Using Ferrozine is one of the most used methods to detect Fe(II) (Im et al., 2013). The ferrozine assay typically detects Fe(II) in the 2–1,000 µM range; however, methodological optimizations can enhance its sensitivity down to approximately 0.5 µM (Riemer et al., 2004), making it precise and cost-effective technique. Ferrozine is relatively inexpensive, and the whole procedure can be performed within 1 h, but it requires complete reduction of ferric ions (Riemer et al., 2004; Tamarit et al., 2006; Im et al., 2013). High concentrations of manganese or copper can lead to underestimated results, It is important to take that into consideration when performing the assay (Dubinsky et al., 2010; Im et al., 2013). The original paper describing this method was published in 1970 by Stookey (1970) who described it as a highly selective, intense purple complex with λ max at ∼562, and recommended 1 min incubation time (Stookey, 1970). After this, the methods have been mostly used for Fe quantification in chemical samples with slight modification, including longer incubation time (Kostka and Luther, 1994; Thamdrup et al., 1994; Riemer et al., 2004). The biological applications have been presented in the 1990s and 2000s (Riemer et al., 2004). In 2004, Riemer et al. adapted the protocol for use in eukaryotic cells, with the protocol including HCl/KMnO₄ pretreatment to release from proteins and achieve full reduction. The redefined protocol recommends protein precipitation to avoid background interference (Riemer et al., 2004). The method has also been widely applied to study bacteria (Dubinsky et al., 2010; Akob et al., 2012; Shin et al., 2021) and yeast (Tamarit et al., 2006). Shin et al., in 2020 used ferrozine to examine the uptake of iron in S. aureus. The cells were Fe deprived by using 2-bipyridyl, and then re-exposed to FeSO₄. Then the cells were lysed, and acidified to release ions. The final reaction mixture contained neocuproine to prevent the interference from copper. This approach enabled us to look into the intercellular activity of iron transport systems under different experimental conditions (Shin et al., 2021). In the study by Dubinsky et al. from 2010 (Dubinsky et al., 2010), ferrozine was used to measure the extracellular iron produced during microbial iron reduction. The ion were released with the use of HCL, and the supernatant was then filtered. The final ferrozine solution has been prepared in HEPES buffer. This simple procedure allowed for examining iron reduction activity in soil, showing the involvement of bacteria in biogeochemical processes (Dubinsky et al., 2010). Nevertheless, the simplicity of the approach allows to study more complex behaviors in symbiotic roots related species (Giacalone et al., 2025). Optimized ferrozine assay is proposed to measurement of redox-active metabolites (RAMs) associated with natural and agricultural systems, a class of secondary metabolites that can help bacteria solubilize phosphorus. Ferrozine assay started as a simple chemical test, and over time evolved into a commonly used method for iron detection in microbiological studies.

1,10-Phenanthroline creates an orange complex with Fe(II) (Harvey et al., 1955). The working range of the dye is 0.2–100 µM and displays a strong absorbance at λ max ≈510–514 nm (Komadel and Stucki, 1988; Fernandes et al., 2023). The advantages of this technique include good repeatability and the possibility of rapid measurement in the field by using a handheld photometer (Fernandes et al., 2023). One of the first protocols was developed by Harvey et al., in 1955 (Harvey et al., 1955), allowing for both Fe(II) and total iron quantification by introducing a chemical reduction step (Harvey et al., 1955). An important feature is their photochemical behavior. Light exposure (<500 nm) can induce reduction from Fe(III) without the need for a reducing agent (Komadel and Stucki, 1988). The downside is lower sensitivity as copper, zinc, or nickel ions form weaker but still absorbent complexes, which might introduce additional steps to the reaction to mask them with agents like neocuproine [23, 26]. Although ferrozine gradually replaced phenanthroline, it is still relevant in field applications because of its easy and robust procedure (Fernandes et al., 2023). Recent technological developments have improved the utility of this method by the design of a self-contained, portable, and compact iron measurement system (IMS) based on spectroscopic absorption for the determination of Fe(II). That allows for detection limits as low as 2.5 µg Fe²⁺/L (Fernandes et al., 2023).

Bathophenanthroline disulfonate is a sulfonated derivative of phenanthroline that is water-soluble, which makes it the most sensitive (µM range) among phenanthroline compounds (Lee and Stumm, 1960; Cowart et al., 1993; Naka et al., 2000). One of the limitations is the higher cost of the reagents. Additionally, in the presence of strong antioxidants, the background signal may be increased due to autooxidation, so samples should be measured as soon after preparation as possible (Cowart et al., 1993). One of the first papers written by Lee and Stumm in 1960 (Lee and Stumm, 1960) introduced the use of bathophenanthroline for Fe determination in water samples, under acidic conditions (pH 3–4), it forms a red complex specifically with Fe(II), without significant interference from Fe (III) (Lee and Stumm, 1960). In 1977, Perry and San Clemente (1977) addressed some limitations of this technique, especially in reducing the Fe(III) to Fe(II) before the reaction. They showed that conventional reduction with hydroxylamine was inefficient. Their approach with the use of L-ascorbic acid, HCL, and heating improved the reaction up to 82%, which significantly improved the reaction, especially for low iron samples like microbiological media (Perry and San Clemente, 1977). Freinbichler et al. (2020) extended the application further in 2019 to the in vivo neurochemical studies, introducing high-performance liquid chromatography using bathophenanthroline disulfonate. This allowed for the detection of Fe in very small volumes (a few microliters). This modification has been very significant for looking into iron dysregulation in neurodegenerative diseases (Freinbichler et al., 2020).

Since the introduction of the ferrozine assay, it has undergone optimization and can now be used to determine Fe concentration in different environments. Meanwhile, older methods, including phenanthroline and bathophenanthroline assays, have not been used as extensively but remain important in a specific setting, e.g., such as anaerobic systems, detection in acidic samples, or microfluidic applications. Moreover, the phenanthroline and ferrozine methods are commonly used to quantify Fe(II) in Fenton reactions, with particular importance for iron cycling and oxygenation processes. However, reaction systems can be affected by the presence of Fe(III). Namely, Fe(III) can interfere by oxidizing heterocyclic amines or by forming its own complexes, potentially leading to overestimate Fe(II) concentrations as (Yang et al., 2020). To avoid this, temperature, pH, and buffer concentration should be carefully controlled. Incubation time must be minimized and standardized across samples. Yang and colleagues proposed fluoride as a remedy of residual ferric contamination (Yang et al., 2020).

In this variant, the sample is nebulized into a mist and aspirated into a flame, where ions get atomized. The concentration is then determined by the change in intensity of the light beam. FAAS offers several advantages, such as high throughput, moderate costs, and a relatively simple procedure. However, its limit of detection typically ranges from 2.5 to 10 μg/L, depending on matrix complexity and instrumentation (Tautkus et al., 2004; Ward and Crichton, 2015). Yaman and Kaya, in their work from 2005, present an approach to distinguishing between iron oxidation levels using solvent extraction. Their methods involve using 1-(2-Pyridylazo)-2-naphthol (PAN), which creates a complex with Fe(II)^, then extracting this complex using chloroform. The remaining Fe(III) requires reduction before the qualification. This technique proved to be relatively simple, selective, and sensitive (Yaman and Kaya, 2005).

Graphite Furnace AAS, also known as Electrothermal AAS, enhances sensitivity by atomizing the sample in a small graphite tube rather than in an open flame (Kragten and Reynaert, 1974; Butcher, 2024). The sample is deposited directly in the furnace, dried, chatted, and atomized in a controlled sequence. GFAAS shows detection limits 20–200 times lower than for FAAS (in the ng/L range) (Butcher, 2024), enabling precise quantification of iron in low-concentration samples such as serum or intracellular extracts. In the work by Miller-Ihli from 1989 (Miller-Ihli, 1989) GFAAS has been presented as a highly versatile method for iron analysis in biological samples. They presented different sample preparation strategies, including direct analysis of fluids, wet ashing with nitric acid and hydrogen peroxide, and dry ashing by high temperature. The method has proved to be applicable in multielement analysis in biological reference materials with high accuracy and precision, with minimal sample contamination and loss (Miller-Ihli, 1989).

Anodic Stripping Voltammetry ASV is the most widely used electrochemical technique for iron detection (Nsabimana et al., 2019; Wygant and Lambert, 2022). It has two major steps. The Fe(III) is reduced to Fe(II) or Fe⁰ and deposited on the electrode surface (Florence, 1970; Van Staden and Matoetoe, 1998; Borrill et al., 2019). Then the deposited iron is oxidized back into the solution while the current is recorded. The peak current is proportional to the iron concentration (Abollino et al., 2019; Nsabimana et al., 2019). This method offers great sensitivity that can reach low nanomolar concentrations. However, it requires careful control of electrode surface conditions, and it can be influenced by other redox reactions (Florence, 1970; Van Staden and Matoetoe, 1998; Nsabimana et al., 2019). In 1970, Florence (Florence, 1970) described the use of ASV to determine the iron by a chemical exchange between FE(III) and a bismuth-EDTA complex. Unlike earlier methods that used lead-EDTA, the bismuth EDTA complex was more stable and selective, which led to a lower detection limit (about 9 nM). This method involved deposition of bismuth onto a rotating glassy carbon electrode with a mercury film, followed by anodic stripping. The reaction has been optimized for a pH of 4 and has been used to detect iron in various water samples and chemicals (Florence, 1970). Van Staden and Matoetoe in a study from 1998 (Van Staden and Matoetoe, 1998) developed a flow-through system using differential pulse anodic stripping voltammetry (DPASV) for the detection of both Fe(II) and Fe(III). They used pyrophosphate buffer at pH 9 to stabilize the two oxidation states. Distinct peaks for Fe(III) and Fe(II) were observed at −0.8 and −0.5. They presented a detection limit of about 10 nM and an SD of less than 4%. The method showed good agreement with spectrophotometric methods (Van Staden and Matoetoe, 1998). A more recent innovation by Han et al. (2021) offers a high surface area, enhanced conductivity, and superior electrocatalytic activity toward the reduction of Fe(III). The use of a micro needle electrode sensor modified with gold nanoclusters immobilized on a conductive polymer film allows for strong reproducibility, selectivity, and a detection limit of 3.1 nM. This system avoids toxic components and does not require an additional complexing agent (Han et al., 2021).

Amperometry is a technique that measures the electric current resulting from the oxidation or reduction of analytes at the surface of an electrode under controlled applied potential (Adeloju, 2004; Mross et al., 2015). When potential is applied, Fe(II) or Fe(III) undergo redox reactions at the electrode, generating a current proportional to their concentration (Adeloju, 2004; Saavedra and Cortón, 2014). Vorlíček and Vydra (1965) showed a biamperometric approach where iron (III) is titrated with EDTA using two graphite electrodes. The current drop at the endpoint serves as a precise indicator of Fe(III) concentration (Vorlíček and Vydra, 1965). Saavedra and Cortón (2014) presented a real-time amperometric sensor suitable for bacterial applications. The system’s cyclic voltammetry scans reveal two distinctive peaks: oxidation of Fe(II) and reduction of Fe(III). This dual detection enables tracking of bacterial bio-oxidation kinetics. The use of calibration curves provides a rapid and relatively interference-free quantification (Saavedra and Cortón, 2014).

Potentiometry is a classical electrochemical technique based on measuring the potential difference between a reference and an indicator electrode, and offers a versatile tool for detecting iron ions (Amos and Brown, 1963; Bralić and Radić, 1999). Amon and Brown in 1963 (Amos and Brown, 1963) established a robust titration procedure capable of determining iron (II) using potassium dichromate as an oxidizing agent (Amos and Brown, 1963). Their method leveraged a lead reductor to reduce iron (III) before titration, and the end point was precisely identified using a platinum electrode versus a saturated calomel reference. Although primarily designed for uranium-iron mixtures, the procedure shows potentiometric titration applicable to microbial samples, especially when interferences may exclude other methods (Amos and Brown, 1963). Then, a flow injection potentiometry (FIP) system for detecting Fe(III) through complexation with fluoride ions was introduced as part of advances in sensor technology (Bralić and Radić, 1999). By incorporating a fluoride ion-selective electrode into a cascade flow cell, the system achieved rapid quantification of iron (III) concentration across a wide dynamic range. The response was directly linked to the kinetics of FeF₂ ⁺ complex formation. This method’s flow design makes it perfect for real-time monitoring of iron bioavailability (Bralić and Radić, 1999). Recently, a Fe(III)-selective ion electrode (ISE) was developed using piperine, an alkaloid derived from Piper nigrum, incorporated into a solvent polymeric membrane (Madhushani and Hasini, 2021). This approach allowed for the concentration range of 1 × 10⁻⁴ to 1 M in citrate buffer (pH 3.1), with a detection limit of 6 × 10⁻⁵ M. Analytical performance was validated against AAS, with no significant differences observed. The sensor remained functional for up to 10 weekends with minimal potential drift. Its simplicity, portability, and low cost make it a promising alternative for in situ iron analysis (Madhushani and Hasini, 2021).

High Performance Liquid Chromatography (HPLC) is used to separate iron-containing compounds, such as siderophores, iron-bound metabolites, or metalloproteins. Detection can then be performed by using variety of spectroscopic methods such as Ultraviolet-visible (UV-Vis) spectroscopy, Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), or electrochemical detection. HPLC methods often involve reversed-phase or ion-exchange columns, with chelators like EDTA to maintain iron in soluble, detectable forms (Proch and Niedzielski, 2021). Work by Şenyuva from 2002 (Şenyuva et al., 2002) described the use of HPLC in post-column derivatization to separate Fe(II) and Fe(III). After separation, the reaction with the use of PAR (4-(2-pyridylazo)resorcinol) led to the creation of complexes that could be detected at 521 nm. This achieved great sensitivity (0.109 ug/L for Fe(II), 0.217 ug/L for Fe(III)) and reproducibility. It requires additional reagents and reaction time, but it is very useful for trace metal detection. The methods showed strong correlations with AAS total iron results (Şenyuva et al., 2002). Proch and Niedzielski (2021) have described the use of HPLC coupled with MIP OES (Microwave-Induced Plasma Optical Emission Spectrometry) and ICP OES (Inductively Coupled Plasma Optical Emission Spectrometry), which enabled the separation and detection of iron without a post-column procedure. Using a cation-exchange column and PDCA-cased eluent, Fe(II) and Fe(III) were resolved in under 5 min. MIP OES is a relatively new approach, using a nitrogen plasma, which demonstrates advantages in cost and simplicity, but it has higher detection limits (∼100 ug/L) than ICP OES (∼6 ug/L). MIP OES still achieved a useful detection limit and showed potential as a green alternative, given that it uses less gas (Proch and Niedzielski, 2021). High-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) has been used to detect hydroxamate siderophores (McCormack et al., 2003). This method involved using polystyrene-divinylbenzene stationary phase and gradient elution with methanol and formic acid, which allowed for the separation of both iron complexes and free ligands. The detection limit was very low, at about 0.23 nM for ferrioxamine. This showed that using HPLC-ESI-MS could overcome limitations of traditional assays by its sensitivity and selectivity (McCormack et al., 2003). HPLC coupled with inductively coupled plasma mass spectrometry (ICP-MS) enables direct detection of iron within organic complexes, offering a robust approach to identify and quantify iron ligands in bacterial cultures (Boiteau et al., 2013). To improve the sensitivity, the authors used iron-FREE HPLC system and minimized interference in ICP-MS with a hexapole collision cell and oxygen in the carrier gas. The method detected iron complexes from cyanobacteria and marine samples, showing its potential in tracking siderophore production and iron in natural environments (Boiteau et al., 2013).

IC allows for the separation of ionic iron species, It is useful in studying iron redox dynamics or the release of iron from siderophores and binding proteins. It is less common than HPLC, but IC can provide insights into environmental samples and support studies of iron metabolism mutants (Schnell et al., 1998; Şenyuva et al., 2002; Michalski, 2009). The study by Schnell et al (1998), about the metabolic pattern of sulfate-reducing bacteria, Fe(III) and Fe(II) are separated on a polymer-coated silica-based cation exchange column, followed by post-column derivatization with 4-(2-pyridylazo)resorcinol (PAR) for spectroscopic detection at 520 nm (Lebel and Fu Yen, 1984; Schnell et al., 1998). The major advantage of IC is the simultaneous determination of Fe (III) and Fe(II) in bacterial cultures and environmental samples. Schnell et al. (1998) Demonstrated that reduction of Fe(III) by the Geobacter metallireducens can be effectively tracked and determined. Their method proved to be effective with good reproducibility and sensitivity, detecting iron at micromolar concentrations with minimal interference from other components (Schnell et al., 1998). One challenge noted by the author is the potential oxidation of Fe (II) during the process. Strategies such as conditioning columns with ascorbic acid and maintaining anoxic conditions for reagents and eluents are the key to ensuring accurate results (Schnell et al., 1998). IC can also be used to remove Fe from media to study iron-deprived bacteria and the expression of iron-regulated membrane proteins (Kadurugamuwa et al., 1987).

Reporter gene assays are fundamental tools for studying the regulation of iron-responsive genes in bacteria (Escolar et al., 1999; Jiang et al., 2008). These assays rely on cloning an iron-regulated promoter upstream of a reporter gene, allowing for assessing promoter activity in response to iron availability or genetic modifications (Cotter et al., 1992; Escolar et al., 1999; New et al., 2003). Common reporter systems include: LacZ (β-Galactosidase), GFP (green fluorescent protein), Lux (bacterial luciferase), and luc (firefly luciferase) (New et al., 2003). These reporters produce measurable outputs that reflect the activity of the iron-responsive promoter, offering a nondestructive and real-time readout. The general procedure consists of cloning iron-regulated gene (Cotter et al., 1992; New et al., 2003) (e.g., fur, feoB, siderophore synthesis genes) upstream of the reporter gene, then the recombinant plasmid is transformed into bacteria, bacteria are grown, and the activity of the reporter is measured (Braun and Burkhardt, 1982; Cotter et al., 1992; Dancis et al., 1992; Escolar et al., 1999). In E. coli, the P_fur::lacZ fusion has been widely used to monitor Fur-dependent repression (Escolar et al., 1999). These fusions have been crucial for discovering new members of the Fur regulon, broadening the understanding of iron global impact of bacterial physiology (Escolar et al., 1999). A lacZ fusion approach has been used to investigate how iron availability affects respiratory gene expression in Escherichia coli. Under iron-limiting conditions, anaerobic respiration genes were selectively downregulated, while aerobic respiration systems showed slight upregulation, revealing additional layers of iron-responsive regulation beyond the canonical Fur pathway (Cotter et al., 1992). Overall, the reporter systems are a very versatile methodology that can be fused to any promoter of interest. Advances in genetic manipulation techniques in recent years have improved their accessibility and made it easier to introduce targeted mutations, even in clinical strains (Strzelecki et al., 2024).

qPCR and RNA-seq are important tools for measuring the expression of iron-regulated genes at the transcriptional level. These techniques enable precise, sensitive detection of mRNA transcripts, allowing for identification of changes in gene expression in response to iron availability or genetic modifications.

Quantitative PCR can be used to detect mRNA levels by amplifying reverse-transcribed cDNA using sequence-specific primers and monitoring the accumulation of PCR product in real time. To study the regulation of iron, bacteria are grown under iron-limited conditions. Total RNA is extracted, converted to cDNA, and iron-regulated genes are quantified (Rocha et al., 2020). The study by Nilsen and Boye in 2005 (Nielsen and Boye, 2005) used this method to analyze gene expression in Actinobacillus pleuropneumoniae under iron-depleted conditions. The authors focused on identifying suitable housekeeping genes for iron studies. They demonstrated significant upregulation of tbpA, exbB, and fhuD genes under iron-limited conditions, confirming their role in iron metabolism (Nielsen and Boye, 2005). Also, the regulatory networks governing iron transport and homeostasis in soil related Pseudomonas fluorescens have been investigated through genes expression analysis (Fortuna et al., 2019). Bacteria were exposed to nanoscale zero-valent iron, and the expression of pvdS (regulator of siderophore pyoverdine synthesis) and bacterioferritin-associated ferredoxin gene (involved in iron storage). The qPCR results were cross-validated with culture-based methods. This work highlighted the importance of these methods in confirming whether tested compounds alter the gene expression of iron-related genes (Fortuna et al., 2019). Quantitative PCR is a sensitive, reproducible, and versatile method for studying bacterial iron acquisition and homeostasis. Whether investigating virulence in pathogens or ecological responses in environmental isolates, qPCR provides high-resolution insight into bacterial adaptation to iron availability. By following best practices such as primer design, reference gene validation, and cross-method verification, researchers can use qPCR to advance our understanding of bacterial iron metabolism.

RNA-seq involves high-throughput sequencing of cDNA libraries prepared from total bacterial RNA. These methods provide global gene expression profiles, identification of novel iron-regulated genes and non-coding RNAs involved in iron metabolism (Butcher and Stintzi, 2013; Liu et al., 2017; Ibraim et al., 2019). Transcriptomics has been applied to investigate gene expression changes in Riemerella anatipestifer under iron-limited conditions (Liu et al., 2017). By culturing bacteria with and without an iron chelator, they identified 463 genes: 80 upregulated (mainly involved in iron acquisition) and 383 downregulated (Liu et al., 2017). Similar experiments have also been done by other authors linking iron regulation to bacterial motility, and discovering non-coding RNAs, many of which were iron or Fur-regulated, suggesting RNA-based regulatory mechanisms in iron homeostasis (Butcher and Stintzi, 2013). The results highlight iron’s central role in bacterial metabolism and demonstrate RNA-seq’s role in expanding the knowledge about iron’s role in biological systems.