Fluorescent probes for cellular assays

A fluorescent probe is a fluorophore designed to localize within a specific region of a biological specimen or to respond to a specific stimulus. Fluorescent probes have been used for nearly a century to study cellular processes due to their exquisite sensitivity and selectivity. Fluorescent probes have also gained in popularity as safety and environmental concerns over the use of radioactive probes have grown. At the same time, cellular assays are being more widely used now than ever before. This review will give a broad overview of types of fluorescent probes, types of fluorescent assays, and their application in cellular assays for a number of pharmaceutically relevant target classes.     

The chronological evolution of small organic molecular fluorescent probes for thiols

Abnormal concentrations of biothiols such as cysteine, homocysteine and glutathione are associated with various major diseases. In biological systems, the structural similarity and functional distinction of these three small molecular thiols has not only required rigorous molecular design of the fluorescent probes used to detect each thiol specifically, but it has also inspired scientists to uncover the ambiguous biological relationships between these bio-thiols. In this minireview, we will discuss the evolution of small organic molecular fluorescent probes for the detection of thiols over the past 60 years, highlighting the potent methodologies used in the design of thiol probes and their particular applications in the semi-quantification of cellular thiols and real-time labelling. At the same time, the present challenges that limit their further application will be discussed. We hope that this minireview will promote future research to enable deeper insight into the crucial role of thiols in biological systems.

The chronological evolution of small organic molecular fluorescent probes for thiols: from separation dependency analysis to cellular specific analysis, what''s next?     

Fluorescent carbohydrate probes for cell lectins

Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe(x)-PAA-flu, Sia2-PAA-flu, GlcNAc2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe(x)-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe(x)-PAA-3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe(x)-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.     

Fluorescent probes for recognition of ATP

Adenosine 5'-triphosphate (ATP) plays an important role in various physiological activities and pathological processes in living cells. Consequently, a large number of fl uorescent sensors for detecting ATP have developed in recent years. In this review, we summarized these fl uorescent sensors, where these sensors were divided into fi ve typed ones according to the structure of probes used.     

Recent advances in organic-dye-based photoacoustic probes for biosensing and bioimaging

Photoacoustic imaging(PAI) is a non-destructive biomedical imaging technology with broad application prospects. PAI combines the advantages of optical imaging and ultrasound imaging with high selectivity and deep penetration to overcome the high scattering limitation of light in tissues. This emerging technology also achieves high-resolution and high-contrast imaging of deep tissue in vivo. Recently, photoacoustic(PA) probes based on organic dyes have emerged prominently in biosensing and bioimaging due to their excellent optical properties and structural adaptability. This paper gives an outline of the basic PAI principles and focuses on the application of organic-dye-based PA probes for molecular detection and in vivo imaging. The advantages of PAI technology and the drawbacks of current PA probes are then summarized. Finally, the prospects for application are evaluated considering the potential challenges in the biomedical fields.     

Fluorescent probes in stomatology

As people pay more and more attention to quality of life, stomatology, as one of the main branches of modern medicine, has experienced rapid development. The research results of many fields, such as molecular biology, analytical chemistry, material chemistry and other disciplines, are gradually applied in the field of stomatology. Due to the advantages of fast response, flexible design, high specificity, high sensitivity and easy operation, fluorescent probe technology is widely used not only in environmental monitoring, food analysis, bioanalysis and life sciences, but also in stomatology as an increasingly powerful analytical and research tool. In this review, we will introduce the uses of fluorescent probes in stomatology for detection, diagnosis, imaging, treatment and screening from five aspects: oral and maxillofacial tumors, dental hard tissue diseases, oral soft tissue diseases, oral related ion diseases, and forensic dentistry. It is hoped that our review may contribute to the further development of fluorescent probes in terms of selectivity, sensitivity, non-toxicity, and stability, thus enhancing the pathogenesis, diagnosis and therapy of stomatological diseases, and laying the foundation for further study of oral disease mechanisms.     

Fluorescent probes for sensing processes in polymers

Fluorescence spectroscopy is an important analytical technique that has been widely used in a variety of applications, such as biomedicine, biology, and science of materials, because it presents some properties which makes it unique, that is, extraordinary sensitivity and selectivity, short delay time (<10(-9) s), and it is neither invasive nor destructive, so it can be used for in situ measurements. Generally, intrinsic fluorescence of many materials, like polymers, is unspecific so it is not useful to analyse their properties or to be correlated to changes in their microenvironment. The incorporation of additives with fluorescent groups would be necessary. When the fluorescence emission of these molecules is sensitive to changes of properties, such as polarity, fluidity, order, molecular mobility, pH, or electric potential, they can be used for detecting such changes in their microenvironment, and they are called fluorescent probes. As long as these probes can follow processes of practical interest, they can be employed as sensors, if the information given by the measure of fluorescence adequately reflects the changes in the system. In addition, a sensor must fulfil some other requirements in order to make them of practical use, the most important being that the material support in which the sensor molecule is inserted. This support should permit a rapid detection of the process and should allow easy processing in a variety of forms. Polymers are well-known systems in which estimation of local parameters are possible by means of fluorimetric techniques. It allows the study of dynamic processes of interest, such as polymerization kinetics and mechanisms, thermal transitions, photodegradation, swelling morphology changes, and so forth.     

Fluorescent molecular rotors as probes for polymers

The use of a molecular rotor (1,1-dicyano-4-(4'-dimethylaminophenyl)-1,3-butadiene) as a fluorescent probe was proved to be of great interest for the study of polymers. First, the rotor can detect the critical time of the glass effect in the bulk polymerization of MMA into PMMA due to viscosity change, this will allow a better control of the process and is complementary to the information issued from the use of the fluorescent pyrene probe which is sensitive to the gel effect. Second, the cinnamylidene rotor was able to detect the formation of hydrophobic microdomains for cationic amphiphilic polymers in their aggregation modes when they were solubilized in water, both polarity and viscosity changes are playing a role. The possibility of incorporation of various molecular fluorescent rotors in polymers beads was also studied.     

Fluorescent hybridization probes for nucleic acid detection

Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.     

Fluorescent probes for visualizing ROS-associated proteins in disease

Abnormal expression of proteins, including catalytic and expression dysfunction, is directly related to the development of various diseases in living organisms. Reactive oxygen species (ROS) could regulate protein expression by redox modification or cellular signal pathway and thus influence the development of disease. Determining the expression level and activity of these ROS-associated proteins is of considerable importance in early-stage disease diagnosis and the identification of new drug targets. Fluorescence imaging technology has emerged as a powerful tool for specific in situ imaging of target proteins by virtue of its non-invasiveness, high sensitivity and good spatiotemporal resolution. In this review, we summarize advances made in the past decade for the design of fluorescent probes that have contributed to tracking ROS-associated proteins in disease. We envision that this review will attract significant attention from a wide range of researchers in their utilization of fluorescent probes for in situ investigation of pathological processes synergistically regulated by both ROS and proteins.

Abnormal proteins, influenced by reactive oxygen species (ROS), are directly related to the development of various diseases.     

Design strategy and bioimaging of small organic molecule multicolor fluorescent probes

Science China Chemistry - Multicolor fluorescent probes based on small organic molecules have the advantages of low cost, good biocompatibility, easily modifiable molecular structures and...     

A modular nanoparticle-based system for reagentless small molecule biosensing

Metalloprotein tethered CdSe nanoparticles have been generated to provide selective and reagentless maltose biosensing. As opposed to cell or protein detection by semiconducting nanoparticle bioconjugates, a modular method for small-molecule detection using semiconducting nanoparticle bioconjugates has been difficult. Here we report a method for reagentless protein-based semiconducting nanoparticle biosensors. This method uses Ru(II) complex-CdSe nanoparticle interactions and the maltose-induced conformation changes of maltose binding protein to alter the CdSe nanoparticle fluorescence emission intensity. In this proof-of-principle system, the maltose-induced protein conformation changes alter the Ru(II) complex-CdSe nanoparticle interaction, which increases the CdSe emission intensity. Altered CdSe emission intensity effects are best described as electron transfer from the Ru(II) complex to the CdSe excited state forming the nonfluorescent CdSe anion. Four surface-cysteine, Ru(II) complex-attached maltose-binding proteins have been studied for maltose dependent alteration of CdSe emission intensities. With 3.0-3.5 nm diameter CdSe nanoparticles, all ruthenated maltose-binding proteins display similar maltose-dependent increases (1.4-fold) in CdSe emission intensity and maltose binding affinities (KA = 3 x 106 M-1). For these four systems, the only difference was the sample-to-sample variation in maltose-dependent responses. Thus, very few surface cysteine mutations need to be examined to find a successful biosensor, as opposed to analogous systems using organic fluorophores. This strategy generates a unimolecular, or reagentless, semiconducting nanoparticle biosensor for maltose, which could be applied to other proteins with ligand-dependent conformation changes.     

Fluorescent probes for microdetermination of inorganic phosphates and biophosphates

This review covers the progress made in the development of fluorescent probes for inorganic and organic phosphates that are of significance in biosciences. Such probes need to work at physiological pH and at room temperature. The various modes of interactions between probe and phosphate species are discussed, not the least with the aim to assist in the design of more selective probes for which there is a substantial need. Correspondence: Otto S. Wolfbeis, Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93040 Regensburg, Germany     

Fluorescent probes for detection and bioimaging of leucine aminopeptidase

Leucine aminopeptidase (LAP) is one of important proteolytic enzymes and closely related with pathogenesis of cancer and liver injury. Determination of LAP activity in serum is used clinically for liver disorder diagnosis. The level of expressed LAP is very low in normal cells, but overexpressed in tumors and liver diseases, especially drug-induced hepatitis. LAP has become a predictive biomarker for many cancers and diverse physiological processes. Therefore, in situ dynamic monitoring and identifying intracellular LAP is imperative for LAP-related disease diagnosis. This review focuses on LAP-specific fluorescence imaging probes for the detection and tracking of intracellular LAP actively in vitro and in vivo. The progress suggests that fluorescence imaging is a vital and rapidly growing technology for early diagnosis of tumors.     

Fluorescent neuroactive probes based on stilbazolium dyes

A set of spectrally diverse stilbazolium dyes was identified in an uptake assay using cultured brainstem and cerebellum cells isolated from e19 chicks. Pretreatment of cells with indatraline, a monoamine reuptake inhibitor, allowed identification of dyes that may interact with monoamine transporters. Two structurally related, yet spectrally segregated, probes, (E)-1-methyl-4-[2-(2-naphthalenyl)ethenyl]-pyridinium iodide (NEP+, 3A) and (E)-4-[2-(6-hydroxy-2-naphthalenyl)ethenyl]-1-methyl-pyridinium iodide (HNEP+, 4A), were selected and further investigated using HEK-293 cells selectively expressing dopamine, norepinephrine or serotonin transporters. HNEP+ was selectively accumulated via catecholamine transporters, with the norepinephrine transporter (NET) giving the highest response; NEP+ was not transported, though possible binding was observed. The alternate modes of interaction enable the use of NEP+ and HNEP+ to image distinct cell populations in live brain tissue explants. The preference for HNEP+ accumulation via NET was confirmed by imaging uptake in the absence and presence of desipramine, a norepinephrine reuptake inhibitor.     

Fluorescent properties of oligonucleotide-conjugated thiazole orange probes

The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophilic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca-2'-deoxyriboadenylate (dA15), are evaluated. Linkage at the phosphate group yields two stereoisomers, S-isomer of the phosphorus chiral center (Sp) and R-isomer of the phosphorus chiral center (Rp); these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange-oligonucleotide conjugates (TO-probes). Quantum yields of fluorescence (phifl) of between 0.04 and 0.07 were determined for the single-stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5'[CACT15CAC3']); (phifl values of between 0.06-0.35 were determined for the double-stranded conjugates. The phifl value (0.17) of thiazole orange, 1-(N,N'-trimethylaminopropyl)-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium iodide (TO-Pro 1) in the presence of the oligonucleotide duplex (TO-Pro 1: dA15.d5'[CACT15CAC3'] (1:1)) is much less than that for some of the hybrids of the conjugates. Our studies, using steady-state and time-resolved fluorescence experiments, show that a number of discrete fluorescent association species between the thiazole orange and the helix are formed. Time-resolved studies on the four double-stranded TO-probes revealed that the fluorescent oligonucleotide-thiazole orange complexes are common, only the distribution of the species varies with the character of the chain and the chirality at the internucleotidic phosphate site. Those TO-probes in which the isomeric structure of the phosphate-chain linkage is Rp, and therefore such that the fluorophore is directed toward the minor groove, have higher phifl values than the Sp isomer. Of the systems studied, thiazole orange linked by an alkyl chain to the internucleotidic phosphate (Rp isomer) has the highest phifl and the greatest fraction of the longest-lived fluorescent thiazole orange species (in the hybrid form).     

Fluorescent probes for cytochrome p450 structural characterization and inhibitor screening

We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by F?rster energy transfer upon binding (Kd = 0.83 muM) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (Kd approximately 0.02 muM) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 A resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the B', F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors.