Singlet Oxygen Detection by Chemiluminescence Probes in Living Cells.

Singlet oxygen is a reactive oxygen species that causes oxidative damage to plant cells, but intriguingly it can also act as a signalling molecule to reprogram gene expression required to induce plant physiological/cellular responses. Singlet oxygen photosensitization in plants mainly occurs in chloroplasts after the molecular collision of ground-state molecular oxygen with triplet-excited-state chlorophyll. Singlet oxygen direct detection through phosphorescence emission in chloroplasts is a herculean task due to its extremely low luminescence quantum yield. Because of this, indirect alternative methods have been developed for its detection in biological systems, for example, by measuring the changes in the EPR signal or fluorescence intensity of singlet oxygen reaction-based probes. The singlet oxygen chemiluminescence (SOCL) is a chemiluminescence probe with high sensitivity and selectivity towards singlet oxygen and promising use to detect it in living cells without the inconvenience of low stability of the EPR signal of spin probes in the presence of redox compounds, spurious light scattering coming from the light source required for the excitation of fluorescence probes or the light emission of endogenous fluorescent molecules like chlorophyll in chloroplasts. The protocol presented in this chapter describes the first steps to characterizing singlet oxygen production within the biological system under study; this is accomplished through monitoring molecular oxygen consumption by SOCL using a Clark-type oxygen electrode and measuring the chemiluminescence generated by SOCL 1,2-dioxetane using a spectrofluorometer. For singlet oxygen detection within living cells, a version of SOCL with increased membrane permeability (SOCL-CPP) is described.

Hyper-Responsive Chemiluminescent Probe Reveals Distinct PYRase Activity in Pseudomonas aeruginosa.

Pyrrolidone carboxyl peptidase, commonly known as PYRase, is an exopeptidase that catalytically cleaves an N-terminal pyroglutamic acid from peptides or proteins. The diverse functions of PYRases in bacterial enzymology have prompted the development of various bacterial diagnostic techniques. However, the specific physiological role and activity of this enzyme across the bacterial kingdom remain unclear. Here, we present a functional phenoxy-1,2-dioxetane chemiluminescent probe (PyrCL) that can selectively detect PYRase activity in both Gram-positive and Gram-negative bacteria. The probe activation mechanism is based on the cleavage of a pyroglutamyl substrate, followed by a release of the phenoxy-dioxetane luminophore, which then undergoes efficient chemiexcitation to emit a green photon. Probe PyrCL exhibits an effective turn-on response with superior detection capability in terms of response time and sensitivity compared to existing fluorescence probes. The superior detection sensitivity of the chemiluminescent probe enables us to reveal previously undetected PYRase activity in Streptococcus mutans. Furthermore, it enables the discrimination of Pseudomonas aeruginosa from other Gram-negative bacteria in the tested panel, based on their distinct PYRase activity. We expect that probe PyrCL will have great value for PYRase-based bacteria diagnosis with use in basic research and clinical applications.

Highly sensitive chemiluminescence sensors for the detection and differentiation of chemical warfare agents.

Highly sensitive chemiluminescence-based probes that effectively detect and differentiate between the extremely toxic real G- and V-type organophosphorus chemical warfare agents (OPCWAs) are presented. This straightforward approach does not require any instrumentation or light source; hence, it appears ideal for the future development of field colorimetric detectors.

Enzymatic Activity Profiling Using an Ultrasensitive Array of Chemiluminescent Probes for Bacterial Classification and Characterization.

Identification and characterization of bacterial species in clinical and industrial settings necessitate the use of diverse, labor-intensive, and time-consuming protocols as well as the utilization of expensive and high-maintenance equipment. Furthermore, while cutting-edge identification technologies such as mass spectrometry and PCR are highly effective in identifying bacterial pathogens, they fall short in providing additional information for identifying bacteria not present in the databases upon which these methods rely. In response to these challenges, we present a robust and general approach to bacterial identification based on their unique enzymatic activity profiles. This method delivers results within 90 min, utilizing an array of highly sensitive and enzyme-selective chemiluminescent probes. Leveraging our recently developed technology of chemiluminescent luminophores, which emit light under physiological conditions, we have crafted an array of probes designed to rapidly detect various bacterial enzymatic activities. The array includes probes for detecting resistance to the important and large class of ß-lactam antibiotics. The analysis of chemiluminescent fingerprints from a diverse range of prominent bacterial pathogens unveiled distinct enzymatic activity profiles for each strain. The reported universally applicable identification procedure offers a highly sensitive and expeditious means to delineate bacterial enzymatic activity fingerprints. This opens new avenues for characterizing and identifying pathogens in research, clinical, and industrial applications.

Spirostrain-Accelerated Chemiexcitation of Dioxetanes Yields Unprecedented Detection Sensitivity in Chemiluminescence Bioassays.

Chemiluminescence is a fascinating phenomenon that involves the generation of light through chemical reactions. The light emission from adamantyl-phenoxy-1,2-dioxetanes can glow from minutes to hours depending on the specific substituent present on the dioxetane molecule. In order to improve the light emission properties produced by these chemiluminescent luminophores, it is necessary to induce the chemiexcitation rate to a flash mode, wherein the bulk of light is emitted instantly rather than slowly over time. We report the realization of this goal through the incorporation of spirostrain release into the decomposition of 1,2-dioxetane luminophores. DFT computational simulations provided support for the hypothesis that the spiro-cyclobutyl substituent accelerates chemiexcitation as compared to the unstrained adamantyl substituent. Spiro-linking of cyclobutane and oxetane units led to greater than 100-fold and 1000-fold emission enhancement, respectively. This accelerated chemiexcitation rate increases the detection sensitivity for known chemiluminescent probes to the highest signal-to-noise ratio documented to date. A turn-ON probe, containing a spiro-cyclobutyl unit, for detecting the enzyme ß-galactosidase exhibited a limit of detection value that is 125-fold more sensitive than that for the previously described adamantyl analogue. This probe was also able to instantly detect and image ß-gal activity with enhanced sensitivity in E. coli bacterial assays.

Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker.

Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer therapeutics, with 14 ADCs already approved worldwide. We developed unique linker technologies for the bioconjugation of drug molecules with controlled-release applications. We synthesized cathepsin-cleavable ADCs using a dimeric prodrug system based on a self-immolative dendritic scaffold, resulting in a high drug-antibody ratio (DAR) with the potential to reach 16 payloads due to its dendritic structure, increased stability in the circulation and efficient release profile of a highly cytotoxic payload at the targeted site. Using our novel cleavable linker technologies, we conjugated the anti-human epidermal growth factor receptor 2 (anti-HER2) antibody, trastuzumab, with topoisomerase I inhibitors, exatecan or belotecan. The newly synthesized ADCs were tested in vitro on mammary carcinoma cells overexpressing human HER2, demonstrating a substantial inhibitory effect on the proliferation of HER2-positive cells. Importantly, a single dose of our trastuzumab-based ADCs administered in vivo to mice bearing HER2-positive tumors, showed a dose-dependent inhibition of tumor growth and survival benefit, with the most potent antitumor effects observed at 10 mg/kg, which resulted in complete tumor regression and survival of 100% of the mice. Overall, our novel dendritic technologies using the protease-cleavable Val-Cit linker present an opportunity for the development of highly selective and potent controlled-released therapeutic payloads. This strategy could potentially lead to the development of novel and effective ADC technologies for patients diagnosed with HER2-positive cancers. Moreover, our proposed ADC linker technology can be implemented in additional medical conditions such as other malignancies as well as autoimmune diseases that overexpress targets, other than HER2.

Chemiluminescent duplex analysis using phenoxy-1,2-dioxetane luminophores with color modulation.

Multiplex technology is an important emerging field, in diagnostic sciences, that enables the simultaneous detection of several analytes in a single sample. The light-emission spectrum of a chemiluminescent phenoxy-dioxetane luminophore can be accurately predicted by determining the fluorescence-emission spectrum of its corresponding benzoate species, which is generated during the chemiexcitation process. Based on this observation, we designed a library of chemiluminescent dioxetane luminophores with multicolor emission wavelengths. Two dioxetane luminophores that have different emission spectra, but similar quantum yield properties, were selected from the synthesized library for a duplex analysis. The selected dioxetane luminophores were equipped with two different enzymatic substrates to generate turn-ON chemiluminescent probes. This pair of probes exhibited a promising ability to act as a chemiluminescent duplex system for the simultaneous detection of two different enzymatic activities in a physiological solution. In addition, the pair of probes were also able to simultaneously detect the activities of the two enzymes in a bacterial assay, using a blue filter slit for one enzyme and a red filter slit for the other enzyme. As far as we know, this is the first successful demonstration of a chemiluminescent duplex system composed of two-color phenoxy-1,2-dioxetane luminophores. We believe that the library of dioxetanes presented here will be beneficial for developing chemiluminescence luminophores for multiplex analysis of enzymes and bioanalytes.

Dual Chemiexcitation by a Unique Dioxetane Scaffold Gated by an OR Logic Set of Triggers.

Chemiexcitation of phenoxy-1,2-dioxetane chemiluminescent luminophores is initiated by electron transfer from a meta-positioned phenolate ion to the peroxide-dioxetane bond. Here we report the development of a unique 1,2-dioxetane chemiluminescent scaffold with chemiexcitation gated by an OR logic dual-set of triggering events. This scaffold is composed of meta-dihydroxyphenyl-1,2-dioxetane-adamantyl molecules, equipped with acrylic acid and chlorine substituents, that chemiexcitation under physiological conditions. A dual-mode chemiluminescent probe, armed with two different triggering substrates designed for activation by the enzymes ß-galactosidase and alkaline phosphatase, was synthesized. The probe emitted intense light signals in the response to each enzyme, demonstrating its ability to serve as a single-component chemiluminescent sensor for dual-analyte detection. We also demonstrated the ability of the probe to detect ß-galactosidase and phosphatase activities in bacteria. This is the first 1,2-dioxetane scaffold capable of responding to two different chemiexcitation events from two different positions on the same dioxetane molecule. We anticipate that the OR-gated mode of chemiexcitation, described herein, will find utility in the preparation of chemiluminescent probes with a dual-analyte detection/imaging mode.

Ultrasensitive chemiluminescent neuraminidase probe for rapid screening and identification of small-molecules with antiviral activity against influenza A virus in mammalian cells.

Influenza A virus is the most virulent influenza subtype and is associated with large-scale global pandemics characterized by high levels of morbidity and mortality. Developing simple and sensitive molecular methods for detecting influenza viruses is critical. Neuraminidase, an exo-glycosidase displayed on the surface of influenza virions, is responsible for the release of the virions and their spread in the infected host. Here, we present a new phenoxy-dioxetane chemiluminescent probe (CLNA) that can directly detect neuraminidase activity. The probe exhibits an effective turn-on response upon reaction with neuraminidase and produces a strong emission signal at 515 nm with an extremely high signal-to-noise ratio. Comparison measurements of our new probe with previously reported analogous neuraminidase optical probes showed superior detection capability in terms of response time and sensitivity. Thus, as far as we know, our probe is the most sensitive neuraminidase probe known to date. The chemiluminescence turn-on response produced by our neuraminidase probe enables rapid screening for small molecules that inhibit viral replication through different mechanisms as validated directly in influenza A-infected mammalian cells using the known inhibitors oseltamivir and amantadine. We expect that our new chemiluminescent neuraminidase probe will prove useful for various applications requiring neuraminidase detection including drug discovery assays against various influenza virus strains in mammalian cells.

Application of Super Photoacids in Controlling Dynamic Processes: Light-Triggering the Self-Propulsion of Oil Droplets.

The dynamic control of pH-responsive systems is at the heart of many natural and artificial processes. Here, we use photoacids, molecules that dissociate only in their excited state and transfer their proton to nearby proton acceptors, for the dynamic control of processes. A problem arises when there is a need to protonate highly acidic acceptors. We solve this problem using super photoacids that have an excited-state pKa of -8, thus enabling them to protonate very weak proton acceptors. The process that we target is the light-triggered self-propulsion of droplets, initiated by an excited-state proton transfer (ESPT) from a super photoacid donor to a surfactant acceptor situated on the surface of the droplet with a pKa of ∼0. We first confirm using steady-state and time-resolved spectroscopy that a super photoacid can undergo ESPT to the acidic surfactant, whereas a "regular" photoacid cannot. Next, we show self-propulsion of the droplet upon irradiating the solvated super photoacid. We further confirm the protonation of the surfactant on the surface of the droplet using transient surface tension measurements. Our system is the first example of the application of super photoacids to control dynamic processes and opens new possibilities in the field of light-triggered dynamic systems.

Enzyme-Activated, Chemiluminescent Siderophore-Dioxetane Probes Enable the Selective and Highly Sensitive Detection of Bacterial Pathogens.

The sensitive detection of bacterial infections is a prerequisite for their successful treatment. The use of a chemiluminescent readout was so far hampered by an insufficient probe enrichment at the pathogens. We coupled siderophore moieties, that harness the unique iron transport system of bacteria, with enzyme-activatable dioxetanes and obtained seven trifunctional probes with high signal-to-background ratios (S/B=426-859). Conjugates with efficient iron transport capability into bacteria were identified through a growth recovery assay. All ESKAPE pathogens were labelled brightly by desferrioxamine conjugates, while catechols were weaker due to self-quenching. Bacteria could also be detected inside lung epithelial cells. The best probe 8 detected 9.1×103  CFU mL-1 of S. aureus and 5.0×104  CFU mL-1 of P. aeruginosa, while the analogous fluorescent probe 10 was 205-305fold less sensitive. This qualifies siderophore dioxetane probes for the selective and sensitive detection of bacteria.

Modular Access to Diverse Chemiluminescent Dioxetane-Luminophores through Convergent Synthesis.

Adamantyl-dioxetane luminophores are an important class of chemiluminescent molecular probes for diagnostics and imaging. We have developed a new efficient synthetic route for preparation of adamantyl-enolether as precursors for dioxetane chemiluminescent luminophores. The synthesis is convergent, using an unusual Stille cross-coupling reaction employing a stannane-enolether, to directly afford adamantyl-enolether. In a following simple step, the dioxetane is obtained by oxidation of the enolether precursor with singlet-oxygen. The scope of this synthetic route is broad since a large number of haloaryl substrates are either commercially available or easily accessible. Such a late-stage derivatization strategy simplifies the rapid exploration of novel luminogenic molecular structures in a library format and simplifies the synthesis of known dioxetane luminophores. We expect that this new synthetic strategy will be particularly useful in the design and synthesis of yet unexplored dioxetane chemiluminescent luminophores.

Self-Immolative Polymers: An Emerging Class of Degradable Materials with Distinct Disassembly Profiles.

Self-immolative polymers are an emerging class of macromolecules with distinct disassembly profiles that set them apart from other general degradable materials. These polymers are programmed to disassemble spontaneously from head to tail, through a domino-like fragmentation, upon response to extremal stimuli. In the time since we first reported this unique type of molecule, several groups around the world have developed new, creative molecular structures that perform analogously to our pioneering polymers. Self-immolative polymers are now widely recognized as an important class of stimuli-responsive materials for a wide range of applications such as signal amplification, biosensing, drug delivery, and materials science. The quinone-methide elimination was shown to be an effective tool to achieve rapid domino-like fragmentation of polymeric molecules. Thus, numerous applications of self-immolative polymers are based on this disassembly chemistry. Although several other fragmentation reactions achieved the function requested for sequential disassembly, we predominantly focused in this Perspective on examples of self-immolative polymers that disassemble through the quinone-methide elimination. Selected examples of self-immolative polymers that disassembled through other chemistries are briefly described. The growing demand for stimuli-responsive degradable materials with novel molecular backbones and enhanced properties guarantees the future interest of the scientific community in this unique class of polymers.

Injectable Nanocomposite Implants Reduce ROS Accumulation and Improve Heart Function after Infarction.

In a myocardial infarction, blood supply to the left ventricle is abrogated due to blockage of one of the coronary arteries, leading to ischemia, which further triggers the generation of reactive oxygen species (ROS). These sequential processes eventually lead to the death of contractile cells and affect the integrity of blood vessels, resulting in the formation of scar tissue. A new heart therapy comprised of cardiac implants encapsulated within an injectable extracellular matrix-gold nanoparticle composite hydrogel is reported. The particles on the collagenous fibers within the hydrogel promote fast transfer of electrical signal between cardiac cells, leading to the functional assembly of the cardiac implants. The composite hydrogel is shown to absorb reactive oxygen species in vitro and in vivo in mice ischemia reperfusion model. The reduction in ROS levels preserve cardiac tissue morphology and blood vessel integrity, reduce the scar size and the inflammatory response, and significantly prevent the deterioration of heart function.

Turn on chemiluminescence-based probes for monitoring tyrosinase activity in conjunction with biological thiols.

We report a chemiluminescent probe (CLPT1) that permits the paired detection of tyrosinase (Tyr) and biological thiols. Tyr only leads to a poor chemiluminescence response, a finding ascribed to the formation of a stable o-benzoquinone intermediate. The addition of glutathione (GSH), or ascorbate to the o-benzoquinone intermediate results in thiol conjugation or reduction to this intermediate, respectively. This produces a strong chemiluminescence response. Thiol co-dependence was demonstrated in live cells using the cell permeable analogue, CLPT3. The present chemiluminescence-based strategy allows the concurrent detection of tyrosinase activity and biological thiols.

Universal Access to Protease Chemiluminescent Probes through Solid-Phase Synthesis.

Protease chemiluminescent probes exhibit extremely high detection sensitivity for monitoring activity of various proteolytic enzymes. However, their synthesis, performed in solution, involves multiple synthetic and purification steps, thereby generating a major limitation for rapid preparation of such probes with diverse substrate scope. To overcome this limitation, we developed a general solid-phase-synthetic approach to prepare chemiluminescent protease probes, by peptide elongation, performed on an immobilized chemiluminescent enol-ether precursor. The enol-ether precursor is immobilized on a 2-chlorotrityl-chloride resin through an acrylic acid substituent by an acid-labile ester linkage. Next, a stepwise elongation of the peptide is performed using standard Fmoc solid-phase peptide synthesis. After cleavage of the peptide-enol-ether precursor from the resin, by hexafluoro-iso-propanol, a simple oxidation of the enol-ether yields the final chemiluminescent dioxetane protease probe. To validate the applicability of the methodology, two chemiluminescent probes were efficiently prepared by solid-phase synthesis with dipeptidyl substrates designed for activation by aminopeptidase and cathepsin-B proteases. A more complex example was demonstrated by the synthesis of a chemiluminescent probe for detection of PSA, which includes a peptidyl substrate of six amino acids. We anticipate that the described methodology would be useful for rapid preparation of chemiluminescent protease probes with vast and diverse peptidyl substrates.

Synthesis and Evaluation of Ubiquitin-Dioxetane Conjugate as a Chemiluminescent Probe for Monitoring Deubiquitinase Activity.

The removal of ubiquitin (Ub) from a modified protein or Ub chain is a process that occurs regularly by the ubiquitin-proteasome system. This process is known to be mediated by various deubiquitinating enzymes (DUBs) in order to control the protein's half-life and its expression levels among many other signaling processes. Since the function of DUBs is also involved in numerous human diseases, such as cancer, there is an obvious need for an effective diagnostic probe that can monitor the activity of these enzymes. We have developed the first chemiluminescence probe for detection of DUBs activity. The probe was prepared by conjugation of the chemically synthesized C-terminally activated Ub(1-75) with a Gly-enolether precursor. Subsequent oxidation, under aqueous conditions, of the enolether conjuagate with singlet-oxygen furnished the dioxetane probe Ub-CL. This synthesis provides the first example of a dioxetane-luminophore protein conjugate. The probe's ability to detect deubiquitinating activity was successfully validated with three different DUBs. In order to demonstrate the advantage of our new probe, comparison measurements for detection of DUB UCH-L3 activity were performed between the chemiluminescent probe Ub-CL and the well-known Ub-AMC probe. The obtained data showed significantly higher S/N, for probe Ub-CL (>93-fold) in comparison to that observed for Ub-AMC (1.5-fold). We anticipate that the successful design and synthesis of the turn-ON protein-dioxetane conjugate probe, demonstrated in this work, will provide the insight and motivation for preparation of other relevant protein-dioxetane conjugates.

Chemiluminescent Protease Probe for Rapid, Sensitive, and Inexpensive Detection of Live Mycobacterium tuberculosis.

Tuberculosis (TB) is a top-ten cause of death worldwide. Successful treatment is often limited by insufficient diagnostic capabilities, especially at the point of care in low-resource settings. The ideal diagnostic must be fast, be cheap, and require minimal clinical resources while providing high sensitivity, selectivity, and the ability to differentiate live from dead bacteria. We describe here the development of a fast, luminescent, and affordable sensor of Hip1 (FLASH) for detecting and monitoring drug susceptibility of Mycobacterium tuberculosis (Mtb). FLASH is a selective chemiluminescent substrate for the Mtb protease Hip1 that, when processed, produces visible light that can be measured with a high signal-to-noise ratio using inexpensive sensors. FLASH is sensitive to fmol of recombinant Hip1 enzyme in vitro and can detect as few as thousands of Mtb cells in culture or in human sputum samples within minutes. The probe is highly selective for Mtb compared to other nontuberculous mycobacteria and can distinguish live from dead cells. Importantly, FLASH can be used to measure antibiotic killing of Mtb in culture with greatly accelerated timelines compared to traditional protocols. Overall, FLASH has the potential to enhance both TB diagnostics and drug resistance monitoring in resource-limited settings.

Chemiluminescence Detection of Hydrogen Sulfide Release by ß-Lactamase-Catalyzed ß-Lactam Biodegradation: Unprecedented Pathway for Monitoring ß-Lactam Antibiotic Bacterial Resistance.

ß-Lactamase positive bacteria represent a growing threat to human health because of their resistance to commonly used antibiotics. Therefore, development of new diagnostic methods for identification of ß-lactamase positive bacteria is of high importance for monitoring the spread of antibiotic-resistant bacteria. Here, we report the discovery of a new biodegradation metabolite (H2S), generated through ß-lactamase-catalyzed hydrolysis of ß-lactam antibiotics. This discovery directed us to develop a distinct molecular technique for monitoring bacterial antibiotic resistance. The technique is based on a highly efficient chemiluminescence probe, designed for detection of the metabolite, hydrogen sulfide, that is released upon biodegradation of ß-lactam by ß-lactamases. Such an assay can directly indicate if antibiotic bacterial resistance exists for a certain examined ß-lactam. The assay was successfully demonstrated for five different ß-lactam antibiotics and eight ß-lactam resistant bacterial strains. Importantly, in a functional bacterial assay, our chemiluminescence probe was able to clearly distinguish between a ß-lactam resistant bacterial strain and a sensitive one. As far as we know, there is no previous documentation for such a biodegradation pathway of ß-lactam antibiotics. Bearing in mind the data obtained in this study, we propose that hydrogen sulfide should be considered as an emerging ß-lactam metabolite for detection of bacterial resistance.

A Functional Chemiluminescent Probe for in Vivo Imaging of Natural Killer Cell Activity Against Tumours.

Natural killer (NK) cells are immune cells that can kill certain types of cancer cells. Adoptive transfer of NK cells represents a promising immunotherapy for malignant tumours; however, there is a lack of methods to validate anti-tumour activity of NK cells in vivo. Herein, we report a new chemiluminescent probe to image in situ the granzyme B-mediated killing activity of NK cells against cancer cells. We have optimised a granzyme B-specific construct using an activatable phenoxydioxetane reporter so that enzymatic cleavage of the probe results in bright chemiluminescence. The probe shows high selectivity for active granzyme B over other proteases and higher signal-to-noise ratios than commercial fluorophores. Finally, we demonstrate that the probe can detect NK cell activity in mouse models, being the first chemiluminescent probe for in vivo imaging of NK cell activity in live tumours.