Get Your Geek On: The Science Behind Food Allergy Testing

Food testing is serious business. It’s also a large and growing one: the market for food testing kits was valued at $1.58 billion in 2016. That figure is expected to climb to $2.38 billion by 2022.

Since the enactment of the Food Safety Modernization Act (FSMA) of 2011, food manufacturers are increasingly implementing comprehensive food testing procedures. Allergen testing has accordingly taken on a more prominent role in food safety plans. Traditionally, food allergen testing has been confined to the lab; but as new technologies emerge, and old technologies evolve, that’s starting to change.

In this post, we break down the most common food allergen detection technologies. We also discuss emerging technologies and approaches (including ours!) and why changes in food allergen detection are on the horizon. Spoiler alert: prepare for some major geeking out!

Liquid Chromatography-Mass Spectrometry (LC-MS)

As its name implies, liquid chromatography-mass spectrometry (LC-MS) is a two-phase test. During the liquid chromatography phase, a food sample is dissolved in a liquid and funneled through a highly-pressurized chromatography column, which separates molecules based on size and structure.

The mass spectrometer measures the mass of each molecule, as well as the masses of any molecular fragments. A molecule’s mass and fragmentation pattern provide identifying information about the molecule.

Caffeine: Mass Spectrum

Mass spectrum fingerprint of caffeine.

Although LC-MS is a highly-selective tool for molecular identification, LC-MS instruments are expensive and large. Even modest instruments can cost tens of thousands of dollars and stand as high and wide as a microwave. Higher-end instruments can be as large as a car! Test times are also relatively long, ranging from 10 to 30 minutes per food sample. Accordingly, these tests are generally confined to lab environments at present.

Ultraviolet, Visible Light, Infrared, and Raman Spectroscopy

These spectroscopic methods rely on light absorption. A molecule’s chemical structure determines which light wavelengths may be absorbed and the degree of absorption. Spectrometers shine a range of wavelengths at a food sample, and a molecule’s relative absorption of those different wavelengths generates an identifying “fingerprint” for that molecule. You can think of spectroscopy as the enLIGHTened approach to molecular detection 😉.

Caffeine: Infrared Spectrum

Infrared spectral fingerprint for caffeine. Peaks and dips signify degree of molecular light absorption.

Spectral fingerprints are ideal for identifying molecules in samples containing only a few ingredients. Spectra can be generated in a span of seconds, with high-resolution versions taking only one to two minutes. However, identifying molecules in complex mixtures like food samples can present serious challenges for spectroscopic methods, as spectral fingerprints are likely to overlap, making individual molecules difficult or impossible to identify—especially in low quantities. Accordingly, spectroscopy does not currently lend itself to allergen detection in food samples. Moreover, any spectrometer that could potentially afford sufficient selectivity for allergen detection would be large and costly.

Immunoassays & ELISA

Immunoassay tests rely on antibodies. Antibodies are naturally-occurring proteins in the body’s immune system designed to recognize and fight potentially harmful foreign materials. Each antibody is formed to recognize a specific target—usually a protein or protein fragment. Since the 1950’s, scientists have cultivated antibodies to function outside of the body. These antibodies led to tests known as immunoassays. There are many variants of immunoassays, including ELISA (enzyme-linked immunosorbent assay) tests, which many food manufacturers use to test for allergens during the manufacturing process.

In a typical immunoassay, a liquid sample suspected of containing a particular allergenic protein is exposed to a test strip containing antibodies, which are formulated to recognize that specific protein. If the target protein is present, the protein will stick to the antibodies on the test strip and a secondary reaction will stain the bound protein, causing the test strip to change color.

Immunoassays are highly selective, portable, and can produce results in as little as a few minutes. However, culturing and harvesting specific antibodies can be expensive. Moreover, antibodies—like most proteins—are sensitive to harsh conditions like high temperatures or extreme pH levels. The integrity of these tests, therefore, depends on adequate storage conditions. Antibodies are also known to have relatively short shelf lives and typically degrade within one year.

PCR and Molecular Beacons

Another technology in the allergen detection field involves identifying DNA sequences from an allergenic ingredient using a combination of a polymerase chain reaction (PCR) and molecular beacons. Don’t worry, it’s not as complicated as it sounds.

One way to test for an allergenic ingredient is to detect DNA segments unique to that ingredient. DNA is made of two complementary strands, and when one strand finds its complement, they bind. Simple enough. PCR uses the complementary nature of DNA to identify and exponentially replicate target DNA strands. This replication makes the DNA strands easier to detect using what are called molecular beacons: specialized molecular tags that turn fluorescent upon binding to a target DNA strand. These illuminated beacons can then be measured with a fluorescence spectrometer. While PCR-based assays are sensitive and selective, these tests are generally better suited for laboratory environments because they require automated laboratory equipment.

Historically, molecular beacons have been used to detect nucleotide chains like DNA; more recently, molecular beacons are being used to bind and stain proteins–including allergens–instead of DNA sequences. In this approach, PCR is not necessary, as the molecular beacons attach directly to the protein. Notably, molecular beacon tags require a fluorescence spectrometer to measure the target allergenic protein or nucleotide sequences.

Molecularly Imprinted Polymers (Allergy Amulet’s Technology!)

Molecularly imprinted polymer (MIP) sensors are an exciting emerging technology. MIPs are highly-specialized plastic films molded to recognize a single target molecule, such as an allergenic protein or a chemical tracer for an allergenic ingredient. Historically, molecularly imprinted polymers have been used for drug separation and delivery. Only recently have MIPs been adapted for use as molecular recognition elements in electronic sensing devices.

Building an MIP is similar in concept to creating a lock for which the target molecule is the key. Our polymer films contain hundreds of trillions of cavities (locks), which recognize a specific target molecule (key) by size, shape, and complimentary electron charge distribution. The molding procedures used for MIPs mean that they can be designed to target a wide variety of molecular targets. Our Scientific Advisor, Dr. Joseph BelBruno, was the first to develop electronic MIP sensors for detecting nicotine and marijuana. Allergy Amulet is the first to develop MIP sensors for detecting allergenic ingredients.

Imprinted cavity molded to bind to a specific target molecule.

Because the core ingredient in a MIP-based sensor is a specialized plastic, MIP films are highly durable and affordable to produce. The high specificity of target binding, coupled with a straightforward electrochemical resistance measurement, allows for rapid and portable testing.

That’s it! Now you know the science behind allergen detection methodologies. We hope you enjoyed geeking out with us for a short while. Until next time!

-        The Allergy Amulet Science Team

These scientific explanations have been simplified to accommodate our nontechnical readership.