Multiplexed Imaging of Trace Residues in a Single Latent Fingerprint

Nov 29, 2016 - College of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of Medicinal Chemical Biology, and Tianjin Key ...
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Multiplexed Imaging of Trace Residues in a Single Latent Fingerprint Yuyan Zhang, Wen Zhou, Yang Xue, Jie Yang, and Dingbin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04077 • Publication Date (Web): 29 Nov 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Analytical Chemistry

Multiplexed Imaging of Trace Residues in a Single Latent Fingerprint Yuyan Zhang,† Wen Zhou,†,‡ Yang Xue,† Jie Yang,† and Dingbin Liu*,†,‡ †

College of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of Medicinal Chemical Biology, and Tianjin Key Laboratory of Molecular Recognition and Biosensing, Nankai University, Tianjin 300071 (China) ‡

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071 (China)

ABSTRACT: The development of highly sensitive, selective, nondestructive, and multiplexed imaging modalities is essential for latent fingerprint (LFP) identification and fingerprint residues detection. Herein, we present a versatile strategy to identify LFPs and to probe the multiple trace residues in a single LFP simultaneously. With the purpose of achieving high sensitivity, we for the first time introduced a polydopamine (PDA)-triggered Au growth method to prepare super-bright and multiplex surface-enhanced Raman scattering (SERS) tags, which were endowed with high selectivity by conjugating with specific antibodies. In combination with a rapid Raman mapping technique, the sensitivity of the SERS probes was down to picogram scale and all the three levels of LFP features can be clearly seen. More significantly, the multiplexed imaging of diverse residues in a single LFP provides more accurate information than that using monochromatic imaging of individuals alone. The high analytical figures of merit enable this approach great promise for use in the fields ranging from chemical detection to molecular imaging.

Fingerprints, the unique impressions of the ridges and furrows on the corrugated skin of human fingers, are utilized as one of the most important means for personal identification in forensic investigation, access control, and medical diagnosis.1 Certain fingerprints can be directly observed if the fingers are contaminated with specific substance such as blood or paint. In many cases, the impressions deposited on the surfaces by the sweat are however invisible to the naked eyes, termed latent fingerprints (LFPs).2 To enable the LFPs visible, numerous methods have been developed and commonly used over the past years, including various fuming strategies, vacuum metal deposition, and powder dusting, etc.3 However, these methods suffer from several drawbacks such as insufficient sensitivity, poor selectivity, and inadequate information. During the past few years, tremendous efforts have been made toward the improvement of identification sensitivity. For instance, electrochemiluminescence and photoluminescence techniques integrated with LFPs identification can remarkably improve sensitivity.4,5 Apart from LFPs identification, much attention has recently been paid on the detection of fingerprint residues because they may provide more useful information about the individuals than the use of LFPs identification alone.6 Fingerprint residues often contain many endogenous metabolites and exogenously doped chemicals. A variety of spectroscopic techniques including mass spectrometry7,8 and vibrational spectroscopy (infrared and Raman)9,10 have been employed to visualize the fingerprints and to detect the residues simultaneously, but they can only probe high-abundance chemicals. Biolabeling techniques show great potentials in that they can combine with other high-sensitivity strategies to offer both increased selectivity and sensitivity. For example, immunological deposits were used to detect the nanogram-level metabolites in fingerprints.3 Aptamer-based reagents have been coupled with nanoparticles for the sensitive and selective

detection of lysozyme and cocaine in the residues.11 However, these approaches can only detect one type of chemicals in single fingerprints. Undoubtedly, highly sensitive, selective, and multiplexed imaging of diverse residues simultaneously in single fingerprints can provide more reliable information than just imaging one type of residues, but has rarely been achieved thus far.12 Herein, we have developed a versatile strategy for identifying the LFPs and multiplexed imaging of several trace residues within a single LFP with sufficient sensitivity and selectivity. This strategy relies on the fabrication of a new type of super-bright surface-enhanced Raman scattering (SERS) tags functionalized with specific antibodies. SERS tags have been widely used in chemical and biological detection because of their distinct properties such as high sensitivity, rich molecular information, narrow characteristic bands, and insusceptibility to photobleaching.13-17 Despite several attempts in using SERS for LFPs identification have been reported,6,18 simultaneous imaging of multiple trace residues in a single LFP has not been accomplished to date. In this study, with the purpose of imaging low-abundance LFP residues, we attempted to fabricate SERS tags with ultrahigh brightness. Conventional strategies for preparing highbright SERS tags focus on creating hot-spot-involved nanostructures, such as gold nanostars (AuNSs)19,20 and nanogap-embedded core-shell nanoparticles21-24. The nanoparticles with in-built hot spots have received considerable attention owing to their high brightness and signal stability to external environments. Unlike conventional strategies choosing spherical gold nanoparticles (AuNPs) as the cores, we herein used AuNSs as the cores to fabricate Raman reporterembedded AuNPs with the purpose of achieving sufficient sensitivity. More importantly, the Raman reporters used in conventional strategies for preparing core-shell NPs should be

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terminated with a high-affinity group (e.g., HS-, for molecule anchoring) and a polar moiety (e.g., -COOH, -NH2, or -SH, for directing the subsequent NP growth).22,25 We herein propose a polydopamine (PDA)-guided growth strategy for enclosing Raman reporters into the AuNPs, regardless of the chemical properties of the reporters, thus greatly extending the versatility of the approach in SERS tag preparation. As shown in Figure 1, we fabricated three types of SERS tags with diverse mercaptobenzene derivatives (NTP, MBN, and ATP) as Raman reporters and allowed them into sensitive and selective detection of different kinds of residues (lysozyme, human IgG, and cotinine) within a single LFP simultaneously.

Figure 1. Schematic illustration of multiplexed imaging of the residues including lysozyme, human IgG, and cotinine in a single LFP. The simultaneous LFP identification and its residues detection is dependent on the combination of immunoassays and Raman mapping technique.

EXPERIMENTAL SECTION Materials and instrumentation. Human immunoglobulin G (IgG) and goat antirabbit IgG were obtained from Beijing Dingguo Changsheng Biotechnology Co. Ltd. Silver nitrate (AgNO3) was purchased from Beijing Chemical Plant Co. Ltd. 4-Mercaptobenzonitrile (MBN), 4-nitrothiophenol (NTP), 4aminothiophenol (ATP), 1,4-diethynylbenzene (DEB), leucocrystal violet (LV), 1-phenyl-2-trimethylsilylacetylene (PST), 3-[(trimethylsilyl)ethynyl]thiophene (TMSET), 2ethynylterephthalic acid (ETPA), CDCl3, 3-mercaptopropionic acid (MPA), dopamine, sodium citrate, bovine serum albumin (BSA), the phosphate-buffered saline (PBS; 0.01 M, pH 7.4), L-ascorbic acid (LAA), glutaraldehyde, (3-aminopropyl) triethoxysilane (APTES) silanization, ethanol (EtOH), hydrogen peroxide (H2O2), tris (hydroxymethyl) aminomethane (Tris), hexadecyl trimethylammonium bromide (CTAB), hydrogen tetrachloroaurate (III) trihydrate (HAuCl4-3H2O), mPEG-NH2 (5 KDa) were all purchased from Sigma-Aldrich. TEM images were obtained by FEI Tecnai G2 F20 S-TWIN at 200 kV. UVvis absorption spectra were measured with U-3010 spectrophotometer (Hitachi, Japan). SERS and Raman mapping images were recorded on an InVia Raman microscope (Renishaw) equipped with research grade Leica DMLM microscope and a 150 mW/cm2 633-nm diode laser. Dynamic light scattering (DLS) and zeta potential (ζ) were performed on a Zeta Sizer Nano ZS (Malvern Zetasizer 3000HS and He/Ne laser at 632.8 nm at scattering angles of 90 at 25 oC). Kinetic absorbance of AuNS solutions in 96-well plates were collected

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at 545 and 740 nm by a Synergy 2 Multi-Mode Microplate Reader (Bio-Tek Instruments, Inc.) Preparation of latent fingerprints on glass slides. The glass slides used in the following procedures were firstly immersed in the solution of H2SO4/H2O2 (v/v 3: 1) for 1 h. Then, the slides were rinsed and ultrasonicated with excess DI water and ethanol prior to use, followed by treating with 10% (v/v) APTES ethanol solution for 1 h at room temperature to produce amines on the glass slides. After that, the amineimmobilized slides were immersed into an ethanol solution containing 5% glutaraldehyde for 2 h. As a result, the aldehyde-functionalized glass slides were obtained. The latent fingerprints were prepared by rubbing fingers against 5 µL residues with desired concentrations and stamping them onto the aldehyde-functionalized glass slides for 1 min. After that, the slides were immersed in a mixture of mPEG-NH2 and PB solution containing 1% BSA for 2 h at 37 oC, so as to avoid the possible non-specific adsorption when incubated with SERS tags. Synthesis of gold nanostars (AuNSs). AuNSs were prepared using a seed-growth method. Briefly, 100 mL HAuCl4 aqueous solution (1 mM) was heated to reflux under magnetic stirring, and a boiling trisodium citrate aqueous solution (10 mL, 38.8 mM) was added rapidly. The mixture was vigorously stirred for another 15 min. 13 nm sized AuNPs were obtained, indicative of the color change of the solution from pale yellow to deep red. Subsequently, 0.2 mL of 100 mM HAuCl4 was added into 60 mL of DI water, followed by adding 80 μL of 1 M HCl and 0.6 mL of 13 nm AuNP seeds (1 nM). The solution was stirred for 2 min, and then 160 μL of 10 mM AgNO3 was injected into the reaction mixture, then, 400 μL of LAA (100 mM) was added rapidly and the solution turned green immediately. At last, 0.6 mL of CTAB (100 mM) was added into the mixture to stabilize the AuNSs. The UV-vis spectrum was collected and the maximal absorbance peak appeared at 725 nm indicated the formation of AuNSs. Synthesis of Au@reporter@Au NPs. Au@reporter/PDA@Au NPs (namely Raman reporterembedded AuNPs) were prepared via a two-step method. Certain Raman reporters with final concentration of 0.1 mM were added into the as-synthesized AuNS solution (80 pM) in Tris·HCl buffer (10 mM, PH 8.5), followed by adding dopamine (0.2 mg/mL). The resulted solution was continuously stirred for 1 h to allow the adequate coating of PDA layer around the surfaces of AuNSs, where Raman reporters were present in the PDA layer. The obtained Au@reporter/PDA NSs were characterized by TEM measurements, DLS, and UV-visible spectroscopy. Next, a desired concentration of HAuCl4 was injected into the as-prepared Au@reporter/PDA NP solution, then the mixture was shaken for 30 min at 37 oC. The color of the solution turned dark purple in the beginning of the reaction, and gradually became red, indicating the formation of spherical AuNPs. MPA (1.5 mM) was employed to stop the reaction at desired time. The reductive degradation of the PDA corona and AuNP growth process were monitored by Raman spectroscopy, TEM measurements, and UV-vis spectroscopy. Preparation of antibody-functionalized SERS tags. The residue-specific SERS tags were prepared by anchoring their corresponding antibodies onto surfaces of Raman reporterembedded AuNPs via electrostatic interactions. In our experiment, 10 μL of specific antibody (1 mg /mL) was added into

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the Au@reporter@Au NPs solution (1 mL). The resulted solution was shaken at 37 oC for 2 h. After that, 100 μL of PB solution containing 1% BSA was added to block the rest active sites of the AuNP surfaces. The solution was shaken for another 2 h at 37 oC to minimize the possible non-specific adsorption of the SERS tags when used for LFP imaging. Finally, the resulted solution was centrifuged at 6000 rpm to remove the free antibodies and BSA, and the precipitates were dispersed with DI water for further use. SERS detection and imaging of LFP residues. The SERS measurements of the probes were conducted by capillaries using Reinshaw InVia Raman spectrometer with 633 nm laser (10 mW) and 50× objective lens, and the data was acquired with an exposure time of 5 s. The large-area SERS mapping images of LFPs on the glass slides were obtained by scanning the Raman regions at 1310-1350, 1410-1450, and 2200-2250 cm-1 respectively. All the images were acquired at an interval of 15 µm with an exposure time of 0.1 s (633 nm excitation, 10 mW, 50× objective lens). RESULTS AND DISCUSSION Rational design of the SERS nanotags. SERS-based assays exhibit several distinct properties when applied for the biomedical imaging and detection.26-30 First, SERS signals could be enhanced for several orders of magnitude when a molecule is located at the “hot spot”, such as the junctions and interstices in the nanostructures.31 Second, a protective layer is essential for maintaining the stability and monodispersity of a SERS probe.32 In our experiment, we designed AuNPs characteristic of embedded electromagnetic fields for achieving both high sensitivity and colloidal stability. AuNSs, with typical sharp tentacles and large surface areas, were chosen as the plasmonic cores because of their strong Raman enhancement.33 The schematic illustration of the stepwise synthesis of AuNP structures is shown in Figure 2a. PDA was coated on AuNSs based on the mussel-inspired polymerization of dopamine in Tris·HCl buffer (10 mM, pH 8.5),34 which directed the coating of the Raman reporters on the AuNSs without consideration of their chemical properties. In addition, PDA triggered the growth of AuNPs because PDA possesses a large amounts of amidogen and the catechol moieties, which could initiate the reduction of Au (III) to Au (0), which deposits on the nanoparticle surfaces and ultimately produce the Raman reporter-embedded AuNPs, along with the release of the PDA corona from the AuNS surfaces. Preparation and characterization of the Raman reporter-embedded AuNPs. In this study, we applied cetyltrimethylammonium bromide (CTAB)-stabilized AuNSs (60 nm in diameter) (Figure S1b in Supporting Information) with sharp tips as the cores of SERS probes. The AuNS cores (80 pM) were incubated with the mixture of dopamine (0.2 mg/mL) and Raman reporters (0.1 mM) in Tris·HCl buffer (10 mM, pH 8.5) for 1 h at room temperature. As a consequence, the AuNS cores were uniformly coated with a layer of PDA (Figure 2b), by which the Raman reporters are grafted on the surfaces of AuNSs regardless of their chemical structures. The subsequent addition of HAuCl4 allows the oxidation of the catechols to quinones, causing the partial disassembly of PDA from Au surfaces due to the weak interaction between quinone and AuNS core.25 Simultaneously, Au (III)-induced oxidation facilitated the anisotropic growth of AuNSs into spherical AuNPs, in which the Raman reporters were embedded. These

Au nanostructures were characterized by various analytical tools including UV-vis absorption spectroscopy and TEM images. With the PDA coating, the maximal absorption band of AuNSs red-shifted from 725 to 740 nm, indicating the assembly of PDA layer on AuNSs (termed Au@reporter/PDA NSs). The reductive growth of Au enables the transformation of particle morphology from AuNSs to spherical AuNPs, indicative of a noticeable color change of the solution from blue to red, combined by the remarkable blue-shift of absorption bands from 740 to 545 nm (Figure S2d in Supporting Information). It is worth noting that Raman reporter-embedded AuNPs could be formed by only adding HAuCl4 into the Au@reporter/PDA NSs solution, without requirement of other additives.

Figure 2. a) Schematic illustration of the procedure for the preparation of Au@reporter@Au NPs (namely reporter-embedded AuNPs). b) TEM images of Au@reporter/PDA NSs and those after addition of HAuCl4 solution (150 µM) and incubation for different time: c) 1 min, d) 3 min, e) 4 min, f) 5 min, g) 10 min, h) 30 min, and i) 120 min. The Au growth was stopped with excess MPA (1.5 mM) at desired time. Insets are corresponding AuNPs solutions. Scale bar, 50 nm.

The dynamic process of PDA-directed Au growth were further monitored by measuring the intermediates with UV-vis absorption and TEM. To do this, HAuCl4 (150 µM) were incubated with the Au@reporter/PDA NSs solution for varied time intervals: 1, 3, 4, 5, 10, 30, and 120 min. 11Mercaptopropionic acid (MPA, 1.5 mM) was employed to stop the AuNP growth at desired time through the formation of less reactive Au (I)-MPA complex.35 As shown from the TEM images, a morphological change of the particles can be clearly observed in 3 min and the Au@reporter/PDA NSs were evolved into spherical AuNPs in 10 min. With longer incubation time even to 120 min, the size of the spherical AuNPs had negligible increase, demonstrating that the Au growth can be completed rapidly. At the same time, the color of the solution changed from blue to purple, and eventually to red in several minutes. In addition, the maximal absorption peak of AuNS narrowed and blue-shifted gradually from 740 to 545 nm (Figure S3 in Supporting Information), along with the increase of absorbance in the visible region. Both the changes of color and UV-vis spectroscopy confirmed the generation of typical spherical AuNPs.

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The kinetic process of Au growth after incubating with varying concentrations of HAuCl4 (50, 100, 150, 200 µM) was further investigated by a Synergy 2 Multi-Mode Microplate Reader through detecting the absorbance variations at 545 and 740 nm every minute. As shown in Figure S4 in Supporting Information, the absorbance at 740 nm decreased and those at 545 nm increased significantly within 15 min, directly proving that the process of AuNS growth mainly occured in initiation of the reaction and the nanoparticle morphology was closely correlated with the growth time and the amount of HAuCl4. To further understand the mechanism, we took advantage of Raman spectroscopy to explore the distribution of PDA around Au surfaces before and after Au growth. To avoid the possible signal interference of Raman reporters with the PDA, PDA alone was assembled on the AuNSs to direct the Au growth. Once PDA coating on the AuNSs, the typical Raman peaks assigned to PDA can be observed clearly. With the addition of HAuCl4 (100 µM), the Raman signals of PDA diminished significantly (Figure S5 in Supporting Information). Since encapsulating the Raman dyes into the obtained spherical AuNPs can significantly improve their intensities (Figure 3), it is hypothesized that the PDA signals should be enhanced if the PDA is embedded in the AuNPs. We thereof reasoned that the decrease of PDA signals was attributed to the reductive release of PDA in the process of Au growth.25

Figure 3. Raman Spectra of Au@reporter/PDA NSs and those of Au@reporter@Au NPs. The Raman reporters are: a) NTP, b) ATP, and c) MBN, which present typical non-overlapping bands at 1330, 1430, and 2225 cm-1 respectively for multiplexed imaging. d) The histogram of the Raman intensity before and after Au growth as described in a)-c).

We next verified the distribution of the Raman reporters whether they are embedded into the AuNPs or adsorbed onto the as-obtained AuNP surfaces. As to the Raman reporters in the presence of thiol moiety, their signals were enhanced significantly after Au growth (Figure 3). However, excess HAuCl4 resulted in the decrease of Raman intensity (Figure S6 in Supporting Information). We reasoned that further Au growth could thicken the Au shell, which could shield the reporters being stimulated by the scatttering light. This is an evidence that the reporters were embedded into the AuNPs. More interestingly, the Raman signals for the reporters in the absence of high-affinity moieties can also be enhanced with various degrees (Figure S7a, c, and d in Supporting Infor-

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mation). Note that the reporters with large Raman scattering cross-sections (like LV and PST) show much enhanced Raman signals after Au growth, while that with small scattering crosssections (like CDCl3) show relatively weak Raman signals, but the enhancement can also be observed after Au growth. This is a convincing evidence that the reporters are enclosed into the AuNPs other than attached on the surfaces because the lack of high-affinity moiety may enable the reporters unstable on the Au surfaces. These results imply that the reporters can be grafted onto surfaces of AuNSs with the assistance of the PDA, without considering their chemical properties. Subsequently, the Au growth process led to the release of PDA and simultaneously left the reporters into the newly-produced AuNPs. Surface functionalization of the SERS tags. As a proofof-concept, three mercaptobenzene derivatives (NTP, ATP, and MBN) were applied for SERS palettes owning to their high brightness, simple Raman peaks, and narrow band emissions from 1300 to 2400 cm-1. More importantly, the peaks being chosen for multiplexed imaging are completely resolved. As shown in Figure 4, the Raman peaks at 1330, 1430, and 2225 cm-1 in the Raman spectra of NTP, ATP, and MBN were selected for simultaneous imaging of the LFP residues including lysozyme, cotinine, and human IgG respectively. The corresponding antibodies were functionalized onto the reporter-embedded AuNPs through electrostatic interactions, and the probes were then blocked with bovine serum albumin (BSA) to eliminate the possible non-specific adsorption when conducting the LFP imaging. The successful antibody functionalization was confirmed by a red-shift of the maximal absorption band from 545 to 548 nm (Figure S8a in Supporting Information). Moreover, the surface modification was further explored by the zeta potential measurements (Figure S8b in Supporting Information). As expected, the zeta potential of the as-obtained AuNPs was positive (24 ± 7 mV) because of the coverage of CTAB. The coating of antibodies and BSA switched the particle charges to be negative (-5 ±1 mV), most likely owing to the widespread existence of the negatively charged groups in both antibodies and BSA. Imaging trace residues in LFPs. With the SERS probes in hand, 5 µL of LFP residues with different concentrations (0, 10-8, 10-7, 10-5, 10-3, and 10-1 mg/mL) were dropped onto the aldehyde-functionalized glass slide. The use of immobilized aldehyde could assist the adhesion of residues in LFPs.36 Subsequently, the LFP surfaces were blocked with mPEG-NH2 (2 mM) and BSA (1 %) solutions. After that, the obtained glass slides were treated with SERS probes respectively. As shown in Figure 4, LFPs contaminated with different concentrations of lysozyme, cotinine, and human IgG were incubated with corresponding SERS probes for 2 h at 37 oC. Finally, SERS imaging of LFPs was carried out on a confocal Raman spectroscopy coupled with a high-speed imaging system. For lysozyme imaging, the correponding SERS signals were scaned at Raman region from 1310 to 1350 cm-1 since the embedded Raman reporters are NTP with typical peak at 1330 cm-1; similarly, the images of cotinine and human IgG were obtained by collecting the Raman signals at the regions 14101450 (assigned to ATP) and 2200-2250 (assigned to MBN) cm-1 respectively. From the images, all the three levels of LFP structures including ridge pattern, ridge termination, and sweat pores37 can be clearly observed for each kind of residue. Impressively, the LFP structures can still be seen even the concentrations of the residue were down to pg/mL levels. The

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lowest concentrations of residues to be clearly imaged were approximately 10-8 mg/mL (50 fg scale). Moreover, the nonspecific adsorption of the SERS probes was verified through incubating the specific probes over the clean LFPs. The results reveal that negligible LFPs can be observed in the absence of LFP residues.

Figure 5. Multiplexed imaging of diverse residues (10-5 mg/mL) in a single LFP. a)-c) are the mapping images for lysozyme, cotinine, and human IgG in the channels of 1330, 1430, and 2225 cm-1 and d) their merged image. The dotted circles show the sweat pores. e)-h) show the gray values mapping across the yellow rectangles in a)-d). Scale bar, 500 µm. Figure 4. SERS imaging of the fingerprints with different concentrations of a) lysozyme (green), b) cotinine (blue), and c) IgG (red). The mapping images in a), b), and c) were obtained by scanning the Raman regions at 1310-1350, 1410-1450, and 22002250 cm-1 respectively. All the images were acquired at an interval of 15 μm with an exposure time of 0.1 s (633 nm excitation, 10 mW, 50× objective lens). Scale bar, 1 mm.

Multiplexed imaging of residues in a single LFP. The detection results above reveal that the proposed immunology/SERS approach could provide sufficient sensitivity and selectivity, which encouraged us to perform the multiplex imaging of diverse residues in a single LFP. To do this, triplex fingermarks of lysozyme, cotinine, and IgG were dropped onto a glass slide and incubated with the mixtures of corresponding SERS probes. To make the multiplexed imaging results convincing, we would like to test the multiplicity of our method at two concentrations (10-5and 10-7 mg/mL). However, the imaging results of the three kinds of residues in a single LFP were compared under the same concentrations. For the slide contaminated with 10-5 mg/mL residue mixtures, the LFP structures can be seen with each channel (1330, 1430, and 2225 cm1 ). The similar results for the 10-7 mg/mL residue mixtures were shown in Figure S9 in Supporting Information. Interestingly, the merged image from the three channels provides more obvious third-level structural information such as sweat pores than those using single channels alone, suggested by the yellow dotted circles in Figure 5a-d. Moreover, we further verified that the spatial pattern of the fingerprint based on triplex-imaging was apparently clearer than monochromatic imaging through comparing the variation of the gray values over several pores (yellow rectangles).38 As shown in Figure 5e-h, variation of the gray values over five sweat pores in the merged SERS image is more uniform than those images collected from single channels. These results indicate that multiplex imaging is more beneficial to individual identification especially for partial or damaged fingerprints in forensic investigations.

CONCLUSION In summary, we have developed a highly sensitive, selective, and multiplex imaging system for simultaneous identification of LFPs and several trace residues in a single LFP. This strategy relies on the PDA-assisted fabrication of super-bright SERS tags, where the Raman reporters were embedded into the AuNPs, without consideration of their chemical properties. Diverse Raman reporters including NTP, ATP, and MBN were chosen and their typical Raman peaks at 1330, 1430, and 2225 cm-1 were completely resolved, enabling the simultaneous imaging of lysozyme, cotinine, and human IgG respectively without any spectral unmixing. Moreover, the triplex SERS imaging-informative analysis could provide more accurate information than the use of monochromatic imaging alone for identification of partial or damaged fingerprints which often occur in crime scene. Despite this study only focused on multiplexed imaging of LFP residues, this system could be principally extended to other biomolecules detection by substituting only the corresponding recognition ligands.

ASSOCIATED CONTENT Supporting Information Supporting Information. Nine additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This study was supported by the National Natural Science Foundation of China (21475066 and 81401463), the Natural Science Foundation of Tianjin City (15JCZDJC65700), the Fundamental Research Funds for Central Universities (China), and the Thousand Youth Talents Plan of China.

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