Glutathione-capped gold nanoclusters as near-infrared-emitting efficient contrast agents for confocal fluorescence imaging of tissue-mimicking phantoms.

An innovative research has been conducted focused on demonstrating the ability of novel dual-emissive glutathione-stabilized gold nanoclusters (GSH-AuNCs) to perform bright near-infrared (NIR)-emitting contrast agents inside tissue-mimicking agarose-phantoms via two complementary confocal fluorescence imaging techniques. First, using a new and fast microwave-assisted approach, we synthesized photostable dual-emitting GSH-AuNCs with an average size of 3.2 ± 0.4 nm and NIR emission quantum yield of 9.9%. Steady-state fluorescence measurements coupled with fluorescence lifetime imaging microscopy (FLIM) assays performed on lyophilized GSH-AuNCs revealed that the obtained GSH-AuNCs exhibit PL emissions at 610 nm (red PL) and, respectively, 800 nm (NIR PL) in both solution and powder solid-state. Time-resolved fluorescence measurements showed that the two PL components are characterized by average lifetimes of 407 ns (red PL) and 1821 ns (NIR PL), respectively. Additionally, due to a partial overlap between the red PL and the absorption of the NIR PL, an energy transfer between the two coexisting emissive centers was discovered and confirmed via steady-state and time-resolved fluorescence measurements. Furthermore, the FLIM analysis performed on powder GSH-AuNCs under 640 nm, an excitation more suitable for bioimaging applications, revealed a homogeneous and photostable NIR PL signal from GSH-AuNCs. Finally, the ability of GSH-AuNCs to operate as reliable NIR-emitting contrast agents inside tissue-mimicking agarose-phantoms was demonstrated here for the first time via complementary FLIM and re-scan confocal fluorescence imaging techniques. In consequence, GSH-AuNCs show great promise for future in vivo imaging applications via confocal fluorescence microscopy.

Cardiac Troponin Biosensor Designs: Current Developments and Remaining Challenges.

Acute myocardial infarction (AMI) is considered as one of the main causes of death, threating human lives for decades. Currently, its diagnosis relies on electrocardiography (ECG), which has been proven to be insufficient. In this context, the efficient detection of cardiac biomarkers was proposed to overcome the limitations of ECG. In particular, the measurement of troponins, specifically cardiac troponin I (cTnI) and cardiac troponin T (cTnT), has proven to be superior in terms of sensitivity and specificity in the diagnosis of myocardial damage. As one of the most life-threatening conditions, specific and sensitive investigation methods that are fast, universally available, and cost-efficient to allow for early initiation of evidence-based, living-saving treatment are desired. In this review, we aim to present and discuss the major breakthroughs made in the development of cTnI and cTnT specific biosensor designs and analytical tools, highlighting the achieved progress as well as the remaining challenges to reach the technological goal of simple, specific, cheap, and portable testing chips for the rapid and efficient on-site detection of cardiac cTnI/cTnT biomarkers in order to diagnose and treat cardiovascular diseases at an incipient stage.

Photoluminescent Histidine-Stabilized Gold Nanoclusters as Efficient Sensors for Fast and Easy Visual Detection of Fe Ions in Water Using Paper-Based Portable Platform.

Herein is presented a novel and efficient portable paper-based sensing platform using paper-incorporated histidine stabilized gold nanoclusters (His-AuNCs), for the sensitive and selective detection of Fe ions from low-volume real water samples based on photoluminescence (PL) quenching. Highly photoluminescent colloidal His-AuNCs are obtained via a novel microwave-assisted method. The His-AuNCs-based sensor reveals a limit of detection (LOD) as low as 0.2 µM and a good selectivity towards Fe ions, in solution. Further, the fabricated portable sensing device based on paper impregnated with His-AuNCs proves to be suitable for the easy detection of hazardous Fe levels from real water samples, under UV light exposure, through evaluating the level of PL quenching on paper. Photographic images are thereafter captured with a smartphone camera and the average blue intensity ratio (I/I0) of the His-AuNCs-paper spots is plotted against [Fe2+] revealing a LOD of 3.2 µM. Moreover, selectivity and competitivity assays performed on paper-based sensor prove that the proposed platform presents high selectivity and accuracy for the detection of Fe ions from water samples. To validate the platform, sensing assays are performed on real water samples from local sources, spiked with 35 µM Fe ions (i.e., Fe2+). The obtained recoveries prove the high sensitivity and accuracy of the proposed His-AuNCs-paper-based sensor pointing towards its applicability as an easy-to-use, fast, quantitative and qualitative sensor suitable for on-site detection of toxic levels of Fe ions in low-volume real water samples.

Gold nanoclusters performing as contrast agents for non-invasive imaging of tissue-like phantoms via two-photon excited fluorescence lifetime imaging.

Recently, gold nanoclusters (AuNCs) have received considerable scientific interest due to their ability to generate intrinsic photoluminescence (PL), making them suitable for a wide range of applications, such as sensing, biolabeling and bioimaging. Fluorescence lifetime imaging microscopy (FLIM) is an extremely promising technique when it comes to tissue imaging, especially once combined with near-infrared two-photon excitation (TPE) due to deep tissue penetration and improved spatial resolution. In this paper, we carried out an innovative study on the ability of bovine serum albumin stabilized gold nanoclusters (BSA-AuNCs) to perform as reliable label-free contrast agents for the visualization of tissue-like agarose phantoms via TPE-FLIM. We prove that BSA-AuNCs exhibit uniform and reproducible TPE PL in the first biological window, when embedded in phantoms, under 820 nm excitation provided by a Ti:Sapphire pulsed laser. The two-photon origin of the emission signal inside the phantom is demonstrated by the quadratic dependence of the PL intensity on the excitation power. Moreover, we focused on the evaluation of BSA-AuNCs' potential as contrast agents at different concentrations inside phantoms, simulating an ex vivo environment, at three NIR excitation wavelengths, in view of defining the optimal experimental conditions for future real-tissue imaging assays. The present study aims at translating our previous results on the successful performance of BSA-AuNCs as contrast agents for in vitro FLIM imaging, using visible light, towards non-invasive ex vivo NIR imaging applications. Besides the advantageous use of the combined techniques TPE-FLIM, the novelty of our work consists of demonstrating for the first time the capacity of BSA-AuNCs to perform as bright contrast agents inside cancer-tissue mimicking phantoms. We prove that BSA-AuNCs show great promise as fluorescent contrast agents for TPE-FLIM towards image-assisted tumor surgery.

Novel (Phenothiazinyl)Vinyl-Pyridinium Dyes and Their Potential Applications as Cellular Staining Agents.

We report here the synthesis and structural characterization of novel cationic (phenothiazinyl)vinyl-pyridinium (PVP) dyes, together with optical (absorption/emission) properties and their potential applicability as fluorescent labels. Convective heating, ultrasound irradiation and mechanochemical synthesis were considered as alternative synthetic methodologies proficient for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent requirements in the synthesis of novel dye (E)-1-(3-chloropropyl)-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium bromide 3d and its N-alkyl-2-methylpyridinium precursor 1c. The trans geometry of the newly synthesized (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium iodide 3b and (E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium tetrafluoroborate 3a' was confirmed by single crystal X-ray diffraction. A negative solvatochromism of the dyes in polar solvents was highlighted by UV-Vis spectroscopy and explanatory insights were supported by molecular modeling which suggested a better stabilization of the lowest unoccupied molecular orbitals (LUMO). The photostability of the dye 3b was investigated by irradiation at 365 nm in different solvents, while the steady-state and time-resolved fluorescence properties of dye 3b and 3a' in solid state were evaluated under one-photon excitation at 485 nm. The in vitro cytotoxicity of the new PVP dyes on B16-F10 melanoma cells was evaluated by WST-1 assay, while their intracellular localization was assessed by epi-fluorescence conventional microscopy imaging as well as one- and two-photon excited confocal fluorescence lifetime imaging microscopy (FLIM). PVP dyes displayed low cytotoxicity, good internalization inside melanoma cells and intense fluorescence emission inside the B16-F10 murine melanoma cells, making them suitable staining agents for imaging applications.

Microfluidic platform for integrated plasmonic detection in laminal flow.

In this work, we propose a novel approach to design robust microfluidic devices with integrated plasmonic transducers allowing portability, reduced analysis time through dynamic measurements and high sensitivity. Specifically, the strategy we apply involves two steps: (i) the controlled deposition of gold bipyramidal nanoparticles (AuBPs) onto a functionalized solid glass substrate and (ii) the integration of the as-fabricated plasmonic substrate into a polydimethylsiloxane (PDMS) microfluidic circuit. The localized surface plasmon resonance (LSPR) sensitivity of the plasmonic-microfluidic device was evaluated by monitoring the optical responses at refractive index changes, proving a bulk sensitivity of 243 nm RIU-1 for the longitudinal LSPR band of isolated AuBPs and 150 nm RIU-1 for the band assigned to end-to-end linked nanoparticles. A strong electric field generated in the gaps between AuBPs-due to the generation of the so-called extrinsic 'hot-spots'-was subsequently proved by the volumetric surface enhanced Raman scattering (SERS) detection of molecules in continuous flow conditions by loading the analyte into the microfluidic channel via a syringe pump. In conclusion, our miniaturized portable microfluidic system aims to detect and identify, in real-time with high accuracy, analyte molecules in laminal flow, thus providing a groundwork for further complex biosensing applications.

Surface-enhanced fluorescence imaging on linear arrays of plasmonic half-shells.

Here, we perform a Surface-Enhanced Fluorescence (SEF) intensity and lifetime imaging study on linear arrays of silver half-shells (LASHSs), a class of polarization-sensitive hybrid colloidal photonic-plasmonic crystal unexplored previously in SEF. By combining fluorescence lifetime imaging microscopy, scanning confocal fluorescence imaging, Rayleigh scattering imaging, optical microscopy, and finite difference time domain simulations, we identify with high accuracy the spatial locations where SEF effects (intensity increase and lifetime decrease) take place. These locations are the junctions/crevices between adjacent half-shells in the LASHS and locations of high electromagnetic field enhancement and strong emitter-plasmon interactions, as confirmed also by simulated field maps. Such detailed knowledge of the distributed SEF enhancements and lifetime modification distribution, with respect to topography, should prove useful for improved future evaluations of SEF enhancement factors and a more rational design of efficiency-optimized SEF substrates. These linear arrays of metal-coated microspheres expand the family of hybrid colloidal photonic-plasmonic crystals, platforms with potential for applications in optoelectronic devices, fluorescence-based (bio)chemical sensing, or medical assays. In particular, due to the polarized optical response of these LASHSs, specific applications such as hidden tags for anti-counterfeiting or plasmon-enhanced photodetection can be foreseen.

Assessment of the photothermal conversion efficiencies of tunable gold bipyramids under irradiation by two laser lines in a NIR biological window.

In this work, we present a thorough study on the evaluation of the photothermal conversion efficiencies of gold nanobipyramids (AuBPs) under irradiation by two phototherapeutic laser lines at 785 and 808 nm. Due to fine tunability of the longitudinal localized surface plasmon resonance (LSPR) of AuBPs along the entire biological window, AuBPs have great potential to be applied as efficient photothermal agents in specific hyperthermia applications. Aiming to identify the most suitable AuBPs for each laser line, here we synthetized AuBPs of six different aspect ratios with longitudinal LSPR ranging from 662 to 929 nm and compared their intrinsic photothermal properties in colloidal solutions under laser irradiation at various experimental parameters such as sample volume, optical density and laser power. In addition, the experimental plasmonic resonances of the as-prepared AuBPs were perfectly simulated and their theoretical extinction and absorption cross-sections provided by finite-difference time-domain technique. Finally, we found photothermal conversion efficiencies ranging from 40% to 97% for all AuBPs systems under both NIR irradiation laser lines concluding that for the 785 nm excitation wavelength the AuBPs with longitudinal LSPR at 802 nm are most efficient, whereas in the case of the 808 nm laser line the AuBPs with optical response at 812 nm exhibit the best thermal performance. These studies are crucial for designing AuBPs as effective phototherapy agents acting alone or in combination with other plasmon-based or plasmon-assisted therapies.

Gold NanoBipyramids Performing as Highly Sensitive Dual-Modal Optical Immunosensors.

In this work, we demonstrate the feasibility of gold bipyramidal-shaped nanoparticles (AuBPs) to be used as active plasmonic nanoplatforms for the detection of the biotin-streptavidin interaction in aqueous solution via both Localized Surface Plasmon Resonance and Surface Enhanced Raman Scattering (LSPR/SERS). Our proof of concept exploits the precise attachment of the recognition element at the tips of AuBPs, where the electromagnetic field is stronger, which is beneficial to the surface sensitivity of longitudinal LSPR on the local refractive index and to the electromagnetic enhancement of SERS activity, too. Indeed, successive red shifts of the longitudinal LSPR associated with increased local refractive index reveal the attachment of para-aminothiophenol (p-ATP) chemically labeled Biotin to the Au surface and the specific capture of the target protein by biotin-functionalized AuBPs. Finite-Difference Time-Domain simulations based on the reconstructed index of refraction confirm LSPR measurements. However, the molecular identification of the biotin-streptavidin interaction remains elusive by LSPR investigation alone. Remarkably, we succeeded to complement the LSPR detection with reliable SERS measurements which permitted to (a) certify the molecular identification of biotin-streptavidin interaction and (b) extend the limit of detection of streptavidin in solution toward 10-12 M. Finally, to further probe the possibility to implement the AuBPs as dual LSPR-SERS based immunoassays in solution for real clinical diagnostics, we additionally investigated the AuBP's performance to transduce the specific antihuman IgG- human IgG binding event, providing thus a reference design for building unique plasmonic immunoassays for dual-optical detection of target proteins in aqueous solution.

Designing Efficient Low-Cost Paper-Based Sensing Plasmonic Nanoplatforms.

Paper-based platforms can be a promising choice as portable sensors due to their low-cost and facile fabrication, ease of use, high sensitivity, specificity and flexibility. By combining the qualities of these 3D platforms with the optical properties of gold nanoparticles, it is possible to create efficient nanodevices with desired biosensing functionalities. In this work, we propose a new plasmonic paper-based dual localized surface plasmon resonance⁻surface-enhanced Raman scattering (LSPR-SERS) nanoplatform with improved detection abilities in terms of high sensitivity, uniformity and reproducibility. Specifically, colloidal gold nanorods (GNRs) with a well-controlled plasmonic response were firstly synthesized and validated as efficient dual LSPR-SERS nanosensors in solution using the p-aminothiophenol (p-ATP) analyte. GNRs were then efficiently immobilized onto the paper via the immersion approach, thus obtaining plasmonic nanoplatforms with a modulated LSPR response. The successful deposition of the nanoparticles onto the cellulose fibers was confirmed by LSPR measurements, which demonstrate the preserved plasmonic response after immobilization, as well as by dark-field microscopy and scanning electron microscopy investigations, which confirm their uniform distribution. Finally, a limit of detection for p-ATP as low as 10-12 M has been achieved by our developed SERS-based paper nanoplatform, proving that our optimized plasmonic paper-based biosensing design could be further considered as an excellent candidate for miniaturized biomedical applications.

Innovative, Flexible, and Miniaturized Microfluidic Paper-Based Plasmonic Chip for Efficient Near-Infrared Metal Enhanced Fluorescence Biosensing and Imaging.

The implementation of metal enhanced fluorescence (MEF) as an efficient detection tool, especially in the near-infrared region of the electromagnetic spectrum, is a rather new direction for diagnostic analytical technologies. In this context, we propose a novel microfluidic plasmonic design based on paper for efficient MEF detection of the "proof-of-concept" biotin-streptavidin recognition interaction. Our design made use of the benefits of gold nanobipyramids (AuBPs), considering the strong enhanced electromagnetic field present at their sharp tips, and filter paper to operate as a natural microfluidic channel due to excellent wicking abilities. The calligraphed plasmonic paper, obtained using a commercial pen filled with AuBPs, was integrated in a robust sandwich optically transparent polydimethylsiloxane chip, exhibiting portability and flexibility while preserving the chip's properties. To place the Alexa 680 fluorophore at an optimal distance from the nanobipyramid substrate, the human IgG-anti-IgG-conjugated biotin sandwich reaction was employed. Thus, upon the capture of Alexa 680-conjugated streptavidin by the biotinylated system, a 1.3-fold average enhancement of the fluorophore's emission was determined by bulk fluorescence measurements. However, the local enhancement factor was considerably higher with values spanning from 5 to 6.3, as proven by mapping the fluorescence emission under both re-scan microscopy and fluorescence lifetime imaging, endorsing the proposed chip's feasibility for bulk MEF biosensing as well as high-resolution MEF bioimaging. Finally, the versatility of our chip was demonstrated by adapting the biosensing protocol for cardiac troponin I biomarker detection, validated using 10 plasma samples collected from pediatric patients and corroborated with a conventional ELISA assay.

Dual-emissive solid-state histidine-stabilized gold nanoclusters for applications in white-light generation.

The majority of present-day white-light emitting devices (WLEDs) are built upon the use of rare-earth elements, which have a short supply, are expensive and can become extremely toxic. Thus, in this work, we synthesized an eco-friendly, efficient and cheap white-light emitting material (WLEM) based on solid-state histidine-stabilized gold nanoclusters (His-AuNCs), obtained through the lyophilization of microwave-synthesized photoluminescent His-AuNCs. Their morphological and structural characterization was followed by thorough evaluation of their intrinsic solid-state photoluminescence properties via steady-state and time-resolved fluorescence spectroscopy and microscopy, at multiple excitation wavelengths. A white-light emission was observed under UV light excitation due to the two-band broad emission, with maxima at 475 and 520 nm, covering a large area of the visible spectrum. In order to evaluate the purity of the white-light emission we calculated the chromaticity coordinates, at different wavelengths, and displayed them on a CIE (Commision Internationale d'Eclairage) diagram. An excellent value of (0.36, 0.33) was found at 420 nm excitation, which falls within the range of pure white-light emission. Moreover, the His-AuNCs show great photo- and thermo-stability, thus proving their ability to perform as a reliable WLEM with potential use in the development of eco-friendly WLEDs.

Probing polyvinylpyrrolidone-passivated graphene oxide nanoflakes as contrast agents inside tissue-like phantoms via multimodal confocal microscopy.

Beside attractive electrical, thermal and mechanical properties, graphene oxide (GO) exhibits visible and near-infrared (NIR) photoluminescence (PL) and well-defined fingerprint Raman bands which are remarkable optical signatures to implement GO as new contrast agent for the visualization of cells or tissue, including cancer tumors. However, the biomedical use of GO as optical contrast agent is to some extent hindered by the intrinsic low emission efficiency especially at neutral pH. Herein, we successfully modulate the PL of GO nanoflakes in acidic and neutral medium by passivating them with polyvinylpyrrolidone (PVP), an amphiphilic and biocompatible polymer, thus improving the PL at pH relevant for biomedical applications. We demonstrate the potential of as-fabricated PVP-GO nanocomposites to operate as dual Raman-PL contrast agents inside tissue-like agarose-phantoms via scanning confocal Raman microscopy (CRM) under excitation at 532 nm. Super-resolution re-scan confocal microscopy (RCM) was further employed to investigate the distribution of PVP-GO inside biological phantoms at 3D level under three excitation lines (405, 488, and 561 nm). Finally, two-photon excited fluorescence lifetime imaging microscopy (TPE-FLIM) at 810 nm excitation reveals the ability of PVP-GO to serve as NIR-activatable contrast agent inside tissue-like phantom. Notably, PVP coating empowers GO nanoflakes not only with enhanced optical signature, but also with excellent dispersibility inside biological phantoms, thus offering improved labeling performance of as-designed imaging contrast agent.

Intrinsic Photoluminescence of Solid-State Gold Nanoclusters: Towards Fluorescence Lifetime Imaging of Tissue-Like Phantoms Under Two-Photon Near-Infrared Excitation.

Gold nanoclusters (AuNCs) have attracted extensive attention as light-emissive materials with unique advantages such as high photostability, large Stoke shifts and low toxicity. However, a better understanding of their solid-state photoluminescence properties is still needed. Herein, we investigated for the first time the intrinsic photoluminescence properties of lyophilized bovine serum albumin stabilized AuNCs (BSA-AuNCs) via fluorescence lifetime imaging microscopy (FLIM) studies performed under both one and two photon excitations (OPE and TPE) on individual microflakes, combined with fluorescence spectroscopic investigations. Both in solution and solid-state, the synthesized BSA-AuNCs exhibit photoluminescence in the first biological window with an absolute quantum yield of 6% and high photostability under continuous irradiation. Moreover, under both OPE and TPE conditions, solid BSA-AuNCs samples exhibited a low degree of photobleaching, while FLIM assays prove the homogeneous distribution of the photoluminescence signal inside the microflakes. Finally, we demonstrate the ability of BSA-AuNCs to perform as reliable bright and photostable contrast agents for the visualization of cancer tissue mimicking agarose-phantoms using FLIM approach under non-invasive TPE. Therefore, our results emphasize the great potential of the as synthesized BSA-AuNCs for ex vivo and in vivo non-invasive NIR imaging applications.

One-photon excited photoluminescence of gold nanospheres and its application in prostate specific antigen detection via fluorescence correlation spectroscopy (FCS).

Gold nanoparticles are known to exhibit appealing intrinsic plasmon-modulated photoluminescence (PL) properties which can be explored in various fluorescence-based sensing applications. In this paper, we evaluate the PL of different-sized gold nanospheres (AuNSs) under one-photon excitation (1PE) and develop a sensitive homogeneous immunoassay for the detection of prostate specific antigen (PSA) in colloidal suspension via fluorescence correlation spectroscopy (FCS). The 1PE PL of AuNSs of three different sizes are evaluated in solution phase under excitation at 405 nm via steady-state fluorescence spectroscopy measurements, while FCS analysis emphasizes the feasibility of using 1PE PL properties to monitor their diffusion behavior. Fluorescence lifetime imaging microscopy (FLIM) assays coupled with PL spectral profile analysis performed on single-particles-like structures conform the plasmonic origin of the detected PL and validate their potential of synthesized AuNSs as fluorescent probes in bioimaging and bioassays. Finally, to the best of our knowledge, we provide the first demonstration of the successful use of the 1PE PL of the synthesized AuNSs as probes for the FCS-based one-step label-free sensitive optical detection of PSA biomarker. The approach consisting in monitoring the diffusion of the AuNSs-oligomers induced by the interaction of anti-PSA-conjugated AuNSs with PSA molecules is successfully validated for the detection of PSA levels as low as 4.4 ng/ml in solution. Considering that the development of rapid, efficient and label-free biosensing methods is of continuous interest nowadays, we are confident that our results may have a strong impact on medicine towards more efficient, sensitive and reliable diagnosis.

Folic acid functionalized gold nanoclusters for enabling targeted fluorescence imaging of human ovarian cancer cells.

Photoluminescent gold nanoclusters have attracted an extensive research interest in bioimaging and therapeutics due to several distinctive advantages such as high fluorescent photostability, good dispersibility, low toxicity and large Stokes shift. However, a better understanding of the correlation between optical properties in various environments and their uptake by specific cancer cells is still needed. Herein, we developed bovine serum albumin stabilized gold nanoclusters (BSA-AuNCs) with an intrinsic tunable photoluminescence emission in the first biological window. The as-synthetized BSA-AuNCs agents consists in protein polymerized-chains dopped with AuNCs with an average size of 2-3 nm and were found to exhibit relevant properties as high photostability, temperature-dependent and excitation induced tunable red photoluminescence. The photostable BSA-AuNCs were functionalized with folic acid (FA-BSA-AuNCs) in order to achieve for the first time an active targeting of NIH:OVCAR-3 human ovarian adenocarcinoma cells, via AuNCs, towards bioimaging applications. After confirming their biocompatibility up to a concentration of 40 mg/ml, the improved cellular uptake and staining ability of FA-BSA-AuNCs compared to the BSA-AuNCs was validated by conventional wide-field epi-fluorescence microscopy, while the intracellular localization was monitored by confocal fluorescence lifetime imaging microscopy (FLIM). Considering their valuable intrinsic photoluminescent properties, the synthesized FA-BSA-AuNCs hold great promise for direct application in cellular imaging as efficient contrast agents towards early cancer diagnosis and image-guided therapy of cancer.

Intracellular Dynamic Disentangling of Doxorubicin Release from Luminescent Nanogold Carriers by Fluorescence Lifetime Imaging Microscopy (FLIM) under Two-Photon Excitation.

There is still a lack of available techniques to follow noninvasively the intracellular processes as well to track or disentangle various signals from the therapeutic agents at the site of action in the target cells. We present here the assessment of the intracellular kinetics of doxorubicin (DOX) and gold nanoparticle (AuNP) carriers by mapping simultaneously fluorescence and photoluminescence signals by fluorescence lifetime imaging microscopy under two-photon excitation (TPE-FLIM). The new nano-chemotherapeutic system AuNPs@gelatin-hyd-DOX has been fabricated by DOX loading onto the surface of gelatin-biosynthesized AuNPs (AuNPs@gelatin) through a pH-sensitive hydrazone bond. The successful loading of DOX onto the AuNPs was studied by spectroscopic methods and steady-state fluorescence, and the nanosystem pH-responsive character was validated under simulated biological conditions at different pH values (i.e., pH 4.6, 5.3, and 7.4). Considering that the fluorescence lifetime of DOX molecules at a specific point in the cell is a reliable indicator of the discrimination of the different states of the drug in the internalization path, i.e., released versus loaded, the kinetics of AuNPs@gelatin-hyd-DOX cellular uptake and DOX release was compared to that of free DOX, resulting in two different drug internalization pathways. Finally, cell viability tests were conducted against NIH:OVCAR-3 cell line to prove the efficiency of our chemotherapeutic nanosystem. TPE-FLIM technique could be considered promising for noninvasive, high-resolution imaging of cells with improved capabilities over current one-photon-excited FLIM.

Multimodal Biosensing on Paper-Based Platform Fabricated by Plasmonic Calligraphy Using Gold Nanobypiramids Ink.

In this work, we design new plasmonic paper-based nanoplatforms with interesting capabilities in terms of sensitivity, efficiency, and reproducibility for promoting multimodal biodetection via Localized Surface Plasmon Resonance (LSPR), Surface Enhanced Raman Spectroscopy (SERS), and Metal Enhanced Fluorescence (MEF). To succeed, we exploit the unique optical properties of gold nanobipyramids (AuBPs) deposited onto the cellulose fibers via plasmonic calligraphy using a commercial pen. The first step of the biosensing protocol was to precisely graft the previously chemically-formed p-aminothiophenol@Biotin system, as active recognition element for target streptavidin detection, onto the plasmonic nanoplatform. The specific capture of the target protein was successfully demonstrated using three complementary sensing techniques. As a result, while the LSPR based sensing capabilities of the nanoplatform were proved by successive 13-18 nm red shifts of the longitudinal LSPR associated with the change of the surface RI after each step. By employing the ultrasensitive SERS technique, we were able to indirectly confirm the molecular identification of the biotin-streptavidin interaction due to the protein fingerprint bands assigned to amide I, amide III, and Trp vibrations. Additionally, the formed biotin-streptavidin complex acted as a spacer to ensure an optimal distance between the AuBP surface and the Alexa 680 fluorophore for achieving a 2-fold fluorescence emission enhancement of streptavidin@Alexa 680 on the biotinylated nanoplatform compared to the same complex on bare paper (near the plasmonic lines), implementing thus a novel MEF sensing nanoplatform. Finally, by integrating multiple LSPR, SERS, and MEF nanosensors with multiplex capability into a single flexible and portable plasmonic nanoplatform, we could overcome important limits in the field of portable point-of-care diagnostics.

Probing cellular uptake and tracking of differently shaped gelatin-coated gold nanoparticles inside of ovarian cancer cells by two-photon excited photoluminescence analyzed by fluorescence lifetime imaging (FLIM).

Nowadays, the non-linear optical effect of two-photon excited (TPE) fluorescence has recently grown in interest in recent years over other optical imaging method, due to improved 3D spatial resolution, deep penetrability and less photodamage of living organism owing to the excitation in near-infrared region (NIR). In parallel, gold nanoparticles (AuNPs) have gain considerable attention for NIR TPE bio-imaging applications due to their appealing ability to generate strong intrinsic photoluminescence (PL). Here, we demonstrate the capability of differently shaped gelatin-coated AuNPs to perform as reliable label-free contrast agents for the non-invasive NIR imaging of NIH:OVCAR-3 ovary cancer cells via TPE Fluorescence Lifetime Imaging Microscopy (FLIM). Examination of the spectroscopic profile of the intrinsic signals exhibited by AuNPs inside cells confirm the plasmonic nature of the emitted PL, while the evaluation of time-dependent profile of the TPE PL signal under continuous irradiation indicates the photo-stability of the signal revealing simultaneously a photo-blinking behavior. Finally, we assess the dependence of the TPE PL signal on laser excitation power and wavelength in view of contributing to a better understanding of plasmonic TPE PL in biological media towards the improvement of TPE FLIM imaging applications based on AuNPs.

Surface Plasmon Resonance or Biocompatibility-Key Properties for Determining the Applicability of Noble Metal Nanoparticles.

Metal and in particular noble metal nanoparticles represent a very special class of materials which can be applied as prepared or as composite materials. In most of the cases, two main properties are exploited in a vast number of publications: biocompatibility and surface plasmon resonance (SPR). For instance, these two important properties are exploitable in plasmonic diagnostics, bioactive glasses/glass ceramics and catalysis. The most frequently applied noble metal nanoparticle that is universally applicable in all the previously mentioned research areas is gold, although in the case of bioactive glasses/glass ceramics, silver and copper nanoparticles are more frequently applied. The composite partners/supports/matrix/scaffolds for these nanoparticles can vary depending on the chosen application (biopolymers, semiconductor-based composites: TiO2, WO3, Bi2WO6, biomaterials: SiO2 or P2O5-based glasses and glass ceramics, polymers: polyvinyl alcohol (PVA), Gelatin, polyethylene glycol (PEG), polylactic acid (PLA), etc.). The scientific works on these materials' applicability and the development of new approaches will be targeted in the present review, focusing in several cases on the functioning mechanism and on the role of the noble metal.