Protein language models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing.

Current base editors (BEs) use DNA deaminases, including cytidine deaminase in cytidine BE (CBE) or adenine deaminase in adenine BE (ABE), to facilitate transition nucleotide substitutions. Combining CBE or ABE with glycosylase enzymes can induce limited transversion mutations. Nonetheless, a critical demand remains for BEs capable of generating alternative mutation types, such as T>G corrections. In this study, we leveraged pre-trained protein language models to optimize a uracil-N-glycosylase (UNG) variant with altered specificity for thymines (eTDG). Notably, after two rounds of testing fewer than 50 top-ranking variants, more than 50% exhibited over 1.5-fold enhancement in enzymatic activities. When eTDG was fused with nCas9, it induced programmable T-to-S (G/C) substitutions and corrected db/db diabetic mutation in mice (up to 55%). Our findings not only establish orthogonal strategies for developing novel BEs but also demonstrate the capacities of protein language models for optimizing enzymes without extensive task-specific training data.

Rational design of a novel acryl-modified CQDs fluorescent probe for highly selective detection and imaging of cysteine in vitro and in vivo.

A novel fluorescent nanoprobe CQDs-O-Acryl has been designed and synthesized to directly and accurately identify Cys over other biothiols in PBS (10 mM, pH 7.4) buffer. The carbon quantum dots (CQDs-OH) (λex/em maxima = 495/525 nm) were fabricated by a solvothermal method using resorcinol as the carbon source. The CQDs-O-Acryl was achieved through covalently grafting the acryloyl group on the surface of carbon quantum dots by nuclear reaction based on static quenching. The structure and morphology of CQDs-OH and CQDs-O-Acryl have been characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and UV-vis absorption spectroscopy. Upon the addition of Cys, the ester bond of CQDs-O-Acryl has been broken, and the free CQDs were released by conjugated addition and cyclization reactions successively, emitting strong green fluorescence at 525 nm (λex = 495 nm). Under the optimized conditions, CQDs-O-Acryl exhibited good sensing of Cys within the range 0.095-16 µM (the LOD of 0.095 µM). Due to the high sensitivity, reliability, fast fluorescence response (10 min), and low toxicity of CQDs-O-Acryl, it was successfully applied to fluorescence imaging of Cys in A549 cells and zebrafish.

A Novel Fluorescence Probe Based on Azamonardine for Detecting and Imaging Cysteine in Cells and Zebrafish with High Selectivity and Sensitivity.

A novel fluorescent probe based on azamonardine (Aza) fluorophore was designed and synthesized for the highly selective detection of cysteine (Cys) in vivo and in vitro. After reacting with acryloyl chloride, the fluorescence of Aza is effectively quenched, resulting in the formation of the Aza-acryl probe. Upon the addition of Cys, the ester bond of Aza-acryl is cleaved, releasing a new compound (Compound 1) with strong fluorescence, thereby achieving fluorescence turn-on detection of Cys. The structure of Aza-acryl was characterized using X-ray crystallography and NMR spectroscopy. Additionally, density functional theory was employed to elucidate the quenching mechanism of the acyl group on the Aza. Aza-acryl exhibits high selectivity towards Cys and distinguishes it from other biothiols such as homocysteine (Hcy) and glutathione (GSH). The mechanism of Aza-acryl for detecting Cys was investigated through HPLC, NMR spectroscopy, high-resolution mass spectrometry, and reaction kinetics experiments. Aza-acryl demonstrates excellent imaging capabilities for Cys in cells and zebrafish, providing a reliable and selectable tool for the detection and imaging of Cys in biological systems.

Photothermometric analysis of bismuth ions using aggregation-induced nanozyme system with a target-triggered surface cleaning effect.

The development of nanozyme-based photothermometric sensing for point-of-care testing (POCT) heavy metal ions is of great significance for disease diagnosis and health management. Considering the low catalytic activity of most nanozymes at physiological pH, we found bismuth ions (Bi3+) could effectively enhance the peroxidase (POX)-like activity of cetyltrimethylammonium bromide and citrate-capped octahedral gold nanoparticle (CTAB/Cit-AuNP) nanozymes. It is mainly based on Bi3+ ions being able to trigger the surface cleaning effect of CTAB/Cit-AuNPs. Because the more active Bi3+ ions could effectively bind with citrate on the gold surface and competitively destroy the electrostatic interaction between citrate and CTAB, resulting in the removal of CTAB ligands from the gold surface. Without the ligand protection, CTAB/Cit-AuNPs aggregated immediately, and further resulted in a significant activation of the POX-like activity of AuNP nanozymes. Based on this principle, we introduced the enzyme substrate 3,3',5,5'-tetramethylbenzidine (TMB) into this aggregation-induced nanozyme system, and rationally designed a photothermometric platform to quickly and sensitively detect Bi3+ ions by using the good photothermal effect of the oxidation product of TMB (oxTMB). The developed photothermometric method only using a common thermometer has a limit of detection (LOD) as low as 45.7 nM for POCT analysis of Bi3+ ions. This study not only provides a more accurate understanding of the aggregation-induced nanozymes based on the surface cleaning principle, but also shows the potential applications of aggregation-induced nanozymes in the POCT field.

Synthesis of three-dimensional Au-graphene quantum dots@Pt core-shell dendritic nanoparticles for enhanced methanol electro-oxidation.

Au-graphene quantum dots (GQDs)@Pt core-shell nanodendrites are synthesized through a two-step reduction approach, in which Au forms the core, GQDs form an intermediate layer and dendritic Pt forms the shell. Among the above synthesized catalysts, the GQDs can manipulate the binding of reaction intermediates on the Pt surface as well as assemble π-π * conjugate bonds, thus forming a dendritic Pt shell instead of a compact Pt shell. The obtained core-shell structure was characterized by transmission electron microscopy, energy-dispersive x-ray and x-ray photoelectron spectroscopy. The methanol electro-oxidation was investigated in alkaline media on the Au-GQDs@Pt modified electrode via cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy analysis. In particular, we discovered that Au-Pt assembled with GQDs could dramatically improve the activity and stability of the catalysts, owing to the synergistic effect raised by the GQDs, which exhibit prominent electron conductivity and great chemical/physical stability. It was also found that the Pt/Au mole ratios could control the Pt shell thickness, which significantly affected the catalytic methanol oxidation activity of the Au-GQDs@Pt nanodendrites. The Au-GQDs@Pt nanodendrites with optimum Pt/Au mole ratios of 1.0 exhibited a 2.5 times increase in electrocatalytic activity toward methanol oxidation compared with the commercial catalyst (Pt/C), and its CO tolerance was also greatly improved. The above results show that the Au-GQDs@Pt nanocatalysts have potential application prospects in direct methanol fuel cells.

Enforced Tubular Assembly of Electronically Different Hexakis(m-Phenylene Ethynylene) Macrocycles: Persistent Columnar Stacking Driven by Multiple Hydrogen-Bonding Interactions.

Hexakis(m-phenylene ethynylene) (m-PE) macrocycles 1-4, sharing the same hydrogen-bonding side chains but having backbones of different electronic properties, are designed to probe the effectiveness of multiple H-bonding interactions in enforcing columnar assemblies. 1H NMR, absorption, fluorescence, and circular dichroism (CD) spectroscopy indicate that, compared with analogous macrocycles that self-associate based on aromatic stacking which is highly sensitive to the electronic nature of the macrocyclic backbones, macrocycles 1-4 all exhibit strong aggregation down to the micromolar (µM) concentrations in nonpolar solvents. Increasing solvent polarity quickly weakens aggregation. In THF and DMF, the macrocycles exist as free molecules. The observed solvent effects, along with the behavior of 5-F6 that cannot self-associate via H-bonding, confirm that H-bonding plays the dominating role in driving the self-association of 1-4. The backbone electronic nature does not change the self-assembling pattern common to 1-4. Fluorescence and CD spectra confirm that macrocycles 1-4 assemble anisotropically, forming helical stacks in which adjacent molecules undergo relative rotation to place individual benzene residues in the favorable offset fashion. Columnar alignment of 1-4 is confirmed by atomic force microscopy (AFM), which resolves single tubes consisting of stacked macrocycles. In addition, macrocycles with backbones of different electronic properties are found to undergo heteroassociation, forming hybrid nanotubes. This study has demonstrated the generality of enforcing the alignment of shape-persistent macrocycles, which represents an invaluable addition to the small number of known tubular stacks capable of accommodating structurally varied molecular components and provides self-assembling nanotubes with inner pores allowing ready structural and functional modification.

[Recent progress in research on oxidative stress in glaucoma].

Apoptosis of retinal ganglion cells and progressive loss of the axons is an important feature of glaucoma, its pathogenesis is not exact. Some experimental and clinical study found that oxidative stress plays an important role in the onset and progression of glaucoma. Oxidative stress markers were found in glaucoma patient's serum and ocular tissue and anti-oxidative stress therapy also had a role in controlling glaucoma. In this review, we will discuss the oxidative stress pathogenesis and the antioxidant therapy in glaucoma.

Protocol to use protein language models predicting and following experimental validation of function-enhancing variants of thymine-N-glycosylase.

Protein language models (PLMs) are machine learning tools trained to predict masked amino acids within protein sequences, offering opportunities to enhance protein function without prior knowledge of their specific roles. Here, we present a protocol for optimizing thymine-DNA-glycosylase (TDG) using PLMs. We describe steps for "zero-shot" enzyme optimization, construction of plasmids, double plasmid transfection, and high-throughput sequencing and data analysis. This protocol holds promise for streamlining the engineering of gene editing tools, delivering improved activity while minimizing the experimental workload. For complete details on the use and execution of this protocol, please refer to He et al.1.

28-day repeated-dose toxicity of orally administered Jinmao Jiedu granule in Sprague-Dawley rats.

Jinmao Jiedu granule is a Chinese medicine preparation consisting of Actinidia valvata Dunn, Salvia chinensis Benth, Iphigenia indica Kunth, and chicken gizzard. For many years, it has been employed in adjuvant therapy for cancer, especially liver cancer. However, the potential toxicity of the granule has not been reported. The present study aimed to assess the repeated-dose toxicity of orally administered Jinmao Jiedu granules for Sprague-Dawley (SD) rats. SD rats were orally administered Jinmao Jiedu granules at doses of 2.85, 5.70, and 11.40 g/kg in a 28-day subchronic toxicity study. No adverse clinical signs associated with treatment were noted throughout the experiment. There were no treatment-related toxicity alterations in body weight, hematology, clinical biochemistry, urinalysis, necropsy, and histopathology in rats compared with the control group. The No Observed Adverse Effect Level (NOAEL) of the Jinmao Jiedu granule was higher than 11.40 g/kg/day in rats.

Observation of CO2 and solvent adduct ions during negative mode electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of monohydric alcohols.

RATIONALE: Monohydric alcohols are common in natural products, bio-oils, and medicine. We have found that monohydric alcohols can form O3 (ions containing three oxygen atoms) and O4 adduct ions in negative electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), which would significantly affect the composition analysis of alcohols, especially in a complex mixture. It is necessary to study the reaction pathways and the method to eliminate or reduce the 'artifact' adducts. METHODS: Octadecanol, cholesterol, squalanol and two complex monohydric alcohol mixtures were selected as model compounds. These samples were subjected to negative ion ESI FT-ICR MS analysis. The composition and formation mechanism of adducts were studied by the ultrahigh-resolution accurate mass measurement for elemental composition, along with the MS(2) isolation and collision-induced dissociation (CID) experiments for structural determination. RESULTS: The reaction pathway of O3 adduct formation is the coupling of a monohydric alcohol ion with a CO2 to form a stable O3 ionic species by likely a covalent bond (source of CO2 is not clear). The O4 species are formed by O3 ionic species adducted with an alcohol molecule of the solvent, such as methanol or ethanol, by likely a hydrogen bond. These adduct ions could be eliminated or reduced by increasing collision energy. However, excessive collision energy would fragment monohydric alcohol ions. CONCLUSIONS: The formation mechanisms of O3 and O4 adducts from monohydric alcohols in negative ion ESI FT-ICR MS were proposed. The solvent adduction effects can be eliminated or reduced by optimizing the collision energy of CID in FT-ICR MS.

Visualizing electron rearrangement in space and time during the transition from a molecule to atoms.

Imaging and controlling reactions in molecules and materials at the level of electrons is a grand challenge in science, relevant to our understanding of charge transfer processes in chemistry, physics, and biology, as well as material dynamics. Direct access to the dynamic electron density as electrons are shared or transferred between atoms in a chemical bond would greatly improve our understanding of molecular bonding and structure. Using reaction microscope techniques, we show that we can capture how the entire valence shell electron density in a molecule rearranges, from molecular-like to atomic-like, as a bond breaks. An intense ultrashort laser pulse is used to ionize a bromine molecule at different times during dissociation, and we measure the total ionization signal and the angular distribution of the ionization yield. Using this technique, we can observe density changes over a surprisingly long time and distance, allowing us to see that the electrons do not localize onto the individual Br atoms until the fragments are far apart (∼5.5 Å), in a region where the potential energy curves for the dissociation are nearly degenerate. Our observations agree well with calculations of the strong-field ionization rates of the bromine molecule.

Baicalin prevents fibrosis of human trabecular meshwork cells via inhibiting the MyD88/NF-κB pathway.

Trabecular meshwork fibrosis contributes to increased aqueous humor outflow resistance, leading to elevated intraocular pressure in primary open-angle glaucoma. Baicalin, an extract from Scutellaria baicalensis Georgi, has shown anti-fibrotic effects in liver, lung, and kidney diseases. However, its anti-fibrotic effect on human trabecular meshwork (HTM) cells has not yet been clarified. In this study, we investigated its effects on TGF-ß2-induced HTM fibrosis as well as the underlying regulatory mechanisms. HTM cells were pretreated with baicalin, TAK-242, and baicalin + TAK-242 for 2 h followed by treatment with or without 5 ng/mL TGF-ß2 for 48 h. Cell viability was assayed using cell counting Kit-8 and fibronectin (FN), laminin (LN), and α-smooth muscle actin (α-SMA) were assessed by western blotting, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry. Further, the protein and gene expression levels of the TLR4/MyD88/NF-κB pathway (TLR4, MyD88, and NF-κB p65) were also examined by western blotting and RT-PCR, respectively. Thus, we observed that high doses of baicalin (40 µM) decreased (p < 0.1) HTM cell viability and 20 µM baicalin pretreatment was identified as the optimum pretreatment concentration. TGF-ß2 upregulated (p < 0.5) the expression of FN, LN, α-SMA, MyD88, NF-κB p65 proteins and mRNA in HTM cells, and these effects were inhibited by baicalin and TAK-242 (p < 0.5). However, western blot analysis showed that baicalin did not repress TLR4 expression in HTM cells. Therefore, our findings suggested that baicalin could prevent TGF-ß2-induced extracellular matrix (FN, LN) deposition and α-SMA expression in HTM cells by inhibiting the MyD88/NF-κB pathway.

Primary lacrimal sac lymphoma: a case-based retrospective study in a Chinese population.

AIM: To determine the clinical characteristics, pathological types, tumor markers, treatments, and outcomes of Chinese patients with primary lacrimal sac lymphoma. METHODS: This case-based retrospective study analyzed 15 Chinese patients with primary lacrimal sac lymphoma. The clinical data collected included gender, age at diagnosis, symptoms, imaging examination results, pathologic diagnosis, pathogen identification, tumor markers, treatments, follow-up, and prognosis. Descriptive statistics were used to characterize the patients. Progression-free survival (PFS) was defined as the time from surgery to the last follow-up, first record of tumor recurrence, or death. RESULTS: There were 7 males and 8 females with unilateral primary lacrimal sac lymphoma in the left eye (n=6) or right eye (n=9). The initial symptom in 13 patients was epiphora, and 2 patients had redness and swelling in the lacrimal sac area. All patients ultimately developed epiphora, and 12 had masses in the lacrimal sac area. Analysis of preoperative plasma tumor markers indicated 14 patients had elevated homocysteine, 9 had elevated ß2-microglobulin, and 2 had elevated lactate dehydrogenase (LDH); 2 patients had elevations of all three markers, and 1 patient had no elevation of any marker. All patients underwent surgical resection and 12 patients received postoperative chemotherapy. The pathological types were DLBCL (n=8), MALT lymphoma (n=5), and NK/T-cell lymphoma, nasal type (n=2). The mean follow-up time was 25.8mo (range: 4-41) and 2 patients died. Seven patients who underwent mass excision combined with dacryocystorhinostomy (DCR) had no postoperative epiphora. Eight patients who only underwent mass excision had varying degrees of postoperative epiphora. Preoperative LDH elevation and NK/T-cell lymphoma, nasal type were associated with poor prognoses. CONCLUSION: Early diagnosis and treatment can lead to a good prognosis for most patients with primary lacrimal sac lymphoma. Mass resection combined with DCR can reduce the occurrence of post-surgical epiphora. The pathology type and tumor marker status are associated with prognosis.

Identification of a novel mutation in the FGF10 gene in a Chinese family with obvious congenital lacrimal duct dysplasia in lacrimo-auriculo-dento-digital syndrome.

AIM: To identify the pathogenic gene variant in a family with lacrimo-auriculo-dento-digital syndrome [LADD (MIM 149730)] showing congenital lacrimal duct dysplasia as the main clinical manifestation and lay the foundation for future research on the pathogenic gene. METHODS: Ophthalmological examinations, including slit-lamp biomicroscopy and lacrimal duct probing, and computed tomography dacryocystography (CT-DCG) were performed for all participants. The family pedigree was drawn, genetic features were analyzed, and the genomic DNA of the subjects was extracted. Pathogenic genes were screened via whole exome sequencing (WES) and confirmed using Sanger sequencing. RESULTS: Six patients belonged to this three-generation family, and their clinical manifestations included congenital nasolacrimal duct obstruction, congenital absence of lacrimal puncta and canaliculi, lacrimal fistulae, and limb deformities. This pattern indicates autosomal dominant inheritance. Diagnosis was based on the clinical characteristics of LADD syndrome, which presented in all the patients in this family. A novel frameshift mutation in the FGF10 gene (NM_004465.1), c.234dupC (p.Trp79Leus*15), was identified in all patients via WES. The variant was confirmed by Sanger sequencing and classified as a "pathogenic mutation" according to the American College of Medical Genetics and Genomics (ACMG) variant interpretation guidelines. CONCLUSION: A novel frameshift mutation in the FGF10 gene is found in all patients. This finding helps this family with LADD syndrome receiving a more accurate clinical diagnosis and genetic counseling by extending the mutation range of the FGF10 gene.

Analysis of saturated hydrocarbons by redox reaction with negative-ion electrospray Fourier transform ion cyclotron resonance mass spectrometry.

A novel technique was developed for characterization of saturated hydrocarbons. Linear alkanes were selectively oxidized to ketones by ruthenium ion catalyzed oxidation (RICO). Branched and cyclic alkanes were oxidized to alcohols and ketones. The ketones were then reduced to alcohols by lithium aluminum hydride (LiAlH(4)). The monohydric alcohols (O(1)) in the products obtained from the RICO and RICO-LiAlH(4) reduction reactions were characterized using negative-ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) for identification of iso-paraffins, acyclic paraffins and cyclic paraffins. Various model saturated compounds were used to determine the RICO reaction and ionization selectivity. The results from the FTICR MS analysis on the petroleum distillates derived saturated fraction were in agreement with those from field ionization gas chromatography time-of-flight mass spectrometry (FI GC-TOF MS) analysis. The technique was also used to characterize a petroleum vacuum residue (VR) derived saturates. The results showed that the saturated molecules in the VR contained up to 11 cyclic rings, and the maximum carbon number was up to 92.

Direct visualization of laser-driven electron multiple scattering and tunneling distance in strong-field ionization.

Using a simple model of strong-field ionization of atoms that generalizes the well-known 3-step model from 1D to 3D, we show that the experimental photoelectron angular distributions resulting from laser ionization of xenon and argon display prominent structures that correspond to electrons that pass by their parent ion more than once before strongly scattering. The shape of these structures can be associated with the specific number of times the electron is driven past its parent ion in the laser field before scattering. Furthermore, a careful analysis of the cutoff energy of the structures allows us to experimentally measure the distance between the electron and ion at the moment of tunnel ionization. This work provides new physical insight into how atoms ionize in strong laser fields and has implications for further efforts to extract atomic and molecular dynamics from strong-field physics.

A novel molecularly imprinted electrochemiluminescence sensor for isoniazid detection.

Based on Ru(bpy)(3)(2+)-Au nanoparticles decorated multi-walled carbon nanotubes composites and a molecularly imprinted polymer (MIP), we propose a novel molecularly imprinted electrochemiluminescence (ECL) sensor to selectively determine isoniazid (INH). The MIP is synthesized through electrochemical copolymerization of acrylamide and N,N'-methylene diacrylamide in the presence of INH template molecules. The enhanced ECL intensity is linear in the range of 0.1 to 110 µg cm(-3) and the detection limit is 0.08 µg cm(-3) (3σ) INH with relative standard deviation 3.8% (n = 6) for 8 µg cm(-3). As a result, the sensor has been successfully applied to the determination of INH in human urine and pharmaceutical samples. Moreover, the possible ECL mechanism is discussed.

Novel cathodic electrochemiluminescence of tris(bipyridine) ruthenium(II) on a gold electrode in acidic solution.

The novel phenomenon of cathodic electrochemiluminescence on a gold electrode in tris(bipyridine) ruthenium(II) (Ru(bpy)(3)(2+)) solution is described for the first time. A cathodic electrochemiluminescence (ECL) was found to mainly occur at 0.4-0.8 V with continuous potential scanning from 0.2-1.4 V and the ECL peak was observed around 0.68 V, which was quite different from generally reported Ru(bpy)(3)(2+) ECL. Our group speculated that Ru(bpy)(3)(2+) possibly reacts with the gold electrode in the acidic phosphate buffer solution (PBS) to generate luminescence. The possible ECL mechanism was discussed according to the presented results. Moreover, it is revealed that the Au as co-reactant in the Ru-system contributed dominantly to the whole ECL. Therefore, the reaction between Ru(bpy)(3)(2+) and the newly formed Au implied that the inert metal Au could become a promising material for ECL investigations.

A red-emitting Europium(III) complex as a luminescent probe with large Stokes shift for the sequential determination of Cu2+ and biothiols in real samples.

In this work, a novel Eu3+-DTPA-bis(AMC) complex with red luminescence was designed and synthesized for sequential detection of Cu2+ and biothiols (Cys/Hcy/GSH) based on the displacement strategy with the good selectivity, high sensitivity, and large Stokes shift (288 nm). The possible detection mechanism was verified by UV-vis, the high-resolution mass spectrometry, and the fluorescence decay curve. The experimental parameters, including the solution pH, the incubation time, the concentration ratio of Eu3+-DTPA-bis(AMC) to Cu2+ and biothiols concentration, were optimized. Under the optimal conditions, it shows a good linear relationship between the concentration (0-10 µM) of Cu2+ and the fluorescence intensity of Eu3+-DTPA-bis(AMC), with a low detection limit of 0.065 µM. The linear range and the limit of detection of the Eu3+-DTPA-bis(AMC)/Cu2+ system for Cys/Hcy/GSH were 2.5-22.5/5-45/5-50 µM and 0.11/0.07/0.05 µM, respectively. Surprisingly, the high or low concentration of Eu3+-DTPA-bis(AMC)/Cu2+ can significantly affect the selectivity of the sensing system to biothiols (Cys/GSH/Hcy). When the concentration of the Eu3+-DTPA-bis(AMC)/Cu2+ system is 10.0 µΜ, it could recognize biothiols (Cys/GSH/Hcy) from other substances, but when the concentration is as low as 3.3 µM, it could further specifically distinguished Cys from Hcy/GSH. Owing to the high anti-interference characteristics, accuracy and specificity, the sensing system was well applied to the cascade detection of Cu2+ in actual environmental samples and Cys in biological and food samples, including FBS, urine, milk, beverage, fresh juice with the satisfactory recoveries from 96.20 to 106.80 %.

One-step microwave synthesis of red-emissive carbon dots for cell imaging in extreme acidity and light emitting diodes.

Red emissive carbon dots (R-CDs) have received great attention in biological fields due to their deep tissue penetrability, great bioimaging capability, low interference from auto-fluorescence, and potential for optoelectronic applications. Herein, excitation-independent, highly acid-sensitive R-CDs were successfully obtained via one-step microwave treatment of o-phenylenediamine (o-PD) and phosphoric acid and carefully purified by column chromatography. The relationship between the fluorescence emission and surface groups of the R-CDs was studied in detail using XPS, NMR, and fluorescence spectroscopy, and the different mechanisms of action of the R-CDs and acid in H2O and ethanol were determined. The excellent anti-interference ability and biocompatibility of the R-CDs were confirmed, and the probes were successfully used for imaging A549 and Escherichia coli (E. coli) cells in extreme acidity. Finally, based on their relatively high quantum yield and long wavelength emission, the application potential of the R-CDs in the fabrication of red light-emitting diodes (LEDs) was investigated.