Absorbance and fluorescence are often considered competing for the quantification of
biomolecules.
They present their own advantages and disadvantages. In fact, both types of tests work equally for newborn screening.
Newborn screening by absorbance is as successful as screening by fluorescence. Indeed, colorimetric tests have all the advantages for an efficient and cost-effective neonatal screening. Here are the reasons why.
Sufficient sensitivity
The concentration of biomarkers in newborn screening (NBS) amounts to a few micrograms per liter, i.e. ranging from milli- to micromolar. Absorbance assays – enzymatic or ELISA – can detect molecules down to the nano-/picomolar level [1]. Although less sensitive than fluorescence, newborn screening by absorbance remains sensitive enough.
Affordable and simple technology
The price of spectrophotometers tends to be lower than that of fluorimeters [2]. Similarly, chromogenic substrates are more affordable. Combined, newborn screening by absorbance appears to be more cost-effective. This is particularly relevant in a market where cost per determination dominates.
Moreover, spectrophotometers are easy to use instruments [2]:
- Software makes them intuitive
- Maintenance is easy since they have few moving parts
Enhanced specificity
As for fluorescence, particular chromogenic substrates are used to quantify the NBS biomarkers: tetrazolium salts for enzymatic assays and TMB for ELISA [3]. The enzymatic treatment leads to color generation. The absorbance signal reading is then specific and proportional to the analyte of interest.
Reduced influence of experimental conditions
Experimental conditions (e.g. pH, temperature, turbidity) influence both absorbance and fluorescence assays. Manufacturers consequently take all the necessary precautions to provide high-quality kits: standardized reagents, well-defined protocol and product quality management. All this helps to avoid measuring errors in newborn screening by absorbance or by fluorescence.
Concretely, why choose absorbance?
ZenTech s.a. offers a wide range of enzymatic or ELISA kits for NBS. They are currently all based on absorbance measurement. Choosing absorbance is interesting since:
- Enough sensitivity and specificity for newborn screening
- Affordable and simple technique
- No particular experimental restrictions
For all these reasons, newborn screening by absorbance has proven to be perfectly adapted.
Bibliography
- [1] Acharya, A. P. et al. (2013) ‘A fluorescent peroxidase probe increases the sensitivity of commercial ELISAs by two order of magnitude’, Chem Commun (Camb), 14, 49(88), pp. 10379–81.
- [2] https://www.biocompare.com/Bench-Tips/173963-Choosing-the-Best-Detection-MethodAbsorbance-vs-Fluorescence/
- [3] Gibbs, J., Vessels, M. and Rothenberg, M. (2011). Selecting the Detection System – Colorimetric, Fluorescent, Luminescent Methods for ELISA Assays (Application note No CLS-DD-AN-458), https://www.corning.com/catalog/cls/documents/application-notes/CLS-DD-AN-458.pdf
