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WIREs Nanomed Nanobiotechnol
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Cell‐surface sensors: lighting the cellular environment

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Abstract Cell‐surface sensors are powerful tools to elucidate cell functions including cell signaling, metabolism, and cell‐to‐cell communication. These sensors not only facilitate our understanding in basic biology but also advance the development of effective therapeutics and diagnostics. While genetically encoded fluorescent protein/peptide sensors have been most popular, emerging cell surface sensor systems including polymer‐, nanoparticle‐, and nucleic acid aptamer‐based sensors have largely expanded our toolkits to interrogate complex cellular signaling and micro‐ or nano‐environments. In particular, cell‐surface sensors that interrogate in vivo cellular microenvironments represent an emerging trend in the development of next generation tools which biologists may routinely apply to elucidate cell biology in vivo and to develop new therapeutics and diagnostics. This review focuses on the most recent development in areas of cell‐surface sensors. We will first discuss some recently reported genetically encoded sensors that were used for monitoring cellular metabolites, proteins, and neurotransmitters. We will then focus on the emerging cell surface sensor systems with emphasis on the use of DNA aptamer sensors for probing cell signaling and cell‐to‐cell communication. WIREs Nanomed Nanobiotechnol 2012, 4:547–561. doi: 10.1002/wnan.1179 This article is categorized under: Diagnostic Tools > Biosensing Therapeutic Approaches and Drug Discovery > Emerging Technologies Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

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Semisynthetic Snifit sensors for monitoring metabolites on cell surface. (a) Design of the sensor and fluorescent signal generation on the cell surface upon interacting with the target. The protein part of the sensor is a fusion protein of SNAP‐tag, CLIP‐tag, and a binding protein (BP, shown as gray). The active semisynthetic sensor protein is obtained by labeling SNAP‐tag with a PEG molecule containing a fluorophore (red rectangle) and a ligand for the binding protein (purple ball) and by labeling CLIP‐tag with a second fluorophore (green rectangle). In the absence of analyte, the intramolecular ligand binds to the binding protein, keeping the sensor protein in a closed conformation. Donor and acceptor fluorophores are in close proximity, resulting in a high FRET efficiency. In the presence of analyte, the intramolecular ligand is displaced, and the sensor protein shifts toward an open conformation. Donor and acceptor fluorophore are more distant from each other than in the closed conformation and FRET efficiency therefore decreases. (b) Kinetic and thermodynamic characterization of the sensor protein SNAP‐CLIP‐BP on the surface of HEK 293T cells. Time course of the perfusion of the labeled sensor with increasing amount of the metabolite, benzenesulfonamide (a = 1 µM, b = 10 µM, c = 50 µM, d = 100 µM, e = 500 µM, f = 1 mM, g = 10 mM). (c) Benzenesulfonamide titration curve of SNAP‐CLIP‐BP on the extracellular surface of HEK 293T cells.

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Glutamate nanosensor (FLIPE). (a) Schematic presentation of the sensor displayed on the cell surface using transmembrane spanning domain of the platelet‐derived growth factor (PDGF). (b) FLIPE construct consisting of a secretion signal at the N‐terminus of FLIPE and a C‐terminal fusion with the PDGF membrane span. (c) Confocal images of hippocampal neurons expressing FLIPE‐600n. (d) A hippocampal cell stained with FM4–64. Synapses stained in red. (e) and (f) FRET change at the surface of hippocampal neurons expressing FLIPE in response to electrical stimulation. (e) Ratiometric images and (f) corresponding quantification of a hippocampal neuron expressing FLIPE after electrical stimulation or perfusion with varying concentrations of glutamate. The presence of glutamate caused a decrease of FRET signal.

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Schematic illustration of representative approaches of cell surface sensor installation. (a) Genetic approach: DNA that can encode a desired protein is inserted into a plasmid DNA and introduced into the cell which is expressed by the cell and transported onto the cell. (b) Covalent coupling: There are two approaches for covalent coupling. In the first one, a biotin is covalently attached onto the cell surface protein to their amine groups using NHS (N‐hydroxysuccinimide)‐biotin followed by coupling with avidin/streptavidin. The surface is then labeled with biotinylated exogenous molecules of interest (i.e., aptamer). In the second one, the surface protein molecules on the cell surface are directly conjugated to NHS‐activated sensing moieties. (c) Hydrophobic interaction: A fatty acid (palmitoyl derivatives)‐conjugated sensing moiety is mixed with cells. The hydrophobic fatty acid tail is incorporated into the cell membrane displaying the sensor part outside the cell membrane. (d) Affinity labeling: Specific ligands such as antibody or aptamer can recognize their targets on the cell surface. The ligand can be pre‐conjugated to other materials such as nanoparticles and enzymes. (e) Enzymatic ligation: Acceptor peptide (AP)‐tagged recombinant protein on the cell surface can be enzymatically biotinylated using special enzyme such as biotin ligase. The biotins are available for interacting with streptavidin modified ligands or any desired species. (f) Metabolic labeling: A functional sugar derivative such as fucose‐azide (FucAz) or keto‐sugar is introduced into cell culture media. The cells use the sugar moieties in their metabolic pathway to be tagged into the glycoproteins and finally transported onto the cell membrane. The functional groups are now available for coupling with other biomolecules of interest.

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Schematic representation of the commonly used cell surface sensor designs. (a) Fluorescent proteins undergo conformational changes upon binding to a specific target inducing FRET.3–5,8 (b) Fluorescence signaling cell surface sensor based on cleavage of the substrate by specific enzymes.9 (c) FRET sensor to monitor cell surface receptors aggregation.10 (d) Luciferase enzyme sensor for ATP monitoring on cell surface.11 Luciferase on the cell surface is activated by ATP which is excreted by cells and cleaves a substrate producing luminescence. (e) Polymer‐based colorimetric cell surface sensor to monitor cell surface perturbation that undergoes color transition.12 (f) Gold nanoparticles with unique surface plasmon resonance properties as probes for monitoring cell surface receptor aggregation.13 (g) FRET‐based sensor using quantum dots to monitor cell surface markers.14 (h) Aptamer sensors on cell surface using FRET mechanism for monitoring cell signaling and cellular microenvironment.7

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Performance of the MSC‐aptamer sensor in living cells and animal. (a) Representative fluorescent microscope images of the MSC‐aptamer sensor before and after adding PDGF. (b) Signal generated by MSC‐aptamer sensor at different concentration of PDGF (signals were defined as the ratio of geometric means (GM) before and after the addition of PDGF). (c) Real‐time sensing of PDGF secretion from neighboring MDA‐MB‐231 cells by sensor‐engineered MSCs. Left panel: Microwells containing different numbers of PDGF‐producing MDA‐MB‐231 cells (green) including sensor‐MSC (red) at time t = 0 (n is the number of MSCs used in the analysis). In this set of experiments, the aptamer was labelled with a red dye (Cy5), and with Iowa Black RQ as a quencher. Right panel: fluorescence of MSC‐aptamer declining during the course of PDGF production. The signal, which is defined as the percentage of MSCs that have fluorescence intensity less than 50% of their initial value at the indicated time, correlates with the number of PDGF‐producing MDA‐MB‐231 cells in the same well as a sensor‐MSC. (d) Bone marrow homing and transmigration of aptamer‐labeled MSCs. (d‐I) Large‐area map of right parietal bone marrow compartments in eight week‐old Balb/c mouse 24 h after injection of native MSC and aptamer‐MSC. Several image stacks were acquired in the right parietal bone ∼200 µm to the right of the sagittal suture. (d‐II) Zoomed‐in image of the area in the white box in d‐I, shows a similar distribution of MSC (green) and aptamer‐MSC (blue) in the vicinity of a large venule (red). (d‐III) Quantification of the average number of cells per z‐stack. (d‐IV) Quantification of percentage of cells positioned outside blood vessels shows no difference between MSC and aptamer‐MSC (P‐value = 0.116). MSC, green; aptamer‐MSC, blue; blood vessels, red; bone, white. Scale bar, 100 µm.

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Cell surface aptamer sensors. (a) Schematic representation of the concept of probing cellular niche environment and signaling using cells engineered with cell surface aptamer sensors. PDGF aptamer sensors immobilized on mesenchymal stem cells (MSCs) (deep yellow indicated as sensor‐cells) recognize signaling molecules (PDGF in this case) secreted by niche cells sitting on the extracellular matrix and produces fluorescent signal. (b) Sequence and design of the PDGF aptamer sensor with fluorophore and quencher pair. 5′‐end of the apatmer sensor is labeled with fluorophore (FAM). The extended 3′‐termini is hybridized with a short capture oligonucleotide which is labeled with biotin for immobilization on the MSCs through streptavidin–biotin method as shown in (d). (c) The solution performance of quenching ability of the aptamer sensor upon addition of PDGF.

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Cell surface sensor for monitoring phospholipid expression on the cell membrane. (a) Design of the sensor and FRET signaling properties after binding to the targets. The sensor is composed of PH (pleckstrin homology) domain that binds to PtdlnsP3 and two FPs CFP and YFP including a rigid helix (EAAAR)n and a gly–gly hinge. The sensor is displayed on the cell surface using MLS (membrane localization sequence). (b) Diagram of the sequence design indicating different domains including the linkers. (c) Subcellular localization of the sensor (fllip‐pm) in CHO cells. The YFP images are shown in green. V, vertical sections. H, horizontal. (d) Color images of the CFP:YFP ratio at different time points (0, 100, 300, and 500 seconds) after addition of 50 ng/mL PDGF.

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