Enzyme substrates are used in a variety of research and clinical applications including enzyme-linked immunosorbent assays (ELISA), blotting techniques (Western, Southern, Northern), in-situ hybridization, plaque/colony lifts, DNA sequencing, and cytometry. The most frequently used enzymes include horseradish peroxidase (HRP), alkaline phosphatase and β-galactosidase because of their high turnover rate, stability, ease of conjugation, and relatively low cost. Giri Diagnostic, Pvt.Ltdis actively involved in developing improved enzyme substrate formulations to be more sensitive, safe, and with wide applicability. There are three types of enzyme substrates being developed at Giri Diagnostic:

1. Chromogenic

2. Fluorogenic

3. Lumigenic

Chromogenic Substrates:-

Chromogenic substrates produce color in the presence of suitable enzymes. For example, p-nitrophenyl phosphate (pNPP) is a chromogenic substrate specific for phosphatases, most commonly alkaline phosphatase. Alkaline phosphatase hydrolyzes pNPP into p-Nitrophenol and inorganic phosphorus, resulting in a yellow solution. The reaction may be followed on a colorimetric spectrometer at 405nm. The color intensity is directly proportional to the amount of alkaline phosphatase, allowing for reliable quantitation.

The reaction can be shown as

Another example of a highly effective chromogenic substrate is TMB

(3,3´,5,5´-tetramethylbenzidine) for HRP. HRP catalyzes the oxidation of TMB in the presence of peroxide, characterized by the following reaction scheme:-

The reaction is stopped by the addition of acid, which results in a yellow solution, measurable at 450nm.

Chromogenic assays are popular because results are observable even without instrumentation (qualitative), or with instrumentation (quantitative). However, chromogenic substrates are the least sensitive of the three mentioned substrate types, requiring a relatively high concentration of enzyme for chromogenic assays.

Fluorogenic Substrates:-

Fluorogenic substrates produce a fluorescent compound in the presence of a suitable enzyme. Fluorescein diphosphate (FDP) is an excellent substrate for alkaline phosphatase, commonly used for ELISAs. Alkaline phosphatase hydrolyzes the non-fluorescent FDP into highly fluorescent fluorescein (excitation/emission = 490/514nm). The high pH used to monitor alkaline phosphatase activity is advantageous since it also enhances fluorescence. The reaction can be shown as:

Fluorogenic substrates are several orders of magnitude more sensitive than chromogenic substrates, but tend to be less sensitive than chemiluminescent substrates.

Lumigenic Substrates:-

Lumigenic substrates refers to substrates that produce light from bioluminescence or chemiluminescence, which are production of light from living organisms or chemical reactions, respectively. Giri Diagnostic specializes in novel formulations of chemiluminescent substrates, such as stabilized 1,2-dioxetanes. Stabilized 1,2-dioxetanes are highly sensitive substrates, which can be formulated to be specific for a variety of enzymes (i.e. alkaline phosphatase or β-galactosidase). The following example exemplifies alkaline phosphatase as the target enzyme. After cleavage of the inorganic phosphate group, the 1,2-dioxetane becomes unstable, producing light through its decomposition. The reaction can be shown as:

The 1,2-dioxetane substrates for alkaline phosphatase can be improved through addition of enhancers and fluorescent molecules results in the ability to detect down to a single molecule of alkaline phosphatase.

>Luminol is a very common chemiluminescent substrate used for detection of HRP. HRP catalyzes the decomposition of luminol in the presence of peroxide to produce a blue light as demonstrated in the following reaction:

Chemiluminescent substrates are the most sensitive substrates available, and are generally more economic than fluorescent substrates. Chemiluminescent substrates are also highly specific with a broad dynamic range to suit a variety of applications.