This PDF file contains the front matter associated with SPIE Proceedings Volume 10892, including the Title Page, Copyright information, Table of Contents, and Author and Conference Committee lists.

Colloidal quantum dots (QDs) emitting in the visible spectrum are of interest for many biomedical applications, including bioimaging, biosensing, drug delivery, and photodynamic therapy. However, a significant limitation is that QDs typically contain cadmium, which is highly cytotoxic and makes prospects for their FDA approval for human treatment very unlikely. Previous work on biocompatible QDs has focused on indium phosphide and zinc oxide as alternative materials for QDs. However, these nanoparticles have also been shown to be cytotoxic. High-efficiency luminescent ZnTe-based QDs could be a reasonable alternative to Cd-containing QDs. We started our recent studies of ZnTe core, zinc chalcogenide shell QDs with synthesis, structural characterization, and investigation of optical properties of ZnTe/ZnSe colloidal QDs that displayed a blue-green photoluminescence under UV excitation. In this paper, the characteristics of ZnTe/ZnS QDs are compared to those of ZnTe/ZnSe QDs. We conclude that ZnTe/ZnS QDs are appealing candidates for various biomedical applications instead of the currently prominent alternative: cadmium-chalcogenide core QDs.

High temperature colloidal doping method has been used to synthesistransition metals (M = Cr, Mn, Co, Ni, Cu) MCdS/ ZnS core/shell nanoparticles. Transition metalsions were incorporated into the CdS quantum dots pursued by 6 mono lyres of the ZnS materials, which was enveloped doped CdS um dots. These doped nanoparticles were further studied using transmission electron microscopy (TEM), time resolved spectroscopy, ultraviolet-visible (UV-VIS) spectroscopy and photoluminescence (PL) spectroscopy measurements. Transition metalsdoped CdS/ZnS nanoparticles confirm the dual-peaks, first peak (sharp emission peak) is due to CdS quantum dot at 435 nm ; second peak (broad emission peak )(510–660 nm) is come from Transition metalions as compared to CdS/ZnS Quantum dots.

In this paper we present an optical characterization of nanoparticle assemblies containing plasmonic gold nanoparticles and fluorescent upconversion nanoparticles in different ratios. Both the two-photon luminescence derived from gold nanoparticles and the fluorescence originated from upconversion nanoparticles are detected. The optical signatures reflect the composition of the assemblies.

Correlating the photoluminescence (PL) properties of nanomaterials like semiconductor nanocrystals (QDs) and lanthanide-based upconversion nanocrystals (UCNPs) at the ensemble and single particle level is increasingly relevant for applications of these nanomaterials in the life sciences like bioimaging studies or their use as reporters in microfluidic assays. Aiming to derive particle architectures well suited for spectroscopic and microscopic applications, we compared the spectroscopic properties of different QDs like II/VI QDs and cadmium-free AIS/ZnS QDs as well as UCNPs of different chemical composition for particle ensembles and single particles. This included the PL spectra, fluorescence quantum yields (ΦF), brightness values, blinking behavior, and PL decay kinetics of these nanomaterials. Special emphasis was focused on measurements of the fluorescence quantum yield (ΦF) and brightness, that determine the signal size and provide a measure for the quality of nanocrystals like QDs and UCNPs which are prone to surface quenching and stabilized with coordinatively bound surface ligands. [1-3] In this respect, the potential of fluorescence correlation spectroscopy (FCS) for relative ΦF measurements of nanoparticles at ultralow concentration was explored exemplarily for ligand-stabilized CdTe nanocrystals in water. [4] References. (1) Grabolle, M.; Spieles, M.; Lesnyak, V.; Gaponik, N.; Eychmüller, A.; Resch-Genger, U., Anal. Chem 2009, 81, 6285-6294. (2) Würth, C.; Grabolle, M.; Pauli, J.; Spieles, M.; Resch-Genger, U., Nat. Prot. 2013, 8, 1535-1550. (3) Kaiser, M.; Würth, C.; Kraft, M.; Hyppanen, I.;Soukka, T.; Resch-Genger, U., Nanoscale 2017, 9, 10051 - 10058. (4) Abbandonato, G.; Hoffmann, K.; Resch-Genger, U., Nanoscale 2018, 10, 7147-7154.

Recently DNA-coated gold nanoparticles have emerged as ideal tools for the detection of mRNA in cells using dye modified oligonucleotides. However, the tracking of the gold core has been hindered by the small size of the particle core. In this work we utilize a home built set up and 43 nm DNA-coated spherical gold nanoparticles for the simultaneous imaging of mRNA detection using fluorescence microscopy and the gold nanoparticle core using two photon photoluminescence (TPPL).

Diatom microalgae represent the most abundant source of mesoporous biosilica in our planet. Their fossil derivative, diatomaceous earth (DE), consisting of diverse algal debris with nanostructured morphologies, is envisaged as a low cost silica support for biological applications. Intriguing features such as high surface/volume ratio and biocompatibility as well as unique absorption and confinement properties, make DE a suitable mesoporous support for biomolecules’ immobilization and stabilization. In this work, the model enzyme laccase was immobilized on DE using a polydopamine (PDA) coating that entraps a layer of protein molecules weakly interacting with DE. The DE/PDA/Laccase material, produced in aqueous solution under mild environment-friendly conditions, was characterized by spectroscopy and microscopy. The kinetic parameters and the recycle of the laccase were evaluated. This new hybrid material is in principle suitable for biomedical applications and for bioremediation in different environments.

Intracellular delivery of molecular cargo is the basis for a plethora of therapeutic applications, including gene therapy and cancer treatment. An efficient method to perform intracellular delivery is the photoactivation of nanomaterials that bear releasable molecular cargo. However, potential in vivo applications of this method are limited by our ability to deliver nanomaterials and light in tissue. In this paper, we will present method to perform intracellular delivery of molecular cargo on live cells and tissue by using a reusable, needle-like optofluidic probe capable of penetrating soft tissue. The probe consists of a dual-core glass fiber, enabling simultaneous light and liquid delivery. First, we used the optofluidic probe to confine an intracellular delivery mixture, composed of 100 nm gold nanoparticles (AuNP) and membrane-impermeable calcein, in the vicinity of cancer cells and mouse retinal explants. Secondly, we delivered nanosecond (ns) laser pulses (wavelength: 532 nm; duration: 5 ns; 30-90 mJ/cm2) using the same probe and without introducing a AuNP cells incubation step. We found that AuNP photo-activation caused localized and reversible disruption of the cell membrane, enabling calcein delivery into the cytoplasm. We measured 67% intracellular delivery efficacy and showed that the optofluidic probe can be used to treat cells with single-cell precision. The method presented here can facilitate in vivo treatments in soft tissue of small animals (e.g. brain, retina), such us nanomaterial-assisted neuro-stimulation, transfection and tumor elimination.

We present a wide-field imaging technique recently developed by us to measure quantitatively the optical extinction cross section σ_ext of individual nanoparticles. The technique is simple, high speed, and enables the simultaneous acquisition of hundreds of nanoparticles in the wide-field image for statistical analysis, with a sensitivity corresponding to the detection of a single gold nanoparticle down to 2nm diameter. Notably, the method is applicable to any nanoparticle (dielectric, semiconducting, metallic), and can be easily and cost-effectively implemented on a conventional wide-field microscope. Of specific significance for accurate quantification, we show that σ_ext depends on the numerical aperture of the microscope illumination due to the oblique incidence, even for spherical particles in an isotropic environment. This "long shadow" effect needs to be taken into account when comparing σ_ext to theoretical values calculated under plane wave illumination at normal incidence. Owing to the accurate experimental quantification of σ_ext, one can then use it to determine the nanoparticle size, as demonstrated here on gold nanoparticles of 30nm nominal diameter. This technique thus has the potential to become a simple and cost-effective new tool for accurate size characterization of single small nanoparticles, complementing time consuming and expensive methods such as electron microscopy.

Comprehensive single nanoparticle analysis of synthetic drug delivery systems, as well as natural occurring particles such as Extracellular Vesicles (EVs), is still a major challenge in the field, and is necessary to enhance their successful design, screening and study towards translational application. Investigating population heterogeneity is essential for nanoparticles, as their behaviour, characteristics and thus applicability are strongly affected by this, and cannot be resolved with conventional bulk analysis techniques. Here, we present a dedicated platform for comprehensive Single Particle Automated Raman Trapping Analysis (SPARTA). Nanoparticles ranging from synthetic polymer particles to liposomes or EVs can be integrally analysed by SPARTA without any modification, to obtain their size, determine functionalisation and composition, and monitor dynamic reactions occurring on their surface. The single nanoparticle nature of this approach allows highly detailed investigation in particle heterogeneity, resolving particle mixtures and tracking sequential functionalisations and dynamics on the particle surface. By using a Raman solution marker we demonstrated for the first time the capability to size single nanoparticles in a trap solely by Raman scattering, while simultaneously obtaining their compositional information, allowing novel insights in size-composition relationships. In addition, SPARTA can be applied to study in great detail the biochemical profiles of single EVs from cancerous and non-cancerous origin, towards the use of EVs as cancerous biomarkers for diagnosis, disease progression and evaluating therapeutic efficacy. SPARTA has great potential to critically impact fields from nano drug delivery system design to cancer biomarker identification and profiling.

Drag-laden magnetic nanoparticles can deliver drugs to a zone of ischemic damage for various purposes of clinical medicine. THz spectroscopy of nanoparticles with adsorbed organic and biological molecules could enable estimation of drug delivery efficiency of the nanoparticles sample and curative effect of delivering chemical substance. The first task of this work was to simulate the contribution of nanoparticles and the shell of organic molecules (glycose) to the dielectric properties of the pressed pellets, consisting of the polyethylene and nanoparticles. The second task of this paper was to study experimentally the possibility of using terahertz radiation for spectral diagnosis of NPs based on iron oxide in a biologically inert shell of silicon dioxide drug-laden with a glycose.

Nanoparticle (NP) bioconjugates have an important role in the development of photothermal (PT) therapy, a promising noninvasive approach wherein the NP acts as a light harvesting antenna to convert light into thermal energy to control cellular function. NP-mediated PT control of cellular membrane potential has gained significant interest in recent years as membrane potential regulates proliferation, migration, action potentials (in neurons), and contraction (in muscle cells). Recently gold nanoparticles (AuNPs) and Au nanorods have been demonstrated to induce action potentials via light-induced thermal activation of membrane tethered NPs. Spherical AuNPs have an efficient plasmonic output and are easily modified to interface with the cell surface. We demonstrate here that 20 nm diameter spherical AuNPs (tethered to the plasma membrane by a cholesterol moiety) transduce incident 532 nm light into proximal membrane heating that induces depolarization of membrane potential. Using these NP bioconjugates, we show the ability to controllably induce action potentials in dorsal root ganglion neurons and to control the membrane potential of rat pheochromocytoma cells. The ability to use light-actuated NP conjugates to control cellular behavior is an emerging research field with implications for neuronal and muscle cell modulation as well as in cancer therapeutics.

Lanthanide doped upconversion nanoparticles (UCNPs) are promising luminescent materials for biomedical applications due to their ability to convert low energy, non-scattering NIR light to higher energy wavelength emissions. Sensing, bioimaging, drug delivery, therapy and photobiomodulation are the expected biomedical fields that will be impacted by the combination of NIR stimulation and upconversion emission. In the case of a typical upconversion from NIR, energy transfer occurs from Yb³⁺ sensitizer ions, which can be excited at 980 nm, to the activator lanthanide ions such as Er³⁺, Tm³⁺, Ho³⁺, Eu³⁺. Synthesis and design of the UCNPs and their introduction into the biological system requires stringent procedures due to the complex nature of biological environment at the cellular level. Our goal in this study is to develop small size, biocompatible UCNPs with a facile microwave assisted synthesis method and utilize them for photobiomodulation of neuronal cells. We aim to elucidate the intracellular mechanisms that are impacted by the upconversion photons emitted from designed nanotransducers towards stimulation of cell function. For this purpose, we sensitized blue emitting NaYF₄ UCNPs and in-vitro laser irradiation experiments are conducted with NG108-15 (neuroblastoma-glioma hybrid) cells. Experiments are designed to further investigate the thermal and chemical effects that contribute to the resulted modifications in the cell function.

In this work, we utilized a unique polyhistidine peptide-DNA to conjugate with DHLA-capped QD625 (QD625) and different lengths of ssDNAs which were complementary to different parts of polyhistidine peptide-DNA to conjugate with Tb, and therefore, Tb are located away from the surface of QD with the length of polyhistidine peptide in addition of the length of ssDNA of 0, 2, 4, 6, 10 and 14 bases, or the lengths of dsDNA of 10, 14, 18, 22 and 26 base pairs, respectively. The lifetime of QD became longer and longer as Tb was moving away from QD. The distances calculated from Tb and QD channels by fitting were in an excellent agreement with the model that demonstrated temporal multiplexing FRET using a single Tb-QD FRET pair is successfully developed and can be used as biosensor.

Time-gated Förster resonance energy transfer (TR-FRET) introduces a time-gate before the detection of the fluorescence spectra or photon count. If the donor is sufficiently long-lived TR-FRET allows for any initial acceptor sensitization to decay before the measurement. TR-FRET in the μs range is particularly advantageous for small molecule assays as it eliminates background fluorescence from screening compounds, which typically have ns lifetimes. The sensor we developed utilizes Terbium (Tb)-labeled antibodies (Ab) that selectively recognizes adenosine diphosphate (ADP). The Tb emitters have fluorescence lifetimes on the ms scale, making them excellent candidates for TR-FRET donors. In an attempt to increase the FRET signal we utilized a semiconductor quantum dot (QD) as an acceptor. The QD presented an ADP modified His6-peptide conjugated to its surface via self-assembly metal-affinity coordination, which bound the Tb labeled Ab to the QD surface. QDs have large extinction coefficients, broad absorption, brightness, and sharp emission peaks, optimal for sensitive and multiplexed detection. By using a QD acceptor the Förster radius was increased by approximately 2 nm as compared to traditional organic dyes. We were able to demonstrate a Tb-to-QD based TR-FRET bioassay for broadly applicable ADP sensing, working at nM concentrations for sensor, analyte, and enzyme. Quantitative values were obtained for the kinetics of a model enzyme (glucokinase). The specific sensor was also capable of discriminating enzyme inhibitor capabilities of structurally similar compounds. The strategy of using modified peptides to present antibody epitopes on QD surfaces is readily transferable to other assays.

An alternative molecular recognition approach was developed for sensing small molecule analytes using the differential binding of an allosteric transcription factor (TF, specifically TetR) to its cognate DNA as the molecular recognition element coupled with fluorescent resonance energy transfer (FRET) to yield an internally calibrated optical signal transduction mechanism. Sensors were evaluated comprising Cy5-modified DNA (FRET acceptor) with either a tdTomato-TetR fusion protein (FP-TF) or quantum dot-TetR conjugate (QD-TF) as the FRET donor by measuring the ratio of acceptor and donor fluorescence intensities (FA/FD) with titrations of a derivative of the antibiotic tetracycline, anhydrous tetracycline (aTc). A proof-of-concept FRET-based biosensor was successfully demonstrated through the modulation of FA/FD signal intensities based on varying analyte concentrations. Sensor design parameters affecting overall signal-to-noise ratio and sensitivity of the sensors are also identified.

In recent years, bioessential element-based chalcogenides, namely Selenium (Se) and Tellurium (Te), have established noted fundamentals as metal-based protective agents. In relation to anti-cancer therapeutics, Se in particular exhibits promising characteristics as potentially effective treatment alternatives due to its notoriety as a highly selective, drug-coordinating element. In addition to their competitive clinical resume, Se nanoparticles packaged as chalcogenides are believed to support anti-inflammatory, antimicrobial and antifungal efforts. Though more is needed to understand the biological effect these materials play within the body, studies postulate that there is significant potential for Se based nanoalloys. Partnering Se with elemental neighbor Te, SexTel-x, these alloys function as target mediators. They are believed to sustain cell viability ARPE-19 cells while initiating apoptotic effects on MDA-MB-453 cancer cells, along with promoting the reduction of reactive oxygen species (ROS) activity. Lastly, cellular integrity is maintained by the lack of DNA fragmentation within normal cells, further supporting the efforts of employing SexTel-x alloys as potential anti-cancer agents. Ultimately, this research will serve as fundamental currency marketing SexTel-x nanoalloys as synergistically compliant anti-inflammatory, anti-cancer therapeutic agents, priming the tone for treatment efficacy.

High atomic number nanomaterials have been explored as a tool for improving cancer therapeutics. Gold nanoparticles are a system that has been introduced as they can act as effective radiation dose enhancers and anticancer drug carries. Gold nanoparticles have unique physiochemical properties that allow them to be probed in cells using techniques such as scanning electron microscopy and hyper spectral imaging. Optimization of gold nanoparticle uptake into 3D in-vitro models is essential to optimizing future cancer therapeutic applications and bridging the gap between in-vitro and in-vivo tumor environments. The uptake of functionalized gold nanoparticles into 2D monolayer and 3D spheroid cell models was tested. Functionalization of the GNPs was confirmed by use of dynamic light scattering, UV-Visible light spectroscopy, the Zeta potential, and imaged with a scanning electron microscope and hyper spectral imaging. These findings suggest that both the size and functionalization of the gold nanoparticles should be considered in future 3D in-vitro studies.

The coupling of a gold nanoparticle to peptides and/or nucleic acids can allow imaging of intracellular events when optically labelled by molecular dyes. The use of a gold bio-optical transponder (BOT) capable of simultaneously reporting the timing of intracellular cargo delivery, cargo release from a gold nanoparticle, and subsequent cellular processing provides direct insight into the endo-lysosomal processing of nano-based delivery probes. The paper will explore use of BOT sensors to monitor peptide dependent uptake of AuNPs into native vs drug resistant melanoma cells; monitor the effect off surface coupling of gene knock-in, and explore the ability to evaluate the evolving intracellular pH (pHi) during endosomal maturation. The BOT is designed around use of multicolor surface energy transfer (SET) to simultaneously track processes and provide an internal reference for ratiometric analysis of events. The use of a BOT based sensor to explore intracellular processing may enable greater insight into our understanding of the fundamental processes of biology.

The quest for new techniques to measure single nanoparticle is a great impetus to research efforts to understand individual behaviors. Herein, we develop an electrochemiluminescence (ECL) microscopy for visualization of stochastic collision electrochemistry of single nano-emitters without the interference of current and optical background. This design uses a water-immersion objective to capture the ECL emission of nanoparticles near the specular electrode surface for enhancing light collection efficiency. The approach enables us to trace the collision trajectory of multiple nanoparticles and spatially distinguish simultaneous collisions. This imaging technique displays great potential for applications in single-particle electrochemical and analytical research. In addition to imaging single nano-emitters, we perform single cell imaging by the homemade ECL microscope. Since cells are immobilized on the electrode surface, the steric hindrance and the insulation from the cells make it difficult to obtain a luminous cell ECL image. To solve this problem, direct ECL imaging of a single cell was investigated and achieved on chitosan and nano-TiO2 modified fluoride-doped tin oxide conductive glass (FTO/TiO2/CS). The permeable chitosan film is not only favorable for cell immobilization but also increases the space between the bottom of cells and the electrode; thus, more ECL reagent can exist below the cells compared with the cells on a bare electrode, which guarantees the high sensitivity of quantitative analysis. The light intensity is correlated with the H2O2 concentration on FTO/TiO2/CS, which can be applied to analyze the H2O2 released from cells at the single-cell level.

Emerging advances in iron oxide nanoparticles exploit their high magnetization for various applications, such as catalysis, bioseparation, hyperthermia, and magnetic resonance imaging (MRI). In contrast to the excellent magnetic performance, their upconverted photoluminescence have not been thoroughly explored, thus limiting their development as a tool in photomedicine. In this work, we develop a seed/growth-inspired synthesis combined with primary mineralization and a ligand-assisted secondary growth strategy to prepare mesostructured α-FeOOH nanorods (NRs). Because α-FeOOH rods are all iron-based composites, they exhibit low cytotoxicity towards cells. Surprisingly, these mesoporous α-FeOOH mesostructures display strong third harmonic generation (THG) signals under near-infrared excited wavelength at 1230 nm. They exhibited a much stronger THG intensity compared to naked α-FeOOH NRs. Using these unique nonlinear optical properties, we demonstrate that α-FeOOH rods can serve as contrast agents in THG microscopy for the cell tracking as well as angiography in vivo. Vessel walls can be revealed after the clearance of particles. Our results provide a new strategy of material synthesis for obtaining high THG imaging contrast.

Nanotherapeutics are destination-aware drug cargo which help improving the treatment duration and reducing drug side effects. Biodegrability and biocompatibility of chitosan nanoparticles (ChNPs) are progressively used for biomedical applications. In this work, ChNPs were prepared by ionotropic gelation technique through electrostatic interaction between chitosan chains (positively charged) and non-toxic polyanions tripolyphosphate (TPP) (negatively charged). The ratio between chitosan and TPP was found to have a crucial role on the size, the poly dispersity index (PDI), and the zeta potential of the prepared ChNPs. Different volumetric ratios of Ch:TPP (1:1, 2:1, and 3:1) were prepared. The optimally prepared ChNPs were used for encapsulating the anticancer drug, 5- Fluorouracil (5-FU) forming (5-FU-ChNPs). ChNPs and 5-FU-ChNPs gave hydrodynamic diameter of 140.4 nm and 293 nm respectively, which were confirmed by transmission electron microscope (TEM). Cytotoxic assay was carried out on hepatocellular carcinoma (HepG2) cell line and it revealed the effectiveness of laser-irradiated 5-FU-ChNPs compared with in absence of laser irradiation. The 5- FU-ChNPs exhibited the desired light absorption causing hydrolysis and secession of the polymer chains at the tumor site. This led to efficient destruction of the cancer cells and verified the effectiveness of 5-FU-ChNPs as drug delivery system assisted by laser irradiation.

The use of microrobotics in biological systems has attracted much attention due to its diverse functionality and controllable motion. Combining magneto-polymer nanocomposite with fluorescent nanoparticles provides new potentials for micro-machining in biomedicine. Due to their contact-free, remote controllable, and biocompatible properties, iron oxide (Fe₃O₄) nanoparticles have been widely used in magnetic resonance imaging (MRI), cell targeting, and drug delivery, and are considered to be an attractive option in further development of micro- scale systems. The fluorescent properties and high photo-stability of semiconductor nanocrystal quantum dots (QDs) have also shown great potential for bio and quantum applications. This work explores the fabrication and manipulation of bimodel fluorescent-magnetic microstructures on a new photo-patternable composite consisting of colloidal semiconductor nanocrystal QDs (CdSe/CdS), superparamagnetic magnetite nanoparticles (Fe₃O₄), and a commercial SU-8 photoresist. Using a mask optical lithography technique, we fabricated 2D microstructures of various shapes and demonstrated their strong response to an externally applied magnetic field. Linear, rotational, and spinning movements are presented. Photo-radiation fluorescent checking was used to map the location of the QDs within the microstructures and strong fluorescent emitters were characterized. Combining Fe₃O₄ nanoparticles, QDs, and SU-8 polymer into a single complex microstructure contributes to a wide range of applications in biomedicine such as biological-labeling, in vivo cargo transportation, and micro-machining, as well as perspectives in quantum technology.

Here, we report the preparation of carbon dots (CDs) and doping with different elements namely boron, nitrogen and phosphorous using facile single step hydrothermal method. We used biopolymers as the source material for CDs synthesis. The prepared carbon dots and elements (B, N and P) doped carbon dots’ physicochemical properties are investigated using different analytical techniques. Several analytical characteristics such as Uv-visible spectroscopy, fluorescent spectroscopy and transmission electron microscopy confirm the doping of element into carbon dots. From DLS analysis it was found that the prepared carbon dots are range from 3-9 nm. Excitation dependent fluorescence with high quantum yields for B and N doped CDs showed 47% and 44%, respectively. The doped CDs impact on cell viability was investigated against neuronal PC12 cells. Interestingly, the prepared carbon dots did not affect the differentiation process of neuronal cells. Hence, the highly fluorescent CDs can be served as excellent materials for neural tissue engineering as well as biomedical engineering applications.