Application of Κ-Carrageenan for One-Pot Synthesis of Hybrids of Curcumin Natural with Iron and Copper: Stability Analysis and Application in Papilloscopy

1. Introduction

Papilloscopy refers to the science dedicated to studying fingerprints, which vary between individuals and even on the same person. This uniqueness has made fingerprints a reliable method of human identification for over a century [1]. In forensic sciences, fingerprints analysis is based on Locard’s exchange principle, which establishes that every contact between two objects results in a transfer of traces between them. Consequently, the act of touching a surface transfers material from the ridges of the fingers onto a surface [2]. In criminal investigations, fingerprints are considered physical evidence, as they not only capture the pattern ridges but can also retain exogenous compounds such as drugs, explosives, and chemicals [3]. Latent fingerprints (LFPs) found at crime scenes are invisible to the naked eye and require specific methods to be visualized, which include optical (ultraviolet laser), chemical (cyanoacrylate fuming), and physical (powder dusting and deposition [4,5]. One of the oldest and still widely used techniques by forensic experts is the powder technique, especially when LFPs are deposited on smooth surfaces such as glass, metal, and plastic. The technique is simple, involving the application of powder with a specific brush onto the surface where the LFP is found, making the ridges patterns and their minutiae visible to the naked eye [6]. An ideal compound for developing fingerprint should provide high contrast, good adhesion properties, and desirable selectivity [1]. In this context, the search for a selective, high-contrast, low-cost, and reliable powder for developing LFPs has been relentless. Most LFP powders available use hazardous chemicals in their synthesis, and continuous use can cause side effects. Therefore, the use of natural products as a raw material source for the development of new LFP developers’ is of great interest, as their use may result in a final product with little to no toxicity [7].

Based on this, curcumin, a natural dye extracted from the rhizomes of Curcuma longa, has emerged as a promising candidate [8]. Its structure comprises a β-diketone fraction, methoxy groups, phenolic hydroxyls, doubly conjugated bonds, and keto-enol tautomerism [9]. Studies reports that the properties found in Turmeric are mainly attributed to curcumin, making the isolated compound of great interest [10]. Interesting properties of isolated curcumin have been reported, which includes high antimicrobial activity [11], antibacterial properties [12], antioxidant features [13], among others. However, isolated curcumin exhibits rapid photodegradation when exposed to light [14] and air [15], which limits its stability and effectiveness. Furthermore, the curcumin molecule is not stable in solution due to the β-diketone fraction of curcumin, which is a hydrolytic center. This hydrolytic center is involved in the elimination of the hydroxyl radical and in redox reactions, leading to the formation of degradation products, that is, minor phenolics [14], such as ferulic acid, vanillic acid and vanillin [16]. To overcome these limitations, hybrid materials can be designed from various components in order to achieve original properties in a single material [17]. The β-diketone groups enable curcumin to chelate metal ions, leading to the formation of hybrid materials, which comprises by a combination of two or more phases, whether organic or inorganic with unique properties [18]. However, the properties of a hybrid material are not merely a sum of the individual contributions of its constituents, depending on the chemical nature of the organic and inorganic compounds. Due to their complexity, they can be classified in different categories. Among all, the most usual classification is performed according to the type of interaction or nature of the chemical bonds between the organic and inorganic components. As mentioned earlier, curcumin contains a fraction that acts as a strong chelating agent, and thus, hybrid materials synthesized from curcumin belong to class II, which consists of materials formed through covalent or ionic bonds [19]. For the synthesis of a hybrid material containing curcumin and a metal as organic and inorganic phases, respectively, a third phase with metal-reducing properties is needed. In this scenario, the use Kappa-carrageenan (κ-car) as a reducer agent has attracted interest [20] κ-car is a polysaccharide extracted from red macroalgae, featuring non-toxic, low-cost, edible, biodegradable and biocompatible properties [21]. The negatively charged surface of κ-car enables easy interaction with positively charged metal ions through electrostatic attraction. Moreover, the presence of hydroxyl (OH) groups facilitates the reduction of these metals [22].

According to what has been reported, in this work, we continue our efforts to develop derivatives of organic compounds with application in papilloscopy [7,23,24,25,26,27,28,29,30,31,32]. Therefore, this study reports the development of a new hybrid material of curcumin and Kappa-carrageenan combined with two low-cost copper and iron metals. The hybrid materials were evaluated for thermal stability and solution behavior. Finally, these hybrid composites were tested as potential LFO developers, offering a novel, low-toxicity alternative for fingerprint analysis.

2. Materials and Methods

The curcumin (diferuloylmethane, 65 % purity) and Kappa-carrageenan were purchased from Sigma-Aldrich (St. Louis, USA). Acetonitrile was purchased from Biograde (North Carolina, USA – HPLC ≥ 99.99%). Tetrahydrofuran (THF) was purchased from Honeywell (North Carolina, USA – HPLC ≥ 99.99%). Fingerprint powder developer Gold® was purchased from Sirchie® (Youngsville, USA). Other chemicals were of analytical grade from commercial sources.

3. Results

The influence of metals on the color of hybrid materials was observed and the images are shown in Figure 1. It was observed that curcumin (Figure 1B) and Kappa-carrageenan (Figure 1C) exhibit saffron yellow and whitish colors, respectively. Additionally, after the addition of Kappa-carrageenan in curcumin (Figure 1E), only the hue was altered, while the overall color remained unchanged, which agrees with the L*, a*, and b* values, as indicated in Table S2 (Supplementary information). In the presence of metals (Figures 1 F and G), significant changes in the color of the materials were clearly noticed. As shown in Table 1, the a* and b* values for the copper hybrid material (a* 9.10 and b* 58.44) and the iron hybrid material (a* 2.41 and b* 11.98) decreased compared to the values obtained for the curcumin/Kappa-carrageenan mixture (a* 16.46 and b* 82.17), suggesting that the red and yellow components were reduced. Among the hybrid materials, the copper hybrid exhibited the highest red and yellow values, indicating that Cu 2+ has a more significant role in enhancing the color of curcumin/Kappa-carrageenan. Additionally, the L* values were reduced for the copper (L* 43.92) and iron (L* 14.33) hybrid materials, indicating that they exhibited a darker color compared to the curcumin/Kappa-carrageenan (L* 63.40), which is consistent with reports from the literature [33]. Additionally, when determining the concentration of Cu and Fe in the hybrid materials, values of 13.31 and 13.51 mg g-1, respectively, were obtained, thus confirming the presence of the metals in their respective hybrid materials.

Conform, Figure 2A show the UV–Vis spectrum of curcumin, Cu and Fe hybrid materials. The curcumin exhibited a maximum absorption at the wavelength of 425 nm and a weak absorption at 262 nm [38]. The samples containing Cu an Fe exhibited more intense absorption bands, with a shift of approximately 6 nm [39]. Furthermore, bands at 419 nm and 255 nm were clearly observed for the Cu hybrid material, while peaks at 428 nm and 257 nm are observed for the Fe hybrid material. The crystal structure of samples was further investigated through XRD and the results are presented in Figure 2B. The XRD pattern of curcumin exhibits its typical crystal structure with intense peaks, especially at angles below 30 degrees, similar to what has been reported in other similar studies [40]. For the Kappa-carrageenan samples an amorphous structure was observed, similar as reported by other authors [41]. The amorphous behavior remained predominant for the hybrid samples, where no peaks corresponding to the crystal planes could be identified. The FT-IR was used to identity the functional groups on the surface of the samples and the results are displayed in Figure 2C and D. The infrared spectra of curcumin show a typical band at 3461 cm-1 corresponding to the phenolic stretching (O-H). Thus, the aromatic stretching (C=C) and stretching vibrations (C=C) were observed at 1629 cm-1 and 1509 cm-1, respectively. The stretching vibrations (C=O) are observed at 1599 cm-1, the bending vibrations (C-H) and aromatic stretching (C-O) vibrations were noticed at 1427 cm-1 and 1280 cm-1, respectively. Finally, the stretching vibrations (C-O-C) are observed at 1025 cm-1 [42]. The FT-IR of the Fe and Cu hybrid materials exhibit similar bands as observed in the curcumin. Furthermore, the vibrations mode suffered shifts in their wavenumber indicating changes in the vibrational state of curcumin upon binding to the metal ions [43,44].

TGA of the hybrid materials, curcumin, and Kappa-carrageenan were performed, and the results are shown in Figure 3. Kappa-carrageenan (Figure 3A) exhibited three stages of weight loss, which is consistent with previous reports [45]. The first thermal event occurred around 218 °C. The second and third thermal event occurred at approximately 531 °C and 747 °C, respectively. The decomposition of curcumin began around 333 °C, with total degradation occurring around 575 °C, reaching 45.43%, which is in accordance with literature reports. The TGA curve for Curcumin/Kappa-carrageenan (Figure 3B) showed three stages of mass losses of 12.43%, 14.29%, and 10.46% at temperatures of 207 °C, 342 °C, and 722 °C, respectively. Finally, two and three stages of mass loss were observed for the iron and copper hybrid materials, respectively. Both samples exhibited the greatest mass loss in the first thermal event, 17.25% and 18.12%, at temperatures of 198 °C and 229 °C, respectively [33].

In the analysis of curcuminoids in a commercial sample of natural curcumin, three distinct peaks were observed, as shown in Figure S1 (Supplementary information). Peak 1 has a retention time of 2.957 minutes, Peak 2 has a retention time of 3.103 minutes, and Peak 3 has a retention time of 3.263 minutes. In the study of curcumin and hybrid material degradation, the samples were injected into the HPLC at different time intervals starting from the moment of dissolution. During this period, the samples were incubated in clear glass vials and left in an unprotected light area throughout the analysis period. Figure 4A shows the chromatograms of the curcumin solution at different time points, while Figure 4B presents the chromatogram of the curcumin solution after 24 hours, where the presence of more than one peak can be noted. In Figure 4C and 4D, the chromatograms of copper and iron hybrid materials, respectively. It can be observed that new peaks were not detected after 24 hours.

In the development of fingerprints using composites generated from hybrid materials, an initial study was conducted with a variety of donors. To convert the visual appearance of the fingerprints developed with powders into numerical values, and recognizing that the researchers of this study are not fingerprint identification experts, a scoring system was employed instead of relying solely on minutiae detection and counting. This scoring system has been in use for over 35 years. The fingerprint evaluation scheme is presented in Table S1 (Supplementary Information). Cu and Fe composites were then applied to develop fingerprints using the physical powdering method on a glass surface. Figure 5 and Figure 6 show the natural and sebaceous fingerprints from four donors, along with their respective scores.

Figure 7 and Figure 8 represent a depletion series from donor 3, developed with copper and iron composites. In Supplementary Figures S2–S7, the depletion series from the other donors developed with both composites can be observed.

Figure 9 shows the same fingerprint (natural and sebaceous) developed on the left with composites and on the right with commercial gold powder. Supplementary Figures S8 and S9 display the development achieved for the same natural and sebaceous fingerprints from the other donors, using both composites and the commercial powder.

The SEM images (Figure 10) provided a detailed analysis of the development of latent fingerprints using both composites. Magnifications of 40x, 600x, and 2000x were applied to investigate the minutiae of the fingerprints, the interaction of the particles with the surface, and the shape and size of the particles. For this analysis, latent fingerprints, both natural and sebaceous, were deposited on glass surfaces and developed with Cu and Fe composites. These magnification levels offered a more precise view of the particle adhesion to the ridges of the fingerprints and the effectiveness of the composites in developing the fingerprints.

4. Discussion

According to the literature, it is known that the conjugated β-diketone portion of curcumin is a strong chelating agent, being capable of interacting with several metal ions through a coordination bond, which can modify the color of the organic phase of curcumin [46,47]. Thus, the colorimetry results indicated the effective formation of hybrid materials, evidenced by the color change after the addition of these metals and confirmed by their concentration found in the materials.

Thus, UV-Vis, X-ray and FT-IR results suggests that the one-pot synthesis was efficient in forming hybrid materials using Kappa-carrageenan as a reducing agent. A strong absorption in the 400-550 nm range of UV-Vis spectrum (Figure 2A) was observed for all samples, corresponding to the blue and green light region, which agrees with the colorimetric results. The shift in wavelength from curcumin to the hybrid materials suggests an interaction between the carbonyl group of curcumin in metal coordination [39]. XRD analysis (Figure 2B) of curcumin confirmed its typical crystalline structure [40], while both Kappa-carrageenan and the hybrid materials exhibited amorphous structures. FT-IR was used to identity the functional groups on the surface of the samples and the results are displayed in Figure 2C and 2D. The infrared spectra of curcumin show a characteristic band at 3461 cm-1 corresponding to the phenolic stretching (O-H). The aromatic stretching of (C=C) and stretching vibrations of (C=C) were observed at 1629 cm-1 and 1509 cm-1, respectively. The stretching vibrations of (C=O) were observed at 1599 cm-1, while the bending vibrations corresponding (C-H) and aromatic stretching of (C-O) vibrations were noticed at 1427 cm-1 and 1280 cm-1, respectively. Finally, stretching vibrations related to (C-O-C) were observed at 1025 cm-1 [42]. The FT-IR of the Fe and Cu hybrid materials shown in Figure 2-C and D, exhibited similar bands as observed in the curcumin, with shifts in wavenumber indicating changes in the vibrational state of curcumin upon binding to the metal ions [43,44].

The thermal stability of hybrid materials is crucial for their practical applications. Kappa-carrageenan exhibited three thermal events (Figure 3A): the first corresponding to moisture loss, while the second and third events were related to the decomposition of the carbohydrate skeleton and complete degradation. Curcumin began with the dehydroxylation process, followed by its total degradation. The Curcumin/Kappa-carrageenan composite showed similar behavior to pure Kappa-carrageenan (Figure 3B). On the other hand, the hybrid materials, displayed differences due to the varying amounts of curcumin present in the samples. Thus, the TGA curves indicate that the hybrid materials possess superior thermal stability than pure curcumin [34].

The presence of three peaks in the chromatogram (Figure S1 – Supplementary information) can be explained by the composition of commercial curcumin (65%), which contains two other curcuminoids: desmethoxycurcumin and bisdemethoxycurcumin [48], representing the remaining 35% of the sample. Thus, the method adapted proved to be efficient, as the three peaks did not overlap. In addition, it provided a fast analysis time of 5 minutes [33]. The curcuminoid diferuloylmethane, commonly known as curcumin, is the largest fraction present in the sample, as curcumin was acquired at 65% purity. Therefore, the peak referring to the retention time of 3.263 minutes, which has the greatest area and intensity, represents curcumin. Desmethoxycurcumin is the second largest fraction of the sample and was identified with a retention time of 3.103 minutes. Finally, bisdemethoxycurcumin, which denotes the smallest fraction of the sample, was identified with a retention time of 2.957 minutes. Several reports described the same order of curcuminoids with bisdemethoxycurcumin being the first peak and curcumin the last peak [49,50], the impurities in curcumin sample, no interference in the formation of hybrid materials were found, since all curcuminoids contain the β-diketone fraction responsible for binding the metals. The absence or presence of methoxy groups (the only difference between the curcuminoids) does not influence the process, as they remain intact after the synthesis of the hybrid materials [18].

Regarding curcumin degradation, studies using curcumin dissolved in ethanol [42] or in Phosphate Buffered Saline (PBS) buffer [16] reported vanillin, vanillic acid, and ferulic acid as degradation products [18,51]. In this study, samples were dissolved in acetonitrile, resulting in degradation after 24 h, as observed by multiple peaks in the chromatogram of Figure 5B, indicating the formation of other molecules, i.e., degradation products. Additionally, a decrease in the curcumin peak intensity from 450 at time zero to 55 after 24 hours, along with a considerable reduction in peak area, confirmed the generation of degradation products. However, the same behavior was not observed in the chromatograms of the copper hybrid materials (Figure 4C) and the iron hybrid materials (Figure 4D), suggesting that the hybrid materials offer superior stability compared to isolated curcumin.

The use of powders to develop fingerprints, it is crucial to address the limitations of curcumin, especially in terms of stability. The previous results demonstrated that the combination of curcumin with Cu and Fe metals has led to a significant increase in stability of the samples, which is one of the key requirements for practical use [52,53]. Based on these results, the potential of these compounds as developers for latent fingerprints was investigated. Composites were applied to fingerprints from various donors, aiming to capture variations related to age, sex, and other characteristics. It is known that factors such as occupation, health (both physical and mental), diet, and medications directly affect the fingerprint composition, both natural and sebaceous fingerprints were evaluated to assess interactions with the water-soluble and lipid-soluble compounds [24,54]. Through the method of Sears et al., (2012) [37], the visual appearance of the fingerprints was converted to numerical values for classification. The results demonstrated that both composites were effective in developing natural and sebaceous fingerprints from all donors, resulting in clear images on glass surfaces. The composites adhered to the fingerprints without having adhesion to the glass, providing good contrast in the images (Figure 5 and Figure 6). In numerical values, the lowest scores, such as zero (no evidence of mark) and one (evidence of contact without ridge details), were not assigned to any fingerprint developed with the composites. Only one natural fingerprint of the Cu hybrid composite and two from the Fe hybrid composite received a score of 2, indicating limited development (with about 1/3 of ridge details present, but insufficient for identification). The remaining fingerprints were rated with scores of 3 or 4, reflecting identifiable and well-developed marks. The Cu composite (Figure 5) showed better performance in developing sebaceous fingerprints, receiving a score of 4 for donors 2, 3, and 4. For natural fingerprints, donor 1 received a score of 4, while donors 3 and 4 received a score of 3. Similarly, the Fe composite (Figure 6) also demonstrated superior performance in sebaceous fingerprints compared to natural ones. Donors 1 and 4 had natural fingerprints rated 3, while all sebaceous fingerprints were rated 3, except for donor 3, who received a score of 4, indicating identifiable and detailed fingerprints for all donors. These findings indicate that, although the same deposition and development method was applied consistently, the influence of the donor on the development process was evident. Nevertheless, the results were consistent and satisfactory, with the Cu composite showing higher contrast and better scores compared to the Fe composite.

To evaluate the sensitivity of the composites in developing fingerprints, depletion series were performed. These series consist of successive contacts of the same finger with the surface, resulting in a progressive reduction in the amount of residue left with each new contact. Thus, the effective development of fingerprints left in the final contact indicate higher sensitivity of the developing powder, as there is less material available for adhesion [37]. Figure 7 and Figure 8 present the depletion series for donor 3, developed with Cu and Fe composites, respectively. Although the Cu composite showed better performance in the previously discussed fingerprints, when comparing the depletion series for donor 3, greater clarity was observed in the revelations made with the Fe composite. This composite demonstrated good development, with excellent sharpness and contrast in most deposits, except for the tenth, where there was a significant decrease in quality. On the other hand, the Cu composite showed better clarity in the first four deposits, but from the fifth onward, there was a progressive decline in the quality of development. This decline may be attributed to the higher contrast provided by the Cu composite, which could have hindered the clarity of the fingerprints in deposits with less sebaceous residue. Similar results were observed for the other donors in the depletion series, as shown in Supplementary Figures 2 to 7. Although the Fe composite offers lower contrast compared to Cu, it demonstrated superior sharpness in the depletion series for all donors, often effectively revealing fingerprints up to the tenth deposit. In contrast, the Cu composite showed satisfactory development with good up to the fourth deposit, followed by a progressive decline in quality from the fifth onward.

It is important to consider that fingerprints exhibit inherent variability in their composition, including factors such as chemical composition, the amount of material deposited, and the pressure applied during deposition. To minimize the impact of these variables on fingerprint development, the use of half-and-half fingerprint tests is an effective approach. In this method, fingerprints are deposited and physically split along the centerline, allowing each half to be developed using different techniques or powders (powdering method) [37]. This "half-and-half" strategy, applied to the comparative analysis between synthesized powders and commercially available powders, is particularly relevant as it allows the evaluation of whether the developed material performs similarly to those currently used by the Federal Police [32]. Figure 9 illustrates the natural and sebaceous fingerprints of donor 4, developed using the half-and-half method. The left halves were developed with Cu or Fe composites, while the right halves were developed with the commercial gold powder. A good development of natural fingerprints was observed with both composites, exhibiting quality comparable to that of the commercial gold powder. In the case of sebaceous fingerprints, the Cu composite showed performance similar to that of the commercial powder, while the Fe composite demonstrated superior sharpness compared to the commercial powder. Figures S8 and S9 (Supplementary information) show the half-and-half fingerprints of the other donors. A similar behavior was observed in the development of the composites, with performance equivalent to or better than the commercial powder. When comparing both composites, the Fe one stood out by providing greater sharpness, resulting in clearer definition of fingerprint minutiae.

So far, hybrid material composites have been evaluated as developers for latent fingerprints using various methods, all applied to glass surfaces. Considering that the surface on which a fingerprint is deposited can significantly influence the effectiveness of its development, the final analysis of these powders included tests on surfaces representative of those encountered in operational work. For this, the same composites were applied to different surfaces commonly found in forensic investigations [37]. Examples of these surfaces can be seen in Figure 11.

Figure 11 illustrates the development of latent fingerprints with both composites on porous and non-porous surfaces. Objects commonly found in real-world scenarios were selected, including glass bottles, metal knife surfaces, plastic knife handles, wooden door surfaces, metal doorknobs, and the glass screens of smartphones. Both composites were effective in enhancing latent fingerprints on both light and dark surfaces, as well as porous and non-porous ones, demonstrating the versatility of the developed powders. The revealed fingerprints exhibited sufficient detail to be used in human identification processes.

The SEM images (Figure 10) provided a detailed analysis of the minutiae in both natural and sebaceous fingerprints developed with both Fe and Cu composites. At 40x magnification, it was observed that the Fe composite offered greater clarity compared to the Cu composite, which is directly related to the strong adhesion of the Fe composite to the fingerprint compounds, while interacting minimally with the glass surface, as evidenced in the 600x magnified images. Regarding morphology, both composites exhibited irregular shapes and heterogeneous particle size [28], ranging from 50 micrometers to less than 10 micrometers. Despite these characteristics, the composites were effective in developing latent fingerprints, providing sufficient detail for human identification.

5. Conclusions

The hybrid copper and iron materials were synthesized via one-pot methodology using natural curcumin and Kappa-carrageenan as a reducing agent. These materials were characterized by different techniques such as FT-IR, UV-Vis, XRD and F AAS. The observed color changes and enhanced stability of these materials can be attributed to the coordination of Cu²⁺ or Fe³⁺ metal ions with curcumin, as well as potential interactions with Kappa-carrageenan. To verify whether curcumin hybridization effectively improved its stability, thermogravimetric analysis was performed, showing that the hybrid materials had greater thermal stability than curcumin. Solution stability studies using liquid chromatography revealed that curcumin showed degradation product peaks after 24 hours of solvent exposure, whereas the hybrid materials remained stable after 24 hours. The hybrid composites were further applied as latent fingerprint developers. The results showed that both materials effectively developed natural and sebaceous fingerprints from various donors, with good clarity and contrast on glass surfaces. In successive contact depletion series, the Fe composite demonstrated higher sensitivity, revealing sharpness fingerprints up to the tenth contact, while the Cu composite showed less quality after the fifth contact. In comparative tests with commercial gold powder, both hybrid materials performed similarly, with the Fe composite standing out for its fingerprint sharpness. Additionally, when applied to surfaces representative of forensic scenarios (such as glass bottles, metal doorknobs, knife surfaces, and smartphones), the composites were versatile, revealing sufficient fingerprint details for human identification on both porous and non-porous surfaces. Overall, these findings highlight the potential of hybrid materials to enhance forensic applications and contribute to the sustainable use of natural resources.