Noncovalently Caged Firefly Luciferins Enable Amplifiable Bioluminescence Sensing of Hyaluronidase-1 Activity in Vivo.

Hyaluronidase 1 (Hyal-1) is an important enzyme involved in intracellular hyaluronic acid (HA) catabolism for performing various physiological functions, and its aberrant level is closely associated with many malignant diseases. Bioluminescence imaging is advantageous for monitoring Hyal-1 activity in vivo, but it remains challenging to design an available probe for differentiating Hyal-1 from other isoforms by a traditional strategy that covalently masks the firefly luciferase substrate. Herein, we, for the first time, present a noncovalently caging approach to construct a Hyal-1-specific bioluminogenic nanosensor by entrapping d-luciferin (d-Luc) inside the cholesterylamine-modified HA (CHA) nanoassembly to inhibit the bioluminescence production. When encountered with intracellular Hyal-1, CHA could be fully dissembled to liberate multiple copies of the loaded d-Luc, thereby emitting light by the luciferase-catalyzed bioluminescence reaction. Because of its cascade signal amplification feature, d-Luc@CHA displayed a remarkable "turn-on" response (248-fold) to 5 µg/mL Hyal-1 with a detection limit of 0.07 ng/mL. Importantly, bioluminescence imaging results validated that d-Luc@CHA could be competent for dynamically visualizing endogenous Hyal-1 changes in living cells and animals and possessed the capability of discriminating between normal and cancer cells, thus offering a promising toolbox to evaluate Hyal-1 roles in biological processes as well as to diagnose Hyal-1-related diseases.

Near-Infrared Fluorescent Furin Probe for Revealing the Role of Furin in Cellular Carcinogenesis and Specific Cancer Imaging.

Furin, an important member in the family of proprotein convertases, is a participant in the activation of various precursor proteins. The expression level of furin stays in a very low range in most normal cells, but elevates with a big margin in many cancer cells. More importantly, furin is closely related to tumor formation and migration. Herein, a furin-activatable near-infrared (NIR) fluorescent probe (HD-F) was first developed that allowed for specific, sensitive detection and imaging of furin both in vitro and in vivo. HD-F consists of a classical NIR fluorophore (HD), a furin-particular polypeptide sequence RVRR, and a self-eliminating linker. Without the interaction with furin, no noticeable fluorescence enhancement was detected, even over 3 days, demonstrating the excellent stability of HD-F. Upon conversion by furin, there was a distinct signal increase around 708 nm. It has achieved assay and visualization of endogenous furin in various cells, tumor tissues, and tumor-bearing mouse models. Importantly, HD-F is well-suited for monitoring the change of furin expression level in the process of hypoxia-inducible factor-1 stabilized by CoCl2. Moreover, HD-F could visualize the divergence in the expression level of furin between normal and cancer cells, indicating its potential in specific cancer imaging. Thus, this novel probe is able to serve as a potential tackle for better understanding of the intrinsic link between a hypoxic physiological environment and cellular carcinogenesis and predicting cancer in preclinical applications.

A bioluminescent probe for imaging endogenous hydrogen polysulfides in live cells and a murine model of bacterial infection.

In this work, we report the first bioluminescent probe BP-PS for detecting H2Sn with high specificity and sensitivity. Owing to the bioluminescence imaging without requiring an excitation light source, tissue autofluorescence is eliminated and BP-PS shows a high signal-to-noise ratio. Moreover, BP-PS was successfully utilized to visualize endogenous H2Sn in live cells and a murine model of bacterial infection.

Engineering of a bioluminescent probe for imaging nitroxyl in live cells and mice.

A bioluminescent probe, BP-HNO, which exhibits a turn-on response to nitroxyl with high sensitivity and selectivity, is reported for the first time in this work. BP-HNO is free from the interference of biological autofluorescence to afford a high signal-to-noise ratio for bioimaging, and was successfully applied to imaging nitroxyl in live cells and mice.

Recent Progress in Small-Molecule Near-IR Probes for Bioimaging.

Small-molecule near-IR (NIR) optical imaging has experienced tremendous advancements over the past two decades, playing important roles in both theory and application in the biomedical field. NIR optical imaging affords improved contrast and depth of tissue penetration by reducing photon scattering, lowering tissue absorption, and minimizing autofluorescence in the NIR window. Moreover, molecular engineering endows small-molecule probes with powerful tunability, providing a valuable means for real-time noninvasive visualization of various biological processes and analytes in vivo with high sensitivity and resolution. In this review, we focus on the most recent advances in the development of small-molecule NIR probes and their applications in bioimaging. We also highlight the challenges and opportunities in this rapidly developing field.

In Situ Imaging of Furin Activity with a Highly Stable Probe by Releasing of Precipitating Fluorochrome.

Furin, a kind of trans-Golgi proprotein convertases, plays important role in various physiological processes. It is overexpressed in many cancers and relates to tumor growth and migration. In situ detection and imaging of furin is of great significance for obtaining real-time information about its activity. However, the previously reported fluorescent probes for furin usually failed to realize in situ detection and long-term bioimaging, because these probes are based on water-soluble fluorophores, which tend to diffuse away from the reaction sites after converted by furin. Such a problem can be addressed by designing a probe, which releases a precipitating fluorophore upon furin conversion. Herein, we developed a probe HPQF for in situ detection of endogenous furin activity and long-term bioimaging by integrating a strictly insoluble solid-state fluorophore 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (Cl-HPQ) with a furin specific peptide substrate (RVRR) through a self-immolative linker. The HPQF probe shows high selectivity and sensitivity to furin. Upon converted by furin, HPQF releases free Cl-HPQ, which precipitates near the enzyme active site. The precipitates emit bright solid-state fluorescence for in situ imaging. HPQF could truly visualize the location of intracellular furin, which was further confirmed by colocalization and immunofluorescence experiments. Excitingly, the long-term bioimaging was also achieved benefiting from its outstanding signal-stability and antidiffusion ability. HPQF was further utilized to monitor the level change of furin under stabilizing of hypoxia-inducible factor (HIF) regulated by cobalt chloride (CoCl2) as well as visualization of furin in MDA-MB-468 cell tumor tissues.

A Bioluminescent Probe for Imaging Endogenous Peroxynitrite in Living Cells and Mice.

Peroxynitrite (ONOO-), an extremely reactive nitrogen species (RNS), is implicated in diverse pathophysiological conditions, including cancer, neurodegenerative diseases, and inflammation. Sensing and imaging of ONOO- in living systems remains challenging due to the high autofluorescence and the limited light penetration depth. In this work, we developed a bioluminescent probe BP-PN, based on luciferase-luciferin pairs and the ONOO--responded group α-ketoamide, for highly sensitive detection and imaging of endogenous ONOO- in living cells and mice for the first time. Attributed to the BL without external excitation, the probe BP-PN exhibits a high signal-to-noise ratio with relatively low autofluorescence. Furthermore, we examine the application of the probe BP-PN using the mice model of inflammation, and BP-PN shows high sensitivity for imaging endogenous ONOO- in inflamed mice. This newly developed bioluminescent probe would be a potentially useful tool for in vivo imaging of ONOO- in wider physiological and pathological processes.

A two-photon fluorescent probe for bio-imaging of formaldehyde in living cells and tissues.

Formaldehyde (FA) plays an important role in living systems as a reactive carbonyl species (RCS). An abnormal degree of FA is known to induce neurodegeneration, cognitive decrease and memory loss owing to the formation of strong cross-link DNA and protein and other molecules. The development of efficient methods for biological FA detection is of great biomedical importance. Although a few one-photon FA fluorescent probes have been reported for imaging in living cells, probes excited by two photons are more suitable for bio-imaging due to their low background fluorescence, less photobleaching, and deep penetration depth. In this study, a two-photon fluorescent probe for FA detection and bio-imaging in living cells and tissues was reported. The detection is based on the 2-aza-Cope sigmatropic rearrangement followed by elimination to release the fluorophore, resulting in both one- and two-photon excited fluorescence increase. The probe showed a high sensitivity to FA with a detection limit of 0.2 µM. Moreover, enabled the two-photon bio-imaging of FA in live HEK-293 cells and tissues with tissue-imaging depths of 40-170 µm. Furthermore, could be applied for the monitoring of endogenous FA in live MCF-7 cells, presaging its practical applications in biological systems.

Quench-Shield Ratiometric Upconversion Luminescence Nanoplatform for Biosensing.

Upconversion nanoparticles (UCNPs) possess several unique features, but they suffer from surface quenching effects caused by the interaction between the UCNPs and fluorophore. Thus, the use of UCNPs for target-induced emission changes for biosensing and bioimaging has been challenging. In this work, fluorophore and UCNPs are effectively separated by a silica transition layer with a thickness of about 4 nm to diminish the surface quenching effect of the UCNPs, allowing a universal and efficient luminescence resonance energy transfer (LRET) ratiometric upconversion luminescence nanoplatform for biosensing applications. A pH-sensitive fluorescein derivative and Hg(2+)-sensitive rhodamine B were chosen as fluoroionphores to construct the LRET nanoprobes. Both showed satisfactory target-triggered ratiometric upconversion luminescence responses in both solution and live cells, indicating that this strategy may find wide applications in the design of nanoprobes for various biorelated targets.

Bispyrene-fluorescein hybrid based FRET cassette: a convenient platform toward ratiometric time-resolved probe for bioanalytical applications.

Pyrene excimer possesses a large Stokes shift and long fluorescence lifetime and has been widely applied in developing time-resolved biosensing systems to solve the autofluorescence interference problems in biological samples. However, only a few of pyrene excimer-based small molecular probes have been reported so far. Ratiometric probes, on the other hand, can eliminate interferences from environmental factors such as instrumental efficiency and environmental conditions by a built-in correction of the dual emission bands but are ineffective for endogenous autofluorescence in biosystems. In this work, by combining the advantages of time-resolved fluorescence technique with ratiometric probe, we reported a bispyrene-fluorescein hybrid FRET cassette (PF) as a novel ratiometric time-resolved sensing platform for bioanalytical applications, with pH chosen as a biorelated target. The probe PF showed a fast, highly selective, and reversible ratiometric fluorescence response to pH in a wide range from 3.0 to 10.0 in buffered solution. By employing time-resolved fluorescence technique, the pH-induced fluorescence signal of probe PF can be well-discriminated from biological autofluorescence background, which enables us to detect pH in a range of 4.0-8.0 in cell media within a few seconds. It has also been preliminarily applied for ratiometric quantitative monitoring of pH changes in living cells with satisfying results. Since many fluorescein-based fluorescence probes have been developed, our strategy might find wide applications in design ratiometric time-resolved probes for detection of various biorelated targets.

A rhodamine-appended water-soluble conjugated polymer: an efficient ratiometric fluorescence sensing platform for intracellular metal-ion probing.

Thewater-soluble CP was conjugatedwith a rhodamine spirolactam for the first time to develop a new FRET-based ratiometric fluorescence sensing platform(CP 1) for intracellular metal-ion probing. CP 1 exhibits excellent water-solubility with twowell-resolved emission peaks, which benefit ratiometric intracellular imaging applications.