Stability of R-phycoerythrin from Furcellaria lumbricalis - Dependence on purification strategies and purity.

R-phycoerythrin (R-PE) is the most abundant, naturally occurring phycobiliproteins found in red algae. The spectroscopic and structural properties of phycobiliproteins exhibit unique absorption characteristics with two significant absorption maxima at 498 and 565 nm, indicating two different chromophores of R-PE, phycourobilin and phycoerythrobilin respectively. This study aimed to clarify how the stability of R-PE purified from F. lumbricalis was affected by different purification strategies. Crude extracts were compared to R-PE purified by i) microfiltration, ii) ultrafiltration, and iii) multi-step ammonium sulphate precipitation followed by dialysis. The stability of the different R-PE preparations was evaluated with respect to pH (2, 4, 6, 7, 8, 10 and 12) and temperature (20, 40, 60, 80 and 100 °C). The absorbance spectra indicated higher stability of phycourobilin as compared to phycoerythrobilin for heat and pH stability in the samples. All preparations of R-PE showed heat stability till 40 °C from the findings of color, concentration of R-PE and fluorescence emission. The crude extract showed stability from pH 6 to 8, whereas R-PE purified by ultrafiltration and multi-step ammonium sulphate precipitation were both stable from pH 4 to 8 and R-PE purified by microfiltration exhibited stability from pH 4 to 10 from the results of color, SDS-PAGE, and concentration of R-PE. At pH 2, the color changed to violet whereas a yellow color was observed at pH 12 in the samples along with the precipitation of the protein.

Properties and potential applications of bioconjugates of R-phycoerythrin with Ag° or CdS nanoparticle synthesized in its tunnel cavity: A review.

Green synthesis is a promising method for the preparation of nanoparticles (NPs) due to its simplicity, low cost, low toxicity, and environmental friendliness. Biosynthesized NPs exhibit multifunctional activity, good biocompatibility, and higher anticancer and antibacterial activity compared to chemically synthesized NPs. R-phycoerythrin, a photosynthetic light-harvesting pigment of protein nature (M.w. 290 kDa), is an attractive platform for the synthesis of small sizes NPs due to its structural features, non-toxicity, water solubility. Photosensitive bioconjugates of R-phycoerythrin with NPs were prepared by synthesizing Ag° and CdS NPs in tunnel cavities of R-phycoerythrin (3.5 × 6.0 nm) isolated from the red seaweed Callithamnion rubosum. The review is devoted to the physical processes and chemical reactions that occur in the native protein macromolecule of a complex structure during the synthesis of a NP in its cavity. The influence of Ago and CdS NPs on the electronic processes caused by the absorption of photons, leading to reversible and irreversible changes in R-phycoerythrin has been analyzed. Properties of R-phycoerythrin bioconjugates Ag° and CdS with NPs combined with the literature data suggest potential applications of Ag°â‹…PE and CdS⋅PE bioconjugates for cancer diagnosis, treatment, and monitoring as well as for realizing theranostic strategy in the future. The use of these bioconjugates in anticancer therapy may have synergistic effects since both R-phycoerythrin and NPs induce cancer cell death.

The Influence of κ-Carrageenan-R-Phycoerythrin Hydrogel on In Vitro Wound Healing and Biological Function.

Wound healing is widely recognized as a critical issue impacting the healthcare sector in numerous countries. The application of wound dressings multiple times in such instances can result in tissue damage, thereby increasing the complexity of wound healing. With the aim of tackling this necessity, in the present study, we have formulated a hydrogel using natural polysaccharide κ-carrageenan and phycobiliprotein R-phycoerythrin from Pyropia yezoensis. The formulated hydrogel κ-Carrageenan-R-Phycoerythrin (κ-CRG-R-PE) was analyzed for its antioxidant and antimicrobial activity. The wound healing potential of the κ-CRG-R-PE was evaluated in Hs27 cells by the wound scratch assay method. The hydrogel showed dose-dependent antioxidant activity and significant antimicrobial activity at 100 µg/mL concentration. κ-CRG-R-PE hydrogels promoted more rapid and complete wound closure than κ-Carrageenan (κ-CRG) hydrogel at 24 and 48 h. κ-CRG-R-PE hydrogels also filled the wound within 48 h of incubation, indicating that they positively affect fibroblast migration and wound healing.

Optimization of R-Phycoerythrin Extraction by Ultrasound-Assisted Enzymatic Hydrolysis: A Comprehensive Study on the Wet Seaweed Grateloupia turuturu.

Enzyme-assisted extraction (EAE) and ultrasound-assisted extraction (UAE) are both recognized as sustainable processes, but little has been done on the combined process known as ultrasound-assisted enzymatic hydrolysis (UAEH), and even less on seaweed. The present study aimed to optimize the UAEH of the red seaweed Grateloupia turuturu for the extraction of R-phycoerythrin (R-PE) directly from the wet biomass by applying a response surface methodology based on a central composite design. Three parameters were studied: the power of ultrasound, the temperature and the flow rate in the experimental system. Data analysis demonstrated that only the temperature had a significant and negative effect on the R-PE extraction yield. Under the optimized conditions, the R-PE kinetic yield reached a plateau between 90 and 210 min, with a yield of 4.28 ± 0.09 mg·g-1 dry weight (dw) at 180 min, corresponding to a yield 2.3 times higher than with the conventional phosphate buffer extraction on freeze-dried G. turuturu. Furthermore, the increased release of R-PE, carbohydrates, carbon and nitrogen can be associated with the degradation of G. turuturu constitutive polysaccharides, as their average molecular weights had been divided by 2.2 in 210 min. Our results thus demonstrated that an optimized UAEH is an efficient method to extract R-PE from wet G. turuturu without the need for expensive pre-treatment steps found in the conventional extraction. UAEH represents a promising and sustainable approach that should be investigated on biomasses where the recovery of added-value compounds needs to be improved.

Intensifying extraction of biomolecules from macroalgae using vortex based hydrodynamic cavitation device.

Macroalgae have a tremendous potential to become an important renewable resource for valuable biomolecules and chemicals. New and improved ways of cell disruption and of enhancing rate as well as yield of extraction of valuable products from macroalgae are needed to fully realise this potential. In this work, hydrodynamic cavitation (HC) was used for intensifying rate and yield of extraction of phycoerythrin, proteins and carbohydrates from marine macroalgae Palmaria palmata. We use vortex-based HC devices which do not use small restrictions like orifice-based HC devices or moving parts like rotor-stator based HC devices. A bench scale setup with a nominal slurry flow rate of 20 LPM was established. Dried and powdered macroalgae was used. Influence of key operating parameters like pressure drop and number of passes on extraction performance (the rate and yield) was measured. A simple, yet effective model was developed and used for interpreting and describing experimental data. The results indicate that there exists an optimum pressure drop across the device at which extraction performance is maximum. The extraction performance with HC was found to be significantly better than the stirred vessels. HC has resulted in 2 to 20 times improvement in the rate of extraction of phycoerythrin (R-PE), proteins and carbohydrates. Based on the results obtained in this work, pressure drop of 200 kPa and number of passes through the HC devices of about 100 were found to be most effective for HC-assisted intensified extraction from macroalgae. The presented results and model will be useful for harnessing vortex-based HC devices for intensifying the extraction of valuable products from macroalgae.

Nanoencapsulation of R-phycoerytrin extracted from Solieria filiformis improves protein stability and enables its biological application as a fluorescent dye.

We aimed to encapsulate R-PE to improve its stability for use as a fluorescent probe for cancer cells. Purified R-PE from the algae Solieria filiformis was encapsulated in polymeric nanoparticles using PCL. Nanoparticles were characterised and R-PE release was evaluated. Also, cellular uptake using breast and prostate cancer cells were performed. Nanoparticles presented nanometric particle size (198.8 ± 0.06 nm) with low polydispersity (0.13 ± 0.022), negative zeta potential (-18.7 ± 1.10 mV), and 50.0 ± 7.3% encapsulation. FTIR revealed that R-PE is molecularly dispersed in PCL. DSC peak at 307 °C indicates the presence of R-PE in the nanoparticle. Also, in vitro, it was demonstrated low release for nanoparticles and degradation for the free R-PE. Finally, cellular uptake demonstrated the potential of R-PE/PCL nanoparticles for cancer cell detection. Nanoparticles loaded with R-PE can overcome instability and allow application as a fluorescent probe for cancer cells.

Three-phase partitioning, a recyclable method for the purification of R-phycoerythrin from a red algae Porphyra yezoensis.

In this study, R-phycoerythrin (R-PE) was isolated and characterized from Porphyra yezoensis by three-phase partitioning (TPP) method. The effects of temperature, time, pH, salt saturation, and volume ratio on the purity and recovery rate of R-PE were studied. The optimum extraction conditions were determined as follows: salt saturation of 70%, temperature of 25 °C, time of 45 min, pH of 7.0, and volume ratio of 1:1. Under the optimal extraction conditions, the purity of R-PE was 3.90. The results of SDS-PAGE showed that R-PE has three bands at 23 kDa, 22 kDa, and 18 kDa, corresponding to its α, ß, γ subunits. The structure and optical activity of R-PE did not change before and after purification based on ultraviolet, infrared, and fluorescence spectra. In addition, the purity and recovery rate of R-PE extracted by tert-butanol were evaluated. The results showed that the extraction performance of tert-butanol for R-PE remained unchanged in three recoveries. These show that TPP is an efficient, green, and recyclable extraction technology.

Extraction and purification of phycobiliproteins from algae and their applications.

Microalgae, macroalgae and cyanobacteria are photosynthetic microorganisms, prokaryotic or eukaryotic, living in saline or freshwater environments. These have been recognized as valuable carbon sources, able to be used for food, feed, chemicals, and biopharmaceuticals. From the range of valuable compounds produced by these cells, some of the most interesting are the pigments, including chlorophylls, carotenoids, and phycobiliproteins. Phycobiliproteins are photosynthetic light-harvesting and water-soluble proteins. In this work, the downstream processes being applied to recover fluorescent proteins from marine and freshwater biomass are reviewed. The various types of biomasses, namely macroalgae, microalgae, and cyanobacteria, are highlighted and the solvents and techniques applied in the extraction and purification of the fluorescent proteins, as well as their main applications while being fluorescent/luminescent are discussed. In the end, a critical perspective on how the phycobiliproteins business may benefit from the development of cost-effective downstream processes and their integration with the final application demands, namely regarding their stability, will be provided.

Single-step purified R-phycoerythrin transmits cellular imaging functionalities in vitro.

A single-step and rapid chromatographic method-based purification of Gracilaria corticata (J. Agardh) R-phycoerythrin (R-PE) was attained using polyacrylamide gel electrophoresis (PAGE) technique without affecting structural integrity. The purified R-PE had a characteristic UV-Vis spectrum with three absorbance maxima at 496, 535, and 565 nm, and fluorescence at 575 nm. R-PE was obtained with a purity index of 4.2 and a recovery yield of 44.3%. SDS-PAGE analysis exhibited three sub-units i.e., 18, 21, and 31 kDa, which corresponds to α, ß, and γ, respectively. This report's purification process was considered less time-consuming and could be efficiently applied to purify phycobiliproteins. The purified R-PE showed optimal stability up to 6 h at pH 7.0 when exposed to light (3000 lx), while the temperature at which the maximum stability was retained was at 20 °C. The cellular imaging property of R-PE was effectively implemented to evaluate its credentials without affecting the cell proliferation of Vero and Hep-2 cell lines with the higher IC50 concentrations in vitro. Under fluorescence microscopy and flow cytometry analysis, purified R-PE displayed the characteristic affinity towards cell imaging functions in preliminary in vitro studies.

Probing the stability of the food colourant R-phycoerythrin from dried Nori flakes.

This study aimed to characterise the stability of R-phycoerythrin (R-PE), a vivid natural colourant with emerging potential for application in the food industry. High-quality (A560/A280 ≥ 5), native (α-helix content 75%) R-PE was purified from commercial dried Nori (Porphyra sp.) flakes. Thermal unfolding revealed two transitions (at 56 and 72 °C), ascribed to different protein subunits. Contrary to elevated temperature, high-pressure (HP) treatment showed significant advantages: The R-PE unfolding was partly reversible and the colour bleaching was minimal. Binding of Cu2+ (6.3 × 105 M-1) and Zn2+ (1.7 × 103 M-1) influenced conformational changes in the protein tetrapyrrole chromophore without affecting R-PE structure and stability (colour). The results give new insights into the stability of R-PE suggesting its usefulness for the replacement of toxic synthetic dyes. Preservation of the red colour of R-PE could be considered in fortified food and beverages by HP processing. R-PE may act as a biosensor for Cu2+ in aquatic systems.

Sustainable valorization of meat processing wastewater with synergetic eutectic mixture based purification of R-Phycoerythrin from porphyrium cruentium.

Wastewater effluent from meat processing industries are enriched with nutrients but remain underutilized. Therefore, an efficient method was administered by employing this resource for the cultivation of Porphyreum cruentium. The cultured biomass was exposed to a one-step disruption and selective purification method using a protic deep eutectic solvent to obtain a commercially significant pigment, R-Phycoerythrin (R-PE). Six protic deep eutectic solvents (pDES) were synthesized and their thermophysical activity determined. A synergy between microwave and cavitation (MACE-DLPME) has been achieved for the effective recovery of these membrane proteins. The addition of pDES provides the selective medium for the concentration of R-PE. Optimization of the MACE-DLPME method yields 95.9% (w/w) of R-PE. Preparative size exclusion chromatography resulted in (28 µg/g) of R-PE. Further ultra-purification by anion exchange chromatography enhances the purity fold of R-PE to 125. The resulting ultrapure fraction exhibits enhanced anti-platelet activity (1.56 mg/g ascorbic acid equivalent).

Difference between mitogen-stimulated B and T cells in nonspecific binding of R-phycoerythrin-conjugated antibodies.

Nonspecific binding of conjugated antibodies represents a critical step which could significantly influence the results of immunostaining or flow cytometry. In this respect, various staining procedures and distinct cell types can alter the results obtained with different fluorochromes. In this study, we analysed nonspecific binding of R-phycoerythrin (R-PE)-conjugated antibodies to mouse mitogen-stimulated B and T lymphocytes. The cells were fixed, permeabilized and stained using isotype control antibodies conjugated with different fluorochromes and assessed by flow cytometry. R-PE-conjugated antibodies bound to LPS-stimulated B cells, in contrast to Con A-stimulated T cells, independently of their specificity. The percentage of R-PE positive B cells varied, according to the used antibodies or the fixation/permeabilization kit. Nevertheless, up to 30% of R-PE+ B cells after staining with R-PE-conjugated isotype control antibodies was detected. Furthermore, LPS-stimulated B cells bound nonspecifically, in a dose-dependent manner, unconjugated R-PE molecules. Con A-stimulated T cells slightly bound R-PE only in high concentrations. Similarly, the antibodies conjugated with other fluorochromes showed less than 1% of nonspecific binding independently of the manufacturer of antibodies or fixation/permeabilization kits. The data demonstrated that LPS-stimulated B cells, in contrast to Con A-stimulated T cells, bind R-PE nonspecifically following formaldehyde or paraformaldehyde fixation. Therefore, the results based on the use of R-PE-conjugated antibodies should be taken with a precaution.

Efficient Purification of R-phycoerythrin from Marine Algae (Porphyra yezoensis) Based on a Deep Eutectic Solvents Aqueous Two-Phase System.

R-phycoerythrin (R-PE), a marine bioactive protein, is abundant in Porphyra yezoensis with high protein content. In this study, R-PE was purified using a deep eutectic solvents aqueous two-phase system (DES-ATPS), combined with ammonium sulphate precipitation, and characterized by certain techniques. Firstly, choline chloride-urea (ChCl-U) was selected as the suitable DES to form ATPS for R-PE extraction. Then, single-factor experiments were conducted: the purity (A565/A280) of R-PE was 3.825, and the yield was 69.99% (w/w) under optimal conditions (adding 0.040 mg R-PE to ChCl-U (0.35 g)/K2HPO4 (0.8 g/mL, 0.5 mL) and extracting for 20 min). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that the purified R-PE contained three main bands. One band was presented after purification in native-PAGE. The UV-vis spectra showed characteristic absorption peaks at 495, 540, and 565 nm. R-PE displayed an emission wavelength at 570 nm when excited at 495 nm. All spectra results illustrated that the structure of R-PE remained unchanged throughout the process, proving the effectiveness of this method. Transmission electron microscope (TEM) showed that aggregation and surrounding phenomena were the driving forces for R-PE extraction. This study could provide a green and simple purification method of R-PE in drug development.

Stability, kinetics, and application study of phycobiliprotein pigments extracted from red algae Gracilaria gracilis.

Phycobiliprotein (PBP) pigments were extracted from red algae Gracilaria gracilis through maceration in phosphate buffer using previously optimized conditions. The stability of PBPs in the extracts was assessed by monitoring the extracts at different pHs and temperatures for 10 days. Since phycoerythrin (PE) is the main PBP present in G. gracilis, PE content was spectroscopically determined and used as a response factor. Kinetic modeling was used to describe PE degradation under different ranges of T and pH. The pigment extracts presented higher stability at pH 6.9 and -20 °C. PE was semipurified by precipitation with ammonium sulphate 65% followed by dialysis against water until a purity index of 0.7. The pigment was successfully applied as colorant in pancakes and yogurts with a pigment concentration of 0.15%. This study highlights the potential of PE pigments extracted from G. gracilis for applications in food products. PRACTICAL APPLICATION: Phycobiliprotein pigments were extracted from red algae Gracilaria gracilis through maceration in phosphate buffer. The stability of the pigment was evaluated at different pHs and temperatures, presenting higher stability at neutral pH and low temperatures. The pigment was successfully applied as colorant in pancakes and yogurts with a low pigment concentration. This study highlights the potential of phycobiliprotein pigments extracted from G. gracilis for applications in food products.

DNAzyme-Functionalized R-Phycoerythrin as a Cost-Effective and Environment-Friendly Fluorescent Biosensor for Aqueous Pb2+ Detection.

The sensitive detection of Pb2+ is of significant importance for food safety, environmental monitoring, and human health care. To this end, a novel fluorescent biosensor, DNAzyme-functionalized R-phycoerythrin (DNAzyme-R-PE), was presented for Pb2+ analysis. The biosensor was prepared via the immobilization of Iowa Black® FQ-modified DNAzyme-substrate complex onto the surface of SPDP-functionalized R-PE. The biosensor produced a minimal fluorescence signal in the absence of Pb2+. However, Pb2+ recognition can induce the cleavage of substrate, resulting in a fluorescence restoration of R-PE. The fluorescence changes were used to measure sensitively Pb2+ and the limit of detection was 0.16 nM with a linear range from 0.5-75 nM. Furthermore, the proposed biosensor showed excellent selectivity towards Pb2+ even in the presence of other metal ions interferences and was demonstrated to successfully determine Pb2+ in spiked lake water samples.

Photobleaching Comparison of R-Phycoerythrin-Streptavidin and Streptavidin-Alexa Fluor 568 in a Breast Cancer Cell Line.

Fluorescent dyes are excited by light and emit light at a longer wavelength. Photobleaching is one the most important obstacles in fluorescent image capturing. Photochemical alteration of a fluorescent dye caused by several excitation/emission cycles results in a fluorophore to be unable to emit light. In this study, R-phycoerythrin (R-PE) and Alexa Fluor 568 were separately conjugated to streptavidin. The efficiency of conjugations, R-PE-streptavidin and streptavidin-Alexa Fluor 568, were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and spectrophotometry, respectively. Herceptin, a humanized therapeutic antibody, was subsequently biotinylated. The reactivity of biotin-labeled Herceptin was examined by enzyme-linked immunosorbent assay. The photobleaching of R-PE-streptavidin and streptavidin-Alexa Fluor 568 were then compared in an immunofluorescent staining on a breast cancer cell line, BT-474. Our data showed that streptavidin-Alexa Fluor 568 was more photostable than R-PE-streptavidin, which provides more time for longer viewing of labeled proteins and image capturing.

Sensitive and selective detection of Cu2+ ions based on fluorescent Ag nanoparticles synthesized by R-phycoerythrin from marine algae Porphyra yezoensis.

In this study, using a natural and green protein R-phycoerythrin (R-PE) extracted from marine Porphyra yezoensis as the stabilizer and reducer, silver nanoparticles (AgNPs) were synthesized. Based on this, a highly sensitive and selective method for the detection of Cu2+ ions was developed using R-PE-AgNPs as fluorescent probe. The interactions between R-PE-AgNPs and Cu2+ ions were systematically characterized by fluorescence spectroscopy, transmission electron microscopy (TEM), elemental mapping and Fourier transform infrared (FTIR). It was found that Cu2+ ions could cause aggregation of the R-PE-AgNPs, accompanied by the greatly increased particle size. Importantly, the method offered a wide linear detection range from 0 µM to 100.0 µM with a detection limit of 0.0190 µM. Moreover, the proposed method was successfully applied to analyze Cu2+ ions in tap water and lake water samples, acquiring satisfactory recovery between 91.6% and 102.2%. Such a green, fast and cost-effective fluorimetric method of the R-PE-AgNPs probe has great potential for tracing Cu2+ ions in diverse aqueous media.

Ag0 Nanoparticles Synthesized in R-phycoerythrin: Change in Bioconjugate Properties upon Ripening of Nanoparticles.

BACKGROUND: Bioconjugates prepared from photoactive molecules and metal nanoparticles are suitable for the development of new optoelectronic devices and for theranostic applications in medicine. METHODS: We produced a bioconjugate of R-phycoerythrin (R-PE), a photosynthetic pigment of red algae, with Ag0 nanoparticles (Ag0 NPs) synthesized in its tunnel cavities by mixing aqueous solutions of AgNO3 and R-PE without exogenous reductant. In freshly prepared bioconjugate (Ag0⋅R-PE), the diameter of Ag0 NPs measured in electron microscopic images was 6.5 ± 0.5 nm, being commensurate with the length of R-PE tunnel cavity, and they were found to emit bright visible fluorescence. RESULT: The initial aggregation temperature (T0) of freshly prepared Ag0⋅R-PE was decreased, compared to that of native R-PE (33 vs. 44°C). Upon the ripening of Ag0 NPs, their fluorescence was almost entirely quenched, and giant surface-enhanced Raman scattering was recorded. The bioconjugate self-assembled into nanorods 25 ± 5 nm long and could withstand heating to 90°C. Further heating (90°C → 100°C) resulted in the formation of filaments which are 0.5 ± 0.2 µm long and spherical aggregates 60 ± 10 nm in diameter. CONCLUSION: These data can contribute to the development of practical applications for the Ag0⋅R-PE bioconjugate.

Purification of R-phycoerythrin from Gracilaria lemaneiformis by centrifugal precipitation chromatography.

Centrifugal precipitation chromatography (CpC) is a powerful chromatographic technique invented in the year 2000 but so far very little applied. The method combines dialysis, counter-current and salting out processes. The separation rotor consists of two identical spiral channels separated by a dialysis membrane (6-8 K MW cut-off) in which the upper channel is eluted with an ammonium sulfate gradient and the lower channel with water, and the mixtures are separated according to their solubility in ammonium sulfate as a chromatographic technique. In the present study, the method was successfully applied for separation and purification of R-phycoerythrin (R-PE), a protein widely used as a fluorescent probe, from the red alga Gracilaria lemaneiformis. The separation was performed with the elution of ammonium sulfate from 50% to 0% in 21.5 h at a flow rate of 0.5 ml/min, while the lower channel was eluted with water at a flow rate of 0.05 ml/min after sample charge, and the column was rotated at 200 rpm. After a single run, the absorbance ratio A565/A280 (a criterion for the purity of R-PE) was increased from 0.5 of the crude to 6.5. The purified R-PE exhibited a typical "three peaks" spectrum with absorbance maximum at 497, 538 and 565 nm. The Native-PAGE showed one single protein band and 20 kDa (subunits α and ß) and 30 kDa (subunit γ) can be observed in SDS-PAGE analysis which were consistent with the (αß)6γ subunit composition of R-PE. The results indicated that CpC is an efficient method to obtain protein with the high purity from a complex source.

Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface.

Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells.