Raman spectrum of carminic acid
The Raman spectrum of carminic acid in solid state was obtained by depositing a solution of this compound on glass (Fig. 1c) and on a rough gold surface (Fig. 1d) in order to reduce the intrinsic fluorescence emission and improve the Raman signal. The analysis of spectra reveals the appearance of bands with an intensity variation depending on the sample preparation (glass or Au surface). Raman of carminic acid in solid state presents the characteristic Raman bands of the protonated molecular form. In general, the most intense bands of the spectrum correspond to the aromatic anthraquinone moiety, undergoing the highest intensification in Raman. For instance, the bands at 1233 and 1471 cm-1 which are assigned to ring nCC stretching coupled to in plane deformations (d) modes of the CH3 and COH fragments. The broad and medium bands at 453 and 554 cm-1 corresponds to a skeletal mode, while the other broad bands at 1306 and 1590 cm-1 are assigned to coupled vibrations of the nCC and dCOH modes25.
The main difference observed between glass and gold Raman spectra (Fig. 1c and 1d) is a group of narrow bands appearing in the 1200-1000 cm-1 range, as well as the bands at 852, 642 and 401 cm-1. The position and broadness of these bands suggest that they are due to the glucose residue of carminic acid. In order to confirm this, we have also registered the Raman of glucose on Au film (Fig. 1e). The good correspondence observed between the bands of carminic acid on gold with those of the Raman of glucose allowed us to suggest that the interaction of carminc acid with Au implies as well an approach of the Glu residue to the surface. On the other hand, these bands are attributed to nCO, nCC, dCOH, dCH and dCCC modes of the sugar moiety. However, the only sugar band that does not appear in the Raman of carminic is that at 930 cm-1, indicating that it is related to the anomeric C atom that links the anthraquinone moiety of carminic which is absent in the colorant. The carminic acid-gold interaction deduced from the spectra is also confirmed by the theoretical model resulting from the calculations (Fig. 1b), also revealing that an effective glucose residue metal surface interaction is taking place.
Raman analysis of fibers
Fig. 2 displays the SERS spectra registered in the analysis of the belt-bag shown in Fig. 2a. This belt-bag dated from the Formative Period corresponding to the Arica Culture and stored at the Precolombine Art Museum in Santiago of Chile. SERS spectra onto the fiber were measured by using AgNS nanoparticles. The advantage of these NPs is that they display a large extinction in the near infrared region (Fig. 3a, black line) due to the oblate arms existing in this NPs (TEM image inserted in Fig. 3a), thus rendering high SERS efficiency when using the 785 nm laser excitation. Conversely, spherical citrate nanoparticles exhibit a lower resonance in the near-infrared region (Fig. 3a, red line).
In particular, the SERS spectra were obtained from the yellow-orange and brown points (Fig. 2b and 2c). These spectra are dominated by some bands that cannot be found in the spectra registered on carminic acid reported in the literature25. This is the case of those bands appearing at 650-660 and 735 cm-1. There is strong evidence that these bands could actually be assigned to nucleobases, specifically ring breathing band of the nucleobases guanine (G) and adenine (A) due to the great similarity with the Raman spectra of these bases34–36. In addition, other bands of these nucleic bases can also be identified in the 1300-1500 cm-1 region, while other less intense ring breathing bands are observed at 770 and 800 cm-1 corresponding to the nucleobases thymine (T) and cytosine (C)37,38. Similar spectra were also obtained from the analysis of many other textile materials from the Arica area in North Chile (results not shown). Moreover, bands appearing at similar wavenumber values were also identified in archaeological textiles dyed with cochineal that were original from Peru7.
The presence of nucleobase Raman bands in the SERS of these archaeological textiles led us to verify this by measuring the SERS of DNA materials. In this case citrate AgNPs and excitation at 514.5 nm were use. The use of these conditions leads to more intense spectra in these cases. Fig. 2d, 2e and 2f display the Raman spectra of different nucleic acids adsorbed on Ag nanoparticles. The SERS spectrum of ct-DNA (Fig. 2e, see structure in Fig 2h) is dominated by the A residue due to the higher affinity of this pyrimidine moiety for the metal 39,40, although a weak G signal is also observed at 660 cm-1. The bands corresponding to G are better observed in the SERS spectrum of the pGC.pCG polymer (Fig. 2d, see structure in Fig. 2i), where G predominates over C due to the higher affinity of the purine residues in comparison with the pyrimidine ones. Both A and G bands are simultaneously observed in the SERS of a plasmid, another DNA with a circular structure (Fig. 2f, see structure in Fig. 2g). In the latter spectrum a doublet is observed at 668 and 687 cm-1, corresponding to the ring breathing bands of G under A- and B-DNA coexisting structures in this polynucleotide41,42. Thus, we have concluded that the presence of these nucleobases in historical textiles is attributed to the dying of fibers with raw cochineal extract, which was directly prepared from the insect containing biological residues from the insect body employed in the colorant fabrication.
Although bands of nucleic bases residue predominate on the spectra of textiles, other bands corresponding to other DNA component, such as phosphate group, can also be seen in the 800-1000 cm-1 spectral range. This is the case of the strong band at 950 cm-1 which is observed at 930 cm-1 in the SERS of pGC-pCG (Fig. 2d). Other bands in the 1500-1600 cm-1 range can be also assigned to the purine vibrations of the G residue.
Nucleic base bands are observed with a higher intensity in SERS spectra than protein or carbohydrate ones, also present in the cochineal extract. This is attributed to the higher affinity of purine and pyrimidine nucleobases for the metal43. The analysis of other complex biological materials by SERS reveals that nucleobases always display the strongest signals in non-labelled SERS analysis of biological material44,45.
Replicas of the archaeological samples were prepared to further demonstrate that the presence of nucleic acids components in cochineal is due to the raw extract of cochineal insects. These samples were prepared by reproducing the methods employed in the past regarding the extraction of the cochineal colorant from the insect according to the scheme displayed in Fig. 4a-d. In order to better analyze the sample, the aqueous suspension obtained from the powdered insect extract was separated in two parts: the aqueous supernatant and the solid residue in the bottom. SERS spectra were measured from these two parts and are shown in Figs. 4e and 4f, respectively. SERS spectrum of the supernatant extract (Fig. 4e) show bands that can clearly be assigned to carminic acid at 1071, 1136, 1290 and 1567 cm-1, and that can also be seen in SERS spectra of the carminic acid previously reported21,25,46. However, the bottom residue gives rise to many bands that can be attributed to the biomolecules predominantly existing in this fraction. In particular, the band at 660 cm-1, due to G, and weaker bands at 737 and 767 cm-1, attributed to A and T 34,47, respectively. Moreover, the bands at 585 and 496 cm-1 could correspond to skeletal structural vibrations in nucleic acids48 or bending NH vibrations in nucleobases49, although some contribution from other related biological compounds such as proteins is also possible50. Furthermore, the broad band at 446 cm-1 is reported as a CO deformation in purine rings of nucleobases such as guanine or xanthin39,43. The bands corresponding to phosphate moieties from nucleic acid chains 48, seen in the 900-1100 cm-1 range also corroborate the presence of these types of biological species in cochineal raw material.
At higher wavenumbers, bands of the anthraquinone moiety of carminic acid are mixed to the purine and pyrimidine rings bands of nucleobases. Even though, the bands appearing at 1349, 1284 and 1190 cm-1, are due to carminic acid and correspond to those seen at 1344, 1294 and 1196 cm-1, in the aqueous supernatant fraction. Other bands at 1136 and 1071 cm-1 are due to the glucose residue in carminic acid also observed in the Raman analysis of the colorant (Fig. 1e). The shifts observed in some bands of the colorant are attributed to the presence of many other molecules that can affect its adsorption on the metal surface.
The extracted cochineal colorant was then employed to dye commercial wool fibers in the fabrication of textile replicas. Fig. 5 shows in the top panel the fibers before dying (Fig. 5a); cochineal dyed fiber (Fig. 5b) and the dyed fiber with AgNS deposited on the surface (Fig. 5c) employed to obtain the SERS spectra. Raman spectroscopy is one of the few techniques that allow the in-situ study of dyed fibers, but the extremely high fluorescence of cochineal makes necessary the use of SERS to quench this fluorescence. The SERS spectra were registered upon deposition of AgNS on wool fibers dyed with cochineal at two different conditions: directly on the fiber (Fig. 5h) and by using Al2(SO4)3 as mordant (Fig. 5g). The latter spectra were compared to the Raman and the SERS spectrum of non-dyed wool fibers (Fig. 5j and Fig. 5i). The Raman of the wool fiber displays typical bands corresponding mainly to the amino acid phenylalanine (1606, 1002, 852 cm-1); tryptophan (1547, 1360 cm-1); tyrosine (1615, 1188, 825 cm-1); cysteine (665, 513 cm-1), as well as bands corresponding to the polypeptide keratin backbone amide III and amide I at 1280 and 1666 cm-1, respectively.
SERS spectrum of directly dyed cochineal fibers (Fig. 5h) displays intense carminic bands at 1290 and 1200 cm-1, but also many of the ring breathing bands of nucleobases observed in the 800-600 cm-1 region, which are also observed in the SERS analysis of historical textiles (Fig. 5f, corresponding the latter to the orange areas of the belt-bag shown in Fig. 3a). In addition, intense structural bands from biomolecules are also seen below 600 cm-1.
The use of Al as mordant leads to a deep modification of the SERS of the resulting fiber, the presence of this metal induces a selective enhancement of the 660 cm-1 band due to the G residue (Fig. 5g), while other bands ascribed to skeletal vibrations below 500 cm-1 are also modified by the mordant effect of the metal. The specific interaction of G with Al could take place through the inner amide moiety in the purine ring (Fig. 5e). This interaction could also explain other changes observed in the high wavenumber region, such as the intensification of other bands attributed to the G residue at 1675, 1602 and 1537 cm-1, although shifted from their usual position in nucleic acids due to an effect of complexation with the metal in the mordant. Since the latter bands are mainly attributable to the C=O stretching of the pyrimidine ring coupled to ring vibrations and the stretching of C=N group in the imidazole ring51, we suggest that an interaction of Al with the ketonic oxygen and the N7 in the imidazole ring is taking place in the metallic complex (Fig. 5e). This selective interaction of G with Al has been also recently described in the literature, involving a strong charge-transfer between Al to the C=O and the N7 atom of the G purine residue52,53.
Al is then interacting very strongly with the carminic acid and thanks to this mordant interaction improves the fixation of the colorant to the wool fiber through the keto and phenol groups of the anthraquinone rings (Fig. 5d), as also deduced from the SERS analysis of this molecule onto metallic nanoparticles 25. As a consequence of this strong interaction, a large shift of the 1290 cm-1 characteristic band of carminic to 1322 cm-1 is observed. The latter shift is very important in the identification of the presence of mordants in historical textiles, as similar effects can be observed in the analysis of many historical cochineal-dyed fibers. This is the case of the SERS of the historical textile fiber shown in Fig. 5f and others previously reported7. The 1290 cm-1 band is attributed to the C-OH deformations coupled to ring stretching vibrations in the anthraquinone ring I 25 (Fig. 1). Therefore, the shift to higher wavenumbers is attributed to a strong interaction of carminic acid with Al.
However, the analysis of archaeological textiles still reveals a large variability regarding the bands intensity of G and A nucleobases, as well as other biomolecule characteristic bands depending on the color of the studied area. This is the case of the belt-bag analyzed in the present work, where different spectra were measured in the red, brown, yellow and orange areas (Fig. 2a) showing bands attributed to A, G, and carminic acid. In addition to all these elements of variability, it is necessary to mention that cochineal can have different colors and structures due to the possible ionization of the carboxylic acid and phenolic OH groups as reveal the UV-vis spectra registered for carminic at different pH (Fig. 3b). We suggest that different colors could be produced in the mordant process by changing the relative amount of metal, and other additives such are organic acids or ammonia, in relation to the raw cochineal material. As a result, different recipes could lead to different purple-red-orange hues observed in the same archaeological material54. Therefore, more investigation on the effect of the metal and the presence of biomolecules should be done in order to better interpret the resulting SERS spectra and correlate them to the analyzed archaeological samples.