Chromogenic Assays: What they are and how they are used in research

Chromogenic assays are detection methods that rely on a color change during the experimental procedure to obtain qualitative and quantitative results. Chromogenic assays are widely used in modern research.

In this article we will learn the basic principle of a chromogenic assay, the biochemical requirements and how they are useful in common research techniques.

In this article:

Basic principle of a chromogenic assay

Chemicals required in a chromogenic assay

Why is colorimetry important in chromogenic assays

What is colorimetry

Types of experiments involving chromogenic assays

Beta-galactosidase assay

Western blot

ELISA

Chromogenic growth media for detecting specific microbes

References

Basic principle of a chromogenic assay

Here is an analogy to demonstrate the basic concept of a chromogenic assay.

Imagine having a magic dart that always hits the target even if you throw it in the dark. Exploiting this property, you throw this dart to detect your target – wherever your dart lands, lies your target.

But now you have a problem – because you are in a dark room, you cannot see your dart, which also means you can’t see where your target is.

However, there is a way around this. You have a spray that lights up when it is in contact with your dart.

With all these great magical tools in hand, here is what you do to detect your target in the dark.

You first throw the dart – it hits the target, but both the dart and the target are still undetected in the dark.

However, when you use the spray, the spray lights up and you can detect the dart-hit target.

Figure 1. Animation demonstrates the Magic Dart analogy that helps describe the underlying principle of a chromogenic assay. In a dark room, while the dart automatically flies to the target, it cannot be seen until a a magic spray reveals the dart upon contact.

Keeping this analogy in mind, let me explain how this works out in the context of a biological experiment.

Suppose you want to detect a biochemical in your sample – for example, a particular protein. This protein is your “target.”

You use a biochemical – say, an antibody – that specifically recognizes and binds to that target protein. This antibody is analogous to the “magic dart.”

But you still cannot visually see and detect this antibody along with the target protein bound to it.

For this, you use a “magic spray” – in this case, a colorless chemical that produces a color once in contact with the antibody dart.

Figure 2. Key comparing illustrations of a chromogenic assay to elements of the magic dart analogy.

This completes the detection process of your target protein.

With the aid of figure 3, here is a little more mechanistic understanding of the process.

The antibody dart is conjugated (attached) to an enzyme. The magic spray chemical is actually a substrate for this enzyme.

This enzyme breaks down the substrate into two products, one of which produces the color change that aids in the final visual detection, which is where we get the term chromogenic.

Chromogenic means related to color. Since this detection assay relies on a visible color change, we get the full term, “chromogenic assay.”

The substrate mentioned is called “chromogen” (produces color), or a “chromogenic substrate.”

Figure 3. Illustration of how a chromogenic assay works.

Chemicals required in a chromogenic assay

Here is the list of chemicals you would need for a chromogenic assay:

• Substrate (chromogen)
• Corresponding enzyme
• Compound for primarily detecting the target

You will need a substrate that can be broken down by an enzyme to produce a visible color change.

This enzyme corresponds with the chosen substrate and breaks it down during the experiment.

The corresponding enzyme is usually conjugated to the compound that detects the target protein. In the above case, this will be an antibody molecule.

Table 1. List of a few popular enzyme-substrate pairs used in chromogenic assays.

Why is colorimetry important in chromogenic assays

In chromogenic assays the final step in detection is facilitated by a color change – due to production of a colored product compound from the chromogenic substrate.

The higher the concentration of the final colored product, the higher the concentration of the target analyte.

In a chromogenic assay, by measuring the concentration of the final-colored product – by colorimetry – the concentration of the analyte is indirectly estimated.

What is colorimetry

Colorimetry is used to measure the concentration of a colored compound.

Every compound with a specific color has a specific wavelength which is its absorption maxima.

To clarify, absorption maxima is the wavelength where a compound has maximum absorbance, that is, the absorbance reaches its peak.

At this wavelength, if the absorbances of two solutions having different concentrations of this same compound are measured, the experimenter can gauge the concentrations of this compound in the two solutions.

Types of experiments involving chromogenic assays

Any technique that involves qualitative and quantitative detection with the help of a visible color change is a chromogenic assay.

Ranging from detecting proteins, to quantitatively assaying enzyme activity to detecting and isolating specific microbes – the utility of chromogenic assays is large in modern bioscience.

In this section, we'll briefly talk about some common experiments involving chromogenic assays.

Beta-galactosidase assay

The enzyme beta-galactosidase, encoded by the lacZ gene, is commonly used as a read-out for many biological parameters:

• Transcription from a promoter
• Translation of a mRNA
• Screening of transformants with the correct recombinant plasmid construct (blue-white screening)

All such analyses are chromogenic assays – beta-galactosidase cleaves a chromogenic substrate (see substrates listed in table 1) to produce a colored compound that can be detected.

Western blot

Western blot is used detect a protein of interest in your sample. Chromogenic assays are a common part of Western blot procedures.

Pretty much akin to the “magic dart and spray” analogy we used, the antibody used for detecting the target protein (analyte) is conjugated to an enzyme – most commonly HRP or ALP.

This enzyme breaks down the chromogenic substrate producing a colored compound that can be visually detected on the Western blot membrane.

ELISA

Similar toWestern blot, ELISA (Enzyme Linked Immunosorbent Assay) detects a protein of interest in your sample by using an antibody targeted against it.

This antibody, in turn, has an enzyme conjugated to it, that cleaves a chromogenic substrate, producing a color change that is detected using colorimetry.

Chromogenic growth media for detecting specific microbes

Chromogenic microbiological media contain chromogenic substrates. These substrates are cleaved by the enzyme produced by the microbial strain. This cleavage results in a visible color change.

This way we can distinguish between two different strains – one which has the enzyme to produce a color change (by breaking down the chromogenic substrate in the media) versus the other which does not produce this enzyme.

References

Bowler et al (2018). Role of chromogenic assays in haemophilia A and B diagnosis. Haemophilia.

Manafi (1996). Fluorogenic and chromogenic enzyme substrates in culture media and identification tests. International journal of food microbiology. 31:1-3(45-58)

Miron et al (2002). A spectrophotometric assay for allicin, alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates. Analytical Biochemistry. 307:1(76-83)

Ngo and Lenhoff (1980). A sensitive and versatile chromogenic assay for peroxidase and peroxidase-coupled reactions. Analytical biochemistry. Analytical biochemistry. 105(1):389-397

Wang et al (2019). A novel alkaline phosphatase assay based on the specific chromogenic interaction between Fe³⁺ and ascorbic acid 2-phosphate. Analytical Methods. 11(2374-2377)