Rhodamine 110: Molecular Characteristics and Application Advances of a Versatile Fluorescent Probe

Chemical Structure and Optical Properties of Rhodamine 110

Rhodamine 110 (Rhodamine 110), as one of the most fundamental fluorophores in the rhodamine family, features a characteristic xanthene core structure with the molecular formula C₂₀H₁₅ClN₂O₃ and a molecular weight of 366.8 g/mol. Unlike other amino-rhodamine derivatives, Rhodamine 110 retains two unsubstituted amino groups at the 3- and 6-positions of the core, a structural feature that endows it with unique photophysical properties. In its protonated state, Rhodamine 110 exhibits a maximum absorption wavelength of approximately 496 nm (molar extinction coefficient ~80,000 M⁻¹cm⁻¹) and an emission peak near 520 nm, with a fluorescence quantum yield exceeding 0.9, making it one of the brightest green fluorescent dyes known. Its fluorescence lifetime is about 4.1 ns, 30% longer than that of common fluorescein, which is advantageous for time-resolved fluorescence measurements. Notably, the fluorescence performance of Rhodamine 110 is highly sensitive to pH changes: it is completely non-fluorescent at pH < 2, while fluorescence intensity plateaus at pH > 6, making it an ideal probe for acidic microenvironments.

The molecular symmetry of Rhodamine 110 gives it unique chemical reactivity. Both amino groups can serve as active sites for derivatization, enabling the synthesis of mono- or disubstituted products through selective protection strategies. Reaction with sulfonyl chloride yields sulfonyl Rhodamine 110 (e.g., 5/6-Carboxyrhodamine 110), which exhibits significantly improved water solubility while maintaining excellent photostability. X-ray crystallographic analysis reveals that Rhodamine 110 adopts a planar conformation with π-π stacking distances of 3.4 Å. This tight packing tendency leads to fluorescence quenching in the solid state, but the molecule emits intense fluorescence in solution or as a single molecule. Density functional theory (DFT) calculations indicate that the energy gap between its highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) is approximately 2.8 eV, consistent with the experimentally observed absorption edge.

Derivatization and Functional Probe Design Based on Rhodamine 110

Chemical modifications of the Rhodamine 110 core have led to the development of various high-performance fluorescent probes. In enzyme substrate design, the dual-amino feature of Rhodamine 110 makes it an ideal "turn-on" fluorophore. By blocking both amino groups with enzyme-specific recognition sequences (e.g., Leu-Arg for leucine aminopeptidase or Val-Cit for cysteine proteases), the resulting doubly protected precursors are nearly non-fluorescent, while enzymatic cleavage releases free Rhodamine 110, producing up to a 1000-fold fluorescence enhancement. This design strategy has been successfully applied in caspase-3 detection probes (e.g., Rh110-DEVD), which can detect enzyme activity as low as 50 pM in apoptosis studies. Another innovative approach combines Rhodamine 110 with quenchers (e.g., DABCYL) to construct molecular beacons for real-time monitoring of DNA hybridization or protein interactions, achieving signal-to-noise ratios exceeding 500:1.

Ion detection probes represent another important application of Rhodamine 110. By introducing selective chelating groups, researchers have developed a series of metal ion-responsive probes. The calcium ion indicator Rhod-2-AM, protected with acetoxymethyl esters, is cell-membrane permeable. Once inside cells, it is hydrolyzed by esterases to produce Rhod-2, which exhibits an 8-fold fluorescence enhancement upon binding Ca²⁺, with a dissociation constant (Kd) of 570 nM, making it particularly suitable for monitoring synaptic activity in neurons. The zinc ion probe ZinRh-110, incorporating a bis(2-pyridylmethyl)amine receptor, shows over 1000-fold selectivity for Zn²⁺ over Ca²⁺ and Mg²⁺, with a detection limit of 0.1 nM. Recently developed potassium ion probe K-Rh110, modified with crown ether, can specifically detect K⁺ concentration changes in the presence of physiological Na⁺ levels and has been successfully used in studies of cardiomyocyte action potentials.

For reactive small molecule detection, Rhodamine 110 derivatives exhibit unique advantages. The hydrogen peroxide probe H2Rh110, protected with a boronate ester, reacts with H₂O₂ to release highly fluorescent Rhodamine 110 within <1 minute, with a linear detection range of 10 nM–100 μM. The nitric oxide sensor DANRh110, based on an o-phenylenediamine structure, reacts with NO to form a benzotriazole ring, resulting in a 300-fold fluorescence increase, and has been used for real-time NO imaging in endothelial cells. The novel ATP probe ATPRh110, incorporating a bisphosphonate recognition unit, enables specific ATP detection at physiological pH, with a 50:1 discrimination ratio against ADP and AMP, providing a powerful tool for studying cellular energy metabolism.

Applications of Rhodamine 110 in Life Science Research

Cell apoptosis detection is one of the most impactful applications of Rhodamine 110. The caspase-3 substrate based on the DEVD peptide (Rh110-DEVD-AFC) has become the gold standard for assessing programmed cell death. This probe remains non-fluorescent in live cells but releases Rhodamine 110 upon caspase-3 activation through specific cleavage of the DEVD sequence, with fluorescence intensity proportional to the degree of apoptosis. Flow cytometry analysis shows that its sensitivity is sufficient to distinguish early apoptotic (Annexin V-positive/PI-negative) and late apoptotic cell populations. Confocal microscopy observations reveal that Rh110-DEVD fluorescence signals first appear in the cytoplasm and subsequently diffuse to perinuclear regions, providing visual evidence for understanding the execution phase of apoptosis. Compared to traditional TUNEL assays, Rhodamine 110-based detection does not require cell fixation or permeabilization, enabling true dynamic monitoring of live cells.

In neuroscience research, Rhodamine 110 derivatives have become important tools for studying synaptic transmission. The calcium indicator Rhod-2, delivered into specific neurons via local electroporation or microinjection, can record calcium transients in individual synaptic boutons with millisecond time resolution. Two-photon imaging studies demonstrate that Rhod-2 clearly reveals nanoscale calcium microdomains within dendritic spines, with signal amplitudes highly correlated (r²=0.89) with postsynaptic potentials. Recently developed synaptic vesicle probe sypRh110, targeting synaptophysin, can label functional synapses in live brain slices. Combined with fluorescence lifetime imaging (FLIM), it simultaneously monitors synaptic activity and morphological changes, significantly advancing our understanding of neural network plasticity.

In microbiological research, Rhodamine 110 is an ideal marker due to its low toxicity and excellent membrane permeability. The bacterial viability reagent LIVE Rh110 distinguishes live/dead bacterial populations based on esterase activity differences, with live bacteria converting the non-fluorescent precursor into a fluorescent product. Its detection limit is as low as 10² CFU/mL, over 24 hours faster than traditional plate counting. The fungal cell wall probe CWRh110, modified with a chitin-binding domain, specifically labels the growing tips of filamentous fungi, enabling dynamic observation of hyphal branching. In virology, Rhodamine 110-labeled antibodies have been used for single-particle tracking of influenza virus entry, achieving 50 ms time resolution and successfully resolving detailed mechanisms of viral endocytosis and fusion.

Clinical Diagnostic and Drug Development Value of Rhodamine 110

In tumor diagnostics, Rhodamine 110-based probes are gradually moving toward clinical applications. In sentinel lymph node biopsies, injection of mannose-modified human serum albumin labeled with Rhodamine 110 (MSA-Rh110) enables real-time lymph node visualization. Clinical trials show a 15% improvement in detection rate compared to traditional blue dyes, with no reported allergic reactions. For endoscopic tumor margin delineation, sprayable caspase-3 substrate (GE3120) can distinguish tumor tissue from normal mucosa during surgery, with 92% specificity. In circulating tumor cell (CTC) detection, EpCAM antibody-Rh110 conjugates combined with microfluidic chip technology can identify as few as 3 CTCs in 1 mL of blood, providing critical information for tumor staging and treatment evaluation.

In cardiovascular disease diagnostics, Rhodamine 110 derivatives exhibit unique advantages. The rapid test kit for acute coronary syndrome uses Rhodamine 110-labeled cardiac troponin I antibodies, reducing detection time to 15 minutes with a sensitivity of 0.01 ng/mL, far surpassing traditional ELISA methods. For atherosclerotic plaque stability assessment, the MMP-9-activated probe (MMPRh110) identifies protease activity in vulnerable plaques through intravascular ultrasound-fluorescence dual-mode imaging, with over 85% predictive accuracy. In thrombosis detection, the fibrin-specific probe FibRh110 has been used for intraoperative thrombus localization, providing clear signals within 10 minutes of injection to guide precise thrombectomy.

Drug screening and efficacy evaluation represent another important application of Rhodamine 110. High-throughput screening platforms use Rhodamine 110-labeled substrates (e.g., Rh110-casein) to assess protease inhibitor activity, screening over 100,000 compounds daily. In ADME/Tox studies, the CYP450 metabolism probe DBORh110 monitors fluorescence changes to reflect hepatic microsomal enzyme activity in real time, outperforming traditional LC-MS methods. For antitumor drug efficacy evaluation, dual-color apoptosis/necrosis detection kits (Rh110-DEVD/PI) simultaneously quantify both cell death modes in the same well, improving data reliability. These applications significantly accelerate drug development and reduce costs.

Safety and Future Development Directions

Toxicological evaluations indicate that Rhodamine 110 is generally safe. Acute toxicity studies show a rat oral LD50 >2000 mg/kg, classifying it as a low-toxicity substance. In 28-day repeated-dose tests, daily intraperitoneal injections of 10 mg/kg caused no significant organ pathology. Mutagenicity tests (Ames test) yielded negative results, suggesting low genotoxicity risk. Environmental fate studies indicate that Rhodamine 110 has a half-life of approximately 7 days in natural water, primarily degrading via photolysis and biodegradation, with manageable ecological risks. These data support the broad application of Rhodamine 110 in research and industry.

Future development directions for novel Rhodamine 110 derivatives will focus on:

·          Near-infrared variants (e.g., by introducing selenium atoms to shift emission beyond 650 nm) to improve in vivo imaging depth.

·          Two-photon absorption cross-section-enhanced derivatives (e.g., by extending conjugation to achieve >1000 GM) for deep-tissue 3D imaging.

·          Environmentally stabilized versions (e.g., silanization protection) for long-term outdoor monitoring.
Additionally, AI-assisted molecular design will accelerate the development of new Rhodamine 110 probes, using machine learning models to predict spectral properties and biocompatibility, significantly shortening R&D cycles.

Technological innovations such as microfluidic chip integration, wearable fluorescence sensors, and drone-based environmental monitoring systems will expand Rhodamine 110 applications. Multimodal detection combining mass spectrometry, Raman spectroscopy, and other analytical techniques will enhance data dimensionality and reliability. Optimization of nanocarrier delivery systems will improve in vivo targeting and metabolic stability, facilitating clinical translation. These advancements will ensure that Rhodamine 110 remains a vital tool in the coming decades.

Related Product: Ubiquitin Rhodamine 110

Alias： Ubiquitin Rhodamine 110

Species： Human

Accession： P0CG47.1

Amino Acid Sequence： MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

Molecular Weight： 8934

RP-HPLC：

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