Biotechnology Journal

Biotechnol. J. 2014, 9, 266–281

DOI 10.1002/biot.201300201

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Review

FRET-based and other fluorescent proteinase probes Hai-Yu Hu1,2,*, Stefanie Gehrig1,3,4,*, Gregor Reither1,3,4, Devaraj Subramanian1, Marcus A. Mall3,4,5,6, Oliver Plettenburg2 and Carsten Schultz1,3,4 1 European

Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Heidelberg, Germany Deutschland GmbH, Diabetes Division, R&D, Industriepark Hoechst, Frankfurt am Main, Germany 3 Translational Lung Research Center (TLRC), Member of the German Center for Lung Research (DZL), University of Heidelberg, Heidelberg, Germany 4 Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory, Heidelberg, Germany 5 Division of Pediatric Pulmonology & Allergy and Cystic Fibrosis Center, Departments of Pediatrics, University of Heidelberg, Heidelberg, Germany 6 Department of Translational Pulmonology, University of Heidelberg, Heidelberg, Germany 2 Sanofi

The continuous detection of enzyme activities and their application in medical diagnostics is one of the challenges in the translational sciences. Proteinases represent one of the largest groups of enzymes in the human genome and many diseases are based on malfunctions of proteolytic activity. Fluorescent sensors may shed light on regular and irregular proteinase activity in vitro and in vivo and provide a deeper insight into the function of these enzymes and their role in pathophysiological processes. The focus of this review is on Förster resonance energy transfer (FRET)-based proteinase sensors and reporters because these probes are most likely to provide quantitative data. The medical relevance of proteinases are discussed using lung diseases as a prominent example. Probe design and probe targeting are described and fluorescent probe development for disease-relevant proteinases, including matrix-metalloproteinases, cathepsins, caspases, and other selected proteinases, is reviewed.

Received 11 AUG 2013 Revised 25 OCT 2013 Accepted 24 DEC 2013

Keywords: Activity-based probes · Cathepsins · Cell targeting · Chronic inflammatory lung disease · Matrix metalloproteinases

1 Introduction

Correspondence: Dr. Carsten Schultz, European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany E-mail: [email protected] Current address: Devaraj Subramanian, SiChem GmbH, Bremen, Germany Abbreviations: ABP, activity-based probes; ACPP, activable cell-penetrating probes; ADAM, a disintegrin-like and metalloproteinase-like proteinase; ADAMTS, a disintegrin-like and metalloproteinase-like proteinase and thrombospondin type 1 motif; CF, cystic fibrosis; CFP, cyan fluorescent protein; COPD, chronic obstructive pulmonary disease; ECM, extracellular matrix; FAP, fibroblast activation protein; FP, fluorescent protein; FRET, Förster resonance energy transfer; MMP, matrix metalloproteinase; NE, neutrophil elastase; PEG, polyethylene glycol; TNF, tumor necrosis factor; YFP, yellow fluorescent protein

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Proteinases are integral to many physiological functions, including digestion, remodeling of the extracellular matrix (ECM), cell migration, induction of apoptosis, trimming of cell surface proteins, intracellular signal transduction, virus maturation, and defense against pathogens. Many proteinases act on specific substrates, but uncontrolled and prolonged proteinase activity are likely to lead to tissue damage, as in chronic inflammation of the lung [1, 2]. A good example of the importance of proteinases in diseases can be observed in chronic obstructive pulmonary disease (COPD), where the release of elastases from white blood cells into the lumen of the airways during chronic inflammation ultimately leads to pul-

* These authors contributed equally to this work.

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Figure 1. Scheme of small-molecule-based probes for the detection of cysteine cathepsin activity. (A) Covalently binding ABP; (B) noncovalent FRET probe that is peptide-based, or (C) non-covalent FRET probe that is based on reverse design by modification of an inhibitor into a proteinase substrate (see text for details).

monary emphysema and death [3–7]. From a clinical standpoint, it is highly desirable to monitor the activity of these proteolytic enzymes, ideally in a quantitative fashion. Such detection of enzyme activities will also help to evaluate inhibitors and drug candidates. Furthermore, monitoring regular proteinase activity and its regulation in an intact cell or organism helps us to better understand the timing and interaction of proteinase function within complex signaling and metabolic networks regulating physiological function, development, growth, and reproduction. In the past, a standard approach for looking at proteinases, and many other classes of enzymes, was the analysis of transcript levels. However, mRNA abundance does not always reflect the number of translated proteins and, more importantly, it does not necessarily reflect the specific activity of the enzyme under a given condition. The latter depends not only on the intrinsic activity of the enzyme, but also on its location, regulation, and, in the case of proteinases, the level of antiproteinase that counteracts proteinase activity. Typical analysis of protein levels in cells and tissue is possible through specific antibodies, but again this will not address differences in enzyme activity. Another elegant way to detect enzyme activity is to use activitybased probes (ABPs) [8, 9]. Because the mode of action for protein cleavage by proteinases is frequently nucleophilic attack of a side-chain group, such as serine or cysteine, towards a peptide bond of the substrate; this reactivity is used to covalently attach a probe to the catalytic site of

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the target enzyme. The three components required are a sufficiently specific recognition element, a reactive group (called a warhead), and a fluorophore (Fig. 1A). Covalent labeling will only be successful if the catalytic site is available and this should correlate to the active protein fraction. This technique permits the generation of snapshot fluorescent images of cells under various conditions or entire tissues, for instance, from a disease model versus a wild-type control animal. While this is a very powerful technique, dynamic processes cannot be followed with respectable time resolution, partly because the probe destroys the enzyme activity upon binding. Some of the most dynamic and promising tools for this purpose are Förster resonance energy transfer (FRET) probes (Fig. 1B), in which the proteolytic activity of a proteinase increases the donor and acceptor fluorophore distance enormously and produces a ratiometric change in the donor and acceptor emission spectra used to measure proteinase activity [10–12]. In cases where the acceptor is a quencher of the donor without emitting photons, the change in the donor emission is used as an intensiometric readout. A ratiometric readout is clearly preferred over an intensity change because the ratio is intrinsically normalized and the readout is independent of the probe concentration. Additionally, the acceptor can be followed independently by direct excitation. Clearly, the probe needs to be a specific substrate for the proteinase of interest. As the catalytic reaction involves hydrolysis, the probe design strives for enzymatic cleavage that separates the

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two fluorophores irreversibly leading to a full loss of FRET (Fig. 1B). As a result, a probe will perform more sensitively if FRET in the intact probe is highly efficient. In designing the probe, care should be taken that the quantum yield of the acceptor is high because this will maximize acceptor emission (sensitized emission) in the intact probe. Upon cleavage, the sensitized emission will be reduced while the donor emission will rise strongly due to a decrease in FRET. Necessarily, the proteinase reaction is irreversible. For that reason, all proteinase FRET probes produce an accumulating signal, in contrast to FRET probes for the detection of intramolecular enzymatic reactions, such as phosphorylation [13]. There, phosphorylation by a kinase usually induces a conformational change that may be reversed by phosphatase activity, opening up the possibility for long-term dynamic monitoring of enzyme activity. However, the accumulating signal is often beneficial because it leads to a detectable change in FRET, even if very few active enzyme molecules are present. It has been demonstrated that in a single cell as few as 50 β-lactamase molecules are sufficient to produce a lasting signal from a FRET probe, given enough time is provided [14]. Finally, with respect to substrate specificity, it might be beneficial to provide probes that are not based on peptides. In an approach termed “reverse design” [15], the probe design employs a specific inhibitor for the enzyme of interest that is converted into a highly specific substrate by adding a peptide bond and equipped with dyes for a fluorescent readout after probe cleavage (Fig. 1C).

2 Example for the relevance of proteinases in chronic inflammatory lung disease The pathogenesis of diseases such as COPD, cystic fibrosis (CF), and asthma remains poorly understood, and diagnostic and therapeutic options remain highly limited. Compelling evidence obtained from gene-targeted mouse models and studies in patients suggests that members of several proteinase families, including matrix metalloproteinases (MMPs), cathepsins, and serine proteinases, play crucial roles in the pathogenesis of COPD, CF, and asthma [1–5]. The bulk of the secreted proteinase forms are released by macrophages and neutrophils that patrol airway surfaces, providing the first line of defense against inhaled pathogens and irritants under healthy conditions, and are recruited into the airspaces of the lung in infection and inflammation. The clinical observation that patients with a genetically determined deficiency of α1-antitrypsin, which is the natural inhibitor of neutrophil elastase (NE), developed early onset and severe emphysema [1] was the basis for initial studies focused on the role of proteinases in elastolytic damage and loss of alveolar structure in the lung. In this context, studies in gene-targeted mouse models

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Figure 2. Experiments to evaluate probe performance. Initially the probe is tested as a substrate in the test tube, usually followed by cell experiments. Ex vivo experiments are often performed with cells from animal models or clinical samples. Probe applications in wild-type, transgenic, or xenographed animals provide the highest content results.

identified macrophage elastase (matrix metalloproteinase 12 (MMP12)) as a key proteinase in cigarettesmoke-induced emphysema in mice [6]. More recent human studies showed elevated levels of MMP12 in airway secretions that were associated with emphysema severity in patients with COPD [16, 17], and that a functional single nucleotide polymorphism in MMP12 (rs2276109) was associated with a positive effect on lung function and a reduced risk of COPD in smokers [18]. These studies confirmed the pathogenetic concept of proteinase in proteinase/antiproteinase imbalance in emphysema formation and identified MMP12 as an important target in this process. In the meantime, it has become increasingly clear that the role of proteinases in chronic inflammatory lung disease is not limited to proteolytic degradation of the ECM, but that proteinases such as MMP12 and NE are involved in multiple disease processes in chronic lung disease, ranging from modulation of inflammation and innate immunity [19], to airway remodeling [20], mucus hypersecretion [21], and dysregulation of airway ion channels, including the epithelial Na+ channel epithelial sodium channel and cystic fibrosis transmembrane regulator Cl– channel, which together cause airway surface hydration, mucus obstruction, and inflammation [22, 23]. These results support the need for better tools to elucidate the (dys)regulation and interaction of proteinases with their target molecules in pulmonary inflammation at the molecular and cellular level. Furthermore, sensitive sensors will help to exploit the potential of proteinase activity as biomarkers and therapeutic targets in chronic inflammatory lung diseases and lung cancer [24]. Therefore, it is of importance that future sensors and reporters are suitable for applications in model animals and in ex vivo samples from patients (Fig. 2).

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3 FRET probe concepts When deciding to develop a proteinase FRET probe, several factors need to be considered. The first decision is between a genetically encoded and a small-moleculebased probe [12]. Genetically encoded FRET probes are based on fusions of target proteins with fluorescent proteins (FPs). Constructs featuring a substrate sequence sandwiched between two FPs have been used for several proteinases (see below). Recently, construct libraries, semiautomated microscopy, and improved analysis software became available to streamline FRET probe development [25–27]. FP reporters are easily expressed in many cell types and usually locate well into the cytosol or when including a short location sequence to a cell membrane of interest. These sensors can usually be applied in transfectable cells or after expressing and isolating the recombinant protein expressed in bacterial cultures. Generation of transgenic animals is required to apply genetically encoded reporters in in vivo applications, potentially in a tissue-specific fashion. During the evaluation, it is important to test the isolated reporter for cleavage by suitable proteinases to determine the maximal FRET change possible. FPs are usually stable against common proteinases such as trypsin [28]. Digestion experiments may also be performed with cell lysates in case the probe is not easily purified. Usually, FRET changes of FP fusions are much lower than those observed with small-molecule-based probes, partly because of the significant size of the β barrel of the FP prohibits maximal FRET. The use of small-molecule probes is mandatory if measurements are to be performed in native tissue or cells that are hard to transfect. Their advantage is usually a large ratio change that easily reaches 30- to 50-fold in the test tube; thus providing an enormous dynamic range and increased sensitivity. The caveat is that synthesis of the probes is often laborious and each alteration or improvement of the probe requires restarting the process. Other frequently encountered problems are stacking of fluorophores and substrate specificity based on a relatively short substrate sequence. Stacking of fluorophores often occurs because the short distance between dyes and spacers is unable to break dye stacking. If the two fluorophores stick, they frequently form nonfluorescent complexes in which the excitation energy is dissipated through pathways that are ineffective for FRET. Upon successful cleavage, the emission of both the donor and acceptor increases, giving only a small ratio change. In our hands, coumarin dyes without charges were the least problematic in this respect. Unfortunately, coumarins usually have short emission wavelengths that are less suitable for applications in tissue and intact organisms, due to higher background fluorescence and shorter penetration depth of the light, relative to dyes emitting longer

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wavelength light. In principle, stacking should also be reduced if two dyes with negative charges are used because the coulomb forces will separate the dyes. Two positively charged dyes are less favorable, especially for cell applications, because they tend to stick to polynucleotides and negatively charged membranes. The greatest challenge for any specific and sensitive probe is for it to reach its target site within a cell, tissue, organ, or animal. Section  4 describes efforts to target probes to specific sites and to improve delivery, and we describe known FRET probes for various classes of proteinases.

4 Targeting As for any other probe, the utility of an ABP is determined by its maximally achievable signal-to-noise ratio. Irreversibly binding probes, in which the ABP is covalently linked to the target of interest, generate only one fluorescent molecule per binding event. The detected fluorescent signal, therefore, is directly proportional to the overall concentration of active proteinase and detection of low-abundance proteins may be challenging. In contrast, turnover-based probes retain the activity of the target enzyme, effectively amplifying the generated signal. However, in this case, the fluorogenic reporter is not attached to the proteinase. Therefore, diffusion dilutes the signal and leads to a loss in spatial information unless measures are taken to prevent diffusion. A variety of methods (frequently called ‘homing’) were developed to (i) enrich the probe at the site of interest; and (ii) ensure cellular uptake, which is important not only for intracellular targets, but also to maintain the chromophore at the site of proteinase activity and prevent diffusion after probe activation. It was observed that cellular uptake of fluorophores released from quenched probes by membrane-tethered MMPs was greater than release by extracellular MMPs [29]. The proximity of the cleaved imaging dye to the membrane seems to facilitate uptake; however, nonspecific activation and high uptake in the liver limit the applicability of some probes. It was reported that certain boron dipyrromethene (BODIPY) dye derivatives displayed more rapid uptake and longer retention in tissue than that of corresponding derivatives using the less permeable dye Sulfo-Cy5.5 [30]. Lipidation is an effective method to allow anchoring of a probe to a cellular membrane. In recent examples by the Schultz group [31, 32], peptidic probes for NE and MMP12 were developed. Attachment of a palmitoic acid chain ensures a long residence time of the probes at the membrane. In the case of MMP12, the probe was only quickly taken up by endocytosis upon enzymatic cleavage (Fig. 3D). Surprisingly, the NE probe retained its plasma membrane location despite a fairly similar peptide sequence and identical fluorophores. In both cases, no transfer of the cleaved probe

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Figure 3. Homing techniques. (A) The liberation of a fluorescently tagged cell-penetrating peptide in the vicinity of the protease-secreting cell might suffice to give local fluorescent staining, as shown by the Tsien group [37]. (B,C) Targeting of a FRET probe by binding to a specific cell surface receptor or (D) general targeting to the cell membrane through lipidation. (B) Subsequent endocytosis may deliver the reporter to the cell interior to monitor intracellular enzyme activity or (C,D) facilitate uptake of the fluorescent dye containing fragment upon cleavage by an extracellular enzyme to prevent signal dilution and produce a memory effect [39].

to neighboring cells was observed. A similar approach was developed for an intracellular probe to detect apoptotic events using a genetically encoded FRET system. A fusion protein of a yellow fluorescent protein (YFP) and a cyan fluorescent protein (CFP) was constructed and tethered to the membrane by a palmitoyl moiety. Upon induction of apoptosis, caspase-mediated cleavage of the connecting sequence DEVT linker occurred, resulting in a loss of energy transfer and significant changes to the fluorescence spectra and lifetimes [33]. An alternative concept to enhance cellular uptake is the utilization of cell-penetrating peptide sequences, mostly based on polycationic amino acid sequences [34]. To examine proteolytic processes in the endosome, Brock and co-workers [35] described quenched cathepsin substrates, which were elongated at the N-terminal end by polyarginine sequences. Different alternative polycationic sequences, such as the HIV-derived TAT sequences, were applied to enhance endocytotic uptake of cargo into the endosome [36]. A similar, but more advanced, targeting concept was developed by Tsien and co-workers [37]. In their approach, a polycationic sequence was tethered to a matching neutralizing polyanionic peptide chain (Fig.  3A). The two charged peptides were linked by a cleavable sequence recognized by the proteinase of interest. Thus, only upon hydrolysis was the anionic chain cleaved, leaving the exposed cationic peptide with its attached cargo and resulting in rapid and efficient cellular uptake. It was shown that activable cell-penetrating probes (ACPPs) displayed improved systemic distribution and reduced toxicity compared with other cell-penetrating probes [38]. Their good spatial resolution and high sensitivity lend themselves to utilization during surgery [40], allowing the efficient and selective identification and resection of cancerous tissue. Notably, this concept was extended to the costaining of neuronal tissues to minimize surgery-

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induced damage to nerves [41]. It can be expected that a combination of activity-based measures with alternative detection methods, such as white-light endoscopy, will improve tumor detection and eventually surgical outcomes, as recently demonstrated in an orthotopic murine model for distal esophageal adenocarcinoma [42]. Another application that has attracted major interest is increased vascular leakiness within the tumor tissue, allowing preferential uptake of probes tethered to nanoparticles into cancer cells. Conjugation of dyes onto the surface of well defined polymer particles does not only allow high loading of the dye, but also the tissue distribution can be fine-tuned by variation of the diameter and polarity of the material [43]. Harris and co-workers [44] describes the synthesis of nanoparticles of about 85 nm in diameter loaded with an average of 60 10 kDa polyethylene glycol (PEG) chains through MMP2-cleavable linkers. Upon MMP2 processing, the PEG chains were successively cleaved, revealing cell-penetrating peptides and leading to increased local uptake of the particles [44]. One example by Lee et al. [45], reported an alternative nanosensor system for the detection of MMP activity. About 30 quenched fluorogenic peptidic MMP substrates are loaded on each chitosan nanoparticle (diameter 250  nm), allowing efficient tumor detection after intravenous application of the probe [45]. It should be mentioned that the mechanism of both types of probes, as well as subsequent ones, are not, or not fully, based on FRET. Again, the Tsien group [46] reported applications of their above-mentioned ACPP approach for monitoring MMP-2 and -9 activities by tagging dendrimeric nanoparticles with an average hydrodynamic diameter of 9 nm, resulting in a 4–15-fold higher uptake into the tumor compared with nonconjugated ACPP. Salthouse [47] reported another novel nanoparticle-based platform. Two different types of fluorophores were grafted on a nanoparticle: an activable one, detecting the specific activity of a given

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proteinase, and a second, nonactivable one to control tissue adsorption and probe delivery. To target atherosclerotic plaques, cathepsin K specific sequences connected to near-infrared fluorescent (NIRF) dyes were grafted on a poly-L-lysine backbone. To ensure long circulation times, stealth copolymers, methoxy(polyethylene glycols), were attached [48]. High density and close proximity of the loaded dye resulted in efficient quenching of fluorescence. After uptake by macrophages through phagocytosis, dequenching of the fluorescent probe by specific proteolysis was monitored. The same platform may also be used for the incorporation of PET or MRI tracers to create multimodal imaging devices [49]. In particular, dextran-coated iron oxide nanoparticles show great utility as carrier systems in this context. Even trimodality-based imaging probes were reported, combining NIRF, MRI, and PET-CT in an activable [50] or nonactivable mode. The most specific way of targeting a certain cell type is the incorporation of homing moieties that bind to a selectively expressed target on the cell surface (Fig. 3B). Upon binding to the receptor, the probe is endocytosed, ensuring highly selective uptake. Generally, antibodies are used for receptor binding; however, due to their high molecular weight and high amounts of antibody being required to achieve the necessary probe concentration, very few approaches use them to target probes to cells. Homing moieties are usually derived from librarybased approaches, like phage display, in which knowledge on the exact target is not essential, or by selection of preferred surface receptors through mining literature data and expression profiles. Subsequently, the result allows the rational design of binders, using natural ligands or medicinal chemistry efforts as starting points, for example. Hamachi et al. [51] described a system targeting the folate receptor and hypoxia-inducible membrane carbonic anhydrase; both are strongly overexpressed in certain cancer cell lines. Furthermore, small molecules are used as homing moieties, such as methotrexate as a known folate receptor binder. In hydrophobicity-based selfassembly of probe molecules, the signal is efficiently selfquenched. Upon receptor recognition, the quenched probe starts to disassemble, resulting in a clear turn-on signal. An example for probing receptor-mediated uptake into macrophages by genetically encoded probes was provided by Umezawa et al. [39]. An inhibitory domain was fused by a linker, cleavable by MMP9, to an ApoB547-735 subunit and by another linker to GFP, which served as the reporter group. Upon encountering an activated macrophage, the linker was cleaved by MMP9 and released the ApoB unit, which in turn was recognized by a scavenger receptor and subsequently transported by receptor-mediated endocytosis. Clearly, this approach does not require FRET, but is fully based on translocation.

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5 FRET probes for MMPs In 1962, the first MMP identified was involved in morphogenesis during embryonic growth, with the ability to degrade collagen. It was named MMP1 or interstitial collagenase [52]. MMPs play an important role in tissue remodeling by degradation of ECM proteins and processing of bioactive molecules, including cell surface receptors, ligands, and cytokines [53, 54]. Thereby, they are involved in cell proliferation and apoptosis, cell migration, and cell differentiation. These are critical processes to control tissue integrity and function. Dysregulation of MMP activity initiates pathological processes that can result in cancer and metastases formation [55–57], cardiovascular diseases [58–61], osteoarthritis [62], and cirrhosis. MMPs are subclassified based on their primary substrate or their cellular localization, covering collagenases, gelatinases, stromelysins, metalloelastase, and membrane-type MMPs, among others. These subclasses share a domain structure consisting of a propeptide, a catalytic domain with a conserved cysteine, a hinge region, and a hemopexin-like domain. Membrane-bound members further include a transmembrane domain or a glycosyl phosphatidylinositol anchor. MMPs are synthesized as zymogens and need to undergo proteolytic processing to mature. For catalysis they are dependent on a metal ion, usually Zn2+, as a cofactor. In tissue, MMP activity is controlled by a class of endogenous MMP inhibitors, named TIMPs (tissue inhibitor of matrix metalloproteineases) [63]. For cancer cells to metastasize they need to detach from the tissue, which is facilitated by the MMP ECM–proteolytic potential. Overexpression of MMP-2 or MMP-7 correlated with a variety of cancers in terms of disease status, progression, and risk. A marker in atherosclerosis is the overexpression and secretion of MMP-9, whereas MMP-13 is highly overexpressed in osteoarthritis [64]. Favorably, the MMP activity functions as an early marker of disease development, before lesions are visible, and therefore, enables early disease treatment to prevent aggravation [63]. For these reasons, it is clearly beneficial to visualize proteinase activity in a temporal and spatial manner [65, 66]. For MMP detection, a variety of chemically synthesized probes with small-molecule fluorophores and/or quenching groups were designed. A detailed description of the probes developed for MMP detection so far can be found in the very recent review by Knapinska and Fields [63]. The broad-range MMP recognition sequence is the Pro-Leu-Gly motif. For instance, the sequence Pro-Leu-Gly~Val-Arg-Gly is cleaved by MMP-2, -3, -7, and -13 [45]. The commercial probe MMPsense, with the sequence Gly-Gly-Pro-Arg-Gln~IleThr-Ala-Gly, detects the activity of MMP-2, -7, and -9 in vivo by fluorescent molecular tomography [67, 68]. An additional approach is the generation of genetically encoded probes with FPs (such as CFP/YFP, mOrange/ mCherry) linked to a substrate peptide [69, 70]. Further,

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bioluminescence resonance energy transfer (BRET) assays were developed by using a bioluminescent protein (luciferase) and a fluorescent acceptor. Bioluminescence does not require donor excitation, and hence, prevents artifacts due to direct excitation of the acceptor. [71]. Conjugation to the cell membrane has been achieved by incorporation of a receptor transmembrane domain (e.g. of the platelet-derived growth factor (PDGF) receptor) [72]. Similarly, membrane targeting was successful by incorporation of a ligand, which allowed cell-surface receptor binding (TF-peptide-apolipoprotein-GFP/luciferase). This probe liberated a fragment that is endocytosed after extracellular cleavage by MMP [39] (Fig. 3C). In this way, the probe is delivered to the site of activity (“homing”) and allows for localization of areas of hyperactivity. Secreted MMPs can also be visualized by membrane-tethered probes to prevent dilution of their signal, thereby lowering the background [37]. Furthermore, signals may be trapped within cells by facilitating cellular uptake of the activated (i.e. cleaved) probe exclusively, by incorporation of a cell-penetrating peptide (often sequences of several arginines as polycations). The latter is shielded by a blocking group (a polyanion, e.g. consecutive glutamates) in the intact probe (Fig. 3A) [73, 74]. Probes for near-infrared (NIR) imaging (wavelength  = 650–900  nm) of MMPs were developed (i.e. using Cy5.5) [75] that allowed deeper tissue penetration and an improved signal-to-noise ratio, while minimizing autofluorescence artifacts [76]. In addition to solely monitoring activity, probes were modified to further include an inhibitor, which was liberated after probe cleavage to only target cells/environments where the enzyme was hyperactive, such as an area of diseased tissue. This was achieved by MMP7-mediated liberation of 1O2 in tumor tissue, inducing apoptosis [77]. Activity-based-probes (ABPs) for MMPs must include a cross-linking group, such as a photoreactive group, because MMPs do not form a covalent intermediate with their substrate during catalysis. Reverse design (see Section 1) was applied using the hydroxamic acid-based MMP inhibitor GM6001 [9, 78–80] and fluorescent labeling. Finally, ABPs were developed based on barbiturates as inhibitor compounds [81]. Due to overlapping sequence specificity of MMPs, one of the key challenges is the development of specific probes to detect an individual MMP member of interest [82, 83]. In this regard, many studies do not evaluate the developed probes for a larger number of MMPs, thereby leading to probes of insufficient specificity. Therefore, it is critical to control probe activity with various proteinase assays. Designing probes to target MMPs by a nonsubstrate sequence might provide improved selectivity when a combination of target sequence with three-dimensional structural preferences is chosen [63]. Potentially, secondary binding sites (exosites) from noncatalytic regions may contribute to recognition [84, 85].

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The family of human metalloproteinases includes about 70 isoforms, among them the secreted proteinases ADAM and ADAMTS: ADAM stands for a disintegrin-like and metalloproteinase-like proteinase [86]; ADAMTS further includes a thrombospondin type  1 motif [87]. Both enzyme classes combined consist of 25 isoforms and are involved in collagen fibril assembly and proteolysis, associated with morphogenesis, angiogenesis, and aberrations in cancer and in arthritis [88]. Furthermore, ADAMTS-13 influences coagulation by cleavage of the von Willebrand factor (vWF) [89]. These enzymes are also called “sheddases” to emphasize their ability to cleave extracellular domains of transmembrane proteins and cell surface bound proteins, including ligands of the epidermal growth factor (EGF) receptor and tumor necrosis factor alpha (TNFalpha) [90, 91]. Compared with imaging tools for MMPs, the number of FRET probes developed to detect ADAM and ADAMTS activity is small. This might be partially due to their highly overlapping substrate specificity in vitro. In vivo specificity might be a result of their tissue-specific expression. A quenched FRET probe and a Venus/Cerulean FP-fusion with vWF as the target sequence were developed for ADAMTS-13 detection [92, 93]. Furthermore, a fragment of TNFalpha was used for the generation of ADAM-9 FRET reporters [94]. To develop probes with increased specificity, the impact of the substrate’s secondary structure was investigated. Distinct triple-helix motifs increased the specificity towards ADAMTS-4 [95]. Additionally, the specificity for ADAM17 was enhanced by combined targeting of the catalytic site and proteolytically inactive binding sites (exocites) [96]. For asthma-associated ADAM-33 [97], a probe was generated based on the cleavage sequence derived from the amyloid precursor protein (APP) [98].

6 FRET probes for cathepsins Cathepsins are lysosomal proteinases that belong to the papain family. The name cathepsin is derived from the Greek kathepsein, meaning to digest [99]. There are 11 human cysteine cathepsins (i.e. cathepsins B, C, F, H, K, L, O, S, V, X, and W), existing at the sequence level; this was confirmed by bioinformatic analysis of the draft sequence of the human genome [100]. The overexpression of these enzymes was implicated in a number of pathological conditions [101]. Consequently, the development of small-molecule fluorescent probes for visualizing papain-like cysteine proteinase activities has gained considerable interest in recent years. Among cysteine proteinases, cathepsins B, L, and S stand out because they are known to participate in the formation, growth, and invasion of tumors. They also exist at high levels in various cancers. Bogyo et al. [102–104] developed a series of ABPs based on the epoxysuccinyl- and acyloxymethyl ketone reactive groups that targeted cathepsin B, L, and

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+ and carboxypeptidase

Cathepsin X

Cathepsin Y, Cathepsin Z, Cathepsin P

unknown

+

Cathepsin V

Extracellular procathepsin X

Endosomes, extracellular

Extracellular, bone[122]

Cytoplasm and nucleus

Cytoplasm, cell membranes, extracellular

Extracellular, pericellular under pathological conditions

Localization

Tumor cells, tumorassociated macrophages

Tumor cells, tumorassociated macrophages

Tumor cells, tumorassociated macrophages, mast cells, endothelial cells

Tumor cells, tumorassociated macrophages, fibroblasts, osteoclasts, myoepithelial cells

Tumor cells, tumorassociated macrophages, fibroblasts, myoepithelial cells, endothelial cells

Tumor cells

Tumor cells, myoepithelial cells

Tumor cells, tumorassociated macrophages, fibroblasts, neutrophils, mast cells, T cells, endothelial cells

Tumor cells, tumorassociated macrophages, fibroblasts, osteoclasts, neutrophils, endothelial cells

Expressed in tumor and tumor-associated cells [111]

Cancer and metastasis

Cancer and metastasis; immune defects

Atherosclerosis; cancer and metastasis; metabolic syndrome; lung diseases; immune defects; rheumatoid arthritis, osteoarthritis

Atherosclerosis; cancer and metastasis; metabolic syndrome; lung diseases; rheumatoid arthritis, osteoarthritis; osteoporosis

Atherosclerosis; cancer and metastasis; metabolic syndrome; lung diseases; immune defects; rheumatoid arthritis, osteoarthritis

Cancer and metastasis; lung diseases

Cancer and metastasis

Immune defects

Cancer and metastasis; lung diseases; rheumatoid arthritis, osteoarthritis

Disease relevance [112]

Abz-Phe-Arg↓Phe (4NO2)-OH [124]

Bz-Phe-ValArg↓NHMec [123]

Z-Phe-Arg↓NHMec; Z-Leu-Arg↓NHMec

Abz-Leu-Glu↓GlnEDDnp [108]

Z-Phe-Arg↓NHMec

Ac-His-Arg-TyrArg↓ACC

Z-Phe-Arg↓NHMec [121]

Arg↓NHMec [120]

Z-Phe-Arg↓NHMec; Z-Leu-Arg↓NHMec [119]

Z-Phe-Arg↓NHMec [117]

Abz-Gly-Ile-Val-Arg↓ Ala-Lys(Dnp)-OH [113]

Optimal substrate/ sequence

ABP [125]

ABP [105]; reverse design [110]

Reverse design [15]

ABP [102, 103, 106, 114]; reverse design[15]

ABP [118]

Peptidic [109]; ABP [102, 103, 114]; FRET [107, 115–117]

Fluorescent probe (FRET, turn-on, peptidic, reverse design, bioluminescent)

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Cathepsin W

+

Cathepsin S

Cathepsin L2, Cathepsin U

unknown

Cathepsin O

+

Cathepsin K

O, O2, X

+

Cathepsin L

+ and aminopeptidase

Cathepsin B3

– amino dipeptidase

Cathepsin H

Cathepsin J Dipeptidyl peptidase I

Cathepsin C

+ and carboxy dipeptidase

+

Cathepsin B1

Cathepsin B

Endopeptidase

Cathepsin F

Synonyms

Enzyme

Table 1. Properties of human cysteine cathepsins and available imaging probes

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S (Fig.  1A). Both quenched and nonquenched versions were able to profile active cathepsins in live cells as well as in vivo imaging of cathepsin  B and L activity in a mouse model of cancer [102–104]. Later, an optimized nonpeptidic cathepsin  S probe was produced that showed improved in vivo properties, relative to their previous generation of peptidic quenched probes, with higher tumor-specific fluorescence in noninvasive optical imaging experiments [105]. Most recently, Dive et al. [106] developed a new benzophenone probe that did not penetrate the cell membrane and was therefore mostly suitable for detection of extracellular levels of active cathepsin L. As an alternative to ABPs, turnover-based probes (Fig. 1B) are particularly useful for the detection of activities from low-abundance enzymes. Good representatives are the substrates AbzGly-Ile-Val-Arg-Ala-Lys(Dnp)-OH for cathepsin  B [107], Abz-Leu-Glu-Gln-EDDnp for cathepsin S [108], and a dityrosine-based substrate DBDY-(Gly-INH)2 [109] for cathepsin B; all developed for in vitro assays. Reverse design was used to develop highly selective cysteine cathepsins probes, in which the optimized cathepsin inhibitor was transferred into a selective probe by replacing the inhibitor warhead with a cleavable peptide bond and subsequently attaching appropriate reporter groups for optical imaging (Fig. 1C) [15, 110]. In these studies, fluorescent probes enabled imaging of cathepsin K, L, and S activities in vitro, and noninvasive optical imaging of cathepsin S activity in a mouse model of paw inflammation. An overview of published cathepsin imaging probes is given in Table 1.

7 FRET probes for caspases Caspases belong to the superfamily of cysteine proteinases. The family of 13 isoforms plays key roles in inflammatory signaling, as well as initiation and execution of apoptosis. A detailed description of function and categorization may be found in the very recent review by McIlwain et al. [126]. The detection and monitoring of caspase activity might be of crucial importance in early diagnosis of pathologies such as Alzheimer’s [127] and other autoimmune diseases and represents an important target for therapeutic approaches in cancer [128]. Expressed as inactive procaspases, activation is triggered by proteolytic cleavage and dimerization [129]. Therefore, the detection of cleaved caspases at the protein level is the classical strategy to follow activation. This approach is applicable to cellular lysates of various origins; thus providing the possibility of easily quantifying the average proteinase activity at different time points in the process (e.g. [130]). A second strategy is to utilize artificial small-molecule substrates. Overview tables on the various probes published are featured in reviews by McIlwain et al. [126] and Poreba et al. [131]; these include a comprehensive

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comparison of the chemical concepts. The development of a broad spectrum of small-molecule probes was pioneered by Thornberry and co-workers in the 1990s [132–134]. Generally, the probes are either colorimetric or fluorogenic and translate progressive caspase activity into an increase in signal. Most of the short substrate peptides are equipped with a fluorophore and a quenching moiety and are usually not cell permeable. Although very popular, due to the more direct activity read-out compared with analysis at the protein expression level, the fluorogenic probes usually measure proteinase activity in cell lysates with the typical loss of single-cell information. For the latter purpose, cell-permeable probes need to be applied and real-time observation of fluorescent substrate accumulation was achieved in living cells (for examples, see [135, 136]). Interestingly, the application of cell-permeable probes is not commonplace. For instance, a cell-permeable caspase-1 probe, a useful tool to study inflammatory processes, was originally applied to the mouse brain [136]. However, only one additional application of the very same probe can be found [137] and the probe is no longer commercially available. In the field of apoptosis, there are a few more examples of small-molecule caspase probes [36, 135, 138–140], but again the list is short compared with impermeable probes. Here, the aim is to develop probes of low cytotoxicity equipped with far-red fluorescent dyes to be applied in intact animals or in whole tissue preparations [36]. However, most probes are equipped with a fluorophore and a quencher; this results in an intensiometric read-out of caspase activity. Those probes might further be improved by modification of the fluorophores to ratiometric FRET pairs, as developed for other proteinase families, such as MMPs (see Section 5). With FRET providing the possibility of a ratiometric read-out, genetically encoded probes are useful to monitor caspase activity on the single-cell level. A substrate peptide, either constructed artificially or a sequencederived from natural target proteins, is sandwiched between two FPs. In most cases so far, CFP was employed as the donor and YFP as the acceptor to read-out proteinase activity by loss of FRET [141]. Furthermore, a tripartite FRET probe, making use of the energy transfer from CFP to YFP to monomeric red fluorescent protein (mRFP), was developed to monitor consecutive activation of caspase-3 and -6, within the cascade during the onset of apoptosis [142]. With progression in the development of FPs concerning brightness, pH, and photostability and with growing simplicity to separate emission spectra, red/green FRET pairs become more attractively utilized. A probe for caspase-3 activation shows a four-fold increase in the green-to-red ratio when used in cells [143]. As an alternative read-out, the measurement of the donor fluorescence lifetime, a prime strategy for absolute quantification of FRET [144], was used [145]. In a similar context, energy transfer between a bioluminescent donor and

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a fluorescent acceptor to achieve BRET was shown to provide good signal-to-noise ratios [146, 147]. In the development of caspase probes, there is the promising tendency to combine robustness and specificity of small, engineered fluorescent probes in cell-permeable versions that are applicable in tissue lysates and cells. The design of protease probes that pass through the cell membrane without using the endocytotic machinery is still a synthetic challenge for chemists. It is useful to follow the activation cascade of caspases in detail, from its onset to apoptotic cell death. This will be particularly revealing at the tissue level, for instance, during β-cell apoptosis in type I diabetes [148, 149]. In the case of inflammatory signaling, the accumulation of the cleaved substrates makes it easy to detect the onset of the signaling cascade, but fails to follow the shutdown of signaling events. Therefore, it would be particularly beneficial to provide substrates, the cleavage products of which are sufficiently detectable, but have a short lifetime to enable monitoring of transient activation of proteinases in living cells.

Figure 4. A genetically encoded probe for HIV-1 proteinase equipped with tandem acceptors.

sis [160, 161]. Most methods used to study legumain function so far have depended on antibodies and genetic modifications. Recently, the Bogyo group [162, 163] developed several activity-based turn-on probes (Fig. 1A) for functional imaging of legumain in cancer.

8 FRET probes for various other proteinases 8.3 Human immunodeficiency virus (HIV) In this section, we briefly report on FRET probes for proteinases that do not fall into the three categories already discussed. The selection is far from complete, but focuses on enzymes that play important roles in diseases, including cancer, HIV, and inflammation.

8.1 Fibroblast activation protein (FAP) FAP is a type  II cell-surface serine proteinase, which belongs to the prolyl-cleaving peptidase family [150]. FAP is expressed exclusively in cancer-associated fibroblasts of epithelial derived tumors, but not in benign tumors or normal adult tissues [151–154]. In tumors, FAP prevents recognition of tumor cells by the immune system. In engineered mice, elimination of FAP-producing stromal cells allowed the immune system to control the growth of malignant tumors [155]. FRET substrate library profiling showed that FAP firmly required proline and glycine at the substrate P1 and P2 sites, respectively [156]. A FAPspecific probe for the diagnosis of FAP-expressing epithelial cancers was presented by Zheng and co-workers [157].

8.2 Legumain Legumain, also known as asparaginyl endopeptidase, is a lysosomal cysteine proteinase. Accordingly, legumain requires an asparagine residue in the P1 site of the substrate [158]. Although legumain plays many important roles in normal physiology, it is associated with a number of diseases, such as atherosclerosis [159] and tumorgene-

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HIV-1 causes acquired immunodeficiency syndrome (AIDS), which still kills a large number of people worldwide. Currently there has been good progress, but so far there is no ideal therapy for HIV/AIDS. Enormous efforts are being made to identify drug targets and effective drugs for HIV/AIDS therapy. One target in this respect is the only proteinase encoded by the HIV genome; an enzyme essential for the cleavage of several viral proteins during virus maturation. The development of FRET-based probes for HIV-1 proteinase may provide information about the timing and location of enzyme activity inside the host cell. So far, the only FRET-based probe is a genetically encoded fusion protein able to monitor HIV-1 proteinase and screen for substrate sequences and inhibitor activity in vivo [164]. The authors used tandem acceptors to enhance the FRET efficiency (Fig. 4).

8.4 Factor Xa Factor Xa is a serine proteinase, which is arginine-specific at the P1 site of the substrate. Factor Xa is involved in the extrinsic and intrinsic blood coagulation pathways [165]. It plays an important role in the proteolysis of prothrombin to generate thrombin during prothrombin complex formation. This specific physiological role makes it a prime target for the development of selective anticoagulants. Several fluorescence-quenched probes were developed for the characterization of human factor Xa substrate-binding domains [166–168].

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Figure 5. Probes for NE detection. (A) Commercial intensiometric aminocoumarin-labeled (AMC) substrate for detection of soluble human NE. (B) NEmo-1 and NEmo-2 reporters for ratiometric detection of soluble and membrane-associated NE activity of mouse and human origin.

8.5 Neutrophil elastase

9 Perspectives

The first chromo- and fluorogenic substrate for the neutrophilic serine proteinase NE (Ala-Ala-Pro-Val~) was developed in the late 1970s [169] (Fig. 5A). It has become widely used to quantify NE, as a marker, in inflammatory lung diseases, such as COPD and CF [20, 170, 171], because NE is involved in the degradation of most ECM proteins, including elastin, collagen and fibronectin, as well as other disease-aggravating processes [4, 172]. NE is expressed as a zymogen and stored and released in its active form from intracellular granules of neutrophils [173]. Therefore, activity-based sensors provide a readout of the active enzyme level. Quenched FRET probes with improved specificity towards human NE (Ala-Pro-GluGlu-Ile~Met-Arg-Gln), compared with the related neutrophilic serine proteinases PR3 and CathG, were developed [174, 175]. Furthermore, a probe that targets both human and mouse NE allows studies of NE activity in mouse models (Gln-Pro-Met-Ala-Val~Val-Gln-Ser-ValPro-Gln) [176]. We used this substrate to generate a set of two ratiometric FRET reporters (Fig.  5B), which monitored NE activity in a spatially resolved fashion in its soluble form (NEmo-1), as well as associated to the plasma membrane by lipidation (NEmo-2) in lung lavages from mice [31]. The substrate sequence of Kalupov et al. [177] was also used by PerkinElmer to bring the in vivo imaging agent NE680 to the market. NE680 is a self-quenched FRET probe with NIR dyes coupled to a pharmacokinetic modifier, resulting in a probe size of 40 kDa. This probe has been instilled into the airways in a lung inflammatory mouse model to visualize NE activity by fluorescence molecular tomography.

It becomes evident from the above discussion that most of the medically relevant proteinase groups are targets for the development of fluorescent probes. When it comes to applications in tissues and organisms, new tricks to improve the performance of the reporters and to provide effective homing to the target cells need to be developed. The possibilities to efficiently use the probes in the clinic are missing. The average hospital unit will not have access to complicated handling of patient tissues and/or other specimens and confocal microscopy. Therefore, probes need to be designed in a way that enzyme activities are measurable on enzyme-producing cells in native tissue, against a large background from other cell types, with easy-to-handle equipment. Alternatively, protocols are needed to isolate single cells from patients. On the equipment level, simple microscopic and microfluidic devices need to be adjusted to measure fluorescent probes, ideally ratiometrically. This provided, physicians will receive additional functional information on proteinase activities in disease processes that may ultimately help to improve diagnosis and monitoring of disease activity, and initiate or adjust to the most effective therapies, and thus, bring the clinic closer to personalized medicine.

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We are grateful to Nicole Heath for critically reading the manuscript. The work of the Schultz lab is funded by the EMBL, LIVIMODE, the DFG (Transregio 83, SPP1623), the Helmholtz Association (LungSysII), and the EU (Integrated Project LIVIMODE and Training Network SPHIN-

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GONET). C.S. and M.M. acknowledge funding from the German Lung Research Center. The authors declare no conflict of interest.

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The Schultz lab at the European Molecular Biology Laboratory (EMBL) is engaged at the interface of chemistry and biology. Among the many tools produced in the lab for answering biological questions, fluorescent reporter molecules are designed to monitor enzyme activities, including those of proteinases, in intact cells and entire animals. For special probe designs and probe targeting, we team up with colleagues from Sanofi in Frankfurt, and for detecting enzyme activities in mouse and patient samples with physicians from the Translational Lung Research Center (TLRC), Heidelberg.

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ISSN 1860-6768 · BJIOAM 9 (2) 171–310 (2014) · Vol. 9 · February 2014

Systems & Synthetic Biology · Nanobiotech · Medicine

2/2014 FRET imaging Synthetic probes Live-cell imaging

Fluorescent Biosensors www.biotechnology-journal.com

The Fluorescent Biosensor special issue of Biotechnology Journal is edited by Dr. May Morris and Prof. Marc Blondel. The cover image is an artistic interpretation of how fluorescent biosensors function as molecular beacons for scientists navigating a sea of molecules. Image courtesy of L. Divita and R. Wintergerst.

Biotechnology Journal – list of articles published in the February 2014 issue. Editorial: Fluorescent biosensors May C. Morris and Marc Blondel http://dx.doi.org/10.1002/biot.201400008 Review Newly engineered cyan fluorescent proteins with enhanced performances for live cell FRET imaging

Review Fluorescent biosensors for high throughput screening of protein kinase inhibitors Camille Prével, Morgan Pellerano, Thi Nhu Ngoc Van and May C. Morris

http://dx.doi.org/10.1002/biot.201300196

Fabienne Mérola, Asma Fredj, Dahdjim-Benoît Betolngar, Cornelia Ziegler, Marie Erard and Hélène Pasquier

Review FRET-based and other fluorescent proteinase probes

http://dx.doi.org/10.1002/biot.201300198

Hai-Yu Hu, Stefanie Gehrig, Gregor Reither, Devaraj Subramanian, Marcus A. Mall, Oliver Plettenburg and Carsten Schultz

Review Decoding spatial and temporal features of neuronal cAMP/PKA signaling with FRET biosensors Liliana R. V. Castro, Elvire Guiot, Marina Polito, Danièle Paupardin-Tritsch and Pierre Vincent

http://dx.doi.org/10.1002/biot.201300202 Review Imaging early signaling events in T lymphocytes with fluorescent biosensors Clotilde Randriamampita and Annemarie C. Lellouch

http://dx.doi.org/10.1002/biot.201300195 Review Deciphering the spatio-temporal regulation of entry and progression through mitosis Lilia Gheghiani and Olivier Gavet

http://dx.doi.org/10.1002/biot.201300194 Review Shining light on cell death processes – a novel biosensor for necroptosis, a newly described cell death program François Sipieter, Maria Ladik, Peter Vandenabeele and Franck Riquet

http://dx.doi.org/10.1002/biot.201300201 Review Genetically encoded reactive oxygen species (ROS) and redox indicators Sandrine Pouvreau

http://dx.doi.org/10.1002/biot.201300199 Technical Report Time-resolved microfluorimetry: An alternative method for free radical and metabolic rate detection in microalgae Amadine Bijoux and Anne-Cécile Ribou

http://dx.doi.org/10.1002/biot.201300217 Research Article Acri-2,7-Py, a bright red-emitting DNA probe identified through screening of a distyryl dye library Delphine Naud-Martin, Xavier Martin-Benlloch, Florent Poyer, Florence Mahuteau-Betzer and Marie-Paule Teulade-Fichou

http://dx.doi.org/10.1002/biot.201300197

http://dx.doi.org/10.1002/biot.201300200 Review Advances in lanthanide-based luminescent peptide probes for monitoring the activity of kinase and phosphatase Elena Pazos and M. Eugenio Vázquez

http://dx.doi.org/10.1002/biot.201300203

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FRET-based and other fluorescent proteinase probes.

The continuous detection of enzyme activities and their application in medical diagnostics is one of the challenges in the translational sciences. Pro...
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