Ratiometric Signal Research Articles

Tetrazine-Based Ratiometric Nitric Oxide Sensor Identifies Endogenous Nitric Oxide in Atherosclerosis Plaques by Riding Macrophages as a Smart Vehicle.

Atherosclerosis (AS) is the formation of plaques in blood vessels, which leads to serious cardiovascular diseases. Current research has disclosed that the formation of AS plaques is highly related to the foaming of macrophages. However, there is a lack of detailed molecular biological mechanisms. We proposed a "live sensor" by grafting a tetrazine-based ratiometric NO probe within macrophages through metabolic and bio-orthogonal labeling. This "live sensor" was proved to target the AS plaques with a diameter of only tens of micrometers specifically and visualized endogenous NO at two lesion stages in the AS mouse model. The ratiometric signals from the probe confirmed the participation of NO during AS and indicated that the generation of endogenous NO increased significantly as the lesion progressed. Our proposal of this "live sensor" provided a native and smart strategy to target and deliver small molecular probes to the AS plaques at the in vivo level, which can be used as universal platforms for the detection of reactive molecules or microenvironmental factors in AS.

Development of a highly sensitive aptamer-based electrochemical sensor for detecting saxitoxin based on K3Fe(CN)6 regulated silver nanoparticles

BackgroundSaxitoxin (STX) is the most toxic marine toxin, which can pose several adverse effects on human health. High sensitivity, fast response, and low-cost detection of STX contamination are of significance to reducing the fishery and seafood industries' loss. Among the various types of biosensors, the electrochemical biosensors have been extensively studied in the detection of STX, but the electrode surface modification material is easy to fall off, resulting in unstable electrochemical signals and poor reproducibility. It is imperative to have a ratiometric electrochemical biosensor for STX. ResultsIn this study, we developed a novel aptamer-based electrochemical sensor (AECs) for the sensitive detection of STX based on a K3Fe(CN)6 regulated silver nanoparticles (Ag NPs) modified with aptamer. The AECs was constructed by immobilizing aptamer on Ag NPs surfaces. Under optimized conditions, the AECs showed a linear response towards STX in the range from 0.04 to 0.15 μM with the regression equation of Y = −8.0 + 233.7 X (R2 = 0.9956). The limit of detection (LOD) was calculated to be 1 nM (based on 3 N/S), which is significantly lower than the regulatory limits for STX in seafood. Moreover, the AECs showed excellent sensitivity, reproducibility and stability, as well as the detection in samples with acceptable recovery ranged from 71.2 % to 93.8 %, demonstrating its broad application prospects in detection of STX in seafood samples. SignificanceThis work proposed an AECs to achieve sensitive detection of STX. A reaction system of K3Fe(CN)6 etched Ag NPs was introduced and used as the signal source to avoid the instability of the electrochemical signal, which can produce a ratiometric electrochemical signal output mode, improving the stability and sensitivity of electrochemical detection of STX.

Endogenous CO imaging in bacterial pneumonia with a NIR fluorescent probe

Bacterial pneumonia is a serious respiratory illness that poses a great threat to human life. Rapid and precise diagnosis of bacterial pneumonia is crucial for symptomatic clinical treatment. Endogenous carbon monoxide (CO) is regarded as a significant indicator of bacterial pneumonia; herein, we developed a near-infrared (NIR) probe for fluorescence and photoacoustic (PA) dual-mode imaging of endogenous CO in bacterial pneumonia. NO2-BODIPY could rapidly and specifically react with CO to produce strong NIR fluorescence as well as ratiometric PA signals. NO2-BODIPY has outstanding features including fast response, fluorescence/PA dual mode signals, good specificity, and a low limit of detection (LOD = 20.3 nM), which enables it to image endogenous CO in cells and bacterial pneumonia mice with high sensitivity and high contrast ratio. In particular, NO2-BODIPY has two-photon excited (1340 nm, σ1 = 1671 GM) NIR fluorescence and has been utilized to image endogenous CO in bacterial pneumonia mice with deep tissue penetration. NO2-BODIPY has been demonstrated a good capability of fluorescence/PA dual-mode imaging of CO in bacterial pneumonia mice, providing a precise manner to diagnose bacterial pneumonia.

Sequence-Specific Relay Recognition of Multiple Anions: An Interplay between Proton Donors and Acceptors.

Ratiometric detection of analyte is highly deserving since the technique is free from background correction. This work reports the design and synthesis of a pyridine-end oligo p-phenylenevinylene (OPV) derivative, 1 and its application in ratiometric dual-mode (both colorimetric and fluorogenic) recognition of dual anions, bisulfate (LOD=12.5 ppb) followed by fluoride (LOD=18.2 ppb) by sequence-specific relay (SPR) technique. The colorless probe turns brown with addition of bisulfate and again becomes colorless with the sequential addition of fluoride ion. In addition to such naked-eye color change, interestingly the ratiometric spectroscopic signals are reversible and evidently, the probe is reusable for several cycles. Besides, in presence of bisulfate, the protonated probe molecules, owing to their larger amphiphilic characteristics, formed self-assembled nanostructures. In addition to colorimetric and fluorescent changes, 1 H NMR titration and systematic DFT study evidently establish the underneath proton transfer mechanisms. Such reusable OPV-based chemosensor particularly with the capability of naked-eye recognition of dual anions using the SPR technique is seminal and possibly the first report in the literature.

A Ratiometric Gene-Switch System for miRNA Sensing and Gene Regulation.

microRNAs (miRNAs) are a class of non-coding, small RNAs that play an important role in diverse biological processes and diseases. By regulating the expression of eukaryotic genes post-transcriptionally in a sequence-specific manner, miRNAs are widely used to design synthetic RNA switches. However, most of the RNA switches are often dependent on the corresponding ligand molecules, whose specificity and concentration would affect the efficiency of synthetic RNA circuits. Here, a fused transcriptional repressor Gal4BD-Rluc based gene-switch system Gal-miR for miRNA visualization and gene regulation is described. By placing a luciferase downstream gene under the control of endogenous miRNA machinery, the Gal-miR system makes the conversion of miRNA-mediated gene silencing into a ratiometric bioluminescent signal, which quantitatively reflected miRNA-206 activity during myogenic differentiation. Moreover, it demonstrates that this gene-switch system can effectively inhibit breast cancer cell viability, migration and invasion under the control of specific miRNAs by replacing the downstream gene with melittin functional gene. The study proposes a powerful modular genetic design for achieving precise control of transgene expression in a miRNA responsive way, as well as visualizing the dynamics of miRNA activity.

A novel dual-mode sensing strategy for the determination of 1,4-dithiothreitol based on tannic acid stabilized copper nanocluster

In this paper, a novel sensing strategy with both ratiometric fluorescence and colorimetric responses towards 1,4-dithiothreitol (DTT) was developed based on tannic acid-templated copper nanoclusters (TA-CuNCs). The one-step reduction approach was utilized to obtain TA-CuNCs with the fluorescence emission peak located at 436 nm. Through the catalytic oxidation of Cu2+, o-phenylenediamine (OPD) could be converted to 2,3-diaminophenazine (DAP) with an obvious fluorescence signal at 557 nm. Additionally, the obtained yellow DAP showed a clear absorption peak at 420 nm, DAP could cause a significant fluorescence quenching of TA-CuNCs on account of inner-filter effect (IFE). The oxidation process between Cu2+ and OPD could be largely prevented by 1,4-dithiothreitol (DTT), whose sulfhydryl groups bound to Cu2+, and the fluorescence intensity of DAP was diminished, whereas the fluorescence of the TA-CuNCs was restored. The ratiometric fluorescence signal and absorbance intensity changed with the concentration of DTT in the linear ranges of 5–45 μM and 5–40 μM, respectively. And the limits of detection (LOD) for two modes were found to be 2.98 and 2.03 μM, respectively. Furthermore, the dual-mode assay method was used for the determination of DTT accurately and reliably in human serum samples.

In Situ Generated Dye@MOF/COF Heterostructure for Fluorescence Detection of Chloroquine Phosphate and Folic Acid via Different Luminescent Channels.

Metal-organic framework (MOF) and covalent-organic framework (COF) hybrid materials can combine the unique properties of MOF and COF components, and their applications in fluorescence sensing have attracted more and more attention. Herein, ZIF-90 is grown on 3D-COF by a simple in situ growing method in which the 7-amino-4-methylcoumarin (AMC) is encapsulated in ZIF-90 to construct a fluorescent sensor. Chloroquine phosphate (CQP) can coordinate with Zn2+ to decompose the ZIF-90 and release AMC. At 365 nm excitation, the ratiometric fluorescence signal AMC/3D-COF (I430/I598) increases linearly with CQP in a linear range of 4 × 10-5 to 4 × 10-4 M in urine. Under 340 nm excitation, quantitative analysis of CQP in the serum (3 × 10-6 to 4 × 10-5 M) is based on the fluorescence intensity of Zn-CQP/3D-COF (I384/I598). In addition, AMC@ZIF-90/3D-COF (1) exhibits high anti-interference and selectivity in sensing of FA with a "turn off" mode, with a correlation range of 1 × 10-5 to 1 × 10-3 M. The fluorescence color changes triggered by CQP under different excitation conditions, and the different fluorescence responses caused by CQP make it a highly secure anticounterfeiting platform. The synthesized dye@MOF/COF hybrids not only provide a new way to integrate multiple emission to design fluorescent probes for differentiation detection but also offer ideas for the design of anticounterfeiting platforms.

Intelligent quantitative recognition of sulfide using machine learning-based ratiometric fluorescence probe of metal-organic framework UiO-66-NH2/Ppix

Sulfides possess either high toxicity or play crucial physiological role such as gas transmitter dependent upon dosage, hence the significant for their rapid sensitive and selective concentration determination. Herein, a machine learning enhanced ratiometric fluorescence sensor was engineered for sulfide determination by incorporating the nanometal-organic framework (UiO-66-NH2) along with protoporphyrin IX (Ppix). The blue fluorescence at 431 nm originated from the moiety of UiO-66-NH2 by 365 nm excitation serves as an internal calibration reference signal, while the red fluorescence at 629 nm from the moiety of Ppix serves as the analytical signal, and the intensity is correlated to the amount of sulfides. The fluorescence color of the sensor gradually varies from blue to red upon sequential addition of copper and sulfide ions, resulting in RGB (Red, Green, Blue) feature values for corresponding sulfide concentrations, which facilities the advanced data processing techniques using machine learning algorithms. On the basis of fluorescence image fingerprint extraction and machine learning algorithms, an online data analysis model was developed to improve the precision and accuracy of sulfide determination. The established model employed Linear Discriminant Analysis (LDA) and was subjected to rigorous cross-validation to ensure its robustness. By analyzing the correlation between RGB feature values and sulfide concentrations, the study highlighted a significant positive relationship between the red feature values and sulfide concentrations. The application of machine learning techniques on the ratiometric fluorescence signal of the UiO-66-NH2/Ppix probe demonstrated its potential for intelligent quantitative determination of sulfides, offering a valuable and efficient tool for pollution detection and real-time rapid environmental monitoring.

A nanozipper structured efficient FRET probe for ratiometric detection of ATP based on target-induced cycling amplification

In the realm of disease diagnostics and prognostics, accurate quantification of adenosine triphosphate (ATP) has become crucial, as the intricate interplay between ATP and cellular metabolism is a fundamental factor in the pathogenesis and progression of many pathological conditions. Herein, a ratiometric fluorescence DNA nanozipper is developed, which enables highly sensitive detection of ATP by employing catalytic hairpin assembly. This innovative approach utilizes target-induced conformational changes in the nanozipper structure to tune the distance between the carboxyfluorescein as donor and tetramethylrhodamine as acceptor around the Förster radius, thereby enhancing the efficiency of fluorescence resonance energy transfer (FRET). In the state of nanozipper tension, the rigid and steric hindrance Y-shaped configuration leads to a considerable distance between two fluorophores, resulting in FRET low efficiency. However, the presence of ATP triggers allosteric configuration of the DNA nanozipper, switching the nanozipper to a relaxed state, which results in a visible fluorescent ratiometric signal. The sensing strategy exhibits a linear response to ATP concentrations in the range of 30 − 600 nM and can detect concentrations as low as 9.6 nM. Additionally, this detection method introduces a novel idea for the design of efficient FRET probes and demonstrates promising application potential in biological samples.

Ratiometric Fluorescence and Afterglow Lifetime Dual-Channel Nanoprobe for Simultaneous Imaging of HOCl and Temperature in Arthritis.

Arthritis is a joint disorder that potentially causes permanent joint damage and eventual disability without effective treatment. Clinical detection methods, including in vitro blood tests and anatomical imaging, still have limitations in achieving real-time in situ early detection of arthritis. In this work, a dual-channel luminescence nanoprobe (AGNPs-Cy7) is reported, which combines a cyanine dye and a photochemical reaction-based afterglow system for real-time in vivo imaging of arthritis. AGNPs-Cy7 simultaneously detect hypochlorous acid (HOCl) and temperature, two important indicators associated with the early development of arthritis, by monitoring the respective changes in independent ratiometric fluorescence and afterglow lifetime signals. The anti-interference properties of both the ratiometric fluorescence signal and afterglow lifetime signal enhance sensing accuracy compared to the single luminescence intensity. The developed probe successfully reveals the simultaneous increase in HOCl concentration and temperature in an arthritis mouse model.

Detection of Per‐ and Polyfluoroalkyl Substances (PFAS) by Interrupted Energy Transfer

AbstractThe ubiquitous presence of per‐ and polyfluoroalkyl substances (PFAS) in aqueous environments has aroused societal concern. Nonetheless, effective sensing technologies for continuous monitoring of PFAS within water distribution infrastructures currently do not exist. Herein, we describe a ratiometric sensing approach to selectively detect aqueous perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) at concentrations of μg ⋅ L−1. Our method relies on the excitonic transport in a highly fluorinated poly(p‐phenylene ethynylene) to amplify a ratiometric emission signal modulated by an embedded fluorinated squaraine dye. The electronic coupling between the polymer and dye occurs through overlap of π‐orbitals and is designed such that energy transfer is dominated by an electron‐exchange (Dexter) mechanism. Exposure to aqueous solutions of PFAS perturbs the orbital interactions between the squaraine dye and the polymer backbone, thereby diminishing the efficiency of the energy transfer and producing a “polymer‐ON/dye‐OFF” response. These polymer/dye combinations were evaluated in spin‐coated films and polymer nanoparticles and were able to selectively detect PFAS at concentrations of ca. 150 ppb and ca. 50 ppb, respectively. Both polymer films and nanoparticles are not affected by the type of water, and similar responses to PFAS were found in milliQ and well water.

Detection of Per- and Polyfluoroalkyl Substances (PFAS) by Interrupted Energy Transfer.

The ubiquitous presence of per- and polyfluoroalkyl substances (PFAS) in aqueous environments has aroused societal concern. Nonetheless, effective sensing technologies for continuous monitoring of PFAS within water distribution infrastructures currently do not exist. Herein, we describe a ratiometric sensing approach to selectively detect aqueous perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) at concentrations of μg ⋅ L-1 . Our method relies on the excitonic transport in a highly fluorinated poly(p-phenylene ethynylene) to amplify a ratiometric emission signal modulated by an embedded fluorinated squaraine dye. The electronic coupling between the polymer and dye occurs through overlap of π-orbitals and is designed such that energy transfer is dominated by an electron-exchange (Dexter) mechanism. Exposure to aqueous solutions of PFAS perturbs the orbital interactions between the squaraine dye and the polymer backbone, thereby diminishing the efficiency of the energy transfer and producing a "polymer-ON/dye-OFF" response. These polymer/dye combinations were evaluated in spin-coated films and polymer nanoparticles and were able to selectively detect PFAS at concentrations of ca. 150 ppb and ca. 50 ppb, respectively. Both polymer films and nanoparticles are not affected by the type of water, and similar responses to PFAS were found in milliQ and well water.

Electroactive poly(thionine) as imprinted polymer and reference probe simultaneously for ratiometric ion imprinted electrochemical Pb2+ sensor

In this work, an electrochemical sensor based on ion-imprinted polymer/Au nanoparticles/porous biochar (IIP/AuNPs/PBC) composite was proposed for the highly selective and sensitive detection of Pb2+. In this work, poly (thionine) (pTHI) served simultaneously as imprinted polymer and reference probe. It could not only realize the specific detection of Pb2+, but also provide an internal reference signal to eliminate the influence of human and environmental factors on the detection signal and further improve the stability of the sensor. In addition, the AuNPs/PBC composite with large specific surface area, excellent electron transport and electrocatalytic performance could effectively enhance the detection signal as a carrier material. At the same time, the AuNPs on the PBC surface would promote the formation of uniform and stable IIP through Au–S bonds. The synergistic effect between IIP, AuNPs/PBC and ratiometric signal mode gave the Pb2+ sensor excellent performance, including a wide linear range (0.1–1000 μg l−1), low detection limit (0.03 μg l−1, S/N = 3), excellent selectivity and stability. All these results indicate that the proposed sensor could provide a meaningful reference for highly selective detection of heavy metal ions (HMIs).

Dye-Triplet-Sensitized Downshifting Nanoprobes with Ratiometric Dual-NIR-IIb Emission for Accurate In Vivo Detection.

Despite the emerging near-infrared-IIb (NIR-IIb, 1500-1700 nm) bioimaging significantly improving the in vivo penetration depth and resolution, quantitative detection with accuracy remains challenging due to its inhomogeneous fluorescence signal attenuation in biological tissue. Here, ratiometric dual-NIR-IIb in vivo detection with excitation wavelengths of 808 and 980 nm is presented using analyte-responsive dye-triplet-sensitized downshifting nanoprobes (DSNPs). NIR cyanine dye IR-808, a recognizer of biomarker hypochlorite (ClO-), is introduced to trigger a triplet energy transfer process from the dye to Er3+ ions of DSNPs under 808 nm excitation, facilitating the formation of an analyte-responsive 1525 nm NIR-IIb assay channel. Meanwhile, DSNPs also enable emitting intrinsic nonanalyte-dependent downshifting fluorescence at the same NIR-IIb window under 980 nm excitation, serving as a self-calibrated signal to alleviate the interference from the probe amount and depth. Due to the two detected emissions sharing identical light propagation and scattering, the ratiometric NIR-IIb signal is demonstrated to ignore the depth of penetration in biotissue. The arthritis lesions are distinguished from normal tissue using ratiometric probes, and the amount of ClO- can be accurately output by the established detection curves.

A fluorescent dual-emitting platform for fluorescent “turn-on” ratiometric detection of ascorbic acid in beverages utilizing luminol-embedded iron-based metal–organic frameworks

A fluorescent dual-emitting platform for fluorescent “turn-on” ratiometric detection of ascorbic acid in beverages was developed utilizing luminol-embedded iron-based metal–organic frameworks (luminol@Fe-DOBDC MOFs). Luminol@Fe-DOBDC MOFs with fluorescent emissions at 430 nm and 540 nm under excitation wavelength of 365 nm were applied to detect ascorbic acid on the basis of ascorbic acid triggering the reduction of Fe3+ into Fe2+. In the presence of ascorbic acid, fluorescent intensity at 540 nm was increased significantly while fluorescent intensity at 430 nm was changed slightly. Two emission peaks separated by 110 nm can eliminate environmental interferences by built-in self-calibration of ratiometric signal, enhancing the sensitivity and accuracy. The increasing ratiometric fluorescent intensity (I540 nm/I430 nm) has linear relationship with the concentration of ascorbic acid from 0.2 to 30 μM with limit of detection of 70 nM. It is an efficient, sensitive and accurate platform to detect ascorbic acid in commercial beverages using transition-metal-based MOFs.

A Readily Available Red‐Emitting Methylthio‐Substituted Salicylaldehyde Azine with AIE Feature for Ratiometric Detection of HClO

AbstractDeveloping a simple, selective, and rapid reliable method to monitor hypochlorous acid (HClO) in vivo is very meaningful due to its important physiological and pathological functions. In this research, a novel AIE based fluorescent probe for ratiometric detection of HClO was fabricated by a simple one‐step synthesis, in which the methyl sulfide group and salicylaldehyde azine serve as the recognition unit and fluorophore, respectively. This probe was visible‐light‐excitable, and showed a palpable aggregation‐induced emission into the red region with high stability to Cu2+ and pH. Oxidation of the methyl sulfide group can be achieved rapidly and specifically by HClO in physiological condition, which enables it to display a rapid, ratiometric fluorescent response to HClO with high selectivity over other biologically pertinent species. By exploiting the probe‐based tool, we successfully validated its practical utility in selectively recognizing HClO in living cells via ratiometric fluorescence signals, thereby providing a potential method for investigating the relevant functions of HClO in biosystems.

Amplification-Free Ratiometric Electrochemical Aptasensor for Point-of-Care Detection of Therapeutic Monoclonal Antibodies.

The rapid quantification of therapeutic monoclonal antibodies (mAbs) is of great significance to their pharmacokinetics/pharmacodynamics (PK/PD) research and the personalized medication for disease treatment. Taking advantage of the direct decoration of tens of redox tags to the target of interest, we illustrate herein an amplification-free ratiometric electrochemical aptasensor for the point-of-care (POC) detection of trace amounts of therapeutic mAbs. The POC detection of therapeutic mAbs involved the use of the methylene blue (MB)-conjugated aptamer as the affinity element and the decoration of therapeutic mAbs with ferrocene (Fc) tags via the boronate crosslinking, in which the MB-derived peak current was used as the reference signal, and the peak current of the Fc tag was used as the output signal. As each therapeutic mAb carries tens of diol sites for the site-specific decoration of the Fc output tags, the boronate crosslinking enabled the amplification-free detection, which is cost-effective and quite simple in operation. In the presence of bevacizumab (BevMab) as the target, the resulting ratiometric signal (i.e., the IFc/IMB value) exhibited a good linear response over the range of 0.025-2.5 μg/mL, and the limit of detection (LOD) of the electrochemical aptasensor was 6.5 ng/mL. Results indicated that the aptamer-based affinity recognition endowed the detection of therapeutic mAbs with high selectivity, while the ratiometric readout exhibited satisfactory reproducibility and robustness. Moreover, the ratiometric electrochemical aptasensor is applicable to the detection of therapeutic mAbs in serum samples. Taking together, the amplification-free ratiometric electrochemical aptasensor holds great promise in the POC detection of therapeutic mAbs.

A single-fluorophore-based self-calibrating supramolecular sensor for the fast and ratiometric detection of permethrin

Permethrin (PMT) is a broad-spectrum synthetic pyrethroid insecticide widely used for treatment of insects in agriculture. Yet the massive use of PMT comes at the cost of its residue ubiquitous in the environment thereby causing a significant risk to human health. Therefore, development of an analytical method for accurate detection of PMT at the point-of-care is of great importance. In this study, we report a novel single-fluorophore-based supramolecular sensor with dual emissions as ratiometric output signals, successfully realizing the fast detection of PMT with the self-calibrating feature. This sensor exhibited several advantages for PMT detection, such as fast response, high sensitivity, high specificity, and excellent anti-interference property. Notably, this sensor could be used for on-site monitoring of PMT residues on the food surface without the help of complicated testing facilities. To our best knowledge, this is the first promising fluorescent method for the fast and ratiometric determination of PMT.

Competitive ratiometric fluorescent lateral flow immunoassay based on dual emission signal for sensitive detection of chlorothalonil

In this study, we develop a competitive ratiometric fluorescent lateral flow immunoassay (CRF-LFIA) based on dual emission fluorescence signal, which has great advantage in visual and quantitative detection of Chlorothalonil (CTN). Red-emitted fluorescent magnetic nanobeads (FMNBs) and green-emitted aggregation-induced emission fluorescent microsphere (AIEFM) are synthesized and conjugated to antibodies and antigens respectively, resulting in competitive binding with the analyte. The ratiometric fluorescence signal which comes from the overlap of these two fluorescence emissions. FMNBs probes also provide immunomagnetic separation (IMS) to enrich the analysts and resist complex matrix effects. This strip generates a visually discernible yellow-to-green fluorescence color change in the presence of CTN (2 ng/mL), which could be incisively observed by naked eye. Moreover, the limit of detection (LOD) reached 0.152 ng/mL by measurement of color (Red-Green-Blue, RGB) signals. Method validation shows a good correlation between CRF-LFIA and LC-MS/MS.

A Ratiometric Sers Probe for Imaging the Macrophage Phenotypes in Live Mice with Epilepsy and Brain Tumor.

Macrophage performs multiple functions such as pathogen phagocytosis, antigen presentation and tissue remodeling by polarizing toward a spectrum of phenotypes. Dynamic imaging of macrophage phenotypes is critical for evaluating disease progression and the therapeutic response of drug candidates. However, current technologies cannot identify macrophage phenotypes in vivo. Here, we develop a surface-enhanced Raman scattering (SERS) nanoprobe, AH1, which enables the accurate determination of physiological pH with high sensitivity and tissue penetration depth through ratiometric Raman signals. Due to the phenotype-dependent metabolic reprogramming, AH1 can effectively identify macrophage subpopulations by measuring the acidity levels in phagosomes. After intravenous administration, AH1 not only visualizes the spatial distribution of macrophage phenotypes in brain tumors and epileptic regions of mouse models, but also reveals the repolarization of macrophages in brain lesions after drug intervention. This work provides a new tool for dynamically monitoring the disease-associated immune microenvironment and evaluating the efficacy of immune-therapeutics in vivo. This article is protected by copyright. All rights reserved.