Conjugate Probe Research Articles

Self-assembled nanobody-DNA nanomachine conjugate for parathyroid glands intraoperative imaging and parathyroid hormone detection

During thyroidectomy, inadvertent parathyroid damage commonly occurs owing to the varying location of the parathyroid glands and their similarity to adipose tissue, lymph nodes, and thyroid nodules. Therefore, in this study, we aimed to develop a novel self-assembled nanobody-DNA nanomachine conjugate probe (PTH-Nb-HCR) for precisely identifying the parathyroid glands and monitoring parathyroid hormone (PTH) levels in patient blood. The probe consists of two components: the first component is the nanobody (Nb)-nucleic acid conjugate PTH-Nb-Trigger, acting as a recognition unit for PTH and capable of initiating the hybridization chain reaction (HCR), whereas the second component comprises the HCR hairpin probes HP1-FAM (with a modified FAM signal molecule at the 5′ end) and HP2. The assembly of the PTH-Nb-HCR fluorescent probe takes only 20 min. Using the probe, the serum PTH level was quantitatively detected in the 0.1–1000 pg/mL range, and the parathyroid glands were rapidly distinguished from adipose, lymphoid, and thyroid tissues within 5 min. We also used this probe for imaging parathyroid glands with hyperparathyroidism. Considering the strong correlation between fluorescence intensity and preoperative serum PTH levels in patients, we anticipate the use of this probe to distinguish normal and hyperfunctional tissues during hyperparathyroidism surgery.

A novel fluorescent probe with a phosphofluorene molecular structure for selective detection of hydrogen sulfide in living cells.

Hydrogen sulfide (H2S) gas plays a significant role in biological regulation. With advancements in technology, H2S has been discovered across diverse fields, necessitating a comprehensive understanding of its physiological functions through monitoring changes in H2S within complex environments and physiological processes. In this study, we designed a phosphofluorene-based conjugate probe PPF-CDNB with an asymmetric π-conjugated phosphine structure and utilized dinitrophenyl ether as the recognition site for H2S. PPF-CDNB exhibited exceptional resistance to interference and demonstrated stability over a broad pH range (3.0-10.0), making it suitable for various environmental conditions. Intracellular experiments revealed that PPF-CDNB effectively monitored both endogenous and exogenous levels of H2S.

A reaction based carbazole-indolium conjugate probe for the selective detection of environmentally toxic ions.

A nucleophilic addition based chemodosimeter was designed and synthesized with a carbazole donor and an indole acceptor. The addition of a cyanide ion to an electron-deficient indole moiety disrupts the acceptor-donor relationship, resulting in noticeable color shifts and spectrum differences in both the absorption and emission profiles. The design has a D-π-A molecular arrangement. Selectivity was investigated in 90% aqueous DMSO solution of probe CI with various anions such as SCN-, PF6-, NO3-, N3-, I-, HSO4-, CN-, H2PO4-, F-, HS-, ClO4-, Cl-, Br-, and AcO-. An intermolecular charge transfer (ICT) band at 506 nm in the UV-visible spectra vanished and the intensity of emission was quenched at 624 nm upon the addition of CN- ions. These outcomes demonstrate the effective nucleophilic addition of cyanide ions to the electron-deficient indole moiety of the probe, resulting in the formation of a new adduct in which the ICT transition is interrupted when π conjugation is blocked. The Job plot, 1H NMR spectroscopy, and HRMS analysis confirmed the formation of a new product. An outstanding response was shown by paper test strips made using probe molecules for the easy detection of cyanide ions in aqueous solutions. Besides, the probe selectively senses cyanide ions in different water samples.

A Switch-On Affinity-Based Iridium(III) Conjugate Probe for Imaging Mitochondrial Glutathione S-Transferase in Breast Cancer Cells.

Glutathione S-transferase is heterogeneously expressed in breast cancer cells and is therefore emerging as a potential diagnostic biomarker for studying the heterogeneity of breast cancers. However, available fluorescent probes for GSTs depend heavily on GSTs-catalyzed glutathione (GSH) nucleophilic substitution reactions, making them susceptible to interference by the high concentration of nucleophilic species in the cellular environment. Moreover, the functions of subcellular GSTs are generally overlooked due to the lack of suitable luminescence probes. Herein, we report a highly selective affinity-based luminescence probe 1 for GST in breast cancer cells through tethering a GST inhibitor, ethacrynic acid, to an iridium(III) complex. Compared to activity-based probes which require the use of GSH, this probe could image GST-pi in the mitochondria by directly adducting to GST-pi (or potentially GST-pi/GS) in living cells. Probe 1 possesses desirable photophysical properties including a lifetime of 911 ns, a Stokes shift of 343 nm, and high photostability. The "turn on" luminescence mode of the probe enables highly selective detection of the GST with a limit of detection of 1.01 μM, while its long emission lifetime allows sensitive detection in organic dye-spiked autofluorescence samples by a time-resolved mode. The probe was further applied to specifically and quantitatively visualize MDA-MB-231 cells via specific binding to mitochondrial GST, and could differentiate breast cell lines based on their expression levels of GST. To the best of our knowledge, this probe is the first affinity-based iridium(III) imaging probe for the subcellular GST. Our work provides a valuable tool for unmasking the diverse roles of a subcellular GST in living systems, as well as for studying the heterogeneity of breast cancers.

Carbon quantum dot fluorescent probe for labeling and imaging of stellate cell on liver frozen section below freezing point

The targeted labeling imaging of stellate cells on liver frozen section by immunofluorescence is a very promising visualization technique to study the distribution of stellate cells in the liver. In this study, water soluble carbon quantum dots that can emit blue, green and yellow fluorescence are synthesized by the hydrothermal method, and their sizes are 3.2, 3.7, and 4.3 nm, respectively. The three carbon quantum dots have good fluorescence stability, and the quantum yields are 36.1%, 26.3% and 21%, respectively. When the mass fraction of KCl in the blue carbon quantum dot dispersion system is 13%, it still maintains the liquid state at −30 °C. The final fluorescent probe is obtained after the carbon quantum dots are coupled with the secondary antibody, spectral characterizations confirm that the conjugate probe still maintains protein immunoactivity and has good stability. Cell experiments prove that the probe has good biocompatibility, the rabbit anti-mouse Desmin antibody is used as the primary antibody, the results of cellular immunofluorescence imaging and flow cytometry show that the probe can specifically label hepatic stellate cell at −20 °C. The results of liver frozen section experiments show that hepatic stellate cell can be specifically targeted and labeled by the fluorescent probe. This labeling technology provides an important technical means for elucidating the structure and function of the liver at the cellular level, exploring the liver pathological change, and designing and developing drug.

Kinetic analysis of highly effective triplex formation between a small molecule-peptide nucleic acid conjugate probe and the influenza A virus RNA promoter region at neutral pH.

In order to overcome the pH limitations of triplex-forming peptide nucleic acid (PNA) in binding to double-stranded RNA (dsRNA), we have recently proposed a new design of triplex-forming PNA-based fluorogenic probes that work at neutral pH for sensing the panhandle structure of the influenza A virus (IAV) RNA promoter region. Our strategy is based on the conjugation of a small molecule (DPQ) capable of selectively binding to the internal loop structure with the triplex-forming forced intercalation of thiazole orange (tFIT) probe with natural PNA nucleobases. In this work, the triplex formation of tFIT-DPQ conjugate probes with IAV target RNA at neutral pH was examined by means of a stopped-flow technique UV melting and fluorescence titration experiments. The obtained results revealed that (i) the conjugation strategy is responsible for the observed strong binding affinity due to a very fast association rate constant and a slow dissociation rate constant; (ii) the binding follows a pattern of the DPQ unit binding first to the internal loop region, followed by the tFIT unit binding to the complementary dsRNA region. Our results emphasize the importance of both the tFIT and the DPQ components of the conjugate probe design and revealed an association mechanism for the tFIT-DPQ probe-dsRNA triplex formation towards the IAV RNA at neutral pH.

Oligonucleotide-Chemosensor Conjugate as a Dual Responsive Detection Platform and Its Application for Simultaneous Detection of ATP and Zn2.

Simultaneous detection, which helps understand complex physiological processes and accurately diagnose diseases, has been achieved using dual responsive probes. The dual responsive probe can ideally distinguish four cases, which are a combination of the absence and presence of two analytes, with characteristic fluorescence emissions. Owing to the demanding conditions of its development, most previous studies have focused on the simple linkage between small-molecule chemosensors that have an individual target and spectral range. In this study, a new dual responsive detection platform, oligonucleotide-chemosensor conjugate, was developed using a linkage between versatile oligonucleotide probes and small-molecule chemosensors to expand the applicable scaffold and detectable target for simultaneous detection. As a proof of concept, the ATP aptamer probe and Zn2+ chemosensor were conjugated as the levels of ATP and Zn2+ are intimately correlated in several signaling pathways and diseases. Each probe could detect an analyte independently within a conjugate probe, and simultaneous detection was also demonstrated without spectral crosstalk or interference between the receptors. In addition, the introduced cholesterol modification allowed the developed probe to detect changes in analytes on the plasma membrane of live cells through flow cytometry and confocal microscopy.

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

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

Bioactivated in vivo assembly (BIVA) peptide-tetraphenylethylene (TPE) probe with controllable assembled nanostructure for cell imaging

The emergence of fluorescent light-up molecular probe, which can specifically turn on their fluorescent in the presence of stimulation factors, has open up a new opportunity to advance biosensing and bioimaging. In this work, we designed and synthesized a peptide-AIE conjugate probe for cell imaging with controlled in situ assembled nanostructures. The modular designed probe is consisted of a self-assembled peptide-tetraphenylethene (TPE) motif, a fibroblast activation protein alpha (FAP-α) responsive motif, a hydrophilic motif and a targeting motif. The probe exhibits typically turn-on fluorescence property specifically triggered by FAP-α, which is a significant overexpressed membrane protein on pancreatic tumor cells. Interestingly, the peptide modified the TPE dramatically impacts the assembled nanostructure, which can be modulated by peptide sequences. As a result, the peptide FF(Phe-Phe) modification of TPE as the self-assembled motif provides a suitable balance of the probe with light-up property and nanofiber assembled structure in situ. Finally, our probe could effectively detect the FAP-α on tumor cells with high specificity. Meantime, the nanofibers in situ assembled on the surface of CAFs enhanced the probe accumulation and prolonged the retention for cell imaging. We envision that this study may inspire new insights into the design of nanostructure controlled AIE light-up bio-probe.

Development of a Lateral Flow Immunoassay using recombinant GRA7 antigen to detect Toxoplasma gondii IgG antibodies

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis and is of medical importance in pregnant women and immunosuppressed patients. In recent years, many methods have been developed for the detection of infection caused by this parasite; however, most of the developed methods are not adequately sensitive. The dense granular antigen (GRA) 7 is a highly immunogenic protein that is used as a specific antigen for the diagnosis of toxoplasmosis. This study was designed to produce recombinant GRA7 (rGRA7) antigen in bacterial system in order to be applied as an antigen for developing a simple and rapid lateral flow immunoassay test strip using a gold nanoparticle-pAb conjugate probe to detect Toxoplasma IgG-specific antibodies in human sera. After the extraction of genomic DNA from RH strain tachyzoites, polymerase chain reaction (PCR) was performed using specific primers considering restriction sites and BglII and XhoI enzymes. Subsequently, the GRA7 gene was cloned in pET-32a (+) expression vector, and then pET-32a(+)-GRA7 was transformed into E. coli Rosetta (DE3). The induction of protein production was accomplished by IPTG, and the product was finally purified by Ni-NTA affinity chromatography. In order to make the strip test, the anti-human gold nanoparticle conjugate was applied on conjugate pad, rGRA7 antigen was immobilized to a nitrocellulose membrane as the capture agent, and sample and absorbance pads were assembled on a backing card. For the analysis of the sensitivity and specificity of the assay, the selected patients’ sera samples were tested by standard chemiluminescence immunoassay (CLIA) method, and then compared with TOXO-IgG strip results. The findings showed that the use of rGRA7 is an accurate, sensitive, and inexpensive technique for the rapid detection of anti-Toxo­plasma IgG in human sera. Therefore, rGRA7 can be applied as a diagnostic agent in laboratories.

Quantum Dots-Based Immunochromatographic Strip for Rapid and Sensitive Detection of Acetamiprid in Agricultural Products.

In this study, a rapid and sensitive immunochromatographic strip (ICS) assay, based on quantum dots (QDs), was developed for the qualitative and quantitative detection of acetamiprid in agricultural samples. Acetamiprid-ovalbumin conjugates (ACE-OVA) and goat anti-mouse IgG were sprayed onto a nitrocellulose membrane as a test and control line. Two kinds of anti-acetamiprid monoclonal antibodies (mAb) obtained in our lab were characterized by the ELISA and surface plasmon resonance assay. The competitive immunoassay was established using a QDs-mAb conjugate probe. The visual detection limit of acetamiprid for a qualitative threshold was set as 1 ng/mL to the naked eye. In the quantitative test, the fluorescence intensity was measured by a portable strip reader and a standard curve was obtained with a linear range from 0.098 to 25 ng/mL, and the half maximal inhibitory concentration of 1.12 ng/mL. The developed method showed no evident cross-reactivities with other neonicotinoid insecticides except for thiacloprid (36.68%). The accuracy and precision of the developed QDs-ICS were further evaluated. Results showed that the average recoveries ranged from 78.38 to 126.97% in agricultural samples. Moreover, to test blind tea samples, the QDs-ICS showed comparable reliability and a high correlation with ultra-performance liquid chromatography-tandem mass spectrometry. The whole sample detection could be accomplished within 1 h. In brief, our data clearly manifested that QDs-ICS was quite qualified for the rapid and sensitive screening of acetamiprid residues in an agricultural product analysis and paves the way to point-of-care testing for other analytes.

Spherical-supported membranes as platforms for screening against membrane protein targets

Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes (“SSBLM”). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment. To provide proof-of-concept, SSBLMs embedding the polytopic bacterial nucleoside transporter NupC were assembled on 100- and 200 nm silica particles. To test specific binding of antibodies, NupC was tagged with a poly-histidine epitope in one of its central loops between two transmembrane helices. Fluorescent labelling, small angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) were used to monitor formation of the SSBLMs. Specific binding of an anti-his antibody and a gold-nitrilotriacetic acid (NTA) conjugate probe was confirmed with ELISAs and cryo-EM. SSBLMs for screening could be made with purified and lipid reconstituted NupC, as well as crude bacterial membrane extracts. We conclude that SSBLMs are a promising new means of presenting membrane protein targets for (biomimetic) antibody screening in a native-like lipid environment.

Protein adsorption/desorption and antibody binding stoichiometry on silicon interferometric biosensors examined with TOF-SIMS

Time-of-flight secondary ion mass spectrometry has been employed to examine, with biomolecular discrimination, sensing arm areas (20 μm × 600 μm) of integrated onto silicon chips Mach-Zehnder interferometers aiming to optimize their biofunctionalization with regard to indirect immunochemical (competitive) detection of ochratoxin A. Sensing areas are examined after: modification with (3-aminopropyl)triethoxysilane, spotting of OTA-ovalbumin conjugate (probe) from solutions with different concentration, blocking with bovine serum albumin, reaction with OTA-specific mouse monoclonal antibody followed by goat anti-mouse IgG secondary antibody. Component mass loadings of all proteins involved in immunodetection are determined from TOF-SIMS micro-analysis combined with ellipsometry of planar surfaces. These data show that partial desorption of surface-bound probe and blocking protein takes place upon primary immunoreaction to a degree that depends on probe concentration in spotting solution. Taking into account this desorption, apparent binding stoichiometry of both antibodies in immune complexes formed onto chip surface is determined more accurately than the respective evaluation based on real-time sensor response. In addition, mass loadings for probe and secondary antibody is observed to saturate for optimum probe concentrations. Also, principal component analysis of TOF-SIMS data could resolve both immunoreactions and biofunctionalization and discriminate surfaces prepared with optimum probe concentrations from those prepared using suboptimum ones.

Contact pin-printing of albumin-fungicide conjugate for silicon nitride-based sensors biofunctionalization: Multi-technique surface analysis for optimum immunoassay performance

Mass fabrication of integrated biosensors on silicon chips is facilitated by contact pin-printing, applied for biofunctionalization of individual Si3N4-based transducers at wafer-scale. To optimize the biofunctionalization for immunochemical (competitive) detection of fungicide thiabendazole (TBZ), Si3N4 surfaces are modified with (3-aminopropyl)triethoxysilane and examined after: immobilization of BSA-TBZ conjugate (probe) from solutions with different concentration, blocking with bovine serum albumin (BSA), and immunoreaction with a mouse monoclonal antibody against TBZ. Nanostructure, surface density, probe composition and coverage uniformity of protein layers are evaluated with Atomic Force Microscopy, Spectroscopic Ellipsometry, Time-of-Flight Secondary Ion Mass Spectrometry and X-ray Photoelectron Spectroscopy. Contact pin-printing of overlapping probe spots is compared with hand spotted areas. Contact pin-printing resulted in two-fold increase of immobilized probe surface density as compared to hand spotting. Regarding BSA-TBZ immobilization, an incomplete monolayer develops into a bilayer as the concentration of BSA-TBZ molecules in the printing solution increases from 25 to 100μg/mL. Upon blocking, however, a complete protein monolayer is formed for all the BSA-TBZ concentrations used. Free surface sites are filled with BSA for low surface coverage with BSA-TBZ, whereas loosely bound BSA-TBZ molecules are removed from the BSA-TBZ bilayer. As a consequence immunoreaction efficiency increases with the printing probe concentration.

Indirect immunoassay on functionalized silicon surface: Molecular arrangement, composition and orientation examined step-by-step with multi-technique and multivariate analysis

The arrangement, composition and orientation of immunoreagents employed in an indirect immunoassay for determination of mycotoxin OchraToxin A (OTA) are specified for Si3N4 substrate, aiming to imitate biosensor transducers made of the same material. Si3N4 surfaces are examined after modification with (3-aminopropyl)triethoxysilane, spotting with OTA-ovalbumin conjugate (probe), blocking with bovine serum albumin, reaction with a mouse monoclonal antibody against OTA and, finally, reaction with a goat anti-mouse secondary antibody. Atomic force micrographs, their autocorrelation and height histogram parameters, show the stepwise development of a multi-component monolayer covered by groups of secondary antibody molecules. Time-Of-Flight Secondary Ion Mass Spectrometry reveals the composition of probe and blocking protein, as well as their partial desorption during the primary immunoreaction. Ellipsometry provides surface amount of all proteins, increasing step-by-step from 0.7 to 6.9mg/m2. In addition, ellipsometry combined with TOF-SIMS reveals the mass loadings of different molecules in the intermediate and the final overlayer. Based on this, some orientations of the immobilized molecules are proposed and a molar ratio of ∼2.5 for secondary to primary antibody is calculated. The orientations of the primary and secondary antibody are further clarified by Principal Component Analysis of TOF-SIMS data, through which a side-on and a head-on orientation is deduced for the primary and the secondary antibody, respectively. These findings demonstrate how the combination of multiple surface analysis techniques can provide insight on the arrangement, composition and orientation of biomolecules in the course of multi-step procedures employed in biosensors.

An activatable fluorescent probe for targeting cellular membrane through the biothiol-mediated hydrazone-to-pyrazole transformation

A non-fluorescent coumarin-hydrazone conjugate probe was transformed into a strongly fluorescent pyrazolyl coumarin through a thiol-mediated cyclization reaction with F/F0 50. Interestingly, the probe was initially expressed at the cellular cytoplasm but translocated into the membrane regions of cells upon reaction with cellular enriched glutathione, which allowed us to obtain clear membrane imaging in the living cells.

All-optical delay of images by backward four-wave mixing in metal-nanoparticle composites

We theoretically study a novel method for all-optical delay of images based on backward four-wave mixing in composites containing metal nanoparticles. In this approach a delayed phase conjugate probe pulse is generated by the interaction of two counter-propagating pump beams and a non-collinear shaped probe pulse in the nanocomposite.The fractional delay and the reflectivity for the phase-conjugate signal pulses are studied as function of the input pump intensity. It is shown that this scheme can be used for delayed imaging combined with the elimination of optical diffraction. The advantages of this method include minituarized design, tunable wavelength range up to the telecommunication range and wide bandwidth.

Real-time analysis of uptake and bioactivatable cleavage of luciferin-transporter conjugates in transgenic reporter mice

Many therapeutic leads fail to advance clinically because of bioavailability, selectivity, and formulation problems. Molecular transporters can be used to address these problems. Molecular transporter conjugates of otherwise poorly soluble or poorly bioavailable drugs or probes exhibit excellent solubility in water and biological fluids and at the same time an enhanced ability to enter tissues and cells and with modification to do so selectively. For many conjugates, however, it is necessary to release the drug/probe cargo from the transporter after uptake to achieve activity. Here, we describe an imaging method that provides quantification of transporter conjugate uptake and cargo release in real-time in animal models. This method uses transgenic (luciferase) reporter mice and whole-body imaging, allowing noninvasive quantification of transporter conjugate uptake and probe (luciferin) release in real time. This process effectively emulates drug-conjugate delivery, drug release, and drug turnover by an intracellular target, providing a facile method to evaluate comparative uptake of new transporters and efficacy and selectivity of linker release as required for fundamental studies and therapeutic applications.

Label- and marker-free gene detection based on hybridization-induced conformational flexibility changes in a ferrocene–PNA conjugate probe

We report a strategy for label-free and marker-free gene detection transducing the hybridization event to an electrochemical signal based on the hybridization-induced conformational flexibility change in probe structure. The probe structure was designed to possess a ferrocene moiety as a reporter part and a cysteine moiety as an anchor part at each end of a peptide nucleic acid (PNA) as a recognition part. Electrochemical examination of probe-modified gold electrodes revealed that the ferrocene moiety was placed at the flexible end of the linear probe chain. Upon hybridization with a complementary target DNA, the resultant rigid duplex restricted the ferrocene motion to the electrode surface, causing a decrease in the observed current. The target DNA was detected with the detection limit of 1.44 x 10(-11) M. Thus the probe functioned as a 'self-reporting probe' and detection of the target DNA was demonstrated without the need for external indicators. Moreover, the sensor electrode was able repeatedly to detect the target DNA by the process of regeneration and could discriminate a mismatched DNA.

A Novel Rhodamine−Riboflavin Conjugate Probe Exhibits Distinct Fluorescence Resonance Energy Transfer that Enables Riboflavin Trafficking and Subcellular Localization Studies

Riboflavin (vitamin B2, RF) is taken up in eukaryotic cells via specialized transport mechanisms. Although RF has fluorescence properties, direct microscopic visualization of RF uptake and trafficking has been complicated by cellular autofluorescence. We describe the synthesis, cellular uptake characteristics, and spectroscopic properties of a novel rhodamine-riboflavin conjugate (RD-RF), including absorption and emission spectra, two-photon excitation spectra, and fluorescence pH dependence. The conjugate has a molar extinction coefficient of 23 670 M(-1) cm(-1) at 545 nm (excitation wavelength) with a fluorescence quantum yield of 0.94. This compound exhibits intramolecular fluorescence resonance energy transfer (FRET). Selective quenching of the FRET signal is observed when RD-RF is bound with high affinity by the chicken riboflavin carrier protein. In addition to the typical rhodamine excitation and emission, FRET provides a secondary signal for conjugate localization and an in situ mechanism for observing riboflavin binding. Solution and in vitro stability determinations indicate that the linkage between riboflavin and rhodamine is stable for the duration of typical pulse--chase and cellular trafficking experiments. The distinct spectroscopic properties of RD-RF together with a comparable affinity for RF-binding proteins render it an excellent tool for the study of RF transport and trafficking in living cells.