TABLE 2.

Comparative overview of analytical methods for iron detection in biological and environmental samples.

Method type	Advantages	Limitations
Colorimetric assays	Widely accessible and cost-effective; suitable for routine screening in microbiology and environmental studies. Broad effective working pH range (pH 3.0–9.0). Evaluation of non-protein-bound iron in plasma, cerebrospinal fluid, or microdialysates. Can also be used indirectly to monitor iron binding in siderophore assays or oxidation assays.	Limited sensitivity and selectivity; prone to interference from other metal ions.
Atomic absorption spectroscopy	High accuracy for total iron. Depending on technique choice enable high-throughput or precise analysis for low-concentration samples (ppb range).	Requires sample digestion, which can be time-consuming and may lead to loss or alteration of iron species; Flame AAS has moderate sensitivity, which may be insufficient for trace iron detection in some microbial or environmental samples; Graphite Furnace AAS improves sensitivity but is slower and requires more specialized equipment and expertise.
Electrochemical methods	High sensitivity; allows real-time detection of trace iron in various matrices.	Susceptible to interference from other redox-active species; requires careful calibration.
Radiometric assays	Exceptional sensitivity and specificity for studying iron uptake and metabolism.	Involve handling radioactive materials, which require specialized facilities and safety protocols. Not suitable for routine or high-throughput analysis due to regulatory and practical constraints.
Fluorescence and chemiluminescence methods	Enables intracellular or in situ detection; rapid and adaptable to various biological contexts. Useful in oxidation-related studies Allows differentiation of Fe(II)/Fe(III).	May be affected by autofluorescence or indirect detection; requires appropriate controls. Chemiluminescence assays are indirect and often measure ROS related to iron catalysis rather than iron itself.
Chromatography	Detailed speciation and separation of iron species. HPLC is suitable for siderophores, iron complexes (e.g., heme) in serum or microbial extracts. IC enabling redox studies, analysis of iron speciation in environmental or clinical samples.	Require careful sample handling to prevent oxidation or alteration of iron states during analysis.
Siderophore assays	Gold standard for siderophore based iron transport studies in cell cultures or animal models. Useful for screening.	Specific to siderophore mediated Fe acquisition systems activity and do not provide information on other iron pools or species.
Molecular biology	Provides insight into iron-regulated gene expression and cellular responses. Identifies both known and novel iron-regulated genes microbial responses to iron limitation/overload.	Indirect measure of iron status; results may be influenced by multiple regulatory factors.