Boronic Acid-Decorated Carbon Dot-Based Semiselective Multichannel Sensor Array for Cytokine Discrimination and Oral Cancer Diagnosis.

Cytokines are essential components of the immune system and are recognized as significant biomarkers. However, detection of a single cytokine is not precise and reliable enough to satisfy the requirements for diagnosis. Herein, we developed a pattern recognition-based method for the multiplexed sensing of cytokines, which involves three-color-emitting boronic acid-decorated carbon dots (BCDs) and arginine-modified titanium carbide (Ti3C2 MXenes) as the sensor array. Initially, the fluorescence signals of the three BCDs were quenched by Ti3C2 MXenes. In the presence of cytokines, the fluorescence intensity of the BCDs was restored or further quenched by different cytokines. The fluorescence response occurs in two steps: first, boronic acid interacts with cis-diol functional groups of cytokines, and second, arginine headgroup selectively interacts with glycans. By exploiting the different competing binding of the BCDs and the cytokines toward Ti3C2 MXenes, seven cytokines and their mixtures can be effectively discriminated at a concentration of 20 ng mL-1. Furthermore, our sensor array demonstrated an excellent performance in classifying human oral cancer saliva samples from healthy individuals with clinically relevant specificity. The noninvasive method offers a rapid approach to cytokine analysis, benefiting early and timely clinical diagnosis and treatment.

Fluorescent Probe for Investigating the Mitochondrial Viscosity and Hydrogen Peroxide Changes in Cerebral Ischemia/Reperfusion Injury.

Cerebral ischemia-reperfusion injury (CIRI), a cause of cerebral dysfunction during cerebral infarction treatment, is closely associated with mitochondrial viscosity and hydrogen peroxide (H2O2). However, the accurate measurement of mitochondrial viscosity and H2O2 levels in CIRI is challenging because of the lack of sufficient selectivity and blood-brain barrier (BBB) penetration of existing monitoring tools related to CIRI, hampering the exploration of the role of mitochondrial viscosity and H2O2 in CIRI. To address this issue, we designed an activatable fluorescent probe, mitochondria-targeting styryl-quinolin-ium (Mito-IQS), with excellent properties including high selectivity, mitochondrial targeting, and BBB penetration, for the visualization of mitochondrial viscosity and H2O2 in the brain. Based on the real-time monitoring capabilities of the probe, bursts of mitochondrial viscosity and H2O2 levels were visualized during CIRI. This probe can be used to monitor the therapeutic effects of butylphthalein treatment. More importantly, in vivo experiments further confirmed that CIRI was closely associated with the mitochondrial viscosity and H2O2 levels. This discovery provides new insights and tools for the study of CIRI and is expected to accelerate the process of CIRI diagnosis, treatment, and drug design.

Antimicrobial Agent Functional Gold Nanocluster-Mediated Multichannel Sensor Array for Bacteria Sensing.

Urinary tract infections (UTIs) have greatly affected human health in recent years. Accurate and rapid diagnosis of UTIs can enable a more effective treatment. Herein, we developed a multichannel sensor array for efficient identification of bacteria based on three antimicrobial agents (vancomycin, lysozyme, and bacitracin) functional gold nanoclusters (AuNCs). In this sensor, the fluorescence intensity of the three AuNCs was quenched to varying degrees by the bacterial species, providing a unique fingerprint for different bacteria. With this sensing platform, seven pathogenic bacteria, different concentrations of the same bacteria, and even bacterial mixtures were successfully differentiated. Furthermore, UTIs can be accurately identified with our sensors in ∼30 min with 100% classification accuracy. The proposed sensing systems offer a rapid, high-throughput, and reliable sensing platform for the diagnosis of UTIs.

General Strategy for Specific Fluorescence Imaging of Homocysteine in Living Cells and In Vivo.

The aberrantly changed level of homocysteine (Hcy) triggers a variety of pathological symptoms and subsequently Hcy-related diseases. Direct and selective visualization of Hcy in biological systems is pivotal to understanding the pathological functions of Hcy at the molecular level. Herein, a general strategy was developed for the specific fluorescence imaging of Hcy through the combination of dual-binding sites and the introduction of a nitro group at the 6-position of the 7-diethylaminocoumarin fluorophore. Also, a series of novel fluorescent probes were exploited for monitoring Hcy with excellent selectivity, high sensitivity, and far-red/near-infrared fluorescence emission. Furthermore, fluorescence imaging of endogenous Hcy dynamics in living cells and in vivo was achieved, providing direct and solid evidence for the increasement of endogenous Hcy in type 2 diabetes mellitus and Alzheimer's disease. This research will greatly advance the development and understanding of the molecular nexus between the Hcy metabolism cascade and the root causes of diseases related to Hcy.

Recognition Engineering-Mediated Multichannel Sensor Array for Gut Microbiota Sensing.

The composition and activity of the gut microbiota are crucial for health management and disease treatment. Herein, we develop a rapid and robust multichannel sensor array via a recognition engineering strategy using antimicrobial agent (vancomycin, bacitracin, and lysozyme) functional gold nanoclusters and gluconamide-modified Ti3C2 MXenes, which provide superior fingerprint patterns to distinguish gut-derived bacteria. The discrimination ability of the sensor array was highly improved via the synergistic recognition between the bacteria and the various antimicrobial agents. Five gut-derived bacteria, including probiotics, neutral, and pathogenic bacteria were clearly differentiated and discriminated from the bacteria mixtures. Furthermore, the sensing system was successfully applied for the accurate classification of human colorectal cancer samples from healthy individuals rapidly (30 min) with clinically relevant specificity. The rapidity, simplicity, and economic cost of this strategy offers a robust platform for gut microbiota analysis.

A sensitive electrochemical sensor for glutathione based on specific recognition induced collapse of silver-contained metal organic frameworks.

An electrochemical sensor capable of detecting glutathione (GSH) with high sensitivity and selectivity was developed based on the unique novel electroactive silver-based metal organic framework (Ag-MOF). The Ag-MOF obtained by silver nitrate and 1,3,5-benzoic acid (H3BTC) was thoroughly characterized and was modified onto the electrode via facile drop-casting method. The electrochemical response of GSH on the Ag-MOF modified electrode showed a significant reduction in the current signal because the Ag-GSH complex had stronger specific affinity than Ag-H3BTC and resulted in the collapse of the Ag-MOF. This sensor demonstrated an extensive linear dynamic range of 0.1 nM-1 µM, along with the low detection limit of 0.018 nM. Additionally, it exhibited good reproducibility, stability, and resistance to interfering compounds. The Ag-MOF modified electrode demonstrated superior performance attributed to its rapid electron transfer rate, outstanding electrochemical redox activity, and specific recognition/competitive reaction. These factors improved both sensitivity and selectivity. The high anti-interference ability allowed for the selective detection of GSH in intricate surroundings. In the real sample testing, the RSD was lower than 3.1% and the recovery was between 98.1 and 103%. This research highlights the potential of Ag-MOFs in developing electrochemical sensors and their promising applications in determining GSH for food screening and early disease diagnosis.

Regulation of the Structure of Zirconium-Based Porphyrinic Metal-Organic Framework as Highly Electrochemiluminescence Sensing Platform for Thrombin.

An electrochemiluminescence (ECL) sensor provides a sensitive and convenient method for early diagnosis of diseases; however, it is still a challenge to develop simple and sensitive sensing platforms based on efficient ECL signals and luminophore groups. Porphyrin-based metal-organic frameworks (MOFs) show great potential in ECL sensing; however, the mechanism and structure-activity relationship, as well as application, are rarely reported. Herein, hydrothermal reactions obtained porphyrin Zr-MOFs (PCN-222) with different specific surface areas, pore sizes, structures, and surface charge states by tuning the reaction time were developed, which served both as the ECL luminophore, coreaction promoter for S2O82-, and a connection in the ECL immunoassay. By progressively controlling the condition of the hydrothermal reaction, PCN-222 with large surface area-abundant micropores can be obtained, which has good conductivity and positively charged surfaces, obtaining excellent ECL performance. The ECL performance and the enhancement mechanism were investigated in detail. Using PCN-222-6h with the best ECL intensity as the immobilization matrix for the aptamer, a highly sensitive and selective assay for thrombin was developed. The decrease of the ECL signal was logarithmically linear with the concentration of thrombin in the range from 50 fg mL-1 to 100 pg mL-1 with a low detection limit of 2.48 fg/mL. This proposed strategy provides a brand new approach for tuning of the structures of MOFs as effective ECL signal probes, thus providing wider possibilities for effective ECL immunoassays in the detection of other biomarkers in diagnosis of diseases.

Self-Cascade Nanoenzyme of Cupric Oxide Nanoparticles (CuO NPs) Induced in Situ Catalysis Formation of Polyelectrolyte as Template for the Synthesis of Near-Infrared Fluorescent Silver Nanoclusters and the Application in Glutathione Detection and Bioimaging.

In this work, near-infrared fluorescent silver nanoclusters (Ag NCs) were prepared based on the in situ formed poly methacrylic acid (PMAA) as the template and stabilizer, which is synthesized by methacrylic acid (MAA) and hydroxyl radical (·OH) that is generated by the cascade nanoenzyme reaction of cupric oxide nanoparticles (CuO NPs). CuO NPs possess the intrinsic glutathione-like (GPx-like) and peroxidase-like (POD-like) activities, which can catalyze glutathione (GSH) and O2 to produce hydrogen peroxide (H2O2), and then transform into ·OH. The fluorescence intensity of Ag NCs decreases with the addition of GSH, because the -SH can easily anchor on the surface, resulting in the PMAA leaving the Ag NCs, and the coeffect of GSH and PMAA results in the aggregation to form larger Ag NPs. A good linear relationship between the fluorescence quenching rate and the GSH concentration was found in the range 0.01-40 µM with the detection limit 8.0 nM. The Ag NCs can be applied in the detection of GSH in the serum, as well as bioimaging of endogenous and exogenous GSH in cells with high sensitivity. Moreover, the normal and cancer cells can be distinguished through bioimaging because of the different GSH levels. The new method for the preparation of biocompatible nanoprobe based on the nanozyme tandem catalysis and the in situ formed template can avoid the direct usage of polymers or protein templates that hinder preparation and separation, providing a reliable approach for the synthesis, biosensing, and bioimaging of nanoclusters.

Novel ratiometric electrochemical sensing platform for uric acid based on electroactive cuprous oxide nanocubes combined with boron carbide.

Electrochemically active oxides play important roles in the fabrication of electrochemical sensing platforms, in which they can be utilized as electrochemical probes or catalysts in electrochemical reactions. Herein, a novel ratiometric electrochemical sensor for uric acid (UA) was developed based on the newly synthesized Cu2O nanocubes with good electrochemical activity combined with boron carbide (B4C) with excellent conductivity. The oxidation peak of Cu2O remained unchanged, which could be used as a reference, while the oxidation peak of UA catalyzed by the modified electrode increased with the concentration of UA. The two signals displayed a large peak-to-peak potential and thus a ratiometric electrochemical sensor for UA was established, which could further reduce the effects of unrelated factors, such as the environment influence. The sensor exhibited good linear ranges of 0.1-100 µM and 100-1000 µM, and showed good sensitivity, selectivity, repeatability, and stability. The sensor was successfully applied in the detection of UA in complex human serum and urine samples.

A fluorescence nanoplatform for the determination of hydrogen peroxide and adenosine triphosphate via tuning of the peroxidase-like activity of CuO nanoparticle decorated UiO-66.

A novel nanocomposite of CuO nanoparticle-modified Zr-MOF (CuO/UiO-66) was synthesized and developed as a fluorescence nanoplatform for H2O2 and adenosine triphosphate (ATP) via the "turn-on-off" mode in the presence of terephthalic acid (TA). The structure of CuO/UiO-66 was thoroughly characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), and other techniques. The CuO/UiO-66 with enhanced peroxidase-like (POD) activity obtained due to the Zr4+ in UiO-66 is beneficial to the aggregation of CuO NPs on its surface. As a result, the strengthened fluorescence at 425 nm with the excitation of 300 nm was found due to the highly fluorescent species of TAOH. This is produced by the oxidation of TA by ·OH that came from the catalysis of H2O2 via the peroxidase mimic of CuO/UiO-66. Hence the modification of CuO NPs on porous UiO-66 can provide a friendly and sensitive physiological condition for H2O2 detection. However, upon addition of ATP, the fluorescence intensity of TAOH at 425 nm effectively declined owing to the formation of complexation of Zr4+-ATP and the interaction of CuO to ATP which hampers the catalytic reaction of CuO/UiO-66 to H2O2. The specific interaction induced "inhibition of the peroxide-like activity" endows the sensitive and selective recognition of ATP. The detection limits were 16.87 ± 0.2 nM and 0.82 ± 0.1 nM, and linear analytical ranges were 0.02-100 µM and 0.002-30 µM for H2O2 and ATP, respectively. The novel strategy was successfully applied to H2O2 and ATP determination in serum samples with recoveries of 97.2-103.8% for H2O2 and 97.6-101.7% for ATP, enriching the avenue to design functional MOFs and providing new avenue of multicomponent bioanalysis.

Self-Catalyzed Surface Reaction-Induced Fluorescence Resonance Energy Transfer on Cysteine-Stabilized MnO2 Quantum Dots for Selective Detection of Dopamine.

A simple one-step ultrasonic method was developed for the synthesis of luminescent MnO2 quantum dots (MnO2 QDs) in the presence of cysteine, in which cysteine acted as the exfoliating agent and stabilization ligand. The cysteine-stabilized MnO2 QDs (Cys-MnO2 QDs) possess a fluorescence quantum yield of 4.7%, and the fluorescence intensity of Cys-MnO2 QDs is sensitive to dopamine (DA). The mechanism by which the Cys-MnO2 QDs catalyzed the self-polymerization of DA to form polydopamine nanoparticles (PDA NPs) and caused the fluorescence resonance energy transfer (FRET) between MnO2 QDs and PDA NPs was revealed. The sensing platform displayed a wide detection range (0.1-200 µM) with a low detection limit of 28 nM for the detection of DA. Moreover, the Michael addition/Schiff base reaction between the PDA NPs and cysteine on MnO2 QDs was demonstrated to facilitate the excellent selectivity toward DA detection in the presence of various interferences. This work not only develops a robust method for the preparation of highly luminescent MnO2 QDs but also provides a universal strategy on the basis of surface chemical reaction-induced FRET for the detection of DA with high sensitivity and selectivity, which is promising in the application of clinical diagnosis, drug delivery, and fluorescence-guided cancer therapy.

Direct Quantification and Visualization of Homocysteine, Cysteine, and Glutathione in Alzheimer's and Parkinson's Disease Model Tissues.

Alzheimer's disease (AD) and Parkinson's disease (PD) are chronic neurodegenerative diseases with high morbidity and mortality. Homocysteine (Hcy), cysteine (Cys), and glutathione (GSH) are closely related to AD and PD. However, the dynamics of Hcy, Cys, and GSH in the brain tissues and the potential pathogenesis between Cys/Hcy/GSH with AD and PD remain unclear. Herein, a novel fluorescent probe 1 with multiple binding sites was rationally designed and exploited for the direct quantification of serum total Hcy and Cys along with superior optical properties. Importantly, differentiation and simultaneity fluorescence imaging of Cys, Hcy, and GSH dynamics were achieved in living cells, tissues, and mouse models of AD and PD with this probe, providing direct evidences for the relationship between Hcy/Cys/GSH and AD/PD for the first time. In addition, pathogenesis studies demonstrated that elevated Hcy and Cys levels are closely related to imbalanced redox homeostasis, increased amyloid aggregates, and nerve cell cytotoxicity. These findings will greatly promote the understanding of the functions of Hcy/Cys/GSH in Alzheimer's and Parkinson's diseases, demonstrating clinical promise for the early diagnosis and prevention of AD and PD.

Fluorometric and Colorimetric Dual-Readout Assay for Histone Demethylase Activity Based on Formaldehyde Inhibition of Ag+-Triggered Oxidation of O-Phenylenediamine.

Histone demethylases (HDMs) are vital players in epigenetic regulation and important targets in cancer treatment, but effective molecular tools for analyzing HDMs activity are still limited. Interestingly, we found that the process of Ag+-triggered oxidation of O-phenylenediamine (OPD) to 2,3-diaminophenazine (OPDox) could be efficiently inhibited by formaldehyde (HCHO), with the decrease of fluorescent and colorimetric signals from OPDox. Accordingly, we developed a novel label-free fluorescent and colorimetric dual-readout assay for HDMs activity based on direct quantitation of HCHO liberated in the demethylation process. On the basis of the excellent performance of the Ag+-OPD-based method for HCHO quantitation, lysine-specific demethylase 1(LSD1) activity was not only successfully detected with a low detection limit of 0.3 nM (fluorescence) and 0.5 nM (colorimetric) but also observed by the naked eye. Moreover, the feasibility of the proposed assay was further expanded to assess the LSD1 activity in cancer cell lysate and its inhibition through a mix-and-readout procedure. This label-free, cost-effective, and highly sensitive dual-readout assay presents a valuable tool for epigenetics research and drug discovery.

Dual-Product Synergistically Enhanced Colorimetric Assay for Sensitive Detection of Lipid Transferase Activity.

Lipid transferase-catalyzed protein lipidation plays critical roles in many physiological processes and it has been an increasingly attractive therapeutic target from cancer to neurodegeneration, while sensitive detection of lipid transferase activity in biological samples remains challenging. Here, we presented an AuNP-based colorimetric method with dual-product synergistically enhanced sensitivity for convenient detection of lipid transferase activity. Homo sapiens N-myristoyltransferase 1 (HsNMT1), a key lipid transferase, was selected as the model. Accordingly, positively charged substrate peptides (Pep) of HsNMT1 can induce the aggregation of AuNPs through disrupting their electrostatic repulsion, while the HsNMT1-catalyzed lipid modification generates aggregated lipidated peptides (C14-Pep) and negatively charged HS-CoA, which will eliminate the disruption and stabilize the AuNPs by the formation of Au-S bonds, respectively. Consequently, charge reversal of the biomolecules and the formation of Au-S bonds synergistically contribute to the stability of AuNPs in the presence of HsNMT1. Therefore, the HsNMT1 activity can be visually detected by the naked eye through the color change of the AuNPs originated from the change in their distance-dependent surface plasmon resonance absorptions. Here, the A520/A610 ratio can sensitively reflect the activity of HsNMT1 in the linear range of 2-75 nM with a low detection limit of 0.56 nM. Moreover, the method was successfully applied for probing the HsNMT1 activities in different cell lysates and inhibitor screening. Furthermore, given the replaceability of the substrate peptide, the proposed assay is promising for universal application to other lipid transferases and exhibits great potential in lipid transferase-targeted drug development.

Bi-Underpotential/PtAu-bulk co-electrodeposition and subsequent Bi dissolution for the electrocatalytic oxidation and amperometric analysis of formaldehyde.

A PtAuBi-UPD composite electrocatalyst modified glassy carbon electrode (GCE) is prepared via the simultaneous underpotential deposition (UDP) of Bi and bulk deposition of Pt and Au, followed by stripping of the accessible Bi, and it shows high performance for the electrocatalytic oxidation and amperometric analysis of formaldehyde.

Hydrogen peroxide sensing in body fluids and tumor cells via in situ produced redox couples on two-dimensional holey CuCo2O4 nanosheets.

A novel nanomaterial of two-dimensional holey CuCo2O4 (2D HCCO) nanosheets was synthesized via a general template-directed method and employed for the first time to construct an effective electrochemical platform for H2O2 sensing with the combination of cerium oxide (CeO2). During the electrocatalytic reduction of H2O2, the synergetic catalysis of CeO2/HCCO/MWCNTs/GCE owing to the naturally holey frameworks and the mediator of CeO2 results in the ultra-sensitive detection of H2O2. The current was greatly enhanced owing to the unique holey structure that can minimize the charge transfer distance and provide more active sites to boost the signals, and the dual oxidation state of Ce3+/Ce4+ on the surface of 2D HCCO nanosheets can promote the in situ production of Cu2+/Cu+ and Cu+/Cu and further amplify the detection signal. The CeO2/HCCO/MWCNTs/GCE showed a wide linear range from 1 µM to 7.31 mM using chronoamperometry at the potential of - 0.25 V and a relatively low detection limit of 0.16 µM in physiological environment, which was also utilized for tracking the trace H2O2 released from Hela cells. This study shows great promise for the emerging application of holey HCCO-based biosensors in bioanalysis and early cancer diagnosis. Graphical abstract.

Chimeric DNA-Functionalized Titanium Carbide MXenes for Simultaneous Mapping of Dual Cancer Biomarkers in Living Cells.

Acquiring multilayer information on diverse biomarkers with different spatial distributions at the cellular level is crucial for monitoring the progression of cancers. Herein, a dual-signal-tagged chimeric DNA-functionalized titanium carbide MXenes nanoprobe (dcDNA-Ti3C2) that responds to biomarkers with different cellular locations from plasma membrane to cytoplasm was designed toward this end. In the presence of cancer biomarkers, including transmembrane glycoprotein mucin 1 (MUC1) and cytoplasmic microRNA-21 (miR-21), the recognition between MUC1 and its aptamer in the dcDNA-Ti3C2 probe induces the separation of TAMRA-MUC1 aptamer from Ti3C2 MXenes, thereby resulting in an increase in red fluorescence; and the hybridization of miR-21 with the hairpin probe triggers the increase of green fluorescence. As a result, dual analysis of MUC1 and miR-21 at low-nanomolar concentrations in vitro, as well as in situ simultaneous imaging of the biomarkers within MCF-7 breast cancer cells, was achieved. The feasibility of the nanoprobe was further demonstrated by monitoring the expression changes of both the biomarkers in cancer cells under different inhibitor combinations. Therefore, this strategy allows us to acquire the expression levels and spatial distributions of different biomarkers in living cells, providing a helpful tool for reliable diagnosis of cancers and basic understanding their progression.

Functional Titanium Carbide MXenes-Loaded Entropy-Driven RNA Explorer for Long Noncoding RNA PCA3 Imaging in Live Cells.

The visualization of the long noncoding RNA of prostate cancer gene 3 (lncRNA PCA3), a specific biomarker for androgen receptor-positive prostate cancer, in living cells not only directly reflects the gene expression and localization but also offers better insight into its roles in the pathological processes. Here, we loaded an entropy-driven RNA explorer (EDRE) on the TAT peptide-functionalized titanium carbide MXenes (Ti3C2-TAT) for the imaging of nuclear lncRNA PCA3 in live cells. The EDRE was condensed on the Ti3C2-TAT (Ti3C2-TAT@EDRE) by electrostatic interaction. Ti3C2-TAT@EDRE enables the entering of cells and release of TAT peptides and EDRE in the cytoplasm by the glutathione (GSH)-triggered cleavage of the disulfide bonds in Ti3C2-TAT. The released EDRE is delivered into the nucleus by the nucleus-targeted guidance of TAT peptides, and initiated by the target lncRNA PCA3, subsequently leading to the continuous accumulation of fluorescence signals. Consequently, fluorescence analysis of lncRNA PCA3 at low-picomolar concentrations in vitro as well as sensitive live cell imaging of lncRNA PCA3 in the nucleus of androgen receptor-positive LNCaP prostate cancer cells were achieved, providing a versatile strategy for the monitoring of nucleic acid biomarkers in the nucleus of living cells.

Click-Type Protein-DNA Conjugation for Mn2+ Imaging in Living Cells.

A click-type protein-DNA conjugation, named as MnDDC (Mn2+-activated DCV-DNA conjunction), is presented, where DCV (rep protein of duck circovirus) and its target DNA work as the modular blocks to rapidly and effectively generate Mn2+-dependent and site-specific protein-DNA linkage. On the basis of MnDCC, a fluorescent Mn2+ biosensor composed of DCV and a molecular beacon, was developed for rapid sensing of Mn2+ within 2 min with nanomolar sensitivity. Using the proposed biosensor, not only analysis of Mn2+ in real samples (e.g., serum and food), but also wash-free fluorescent imaging of Mn2+ in extracellular environment and cytoplasm have been achieved. Moreover, the MnDDC-based sensor was proved to be a powerful tool for visualization of Mn2+ during exploration of the associated cytotoxicity in living neural cells, which is helpful to reveal the cellular responses toward the disordered homeostasis of Mn2+ in both extracellular and intracellular microenvironments.

DNA mimics of red fluorescent proteins (RFP) based on G-quadruplex-confined synthetic RFP chromophores.

Red fluorescent proteins (RFPs) have emerged as valuable biological markers for biomolecule imaging in living systems. Developing artificial fluorogenic systems that mimic RFPs remains an unmet challenge. Here, we describe the design and synthesis of six new chromophores analogous to the chromophores in RFPs. We demonstrate, for the first time, that encapsulating RFP chromophore analogues in canonical DNA G-quadruplexes (G4) can activate bright fluorescence spanning red and far-red spectral regions (Em = 583-668 nm) that nearly match the entire RFP palette. Theoretical calculations and molecular dynamics simulations reveal that DNA G4 greatly restricts radiationless deactivation of chromophores induced by a twisted intramolecular charge transfer (TICT). These DNA mimics of RFP exhibit attractive photophysical properties comparable or superior to natural RFPs, including high quantum yield, large Stokes shifts, excellent anti-photobleaching properties, and two-photon fluorescence. Moreover, these RFP chromophore analogues are a novel and distinctive type of topology-selective G4 probe specific to parallel G4 conformation. The DNA mimics of RFP have been further exploited for imaging of target proteins. Using cancer-specific cell membrane biomarkers as targets, long-term real-time monitoring in single live cell and two-photon fluorescence imaging in tissue sections have been achieved without the need for genetic coding.