Luminescent semiconductor nanocrystals, also known as quantum dots (QDs), have had a major impact on applications of optical techniques in the life sciences, such as biosensing and bioimaging. Their unique optical properties, such as broad absorption bands with high molar extinction coefficients, together with their narrow size-dependent photoluminescence (PL) bands, high PL quantum yields (QYs), and a high photostability, are unique advantages for QDs as optical reporters in immunoassays, theranostics, in vitro/in vivo imaging, and flow cytometry. This also laid the foundation for numerous applications of optical multiplexing and barcoding. The most popular QD element compositions are based on heavy-metal elements, such as cadmium or lead, which pose a high toxic risk and are meanwhile restricted in Europe by REACH regulation. Increasing environmental concerns and the growing interest in and need for nanomaterials accessible by sustainable synthesis routes triggered the search for more eco-friendly QDs in the last years. Promising heavy-metal free candidates are ternary AgInS2 QDs, which can be synthesized with high-quality optical properties using aqueous synthesis methods. However, the use of conventional heating mantles or oil baths to heat the reaction mixture to the desired reaction temperature can lead to significant batch to batch variations caused by an inhomogeneous heat diffusion influencing the nucleation and growth of the QDs. A solution to this problem is the use of microwave-assisted heating. This enables a fast and homogeneous heat distribution throughout the entire reaction vessel, resulting in high-quality nanomaterials prepared with high batch-to-batch reproducibility. In this chapter, we will describe the synthesis steps for the preparation of AgInS2/ZnS QDs using microwave-assisted heating. The influence of the stabilizing ligands, variation of precursor concentrations, and the pH of the reaction solution will be detailed in the note section.

Knowledge of the structure-property relationships of functional nanomaterials, including, for example, their size- and composition-dependent photoluminescence (PL) and particle-to-particle variations, is crucial for their design and reproducibility. Herein, the Angstrom-resolution capability of an analytical ultracentrifuge combined with an in-line multiwavelength emission detection system (MWE-AUC) for measuring the sedimentation coefficient-resolved spectrally corrected PL spectra of dispersed nanoparticles is demonstrated. The capabilities of this technique are shown for giant-shell CdSe/CdS quantum dots (g-QDs) with a PL quantum yield (PL QY) close to unity capped with oleic acid and oleylamine ligands. The MWE-AUC PL measurements are calibrated and validated with certified fluorescence standards. The spectrally corrected and size-dependent PL spectra of the g-QDs derived from a single MWE-AUC experiment are then analyzed and compared with the results of single-particle spectroscopic studies, yielding the PL spectra, decay kinetics, and blinking behavior of individual g-QDs. This study underlines the vast potential of MWE-AUC with in-line optical detection for the characterization of advanced nanomaterials with a complex structure.

Semiconductor quantum dots (QDs) possess unique electronic and optical properties, making them promising candidates for applications in light-emitting diodes, solar cells, bioimaging, and photocatalysis. Precise control over their size, shape, and chemical and electronic structure is crucial to ensure the desired functional properties and optimize device performance. However, challenges in QD synthesis and post-synthesis modification persist, especially in large-scale production. This study addresses the classification of QDs synthesized in a tubular flow reactor consisting of a mixture of the desired InP/ZnS core-shell QDs and QDs made from the shell material, i.e., here ZnS QDs formed as a byproduct during the formation step of the ZnS shell. The homogeneous nucleation of ZnS nanoparticles from the shelling material introduces a heterogeneity in size and composition and affects the optical properties of the resulting QDs. To address this issue, we developed a size-selective agglomeration (SSA) technique by incrementally introducing ethanol as a poor solvent and classified the synthesized QDs into 13 distinct fractions. These 13 fractions are sorted into three distinct groups: (i) larger InP/ZnS QDs, (ii) a combination of smaller InP/ZnS QDs and larger ZnS QDs, and (iii) predominant ZnS QDs with some very tiny InP/ZnS QDs. The comprehensive characterization of the fractions was conducted using UV-visible absorption spectroscopy, photoluminescence spectroscopy, high-resolution scanning transmission electron microscopy, energy-dispersive X-ray spectroscopy, total reflection X-ray fluorescence, and analytical ultracentrifugation. We could demonstrate that our method effectively separated unwanted ZnS QDs from the target InP/ZnS QDs. In addition, the fractions enriched in smaller InP/ZnS QDs exhibited a higher photoluminescence quantum yield compared to the fractions with larger QDs. This demonstrates the efficacy of SSA in fine-tuning the composition of QD mixtures produced on a larger scale to improve their functional properties. This approach provides fundamental understanding toward the development of a scalable two-dimensional classification process for such ultrasmall nanoparticles by particle size and composition.

Hydrothermal-based synthetic methods of quantum dots allow for the exploration of reaction parameters normally inaccessible to typical aqueous-based batch reactions, such as elevated reaction temperatures (>100 °C) and reaction pressures above atmospheric pressure. Coupled with microwave heating, new instantaneously bio-compatible quantum dots (QDs) with enhanced opitcal properties can be yielded. As of today, aqueous-based synthetic methods often lag behind their organic analogues in terms of the photophysical properties of the QDs obtained and the ease of modulation of both the emission wavelength and crystallite size. Using a novel microwave-assisted hydrothermal approach, the synthesis of silver sulphide (Ag2S) QDs exhibiting NIR emission spanning the biological transparency windows via modulation of the reaction parameters has been developed. The intrinsic link between their optical and structural properties is explored via laboratory and synchrotron-based structural analysis techniques. Their toxicity towards a hepatic cell line was assessed, and related back to their structure and size. Overall this work aims to not only further develop the repertoire of synthetic methods for the synthesis of Ag2S QDs, but also paves the way for the development of safer QDs suitable for future clinical applications.

Real-time imaging of antibody-drug conjugates (ADCs) offers valuable insights for assessing tumor targeting specificity, monitoring therapeutic efficacy, and detecting off-target accumulation that may cause adverse effects. To enable precise tracking, we developed a versatile fluorescent platform based on an NIR-II emitting aza-BODIPY dye, which can be site-specifically grafted onto an IgG1 antibody to generate well-defined fluorescent ADCs. As a proof of concept, we synthesized an HER2-targeting trastuzumab immunoconjugate bearing a NIR-II aza-BODIPY fluorophore. The cytotoxic monomethyl auristatin E (MMAE) payload was introduced in the final step, resulting in a trackable and homogeneous ADC suitable for both in vitro and in vivo investigations. The resulting Trastu-azaNIRII-MMAE selectively accumulated in HER2-positive subcutaneous tumors, significantly reducing the tumor growth. Using NIR-II optical imaging, a single injection of the NIR-II-ADC allowed for the detection of the conjugate over a period of more than one month, highlighting its potential for long-term tracking and therapeutic applications.

Understanding how structural and optical properties of metallic nanoclusters can be tuned by proteins is crucial for the use of these hybrid molecules in biomedical applications. The interaction of proteins with ultrasmall, atomically-precise gold nanoclusters (Au-NCs) has been mainly investigated in the context of structured proteins, while their behavior with intrinsically disordered proteins (IDPs) remains unexplored. This work examines the structural and optical properties of Au-NCs interacting with bioengineered IDPs containing up to three cysteines. We show that, by exploiting the conformational flexibility of cysteine-containing IDPs, we can anchor proteins to Au-NCs in a position-specific manner, leading to new bioconjugates with properties that differ from those of the individual components. We observed an up to 15-fold photoluminescence enhancement depending on the number of cysteines anchored. By combining mass spectrometry, small-angle X-ray scattering (SAXS), and computational modelling, the ensemble structures of nine bioconjugates with different stoichiometries were elucidated, indicating their overall compactness. Our results suggest that the interface between these atomically-precise species and the conformationally fluctuating protein is responsible for the optical properties of these nanobioconjugates. This research improves our understanding of Au-NC– protein interactions, paving the way to novel nano-molecular hybrid conjugates with tunable properties for bioimaging and therapeutic applications. ### Competing Interest Statement The authors have declared no competing interest. * NC : nanoclusters IDP : intrinsically disordered protein NIR : near-infrared pMBA : para-mercaptobenzoic acid cys : cysteine R g : gyration radius PAGE : Polyacrylamide Gel Electrophoresis MS : Mass Spectrometry MW : Molecular Weight PL : photoluminescence SAXS : Small-Angle X-ray Scattering SEC : Size-Exclusion Chromatography EOM : Ensemble Optimization Method ICT : intramolecular charge transfer BSA : Bovine Serum Albumin. Agence Nationale de la Recherche, ANR-22-CE29-0022, ANR-20-CE92-0039-01 Deutsche Forschungsgemeinschaft, DFG RE 1203/38-1

InP/ZnS core/shell quantum dots (QDs), recognized as highly promising heavy-metal-free emitters, are increasingly being utilized in lighting and display applications. Their synthesis in a tubular flow reactor enables production in a highly efficient, scalable, and reproducible manner, particularly when combined with a single-source shell precursor, such as zinc diethyldithiocarbamate (Zn(S2CNEt(2))(2)). However, the photoluminescence quantum yield (PLQY) of QDs synthesized with this route remains significantly lower compared with those synthesized in batch reactors involving multiple steps for the shell growth. Our study identifies the formation of absorbing, yet nonemissive ZnS nanoparticles during the ZnS shell formation process as a main contributing factor to this discrepancy. By varying the shelling conditions, especially the shelling reaction temperature and InP core concentration, we investigated the formation of pure ZnS nanoparticles and their impact on the optical properties, particularly PLQY, of the resultant InP/ZnS QDs through ultraviolet-visible (UV-vis) absorption, steady-state and time-resolved photoluminescence (PL) spectroscopy, scanning transmission electron microscopy (STEM), and analytical ultracentrifugation (AUC) measurements. Our results suggest that process conditions, such as lower shelling temperatures or reduced InP core concentrations (resulting in a lower external surface area), encourage homogeneous nucleation of ZnS. This reduces the availability of shell precursors necessary for effective passivation of the InP core surfaces, ultimately resulting in lower PLQYs. These findings explain the origin of persistently underperforming PLQY of InP/ZnS QDs synthesized from this synthesis route and suggest further optimization strategies to improve their emission for lighting and display applications.

Semiconductor quantum dots (QDs) possess unique electronic and optical properties, making them promising candidates for applications in light emitting diodes (LEDs), solar cells, bioimaging, and photocatalysis. Precise control over their size, shape, and chemical and electronic structure is crucial to ensure the desired functional properties and optimize device performance. However, challenges in QD synthesis and post-synthesis modification persist, especially in large-scale production. This study addresses the classification of QDs synthesized in a tubular flow reactor consisting of a mixture of the desired InP/ZnS core-shell QDs and QDs made from the shell material, i.e., here ZnS QDs formed as a byproduct during the ZnS shell formation step. The formation of nanoparticles from the shelling material introduces a heterogeneity in size and composition and affects the optical properties of the resulting QDs. To address this issue, we developed a size-selective agglomeration (SSA) technique by incrementally introducing ethanol as a poor solvent and classified the synthesized QDs into 13 distinct fractions. These 13 fractions sorted into 3 distinct groups: (i) larger InP/ZnS QDs, (ii) a combination of smaller InP/ZnS QDs and larger ZnS QDs, and (iii) predominant ZnS QDs with some very tiny InP/ZnS QDs. The comprehensive characterization of the fractions was conducted using UV-visible (UV-vis) absorption spectroscopy, photoluminescence (PL) spectroscopy, high-resolution scanning transmission electron microscopy (HR-STEM), energy-dispersive X-ray spectroscopy (EDXS), total reflection X-ray fluorescence (TXRF), and analytical ultracentrifugation (AUC). We could demonstrate that our method effectively separated unwanted ZnS QDs from the target InP/ZnS QDs. In addition, the fractions enriched in smaller InP/ZnS QDs exhibited a higher photoluminescence quantum yield (PLQY) compared to the fractions with larger QDs. This demonstrates the efficacy of SSA in fine-tuning the composition of QD mixtures produced on a larger scale to improve their functional properties. In the future, this approach can pave the way towards a scalable two-dimensional classification process for such ultra-small nanoparticles by particle size and composition.

Heterogeneous sandwich immunoassays are widely used for biomarker detection in bioanalysis and medical diagnostics. The high analyte sensitivity of the current "gold standard" enzyme-linked immunosorbent assay (ELISA) originates from the signal-generating enzymatic amplification step, yielding a high number of optically detectable reporter molecules. For future point-of-care testing (POCT) and point-of-need applications, there is an increasing interest in more simple detection strategies that circumvent time-consuming and temperature-dependent enzymatic reactions. A common concept to aim for detection limits comparable to those of enzymatic amplification reactions is the usage of polymer nanoparticles (NP) stained with a large number of chromophores. We explored different simple NP-based signal amplification strategies for heterogeneous sandwich immunoassays that rely on an extraction-triggered release step of different types of optically detectable reporters. Therefore, streptavidin-functionalized polystyrene particles (PSP) are utilized as carriers for (i) the fluorescent dye coumarin 153 (C153) and (ii) hemin (hem) molecules catalyzing the luminol reaction enabling chemiluminescence (CL) detection. Additionally, (iii) NP labeling with hemin-based microperoxidase MP11 was assessed. For each amplification approach, the PSP was first systematically optimized regarding size, loading concentration, and surface chemistry. Then, for an immunoassay for the inflammation marker C-reactive protein (CRP), the analyte sensitivity achievable with optimized PSP systems was compared with the established ELISA concept for photometric and CL detection. Careful optimization led to a limit of detection (LOD) of 0.1 ng/mL for MP11-labeled PSP and CL detection, performing similarly well to a photometric ELISA (0.13 ng/mL), which demonstrates the huge potential of our novel assay concept.

The development of new fluorescent organic probes effective in the NIR-II region is currently a fast-growing field and represents a challenge in the domain of medical imaging. In this study, we have designed and synthesized an innovative series of aza-boron dipyrromethenes emitting in the NIR-II region. We have investigated the effect of different water-solubilizing groups not only on the photophysical properties of the compounds but also on their in vitro and in vivo performance after bioconjugation to the antibody trastuzumab. Remarkably, we discovered that the most lipophilic compound unexpectedly displayed the most favorable in vivo properties after bioconjugation. This underlines the profound influence that the fluorophore functionalization approach can have on the efficiency of the resulting imaging agent.

Highly emissive Ag2S nanocrystals (NCs) passivated with a gradated shell incorporating Se and Zn were synthesized in air, and the temperature dependence of their photoluminescence quantum yield (PLQY) was quantified in both organic and aqueous media at similar to 1200 nm. The relevance of this parameter, measured at physiological temperatures, is highlighted for applications that rely on the near infrared (NIR) photoluminescence of NCs, such as deep NIR imaging or luminescence nanothermometry. Hyperspectral NIR imaging shows that Ag2S-based NCs with a PLQY in organic media of about 10% are inefficient for imaging at 40 degrees C through 20 mm thick tissue with low laser irradiation power densities. In contrast, water-transferred Ag2S-based NCs with an initial PLQY of 2% in water exhibit improved robustness against temperature changes, enabling improved imaging performance.

Luminophore stained micro- and nanobeads made from organic polymers like polystyrene (PS) are broadly used in the life and material sciences as luminescent reporters, for bead-based assays, sensor arrays, printable barcodes, security inks, and the calibration of fluorescence microscopes and flow cytometers. Initially mostly prepared with organic dyes, meanwhile luminescent core/shell nanoparticles (NPs) like spherical semiconductor quantum dots (QDs) are increasingly employed for bead encoding. This is related to their narrower emission spectra, tuneability of emission color, broad wavelength excitability, and better photostability. However, correlations between particle architecture, morphology, and photoluminescence (PL) of the luminescent nanocrystals used for encoding and the optical properties of the NP-stained beads have been rarely explored. This encouraged us to perform a screening study on the incorporation of different types of luminescent core/shell semiconductor nanocrystals into polymer microparticles (PMPs) by a radical-induced polymerization reaction. Nanocrystals explored include CdSe/CdS QDs of varying CdS shell thickness, a CdSe/ZnS core/shell QD, CdSe/CdS quantum rods (QRs), and CdSe/CdS nanoplatelets (NPLs). Thereby, we focused on the applicability of these NPs for the polymerization synthesis approach used and quantified the preservation of the initial NP luminescence. The spectroscopic characterization of the resulting PMPs revealed the successful staining of the PMPs with luminescent CdSe/CdS QDs and CdSe/CdS NPLs. In contrast, usage of CdSe/CdS QRs and CdSe QDs with a ZnS shell did not yield luminescent PMPs. The results of this study provide new insights into structure-property relationships between NP stained PMPs and the initial luminescent NPs applied for staining and underline the importance of such studies for the performance optimization of NP-stained beads.

The 2023 Nobel Prize in Chemistry was awarded to Aleksey I. Ekimov (prize share 1/3), Louis E. Brus (prize share 1/3), and Moungi G. Bawendi (prize share 1/3) for groundbreaking inventions in the field of nanotechnology, i.e., for the discovery and synthesis of semiconductor nanocrystals, also termed quantum dots, that exhibit size-dependent physicochemical properties enabled by quantum size effects. This feature article summarizes the main milestones of the discoveries and developments of quantum dots that paved the road to their versatile applications in solid-state lighting, display technology, energy conversion, medical diagnostics, bioimaging, and image-guided surgery.

Quantum dots (QDs) possess unique optoelectronic properties, which make them very attractive to be used as optical probes in biosensing and bioimaging applications. The strong absorbance and light scattering of biological compounds like tissue and blood in the visible range pose a problem. However, if optical probes emitting in the near-infrared (NIR) range are used, scattering, absorption, and autofluorescence of biological components are strongly reduced. This allows for an increased light penetration depth, higher spatial and temporal resolution for the investigation of biological processes. The synthesis and application of NIR emitting QDs is a fast-growing research field and the benefits of using QDs were demonstrated for a variety of applications like as photoelectrochemical biosensor, in vivo vascular imaging, and fluorescence-guided surgery. This article reviews the state-of-the-art developments in the preparation of NIR/IR QDs and highlights the latest research about their utilization in biosensing and bioimaging applications.

Using fluorescence-guided surgery (FGS) to cytoreductive surgery helps achieving complete resection of microscopic ovarian tumors. The use of visible and NIR-I fluorophores has led to beneficial results in clinical trials; however, involving NIR-II dyes seems to outperform those benefits due to the deeper tissue imaging and higher signal/noise ratio attained within the NIR-II optical window. In this context, we developed NIR-II emitting dyes targeting human epidermal growth factor receptor 2 (HER2)-positive ovarian tumors by coupling water-soluble NIR-II aza-BODIPY dyes to the FDA-approved anti-HER2 antibody, namely, trastuzumab. These bioconjugated NIR-II-emitting dyes displayed a prolonged stability in serum and a maintained affinity toward HER2 in vitro. We obtained selective targeting of HER2 positive tumors (SKOV-3) in vivo, with a favorable tumor accumulation. We demonstrated the fluorescence properties and the specific HER2 binding of the bioconjugated dyes in vivo and thus their potential for NIR-II FGS in the cancer setting.

Gold-based nanoparticles below 2 nm in size are promising as luminescent probes for in vivo bioimaging, owing to their brightness and rapid renal clearance. However, their use as contrast agents in the near-infrared II (NIR-II, 1000-1700 nm) range remains challenging due to their low photoluminescence (PL) quantum yield. To address this, PL enhancement can be achieved by either rigidifying the ligand-shell structure or increasing the size of the ligand shell. In this study, we synthesized ultra-small gold nanoparticles stabilized by co-ligands, namely monothiol and short dithiol molecules. By precisely controlling the amount of reducing agent used during particle preparation, we successfully modulated the physicochemical properties of the co-ligand shell, including its size, composition, and structure. Consequently, we achieved a remarkable 60-fold increase in the absorption cross-section at 990 nm while maintaining the small size of the 1.5-nm metal core. The analytical and optical characterization of our thiol-capped gold nanoparticles indicates that the ligand shell size is governed by the quantity of the reducing agent, which, in turn, impacts the balance between radiative and non-radiative processes, thereby influencing the PL quantum yield. Near-infrared absorption and photoluminescence in the second biological window of ultra-small gold nanoparticles could be tuned by controlling the physicochemical properties of the ligand shell.

Quantum dots (QDs) are widely used in optoelectronics, lighting, and photovoltaics leading to their potential release into the environment. The most promising alternative to the highly toxic cadmium selenide (CdSe) QDs are indium phosphide (InP) QDs, which show reduced toxicity and comparable optical and electronic properties. QD degradation leads to the release of toxic metal ions into the environment. Coating the QD core with robust shell(s) composed of another semi-conductor material enhances their properties and protects the QD from degradation. We recently developed double-shelled InP QDs, which proved to be less toxic than single-shell QDs. In the present study, we confirm their reduced cytotoxicity, with an LC50 at 77 nM for pristine gradient shell QDs and >100 nM for pristine thin and thick shell QDs. We also confirm that these three QDs, when exposed to simulated sunlight, show greater cytotoxicity compared to pristine ones, with LC50 ranging from 15 to 23 nM. Using a combination of spectroscopic and microscopic techniques, we characterize the degradation kinetics and transformation products of single- and double-shell QDs, when exposed to solar light at high temperature, simulating environmental conditions. Non-toxic pristine QDs degrade to form toxic In–phosphate, In–carboxylate, Zn–phosphate, and oxidized Se, all of which precipitate as heterogeneous deposits. Comparison of their degradation kinetics highlights that the QDs bearing the thickest ZnS outer shell are, as expected, the most resistant to photodegradation among the three tested QDs, as gradient shell, thin shell, and thick shell QDs lose their optical properties in less than 15 min, 60 min, and more than 90 min, respectively. They exhibit the highest photoluminescence efficiency, i.e., the best functionality, with a photoluminescence quantum yield in aqueous solution of 24%, as compared to 18% for the gradient shell and thin shell QDs. Therefore, they can be considered as safer-by-design QDs.

Binary photoluminescent semiconductor nanocrystals (quantum dots, QDs) are one of the best studied fluorescent nanomaterials, and their unique optoelectronic properties paved the road to many applications in (bio)nanophotonics, optoelectronics, and photovoltaics. However, concerns related to their toxic constituents like cadmium or lead and the emerging interest in greener chemistry synthesis approaches hamper their future applicability. Interesting alternatives for some applications like biosensing or bioimaging are heavy-metal-free ternary QDs like AgInS2 (AIS), CuInS2 (CIS), and quaternary QDs such as AIS-ZnS (ZAIS). In this context, we explored the effect of ligand denticity on the organic-to-aqueous phase transfer of oleylamine-stabilized ZAIS QDs with the hydrophilic ligands mercaptopropionic acid (MPA), dihydrolipoic acid (DHLA), and 3-mercapto-2,2-bis(mercaptomethyl)propanoic acid (3MPA), bearing mono-, bi-, and trialkyl thiol groups. Spectroscopic studies of the resulting water-dispersible ZAIS QDs revealed a considerable influence of ligand denticity and ligand-to-QD ratio on the spectral position and width (FWHM; full width at half-maximum) of the photoluminescence (PL) bands, the PL quantum yields (PL QY), and the PL decay kinetics. Thiol capping and phase transfer resulted in a loss in PL by at least a factor of 2. The ligand-induced PL quenching observed particularly for ligands bearing two or three thiol groups was attributed to the facilitated formation of surface-bound disulfides. The best colloidal stability under high dilution conditions was observed for 3MPA.

Most studies about the interaction of nanoparticles (NPs) with cells have focused on how the physicochemical properties of NPs will influence their uptake by cells. However, much less is known about their potential excretion from cells. However, to control and manipulate the number of NPs in a cell, both cellular uptake and excretion must be studied quantitatively. Monitoring the intracellular and extracellular amount of NPs over time (after residual noninternalized NPs have been removed) enables one to disentangle the influences of cell proliferation and exocytosis, the major pathways for the reduction of NPs per cell. Proliferation depends on the type of cells, while exocytosis depends in addition on properties of the NPs, such as their size. Examples are given herein on the role of these two different processes for different cells and NPs.

The synthesis of two new families of ZnSe magic-sized clusters (MSCs) is achieved using the thiol ligand 1-dodecanethiol in a simple one-pot heat-up approach. The sizes of the MSCs are controlled with the thiol ligand concentration and reaction temperature.