RESUMO
Mechanoluminescence, or the generation of light from materials under external force, is a powerful tool for biology and materials science. However, direct mechanoluminescence from polymers remains limited. Here, we report a novel design strategy for mechanoluminescent polymers that leverages the synergy between a masked 2-furylcarbinol mechanophore for mechanically triggered release and an adamantylidene-phenoxy-1,2-dioxetane chemiluminophore payload. Ultrasound-induced mechanochemical activation of polymers, in both organic and aqueous solutions, triggers a cascade reaction that ultimately results in bright green light emission. This novel strategy capitalizes on the modularity of the masked 2-furylcarbinol mechanophore system in combination with advances in the design of exceptionally bright and highly tunable adamantylidene-1,2-dioxetane chemiluminophores. We anticipate that this chemistry will enable diverse applications in optoelectronics, sensing, bioimaging, optogenetics, and many other areas.
RESUMO
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.
Assuntos
Bactérias , Enzimas , Medições Luminescentes , Bactérias/classificação , Enzimas/químicaRESUMO
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.
Assuntos
Antibacterianos , Pseudomonas aeruginosa , Bactérias Gram-Negativas , Bactérias Gram-Positivas , ProteínasRESUMO
The chemiluminescent light-emission pathway of phenoxy-1,2-dioxetane luminophores attracts growing interest within the scientific community. Dioxetane probes undergoing rapid flash-type chemiexcitation exhibit higher detection sensitivity than those with a slow glow-type chemiexcitation rate. We discovered that dioxetanes fused to non-strained six-member rings, with hetero atoms or inductive electron-withdrawing groups, present both accelerated chemiexcitation rates and elevated chemical stability compared to dioxetanes fused to four-member strained rings. DFT computational simulations supported the chemiexcitation acceleration observed by spiro-fused six-member rings with inductive electron-withdrawing groups of dioxetanes. Specifically, a spiro-dioxetane with a six-member sulfone ring exhibited a chemiexcitation rate 293-fold faster than that of spiro-adamantyl-dioxetane. A turn-ON dioxetane probe for the detection of the enzyme ß-galactosidase, containing the six-member sulfone unit, exhibited a S/N value of 108 in LB cell growth media. This probe demonstrated a substantial increase in detection sensitivity towards E. coli bacterial cells expressing ß-galactosidase, with an LOD value that is 44-fold more sensitive than that obtained by the adamantyl counterpart. The accelerated chemiexcitation and the elevated chemical stability presented by dioxetane containing a spiro-fused six-member ring with a sulfone inductive electron-withdrawing group, make it an ideal candidate for designing efficient turn-on chemiluminescent probes with exceptionally high detection sensitivity.
RESUMO
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.
Assuntos
Fosfatase Alcalina , Medições Luminescentes , Medições Luminescentes/métodos , beta-Galactosidase , Corantes , FenóisRESUMO
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 â CFU mL-1 of S. aureus and 5.0×104 â 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.
Assuntos
Sideróforos , Staphylococcus aureus , Bactérias , Ferro , Pseudomonas aeruginosaRESUMO
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.
Assuntos
Sondas Moleculares , Oxigênio Singlete , Medições LuminescentesRESUMO
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.
RESUMO
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.
Assuntos
Endopeptidases , Peptídeo Hidrolases , Técnicas de Síntese em Fase SólidaRESUMO
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.
Assuntos
Endopeptidases , Processamento de Proteína Pós-Traducional , Humanos , UbiquitinaRESUMO
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.
Assuntos
Biocatálise , Farmacorresistência Bacteriana , Sulfeto de Hidrogênio/metabolismo , beta-Lactamases/metabolismo , beta-Lactamas/metabolismo , beta-Lactamas/farmacologia , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Medições LuminescentesRESUMO
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 inâ vivo. Herein, we report a new chemiluminescent probe to image inâ situ the granzymeâ B-mediated killing activity of NK cells against cancer cells. We have optimised a granzymeâ 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 granzymeâ 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 inâ vivo imaging of NK cell activity in live tumours.
Assuntos
Corantes Fluorescentes/metabolismo , Granzimas/metabolismo , Células Matadoras Naturais/metabolismo , Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Granzimas/química , Humanos , Células Matadoras Naturais/patologia , Medições Luminescentes , Camundongos , Estrutura Molecular , Neoplasias/diagnóstico por imagem , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Imagem ÓpticaRESUMO
The prostate specific antigen (PSA), a serine protease with chymotrypsin-like activity, is predominantly expressed in the prostate and is considered as the most common marker in use to identify and follow the progress of prostate cancer. In addition, it is also now accepted as a marker for detecting semen in criminal cases. Here, we describe the design, synthesis, and evaluation of the first chemiluminescence probe for detection of PSA enzymatic activity. The probe activation mechanism is based on a catalytic cleavage of a specific peptidyl substrate, followed by a release of a phenoxy-dioxetane luminophore, that then undergoes efficient chemiexcitation to emit a green photon. The probe exhibits a significant turn-on response upon reaction with PSA and produces strong light emission signal with an extremely high signal-to-noise ratio. Comparison of the chemiluminescence probe with an analogous fluorescence probe showed superior detection capability in terms of response time and sensitivity. In addition, the probe was able to efficiently detect and image human semen traces on fabric, even after 3 days from sample preparation. The advantageous sensitivity and simplicity of a chemiluminescence assay to detect seminal fluid was effectively demonstrated by on-site measurements using a small portable luminometer. It is expected that the new chemiluminescence probe would be broadly useful for numerous applications in which PSA detection or imaging is required.
Assuntos
Medicina Legal , Sondas Moleculares/química , Antígeno Prostático Específico/metabolismo , Sêmen/metabolismo , Humanos , Limite de Detecção , Luminescência , Medições Luminescentes , Masculino , Proteólise , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
Functional molecular scaffolds comprised of self-immolative adaptors are being used in widespread applications in the fields of enzyme activity analyses, signal amplification, and bioimaging. Optically detected chemical probes are very promising compounds for sensing and diagnosis, since they present several attractive features such as high specificity, low detection limits, fast response times, and technical simplicity. During the last two decades, we have developed several distinct molecular scaffolds that harness the self-immolative disassembly feature of these adaptors to amplify chromogenic output for diagnosis and drug delivery applications. In order to study the molecular behavior of the various amplification systems, an optical output, used to monitor the progress of the disassembly pattern, was required. Therefore, over the course of our research, diverse molecular scaffolds that produce an optical signal in response to a disassembly step, were evaluated. These optically active scaffolds have been incorporated into self-immolative dendrimers and self-immolative polymers to implement unique disassembly properties that result with linear and exponential signal amplification capabilities. In addition, some scaffolds, aimed for linker technology, were used in delivery systems to monitor release of drug molecules. The optical signal used to monitor the release event could be produced by analysis of reporter molecules with chromogenic or fluorogenic properties. Recently, we have also developed molecular scaffolds modified to produce a chemiluminescent signal to monitor the self-immolative disassembly step. The main advantage of these scaffolds over others is the use of chemiluminescence as an output signal. It is well-known that chemiluminescence is considered as one the most sensitive diagnostic methods due to its high signal-to-noise ratio. The unique structures of the self-immolative chemiluminescence scaffolds have been used in the design of three different distinctive concepts: self-immolative chemiluminescence polymers, auto-inductive amplification systems with chemiluminescence signal and monitoring of drug release by a chemiluminescence output. Furthermore, we reported the design and synthesis of the first theranostic prodrug for the monitoring of drug release achieved by a chemiluminescence mode of action. Quinone-methide elimination has proven to serve as a valuable functional tool for composing molecular scaffolds with self-immolative capabilities. Such scaffolds function as molecular adaptors that can almost simultaneously release a target molecule with an accompanied emission of a light signal that is used to monitor the release event. We anticipate that these self-immolative scaffolds will continue to find utility as functional linkers in various chemical and biological research areas such as drug delivery, theranostic applications, and as molecular sensors with signal amplification.
Assuntos
Fenômenos Ópticos , Espectrometria de Fluorescência , Animais , Liberação Controlada de Fármacos , Humanos , Polímeros/químicaRESUMO
Carbapenemase-producing organisms (CPOs) pose a severe threat to antibacterial treatment due to the acquisition of antibiotic resistance. This resistance can be largely attributed to the antibiotic-hydrolyzing enzymes that the bacteria produce. Current carbapenem "wonder drugs", such as doripenem, ertapenem, meropenem, imipenem, and so on, are resistant to regular ß-lactamases, but susceptible to carbapenemases. Even worse, extended exposure of bacteria to these drugs accelerates the spread of resistance genes. In order to preserve the clinical efficacy of antibacterial treatment, carbapenem drugs should be carefully regulated and deployed only in cases of a CPO infection. Early diagnosis is therefore of paramount importance. Herein, we report the design, synthesis, and activity of the first carbapenemase-sensitive chemiluminescent probe, CPCL, which may be used to monitor CPO activity. The design of our probe enables enzymatic cleavage of the carbapenem core, which is followed by a facile 1,8-elimination process and the emission of green light through rapid chemical excitation. We have demonstrated the ability of the probe to detect a number of clinically relevant carbapenemases and the successful identification of CPO present in bacterial cultures, such as those used for clinical diagnosis. We believe that our use of "turn-on" chemiluminescence activation will find significant application in future diagnostic assays and improve antibacterial treatment.
Assuntos
Antibacterianos/farmacologia , Bactérias/genética , Proteínas de Bactérias/química , Carbapenêmicos/química , Imipenem/química , Meropeném/química , beta-Lactamases/química , Antibacterianos/química , Bactérias/química , Humanos , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Selective and sensitive molecular probes for hydrogen peroxide (H2 O2 ), which plays diverse roles in oxidative stress and redox signaling, are urgently needed to investigate the physiological and pathological effects of H2 O2 . A lack of reliable tools for in vivo imaging has hampered the development of H2 O2 mediated therapeutics. By combining a specific tandem Payne/Dakin reaction with a chemiluminescent scaffold, H2 O2 -CL-510 was developed as a highly selective and sensitive probe for detection of H2 O2 both in vitro and in vivo. A rapid 430-fold enhancement of chemiluminescence was triggered directly by H2 O2 without any laser excitation. Arsenic trioxide induced oxidative damage in leukemia was successfully detected. In particular, cerebral ischemia-reperfusion injury-induced H2 O2 fluxes were visualized in rat brains using H2 O2 -CL-510, providing a new chemical tool for real-time monitoring of H2 O2 dynamics in living animals.
Assuntos
Peróxido de Hidrogênio/metabolismo , Luminescência , Sondas Moleculares/metabolismo , Animais , Encéfalo/metabolismo , Humanos , Limite de Detecção , Sondas Moleculares/química , Ratos , Bibliotecas de Moléculas Pequenas/metabolismo , Células THP-1RESUMO
Chemiluminescence is being considered an effective imaging modality as it offers low background and high sensitivity. Recent discovery by our group has led to development of new phenoxy-dioxetane chemiluminescence luminophores, which are highly bright under physiological conditions. However, the current scope of probes based on these luminophores is limited, as they can only be turned on by phenol protecting group removal. Here we present a new chemiluminescence resonance energy transfer (CRET) system, Glow-CRET, in which light emission is triggered by proteolytic cleavage of a peptide substrate that links a dioxetane luminophore and a quencher. In order to compose such system, a new phenoxy-dioxetane luminophore, 7-HC-CL, was developed. This luminophore exhibits intense and persistent glow chemiluminescence; it undergoes very slow chemiexcitation, and it has the highest chemiluminescence quantum yield ever reported under physiological conditions. Based on 7-HC-CL, a Glow-CRET probe for matrix metalloproteinases, MMP-CL, was synthesized. Incubation of MMP-CL with its cognate protease resulted in 160-fold increase in chemiluminescence signal. MMP-CL was also able to detect matrix metalloproteinase activity in cancer cells with significantly higher signal-to-background ratio than an analogous fluorescence resonance energy transfer (FRET)-based probe. This work is expected to open new horizons in chemiluminescence imaging, as it enables to use the dioxetanes in ways that had not been possible. We anticipate that 7-HC-CL and future derivatives will be utilized not only for the construction of further Glow-CRET probes, but also for other applications, such as chemiluminescence tagging of proteins.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Peptídeo Hidrolases/metabolismo , Linhagem Celular Tumoral , Cumarínicos/química , Humanos , Cinética , Medições Luminescentes , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismoRESUMO
A new signal amplification probe with a linear chain reaction amplification mechanism and distinct chemiluminescence output was developed. The probe is composed of a unique structural motif that combines a chemiexcitation mechanism with an intramolecular transesterification into a single molecular structure. As demonstrated with a probe designed to detect hydrogen peroxide, an auto-inductive chemiluminescence signal amplification was obtained through methanol release by an intramolecular transesterification of the generated 2-hydroxymethylbenzoate derivative. The methanol was then oxidized by alcohol oxidase to regenerate the analyte of interest, hydrogen peroxide. Our probe enabled direct measurement of light emission with a limit of detection of 2.5 µM, whilst the assay was rapidly completed within 14 to 150 minutes. Such molecular probes with chemiluminescence signal enhancement through analyte amplification could be used for the detection of other chemical and biological analytes.
RESUMO
A chemiluminescence probe for singlet oxygen 1O2 (SOCL) was investigated in phosphate buffer saline (PBS), either in the absence of proteins or containing bovine serum albumin (BSA). In the protein-free PBS, the reactivity of SOCL for methylene blue (MB)-photosensitized 1O2 was found to be moderate or low. The reaction yield increased with temperature and/or concentration of dissolved molecular oxygen. Unexpectedly, the presence of BSA boosted both the emissive nature and the thermal stability of the phenoxy-dioxetane intermediate formed in the chemiexcitation pathway. Isothermal titration calorimetry showed that SOCL has a moderate binding affinity for BSA and that entropy forces drive the formation of the SOCL-BSA complex. A model with two identical and independent binding sites was used to fit the binding isotherm data. Co-operative binding was observed when MB was present. Local viscosity factors and/or conformational restrictions of the BSA-bound SOCL phenoxy-dioxetane were proposed to contribute to the formation of the highly emissive benzoate ester during the chemically initiated electron exchange luminescence (CIEEL) process. These results led us to conclude that hydrophobic interactions of the SOCL with proteins can modify the emissive nature of its phenoxy-dioxetane, which should be taken into account when using SOCL or its cell-penetrating peptide derivative in living cells.
Assuntos
Medições Luminescentes , Modelos Químicos , Modelos Moleculares , Sondas Moleculares/química , Soroalbumina Bovina/química , Oxigênio Singlete/química , Animais , BovinosRESUMO
Detection of Salmonella and L.â monocytogenes in food samples by current diagnostic methods requires relatively long time to results (2-6â days). Furthermore, the ability to perform environmental monitoring at the factory site for these pathogens is limited due to the need for laboratory facilities. Herein, we report new chemiluminescence probes for the ultrasensitive direct detection of viable pathogenic bacteria. The probes are composed of a bright phenoxy-dioxetane luminophore masked by triggering group, which is activated by a specific bacterial enzyme, and could detect their corresponding bacteria with an LOD value of about 600-fold lower than that of fluorescent probes. Moreover, we were able to detect a minimum of 10 Salmonella cells within 6â h incubation. The assay allows for bacterial enrichment and detection in one test tube without further sample preparation. We anticipate that this design strategy will be used to prepare analogous chemiluminescence probes for other enzymes relevant to specific bacteria detection and point-of-care diagnostics.