Mechanistic insight into the fluorescence activity of forensic fingerprinting reagents

The use of fingerprint development for forensic detection is a powerful investigative tool, providing robust evidence leading to the identification of individuals of interest in crime scene investigations.¹ The application of forensic fingerprinting reagents (FFRs) to the development of latent fingermarks on porous surfaces is a commonly encountered process in forensic chemistry and is of continuous interest to advance alongside the continued evolution of chemical analysis techniques. With the use of computational chemistry methods emerging as an effective tool in informing development processes in chemical sciences,^2–4 it is of interest to apply such methods to forensic chemistry. The subsequent work, reported here, is aimed at informing the design process of fluorescent-inducing amino-acid FFRs and investigates the structural and electronic properties of the chemical products formed when developing fingerprints using 1,8-diazafluoren-9-one (DFO), a fluorescent FFR, and 2,2-dihydroxyindane-1,3-dione (ninhydrin), a non-fluorescent FFR (Fig. 1).

Ninhydrin and DFO are commonly utilized as FFRs for the development of latent fingerprints on porous surfaces.⁵ Both reagents react with trace amounts of amino acids within the fingermark residue to form chromophoric chemical products.^6,7 Only that of DFO exhibits fluorescence emission upon light excitation during fingerprint visualization, while the ninhydrin product requires the addition of ZnCl₂ to produce a fluorescent product.⁸ Fluorescence is a desirable property for developed fingerprints to maximize the observed contrast between the fingerprint pattern and background surface, increasing the detail observed and detected.¹ Obtaining this in a single reaction step provides forensic investigators with a more efficient fingerprinting method and reduces the required chemical resources over those requiring additional reagents or treatments.

Ninhydrin, when reacting with amino acids, forms “Ruhemann’s Purple” (1), as shown in Fig. 2, although it is the protonated form, diketohydrindylidenediketohydrindamine (DYDA), that is responsible for the observed characteristics of the developed fingerprint.^9,10 This structure was elucidated through x-ray crystallography and ¹H nuclear magnetic resonance (NMR), although there are inconsistencies in the reported site of protonation resulting in possible isomers, 2 and 3, respectively. Furthermore, a computational study employing a semi-empirical method suggested that 4 showed closest agreement between the calculated absorption properties and that observed experimentally (a single absorption peak with a λ_max of 485 nm).^9,11

DFO reacts with amino acids during fingerprint development to form a fluorescent product reported to exhibit excitation with λ_max at 470 nm and emission λ_max at ∼570 nm.⁷ The crystal structure of the DFO product was determined to be 5, although it was proposed that the proton is mobile with respect to the remaining nitrogens within the structure, resulting in 6.¹² 

Currently, the most efficient fluorescence-inducing FFR recommended for fingerprint development is 1,2-indanedione, reported to produce brighter fluorescent fingerprints than DFO.^13–15 However, structure elucidation of the fluorescent product reported in the literature has yielded conflicting results leading to no conclusive solid-state structure.^16,17 This work has therefore focused on the DFO product to investigate fluorescent properties.

Previous attempts at the development of new, more effective FFRs have predominantly been based on experimental synthesis with little success reported.^18–21 Computational studies investigating the energetics of the amino acid reaction and wavelength and oscillator strength of absorption for DYDA analogs also resulted in no new developments, highlighting the need for an alternative computational approach.^22–24 

The mechanism of fluorescence is extremely challenging to incorporate into computational design processes due to the complex relationship between the structure and observed properties in solid state materials. However, investigation into the potential energy surface (PES) of non-luminescent compounds can reveal non-radiative processes such as those illustrated by Fig. 3.^25,26 It is the topologies of the S₀ and lowest excited state PES that fundamentally determines the observed fluorescence activity, or inactivity, of a chemical system. A conical intersection (CI), represented by (i) in Fig. 3, is a dominant non-radiative feature in non-fluorescent materials, playing a major role in the de-excitation process.²⁷ This has been demonstrated for solid-state materials using ab initio calculations.^28,29

In this work, we examine the calculated absorption and emission properties of the proposed isomeric structures 2–4 (DYDA) and 5 and 6 (DFO product) to indicate the most likely structures responsible for the reported experimentally observed optical properties. The S₀ and S₁ state PESs representing torsion angle rotations in those identified most likely are also explored to rationalize the observed non-fluorescent and fluorescent properties of the DYDA and the DFO product structures.

S₀ and S₁ minima of all molecules studied were optimized with density functional theory (DFT) and time-dependent density functional theory (TDDFT), respectively, using the CAM-B3LYP functional^30–32 and 6-31G(d) basis set.^33–35 Here, the range-separated functional CAM-B3LYP was employed in order to avoid spurious charge transfer excited states³⁶ between the two units of the molecules studied, which were observed in preliminary calculations. No dispersion correction was used as dispersion is expected to have only a minor influence on these planar structures. Calculations were performed in vacuum as there is no straightforward way of including the solid-state environment of interest. The steepest decent search algorithm was initially employed for S₁ state optimizations, followed by the GEDIIS algorithm for the relaxed scan of torsion angles.³⁷ All calculations were carried out in the gas phase using the Gaussian09 program.³⁸ Natural transition orbitals (NTOs)³⁹ were visualized using the GaussView 5.0 software.⁴⁰ The crystal structure of DYDA was obtained from the Cambridge Structural Database and analyzed using the Mercury visualization program.^41,42

The absorption wavelength (λ_abs) of DYDA, the non-fluorescent product of the reaction of ninhydrin with amino acids, has been experimentally determined at 485 nm.⁹ There have been conflicting reports of structure elucidation resulting in three possible structural isomers (2–4), differing by the site of protonation.^9–11 Considering the conformational change, 4 may exhibit two conformational isomers, 4a or 4b (Fig. 4).

The measured absorption wavelength (λ_abs) and emission wavelength (λ_em) of the materials investigated are reported in Table I alongside the calculated λ_abs of the three lowest energy singlet transitions, calculated λ_em, and corresponding oscillator strengths (f). To calculate the emission properties for each structure, an S₁ energy minimization was initiated from the S₀ minimized geometry. The calculated λ_abs of 1 suggests an absorption of 515.36 nm with high f (0.2314), while two dark states are present at 410.9 and 403.1 nm. The computed maximum is somewhat blueshifted compared to the experimental peak at 579 nm. This shift of about 0.25 eV may be attributed in part to the shift between vertical excitation and band maximum for simulated spectra⁴³ and generally lies within the expected accuracy of TDDFT. The properties of the S₀–S₁ transition in the DYDA isomers suggest that 4a, the O-protonated isomer, shows the closest agreement with the experimental absorption wavelength of 485 nm, with a calculated λ_abs of 489.9 nm and f of 0.1092. The first excited states for isomers 3 and 4b are in a similar energy range, but the calculated oscillator strengths show that these are dark states, which are probably not observed in the spectrum. 2 and 4b feature bright states at 399 and 417 nm, significantly blue-shifted when compared to the experimental absorption maximum. Despite 4a showing the closest agreement with experimental absorption, the experimental λ_abs was measured in CHCl₃, and so, the conclusion of which isomer is most likely to occur based on the comparison of λ_abs is made with caution due to the in vacuo environment of the simulations. The calculated λ_em of DYDA isomers 3–4b show zero f for the lowest energy transition of 2 and 3 and weak f (0.0799 and 0.0916) for 4a and 4b, respectively. The assigned character of the natural transition orbitals (NTOs) of S₁ minimized geometries also suggests n → π^* character. The non-radiative hole–electron NTO pair calculated for isomer 2 (Fig. 6) supports the non-fluorescent behavior of the solid-state structure of DYDA. Both of the observed characteristics of emission for the DYDA isomers are suggestive of non-radiative behavior for all possible isomers.

To gain insight into the relative stability of each isomer, relative energies of the S₀ and S₁ optimized structures under investigation have been calculated and are shown in Table II. Interestingly, 4a is shown to be the least stable (+34.09 kJ mol⁻¹) despite showing the closest match with experiment, while 2, the isomer representing the x-ray crystal structure, is the most stable.

The calculated ΔE for the S₁ minimized geometries of 2–4b (Table II) suggest that 2 and 4b are most stable with similar energies (1.79 kJ mol⁻¹ difference), while the instability of 4a with respect to the remaining isomers (+70.49 kJ mol⁻¹) is further increased in comparison to that in the S₀ state. The difference in isomer stability between S₀ and S₁ states suggests that isomerization may be favored in the excited state between 2 (N-protonated) and 4b (O-protonated). Visualization of the x-ray crystal structure (Fig. 5), indicative of the likely stabilizing intermolecular interactions present within the solid-state fingerprint material, displays intermolecular hydrogen bonding between an N–H proton and carbonyl-O simultaneously across two DYDA molecules, resulting in dimers throughout the structure. This interaction indicates a possibility of concurrent intermolecular proton transfer to occur, representing isomerization between structures 2 and 4b.

The calculated properties of both DFO product isomers 5 and 6, shown in Table I, show non-zero f for the lowest energy absorption transitions (0.3567 and 0.8994). Comparison of the corresponding λ_abs (452.7 nm for 5 and 471.9 nm for 6) indicates that 6 matches more closely with experiment. The calculated λ_em of 6 (562.8 nm) also shows closest agreement with the experimental λ_em of 570 nm in comparison to 5 (555.9 nm). The corresponding f also indicates that this transition has near-zero possibility in 5, whereas 6 has a comparatively high f. π → π^* character is assigned to both S₁ minimized structures indicated by hole–electron NTOs. In Fig. 6, the hole–electron NTOs for structures 2 and 6 are shown to support character assignments. More details can be found in the supplementary material. Calculated ΔE of the S₀-minimized structures of 5 and 6 suggest that their stabilities are similar (0.75 kJ mol⁻¹ difference), whereas 6 is 6.81 kJ mol⁻¹ more stable for the S₁ minimized structures. Both the calculated transition properties and relative stabilities suggest that 6 is more likely to be representative of the structure displaying the observed absorption and emission during fingerprint visualization. It should be noted that the crystal structure reported was a hydrated structure and is most likely not representative of that present in the developed fingerprint as the DFO fingerprint development process involves thorough drying of the fingerprint sample in an oven prior to visualization.

To gain further insight into the underlying mechanisms of fluorescence activity in both DYDA and DFO, it is of interest to explore the PES topologies to identify the presence of non-radiative processes in that of DYDA to rationalize the non-fluorescent properties observed beyond that reported already here.

Non-radiative processes can be identified from the investigation of the ground and excited state PESs representing mechanisms or structural change exhibited by the material.^25,26,28,29 In this present work, the investigation of the S₀ and S₁ PESs representing a central C–C–N–C torsion angle rotation of 4a and 5 is reported for the excited state. Isomer 4a was chosen for this investigation due to a closer resemblance than 4b of the geometry exhibited in the x-ray crystal structure and will therefore give more relevant insight into the hypothesized isomerization between 2 and 4b suggested by the calculated S₀ and S₁ energetics.

A relaxed scan of the C=N–C–C torsion angle of 4a and 5 (Fig. 7) was initiated from the optimized S₁ geometries and carried out in 10° increments for 4a and 20° increments for 5. Smaller increments were explored for 4a as an initial 20° scan did not provide a clear topology, prompting a more detailed investigation. The adjacent C–C=N–C torsion angle was fixed at the angle exhibited in the optimized S₁ geometry (17.5° for 4a, −0.2° for 5).

The plotted energies of the S₁ minimized geometry and the relative S₀ energy indicated by the S₀–S₁ transition with respect to the varied torsion angle at each step in the scan are displayed in Fig. 7. The resultant PESs, plotted relative to the most stable S₀ geometry in each case, reveal contrasting topologies for 4a and 5. Of particular significance is the CI suggested by the PES of 4a occurring at a torsion angle of −62.3°, a 10° rotation from the unrestricted S₁ minimized geometry, which is lower in energy. The existence of this CI is further evidenced by the suggestive relative S₀ and S₁ PES topologies between −72.3°and −112.3°. The S₁ PES also indicates a more stable geometry at −82.3°, suggesting that the S₁ minimized geometry at −52.3° is a local minimum, although closer in conformation to the S₀ minimized geometry.

The relative S₀ and S₁ PES topologies of the relaxed torsion angle scan of 5 resemble an avoided crossing (described previously in Fig. 3), suggesting a lower energy S₁ minimized structure at 99.8°. The relative S₀ energy is higher for this geometry compared to that at −0.2° (the unrestricted S₁ minimized geometry). In contrast to the relaxed torsion scan of 4a, there is no evidence of a CI in that of 5. The discussed avoided crossing suggests the existence of a nearby non-radiative mechanism when the torsion angle is rotated to 99.8°, although considering the solid-state nature of the fingerprint material, this conformational change is assumed to be hindered by intermolecular bonding networks.

Visualization of the reported crystal structure of DYDA (Fig. 5) displays hydrogen bonded dimers, exhibiting two simultaneous N–H–O interactions.¹⁰ Although the structure indicates the existence of form 2, we propose that intermolecular proton transfer occurs in the solid state material to form 4a, explaining the agreement of the calculated λ_abs with experiment for the latter. Since the crystal packing does not exhibit long hydrogen-bonded networks, but dimer units, it is hypothesized that this less rigid structure allows for the rotation of the torsion angle that has been investigated here, likely to occur through vibrational modes. The reported crystal structure of the DFO product resembles isomer 5, although in hydrated form (Ref. 12). Since the suggested fingerprint development process using DFO requires heating of the sample to dry thoroughly prior to visualization,¹ it is assumed that the crystal packing is unlikely to represent that present in the visualized fingerprint material. Further work is proposed to investigate computationally the geometries and hydrogen bonding networks of 4a and 5 in the solid form to provide further insight into the proposed mechanistic processes.

In this work. we have applied DFT and TDDFT in the investigation of the isomeric structures of the forensic fingerprinting reagent products of ninhydrin and DFO and provide mechanistic insight into their fluorescence activity based on the conformational change.

The most likely isomeric forms of the non-fluorescent ninhydrin product, DYDA, and the fluorescent DFO product that exhibit the previously reported observed optical properties following fingerprint development have been discussed. The calculation of absorption properties using DFT and TDDFT indicate that 4a (λ_abs of 489.9 nm and f of 0.1092) shows highest agreement with the experimentally reported λ_abs (485 nm). Despite the reported crystal structure of DYDA indicating protonation at the central N atom (form 2), our calculated λ_abs (471.9 nm) and weak oscillator strength (0.0001) suggest protonation at the carbonyl (C=O) to be more likely. Calculated ΔE suggests that 2 (N-protonated) and 4b (O-protonated) are most stable in S₀ and S₁ states, respectively, suggesting that a proton-transfer isomerization process may be favorable. The interactions present in the x-ray crystal structure suggest that this could occur intermolecularly via isomer 4a. In the DFO case, both the calculated λ_abs and λ_em of 6 (471.9 and 562.8 nm, respectively) show close agreement with the reported experimental values of the DFO product (470 and 570 nm). Calculated energetics suggest similar S₀ stabilities for 5 and 6, with the latter being 6.81 kJ mol⁻¹ more stable in the excited state.

A relaxed S₁ scan representing a torsion angle rotation in 4a and 5 revealed contrasting topologies of the plotted PESs. Initiated from the S₁ minimized geometries, a suggestive conical intersection is observed upon 10° rotation from the S₁ minimized geometry in 4a. A 10° torsion rotation is considered feasible in the solid form of 4a, hypothesized to be exhibited by vibrational modes. It is proposed that isomerization occurs between forms 2 and 4a via a simultaneous intermolecular proton transfer across dimers in the solid-state, explaining the observation of form 2 in the reported crystal structure. On the contrary, for the DFO product, an avoided crossing upon a 100° torsion angle rotation is observed in 5 with no indication of a potential conical intersection. This mechanistic insight from simulation reflects the strong fluorescence exhibited by the DFO product experimentally. Further solid-state calculations are suggested to confirm the nature of the crystal packing of 4a and 5 and to gain insight into the relative energetics of the possible isomeric forms to further evidence the mechanistic process proposed here. We should note that in a developed fingerprint sample, there would be other materials present (from the fingerprint residue/sweat, impurities, and the surface it is developed on). Including the interactions with these components would be useful, but it is impossible to know what exactly they would be considering as the impurities such as dirt and cosmetic product that could be present. Furthermore, fingerprint residue varies from person-to-person and day-to-day and contains both hydrophilic and hydrophobic components. Interactions with water could be modeled but could be misleading and therefore has not been included in this study.

The results presented here have demonstrated the successful application of TDDFT to calculate the absorption and emission properties of the products formed from the well-known FFRs and suggest strong correlation between the modeled S₁ electronic properties and the observed fluorescence activity. The ability to describe the accessibility of non-radiative processes in the materials reported here offers the first step toward the design of new fluorescence-inducing reagents for fingerprint development from first principles.

See the supplementary material for the computed natural transition orbital diagrams, a schematic of how the torsion angle is investigated, and the energy, wavelength, and oscillator strengths of the S₀ and S₁–S₃ transitions for the S₀ minimized structures and the S₁ minimized structures.

L.M.H. and P.G. would like to dedicate this work to several generations of strong women, who through their resilience have paved the way for L.M.H. and P.G. to successfully pursue their journeys to become scientists of the modern world. There is much more inclusivity and respect than ever before. There is still much to do for the generations to come.

We acknowledge the EPSRC Doctoral Training Partnership in conjunction with Loughborough University for funding the Ph.D of L.M.H. The authors also acknowledge the use of the “Hydra” High Performance System at Loughborough University. Finally, L.M.H. and P.G. thank the Defence Science and Technology Laboratory Staff (DSTL), Matthew Brooks, and Helen Bandey for useful discussions.

The data that support the findings of this study are available within the article and its supplementary material and are also available from the corresponding authors upon reasonable request.