Luminescence Resonance Energy Transfer Research Articles

Targeted and imaging-guided chemo-photothermal ablation achieved by combining upconversion nanoparticles and protein-capped gold nanodots

For precise targeting therapy, nanocarriers are typically employed to realize tumor-guided delivery of chemotherapeutic drugs, which should ideally kill cancer cell without side effect under controlled release. Besides, as the temperature is fatal for photothermal therapy (PTT), enhancing photothermal conversion efficiency is essential for improving PTT efficacy. Moreover, as PTT is a localized therapy technique, maintaining high penetration depth and simultaneously achieving multimodal imaging during theranostics are conducive to clinical application. Aiming at resolving these plaguing challenges, here we developed a novel anti-tumor platform by combining upconversion nanoparticles with protein-capped gold nanodots through a facile route, following by loading chemotherapeutic drug (doxorubicin, DOX) and conjugating targeting agent (folic acid, FA). Such nanoplatform with luminescence resonance energy transfer (LRET) effect showed enhanced PTT efficacy under near-infrared (NIR) laser irradiation, and FA conjugation is capable of further targeting cancer cells, namely the focus location in vivo. Meanwhile, the nanocarrier with pH-sensitive DOX release ability would contribute to intensive release responding to acidic tumor microenvironment assisted by NIR light excitation induced heat effect, further improving in vivo anti-tumor efficiency. Additionally, by combining our upconversion/Au nanohybrid with multimodal imaging, direct cancer cell killing responses could be obtained at a deeper penetration depth, promising for completely inhibiting tumor proliferation.

Functional and structural comparison of the ABC exporter MsbA studied in detergent and reconstituted in nanodiscs

Purified membrane proteins are most frequently studied solubilized in detergent, but the properties of detergent micelles are very different from those of lipid bilayers. Therefore, there is an increasing interest in studying membrane proteins under conditions that resemble the membrane protein native environment more closely. Although there are indications of differences between membrane proteins in detergent and in lipid bilayers, direct functional and structural comparisons are very hard to find. Nanodiscs have been established as a new platform that consists of two molecules of a membrane scaffold protein that surround a small lipid-bilayer patch. Here, we undertook the task of comparing the function and conformational states of the transport protein MsbA in detergent and nanodiscs using ATPase activity and luminescence resonance energy transfer (LRET) measurements to assess differences in activity and conformational states, respectively. MsbA is a prototypical member of the ATP binding cassette protein superfamily. MsbA activity was higher in nanodiscs vs detergent, which had clear structural correlates: an increase in the fraction of molecules displaying closed nucleotide-binding domain dimers in the apo state, and a decrease in the distance of the “dissociated” nucleotide-binding domains. Our LRET studies support the notion that the widely separated nucleotide binding domains observed in the MsbA x-ray structures in detergent do not correspond to physiological conformations. Although our studies focus on a particular ABC exporter, the possibility of similar environment effects on other membrane proteins should be carefully considered.

Downconversion Luminescence-Based Nanosensor for Label-Free Detection of Explosives.

We report a selective and sensitive nanosensor probe based on polyethylenimine (PEI)-capped downconverting nanophosphors β-NaYF4:Gd3+,Tb3+@PEI for the detection of 2,4,6-trinitrotoluene (TNT), both in water and buffer media. These downconverting phosphors were synthesized via a hydrothermal route and are known to show excellent chemical, thermal, and photostability. They emit sharp emission peaks centered at ∼488, 544, 584, and 619 nm, among which the peak at ∼544 nm was remarkably quenched (∼90%) by the addition of TNT without giving any new emission peak. The sensing mechanism is based on the formation of a Meisenheimer complex between the electron-rich amine-functionalized β-NaYF4:Gd3+,Tb3+ nanophosphors and electron-deficient TNT molecule, which was prominently visualized by the change in the color of the solution from whitish to brownish yellow, enabling visual detection, followed by luminescence resonance energy transfer between the nanophosphors and the complex. A linear range for TNT detection was obtained from 0.1 to 300 μM with a limit of detection as low as 119.9 nM. This method displayed excellent selectivity toward TNT over other nitroaromatic compounds, which had no influence on the detection. Moreover, various other classes of analytes, viz., amino acids, pesticides, and sugars, did not quench the luminescence intensity of the nanophosphors. This developed nanosensor probe possesses high, stable fluorescence brightness and capability for the selective and sensitive on-site recognition of TNT molecules in aqueous media, avoiding complicated strategies and instruments. Thus, this work promises to pave ways to many applications in the detection of ultratrace analytes.

Synchronous detection of glutathione/hydrogen peroxide for monitoring redox status in vivo with a ratiometric upconverting nanoprobe

Cellular redox status presents broad implications with diverse physiological and pathological processes. Simultaneous detection of both the oxidative and reductive species of redox couples, especially the most representative pair glutathione/hydrogen peroxide (GSH/H2O2), is crucial to accurately map the cellular redox status. However, it still remains challenging to synchronously detect GSH/H2O2in vivo due to lack of a reliable measuring tool. Herein, a ratiometric nanoprobe (UCNP-TB) possessing simultaneous delectability of GSH/H2O2 is established based on a multi-spectral upconverting nanophosphor (UCNP-OA) as the luminescence resonance energy transfer (LRET) donor and two dye molecules as the acceptors, including a GSH-sensitive dye (TCG) and a H2O2-sensitive dye (BCH). With the as-prepared UCNP-TB, real-time and synchronous monitoring the variation of GSH and H2O2in vitro and in living mice can be achieved using the ratio of the upconversion luminescence (UCL) at 540 and 650 nm to that at 800 nm as the detection signal, respectively, providing highly inherent reliability of the sensing results by self-calibration. Moreover, the nanoprobe is capable of mapping the redox status within the drug-resistant tumor and the drug-induced hepatotoxic liver via ratiometric UCL imaging. Thus, this nanoprobe would provide a reliable tool to elucidate the redox state in vivo.

Facile preparation of pyrenemethyl ester-based nanovalve on mesoporous silica coated upconversion nanoparticle for NIR light-triggered drug release with potential monitoring capability

A novel light-responsive nanovalve is facilely prepared on mesoporous silica (mSiO2) coated upconversion nanoparticles (UCNP) through modification of photo-cleavagable pyrenemethyl ester (Py) molecules on its surface. These Py molecules can associate with β-cyclodextrin (β-CD) molecules as gatekeepers to block the mSiO2 pore channels and entrap the loaded drugs. Upon NIR light irradiation, NIR light is upconverted to ultraviolet (UV) and visible (Vis) light by UCNP, and the modified Py molecules are cleaved by the upconverted UV light leading to the detachment of β-CD gatekeepers to trigger drug release. For the fabrication of a monitoring system, β-CD is covalently conjugated with the fluorescein isothiocyanate (FITC) as drug release indicator (FITC-β-CD). The luminescence resonance energy transfer (LRET) from UCNP to Py/FITC-β-CD before drug release results in the quenching of Vis emission of UCNP while the detachment of Py/FITC-β-CD nanovalves after drug release diminishes the LRET between UCNP and FITC-β-CD which recovers the luminescence of UCNP, the change in luminescence provides a potential capability for one to quantitatively monitor the drug release process. in vitro study shows a remarkable killing ability on HeLa cancer cell of this system.

Simulation Guided Design of Spectroscopy Experiments via Maximizing Kinetic Information Gain

We present a computational tool called Optimal Probes that uses protein molecular dynamics (MD) simulation datasets to address a systematic issue in designing spectroscopic experiments. A common problem faced in experiments that measure relaxation of a pair of residues is narrowing down the set of residue pairs to site-specifically label among a large pool of possibilities. Our method ranks every possibility based on a quantitative measure, Generalized Matrix Rayleigh Quotient (GMRQ) score for a Markov state model (MSM) of protein conformational dynamics[1]. We optimize the choice of residue pairs in the protein to build an MSM that can achieve a high GMRQ score, thus capturing slow motions of the protein's dynamics. Once all residue pair sets are scored, the highest scoring inter-residue pairs can be probed to investigate the protein experimentally. We show that incorporating protein dynamics from simulations in such a manner maximizes information gain from experiments. We illustrate that Optimal Probes can make better choices than experimental choices based on human intuition or limited structural information such as distance difference between two known stable structures of the protein. The tool can also be used to predict optimal experiment choices considering previous experiments that have already been performed on the protein of interest. We demonstrate the use of Optimal Probes as an intuitive and user-friendly tool on cytoplasmic and transmembrane proteins for four experimental techniques, Double Electron-Electron Resonance[2], Trp-Tyr quenching, Luminescence Resonance Energy Transfer, and Triplet-Triplet Energy Transfer. In conclusion, Optimal Probes provides an automated prediction of residue pairs for biophysical experiment techniques. [1] Mittal and Shukla. J Phys Chem B, 2017 DOI: 10.1021/acs.jpcb.7b04785 [2] Selvam, Mittal, and Shukla. ACS Central Science, 2018 DOI: 10.1021/acscentsci.8b00330.

Multifunctional Ag@NaGdF4:Yb3+, Er3+ core-shell nanocomposites for dual-mode imaging and photothermal therapy

Multifunctional core-shell nanostructure, which can be applied on bio-imaging and photothermal therapy (PTT), has attracted tremendous interest in recent years. Here, Ag@NaGdF4:Yb3+, Er3+ core-shell nanostructure incorporating luminescent, magnetic and photothermal conversion properties was prepared via a simple method. The as-prepared nanocomposites (NCs) exhibit uniform core-shell sphere morphology, green up-conversion luminescence (UCL), good paramagnetic and photothermal conversion behaviors. The luminescence resonance energy transfer (LRET) between Ag nanoparticles (NPs) and NaGdF4:Yb3+, Er3+ NPs were researched. The cytotoxicity test on HeLa cells using methyl thiazolyltetrazolium (MTT) assay shows good biocompatibility of the core-shell NCs. Moreover, in vitro computed tomography (CT) and UCL imaging studies and in vitro PTT of living assay suggest that the core-shell NCs can be used as theranostic nanoagents for dual-mode imaging guided PTT for tumor.

Luminescence resonance energy transfer in hybrid materials based on terbium(iii) complex, rhodamine B and nanoclay

We prepared a novel kind of luminescent hydrogel based on the combination of terbium(iii) complexes and rhodamine B dye with the LAPONITE® nanoclay.

Responsive Upconversion Nanoprobe for Background-Free Hypochlorous Acid Detection and Bioimaging.

Responsive nanoprobes play an important role in bioassay and bioimaging, early diagnosis of diseases and treatment monitoring. Herein, a upconversional nanoparticle (UCNP)-based nanoprobe, Ru@UCNPs, for specific sensing and imaging of hypochlorous acid (HOCl) is reported. This Ru@UCNP nanoprobe consists of two functional components,, i.e., NaYF4 :Yb, Tm UCNPs that can convert near infrared light-to-visible light as the energy donor, and a HOCl-responsive ruthenium(II) complex [Ru(bpy)2 (DNCH-bpy)](PF6 )2 (Ru-DNPH) as the energy acceptor and also the upconversion luminescence (UCL) quencher. Within this luminescence resonance energy transfer nanoprobe system, the UCL OFF-ON emission is triggered specifically by HOCl. This triggering reaction enables the detection of HOCl in aqueous solution and biological systems. As an example of applications, the Ru@UCNPs nanoprobe is loaded onto test papers for semiquantitative HOCl detection without any interference from the background fluorescence. The application of Ru@UCNPs for background-free detection and visualization of HOCl in cells and mice is successfully demonstrated. This research has thus shown that Ru@UCNPs is a selective HOCl-responsive nanoprobe, providing a new way to detect HOCl and a new strategy to develop novel nanoprobes for in situ detection of various biomarkers in cells and early disgnosis of animal diseases.

Using optical tweezers to construct an upconversion luminescent resonance energy transfer analytical platform

We report a new upconversion nanoparticles (UCNPs) based luminescent resonance energy transfer (LRET) analytical platform by making use of optical tweezers technology. The LRET model is designed by simultaneously conjugating Yb3+ and Er3+ co-doped UCNPs (as the donors) and tetramethyl rhodamine (TAMRA) molecules (as the acceptors) on microspheres to fabricate complex microspheres. Upon a single complex microsphere entering the three-dimensional potential well formed with a tightly focused 980 nm Guassian-shaped laser beam, it is optically trapped and concurrently the upconversion emission is excited, whereby the donor signals are transferred to the acceptors. As a proof-of-concept investigation, microRNA-21 sequences are selected as the targets, by which the distance between the two perfectly matched luminophors is controlled to several nanometers via nucleic acid hybridization. Without the involvement of luminescence amplification strategies, the proposed single microsphere based LRET method shows highly competitive sensitivity with a limit of detection down to 114 fM and satisfactory specificity towards microRNAs detection. Moreover, its practical working ability is demonstrated by credibly quantifying the absolute contents of miRNA-21 sequences in three cancer cell lines and even tracing the targets in as few as 100 cancer cells. Thus, this favorable analytical methodology provides an alternative for bioassays and holds certain potential in biomedical applications.

Lysosome-Assisted Mitochondrial Targeting Nanoprobe Based on Dye-Modified Upconversion Nanophosphors for Ratiometric Imaging of Mitochondrial Hydrogen Sulfide.

Hydrogen sulfide (H2S) is a versatile modulator in mitochondria and involved in numerous diseases caused by mitochondrial dysfunction. Therefore, many efforts have been made to develop fluorescent probes for mitochondrial H2S detection. However, these cationic small molecule probes are inapplicable for in vivo imaging because of the shallow tissue penetration and poor biostability. Herein, a ratiometric upconversion luminescence nanoprobe with an acid-activated targeting strategy is developed for detecting and bioimaging of mitochondrial H2S. The merocyanine triphenylamine-merocyanine (TPAMC)-modified upconversion nanophosphors, acting as the targeting and response component, are encapsulated into a pH-sensitive husk, composed of 1,2-distearoyl- sn-glycero-3-phosphoethanolamine- N-[methoxy-(poly(ethylene glycol))-2000] (DSPE-PEG) and poly(l-histidine)- b-PEG, which improved the nanoprobe's stability during transport in vivo. Under lysosomal pH, the PEG shell is interrupted and the targeting sites are exposed to further attach to mitochondria. Taking advantage of the luminescence resonance energy transfer process between TPAMC and upconversion nanophosphors, the ratiometric detection of mitochondrial H2S can be achieved with high selectivity and sensitivity. Cellular testing reveals the precise targeting to mitochondria via a lysosome delivery process. Importantly, the nanoprobe can be used for monitoring mitochondrial H2S levels in living cells and colon cancer mouse models.

AgInS2-Coated Upconversion Nanoparticle as a Photocatalyst for Near-Infrared Light-Activated Photodynamic Therapy of Cancer Cells.

The development of high-performance near-infrared (NIR) photocatalysts with long-term stability and the elucidation on the working mechanism along with multifunctional activity toward biomedical applications have not been explored sufficiently. Herein, a novel hybrid material of an upconversion nanoparticle (NaYF4/Yb3+,Er3+) (UCN) coated with a ternary semiconductor (AgInS2, AIS) has been synthesized by a simple and robust two-step solvothermal route for NIR light active photocatalysis. Preparation of oleic acid-capped spherical UC nanoparticles (NPs) (∼25 nm) followed by solvothermal decomposition of two precursor complexes Ag(acda) and In(acda)3 resulted in the formation of well-defined NaYF4/Yb3+,Er3+@AgInS2 core-shell nanoparticles (UCN@AIS NPs) (∼90 nm). It has been found that effective energy transfer occurred from NaYF4/Yb3+,Er3+ to AgInS2 by a nonradiative luminescence resonance energy transfer process. Superior photocatalytic decomposition activity was validated in terms of the degradation of methylene blue dye under the exposure of 980 nm NIR laser light with the presence of a UCN@AIS NP catalyst. The degradation process was mediated primarily owing to the formation of a cytotoxic reactive oxygen species (ROS) by the hybrid material under NIR light irradiation, in which UCN performs as a transducer to sensitize AIS and trigger the ROS generation. In vitro cancer cell imaging potentiality of the UCN@AIS NPs was then studied on cervical cancer cells (HeLa cells). The UCN@AIS NPs induced in vitro cervical cancer cell death (photodynamic therapy) with ∼27% efficiency as measured by the MTT assay and thus proved to be a decent candidate for NIR active photocatalysts for biomedical applications.

Magnetic Targeting of Nanotheranostics Enhances Cerenkov Radiation-Induced Photodynamic Therapy.

The interaction between radionuclides and nanomaterials could generate Cerenkov radiation (CR) for CR-induced photodynamic therapy (PDT) without requirement of external light excitation. However, the relatively weak CR interaction leaves clinicians uncertain about the benefits of this new type of PDT. Therefore, a novel strategy to amplify the therapeutic effect of CR-induced PDT is imminently required to overcome the disadvantages of traditional nanoparticulate PDT such as tissue penetration limitation, external light dependence, and low tumor accumulation of photosensitizers. Herein, magnetic nanoparticles (MNPs) with 89Zr radiolabeling and porphyrin molecules (TCPP) surface modification (i.e., 89Zr-MNP/TCPP) were synthesized forCR-induced PDT with magnetic targeting tumor delivery. As a novel strategy to break the depth and light dependence of traditionalPDT, these 89Zr-MNP/TCPP exhibited high tumor accumulation under the presence of anexternal magnetic field, contributing to excellent tumor photodynamic therapeutic effect together with fluorescence, Cerenkov luminescence (CL), and Cerenkov resonance energy transfer (CRET) multimodal imaging to monitor the therapeutic process. The present study provides a major step forward in photodynamic therapy by developing an advanced phototherapy tool of magnetism-enhanced CR-induced PDT for effective targeting and treatment of tumors.

Maximizing Kinetic Information Gain of Markov State Models for Optimal Design of Spectroscopy Experiments.

Spectroscopic techniques such as Trp-Tyr quenching, luminescence resonance energy transfer, and triplet-triplet energy transfer are widely used for understanding the dynamic behavior of proteins. These experiments measure the relaxation of a particular labeled set of residue pairs, and the choice of residue pairs requires careful thought. As a result, experimentalists must pick residue pairs from a large pool of possibilities. In the current work, we show that molecular simulation datasets of protein dynamics can be used to systematically select an optimal set of residue positions to place probes for conducting spectroscopic experiments. The method described in this work, called Optimal Probes, can be used to rank trial sets of residue pairs in terms of their ability to capture the conformational dynamics of the protein. Optimal probes ensures two conditions: residue pairs capture the slow dynamics of the protein and their dynamics is not correlated for maximum information gain to score each trial set. Eventually, the highest scored set can be used for biophysical experiments to study the kinetics of the protein. The scoring methodology is based on kinetic network models of protein dynamics and a variational principle for molecular kinetics to optimize the hyperparameters used for the model. We also discuss that the scoring strategy used by Optimal Probes is the best possible way to ensure the ideal choice of residue pairs for experiments. We predict the best experimental probe positions for proteins λ-repressor, β2-adrenergic receptor, and villin headpiece domain. These proteins have been well-studied and allow for a rigorous comparison of Optimal Probes predictions with already available experiments. Additionally, we also illustrate that our method can be used to predict the best choice for experiments by including any previous experiment choices available from other studies on the same protein. We consistently find that the best choice cannot be based on intuition or structural information such as distance difference between few known stable structures of the protein. Therefore, we show that incorporating protein dynamics could be used to maximize the information gain from experiments.

Breaking Through the Signal-to-Background Limit of Upconversion Nanoprobes Using a Target-Modulated Sensitizing Switch.

Although lanthanide-doped upconversion nanoparticles (UCNPs) have shown great promise in biosensing and bioimaging owing to their excellent photophysical properties, researchers are facing a bottleneck of upconversion (UC) probes which is the limited signal-to-background ratio (SBR). Since UC nanoprobes are basically constructed with a luminescence resonance energy transfer (LRET) process to provide "off-on" signals, the SBR level is principally decided by the luminescence quenching efficiency which is very difficult to further improve through existing approaches. Herein, we put forward a new strategy for fabricating UC nanoprobes using an organic dye as target-modulated sensitizing switch. The dye functions as both the recognition unit for target and a potential sensitizer for upconversion luminescence (UCL). The reaction of the dye with target modulates its photophysical properties, which switches on the sensitization and affords a significantly improved SBR. The idea is validated with a proof-of-concept UC nanoprobe for glutathione (GSH) detection with the SBR of ∼30 (versus a SBR of less than 10 for most current UC nanoprobes). This probe showed good performance in GSH sensing both in vitro and in vivo. Our results indicate that the target-modulated sensitization is a useful new strategy to build UC nanoprobes. And we can reasonably expect that the breakthrough of SBR limit will make UC nanoprobe a more powerful tool in future studies.

Lysosome-targeting NIR ratiometric luminecent upcoversion nanoprobe toward arginine

Near-infrared (NIR) fluorescent sensors have been actively pursued due to organisms exhibiting little autofluorescence in near-infrared regions. Capable of emitting luminesence under 980 nm excitation, upconversion nanoparticles have shown promise as optical agents for in vivo bioimaging. Based on luminescence resonance energy transfer (LRET), a NIR luminescent Arg-nanoprobe was developed by the decoration of upconversion nanoparticles (UCNPs) (NaYF4:Yb,Er) with organic molecules. The as-developed nanoprobe could quantitatively detect concentrations within the range of 528–1250 μM, and had high sensitiviy (the limit of detection, LOD, being 15.6 μM) and good photostability. Because the probe could target lysosomes, intracellar detection of Arg behavior was demonstrated via upconversion luminescence (UCL) imaging.

LRET-based functional persistent luminescence nanoprobe for imaging and detection of cyanide ion

Cyanide anion is an inhibitor of the enzyme cytochrome c oxidase in the fourth complex of the electron transport chain, and usually found in the membrane of the mitochondria of eukaryotic cells, thus exploration of novel analytical methodology for sensitive and accurate detection of cyanide ion in cells is vital and urgent. Herein, we successfully prepared and surface-modified ZnGa2O4:Mn2+ PLNPs with excellent afterglow properties, small size and preferable dispersibility. Furthermore, we report a rational design and fabrication of a functional PL nanoprobe based on luminescence resonance energy transfer (LRET), which for the first time demonstrates detection of cyanide ion without external illumination. The nanoprobe is composed of two main moieties: PLNPs with excellent afterglow properties as energy donor; and DEHSPI, a chromophore with tunable light absorption, as both energy acceptor and CN− recognition element. PL can be effectively quenched by the chromophores assembled on the surface of PLNPs through a LRET process and subsequently recovered upon the addition of CN−. Importantly, the persistent afterglow nature highlights the merits of employing PLNPs as energy donor to eliminate the background noise under in situ excitation. Moreover, the nanoprobe exhibits excellent analytical properties and low cytotoxicity for bio-imaging of CN− in living cells.

Lighting Up MicroRNA in Living Cells by the Disassembly of Lock-Like DNA-Programmed UCNPs-AuNPs through the Target Cycling Amplification Strategy.

Intracellular microRNAs imaging based on upconversion nanoprobes has great potential in cancer diagnostics and treatments. However, the relatively low detection sensitivity limits their application. Herein, a lock-like DNA (LLD) generated by a hairpin DNA (H1) hybridizing with a bolt DNA (bDNA) sequence is designed, which is used to program upconversion nanoparticles (UCNPs, NaYF4 @NaYF4 :Yb, Er@NaYF4 ) and gold nanoparticles (AuNPs). The upconversion emission is quenched through luminescence resonance energy transfer (LRET). The multiple LLD can be repeatedly opened by one copy of target microRNA under the aid of fuel hairpin DNA strands (H2) to trigger disassembly of AuNPs from the UCNP, resulting in the lighting up of UCNPs with a high detection signal gain. This strategy is verified using microRNA-21 as model. The expression level of microRNA-21 in various cells lines can be sensitively measured in vitro, meanwhile cancer cells and normal cells can be easily and accurately distinguished by intracellular microRNA-21 imaging via the nanoprobes. The detection limit is about 1000 times lower than that of the previously reported upconversion nanoprobes without signal amplification. This is the first time a nonenzymatic signal amplification method has been combined with UCNPs for imaging intracellular microRNAs, which has great potential for cancer diagnosis.

Rational Engineering a Multichannel Upconversion Sensor for Multiplex Detection of Matrix Metalloproteinase Activities.

Optical sensing of cancer-relevant protease is of great value for cancer diagnostics, prognosis, and drug discovery. Multiplex sensing is known to improve predicative accuracy yet remains challenging because of severe fluorescence signal crosstalk in a single assay. Herein, we developed a multichannel optical sensor based on upconversion nanoparticles for multiplex ratiometric sensing of proteolytic activities of two matrix metalloproteinases (MMP-2 and MMP-7). To this end, we rationally designed a NaYF4:Gd3+/Yb3+@NaYF4:Yb3+/Tm3+/Er3+ core-shell structure that favors multicolor narrow-band emission of both Tm3+ and Er3+ dopants and efficient luminescence resonance energy transfer (LRET) between the dopants in the shell and the fluorophores on the particle surface. The sensor was constructed via a facile phase transfer protocol using two polyhistidine-containing peptides conjugated with different fluorophores (FITC and TAMRA) as coligands. The blue and green emission could be specifically activated by MMP-7 and MMP-2, respectively, upon peptide cleavage, and the red emission could serve as an internal reference for ratiometric sensing. The sensor exhibits high specificity and sensitivity toward both targets with little signal crosstalk and cross-reactivity. It could potentially serve as a general platform for multiplex detection of various types of proteases.

Multicolor Upconversion Nanoprobes Based on a Dual Luminescence Resonance Energy Transfer Assay for Simultaneous Detection and Bioimaging of [Ca2+ ]i and pHi in Living Cells.

Intracellular [Ca2+ ]i and pHi have a close relationship, and their abnormal levels can result in cell dysfunction and accompanying diseases. Thus, simultaneous determination of [Ca2+ ]i and pHi can more accurately investigate complex biological processes in an integrated platform. Herein, multicolor upconversion nanoparticles (UCNPs) were prepared with the advantages of no spectral overlapping, single NIR excitation wavelengths, and greater tissue penetration depth. The upconversion nanoprobes were easily prepared by the attachment of two fluorescent dyes, Fluo-4 and SNARF-4F. Based on the dual luminescence resonance energy transfer (LRET) process, the blue and green fluorescence of the UCNPs were specially quenched and selectively recovered after the detachment and/or absorbance change of the attached fluorescent dyes, enabling dual detection. Importantly, the developed nanoprobe could successfully be applied for the detection of [Ca2+ ]i and pHi change in adenosine triphosphate (ATP) and ethylene glycol tetraacetic acid (EGTA) stimulation in living cells.