There are other more sophisticated techniques, such as microscale thermophoresis, which combine the precision of fluorometry with the sensitivity and versatility of thermophoresis, resulting in a fast, robust, and flexible platform
[72][46]. Among its characteristics, it stands out that it requires sample volumes of less than 10 µL and can analyze multiple substrates simultaneously
[73][47]. Although the advantages of these tests are remarkable, their implementation is limited by high equipment and operational costs, making it difficult to implement them on a large scale and in poorly equipped laboratories.
Despite the fact that the options are vast, the selection of the method depends on multiple factors that must be previously analyzed by the researchers. For example, if an affordable method for the detection of abundant biomarkers with relative accuracy is required, a colorimetric assay could be a suitable option. On the other hand, if the biomarker is present in lower concentration and high precision is required, the most viable options would be to use fluorescent, luminescent, or microscale thermophoresis methods.
2.3. Method Development and Standardization-Related Challenges
As the star players of the game, enzymes are the first factor to consider in the development of enzyme assays. The enzyme selection obviously depends on the biomarker to be quantified; however, this choice involves other important details. It is widely known that enzymes display their maximum activity under certain environmental conditions, and disturbances in these states significantly affect their activity
[74][48]. This atmosphere includes factors such as pH, temperature, ionic strength, concentrations of substrate and enzymes, and interferents. All these parameters have a significant impact on the correct performance of these biomolecules and must be carefully analyzed.
Ionic strength is another factor that must be closely considered in enzyme-based assays. This characteristic is given as a function of the concentration of all the ions present in the solution
[86][49].
Multiple enzymes (especially mammalian-derived ones) have an optimum pH, temperature, and ionic strength close to the physiological conditions (pH of 7.4, temperature of 37 °C, and ionic strength of 0.15 M)
[76,77,78][50][51][52]. In saliva, the normal pH ranges from 6.2 to 7.6 (average pH of 6.7)
[38][19]; these pH values are below blood values, and their effect on enzymes should be studied. This phenomenon has been widely documented.
Similar to the pH, the temperature is a parameter that has a known influence on enzyme activity. As in any chemical reaction, the rate of the enzyme-mediated reaction is strongly influenced by temperature, (generally increasing the reaction as the temperature increases). However, higher temperatures (over about 55 °C) lead to the denaturation of enzymes and the loss of their activity. It is important to mention that this depends closely on the enzyme selected, as some enzymes have a higher optimum temperature, such as polymerase and helicase (optimum temperature of 70–80 °C)
[84][53].
In enzymatic assays, the balance between substrate concentrations, enzymes, and interferents occupies a central place. This is perhaps one of the main challenges that must be faced when using saliva as a sample as the concentrations of biomarkers are usually low, and it contains multiple compounds that can act as interferers
[12][54].
Many other factors are not discussed in this review. However, they must be meticulously considered in enzymatic assays, such as the correct handling of the sample and methodological aspects related to sample preparation, choice of blanks, incubation and reading times, as well as data processing. All these elements taken together can lead to discrepancies in the intra-day and intra-experiment results, thus the importance of establishing a simple, replicable, and robust protocol.