Hemin as a Molecular Probe for Nitric Oxide Detection in Physiological Solutions: Experimental and Theoretical Assessment.

Given its pivotal role in modulating various pathological processes, precise measurement of nitric oxide (●NO) levels in physiological solutions is imperative. The key techniques include the ozone-based chemiluminescence (CL) reactions, amperometric ●NO sensing, and Griess assay, each with its advantages and drawbacks. In this study, a hemin/H2O2/luminol CL reaction was employed for accurately detecting ●NO in diverse solutions. We investigated how the luminescence kinetics was influenced by ●NO from two donors, nitrite and peroxynitrite, while also assessing the impact of culture medium components and reactive species quenchers. Furthermore, we experimentally and theoretically explored the mechanism of hemin oxidation responsible for the initiation of light generation. Although both hemin and ●NO enhanced the H2O2/luminol-based luminescence reactions with distinct kinetics, hemin's interference with ●NO/peroxynitrite- modulated their individual effects. Leveraging the propagated signal due to hemin, the ●NO levels in solution were estimated, observing parallel changes to those detected via amperometric detection in response to varying concentrations of the ●NO-donor. The examined reactions aid in comprehending the mechanism of ●NO/hemin/H2O2/luminol interactions and how these can be used for detecting ●NO in solution with minimal sample size demands. Moreover, the selectivity across different solutions can be improved by incorporating certain quenchers for reactive species into the reaction.

A Novel Substrate-Inspired Fluorescence-Based Albumin Detection Improves Assessment of Clinical Outcomes in Hemodialysis Patients Receiving a Nursing Nutrition Intervention.

BACKGROUND Albumin level does not precisely reflect nutritional status. We aimed to investigate the impact of a nutrition intervention on hemodialysis patients by use of fluorescence-based plasma albumin (FPA) detection. MATERIAL AND METHODS Eighty patients underwent maintenance hemodialysis for more than half a year and had a mean albumin <3.5 g/dL for over 3 months. The subjects were randomly divided into either a Control Group (CG) or an Intervention Group (IG). The IG received nutritional supplementation, and the CG group received routine nutritional support for 12 months. FPA and plasma albumin (PA) concentrations were measured. The fluorescence probe 1,3-Dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone methyl biphenyl benzoate was used in FPA detection. Quality of life was estimated using WHOQOL-BREF (Quality of Life Scale developed through the World Health Organization), the 36-Item Short-Form Survey (SF-36), and the 6-minute walking test (6MWT). RESULTS After a 6-month and a 12-month intervention, PA and FPA concentrations increased, and the increase in FPA concentration was higher than that of PA in the IG group (P<0.05). Comparatively, the parameters of quality of life and 6MWT were improved in the IG group (P<0.05) but there were only minor changes in the CG group (P>0.05). There is an obvious association between the changes in FPA concentration and the parameters of quality of life and 6MWT but not PA. CONCLUSIONS Use of the fluorescence probe improves the detection sensitivity of plasma albumin and provides a potential method to assess clinical outcomes in hemodialysis patients.

In vivo and ex vivo assessment of bladder hyper-permeability and using molecular targeted magnetic resonance imaging to detect claudin-2 in a mouse model for interstitial cystitis.

OBJECTIVES: To determine if the URO-MCP-1 mouse model for bladder IC/BPS is associated with in vivo bladder hyper-permeability, as measured by contrast-enhanced MRI (CE-MRI), and assess whether molecular-targeted MRI (mt-MRI) can visualize in vivo claudin-2 expression as a result of bladder hyper-permeability. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects primarily women. It is known that permeability plays a substantial role in IC/BPS. Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. Claudin-2 is a molecular marker that is associated with increased hyperpermeability in the urothelium. MATERIALS AND METHODS: CE-MRI was used to measure bladder hyper-permeability in the URO-MCP-1 mice. A claudin-2-specific mt-MRI probe was used to assess in vivo levels of claudin-2. The mt-MRI probe consists of an antibody against claudin-2 conjugated to albumin that had Gd-DTPA (gadolinium diethylenetriamine pentaacetate) and biotin attached. Verification of the presence of the mt-MRI probe was done by targeting the biotin moiety for the probe with streptavidin-horse radish peroxidase (SA-HRP). Trans-epithelial electrical resistance (TEER) was also used to assess bladder permeability. RESULTS: The URO-MCP-1 mouse model for IC/BPS was found to have a significant increase in bladder permeability, following liposaccharide (LPS) exposure, compared to saline-treated controls. mt-MRI- and histologically-detectable levels of the claudin-2 probe were found to increase with LPS -induced bladder urothelial hyper-permeability in the URO-MCP-1 IC mouse model. Levels of protein expression for claudin-2 were confirmed with immunohistochemistry and immunofluorescence imaging. Claudin-2 was also found to highly co-localize with zonula occlidens-1 (ZO-1), a tight junction protein. CONCLUSION: The combination of CE-MRI and TEER approaches were able to demonstrate hyper-permeability, a known feature associated with some IC/BPS patients, in the LPS-exposed URO-MCP-1 mouse model. This MRI approach could be clinically translated to establish which IC/BPS patients have bladder hyper-permeability and help determine therapeutic options. In addition, the in vivo molecular-targeted imaging approach can provide invaluable information to enhance our understanding associated with bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing further therapeutic strategies.

Functional Imaging for Therapeutic Assessment and Minimal Residual Disease Detection in Multiple Myeloma.

Serum markers and bone marrow examination are commonly used for monitoring therapy response in multiple myeloma (MM), but this fails to identify minimal residual disease (MRD), which frequently persists after therapy even in complete response patients, and extra-medullary disease escape. Positron emission tomography with computed tomography using 18F-deoxyglucose (FDG-PET/CT) is the reference imaging technique for therapeutic assessment and MRD detection in MM. To date, all large prospective cohort studies of transplant-eligible newly diagnosed MM patients have shown a strong and independent pejorative prognostic impact of not obtaining complete metabolic response by FDG-PET/CT after therapy, especially before maintenance. The FDG-PET/CT and MRD (evaluated by flow cytometry or next-generation sequencing at 10-5 and 10-6 levels, respectively) results are complementary for MRD detection outside and inside the bone marrow. For patients with at least a complete response, to reach double negativity (FDG-PET/CT and MRD) is a predictive surrogate for patient outcome. Homogenization of FDG-PET/CT interpretation after therapy, especially clarification of complete metabolic response definition, is currently underway. FDG-PET/CT does not allow MRD to be evaluated when it is negative at initial workup of symptomatic MM. New PET tracers such as CXCR4 ligands have shown high diagnostic value and could replace FDG in this setting. New sensitive functional magnetic resonance imaging (MRI) techniques such as diffusion-weighted MRI appear to be complementary to FDG-PET/CT for imaging MRD detection. The goal of this review is to examine the feasibility of functional imaging, especially FDG-PET/CT, for therapeutic assessment and MRD detection in MM.

Advancements in SPR biosensing technology: An overview of recent trends in smart layers design, multiplexing concepts, continuous monitoring and in vivo sensing.

Inspired by the rapid progress and existing limitations in surface plasmon resonance (SPR) biosensing technology, we have summarized the recent trends in the fields of both chip-SPR and fiber optic (FO)-SPR biosensors during the past five years, primarily regarding smart layers design, multiplexing, continuous monitoring and in vivo sensing. Versatile surface chemistries, biomaterials and nanomaterials have been utilized thus far to generate smart layers on SPR platforms and as such achieve oriented immobilization of bioreceptors, improved fouling resistance and sensitivity enhancement, collectively aiming to improve the biosensing performance. Furthermore, often driven by the desires for time- and cost-effective quantification of multiple targets in a single measurement, efforts have been made to implement multiplex bioassays on SPR platforms. While this aspect largely remains difficult to attain, numerous alternative strategies arose for obtaining parallel analysis of multiple analytes in one single device. Additionally, one of the upcoming challenges in this field will be to succeed in using SPR platforms for continuous measurements and in vivo sensing, and as such match up other biosensing platforms where these goals have been already conquered. Overall, this review will give insight into multiple possibilities that have become available over the years for boosting the performance of SPR biosensors. However, because combining them all into one optimal sensor is practically not feasible, the final application needs to be considered while designing an SPR biosensor, as this will determine the requirements of the bioassay and will thus help in selecting the essential elements from the recent progress made in SPR sensing.

FRET efficiency measurement in a molecular tension probe with a low-cost frequency-domain fluorescence lifetime imaging microscope.

We demonstrate the possibility of measuring FRET efficiency with a low-cost frequency-domain fluorescence lifetime imaging microscope (FD-FLIM). The system utilizes single-frequency-modulated excitation, which enables the use of cost-effective laser sources and electronics, simplification of data acquisition and analysis, and a dual-channel detection capability. Following calibration with coumarin 6, we measured the apparent donor lifetime in mTFP1-mVenus FRET standards expressed in living cells. We evaluated the system's sensitivity by differentiating the short and long lifetimes of mTFP1 corresponding to the known standards' high and low FRET efficiency, respectively. Furthermore, we show that the lifetime of the vinculin tension sensor, VinTS, at focal adhesions (2.30 ± 0.16 ns) is significantly (p < 10 - 6) longer than the lifetime of the unloaded TSMod probe (2.02 ± 0.16 ns). The pixel dwell time was 6.8 µs for samples expressing the FRET standards, with signal typically an order of magnitude higher than VinTS. The apparent FRET efficiency (EFRETapp) of the standards, calculated from the measured apparent lifetime, was linearly related to their known FRET efficiency by a factor of 0.92 to 0.99 (R2 = 0.98). This relationship serves as a calibration curve to convert apparent FRET to true FRET and circumvent the need to measure multiexponential lifetime decays. This approach yielded a FRET efficiency of 18% to 19.5%, for VinTS, in agreement with published values. Taken together, our results demonstrate a cost-effective, fast, and sensitive FD-FLIM approach with the potential to facilitate applications of FLIM in mechanobiology and FRET-based biosensing.

A highly selective and sensitive Tb3+-acetylacetone photo probe for the assessment of acetazolamide in pharmaceutical and serum samples.

A novel, simple, sensitive and selective spectrofluorimetric method was developed for the determination of Acetazolamide in pharmaceutical tablets and serum samples using photo probe Tb3+-ACAC. The Acetazolamide can remarkably quench the luminescence intensity of Tb3+-ACAC complex in DMSO at pH 6.8 and λex = 350 nm. The quenching of luminescence intensity of Tb3+-ACAC complex especially the electrical band at λem = 545 nm is used for the assessment of Acetazolamide in the pharmaceutical tablet and serum samples. The dynamic range found for the determination of Acetazolamide concentration is 4.49 × 10-9-1.28 × 10-7 mol L-1, and the limit of detection (LOD) and limit of quantification (LOQ) are (4.0 × 10-9 and 1.21 × 10-8) mol L-1, respectively.

Objective, Quantitative, Data-Driven Assessment of Chemical Probes.

Chemical probes are essential tools for understanding biological systems and for target validation, yet selecting probes for biomedical research is rarely based on objective assessment of all potential compounds. Here, we describe the Probe Miner: Chemical Probes Objective Assessment resource, capitalizing on the plethora of public medicinal chemistry data to empower quantitative, objective, data-driven evaluation of chemical probes. We assess >1.8 million compounds for their suitability as chemical tools against 2,220 human targets and dissect the biases and limitations encountered. Probe Miner represents a valuable resource to aid the identification of potential chemical probes, particularly when used alongside expert curation.

A Printed Multicomponent Paper Sensor for Bacterial Detection.

We present a simple all-in-one paper-based sensor for E. coli detection using a composite ink made of a fluorogenic DNAzyme probe for bacterial recognition and signal generation, lysozyme that lyses whole bacterial cells, and pullulan/trehalose sugars that stabilize printed bioactive molecules. The paper sensor is capable of producing a fluorescence signal as a readout within 5 minutes upon contacting E. coli, can achieve a limit of detection of 100 cells/mL, in a variety of sample matrixes, without sample enrichment, and remains stable for at least 6 months when stored at ambient temperature. Therefore, this simple paper sensor provides rapid bacterial testing on site, and can be shipped and stored under ambient conditions to benefit users living in resource-limited regions.

Synthesis and Functional Assessment of a Novel Fatty Acid Probe, ω-Ethynyl Eicosapentaenoic Acid Analog, to Analyze the in Vivo Behavior of Eicosapentaenoic Acid.

Eicosapentaenoic acid (EPA) is an ω-3 polyunsaturated fatty acid that plays various beneficial roles in organisms from bacteria to humans. Although its beneficial physiological functions are well-recognized, a molecular probe that enables the monitoring of its in vivo behavior without abolishing its native functions has not yet been developed. Here, we designed and synthesized an ω-ethynyl EPA analog (eEPA) as a tool for analyzing the in vivo behavior and function of EPA. eEPA has an ω-ethynyl group tag in place of the ω-methyl group of EPA. An ethynyl group has a characteristic Raman signal and can be visualized by Raman scattering microscopy. Moreover, this group can specifically react in situ with azide compounds, such as those with fluorescent group, via click chemistry. In this study, we first synthesized eEPA efficiently based on the following well-known strategies. To introduce four C-C double bonds, a coupling reaction between terminal acetylene and propargylic halide or tosylate was employed, and then, by simultaneous and stereoselective partial hydrogenation with P-2 nickel, the triple bonds were converted to cis double bonds. One double bond and an ω-terminal C-C triple bond were introduced by Wittig reaction with a phosphonium salt harboring an ethynyl group. Then, we evaluated the in vivo function of the resulting probe by using an EPA-producing bacterium, Shewanella livingstonensis Ac10. This cold-adapted bacterium inducibly produces EPA at low temperatures, and the EPA-deficient mutant (ΔEPA) shows growth retardation and abnormal morphology at low temperatures. When eEPA was exogenously supplemented to ΔEPA, eEPA was incorporated into the membrane phospholipids as an acyl chain, and the amount of eEPA was about 5% of the total fatty acids in the membrane, which is comparable to the amount of EPA in the membrane of the parent strain. Notably, by supplementation with eEPA, the growth retardation and abnormal morphology of ΔEPA were almost completely suppressed. These results indicated that eEPA mimics EPA well and is useful for analyzing the in vivo behavior of EPA.

Optical properties of biomimetic probes engineered from erythrocytes.

Light-activated theranostic materials offer a potential platform for optical imaging and phototherapeutic applications. We have engineered constructs derived from erythrocytes, which can be doped with the FDA-approved near infrared (NIR) chromophore, indocyanine green (ICG). We refer to these constructs as NIR erythrocyte-mimicking transducers (NETs). Herein, we investigated the effects of changing the NETs mean diameter from micron- (≈4 µm) to nano- (≈90 nm) scale, and the ICG concentration utilized in the fabrication of NETs from 5 to 20 µM on the resulting absorption and scattering characteristics of the NETs. Our approach consisted of integrating sphere-based measurements of light transmittance and reflectance, and subsequent utilization of these measurements in an inverse adding-doubling algorithm to estimate the absorption (µ a) and reduced scattering (µ s') coefficients of these NETs. For a given NETs diameter, values of µ a increased over the approximate spectral band of 630-860 nm with increasing ICG concentration. Micron-sized NETs produced the highest peak value of µ a when using ICG concentrations of 10 and 20 µM, and showed increased values of µ s' as compared to nano-sized NETs. Spectral profiles of µ s' for these NETs showed a trend consistent with Mie scattering behavior for spherical objects. For all NETs investigated, changing the ICG concentration minimally affected the scattering characteristics. A Monte Carlo-based model of light distribution showed that the presence of these NETs enhanced the fluence levels within simulated blood vessels. These results provide important data towards determining the appropriate light dosimetry parameters for an intended light-based biomedical application of NETs.

A contaminant-free assessment of Endogenous Retroviral RNA in human plasma.

Endogenous retroviruses (ERVs) comprise 6-8% of the human genome. HERVs are silenced in most normal tissues, up-regulated in stem cells and in placenta but also in cancer and HIV-1 infection. Crucially, there are conflicting reports on detecting HERV RNA in non-cellular clinical samples such as plasma that suggest the study of HERV RNA can be daunting. Indeed, we find that the use of real-time PCR in a quality assured clinical laboratory setting can be sensitive to low-level proviral contamination. We developed a mathematical model for low-level contamination that allowed us to design a laboratory protocol and standard operating procedures for robust measurement of HERV RNA. We focus on one family, HERV-K HML-2 (HK2) that has been most recently active even though they invaded our ancestral genomes almost 30 millions ago. We extensively validated our experimental design on a model cell culture system showing high sensitivity and specificity, totally eliminating the proviral contamination. We then tested 236 plasma samples from patients infected with HIV-1, HCV or HBV and found them to be negative. The study of HERV RNA for human translational studies should be performed with extensively validated protocols and standard operating procedures to control the widespread low-level human DNA contamination.

Colorimetric assessment of BCR-ABL1 transcripts in clinical samples via gold nanoprobes.

Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome.

Linear quantification of a streptavidin-alkaline phosphatase probe for enzyme-linked immuno mass spectrometric assay.

The alkaline phosphatase-streptavidin (AP-SA) probe released adenosine (∼267.2 Da) from the substrate adenosine monophosphate (AMP), where a signal may be detected from as little as 0.5 µl of a 0.1-pg/ml dilution of the probe (2.6 × 10(-22) mol). The signal from the AP-SA probe was linear from 1 to 50 pg/ml by monitoring adenosine release at 268 m/z (M + H) with liquid chromatography, electrospray ionization, and quadrupole mass spectrometry (LC-ESI-MS). The safe limit of detection and quantification of the AP-SA probe was approximately 0.5 pg/well or 5 pg/ml. Enzyme-linked immuno mass spectrometric assay (ELIMSA) using the AP-SA probe provided a linear signal response for prostate-specific antigen (PSA) against external standards from 1 to 500 pg/ml. The ELIMSA showed a safe limit of detection and quantification at 5 pg PSA/well or 50 pg/ml (false positive detection rate P ≤ 0.01). Female samples of 100 µl plasma/well were read against standards and blanks made in normal female plasma, and the lowest sample quantified was approximately 9.8 pg/well or 98 pg/ml. Here ELIMSA was applied to measure PSA in plasma from female, normal male, prostatectomy patient, and cancer patient samples that showed significant differences by analysis of variance (ANOVA).

Computational prediction and validation of an expert's evaluation of chemical probes.

In a decade with over half a billion dollars of investment, more than 300 chemical probes have been identified to have biological activity through NIH funded screening efforts. We have collected the evaluations of an experienced medicinal chemist on the likely chemistry quality of these probes based on a number of criteria including literature related to the probe and potential chemical reactivity. Over 20% of these probes were found to be undesirable. Analysis of the molecular properties of these compounds scored as desirable suggested higher pKa, molecular weight, heavy atom count, and rotatable bond number. We were particularly interested whether the human evaluation aspect of medicinal chemistry due diligence could be computationally predicted. We used a process of sequential Bayesian model building and iterative testing as we included additional probes. Following external validation of these methods and comparing different machine learning methods, we identified Bayesian models with accuracy comparable to other measures of drug-likeness and filtering rules created to date.

A phosphomolybdic acid anion probe-based label-free, stable and simple electrochemical biosensing platform.

A versatile label-free, stable, low-cost and simple electrochemical biosensing platform has been developed based on a phosphomolybdic acid anion probe by jointly taking advantages of its native electronegativity, electrochemical activity and chemisorption with graphene oxide.

A Novel Method for the Assessment of Cortisol Hormone in Different Body Fluids Using A New Photo Probe Thiazole Derivative.

A low cost and accurate method for the detection and analytical determination of the cortisol in pharmaceutical preparation, blood serum and urine was developed. The method was based upon the enhancement of fluorescence intensity of the band at 424 nm of the photo probe by different cortisol concentrations in acetonitrile at (pH 5.7, λex = 320 nm). The influence of the different parameters, e.g. pH, solvent, cortisol concentration and foreign ions concentrations that control the enhancement process of fluorescence intensity of the band of photo probe was critically investigated. The remarkable enhancement of the fluorescence intensity at 424 nm in acetonitrile by various concentrations of cortisol was successfully used as a photo- probe for the assessment of cortisol concentration. The calibration plot was achieved over the concentration range 8.0 × 10(-6)-5.5 × 10(-9) mol L(-1) cortisol with a correlation coefficient of 0.998 and a detection limit of 4.7 × 10(-9) mol L(-1). The developed method is simple and proceeds without practical artifacts compared to the other determination methods.

A multivalent approach of imaging probe design to overcome an endogenous anion binding competition for noninvasive assessment of prostate specific membrane antigen.

2[(3-Amino-3-carboxypropyl)(hydroxy)(phosphinyl)methyl]pentane-1,5-dioic acid) (GPI) is a highly potent inhibitor of prostate specific membrane antigen (PSMA) with a rapid in vivo clearance profile from nontarget organs including kidneys, but its use for imaging of PSMA is impeded by an endogenous anion (serum phosphate) competition, which compromises its specific binding to the antigen. Multipresentation of a targeting molecule on a single entity has been recognized as a practical way for imaging sensitivity enhancement. Herein, we demonstrate a multivalent approach based on a (64)Cu-specific bifunctional chelator scaffold to overcome the endogenous phosphate competition thus enabling the utility of GPI conjugates for in vivo detection of PSMA and imaging quantification. Both monomeric (H2CBT1G) and dimeric (H2CBT2G) conjugates were synthesized and labeled with (64)Cu for in vitro and in vivo evaluations. A 4-fold enhancement of PSMA binding affinity was observed for H2CBT2G as compared to H2CBT1G from the PSMA competitive binding assays performed on LNCaP cells. In vivo PET imaging studies were conducted on mouse xenograft models established with a PSMA(+) cell line, LNCaP, and PSMA(-) PC3 and H2009 cell lines. (64)Cu-CBT2G showed significantly higher LNCaP tumor uptake than (64)Cu-CBT1G at 1, 4, and 24 h postinjection (p.i.) (p < 0.05). In addition, tumor uptake of (64)Cu-CBT2G remained steady out to 24 h p.i. (1.46 ± 0.54, 1.12 ± 0.56, and 1.00 ± 0.50% ID/g at 1, 4, and 24 h p.i., respectively), while (64)Cu-CBT1G showed a great decrease from 1 to 4 h p.i. The PSMA imaging specificity of both H2CBT1G and H2CBT2G was demonstrated by their low uptake in PSMA(-) tumors (PC3 and H2009) and further confirmed by a significant signal reduction in PSMA(+) LNCaP tumors in the blockade study. In addition, the LNCaP tumor uptake (% ID/g) of (64)Cu-CBT2G was found to be in a positive linear correlation with the tumor size (R(2) = 0.92, 0.94, and 0.93 for 1 h, 4 h, and 24 h p.i.). This may render the probe with potential application in the management of patients with prostate cancer.

Assessment of physiological redox state with novel FRET protein probes.

SIGNIFICANCE: Development of redox-sensing fluorescent proteins (redox probe proteins) have enabled live imaging of the physiological redox state within a cell, generating new strategies for detecting changes in the redox state during developmental, pathogenic, and aging processes. Several of the probe proteins utilize their characteristic redox-sensing segments as linkers in between two fluorophores, where structural alternations of the segments lead to changes in FRET efficiencies across the fluorophores. In this review we summarize two types of novel FRET-based redox probe proteins, namely redox linker (RL)-derived probes and Redoxfluor. RECENT ADVANCES: After these FRET-based redox probe proteins were generated, their responsiveness toward redox-related compounds as well as toward reactive oxygen species or reducing stimuli was investigated in vitro. Notably, both the RL-derived probe and Redoxfluor were found to directly respond to the redox state of glutathione, a main redox-formulating compound, showing a promising property for their use in subsequent in vivo analyses. Redoxfluor was not only used for redox sensing in the cytoplasm, but also utilized for assessing the redox state within peroxisomes. CRITICAL ISSUES: In contrast to "one-fluorophore" redox probes such as roGFP and rxYFP proteins, whose usage has been established and widely expanded to various experimental systems, FRET-based redox probes were invented very recently and their applications to in vivo studies are still in their infancy. FUTURE DIRECTIONS: FRET-based redox probes provide novel approaches for redox sensing that are complementary to other methodologies.