen tracer at 0.6 V vs Ag/AgCl. As the primary source of soy in
these foods is soy lecithin, these results were consistent with our earlier data obtained from soy lecithin meas-
urements. While the LFD tests on the Captain’s Wafers Cream Cheese & Chives and Ritz Crackers with Cheese
confirmed the absence of soy allergen in each food, the immunoassay reported a slightly positive readout for
Toast Chee Peanut Butter Crackers samples (Supplementary Fig. S1L). This result was unexpected, as the only
soy-based ingredient in Toast Chee Peanut Butter Crackers identified by the manufacturer and listed on a food
label was soy lecithin. The faint positive signal recorded in LFD analysis of Toast Chee Peanut Butter Crackers
may be due to the “fatty or smeary” nature and texture of the food. These compositional and textural charac-
teristics have been already reported to influence the accuracy of LFDs in detecting allergens, giving rise to false
positive ­readouts30.
Lemon Flavor Crème Oreo is another food containing soy lecithin as its soy source. DPV measurements
of Lemon Flavor Crème Oreo sandwich cookies did not report on the presence of soy allergen tracer at 0.6 V,
but instead a weak anodic signal was observed at 0.5 V vs Ag/AgCl. The recorded electrochemical signal at a
more cathodic potential may be due to the presence of annatto extract in tested cookies. Annatto is a common
food dye known to contain redox-active p ­ olyphenols31. The LFD measurements also did not produce a positive
response during testing of Lemon Flavor Crème Oreo sandwich cookies samples, indicating the high accuracy
of our MIP-based sensors. Subsequently, we subjected MIP-coated electrodes to food measurements with two
soy-containing solid food products: soy burger patties and granola protein bars. Soy flour and soy protein isolate
were identified as the primary sources of soy in each respective formulation. Using Allergy Amulet and LFD
immunoassays, we correctly identified the presence of soy in both tested samples. These findings were consistent
with our earlier test results on soy flour and soy protein isolate. We then tested highly complex semi-liquid food
samples (texture scale: 2) including Roasted Garlic Parmesan Sauce and Thousand Island dressing, consisting of
25 and 36 individual ingredients, respectively. The food labels indicated that both of these foods contained soy
in the form of a soybean oil. Electrochemical and LFD measurements of these foods demonstrated a negative
readout, matching those results obtained from earlier analysis on soybean oil samples.
Polyphenols, like genistein, are well-known antioxidant molecules found in several foods, herbs, and spices.
These species are known to define some of the organoleptic characteristics of foods—e.g., color, flavor, and bitter-
ness. The presence of phenolic moieties in polyphenols, being the main constituents of these compounds, confers
their inherent redox activity and their antioxidant characteristics. As these molecules can demonstrate redox
activity during electrochemical analysis, we investigated the detection capability of the MIP-based electrodes
on molecules that are structurally similar to the soy allergen tracer that are present in significant quantities in
foods (Supplementary Fig. S6). We then continued this investigation on a range of soy-free foods known to be
rich in polyphenols, including coffee, nut milks (macadamia milk, flax milk and cashew milk), fish sauce, and
sesame ­seeds26,32–39. Indeed, electrochemical measurements of these foods reported on the presence of anodic
peaks at 0.1 V, 0.5 V, 0.8 V and 0.9 V vs Ag/AgCl reference electrode, respectively (Fig. 2). No characteristic signal
associated with the presence of soy allergen tracer (0.6 V) was detected during testing of soy-free foods with high
polyphenol content. LFD analysis also reported on the negative response, indicating that such polyphenols had
minimal interference on the accuracy of MIP-based sensors. These findings demonstrate that the differences
in redox potentials of polyphenolic molecules make it possible to distinguish these analytes electrochemically
from soy allergen tracer (Fig. 2). While other soy-free store purchased products including almond milk, rice

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milk, red wine vinegar, peanut oil, whole milk, Coffee Mate creamer, Pure butter short bread, raisins, and Zante
currants may contain small levels of polyphenolic compounds, these foods did not produce any voltametric
response in the investigated potential range (0–1 V). This effect might be caused by insufficient levels of these
compounds to produce anodic signals in the extracted sample, as well as their redox processes taking place at
more negative or positive potentials which fall outside the measurement range. These results demonstrate that
our imprinted polymer-based sensors can successfully differentiate soy allergen tracer from other redox active
species typically found in foods.

Restaurant dishes. Restaurant dishes are typically more complex than homemade foods or store-bought
products in terms of their number of ingredients and textures. They are also an important category for valida-
tion due to the risk restaurant dining poses to allergy sufferers. Therefore, we selected one restaurant dish that
was prepared without soy (garlic ginger bok choy) and two dishes prepared with soy (duck fried rice and veggie
filled Ming’s Bings Veggie-Filled Bing patty). Each dish was made up of at least seven individual ingredients and
represented different cooking and processing methods (see Supplementary Table S1 for breakdown of ingredi-
ents). During garlic ginger bok choy measurements, the Allergy Amulet correctly reported on the absence of soy
allergen tracer in tested foods subjected to immunoassay measurements (Table 1). Although, both duck fried
rice and Ming’s Bing patty have listed soy sauce as one of their ingredients, the Allergy Amulet and LFDs only
reported a positive response for the Ming’s Bing patty (Table 1). These results may be explained by the differences
in the t­ ype25 and amount of soy sauce used for preparing each individual dish.
After the original soy-free food samples were tested, they were “spiked” with a 10 ppm solution of the aller-
genic tracer. The tests were then repeated to confirm the efficacy of the device in the food matrix now contain-
ing the allergen. In each case, our detection platform correctly identified the presence of soy allergen tracer in
soy-spiked foods, showing minimal background interferences (data not shown).

Conclusion
In this work, we confirmed the feasibility of MIP-based sensors for soy allergen detection in complex foods. We
selected food products that represented a wide range of sources (e.g., store-bought and restaurant dishes) and
chose foods that ensured we could distinguish between those dishes containing soy and those that did not. For
every food that was known to contain soy, Allergy Amulet correctly detected its presence at clinically relevant
levels. To confirm that our sensor was performing at least as well as existing commercial allergen detectors, we
then tested those same foods against a lateral flow device (LFD)—one of the key methods for testing allergenic
ingredients in commerce. An exact binomial test was used to compare the binary accuracies of the MIP sensor
and the LFD kit in detecting the presence of soy allergen, which confirmed the higher degree of accuracy of the
MIP (P = 0.007). In particular, our sensors appeared to be superior in testing of highly concentrated soy-based
products and foods with higher fat content. Therefore, we have determined that our MIP-based sensors are not
only a suitable alternative to other analytical methods frequently used for food allergen testing, but also offers
advantages in personal food allergen detection applications and food safety control that allow for detection in a
broader range of conditions than were previously deemed possible.

Received: 13 June 2021; Accepted: 6 October 2021

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Acknowledgements
We would like to thank Blue Dragon restaurant in Boston, MA for providing restaurant dishes used in this work.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-
profit sectors.

Author contributions
M.S. wrote the main manuscript text and prepared figures. M.S., S.R.A., and N.D.K. performed experiments.
M.S., A.B., J.B., and L.M. conceived the experimental design and commented on manuscript text. All authors
reviewed the manuscript.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​021-​00241-6.
Correspondence and requests for materials should be addressed to L.M.
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