Materials. Lanthanide oxide Lu2O3 (99.999%), Yb2O3 (99.999%) and Tm2O3 (99.99%) are purchased from STREM Chemicals, Inc. USA. Lanthanide chlorides (LnCl3, Ln: Lu, Yb and Tm) are prepared by dissolving the corresponding metal oxide in HCl solution at elevated temperature and then evaporating the water completely under reduced pressure. 1-octadecene (ODE), NH4NO3, and NH4F are purchased from Alfa Aesar Chemical Co. Ltd. Na2SO4, KCl, CaCl2, Mg(NO3)2, NaCl, ZnCl2, KBr, Na3PO4, Na2CO3, NaOH, CHCl3, H2SO4 solution, H3PO4 solution, H2O2 solution, citric acid, sodium citrate, ethanol, ethyl acetate, cyclohexane, oleic acid (OA), hexadecyl trimethyl ammonium bromide (CTAB), L-amino acid oxidase (L-AAO, from crotalus adamanteus, type IV), horseradish peroxidase (HRP, type VI), O-dianisidine, coomassie brilliant blue (CBB), human serum albumin (HSA), carbamide, octanoic acid (OOA), decanoic acid (DOA), palmitic acid (PTA), octadecanoic acid (ODOA), arachidic acid (ADA), α-keggin silicomolybdate (SiMo), ascorbic acid (ACA), 2-hydroxypropanoic acid (2-HA), glucose (Glc), tetraethyl orthosilicate (TEOS), (NH4)2MoO4 and L-amino acid (L-AA) are purchased from Sigma Aldrich. All chemical reagents are of analytical grade and are used directly without further purification. Deionized (DI) water is used throughout.
Synthesis of NaLuF4:Yb,Tm. In a typical experiment, a mixture of 1 mmol LnCl3 (Ln: 80%Lu, 19%Yb, 1%Tm), 15 mL OA, and 15 mL ODE are added into a 100 mL three-necked flask. Under the vacuum, the mixture is heated to 160 oC to form a clear solution, and then cooled to room temperature. After the solution cooling down, 0.025 mmol NaOH (0.1 g) and 0.04 mmol NH4F (0.1481 g) are added into the flask directly and stirred for 30 minutes. The solution is slowly heated with gently stirred, degassed at 100 oC, and then heated to 300 oC and maintained for 1 hour under the Argon atmosphere. After the solution is cooled naturally, the NaLuF4:Yb,Tm are separated via centrifugation (10000 rpm) and washed with ethanol/cyclohexane (1:1 v/v) three times. The product is stored under room temperature in cyclohexane.18
Synthesis of NaLuF4:Yb,Tm@NaLuF4. In a typical experiment, a mixture of 1 mmol LuCl3, 15 mL OA, and 15 mL ODE are added into a 100 mL three-necked flask. Under the vacuum, the mixture is heated to 160 oC to form a clear solution, and then cooled to room temperature. After the solution cooling down, 0.025 mmol NaOH (0.1 g), 0.04 mmol NH4F (0.1481 g), and as-prepared NaLuF4:Yb,Tm nanoparticles are added into the flask directly and stirred for 30 minutes. The solution is slowly heated with gently stirred, degassed at 100 oC, and then heated to 300 oC and maintained for 1.5 hour under the Argon atmosphere. After the solution is cooled naturally, the NaLuF4:Yb,Tm@NaLuF4 are separated via centrifugation (10000 rpm) and washed with ethanol/cyclohexane (1:1 v/v) three times. The product is stored under room temperature in cyclohexane.
Synthesis of NaLuF4:Yb,Tm@NaLuF4@mSiO2. In a typical experiment, 250 μL CHCl3 containing 5 mg as-prepared NaLuF4:Yb,Tm@NaLuF4 are mixed with 2.5 mL CTAB solution (37.5 mg mL-1) under ultrasonic for 5 minutes. The mixture is transformed to a 100 mL three-necked flask and heated in 60 oC water-bath to form a clear solution. Then, 22.5 mL DI water is added and the solution is heated to 70 oC before 0.3 mmol NaOH (0.012 g) is added directly. After 20 minutes stirring under the speed of 600 rpm, 120 μL TEOS is dropped slowly and 0.5 mL ethyl acetate is added within 30 seconds. Finally, the solution is continuously stirred in 70 oC water-bath for another 3 hours to enhance the stability of mSiO2 shell. After the solution is cooled naturally, the NaLuF4:Yb,Tm@NaLuF4@mSiO2 are separated via centrifugation (8000 rpm) and washed with ethanol and DI water three times, respectively. The as-obtained NaLuF4:Yb,Tm@NaLuF4@mSiO2 is then suspended in 50 mL ethanol containing 0.3 g NH4NO3 upon stirring for 1 hour at 60 oC. The CTAB-removed NaLuF4:Yb,Tm@NaLuF4@mSiO2 are separated via centrifugation (8000 rpm) and washed with DI water three times. The product is stored under 4 oC in DI water.19
Synthesis of NaLuF4:Yb,Tm@NaLuF4@mSiO2-SiMo (Tm-SiMo). In a typical experiment, 1 mL H2SO4 solution is added to 5 mL DI water containing 5 mg as-prepared NaLuF4:Yb,Tm@NaLuF4@mSiO2. After stirring under room temperature for 10 minutes, 0.5 mL DI water containing 0.05 mmol (NH4)2MoO4 (0.0098 g) is added and the mixture is then dried for 240 min at 100 oC to stabilize the SiMo modification. The Tm-SiMo are separated via centrifugation (8000 rpm) and washed with DI water three times. The product is stored under 4 oC in DI water.20
Synthesis of reduced Tm-SiMo (Tm-rSiMo). In a typical experiment, 5 mg as-obtained Tm-SiMo are suspended in 5 mL DI water containing 5 mmol ACA (0.088 g) upon stirring for 20 minutes at room temperature. The Tm-rSiMo are separated via centrifugation (8000 rpm) and washed with DI water three times. The final product is stored under 4 oC in DI water.
Modification of L-AAO on Tm-rSiMo (Tm-rSiMo-AAO). In a typical experiment, 100 μL L-AAO solution (1 unit mL-1) is added to 5 mL DI water containing 5 mg as-prepared Tm-rSiMo. After stirring under 4 oC for 120 minutes, the Tm-SiMo-AAO are separated via centrifugation (8000 rpm) and washed with DI water three times. The product is stored under -20 oC in DI water.
Characterization. The sizes and morphologies of nanoprobes within layer-by-layer coating procedure are determined using a FEI Tecnai G2F30 transmission electron microscope (TEM). Samples of the above nanoparticles are dropped on the surface of a copper grid. Energy-dispersive X-ray (EDX) mapping and selected area electron diffraction (SAED) pattern of the samples are also performed during TEM measurements. The size distribution is counted and calculated from TEM images (α = 0.90, >500 particles are measured). Solid UV-vis-NIR absorbance spectra are obtained on a Shimadzu SolidSpec-3700 UV-vis-NIR spectrophotometer. Aqueous UV-vis-NIR absorbance spectra are determined on a Shimadzu UV3600 UV-vis-NIR spectrophotometer. 95Mo nuclear magnetic resonance (NMR) spectra are determined by a Bruker Avance 600 liquid nuclear magnetic resonance spectrometer. Powder X-ray diffraction (XRD) pattern is measured with a Brucker D8 advance X-ray diffractometer from 5o to 70o (Cu Kα radiation, λ = 1.54 Å). X-ray photoelectron spectroscopy (XPS) spectra are performed on Thermo escalab 250Xi. The sample for XRD and XPS determination is previously dried in nitrogen atmosphere at 100 oC. Electron paramagnetic resonance (EPR) spectra are determined on a Bruker A300 electron paramagnetic resonance spectrometer. Dynamic light scattering (DLS) experiments are carried out on an ALV-5000 spectrometer goniometry equipped with an ALV/LSE-5004 light scattering electronic and multiple tau digital correlator and a JDS Uniphase He–Ne laser (533 nm) with an output power of 22 mW. The hydrodiameter distribution is measured at 25 oC with a detection angle of 90o. Electrochemical measurements are carried out by a Bio-Logic VMP3 multichannel potentiostat at ambient temperature. Cyclic voltammetry (CV) curves are obtained by electrochemical station (Chenhua Instruments Co. CHI600). A three-electrode system is made up of a glassy carbon electrode (GCE, 3 mm in diameter) as the working electrode, an Ag/AgCl electrode (saturated KCl) as reference electrode, and a Pt wire as counter electrode. The near-infrared (NIR) and short-wave infrared (SWIR) luminescence spectra are taken on a FLS980 lifetime and steady state spectrometer (Edinburgh Instruments) equipped with an external 0-7 W 808 nm and 980 nm adjustable laser as the excitation source. Inductively coupled mass spectroscopy (ICP-MS) analysis is performed on Agilent 7500ce ICP-MS. Enzymatic activity is performed by using standard protocol and monochromator-based multifunction microplate reader (Tecan Infinite M200). NIR and SWIR images are collected by a self-developed small animal luminescence imaging system equipped with Andor iXon Ultra EMCCD (Si-based CCD) and Princeton Instrument NIRvana 640 CCD (InGaAs-based CCD) camera, as well as an external 0-8 W adjustable CW infrared laser (Hide-Wave Co., China). The NIR images are obtained by Si-based CCD using 750-900 nm band-pass filter. The SWIR images are obtained by InGaAs CCD using 1300 nm long-pass filter. The NIR/SWIR image and signal intensity are obtained by analysing NIR and SWIR images using professional software provided by Andor.
Stability and activity of Tm-rSiMo-AAO after storage. The Tm-rSiMo-AAO is stored under -20 oC in DI water within 60 days. The protein-staining solution is freshly prepared by dissolving 20 mg CBB in a mixture of 50 mL ethanol, 100 mL H3PO4 and 850 mL DI water. In a typical experiment, the freshly-prepared and stored Tm-rSiMo-AAO are separated via centrifugation (8000 rpm) and washed with DI water three times. The supernatants and washing solutions are collected and 5 mL protein-staining solution is added, respectively. After 30 minutes, the absorbance of the above solutions at 595 nm (Abs.595) is determined to evaluate the stability of L-AAO on Tm-rSiMo. The freshly-prepared and stored Tm-rSiMo-AAO separated by centrifugation is then dispersed in 5 mL DI water and their absorbance at 800 nm (Abs.800) are determined respectively to evaluate the oxidation degree. To evaluate the activity of AAO on Tm-rSiMo after storage, 1 mL mimic L-AA working solution is added to both freshly-prepared and stored Tm-rSiMo-AAO dispersion, respectively. After 40 minutes, the absorbance of Tm-rSiMo-AAO dispersions at 800 nm (Abs.800) is determined.
Influence of pH on activity of L-AAO. The pH of L-AAO solution is modulated by phosphate and diluted citric acid solution. The mimic L-AA working solution in freshly prepared according to a previous reported prescription (Supplementary table 1). In a typical experiment, 10 μL L-AAO solution (0.02 unit mL-1) with various pH (4.0-7.0, at 0.5 intervals) is added to 90 μL mimic L-AA working solution, HRP (3 units mL-1) and O-dianisidine (10 μM). After 40 minutes, absorbance at 436 nm (Abs.436) of all samples is determined by monochromator-based multifunction microplate reader to evaluate the L-AAO activity, respectively.
Selective determination of L-AA by Tm-rSiMo-AAO. The control solution of ions (including Na+, K+, Ca2+, Mg2+, Zn2+, Cl-, Br-, PO43- and CO32-) and biomolecules (including HSA, OOA, DOA, PTA, ODOA, ADA, 2-HA and Glc) is freshly prepared and the concentration is 10 times higher than that of L-AA. The dissolution of fatty acids in DI water (including OOA, DOA, PTA, ODOA and ADA) is assisted by ethanol and ultrasonic. In a typical experiment, 1 mL mimic L-AA working solution and control solutions are added to 1 mL DI water containing Tm-rSiMo-AAO (2 mg mL-1), respectively. After 40 minutes, the luminescence spectra of all samples are determined and analysed.
Ethics Statement. The fingerprints are collected from 10 males and 10 females in accordance to the official requirement of obtaining informed consent, in the form of a signature from each volunteer, acknowledging that they are aware of the procedure that will take place, any risks or benefits that may accompany the study, as well as acknowledging that they will not receive any payment for their participation. Informed consent from all volunteers who participates in this research study is obtained. After experiments, all fingerprints are thoroughly destroyed by dissolving to keep personal security.
Determination of L-AA extracted from fingerprints. The extraction of L-AA from fingerprints is performed by using a previous reported thermal/acid leaching protocol with reasonable modification.21 In a typical experiment, 120 μL H2SO4 solution (0.01 M) is directly placed onto the fingerprint on a polyethylene chip. The chip is then heated to 40 oC for 20 minutes. The leaching solution is collected off of the chip, diluted to 1 mL and used as the sample for determination after adjusting pH to 5.0. The determination process for fingerprint extraction is same to that for mimic L-AA working solution.
Luminescence imaging of latent fingerprints. The Tm-rSiMo-AAO staining solution is freshly prepared by dispersing Tm-rSiMo-AAO in buffer solution of sodium citrate (SSC, pH = 5.0) containing Tween 20. The polyethylene chips for luminescence imaging are cleaned with DI water and dried in air before use. The fingerprints are used for staining procedure within 12 hours after collection. The fingerprints staining with Tm-rSiMo-AAO is referring to a typical protocol with modification. In a typical experiment, the samples are rinsed with DI water for 1 minute and then immersed in Tm-rSiMo-AAO staining solution under 40 oC for 50 minutes with rotation. Then, NIR and SWIR imaging are performed and signals are analysed.8
Statistical Analysis. The NIR/SWIR ratiometric luminescence method is used to analyze 10 fingerprints collected from male and 10 fingerprints collected from female. Receiver operating characteristic (ROC) analysis was utilized to determine the forensic gender identification potential that the ability to correctly differentiate males and females by determination L-AA content in fingerprints. The ROC analysis captures the trade-off between sensitivity and specificity while changing a discrimination threshold, but it can be summarized as a single measurement (AUC). The sensitivity (true positive rate) was plotted against the specificity (true negative rate) in the ROC curve as a function of a variety of thresholds of class prediction probabilities. The overall accuracy depends on the overlap of the output signal distributions for the two classes, in this case, fingerprints of males and females. Values range between 0.5 and 1.0, where a value of 0.5 indicates that the two distributions are identical and a value of 1.0 indicates that there is no overlap in the distributions of output signals for the two classes. The AUC is used as a lone measure of evaluating the efficiency of the model ranked subjects according to the probability assigned to the positive class. The AUC of the ROC curve was calculated by the trapezoidal method of integration with the corresponding 95% confidence intervals (CI).