One of the key phenomena that determine the fluorescence of nanocrystals is the nonradiative Auger-Meitner recombination of excitons. This nonradiative rate affects the nanocrystals' fluorescence intensity, excited state lifetime, and quantum yield. Whereas most of the above properties can be directly measured, the quantum yield is the most difficult to assess. Here we place semiconductor nanocrystals inside a tunable plasmonic nanocavity with subwavelength spacing and modulate their radiative de-excitation rate by changing the cavity size. This allows us to determine absolute values of their fluorescence quantum yield under specific excitation conditions. Moreover, as expected considering the enhanced Auger-Meitner rate for higher multiple excited states, increasing the excitation rate reduces the quantum yield of the nanocrystals.

Type-II ZnSe/CdS voltage-sensing seeded nanorods (vsNRs) were functionalized with α-helical peptides and zwitterionic-decorated lipoic acids (zw-LAs). Specific membrane targeting with high loading efficiency and minimal nonspecific binding was achieved. These vsNRs display quantum yield (QY) modulation as a function of membrane potential (MP) changes, as demonstrated at the ensemble level for (i) vesicles treated with valinomycin and (ii) wild-type HEK cells under alternating buffers with different [K+]. ΔF/F of ∼ 1% was achieved.

We optimized the performance of quantum confined Stark effect, QCSE, based voltage nanosensors. A high throughput approach for single particle QCSE characterization was developed and utilized to screen a library of such nanosensors. Type II ZnSe CdS seeded nanorods, synthesized by a robotic colloidal synthesizer, were found to have the best performance among the different nanosensors evaluated in this work. The degree of correlation between intensity changes and spectral changes of the excitons emission under applied field was characterized. An upper limit for the temporal response of individual ZnSe CdS nanorods to voltage modulation was characterized by high throughput, high temporal resolution intensity measurements using a novel photon counting camera. The measured 3.5 microsecond response time is limited by the voltage modulation electronics and represents 30 times higher bandwidth than needed for recording an action potential in a neuron.

We have been developing targetable voltage sensing inorganic nanoparticles that are designed to self-insert into the cell membrane and non-invasively optically record, via the quantum confined Stark effect, action potential on the single-particle level, at multi-sites and in a large field-of-view. We synthesized a library of such voltage sensing nanosensors (vsNSs) with different compositions and shapes and developed a high-throughput screen for optimization of their performance. We improved on surface functionalization that is based on adsorption of engineered trans-membrane peptides to the surface of the nanoparticles. This coating imparts vsNSs with membrane-protein like properties and affords their insertion into the membrane in the correct orientation. Membrane voltage sensing by membrane-inserted vsNSs was demonstrated for membrane voltage modulated WT HEK cells using valinomycin and modulated concentration of potassium ions in a microfluidic chamber, and for patch-clamped primary cultured cortical neurons. These novel voltage nansensors hold great promise for electrophysiological investigations of the nervous system.

We optimized the performance of quantum confined Stark effect QCSE based voltage nanosensors. A high throughput approach for single particle QCSE characterization was developed and utilized to screen a library of such nanosensors. Type II ZnSe CdS seeded nanorods were found to have the best performance among the different nanosensors evaluated in this work. The degree of correlation between intensity changes and spectral changes of the excitons emission under applied field was characterized. An upper limit for the temporal response of individual ZnSe CdS nanorods to voltage modulation was characterized by high throughput, high temporal resolution intensity measurements using a novel photon counting camera. The measured 3.5 us response time is limited by the voltage modulation electronics and represents about 30 times higher bandwidth than needed for recording an action potential in a neuron.

We developed membrane voltage nanosensors that are based on inorganic semiconductor nanoparticles. We provide here a feasibility study for their utilization. We use a rationally designed peptide to functionalize the nanosensors, imparting them with the ability to self-insert into a lipid membrane with a desired orientation. Once inserted, these nanosensors could sense membrane potential via the quantum confined Stark effect, with a single-particle sensitivity. With further improvements, these nanosensors could potentially be used for simultaneous recording of action potentials from multiple neurons in a large field of view over a long duration and for recording electrical signals on the nanoscale, such as across one synapse.

Monitoring membrane potential in neurons requires sensors with minimal invasiveness, high spatial and temporal (sub-ms) resolution, and large sensitivity for enabling detection of sub-threshold activities. While organic dyes and fluorescent proteins have been developed to possess voltage-sensing properties, photobleaching, cytotoxicity, low sensitivity, and low spatial resolution have obstructed further studies. Semiconductor nanoparticles (NPs), as prospective voltage sensors, have shown excellent sensitivity based on Quantum confined Stark effect (QCSE) at room temperature and at single particle level. Both theory and experiment have shown their voltage sensitivity can be increased significantly via material, bandgap, and structural engineering. Based on theoretical calculations, we synthesized one of the optimal candidates for voltage sensors: 12 nm type-II ZnSe/CdS nanorods (NRs), with an asymmetrically located seed. The voltage sensitivity and spectral shift were characterized in vitro using spectrally-resolved microscopy using electrodes grown by thin film deposition, which "sandwich" the NRs. We characterized multiple batches of such NRs and iteratively modified the synthesis to achieve higher voltage sensitivity (Delta F/F>10%), larger spectral shift (>5 nm), better homogeneity, and better colloidal stability. Using a high throughput screening method, we were able to compare the voltage sensitivity of our NRs with commercial spherical quantum dots (QDs) with single particle statistics. Our method of high throughput screening with spectrally-resolved microscope also provides a versatile tool for studying single particles spectroscopy under field modulation.

Unraveling emergent brain activities requires simultaneous recording of action potentials from a large number of neurons. Electrical recording methods such as patch clamp and optical recording by voltage sensing dyes and proteins have been developed for years and are widely utilized. However, such techniques have insufficient spatial and/or temporal resolutions and/or suffer from poor photostability, posing a need for probes that circumvent these limitations. Improved probes, with high sensitivity and photostability, could afford the study of large neural networks (in a large filed-of-view) and/or at very high spatial resolution. Using bandgap-engineering and colloidal synthesis methods, we have synthesized seeded semiconductor (SC) nanorods (NRs) with Type-II heterojunctions that exhibit a large Quantum Confined Stark Effect (QCSE) at room temperature (1). For using these NRs as voltage sensors, however, one needs to impart them with membrane-protein like properties so that they can be stably inserted into the membrane. We report here spontaneous insertion of SC NRs into liposomes and cell membranes by functionalizing them with specially designed peptides. We provide evidences for insertion from cryo transmission electron microscopy (TEM) and polarized light microscopy. We also report on first attempts to sense membrane potential with these particles with single-particle sensitivity. With further improvements, SC NRs could potentially be used to study signals from whole neural networks in a large field-of-view. Moreover, successful implementation of SC NRs would allow for the analysis of voltage signals at the nano- (single synapse-) scale. ∗Equal contributions (1) Park, K.; Deutsch, Z.; Li, J. J.; Oron, D.; Weiss, S., Single Molecule Quantum-Confined Stark Effect Measurements of Semiconductor Nanoparticles at Room Temperature. ACS Nano 2012,6 (11), 10013-10023.

We measured the quantum-confined Stark effect (QCSE) of several types of fluorescent colloidal semiconductor quantum dots and nanorods at the single molecule level at room temperature. These measurements demonstrate the possible utility of these nanoparticles for local electric field (voltage) sensing on the nanoscale. Here we show that charge separation across one (or more) heterostructure interface(s) with type-II band alignment (and the associated induced dipole) is crucial for an enhanced QCSE. To further gain insight into the experimental results, we numerically solved the Schrödinger and Poisson equations under self-consistent field approximation, including dielectric inhomogeneities. Both calculations and experiments suggest that the degree of initial charge separation (and the associated exciton binding energy) determines the magnitude of the QCSE in these structures.

We developed a new peptide, natural phytochelatin (PC), which tightly binds to CdSe/ZnS quantum dots' (QDs) surfaces and renders them water-soluble. Coating QDs with this flexible and all-hydrophilic peptide offers high colloidal stability, adds only 0.8-0.9 nm to the radius of the particles (as compared to their original inorganic radius), preserves very high quantum yield (QY) in water, and affords facile bioconjugation with various functional groups. We demonstrate specific targeting (with minimal nonspecific binding) of such fluorescein-conjugated QDs to ScFv-fused mouse prion protein expressed in live N2A cells. We also demonstrated homogeneous in vivo biodistribution with no significant toxicity in live zebrafish.

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.

Since their first appearance as optical probes in biological imaging [1, 2], qdots have been applied in most biotechnological applications using fluorescence, including DNA array technology, immunofluorescence assays (reviewed in [3]), and cell and animal biology (reviewed in [4, 5]. Qdots have gained wide acceptance by the scientific community and the biotechnology industry as new fluorescent, nonisotopic labels of unmatched potentials. Most appealing to the biosciences are the high brightness, high resistance to photobleaching, and the ability to size-tune fluorescent emission of these inorganic-biological hybrid nanostructures. Qdots have also proved to be excellent probes for two-photon confocal microscopy and deep-tissue imaging given their large two-photon absorption cross-section [6–9]. But it is when combined with ultrasensitive microscopy techniques that qdots have shown their true potential by allowing visualization of cellular processes down to the molecular scale [10–13]. The enhanced photophysical properties of qdots can fulfill the stringent requirements needed to provide spatial, temporal, and structural information at all length scales: from the whole body down to the nanometer resolution with a single probe. Fluorescent nanocrystals of semiconductor material, which are generally synthesized in solutions of nonpolar organic solvents using colloidal chemistry [14], require additional chemical modifications to be solubilized in aqueous buffers and functionalized for biological applications (see Chapter 1, “Colloidal Quantum Dots: Synthesis, Photophysical Properties, and Biofunctionalization Strategies,” by Susumu et al. for an in-depth discussion). This can be achieved by either surfactant exchange, a process primarily driven by mass-action in which the native TOP/TOPO hydrophobic surface ligands are substituted with bifunctional amphiphilic ones, or by insulation of the original hydrophobic qdot within a heterofunctional amphiphilic coating [4, 5]. These various qdot solubilization strategies have included the following: (1) ligand exchange with small thiol-containing

Quantum dots (QDOTs) have been widely recognized by the scientific community and the biotechnology industry, as witnessed by the exponential growth of this field in the past several years. We describe the synthesis and characterization of visible and near infrared QDots-a critical step for engineering organic molecules like proteins and peptides for building nanocomposite materials with multifunctional properties suitable for biological applications

We have synthesized high quality type-II CdTe/CdSe near infrared quantum dots using successive ion layer adsorption and reaction chemistry. Transmission electron microscopy reveals that CdTe/CdSe can be synthesized layer by layer yielding quantum dots of narrow size distribution. Excitation and photoluminescence spectra reveal discrete type-II transitions, which correspond to energy lower that type-I bandgap. We have used a peptide coating technique on type-II and commercial near infrared quantum dots for delivery in live animals and cultured cells.

Colloidal gold nanocrystals were found to be efficient photocatalysts for the oxidation of thiols to the corresponding disulfide in aqueous solution under UV radiation, although no noticeable catalytic effect of gold nanocrystals was observed under dark conditions for the same process. The turnover number per particle increased with the size of the nanocrystals and reached 1200 molecules/s for 45-nm gold nanocrystals. The photochemical instability of colloidal gold nanocrystals coated by thiol ligands was found to be determined by the photocatalytic oxidation of the thiol ligands.

The surface ligands, generation-3 (G3) dendrons, on each semiconductor nanocrystal were globally cross-linked through ring-closing metathesis (RCM). The global cross-linking of the dendron ligands sealed each nanocrystal in a dendron box, which yielded box-nanocrystals. Although the dendron ligands coated CdSe nanocrystals (CdSe dendron-nanocrystals) were already quite stable, the stability of CdSe box-nanocrystals against chemical, photochemical, and thermal treatments were dramatically improved in comparison to that of the original dendron-nanocrystals. Furthermore, the box structure of the ligands monolayer coupled with the stable inorganic CdSe/CdS core/shell nanocrystals resulted in a class of extremely stable nanocrystal/ligands complexes. The band edge photoluminescence of the core/shell dendron-nanocrystals and box-nanocrystals were partially remained, and could be further brightened through controlled chemical oxidation or photooxidation. Practically, the stability of the box-nanocrystals is sufficient for most fundamental studies and technical applications. The box-nanocrystals may represent a general solution for the commonly encountered instability for many types of colloidal nanocrystals. The size distribution of the empty dendron boxes formed by the dissolution of the inorganic nanocrystals in concentrated HCl was very narrow. The empty boxes as new types of polymer capsules are soluble in solution, mesoporous, and with a very thin but stable peripheral. Those nanometer-sized cavities should be of interest for many purposes in the field of solution host-guest chemistry.

Dünne CdSe-Schichten („Quantenschalen“) wurden epitaktisch auf CdS-Nanokristalltemplaten gezüchtet. Ihre optischen Eigenschaften werden trotz unterschiedlicher Bandlücken und Templatgrößen fast ausschließlich von der Schalendicke bestimmt. Durch Variieren der Schalendicke kann die Emission der CdSe-Quantenschalen zwischen 520 und 650 nm abgestimmt werden (siehe Bild); die Quantenausbeute lässt sich durch epitaktische Beschichtung der Quantenschalen mit dünnen CdS-Schichten auf über 40 % steigern.