Rhodamine 110: A Versatile Fluorescent Probe for Life Science Research and Clinical Applications

Concept

Rhodamine 110, a core fluorophore of the rhodamine family with the molecular formula C₂₀H₁₅ClN₂O₃ and a molecular weight of 366.8 g/mol, is defined by its unsubstituted 3,6-amino groups that grant it extraordinary photophysical characteristics. As one of the most luminous green fluorescent dyes, it has a maximum absorption wavelength at approximately 496 nm (molar extinction coefficient ≈80,000 M⁻¹cm⁻¹) and an emission peak at around 520 nm, with a fluorescence quantum yield surpassing 0.9. Its fluorescence lifetime of 4.1 ns—30% longer than that of fluorescein—renders it well-suited for time-resolved detection, while its marked pH sensitivity (fluorescence is quenched at pH<2 and stable at pH>6) makes it an excellent probe for analyzing acidic microenvironments.

With a symmetric molecular structure and two reactive amino sites, Rhodamine 110 acts as a flexible scaffold for chemical derivatization. This allows for the design of a wide variety of functional fluorescent probes for ion detection, enzyme activity assays, small molecule sensing and more, establishing it as an essential tool in life science research, clinical diagnosis and drug development.

Research Frontiers

Recent progress in fluorophore chemistry and molecular probe design has positioned Rhodamine 110 at the cutting edge of fluorescent imaging and detection technology. Breakthroughs in its derivatization strategies and application expansion are driving innovation across numerous research fields. State-of-the-art studies have optimized the chemical modification of Rhodamine 110’s xanthene core, developing site-specific derivatization methods that retain the fluorophore’s excellent optical properties while introducing target-specific recognition moieties. This has led to the creation of high-performance probes with ultra-high sensitivity, selectivity and biocompatibility.

A major research focus is the development of Rhodamine 110-based "turn-on" fluorescent probes for real-time, in situ detection of biological targets. By blocking the fluorophore’s amino groups with enzyme-specific peptide sequences or ion-chelating groups, researchers have engineered activatable probes that show negligible background fluorescence and produce up to a 1000-fold fluorescence enhancement upon target binding or enzymatic cleavage. These probes enable the detection of low-abundance biomolecules (e.g., caspases, metal ions) at picomolar to nanomolar levels. Additionally, translational research is advancing the clinical application of Rhodamine 110 derivatives, with novel probes being developed for sentinel lymph node biopsy, tumor margin delineation and circulating tumor cell detection—addressing unmet clinical needs for rapid, accurate and non-invasive diagnostic tools. Concurrently, AI-assisted molecular design is emerging as a transformative approach for creating new Rhodamine 110 variants, with machine learning models predicting spectral properties and biocompatibility to accelerate probe development and shorten R&D cycles.

Research Significance

Exploring the molecular characteristics, derivatization strategies and applications of Rhodamine 110 carries profound scientific and practical importance for life science research, clinical diagnostics and pharmaceutical development. At the basic research level, Rhodamine 110 serves as a model fluorophore for investigating the photophysical properties of xanthene-based dyes, advancing our understanding of structure-optical property relationships and guiding the design of next-generation fluorescent probes with enhanced performance. Its diverse derivatized forms allow for the precise detection and dynamic monitoring of a broad range of biological targets—including enzymes, metal ions, reactive small molecules and biomacromolecules—providing powerful tools for unraveling the molecular mechanisms of cellular processes such as apoptosis, synaptic transmission, energy metabolism and signal transduction.

Clinically, Rhodamine 110-based probes address critical demands for rapid, sensitive and specific diagnostic assays, with applications spanning tumor diagnostics, cardiovascular disease detection and intraoperative imaging. These probes enable early disease diagnosis, precise pathological staging and real-time intraoperative guidance, improving clinical decision-making and patient outcomes. In drug development, Rhodamine 110 derivatives form the basis of high-throughput screening platforms for evaluating drug efficacy and toxicity, accelerating the identification of lead compounds and reducing the time and cost of preclinical research. Furthermore, Rhodamine 110’s low toxicity, excellent biocompatibility and environmental degradability make it a sustainable tool for both in vitro and in vivo applications, with vast potential for translation from laboratory to clinical practice. Beyond life sciences, Rhodamine 110 is also widely used in environmental monitoring and material science, highlighting its cross-disciplinary research value.

Core Properties, Derivatization and Product Applications

Key Molecular and Optical Properties of Rhodamine 110

1. Structural and chemical features: Rhodamine 110 has a planar xanthene core with two unsubstituted amino groups at the 3- and 6-positions, giving it high molecular symmetry and dual reactive sites for selective derivatization. Reaction with sulfonyl chloride yields sulfonyl Rhodamine 110 derivatives with significantly improved water solubility and retained photostability. X-ray crystallographic analysis shows it adopts a planar conformation with a π-π stacking distance of 3.4 Å, leading to solid-state fluorescence quenching but intense fluorescence in solution or as a single molecule.
2. Superior optical performance: It exhibits a high molar extinction coefficient (≈80,000 M⁻¹cm⁻¹) at 496 nm and a high fluorescence quantum yield (>0.9) at 520 nm, making it one of the brightest green fluorescent dyes. Its 4.1 ns fluorescence lifetime supports time-resolved fluorescence measurements, and density functional theory (DFT) calculations confirm a HOMO-LUMO energy gap of approximately 2.8 eV, consistent with experimental absorption edge data.
3. pH-sensitive fluorescence: Rhodamine 110’s fluorescence is highly dependent on pH; it is completely non-fluorescent in strongly acidic conditions (pH<2) and its fluorescence intensity plateaus in neutral to alkaline environments (pH>6), making it an ideal probe for monitoring acidic microenvironments in cells and biological systems.
4. Low toxicity and biocompatibility: Acute and chronic toxicological evaluations confirm Rhodamine 110 is a low-toxicity substance (rat oral LD50 >2000 mg/kg), with no significant organ pathology observed in 28-day repeated-dose tests. Negative Ames test results indicate low genotoxicity risk, supporting its safe use in in vitro and in vivo biological research.

Derivatization Strategies and Functional Probe Design of Rhodamine 110

The dual reactive amino groups of Rhodamine 110 make it an ideal scaffold for the design of functional fluorescent probes, with site-specific derivatization strategies enabling the development of probes for diverse biological detection needs:

1. Enzyme activity detection probes: Blocking both amino groups with enzyme-specific recognition peptide sequences creates nearly non-fluorescent precursors. Enzymatic cleavage releases free Rhodamine 110, generating up to a 1000-fold fluorescence enhancement. This "turn-on" design has been successfully applied to caspase-3 detection probes (e.g., Rh110-DEVD) with a detection limit as low as 50 pM, a gold standard for apoptosis research.
2. Ion-responsive fluorescent probes: Introducing selective metal ion-chelating groups yields highly specific ion detection probes. Rhod-2-AM (a calcium ion indicator) is cell-membrane permeable and exhibits an 8-fold fluorescence enhancement upon Ca²⁺ binding (Kd=570 nM), ideal for monitoring neuronal synaptic activity. ZinRh-110 shows over 1000-fold selectivity for Zn²⁺ over Ca²⁺ and Mg²⁺ (detection limit=0.1 nM), while K-Rh110 enables specific K⁺ detection under physiological Na⁺ levels.
3. Reactive small molecule sensors: Modifying Rhodamine 110 with specific reactive groups creates probes for detecting cellular reactive small molecules. H2Rh110 (boronate ester-modified) reacts with H₂O₂ in <1 minute for quantitative detection (10 nM–100 μM linear range). DANRh110 enables real-time NO imaging in endothelial cells with a 300-fold fluorescence increase, and ATPRh110 shows 50:1 discrimination for ATP over ADP/AMP, a powerful tool for studying cellular energy metabolism.
4. Molecular beacons for biomolecular interaction: Conjugating Rhodamine 110 with quenchers (e.g., DABCYL) constructs molecular beacons for real-time monitoring of DNA hybridization and protein-protein interactions, achieving a signal-to-noise ratio exceeding 500:1.

Broad Applications of Rhodamine 110 in Life Science Research

1. Cell apoptosis detection: Rhodamine 110-based caspase-3 substrates (e.g., Rh110-DEVD-AFC) are the gold standard for programmed cell death assessment. The probe is non-fluorescent in live cells and releases Rhodamine 110 upon caspase-3-mediated DEVD sequence cleavage, with fluorescence intensity proportional to apoptosis levels. It can distinguish early and late apoptotic cell populations via flow cytometry and enables live-cell dynamic monitoring without fixation or permeabilization—an advantage over traditional TUNEL assays.
2. Neuroscience research: Rhodamine 110 derivatives are key tools for studying synaptic transmission. Rhod-2 can record calcium transients in individual synaptic boutons with millisecond time resolution and reveal nanoscale calcium microdomains in dendritic spines via two-photon imaging. The synaptic vesicle probe sypRh110 labels functional synapses in live brain slices, and combined with fluorescence lifetime imaging (FLIM), it simultaneously monitors synaptic activity and morphological changes, advancing understanding of neural network plasticity.
3. Microbiological and virological research: Rhodamine 110’s low toxicity and excellent membrane permeability make it an ideal microbial marker. LIVE Rh110 distinguishes live/dead bacteria based on esterase activity with a detection limit of 10² CFU/mL, 24 hours faster than traditional plate counting. CWRh110 specifically labels the growing tips of filamentous fungi for dynamic hyphal branching observation. Rhodamine 110-labeled antibodies enable 50 ms time-resolution single-particle tracking of influenza virus entry, resolving viral endocytosis and fusion mechanisms.

Clinical Diagnostic and Drug Development Applications of Rhodamine 110

1. Tumor diagnostics: Rhodamine 110-based probes are advancing toward clinical translation. Mannose-modified human serum albumin labeled with Rhodamine 110 (MSA-Rh110) improves sentinel lymph node detection rates by 15% compared to traditional blue dyes with no allergic reactions. The sprayable caspase-3 substrate GE3120 delineates tumor margins during surgery with 92% specificity, and EpCAM antibody-Rh110 conjugates combined with microfluidic chips detect as few as 3 circulating tumor cells (CTCs) in 1 mL of blood, providing critical tumor staging and treatment evaluation data.
2. Cardiovascular disease diagnosis: Rhodamine 110 derivatives enable rapid and sensitive cardiovascular disease detection. Rhodamine 110-labeled cardiac troponin I antibodies reduce acute coronary syndrome detection time to 15 minutes with a sensitivity of 0.01 ng/mL, outperforming traditional ELISA. MMPRh110 assesses atherosclerotic plaque stability via intravascular ultrasound-fluorescence dual-mode imaging with over 85% predictive accuracy, and FibRh110 enables intraoperative thrombus localization for precise thrombectomy guidance.
3. Drug screening and efficacy evaluation: Rhodamine 110-labeled substrates (e.g., Rh110-casein) power high-throughput screening platforms for protease inhibitor activity, capable of screening over 100,000 compounds daily. DBORh110 monitors CYP450 metabolic activity in real time for ADME/Tox studies, outperforming LC-MS methods. Dual-color apoptosis/necrosis detection kits (Rh110-DEVD/PI) simultaneously quantify two cell death modes in the same well, improving preclinical drug efficacy evaluation data reliability and accelerating drug development.

Future Development Directions and Safety of Rhodamine 110

Toxicological and environmental studies confirm Rhodamine 110’s safety and low ecological risk: it has an oral LD50 >2000 mg/kg in rats, no significant organ damage in repeated-dose tests, and low genotoxicity. In natural water, it has a half-life of approximately 7 days, degrading primarily via photolysis and biodegradation, with manageable environmental impact.

Future development of Rhodamine 110 derivatives will focus on three core directions:

1. Near-infrared and deep-tissue imaging variants: Introducing selenium atoms or extending molecular conjugation shifts emission beyond 650 nm, improving in vivo imaging depth, and enhancing two-photon absorption cross-sections (>1000 GM) for 3D deep-tissue imaging.
2. Stabilized and functionalized derivatives: Silanization protection and other modifications create environmentally stabilized Rhodamine 110 probes for long-term outdoor environmental monitoring, while targeted modification improves in vivo metabolic stability and tissue specificity.
3. AI-assisted rational design: Machine learning models predict the spectral properties, biocompatibility and target binding affinity of novel Rhodamine 110 derivatives, significantly shortening probe R&D cycles and accelerating the development of high-performance functional probes.

Technological innovations such as microfluidic chip integration, wearable fluorescence sensors and multimodal detection (combining fluorescence with mass spectrometry/Raman spectroscopy) will further expand Rhodamine 110’s applications. Nanocarrier delivery system optimization will improve the in vivo targeting and bioavailability of Rhodamine 110 probes, facilitating their clinical translation and ensuring Rhodamine 110 remains a core tool in life science research for decades to come.

Applications of ANT BIO PTE. LTD. Products in Rhodamine 110-Based Research

As a leading provider of life science research reagents, ANT BIO PTE. LTD.’s UA sub-brand—specializing in high-quality recombinant proteins—offers a premium Rhodamine 110-conjugated product, Ubiquitin Rhodamine 110 (UA080158), which is a key research tool for Rhodamine 110-based fluorescence imaging and biomolecular interaction studies.

This product is a human ubiquitin protein covalently conjugated with Rhodamine 110, retaining the native biological activity of ubiquitin while possessing the excellent fluorescent properties of Rhodamine 110. It features high purity (verified via RP-HPLC), stable fluorescence performance and excellent biocompatibility, enabling diverse applications in life science research:

• Ubiquitination dynamics research: The Rhodamine 110 conjugate enables real-time live-cell imaging of ubiquitin localization and ubiquitination processes, providing visual insights into the molecular mechanisms of protein ubiquitination in cellular homeostasis, apoptosis and signal transduction.
• Protein-protein interaction analysis: It serves as a fluorescent probe for studying the interaction between ubiquitin and its binding partners (e.g., E3 ligases, deubiquitinases), enabling quantitative and qualitative analysis of these interactions via fluorescence spectroscopy, pull-down assays and fluorescence resonance energy transfer (FRET).
• Ubiquitin-related enzyme activity assays: The conjugated product can be used to develop "turn-on" fluorescence assays for detecting the activity of deubiquitinases and other ubiquitin-modifying enzymes, with high sensitivity and specificity for basic research and drug screening of ubiquitin pathway modulators.
• Cellular localization and trafficking studies: It enables high-resolution imaging of ubiquitin’s subcellular localization in live and fixed cells via fluorescence microscopy and confocal imaging, revealing the spatial and temporal distribution of ubiquitin in normal and pathological cellular states.

Complementing this core Rhodamine 110-conjugated product, ANT BIO PTE. LTD.’s Absin sub-brand offers a comprehensive range of general reagents and kits for fluorescence imaging, cell culture and biomolecular analysis, while the Starter sub-brand provides high-specificity antibodies for co-staining and multi-labeling experiments with Rhodamine 110 probes. This integrated product portfolio creates a one-stop research solution for Rhodamine 110-based fluorescence research, supporting scientists in diverse fields such as cell biology, molecular biology, neuroscience and clinical research. ANT BIO PTE. LTD. also provides detailed product validation data, optimized experimental protocols and professional technical support to ensure reliable and reproducible research results for customers.

Related Products from ANT BIO PTE. LTD.

ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.

Disclaimer

This article was partially created with the assistance of artificial intelligence. If any content involves copyright or intellectual property issues, please inform us, and we promise to verify and remove it immediately.