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Review
. 2019 Jun 3;9(2):76.
doi: 10.3390/bios9020076.

In Vivo Biosensing Using Resonance Energy Transfer

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Free PMC article
Review

In Vivo Biosensing Using Resonance Energy Transfer

Shashi Bhuckory et al. Biosensors (Basel). .
Free PMC article

Abstract

Solution-phase and intracellular biosensing has substantially enhanced our understanding of molecular processes foundational to biology and pathology. Optical methods are favored because of the low cost of probes and instrumentation. While chromatographic methods are helpful, fluorescent biosensing further increases sensitivity and can be more effective in complex media. Resonance energy transfer (RET)-based sensors have been developed to use fluorescence, bioluminescence, or chemiluminescence (FRET, BRET, or CRET, respectively) as an energy donor, yielding changes in emission spectra, lifetime, or intensity in response to a molecular or environmental change. These methods hold great promise for expanding our understanding of molecular processes not just in solution and in vitro studies, but also in vivo, generating information about complex activities in a natural, organismal setting. In this review, we focus on dyes, fluorescent proteins, and nanoparticles used as energy transfer-based optical transducers in vivo in mice; there are examples of optical sensing using FRET, BRET, and in this mammalian model system. After a description of the energy transfer mechanisms and their contribution to in vivo imaging, we give a short perspective of RET-based in vivo sensors and the importance of imaging in the infrared for reduced tissue autofluorescence and improved sensitivity.

Keywords: BRET; CRET; FRET; NIR; biosensor; luciferase; nanoparticles; quantum dot; real-time imaging.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic illustration of FRET, BRET and CRET-based systems, which can be injected in mice for RET-based imaging and biosensing. FRET, BRET, and CRET exploit the non-radiative energy transfer from an excited donor to an acceptor molecule in the ground-state when they are in close proximity, typically 1–10 nm. In FRET, the donor excited state occurs through external optical excitation, whereas in BRET and CRET, the donor is excited through a biochemical or chemical reaction that generates bioluminescence or chemiluminescence, respectively.
Figure 2
Figure 2
BRET in nature. Both the Ca2+-dependent blue-emitting protein aequorin and green fluorescent protein (GFP) are naturally co-localized in rings around the bell of the jellyfish Aequorea victoria. When aequorin is excited in response to a pulse of Ca2+ flooding the area, the resulting energy can be transferred to nearby GFP, shifting the natural luminescence of the organism from blue to green through BRET. Reproduced with permission from [32]. Copyright 2010 Springer Nature.
Figure 3
Figure 3
Comparison between different BRET systems. (A) BRET1 with Rluc8 mutant of Rluc as BRET donor and enhanced yellow fluorescent protein (eYFP) as BRET acceptor. (B) BRET2 with Rluc8 coupled with the substrate DeepBlueC (DBC) as the BRET donor and a green fluorescent protein variant (GFP2) as the BRET acceptor. (C) BRET3 using Rluc8/mOrange BRET-pair with coelenterazine as the substrate. (D) NanoBRET uses Nluc with a novel coelenterazine derivative, furimazine, and a chloroalkane derivative of nonchloro TOM (NCT) dye, which covalently binds to a HALO tag fused to Nluc. Adapted from [19,36].
Figure 4
Figure 4
Extinction coefficient values of water and oxy- and deoxyhemoglobin across wavelengths of light used for imaging. Reprinted with permission from [44]. Copyright 2010, American Chemical Society.
Figure 5
Figure 5
MMP overexpression in tumors detected with activatable NIR probe. (A) Schematic of the activatable probe. A type-V collagen sequence containing repeating Gly-Pro-4-hydroxy-l-proline Gly-Pro-4-hydroxy-l-proline (GPO) triplets conjugated to the NIR dye LS276 forms a triple helical peptide (THP) structure, quenching the dye emission through homoFRET. In the presence of MMP-2 or MMP-9, the THP is fragmented through enzymatic cleavage, resulting in enhanced brightness from the NIR probe. Images taken 24 h after i.p. injection of (B) LS276-THP; (C) LS276-THP with the MMP inhibitor Ilomastat; or (D) commercially available MMPSense 680. (BD) The tumors are represented by an arrow and the kidney marked with the letter “K”. (E) Time-dependent evolution of NIR fluorescence shown by plotting the tumor contrast ratio, i.e., the ratio of the fluorescence intensity in the tumor and in a region of interest on the contralateral flank. (F) Plot of ex vivo fluorescence intensities by organ showing lower fluorescence intensity in the tumor when the inhibitor is present; other organs like the liver and kidney were not similarly impacted by the inhibitor. Fluorescence images were recorded at 830 ± 75 nm using excitation of 755 ± 35 nm for LS276 and 680 nm excitation and 720 nm emission for the MMPSense. Reprinted with permission from [110]. Copyright 2012 American Chemical Society.
Figure 6
Figure 6
In vivo monitoring of inflammation-associated NO concentration. (A) Schematic of NO sensor. (B) Left foot treated with Freund’s adjuvant to induce inflammation; right foot is the inflammation-free control. (C) Comparison of the relative fluorescent intensity in the NO-rich inflamed foot and normal foot after intravenous injection of the probe. An 8-fold higher fluorescence intensity was observed in the inflamed area compared to the control area within 10 min post injection. Fluorescence signals were collected at 600 nm after 470 nm excitation. Reprinted from [114]. Distributed under a Creative Commons Attribution 3.0 Unported License (CC BY 3.0).
Figure 7
Figure 7
Using FRET to monitor the in vivo fate of NPs in the liver of mice following intravenous injection. (A) Self-assembly of SQGem, SQCy5.5 and SQCy7.5 to form a nanoparticle. Nanoparticle integrity verified by FRET by observing the transition from FRET NPs to disassembled NPs. (B) Donor channel signal collection at 697–770 nm. At 0.58 h, FRET NPs in this channel exhibited lower signal intensity than D-NPs, confirming FRET-based quenching. (C) FRET-induced acceptor emission collected between 810–875 nm. At 0.58 h, the high acceptor emission intensity indicates that the FRET NPs retained their integrity in the liver. Comparing (B,C) at 2 h post-injection, the increase in signal in the donor channel and a decrease in signal in the acceptor channel indicates disassembly of the NPs. The images demonstrate the timing of NP disassembly through the reduction in the FRET-induced acceptor emission intensity. Reproduced with permission from [87]. Copyright 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Figure 8
Figure 8
Fluorescence detection of miRNA-122 using a nucleic acid hybridization-based FRET construct with UCNPs. (A) Schematic of the hybridization assay. DNA-labeled UCNPs and DNA-labeled TAMRA are brought into close proximity upon binding to distinct complimentary regions of miRNA-122. Following excitation at 980 nm, the upconversion emission of the UCNP at 545 nm is transferred to the nearby TAMRA dye via FRET, reducing the emission at 545 nm and inducing sensitized emission at 580 nm. (B) Nude mice with human liver cancer HepG2 cells with subcutaneous injection of (a) 100 µL PBS; (b) 50 µL of 20 mg/mL captureDNA-UCNPs + 50 µL of 5 µM DNA-TAMRA; (c) 50 µL of captureDNA-UCNPs + 30 µL of 5µM miRNA-122; (d) 50 µL of captureDNA-UCNPs + 50 µL of DNA-TAMRA + 5 µL of 5 µM miR-122; same concentrations of probe + (e) 7.5 µL miRNA-122 (f) 15 µL miRNA-122 (g) 30µL miRNA-122. (C) Healthy livers of nude mice after tail vein injection of 200 µL of PBS; captureDNA-UCNPs + 100 µL PBS; and 100 µL of 20 mg/mL captureDNA-UCNPs + 100 µL of captureDNA-TAMRA. Adapted with permission from [88]. Copyright 2018, American Chemical Society.
Figure 9
Figure 9
Detection of MeHg+ accumulated in mouse livers in vivo using hCy7-UCNPs. (A) Schematic of the hCy7-UCNP probe. UCNPs decorated with hCy7 exhibit quenching of their emission at 660 nm; exposure to MeHg+ converts hCy7 to hCy7’, which quenches the UCNP emission at 800 nm instead. (B) Titration of MeHg+ to hCy7 reduces the absorbance peak at 670 nm while enhancing absorbance centered at 845 nm. In this titration, the MeHg+ concentration was titrated from 0 to 56 µM. The colored bands indicate overlap with the emission peaks from the UCNPs. (C) Upconversion emission of the UCNPs (black) is present in three major bands around 540 nm (from Er3+), 660 nm (from Er3+), and 800 nm (from Tm3+). In the absence MeHg+, the peak at 660 nm is quenched by hCy7 (blue). In the presence of MeHg+, the peak at 800 nm is quenched by hCy7’ (red). (D) In vivo imaging of hCy7-UCNPs. Top: Mice were IV injected with 40 µg of the hCy7-UCNP probes followed by IV injection of 200 µL 0.1 mM MeHg+ solution (right mouse) or saline solution (left mouse). Luminescent images recorded at 800 ± 12 nm after 980 nm excitation indicate 50% quenching of the upconversion luminescence (UCL) at 800 nm observed for the mouse treated with MeHg+. Bottom: Ex vivo images of the liver confirming accumulation and quenching of the probe. Adapted with permission from [131]. Copyright 2013, American Chemical Society.
Figure 10
Figure 10
In vivo comparison of Antares and FLuc BRET emission intensities using the substrates furimazine and luciferin, respectively. (A) Antares results in a higher BRET emission when intravenously injected with 0.33 µmol furimazine (FRZ) as compared to FLuc when injected i.v. or i.p. with 9.4 µmol of luciferin. (B) Normalized emission from Antares and FLuc from multiple mice; n = 5 for Antares; n = 6 for FLuc i.v. and n = 16 for FLuc i.p. Adapted with permission from [141]. Copyright 2016, Springer Nature.
Figure 11
Figure 11
BRET sensor for protein–protein interactions in the lungs of mice. (A) Schematic of the BRET system. The proteins FRB and FKBP12 form a heterodimer upon addition of the small molecule rapamycin. As the FRB and FKB12 used are chimeras with the luciferase RLuc8.6 and the fluorescent protein Turbo-FP635, respectively, the dimerization brings the BRET donor and acceptor into close proximity, facilitating energy transfer. (B) Dose–response curve of 1 × 105 HT1080 cells expressing the BRET-CID system 6 h after addition of rapamycin with an estimated EC50 of 0.7 ± 0.2 nM. (C) Influence of the inhibitor FK506 on the BRET ratios demonstrating the specificity of the BRET6 sensor. (D) Bioluminescence images of control (top) and rapamycin-dosed mice (bottom) after CLZ injection using open, donor, and acceptor filters. (E) Comparison of the BRET-ratios of control groups, which include the BRET donor only (FRB-RLuc8.6) and the donor–acceptor pair in the absence of the CID-inducer (BRET6 FRB/FKBP12 sensor, DMSO), with the BRET donor–acceptor pair in the presence of the CID-inducer (BRET6 FRB/FKBP12 sensor, rapamycin) [146]. Copyright 2011 Dragulescu-Andrasi et al.
Figure 12
Figure 12
In vivo BRET-based imaging of labeled C6 glioma cells in mice. (A) Schematic of a QD covalently coupled to the BRET donor, RLuc8. The bioluminescence energy of RLuc8-catalyzed oxidation of coelenterazine is transferred to the quantum dots, resulting in QD emission. (B) Absorption and emission spectra of QD655 (λex = 480 nm) and spectrum of the bioluminescent light emitted in the oxidation of coelenterazine catalyzed by RLuc8. (C) Bioluminescence emission spectrum of QD655-Luc8 in mouse serum and in mouse whole blood. (DF) Bioluminescence and fluorescence imaging of QD655-Luc8 and Luc8 injected subcutaneously (I and II) and intramuscularly (III and IV) at indicated sites in a mouse (I and III, QD655-Luc8, 5 pmol; II and IV, Luc8, 30 pmol). (D) Bioluminescence image taken without filters. (E) Bioluminescence image taken with 575-650-nm filter. (F) Fluorescence imaging of the same mouse using 503–555 nm excitation filter. Adapted by permission from [26]. Copyright 2006, Springer Nature.
Figure 13
Figure 13
In vivo demonstration of BRET-mediated photodynamic therapy (PDT) using PLGA nanoparticles loaded with rose bengal (RB) and FLuc. (A) Tumor growth over time when treated with the PLGA NPs. Only the pink and green traces show data from animals dosed with all the elements necessary for PDT. The pink group was exposed to external excitation light as a more traditional approach to PDT, while BRET-mediated local excitation of the construct was used for PDT in the green group. (B) Tumor volume on day 0 and day 14 after PDT treatment. (C) Body weight change after intratumoral injections of the NPs showed no significant change, suggesting low toxicity of the PDT treatment. (D) Excised tumors from the five treated groups on day 14. Reprinted with permission from [157]. Copyright 2018, American Chemical Society.
Figure 14
Figure 14
BLI, CRET and micro-computed tomography of the presence of MPO activity in MDA-MB-231-luc2 tumor metastases. (a) BLI of the tumor metastases 3 weeks after intracardiac injection of tumor cells. (bd) Micro-CT imaging showing osteolytic lesions indicated by the red arrows at the metastasis tumor sites. (e) MPO activity in these lesions analyzed with Luminol-R. (fi) Immunohistological analysis. (f,g) Tumor sites and (g,i) healthy tissues from a control mouse from a similar location as the metastases. (f,h) correspond to the staining of neutrophils and (g,i) to the staining of MPO. (j) Recording of 3-D CLI and micro-CT imaging showing the metastatic lesions in an anatomical configuration. Tumor depths measured with the reconstruction algorithm DLIT. Reprinted with permission from [159]. Copyright 2013, Springer Nature.
Figure 15
Figure 15
Hybrid nanoparticles (HNPs) with a conjugated luminol derivative (L012) produce NIR QD emission in response to reactive oxygen species in vivo. (A) Schematic representation of HNPs used for the detection of overproduced H2O2 in various diseases states. CL signals generated by CRET in presence of hydrogen peroxide in mice bearing (B) tumor, (C) acute inflammation, and (D) arthritis. CL signal was emitted from the HNPs in all three disease models, each of which involves the overproduction of H2O2. CL was not observed form either L012 or QDs alone. (EG) Quantification of the CRET signal intensities. Higher CRET signals were obtained from the late stage inflammation model (D,G) than the early stage inflammation model (C,F), suggesting higher and long lasting ROS in the arthritis model. Adapted with permission from [161]. Copyright 2016, The Royal Society of Chemistry.
Figure 16
Figure 16
Semiconducting polymer nanoparticles emitting in the NIR (SPN-NIR) developed for CRET-based imaging of H2O2. (a) Schematic of the SPN-NIR. The polyfluorene-based semiconducting polymer PFPV, chemiluminescent substrate TCPO, and naphthalocyanine dye NIR775 were co-precipitated with the amphiphilic triblock copolymer PEG-b-PPG-b-PEG to create SPN-NIR particles with hydrodynamic diameters of 15–25 nm. (b) Chemiluminescence and fluorescence spectra of SPN-NIR. Chemiluminescence was induced by the addition of excessive H2O2 (10 mM). (c) Representative chemiluminescence and fluorescence images of SPNs (18 μg/mL, 0.1 mL) in the presence of H2O2 (10 mM). The fluorescence signals were detected at 520 or 780 nm; the chemiluminescence signals were detected with open filter or at 780 nm. (d) In vivo imaging of exogenous H2O2 using SPN-NIR. Representative chemiluminescence image of mouse with the subcutaneous implantation of (1) H2O2 (8 nM) + SPN-NIR (0.1 mg/mL, 0.1 mL) and (2) SPN-NIR (0.1 mg/mL, 0.1 mL). (e) In vivo chemiluminescence intensities of the subcutaneous inclusion of SPN-NIR as a function of the concentration of H2O2. Values are the mean ± s.d. for n = 3 mice. Reprinted with permission from [162]. Copyright 2016, American Chemical Society.
Figure 17
Figure 17
Combined CRET/FRET sensor for in vivo detection of drug-induced hepatotoxicity. (A) Molecular components of CF-SPN are the NIR fluorescent semiconducting polymer PFODBT, a PEG-grafted poly(styrene) copolymer conjugated to galactose for hepatocyte targeting (PS-g-PEG-Gal), the H2O2-specific chemiluminescent substrate CPPO that serves as CRET energy donor, and the FRET acceptor IR775S that degrades after oxidation by ONOO or OCl (dark green). PFODBT serves as the CRET energy acceptor and the FRET energy donor. (B) Illustration of the mechanism of simultaneous and differential detection of ONOO or OCl and H2O2 by CF-SPN. After drug challenge to the liver, CF-SPN report via the chemiluminescent and fluorescent channels the generation of radical metabolites at safe (left) and toxic (right) drug doses. (C) Hydrodynamic diameter distribution of CF-SPN, determined by dynamic light scattering. (D) Transmission electron micrograph of CF-SPNs. Adapted with permission from [163]. Copyright 2014, Springer Nature.

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