Fluorescent‐Labeled Octasilsesquioxane Nanohybrids as Potential Materials for Latent Fingerprinting Detection

Abstract The recent demand for fluorescent‐labeled materials (FLMs) in forensic security concepts such as latent fingerprints (LFs) that encode information for anti‐counterfeiting and encryption of confidential data makes necessary the development of building new and innovative materials. Here, novel FLMs based on polyhedral oligomeric silsesquioxanes (POSS) functionalized with fluorophores via “click” reactions have been successfully synthesized and fully characterized. A comprehensive study of their photophysical properties has displayed large Stokes's shift together with good photostability in all cases, fulfilling the fundamental requisites for any legible LF detection on various surfaces. The excellent performance of the hetero‐bifunctional FLM in the visualization of LF is emphasized by their legibility, selectivity, sensitivity and temporal photostability. In this study, development mechanisms have been proposed and the overall concept constitute a novel approach for vis‐à‐vis forensic investigations to trace an individual's identity.

Abstract: The recent demand for fluorescent-labeled materials (FLMs) in forensics ecurity concepts such as latent fingerprints (LFs) that encode information for anti-counterfeiting and encryption of confidential data makes necessary the development of buildingn ew andi nnovative materials. Here, novel FLMs based on polyhedralo ligomeric silsesquioxanes (POSS) functionalized with fluorophoresv ia "click" reactionsh ave been successfully synthesized and fully characterized. Ac omprehensive study of their photophysical properties has displayed large Stokes's shift together with good photostability in all cases,f ulfilling the fundamentalrequisitesfor any legible LF detection on various surfaces. The excellent performance of the hetero-bifunctional FLM in the visualization of LF is emphasizedb y their legibility,s electivity,s ensitivity and temporal photostability. In this study,d evelopment mechanisms have been proposed and the overall concept constitute an ovel approach for vis-à-visf orensic investigationst ot race an individual's identity.
Fluorescent-labeledmaterials (FLMs) are very important entities with widespread applicability, ranging from white light emitting diodes (WLED), [1] molecular imaging, [2] in vivo and in vitro cellular targeting and imaging, [3] to detection of latent fingerprints (LFs) in the forensic sector. [4][5][6][7][8][9] Within this context, FLMs possess better featurest han classical fluorescent organic dyes, [3,10] including highera bsorption coefficientsa nd sensitivity,a voiding possible photobleaching, larger emission lifetimes and very low toxicitya llowing them to be applied in vitro and in vivo analysis. [11] Public security remains ag lobal challenge and the utilization of LFs, bar-codesf or anti-counterfeiting, and confidential data encryption, among others, are being currently appliedb yf orensic institutions. [5][6][7][8][9] In particular,L Fs appears to be the best optionf or personnel identification in forensic investigations due to their high stability,u niquenessa nd complexityo fr idges patterns. [9,12,13] In an attempt to improve the traditional fingerprint detection methods, [14] fluorescent nanomaterials have been found to be suitable platforms for the development of LFs. [14] Amongc o-doping of 2D nanostructures, ar igid 3D hetero-structural scaffold could be ap romising candidate due to its efficiency,p hotostabilitya nd effectivity.
In this regard, polyhedralo ligomeric silsesquioxanes (POSS) have emergeda savaluableg roup of 3D nano-building-blocks for the fabrication of av ariety of hybrid functional materials. [15][16][17][18][19] Several prime qualifications make POSS the appropriate choice for its use as principal core:( i) it exhibits high stability and excellent biocompatibility in ab iological environment, [20] (ii)the inorganic cage providesn ot only protection to the covalently attached dye from stabilityb ut also ac onvenient framework for 3D multivalent display of pendant epitopes, [21] (iii)facile functionalization. [22] The highly symmetrical and topologically ideal cubic-octameric framework (T8), with general formula (RSiO1.5) 8 and ac age size of approximately 0.5-0.7 nm has been reported as suitable precursor. [15] Based on literature data, [23][24][25] this speciesc an be easily functionalized through a" click" reactionw ithout compromising its structural and functional integrity.H owever,a sf ar as we are aware, examples of hybrid POSS materials bearing fluorophores as powerful tools for effective LFs detectionh ave not been reported yet.
In this work, novelF LMs based on the 3D POSS containing fluorescent dyes have been synthesized in order to develop LF visualization materials. The synthetics trategy has followed the classical "click" reaction where the Cu I -catalyzed [3+ +2] cycloaddition between the POSS precursor with an azide group (PAZ, see Scheme 1) and the corresponding alkyne-like fluorophore has been carried out (see the SI for details). It is worth mentioning that the use of "click" chemistry has been provedt ob e aversatile tool for preparingalarge variety of POSS conjugates for different applications, [21,22] includingP OSS-dye derivatives. [26][27][28][29] Herein,f our differentm aterials relying on the fluorophore nature (pyrene = PAP ;d ansyl = PAD ;b romo-naphthalic anhydride = PANA;p iperazine-naphthalic = PANP)h ave been obtained (Scheme 1). Their photophysical properties as well as measurements on the photostability of these speciesh ave been investigated in detail and,f inally,t hese FMLs have been successfully tested for the detection of LFs. The polyaromatic dyes were selected based on its commercial availability and magnificient fluorescenceproperties. [30] Prior to the synthesis of the corresponding FLMs, the precursors, PAZ, [25] and the alkyne-like fluorophores AP, [31] AD [32] and ANA [33] were prepared following previouslyr eported procedures (see ESI for details) where spectrald ata were consistent accordingt ol iterature.
Next, coupling reaction between PAZ and the fluorophore derivatives, respectively,t hrough at ypical "click" reaction allowed the formation of PAP, PAD, PANA and PANP from goodto-excellent isolated yields (ca. 81 %, 74 %, 91 %a nd 68 %, respectively). Optimized conditions included the presence of CuBr/PMDETAa sc atalyst system using tetrahydrofuran (THF)/ dimethylformamide (DMF) (1/1 v/v) as solvent after 18 h (Table S1). The role of PMDETA was pivotala si ts cavenged in-trudingC u II ,w hereas the use of THF justified the good solubility of PAZ in this solvent. [25] Variation on the catalyst system, solventm ixture and reaction time did not improve the results (Table S1). Moreover,ad ifferent procedure based on direct aminolysis of PANA with piperazine was also attempted (Scheme 1), albeit the isolated yield was even lower (44 %) with this strategy.T he molecular structures of PAP, PAD, PANA and PANP were characterized by 1 Ha nd 29 Si NMR,F TIR and mass spectrometry.
The presenceo ft he triazole proton, the methylene group in alpha position related to the triazole moiety for PAD, PANA and PANP and the iso-butyl protons from the PAZ core evidenced the successful "click" reaction ( Figures S1-S4). From the 29 Si NMR spectra( Figure S5A), two peaks were detected in all cases which indicated the corresponding bi-functionalization of the POSS material with Si atoms containing two different organic groups (Si-iso-butyl and Si-(CH 2 ) 3 -triazole-fluorophore). Further,t hese chemical shifts were in agreement not only with the molecular symmetryc onsiderations [21,23] but also within the expected region for an alkyl-substituted cubic POSS (ca. d = À65 to À70 ppm), [21,24] supporting the T8 cage structural scaffold. All hybrid materials were analyzed by mass spectrometry.T he resultss howed molecular ion peaks at 1126, 1188, 1215, and 1218 (m/z)f or PAP, PAD, PANA and PANP,r espectively,w ith appropriate supportive fragmentation pattern ( Figure S5B). Finally,r egarding FTIR measurements, Figure S6 shows ar epresentativee xample of the assembling between PAZ and the alkyne-like pyrene derivative where the signal of the azide group at 2050 cm À1 clearly disappeared together with the appearance of the typical C=C( 1740 cm À1 )o ft he pyrene. Indeed, the SiÀOÀSi bond signals (1134-1022 cm À1 )o f the T8 silsesquioxane core were also detectedi nt he PAP spectrum confirming that the T8 cage wasnot affected by the reaction conditions or by the strongn ucleophilic N 3 . [25] Once the structural elucidation of PAP, PAD, PANA and PANP was fully detailed, we next investigated their photophysical properties in different solvents which included absorption/ emission bands, molar absorption coefficient, Stoke's shifts, fluorescenceq uantum yields, emission rate constants and singlet energies and lifetimes (Figures S7-S11a nd Ta bles S2-S5).
Accordingly,t he alkyne-like fluorophores were also studied in order to observe the 3D POSS core influence. To note that the presence of heavy atoms on the fluorophore moiety drops strongly the emission quantum yield as in the case of PANA (Table S5)a nd therefore this materialw as ruled out for further spectroscopic analysis.
Commencingw ith AP and PAP ( Figure S7 andT ables S2A B), the UV-vis absorption spectrum of AP showedt hree characteristic peaks in all solvents which was typicallya scribed to pyrene chromophore;h owever,t his well-structured absorption band was not defined in the case of PAP where it presented only one maximum absorption. [34,35] Ther eason of this broad band could be only explained by the presence of the 3D POSS materiala nchorage to the pyrene moiety affecting the S0!S1 and S0!S2 transitions. Additionally,t he most significant effect was detected on the Stoke's shifts values which werem arkedly higher in the case of PAP in comparison with AP.T hese data suggested the positive influenceo ft he triazolyl-POSS scaffold which promoted energy transfer between the donor and acceptor.C oncerning the singlet lifetimes (Table S2B), it was clear that two componentsa ppeared at moderate-to-non polar solvents, indicating the contribution of two species to the emission presumably both the monomer anda ne xcimer species ( Figure S12). [36] Regarding AD and PAD ( Figure S8 and Ta ble S3AB), remarkably differences were observed in the molara bsorptionc oefficient (e)w here it seemed clear that e was solvent-dependent only in the case of PAD.T he emission spectra in various solvents with different polarity revealed ar ed shift on the maximum. Thus, the emission maximum appeared at 450 nm in non-interacting solvents (hexane, cyclohexane), whereas in moderately polar solvents such as dichloromethane (DCM)o r THF the maximum was at 490-500 nm. However, the maximum red shift of the emission was provided in highly polar solvents [acetonitrile (ACN), DMF or dimethylsulfoxide (DMSO)] at ca. 520 nm where an intramolecular charge transfer (ICT) characterc ould be facilitated. [37] High Stoke's shift values were obtaineda st ypical of dansylated compounds [38] and, from the emission decay traces, ab i-exponential fitting in all cases were achieved ( Table S3B) two emission lifetimes were detected in all cases.T hey could be ascribed to the ICT state (shorter lifetimes) and possible aggregates (longerl ifetimes). As ar eference, PAD emission spectra show ashorter wavelength shoulder band at % 400 nm in highlyp olar solvents ( Figure S7). [39] Althoughc ontribution of the longer lifetimec omponent was found to be clearly in am inor extend, it was higheri nh ighly polar solvents than in non-polar solvents. [40] Finally, ANP and PANP were also submitted to photophysical studies ( Figure S9 and Ta bles S4A B). The mostr elevant difference was observed in the molar absorption coefficients, being 3-fold higher in PANP and, therefore, the presence of the 3D POSS material strongly affected to the solubility of the naphthalimide derivative. Again, both ANP and PANP possessed emission bands that were found to be highlyr ed shifted ( % 54 nm and % 54 nm, respectively) from the non-interacting solventh exane to polar DMF and ad ecrease of the fluorescence quantum yield was obtained for PANP especially in nonpolar solvents;t his fact could be attributed to the better solubility of PANP that would facilitate the formation of complexes in the ground state or internal transferp rocesses. In this context, choice of PANP insteado fPANA gave rise to a" OFF-ON" irreversible switch system possessing "receptor-fluorophore-receptor" architectural model ( Figure S13). [41] Furthermore, computational calculations based on TDDFT//B3LYP/6-31G(d) level of theory [42] supported the use of the piperazine moiety (Table 1). Hence, the heavy atom Br presenta tt he naphthalimide fluorophorei nPANA inducedanon-radiative transition with oscillator strength (f)o f0 .0028 justifying its dark state forbidden S 0 !S 1 transitionw hich was non-fluorescent. The high theoretical oscillator strength (f = 0.32) obtained for PANP was corroborated by as ignificantly high fluorescencer adiative factor (R fl = 5.6 10 8 )a nd quantumy ield (f fl = 0.34). Whereas, a non-radiative PANA displayed negligible zero R fl and quantum yield (f fl = 0.026)i nD CM. [43] After nucleophilic substitution of Br by piperazine ar adiativet ransition S 0 !S 1 ,i dentified as the HOMO!LOMO was allowed as ar esult of electron jump within the architectural model.T herefore, the presence of emissive S1 which depends on solvents dielectric underpins the strong fluorescence of PANP,w hich is classified as polar in its excited state. Thus, the theoretical indication furthers upports PET mechanism. Indeed, we termt his an irreversible "OFF-ON system as there is no future reversiblep hoto-damaged sensing program for PANP as reported for most metal- [44] and thiol-induced [45] sensors.I ndeed, terminal hydrogen of piperazinei satarget for our prospective fingerprintingd etection. [46,47] To check whether PAP, PAD and PANP were or were not stable after photolysis, absorption spectra of the corresponding materials were recorded before and after monochromatic light irradiation. As depicted in Figure 1, all these compounds exhibited am agnificent photostability even after 60 minutes of continuousirradiation. As amatter of fact, the 3D POSS scaffold linked to the corresponding fluorophores together with the triazole bridge [48] played ac rucial role for further real applications such as LFs. In this vein, similari nvestigations for the alkyne-like fluorophores were carried out. In contrast to their analogousm aterials, they presented poor stability after prolonged exposure of light as monitored by the absorption and emission spectra ( Figure S14). Some conclusions were drawn from the photophysical data: large Stoke'ss hift were obtained in general,h igh fluorescence quantum yields were observed in some cases and an ideal photostability made these FLMs (PAP, PAD and PANP)p otential candidates to be successfully applied as fluorescencebased fingerprinting detection materials. In general, many methods reported for fingerprinting detection depend on the affinity between the amino acid-based oily components of the fingerprints and the hydrophobic compounds used in the developing reagents. [49] In order to test the potentialo fo ur hybrid materials for this important application, we adopted two methods for fingerprints deposition:( 1) direct stamping of forehead-rubbed fingerprint on both smooth and porouss ubstrates (e.g.,g lass, plastic, leather,p aper), and (2) lifting up the fingerprints using the sticky side of an adhesive tape. Figure 2d isplays fresh (0 days) and aged fingerprint images (stored at RT for 28 or 59 days). The undeveloped fingerprint patterns wereh ardly visible regardless the illumination (UV or vis light). In contrast, those developedu nder diluted solution of the FLMs apparently displayed enhanced legibility due to the greater contrast between the fluorescent ridge and nonfluorescent furrow.The brightness, contrast and visual legibility remained the same for at least three months for PAD and PANP.H owever,d immer fingerprintf luorescencew as observed with PAP after one month, probablyd ue to loss of some components on ther idgeso ver time. The longer temporal stability observedf or PAD and PANP was ascribed to their respective structural outfit bearing functional groups capable of providing hydrogen-bonding with the residual aminoa cids in the fingerprints( e.g. (PAD)S=O···HÀN( amino acid), (PANP)NÀH···O(amino acid)). In addition, Na toms also contribute forming strong N···HÀOi nteractions. [14,[50][51][52][53][54] Furthermore, detailedo bservation of the friction ridge features (see enlarged areas in Figure2), showed whorl, bifurcation, and ridge ending, which satisfyt oalarge extendt he requirements of fingerprint identification.I nterestingly,o ur fingerprint development process also offered distinguished visual legibility on other surfaces, including handset phones urfaces ( Figure S15). Importantly,r elatively weak fluorescencew as observed when thump-printed fingerprints were rinsed with organic solvents such as CH 2 Cl 2 ,T HF and acetone, since these solvents could wash off the oily fingerprint residue. In contrast, samples rinsed with water exhibited intense fluorescence. This observation furtheru nderpins hydrophobic interactions as the first developingm echanism for the tested FLMs in the absence of any additives. [49,55,56] In summary,n ovel fluorescent labeled nanohybrids based on polyhedralo ligomeric silsesquioxanes (POSS) linked to appropriate fluorophores (pyrene, dansyl or naphthalimide) have been synthesized, fully characterized and their photophysical properties have been investigated in detail using different solvents.I np articular, PAP, PAD and PANP displayed adequate physical characteristics and convenient photostability in order to be successfully appliedi nf ingerprinting detection. In fact, PAP, PAD and PANP were successfully used to visualize latent fingerprints on several surfaces, providing reasonablel egibility pattern thats atisfy the needs of fingerprinting identifications in forensic technology.