In our previous work, we reported on the tendency of DFO molecules to aggregate [
12] and investigated the potential for revealing dactyloscopic traces via such aggregates [
11]. Importantly, we demonstrated that the enhancement of fingerprint traces can be achieved through the use of DFO aggregates along with DFO-α-amino acid complexes, which represents a novel approach in the field of forensic science. Our current study builds upon this earlier work and provides further evidence that dactyloscopic traces can be visualized using an effective, non-toxic DFO solution. We also identified the specific form of the aggregate responsible for the observed increase in the luminescence efficiency. The research conducted in this paper demonstrates that the most suitable medium for the formation of the new DAK DFO molecule is a rigid matrix (i.e., paper or a dried PVA polymer film). Accordingly, we present two experimental models: laboratory model rigid media (polymer PVA) and application model rigid media (thermal paper/PVP/ethanol)-
Figure 1.
The laboratory model can provide a detailed description of the fluorescence of DAK DFO, including the UV-Vis absorption and fluorescence spectra and intensity decays (fluorescence lifetimes). Using this model, we can better understand the mechanisms that underlie this phenomenon. Furthermore, the specific processes can be verified and elucidated through the use of computational methods. On the other hand, the application model mainly focuses on visual observations of DAK DFO fluorescence.
2.1. The Laboratory Model of the Rigid Matrix in the Form of PVA Polymer
Based on our previous findings, which suggest that the aggregation processes of DFO are highly susceptible to external environmental factors [
12], we needed a medium that would allow us to precisely control the experimental conditions—especially the rigidity of the medium and the DFO concentration. We selected a matrix of polymerized polyvinyl alcohol (PVA) as an ideal medium because it contains -OH groups in the monomer that help disperse DFO inside the polymer network. Moreover, changing the concentration of PVA affects the rigidity of the medium.
The absorption and fluorescence spectra were measured to provide evidence of the formation of a new DAK DFO structure in the PVA matrix. The absorption spectra obtained for different concentrations of DFO in PVA are presented in
Figure 2a. The most significant changes were observed at concentrations of 1 × 10
−3 [M] and 5 × 1 × 10
−3 [M], where an additional absorption band with well-defined maxima around 520 nm and 570 nm was detected, suggesting the presence of structural forms other than DFO, which exhibited maximum absorption at around 380 nm. In
Figure 2b–e, the dependence of the emission of the DFO/PVA films obtained on the excitation wavelength is shown. Surprisingly, all samples with a concentration of 5 × 10
−3 [M] exhibited fluorescence at a maximum of around 580 nm, despite using only the DFO molecule to obtain them, which should theoretically emit at around 450 nm. These results clearly suggest the formation of new structural forms that are stable despite the changes in the excitation wavelength. The new forms exhibit different emission properties from those of the original molecule.
Figure 2c shows a noteworthy observation that at higher concentrations, a new fluorescence band emerged for excitation wavelengths of 440 nm and above. This observation indicates the formation of a structured form that remains stable at an excitation wavelength of 440 nm or higher. Moreover, the time-resolved fluorescence spectra for the initial DAK DFO concentration of 5 × 10
−3 [M] (
Figure 3b) fully illustrate the stability of the new chemical formulation obtained. The emission maximum did not shift with time, and no new short-lived forms appeared. This suggests the existence of a single (one type) emitting center, rather than an aggregate of several centers.
Spectroscopic analysis of the rigid matrix model described above indicates that we can create a new chemical structure, an aggregate, through “pure chemistry” by adjusting the concentration of DFO and the rigidity of the matrix. Since the newly formed molecule was very stable over time, both in PVA and in the structure of thermal paper, we postulate that two DFO molecules were joined in the cycloaddition reaction pathway, which is well known for this type of chemical structure [
13,
14,
18] (more details are provided in
Section 2.3). However, our approach to constructing the synthesis pathway and fulfilling the technical requirements of the reaction is entirely novel.
Figure 2 confirms that DAK DFO is an excited cyclodimer, which is formed only when the dimer components are in rigid media at a high concentration of 5 × 10
−3 [M]. Therefore, its existence can only be proven based on fluorescence measurements. The fluorescence wavelength of the excited cyclodimer is longer (lower energy) than the emission of the excited monomer. Furthermore, the formation of the excited cyclodimer DAK DFO is dependent on molecular interactions. In our experiment, we found that the optimal concentration for DAK DFO formation occurred at 5 × 10
−3 [M] due to the high density of the monomer. At an excitation wavelength of 455 nm, DAK DFO appeared to emit an orange light, but at an excitation wavelength of 560 nm, higher aggregates of DFO appeared to emit an orange light (
Figure 2f).
To further confirm the presence of the cyclodimer DAK DFO in a rigid matrix, we investigated the fluorescence properties of DFO under different excitation conditions and concentrations. Our results showed that the optimal excitation wavelength for the highest fluorescence intensity was 440–530 nm, with the maximum fluorescence band occurring at 580 nm (
Figure 2e). In contrast, a significant fluorescence intensity was observed at an excitation wavelength of 560 nm, with the maximum fluorescence band occurring at 620 nm for the same dye concentration 5 × 10
−3 [M], which was attributed to the formation of higher-order aggregates. At low concentrations, we observed the fluorescence lifetime characteristic of the DFO monomer, whereas the fluorescence lifetimes corresponding to DAK DFO were observed at the highest concentration. The fluorescence intensity decay was observed for each excitation wavelength in at least two forms. For instance, upon 380 nm excitation, we observed fluorescence intensity decay for both the DFO monomer and the forms formed by interaction with the OH groups present in the PVA polymer. The solvatochromic susceptibility of DFO was confirmed earlier. In contrast, upon excitation at 455 nm and 560 nm, we observed fluorescence intensity decays from different forms compared to 380 nm excitation; along with the monomer form of DFO, we observed the formation of DAK DFO. We chose 455 nm as the excitation limit based on the results of the DAK DFO fluorescence observation in our application model—thermal paper.
Raman spectroscopy investigations have affirmed the structure of the dimer proposed through spectrophotometric and spectrofluorimetric measurements in both steady-state and time-resolved modes, as well as quantum mechanical calculations. The study presents results in the form of Raman scattering ranging from 2000 cm
−1 to 3200 cm
−1 (see
Figure 3a), revealing a distinctive “fingerprint” for the DAK DFO dimer molecule. Particularly for DAK DFO, we observed a robust capability for fluorescence within the VIS range, thereby measurements from 0 to 2000 cm
−1 were significantly affected by this phenomenon, and interpretation within this range was notably disrupted by the fluorescence background [
19].
Conversely, in the range of 2000 cm
−1 to 3200 cm
−1, the majority of the observed Raman scattering lines pertain to the chromophore of the dimer [
20]. In this region, the spectra of the monomer and DAK DFO in their ground states diverge significantly. For the DAK DFO dimer, characteristic doublet lines emerged, indicating the presence of various C-H bond conformations within the cyclobutane plane that form the connection between the monomer structures, ultimately yielding the DAK DFO dimer [
19,
20]. Raman spectra, particularly focusing on the C-H, N-H, and O-H stretching regions (2700–3600 cm
−1), provide conclusive evidence of the cyclodimer structure. This analysis confirms the presence of C-H groups in the cyclobutane group, a structure that forms during the cycloaddition of DFO molecules [
21,
22,
23,
24]. Nevertheless, since our experimental PVA matrices incorporate water into their structure, FTIR experimental measurements are heavily distorted and the interpretation of the study results is subject to substantial error. Thus, the quantum mechanical simulations of IR spectra, which are not subject to this error, are presented in this work (see
Supplementary Materials, Table S2).
The overarching molecular theory concerning the complementarity of IR absorption spectra and Raman scattering also validated the presence of C-H vibrations of varying conformations in the plane in the previously postulated “fingerprint” region of the DAK DFO dimer. These C-H vibrations in the cyclobutane belonging to the DAK DFO structure and their absence in the monomer structure are outlined (see
Table S2 and the IR spectrum in
Figure 4b).
Moreover, the stable structure of the dimer was also corroborated by time-resolved fluorescence spectra, where, despite changes in the excitation wavelength ranging from 450 nm to 520 nm, a consistent fluorescence spectrum was obtained. Furthermore, the time-resolved spectral evolution demonstrated a uniform fluorescence structure, as both the optical contour of the band and the maximum fluorescence band remained unaltered over time, as illustrated in
Figure 3b.
2.2. The Theory of Equilibrium States in Time-Resolved Spectra
As the excitation wavelength increased, we observed alterations in average lifetimes along with concentration changes. These alterations, however, exhibit a linear dependency. Moreover, the determined average fluorescence lifetimes (
Table S1), obtained through an exp = 2 fitting, affirm the presence of two forms, namely the DFO monomer and the DAK DFO dimer. We illustrated these dependencies in
Figure 3c, where the formation of a point was observed for the linear fitting of the dependence:
It was found that if the set of f(t) = c relationships intersect at a single point, representing a true equilibrium, the point can be identified by analyzing the fluorescence intensity decays for different excitation wavelengths of 440 nm, 485 nm, and 560 nm observed at the same emission band maximum of 580 nm (postulated for the DAK DFO dimer). There is reasonable evidence that emitting species are present in the studied system, including DFO monomer (after excitation wavelength 380 nm), DAK DFO dimer (after excitation wavelength 440–485 nm and still present after excitation wavelength 530 nm), and higher aggregates (after excitation wavelength 560 nm), and that the determined point represents the optimal concentration of the first DAK DFO dimer in the rigid material under study.
In the case of molecules forming within rigid matrices and possessing short lifetimes, only the time-resolved spectra and decay of fluorescence band intensity can confirm their presence, creating dependencies as depicted in
Figure 3c. Stationary spectra, burdened with prolonged measurement times, preclude the separation and presentation of equilibrium states present in rigid materials. While stationary spectra provide our initial premise for the formation of aggregate forms, our conclusive argument is gleaned from the time-resolved spectra.
2.3. Explanation of the Underlying Chemistry Using Computational Chemistry Methods
Based on previous studies on the DFO molecule, we have gained extensive knowledge of its chemical behavior in various environments, including its photophysical [
9,
10,
11,
12] and application properties [
11]. Our investigations revealed clear distinctions between the DFO monomer molecule, its complex with α-amino acids, and the aggregate formed as the postulated cyclodimer. In a rigid matrix, the emission spectrum of DAK DFO at 455 nm excitation (with the highest fluorescence intensity) has a fluorescence maximum of 580 nm. The position of the band maximum for DAK DFO’s fluorescence is similar to that of the DFO complex with glycine. However, the optimal excitation wavelength for the DFO-glycine complex is 530 nm, indicating that the emission does not originate from the complex but from the newly formed molecule. Based on the observed stability of the new molecule over time in both PVA and the structure of thermal paper, we propose that two DFO molecules underwent a cycloaddition reaction pathway to form the new DAK DFO molecule.
To identify the reaction mechanism and possible transition states, we performed quantum chemical calculations using the Nudged Elastic Band [
25] (
Figure 4a). In typical reaction pathways, a cycloaddition reaction to the C=C bond is expected. However, in the case of a DFO molecule that contains an N=C double-bond mode with N embedded in the ring, a preference for cycloaddition to C involved in the N=C bond was observed, leading to the formation of a cyclodimer. Standard organic syntheses conducted under controlled laboratory conditions have already demonstrated the possibility of various cycloaddition reactions occurring through the excited states of molecules [
15,
16]. In our research, we observed the formation of the proposed DAK DFO compound in favor of the ortho adduct over the meta adduct, following the region-selectivity rule. This is due to an increase in the amount of charge transfer occurring in the excited DAK DFO cyclo-dimer. The transition form is formed by regrouping electrons around the nitrogen atom, resembling the ionic-type reaction of molecules in the ground state. The final product is formed as a consequence of the circular displacement of pi electrons. Two unsaturated DFO molecules underwent dimerization at positions 7 and 8 in 1,8-diazafluoren-9-one (see
Supplementary Materials—Video S1 and Figure S1). The DFO molecule changes the electronic distribution around the positional skeleton of the molecule, resulting in the observation of DAK DFO dimers at high concentrations.
In
Figure 4c, we presented numerically calculated Jablonski diagrams for two chemical compounds, DFO and DAK DFO, in a polar environment of ethanol. Based on these diagrams, we can see that the energy donors are in the triplet state, which can sensitize the cycloaddition reaction. Therefore, we hypothesize that the reaction most likely occurs in the triplet state of the molecule. It is worth noting that the substances that quench the triplet states do not reduce the quantum yield of fluorescence, suggesting that regrouping to the cyclodimer must be a very fast process. Our analysis revealed that DAK DFO exhibits greater photochemical competitiveness than DFO. The difference between the ground state and the first excited state is smaller for DAK DFO, meaning that in an environment favorable for the formation of DAK DFO, these molecules will absorb light radiation more easily compared to DFO. In other words, less energy is required to excite DAK DFO, as the light of a shorter wavelength is sufficient to induce excitation. Notably, experimental investigations conducted on authentic thermal papers revealed that the most optimal visualization of dactyloscopic traces was achieved utilizing an excitation wavelength of 455 nm (see
Section 2). This particular wavelength corresponds to the theoretically determined energy difference between the ground and excited states in DAK DFO, which has been estimated to be 2.73 eV.
To confirm the structure of DAK DFO, quantum chemical calculations with B3LYP hybrid density functional [
26] were carried out for optimization and to model its vibrational spectra (see
Figure 4b). The figure shows that the analyzed molecules exhibit similar optical properties in the oscillatory–rotational absorption range. Nevertheless, some important differences can be noticed. The N-H bond is less polar than the OH bond, which leads to a weaker absorption band for N-H, with a narrower width and a position at 3047.7 cm
−1, 3093.4 cm
−1, and 3000 cm
−1 for DAK DFO. Furthermore, the C=O stretching of the ketone group is observed at approximately 1863.8 cm
−1 for DFO and 1859.7 cm
−1 for DAK DFO. Other double bonds, such as C=C and C=N, exhibit absorbance in the lower frequency range of approximately 1550–1650 cm
−1. The C=C stretching of the benzene ring is indicated by two sharp absorption bands, one at ~1441.9 cm
−1 and one at 1327.71 cm
−1 for DFO, and 1438.8 cm
−1, 1328.9 cm
−1, and 1280 cm
−1 for DAK DFO.
It is important to note that the IR spectrum region with lower frequencies between 400 and 1400 cm−1 is known as the fingerprint region. Similar to a human fingerprint, the pattern of absorbance bands in this region is a unique characteristic of the compound as a whole. If two different molecules have the same functional groups, their IR spectra will still differ and this difference will be reflected in the bands in the fingerprint region. This allows for IR spectra of unknown samples to be compared to a database of IR spectra of known standards, providing a means of confirming the identification of the unknown sample.
Our experimental and computational analyses have converged to the conclusion that we have discovered a novel DFO dimer resulting from cycloaddition to the C=N bond in the aromatic ring. Notably, the cyclodimer or its potential formation has never been reported before in the context of revealing dactyloscopy traces on paper using a solution with DFO. Our work presents the first evidence of its existence. Previous challenges in interpretation arose from the striking structural similarity between the DFO complex with glycine and the DAK DFO cyclodimer, including the presence of four nitrogens in the aromatic ring and the same number of aromatic rings with a comparable electron configuration, specifically the chromophore groups. As a result, we observed a similar excitation energy value for the DFO complex with glycine and the DAK DFO dimer. This argument provides strong evidence for comparable spectroscopic properties, particularly the fluorescence characteristics of these two species. Our findings offer valuable insights into the chemical behavior of DFO and expand our understanding of its potential applications.
2.4. Practical Application in Friction Ridge Analysis
The findings from our laboratory model and theoretical calculations have helped to elucidate the phenomena that occur when using DFO in practical applications. We have observed that in a rigid matrix, a stable cyclodimeric form is generated at a concentration of 0.001 [M] DFO/PVA, with an absorption and emission spectrum that closely resembles the DFO complex with α-amino acids. This novel form of DFO has also been detected when revealing fingerprints on paper and thermal paper. These results can be attributed to the naturally rigid and porous nature of paper, where a proper concentration of DFO and the use of PVP as a stabilizing polymer provide suitable conditions for the formation of DAK DFO.
Practical observations made during the visualization of fingerprints on thermal paper, as illustrated in the schematic and the fingerprint in
Figure 1, align with the results shown in
Figure 2b for the laboratory model. The optimal visualization effect, marked by the highest fluorescence intensity from the fingerprint surface, was obtained using a forensic illuminator with an excitation wavelength of 455 nm. Although there were differences in the model composition, the overall conditions for the formation of the new aggregate form were similar.
DAK DFO is a highly effective method for revealing fingerprints on thermal paper, owing to its spectroscopic properties as well as its chemical structure. Thermal paper is known for its complex structure (refer to
Figure 1). Modern thermal papers are typically based on leuco-dye chemistry, as described in detail by Kim et al. (2018) [
27]. When a developer (usually Bisphenol A or Bisphenol S) reacts with the fluoran ring, it forms a visible black dye. DAK DFO features an amine group that has both alkaline properties and polarity. This compound is designed to prevent ring banding in thermal dyes due to its alkaline properties while also facilitating a smooth paper coating structure because of its greater polarity than DFO. The addition of an amino group to the ring structure significantly stabilizes the molecule during interactions with α-amino acids or leuco dyes, where it serves as their deactivator.