Fluorescent Nanosensors Based on Fluorescence Resonance Energy

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Fluorescent Nanosensors Based on Fluorescence Resonance Energy Transfer (FRET) Gengwen Chen, Fengling Song,* Xiaoqing Xiong, and Xiaojun Peng* State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Hi-tech Zone, Dalian 116024, People’s Republic of China ABSTRACT: Fluorescence resonance energy transfer (FRET) has been widely used as a spectroscopic technique in various areas such as structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting, and metallic nanomaterials. Meanwhile, based on the mechanism of FRET, a series of FRET nanomaterials systems have been recently developed as novel chemical sensors and biosensors. Compared with those based on small molecules traditional FRET systems, the surface chemistry of nanomaterial has encouraged the development of multiple probes based on linked recognition molecules such as peptides, nucleic acids, or smallmolecule ligands. This critical review highlights the design and the applications of sensitive and selective ratiometric nanoprobes based on FRET. We focus on the benefits and limitations of nano-FRET systems and their applications as chemical sensors and biosensors. will not affect the ratio between the two fluorescence intensities.4 Conventionally, the FRET-based sensing systems have been designed in the form of small-molecular dyads, which contain two fluorophores connected by a spacer through covalent links. However, such fluorescent molecular sensors are difficult to be prepared and still suffer from photobleaching and interference.5 The versatile composition and architecture of nanoparticles enables the preparation of ratiometric nanosensors that overcome these problems. Recently, FRET mechanism has been used for fabrication of nanosensors. A series of fluorescence nanoparticles were reported, such as gold nanoparticles, quantum dots, dye-doped silica nanoparticles, rare-earth upconversion nanophosphors, carbon nanodots, dye labeled microspheres, and polymers. This tutorial review will classify nanosensors based on nanoparticle types and elucidate the design and application of fluorescent nanosensors based on FRET in detecting DNA, metal ions, small molecular, and other analytes.

1. INTRODUCTION Fluorescent labels have been widely used for biological applications, primarily in imaging and assays. Traditional fluorophores such as fluorescent dyes have several drawbacks such as photobleaching and high sensitivity to environmental factors. In the era of nanotechnology, fluorescent nanomaterials are emerged as an integrated research field because they offer superior optical properties, such as brighter fluorescence, wider selections of excitation and emission wavelengths, higher photostability, etc. Their size- or shape-controllable optical characteristics also facilitate the selection of diverse probes for higher assay throughput. Furthermore, the nanostructure can provide a solid support for sensing assays with multiple probe molecules attached to each nanostructure, simplifying assay design and increasing the labeling ratio for higher sensitivity.1 Fluorescence resonance energy transfer (FRET) is a nonradiative process whereby an excited state donor D (usually a fluorophore) transfers energy to a proximal ground state acceptor A through long-range dipole−dipole interactions.2 The acceptor must absorb energy at the emission wavelength(s) of the donor but does not necessarily have to remit the energy fluorescently itself. The rate of energy transfer is highly dependent on many factors, such as the extent of spectral overlap, the relative orientation of the transition dipoles, and, most importantly, the distance between the donor and acceptor molecules.3 FRET usually occurs over distances comparable to the dimensions of most biological macromolecules, that is, about 10 to 100 Å. FRET is very appealing for bioanalysis because of its simpleness of building ratiometric fluorescent systems. Unlike those one-signal sensors, the ratiometric sensors contain two different chromophores and use the ratio of the two fluorescence intensities to quantitatively detect the analytes, and they can eliminate most ambiguities in the detection by self-calibration of two emission bands. Those external factors, such as excitation source fluctuations and sensor concentration, © XXXX American Chemical Society

2. GOLD NANOPARTICLE 2.1. Advantages of Gold Nanoparticle Based FRET. The scientific synthesis of colloidal gold can date back to Michael Faraday’s work in 1857, in which the gold hydrosols were prepared by reduction of an aqueous solution of chloroaurate with phosphorus dissolved in carbon disulfide.6 During the last two decades, considerable effort has been devoted to synthesis of gold nanoparticles (AuNPs), focusing on controlling their size, shape, solubility, stability, and Special Issue: Multiscale Structures and Systems in Process Engineering Received: December 18, 2012 Revised: January 22, 2013 Accepted: January 24, 2013

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dx.doi.org/10.1021/ie303485n | Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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functionality. It is worth noting that the term colloid and cluster are frequently and interchangeably used; the former generally refers to particles with diameters more than 10 nm, while the latter commonly with smaller particles.7 AuNPs have many unique properties such as colorimetry, conductivity, and nonlinear optical properties that make them excellent scaffolds for the fabrication of novel chemical and biological sensors.8 First, AuNPs can be synthesized in a straightforward manner and can be made highly stable. Second, they possess unique optoelectronic properties. Third, these properties of AuNPs can be readily tuned by varying their size, shape, and the surrounding chemical environment. Finally, AuNPs offer a suitable platform with a wide range of organic or biological fluorescent ligands for the selective binding and detection of small molecules and biological targets. So, it is also a suitable platform for FRET system. Although FRET technology is very convenient and can be applied routinely at the single molecule detection limit, the efficiency of FRET is very sensitive to the distance between the donor and an acceptor. The length scale for the detection based on FRET is limited by the nature of the dipole−dipole mechanism, which effectively constrains the length scales to distances on the order of 12-fold change between pH 6 and 8. The modularity of the sensor enables customization to specific biological applications through genetic engineering of the FPs, as illustrated by the altered pH range of the sensor through mutagenesis of the fluorescent protein. The QD-FP sensors facilitate visualization of the acidification of endosomes in living cells following polyarginine-mediated uptake. Another approach is based on FRET between CdSe/ZnS QDs encapsulated within an amphiphilic polymer and a pHsensitive squaraine dye conjugated on the cap surface. As observed for the free dye in homogeneous solution, the dye absorption band is suppressed under basic conditions. Consequently, energy transfer from the nanocrystal (NC) to the dye is inefficient, and the emission spectrum is dominated by the NC at 613 nm. As pH is lowered, the absorption cross section of the dye is increased, and FRET from the NC to the dye becomes more efficient; emission from the NC−dye conjugate is now dominated by that of the dye at 650 nm (Figure 7).30 3.2. Heavy Metal Ion Sensors. Sensing of ions via analyteinduced changes in photoluminescence of QDs based on FRET is a very active research field.31 Li and co-workers developed a FRET-based system. TGA (thioglycolic acid)-CdTe QDs and the acceptor butyl-rhodamine B were introduced in the presence of a surfactant (cetyltrimethylammonium bromide) to increase FRET efficiency.32 Upon addition of Hg2+, the fluorescence of both donor and acceptor was quenched. Since E



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form a compact hybrid assembly through amine−carboxyl attractive interaction, which leads to a high-efficiency (>92%) FRET from QDs to AuNRs and an almost complete emission quenching. Added TNT molecules break the preformed assembly because they can replace the QDs around AuNRs, based on the specific reaction of forming Meisenheimer complexes between TNT and primary amines. Thus, the FRET is switched off, and a more than 10 times fluorescent enhancement is obtained (Figure 9). The fluorescence turn-on

Figure 11. Schematic representation of the method for Maltose determination based on FRET.

displaces the β-CD−QSY9 conjugate to restore NC emission. In their second strategy, the CdSe/ZnS−MBP construct is labeled with two different cyanine dyes: Cy3, which is bound directly to the MBP, and a β-CD conjugated to a Cy3.5, docked to the MBP binding site.37 Prior to maltose binding, Cy3.5 emission prevails by a two-steps energy transfer from Cy3, which in turn accepts energy from CdSe. Upon binding to maltose, the β-CD−Cy3.5 conjugate is displaced and, in the absence of the terminal acceptor, Cy3 is the predominant emitting species. 3.3. Detection of Biomolecules. DNA hybridization and cleavage processes have also been studied. The hybridization was monitored by following FRET between QDs and a molecular fluorophore,38 whereas treatment of the QD/dyeDNA structure with deoxyribonuclease (DNase I) cleaved the DNA duplex and restored the fluorescence properties.39 The dynamics of DNA replication and telomerisation have also been reported. CdSe/ZnS QDs were modified with the thiolated oligonucleotide and later incubated with a mixture of deoxyribonucleotides with an organic fluorescent dye in the presence of telomerase. The fluorescence emission of the QDs decreased as telomerisation proceeded, with the concomitant increase of the characteristic emission of the fluorophore by FRET from the QDs to the dye molecules incorporated into the telomeric units by telomerase. Using a similar procedure, but in the presence of a polymerase, the replication process could be followed by FRET from the QDs to the incorporated dye unit and allowed the detection of a viral DNA. Unlike these reports, there are also FRET-based DNA hybridization sensors that do not require covalent immobilization of the sensor molecules, minimizing in this way DNA modification. Water-soluble CdTe QDs were prepared, and this negatively charged QDs were further incorporated in a cationic polymer solution of poly(diallyldimethylammonium) as the electrostatic linker. The resultant positively charged QDs acted as FRET donors to dye acceptor-labeled ssDNA. The hybridization recognition was based on the interaction of ssDNA and dsDNA with the functionalized QDs leading to differential changes of FRET efficiency (Figure 12).40

Figure 9. Schematic representation of the method for TNT determination based on FRET between QDs and AuNPs.

is immediate, and the limit of detection for TNT is as low as 0.1 nM. Importantly, TNT can be well distinguished from its analogues due to their electron deficiency difference. The developed method is successfully applied to TNT sensing in real environmental samples. A competitive QD-based assay for the detection of the explosive trinitrotoluene (TNT) has also been developed. CdSe/ZnS QDs were functionalized with a single-chain antibody fragment that selectively binds TNT.35 The analogous substrate trinitrobenzene (TNB) was covalently linked to the quencher dye BlackHole Quencher-10 (BHQ10) and was bound to the QD/antibody conjugate; the associated substrate quenched the fluorescence of the QDs. In the presence of the TNT analyte, the quencher TNB-BHQ10 conjugate was competitively displaced. This eliminated the FRET process between the QD and the dye and switched on the fluorescence of the QDs (Figure 10).

Figure 10. Schematic representation of the method for TNT determination based on FRET.

Another method for FRET-based biosensing involves the structural changes of proteins upon interaction with their substrates.36 This method was used for the assembly of a reagent-free QD-based sensor for maltose. Mattoussi and coworkers have developed a sensor for maltose by adapting their CdSe−MBP (maltosebinding protein) conjugates for both analyte-displacement strategies depicted by Figure 11.37 In the first construct, a β-cyclodextrin (β-CD) conjugated to a nonfluorescent QSY9 quencher dye was docked to the MBP saccharide binding site of the CdSe/ZnS−MBP. Maltose

Figure 12. Principle of DNA hybridization-detection system based on the QD/Cy3-labeled DNA FRET. F



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with the QDs lack absorbance in the visible spectral region and thus do not quench the QDs. As a result, the reduction of the capping layer by the NAD(P)H cofactors activates the fluorescence of the QDs and provides a path for the optical detection of NADH (Figure 15). 3.3. Detection of Proteins and Enzymes. The first report on the conjugation of protein molecules to luminescent CdSe/ ZnS QDs arose from Mattoussi’s group. Many of the possible applications of peptide capped-QDs include monitoring alterations of enzymatic activities, which can be related to important biological processes and to diseases. The hydrolytic functions of a series of proteolytic enzymes were analyzed by the application of QD-modified dye-labeled peptides that acted as reporter units for the biocatalytic transformations and used the FRET process as a readout mechanism. CdSe QDs were modified with different peptides that included peptide sequences with specific cleavage sites for different proteases, and quencher units were tethered to the termini of the peptides37,44 (Figure 16). The fluorescence of the QDs was quenched in the presence of the quencher−peptide capping layer. The sequence-selective hydrolytic cleavage of the peptides by the respective protease resulted in the removal of the quencher units, and this restored the fluorescence of the QDs. For example, collagenase was used to cleave the rhodamine Red-X dye-labeled peptide linked to CdSe/ZnS QDs.45 Whereas the tethered dye quenched the fluorescence of the QD, the hydrolytic scission of the dye and its corresponding removal restored the fluorescence of the QDs. In a further example, Prasuhn et al. functionalized QDs with dye-labeled peptides using two different linkage chemistries to yield fluorescence resonance energy transfer (FRET)-based sensors capable of monitoring either enzymatic activity or ionic presence.46 The first sensor targets the proteolytic activity of caspase 3, a key downstream effector of apoptosis. This QD conjugate utilized carbodiimide chemistry to covalently link dye-labeled peptide substrates to the terminal carboxyl groups on the QD’s surface hydrophilic ligands in a quantitative manner. Caspase 3 cleaved the peptide substrate and disrupted QD donor-dye acceptor FRET providing signal transduction of enzymatic activity and allowing derivation of relevant Michaelis−Menten kinetic descriptors. The second sensor was designed to monitor Ca2+ ions that are ubiquitous in many biological processes. For this sensor, Cu+-catalyzed azide−alkyne cycloaddition was exploited to attach a recently developed azide-functionalized CalciumRuby-Cl indicator dye to a cognate alkyne group present on the terminus of a modified peptide. The labeled peptide also expressed a polyhistidine sequence, which facilitated its subsequent metalaffinity coordination to the QD surface establishing the final FRET sensing construct. Adding exogenous Ca2+ to the sensor

A FRET mechanism between QDs and organic dyes has been successfully employed in RNA-peptide interaction studies.41 The specific interaction between biotinylated RNA and the cyanine dye-labeled peptide of interest originated a complex that was then assembled with streptavidin-QDs in a way that QDs functioned both as nanoscaffold and FRET donor (Figure 13). The hybrid QD-DNA-dye molecular

Figure 13. Conceptual scheme of the QD-based nanosensor for Rev− RRE interaction assay based on FRET between 605QD and Cy5.

beacon was attained using a thiol-reactive hexahistidine peptidic linker that was chemically attached to thiolated-DNA oligomers (Figure 14).42 An efficient FRET between the QD-donor and the dye-acceptor was based on a labeled hairpin DNA stem structure bringing both in close proximity. This distance was altered and FRET efficiency disrupted in the presence of complementary DNA by unwinding the stem-loop structure so that specific and nonspecific target DNA could be discriminated. The binding of a Cy5-labeled Rev to a biotinylated RRE IIB RNA formed a Rev−RRE complex, which was caught on the surface of a 605QD to form a 605QD/Rev−RRE/Cy5 assembly through specific streptavidin−biotin binding. FRET occurred between the 605QD and Cy5 upon illumination of the 605QD/Rev−RRE/Cy5 assemblies with an excitation wavelength of 488 nm. (Rev is an important HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the enV gene of HIV-1 RNA genome.) Freeman et al. built a novel FRET system to sensor intracellular metabolic pathways.43 In the system CdSe/ZnS, quantum dots was functionalized by Nile-blue as a hybrid material to biosense 1,4-dihydronicotinamide adenine dinucleotide (phosphate) cofactors, NAD(P)H. Nile blue was covalently linked to the BSA layer as an electron mediator for the oxidation of the NAD(P)H cofactors. The fluorescence of the QDs is quenched by Nile-blue through FRET quenching. In the presence of NADH, the reduced Nile-blue associated

Figure 14. Schematic depiction of His6−peptide-linker facilitated self-assembly of labeled DNA onto QDs (left) and establishment of a hybrid molecular beacon structure (right). G



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Figure 15. Sensing of NADH by Nile-blue-functionalized CdSe/ZnS QDs.

Nanoparticle-based labels are emerging as simpler and more sensitive alternatives to traditional fluorescent small molecules and radioactive reporters in biomarker assays. Stuart B. Lowe and co-workers have designed a multicomponent, solutionbased assay with sensitivity to both protease and kinase activities.47 Enzyme specificity was realized by synthesizing peptides with amino acid recognition sequences based on enzyme substrate interaction studies. Activity determination is reliant on self-assembly of the peptides to specific motifs on the QD surface. Signal independence for each target was achieved by using two QD populations with different emission wavelengths and orthogonal surface functionalizations. In contrast with other QD-based activity assays, the system is able to multiplex more than one family of enzymes. Protein kinases are an important family of enzymes that phosphorylate serine, threonine, or tyrosine side chains and are critical in cell signaling and cancer biology, but their biomedical

Figure 16. Principle of quantum dots-based enzymatic activity sensors.

solution increased the dyes fluorescence, altering the donor− acceptor emission ratio and manifesting a dissociation constant similar to that of the native dye (Figure 17). These results highlight the potential for combining peptides with QDs using different chemistries to create sensors for monitoring chemical compounds and biological processes.

Figure 17. Schematic showing FRET-based sensing for Ca2+ sensor. H



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tion and nonspecific binding on the silica’s surface have been observed and will need to be solved before the full potential of silica nanoparticles in biosensing can be realized. Studies have shown that a ratio of inert to active functional groups on the surfaces of silica nanoparticles that results in a high ζ-potential is critical to maintaining a well-dispersed nanoparticle suspension and reducing nonspecific binding.1a Silica-based nanoparticles/nanobeads are very useful in bioanalysis once conjugated with biological entities (such as DNA or antibodies) for analyte recognition and/or signal generation.51 The surface of such nanoparticles must maintain a stable store of bioligands, which are fixed/conjugated by biochemical coupling reactions and which maintain biological activity in terms of the potential interaction between the recognition element and the target molecule. A variety of crosslinkage methods have been reported to synthesize biomolecule/dye-doped silica nanoparticles.1e,52 Commonly, the surface of the raw silica nanoparticle is terminated with silanol groups, which then act as active positions for subsequent functionalization. For nanoparticles prepared by the Stöber method, this is usually achieved by growing a stable additional silica shell that contains the required functional group for coupling to the required biomolecule and fluorescent sensors.53 More specifically, the fluorophores have been conjugated to NPs whose surface has been modified (Figure 19) with (a) amino groups, (b) azido groups, and (c) alkyne groups and other groups. So, it is a useful tool to build FRET systems between dyes in core and shell.

and pharmaceutical significance, their activity has been little explored using quantum dot technology. Molly M. Stevens demonstrated that self-assembled peptide quantum dot conjugates can serve as surrogate substrates in a simple homogeneous assay for protein kinase activity (Figure 18).48

Figure 18. Schematic representation of kinase-mediated phosphorylation of peptide−QD conjugates, antibody recognition of phosphopeptide, and FRET detection.

Enzymatic phosphorylation of the peptide conjugates is detected by means of a complementary FRET-acceptor labeled antiphosphotyrosine antibody, with formation of the immune complex resulting in energy transfer between the quantum dot and FRET acceptor molecules.

4. DYE-DOPED SILICA NANOPARTICLES Traditionally, silica nanoparticles have been prepared either by using a microemulsion mediated method or the sol gel process (i.e., Stö ber’s method). While in the Stö ber’s method, alkoxysilane compounds (e.g., tetraethylorthosilicate (TEOS), tetramethylorthosilicate (TMOS), and their derivatives etc.) undergo base-catalyzed hydrolysis and condensation reaction in ammonia−ethanol−water mixture, similar reaction can takes place in the nanocores of the microemulsion system.1b,49 In the case of dye-doped silica nanoparticles, the dye is encapsulated within a silica shell. Dye encapsulation is achieved by either covalent attachment of the dye with silica precursors (e.g., with 3-(amino-propyl) triethoxysilane, APTS) before the hydrolysis in StÖ ber’s method or by first solubilizing the dye in the core of the microemulsion and then carrying out the polymerization. This type of nanomaterial has the following advantages in biosensing: (1) silicon is abundant and nontoxic; (2) the high surface-to-volume ratio of the nanoparticles facilitates their binding to biomolecules; (3) the inclusion of a large number of fluorescent dye molecules inside each nanoparticle results in excellent photostability due to the ability of the silica matrix to shield from molecular oxygen, and the inclusion also dramatically increases the dye-to-biomolecule labeling ratio, leading to high signal amplification factors during detection; and (4) silica is relatively inert in chemical reactions but still allows surface modification with well established chemistries. Compared to QDs, fluorescent silica nanoparticles have a wider size range, spanning from a few to hundreds of nanometers; they require less strict size control and exhibit better water solubility.1b,50 However, problems related to particle aggrega-

Figure 19. Silica nanoparticles functionalized with different groups such as amine, car-boxy, aldehyde, epoxy, thiol, and heterobifunctional cross-linkers.

4.1. FRET Nanosensor Based on Dye-Doped Silica Nanoparticles. This system has been applied to detect TNT by utilizing the specific binding between TNT and amines.54 A strong charge transfer interaction between the electrondeficient aromatic ring of TNT and the electron-rich amino group coupled to the silica particle surface resulted in strong absorption at 520 nm and relatively weak absorption at 630 nm. Therefore, the TNT−amine complex could act as the energy acceptor for fluorescein 5(6)-isothiocyanate (FITC) conjugated in the proximity of the amine groups. The silica particles could be deposited into the microwells on a silicon wafer to form a convenient device for detecting trace TNT in solutions or air vapor (Figure 20). Similarly, a label-free DNA detection apparatus was constructed by conjugating the capture DNA sensor onto the surfaces of silica particles.55 Hybridization of the target DNA allowed intercalation of ethidium bromide (EB)the FRET acceptor and electrostatic binding of I



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Figure 20. Schematic illustration of the FRET-based silica nanoparticle sensors for TNT detection. Figure 22. Schematic representation of photodynamic therapy.

tetrahedralfluorenethe FRET donor. Distinguishable fluorescence signals from EB were observed from the perfect match strand and from strands with single- or two-base mutations (Figure 21). Prasad et al. reported the development of energy-transferring, organically modified silica nanoparticles for two-photon photodynamic therapy (PDT).56 In that study, two-photon fluorescent dye nanoaggregates were incorporated into the NPs as an energy up-converting donor, and a photosensitizing PDT drug, 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide, was used as an acceptor. Both species were codoped into the silica nanoparticles. FRET between these nanoaggregates and a photosensitizer is used to excite the photosensitizer indirectly via efficient two-photon excited intraparticle energy transfer from the dye aggregates in the intracellular environment of tumor cells. Consequently, the excited photosensitizer generates singlet oxygen, which in turn induces in vitro cytotoxic effects in the tumor cells. The fluorescence imaging of HeLa cells after the uptake of these particles clearly demonstrated morphological changes indicative of cell necrosis induced by the reactive oxygen species (Figure 22). Enrico Rampazzo and co-workers have recently developed a new one pot approach for the synthesis of core−shell dyedoped silica nanoparticles. Most interestingly, they have demonstrated the possibility of also hosting water-insoluble fluorescent sensors in the outer shell, which are able to give rise to very efficient energy transfer processes with the molecules hosted in the core. The system shown an enhanced detection capability for intracellular Cu+ either in the absence or at low concentrations of exogenous metal ion (Figure 23).57

Recently, Liu and co-workers reported a new strategy for constructing a FRET based ratiometric sensor for Hg2+ detection in water with multilayered silica NPs as the scaffolds.58 In this approach, a nitrobenzoxadiazolyl derivative (NBD) was covalently confined into a thin layer in the particles as the donor, and a spirolactam rhodamine derivative (SRhB) was covalently linked onto the particle surface as the mercury ion sensor. For this NP-based sensing system, the presence of Hg2+ can trigger an efficient ring-opening reaction of the spirolactam rhodamine, affording the system efficient FRETbased ratiometric detection for mercury ions in water (Figure 24). Photo-switchable fluorescent SiO2 nanoparticles have been synthesized recently.59 The fluorescent silica nanoparticles have been prepared by covalently incorporating a fluorophore (rhodamine B) and a photochromic compound (spiropyran) inside the particle core.59b The fluorescence can be switched reversibly between an on and off-state via energy transfer. Another FRET system was also build between a photochromic diarylethene and a rhodamine fluorescent dye (Figure 25). Lei and co-workers build a ratiometric pH sensor based on mesoporous silica nanoparticles and Forster resonance energy transfer.60 The pH sensitive dye (fluorescein isothiocyanate) and reference dye (rhodamine B isothiocyanate) composed the FRET pair and were covalently anchored into the mesoporous pore channel in order to prevent leaching during optical pH measurements. The sensitivity and pH sensing-range of the dye-doped mesoporous silica nanoparticles can be varied by

Figure 21. Schematic illustration of the label-free SNP DNA detection strategy. J



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Figure 23. Schematic representation of the proposed transduction mechanism.

5. DYE LABELED MICROSPHERES AND POLYMERS Fluorescent polymers are a related class of fluorophores that can be either intrinsically fluorescent or functionalized with multiple fluorophores.63 Disadvantages for their use as fluorescent labels for bioconjugation are their size and polydispersity.64 The emission of fluorescent polymers is not localized, since energy transfer occurs along the whole chain, and thus, the emission is diffuse. Polymers therefore cannot be used as point donors in FRET systems. However, fluorescent polymers are also provided with a variety of surface functionalities (e.g., biotin, avidin, collagen, amines, aldehydes, sulfates, and carboxylates) that allow facile bioconjugation to targets of interest. Functionalized spheres can also be purchased and soaked with fluorescent sensor. Fluorescent polymers have been extensively employed in FRET-based analytical assays. Such as DNA hybridization, protease detection, pH, and heavy metal detection. 5.1. Heavy Metal Ion Sensors. Many of the current FRET-based sensors are created in the form of small molecular dyads or triads; they usually need synthetic efforts to covalently link the donor with the acceptor. In addition, for the dyad or triad systems, some exhibit low water solubility, making them usable only in organic solvent(s). Thus, the quest to develop FRET-based sensors, which are nontoxic, usable in aqueous media, and easy to fabricate, is of great importance. Recently, Ma reported a facile and alternative strategy for building a FRET-based ratiometric fluorescent sensing system for ferric ion detection in aqueous media by using the amphiphilic diblock copolymer micelles as the scaffold. In this detection system, a hydrophobic fluorescent dye nitrobenzoxadiazolyl derivative (NBD, as the donor) and a spirolactam rhodamine derivative (SRhB−OH) chosen as the sensitive and selective recognition element for Fe3+ ions.65 Another FRET-system was also build by Boling Ma to detect Hg2+ ions.66 To enhance the quantum yield of the donor, they obtained a hydrophobically modified fluorescein (FLS-C12) instead of NBD dye as donor. A thiocarbonyl-containing spirolactam-rhodamine derivative (RhB-CS) was synthesized as the sensor element for Hg2+ ions (Figure 27). Zhu and co-workers reported A novel conjugated polymer (RB-PPETE) of poly[p-(phenylene ethynylene)-alt-(thienylene ethynylene)] (PPETE) bearing covalently linked thienylene rings and Rhodamine B units to detect Hg2+ ions.67 The fluorescence resonance energy transfer (FRET) was demon-

Figure 24. Schematic illustration for formation of a FRET-based ratiometric Hg2+ sensor with multilayered silica particles as the scaffold.

simply doping the dual-FRET dye pair with different ratios of the dyes. Hypochlorite acid (HOCl) is a power oxidant that functions as antimicrobial agent in water treatment and living organisms, and it plays a critically important role in the immune system. Recently, Chen and co-workers build a specific ratiometric nanosensor for hypochlorite was constructed as a paradigm of FRET spectral unmixing.61 The separation of FRET pairs’ emissions reaches to 175 nm, which ensures the FRET probing more accurate. Efficient FRET occurs between Rhodamine B and one aminocyanine dye on a nanoparticle with a sole nearinfrared (NIR) emission before sensing (Figure 26). Ratiometric fluorescent response appeared with the addition of hypochlorite, and the signal of Rhodamine B was detected. This new nanosensor shows high selectivity and potential in biological systems. The other application is to dope multiple energy transfer fluorescent dyes into silica NPs. By varying the doping ratios of the three tandem dyes, carboxylfluorescein (FITC), carboxylrhodamine 6G (R6G), and 6-carboxy-X-rhodamine (ROX), FRET-mediated emission signatures can be identified, and the silica particles can emit multiple colors at a single excitation wavelength.62 Such particles were applied to specifically detect three pathogenic bacteria species (Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus) in solution. K



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Figure 25. Chemical structure of the fluorescent molecular switch Rh-AA-DAE and the photochromic reaction responsible for the fluorescence modulation. The fluorophore moiety is excited with green light. Red light is emitted in the on state, while resonant energy transfer prevents this emission in the off state.

pH-insensitive semiconducting polymer. This approach offers a rapid and robust sensor for pH determination using the ratiometric methodology where excitation at a single wavelength results in two emission peaks, one of which is pH sensitive, the other of which is pH insensitive for use as an internal reference. Another nanosensor was built for pH detection through the same way. The surface of azide-coated polystyrene was functionalized via CuAAC with fluorescent dyes in an all water-based process. The system contains two FRET partners: dansyl units that act as the donor and pH responsive fluorescein units that act as the acceptor in its dianionic form. Chitosan (CS), a cationic polysaccharide, has been used extensively for various biomedical applications. In aqueous media at pH 7.4, CS forms dissociated precipitates because the aggregation of CS polymers occurs too rapidly and locally.71 Based on the property, Chen and co-workers developed a pHresponsive NPCS NPs encapsulated with doxorubicin (DOX) as an anticancer delivery device meanwhile, the drug release mechanism was intracellularly studied. Recently, Zhou reported a general strategy to produce pHtunable,72 highly activatable multicolored fluorescent nanoparticles using commonly available pH-insensitive dyes with emission wavelengths from green to near IR range. The system exhibits more sensitive than pervious nanosensors. The authors also reported that homo-Förster resonance energy transfer (homoFRET)-enhanced decay was more facile strategy to render ultra-pH response over the H-dimer and photoinduced electron transfer (PeT) mechanisms (Figure 28).

Figure 26. Schematic illustration for formation of a FRET-based ratiometric HOCl sensor.

strated in the polymer, and in the presence of Hg2+ ions the excitation energy along the backbone of the conjugated polymer is transferred to the energy acceptor (Rhodamine B), which leads to a visual color change of the solution from slight yellow to orange. In the same way, four FRET detection systems for mercury ions have been developed.68 Compared with pervious papers, the energy acceptor (Rhodamine B) was modified and the systems became more sensitive. 5.2. pH Sensor. Knowing the local pH within a subcellular organelle is a prerequisite to understand the physiological processes that take place in these small volumes because changes in pH can have a drastic effect on biomolecular structure and enzyme activity.69 Recently, Chan described the development of poly(2,5-di(3′,7′-dimethyloctyl)phenylene-1,4ethynylene) (PPE) polymer dots (Pdots) as a platform for designing FRET-based ratiometric pH nanosensors.70 The pHsensitive dye, fluorescein, was doped to PPE Pdots, which is a L



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Figure 27. Formation of a FRET-based system with polymeric nanoparticle as the scaffold and its application as ratiometric fluorescence sensor for mercury ion.

the emission of polyfluorene, which encourages efficient FRET from the fluorene units to the fluorescein. Oxygen is a critical component for many physical course of a variety of diseases. Therefore, the measurement and imaging of oxygen levels in live cells and tissues are challenging for us.

Figure 28. Chemical structures of PBDA-BDY, PDPA-TMR, PC7AC55, and PC6A-C75 and the representative fluorescent images of their aqueous solutions at the same polymer concentration (100 μg/mL) but different pH values were shown.

5.3. Reactive Oxygen Species (ROS) Detection. Fluorescent reactive oxygen species (ROS) sensors were covalently linked to the polymers as the sensors. He and coworkers reported new water-soluble cationic polyfluorene with boronate-protected fluorescein (peroxyfluor-1) covalently linking to the polymer backbone (PF-FB) was synthesized as a fluorescence sensor to optically detect hydrogen peroxide (H2O2) and glucose in serum.73 The peroxyfluor-1 exists as a lactone form that is colorless and nonfluorescent. Without addition of H2O2, the fluorescence resonance energy transfer (FRET) from fluorene units (donor) to peroxyfluor-1 (acceptor) is absent and only blue donor emission is observed upon excitation of the fluorene units. In the presence of H2O2, the peroxyfluor-1 can specifically react with H2O2 to deprotect the boronate protecting groups and to generate green fluorescent fluorescein (Figure 29). The absorption of fluorescein overlaps

Figure 29. Schematic representation of the H2O2 assays. M



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nucleotide, both sensors bind to their respective complementary sequences, and the fluorescent labels are brought close enough for energy transfer to take place. The intensity of the emission spectra is linearly dependent on the number of such donor−acceptor pairs, which is in turn directly proportional to the target oligonucleotide concentration. In the absence of target oligonucleotides, the labeled sensors in dilute solution are not close enough for energy transfer to take place and thus NIR radiation results in minimal luminescence in the specific emission wavelengths. The most significant feature of this type of nucleotide sensor is the high sensitivity of detection because of the lack of autofluorescence upon NIR excitation. Using a 50 mW excitation power, the detection limit for the target DNA nucleotide was calculated to be 1.3 nm (based on a signal-tonoise ratio of S/N = 3). Similarly, a competitive model assay for biotin was constructed by another group using streptavidinconjugated UCNPs as a donor and biotinylated phycobiliprotein as an acceptor, which showed a lower limit of detection in the subnanomolar concentration range (Figure 31).78 Two

Recently, a lot of nanoparticle-based optical oxygen sensoological and pathological processes in living cells.74 Tissue hypoxia has been found to be closely related to the clinical were reported. Wu and co-workers described a novel nanoparticle architecture for oxygen sensing that consists of p-conjugated polymer molecules doped with an oxygen-sensitive phosphorescent dye.75 Upon light excitation, the polymer efficiently transfers energy to the phosphorescent dye, which results in bright phosphorescence that is highly sensitive to the concentration of dissolved oxygen (Figure 30). The conjugated

Figure 30. Schematic illustration of the formation of conjugated polymer dots for oxygen sensing. Figure 31. Scheme of the FRET system, with phosphor-biotin nanoparticles as energy donors and AU-biotin nanoparticles as energy acceptors, in the analysis of avidin. ET = energy transfer. (Reproduced with permission from ref 76. Copyright 2012, RSC Publishing.)

polymers employed as the doping host are the polyfluorene derivatives poly(9,9-dihexylfluorene) (PDHF) and poly(9,9dioctylfluorene) (PFO). Platinum(II) octaethylporphine (PtOEP) served as the oxygen sensitive dye. Wang and coworkers developed a similar system to detect oxygen.74

other groups have also reported FRET-based systems with UCNPs as energy donors and applied this system to detect avidin and estradiol in the buffer.79 The latest paper reports a sandwich-type immunoassay of goat antihuman IgG based on FRET, using a UCNP as a donor and a gold nanoparticle (AuNP) as an acceptor, because AuNPs have an absorption peak at 530 nm that coincides with the green emission peak (542 nm) of the UCNPSs.80 Human IgG-UCNPs and rabbit antigoat Ig AuNPs came in close proximity when goat antihuman IgG was added to the mixture, because sandwichtype immunoreactions between the goat antihuman IgG and the two different proteins cross-bridge the donors and acceptors and thus shorten their spacing (Figure 32). As a result, the quenching of the NIR-excited luminescence of the UCNPs is linearly correlated to the concentration of the goat

6. RARE-EARTH UPCONVERSION NANOPHOSPHORS (UCNPS) AND CARBON NANODOTS (C-DOTS) Rare-earth upconversion nanophosphors (UCNPs), when excited by continuous-wave near-infrared light, exhibit a unique narrow photoluminescence with higher energy. Such special upconversion luminescence makes UCNPs promising as bioimaging sensors with attractive features, such as no autofluorescence from biological samples and a large penetration depth. UCNPs based FRET typically rely upon FRET coupling of UCNPs and a downconverting fluorophore, where UCNPs is the donor. The fluorophore is chosen such that the emission spectra of the UCNPs overlap the excitation spectra of the downconverting fluorophore. Excitation of the UCNPs with NIR results in emission in the visible range necessary for emission from the conventional fluorophore, when in close proximity. It is advantageous for FRET to use UCNPs as a donor because NIR excitation light does not excite the downconverting acceptor fluorophores.76 This results in good sensitivity of detection of the signal from the acceptor. Moreover, the extremely narrow and sharp lanthanide emission band (from the donor) is easily distinguished from the emission wavelength of the acceptor. Their photostability, low cytotoxicity, good light penetration depth, and minimum photodamage enable prolonged FRET sensing. Some preliminary results of using UCNPs for FRET have been published. Zhang, et al. have developed a highly sensitive and specific UCNPs-FRET-based single-stranded nucleotide sensor.77 Two short oligonucleotide strands labeled with a UCNPS and a fluorophore, respectively, are used as sensors to capture the longer target oligonucleotide. In the presence of the target

Figure 32. Schematic illustration for the LRET process between NaYF4:Yb, Er UCNPs (Donor) and AuNPs (Acceptor). N



Review

antihuman IgG (in the range of 3−67 μg/mL). While FRETbased systems have distinct advantages over current ones, more data on the use of upconversion FRET is required for use in a wider variety of applications. Zhang et al.81 reported results obtained from the core/shell NaYF4:Yb,Er/Tm nanospheres with silica coating. The multicolor UCL are produced by encapsulating organic dyes or QDs into the silica shell, based on FRET from the UCNPs to the organic dyes or QDs. Similarly, Kim et al.82 reported multiplexed imaging complementarily using different QDs and NaYF4:Yb,Tm nanosensors by alternating the excitation wavelengths and unmixing the emissions. Wolfbeis et al.83 tuned the emissions of NaYF4:Yb,Er/Tm nanoparticles by covalently modifying them with amino-reactive organic dyes whose absorption spectra overlapped only one of the dual emissions of the NaYF4:Yb,Er/Tm UCNPs and had high extinction coefficients. Li et al.84 tuned the multicolor of NaYF4:Yb,Er/Tm nanoparticles by FRET from the nanoparticles to the organic dyes, which were loaded in amphiphilic polymer. Carbon nanodots (C-dots) are a new class of carbon nanomaterials with sizes below 10 nm, first obtained during purification of single-walled carbon nanotubes through preparative electrophoresis in 2004.85 Compared to traditional semiconductor quantum dots (QDs) and organic dyes, photoluminescent C-dots are superior in terms of high aqueous solubility, robust chemical inertness, easy functionalization, high resistance to photobleaching, low toxicity, and good biocompatibility.86 As a result, much attention has also been paid to their potential applications in biological labeling, bioimaging, and drug delivery. Of particular interest and significance is the recent finding that C-dots can exhibit PL emission in the near-infrared (NIR) spectral region under NIR light excitation. It should be noted that NIR PL emission of Cdots excited by NIR excitation is particularly significant and useful for in vivo bio-nanotechnology because of the transparency of body tissues in the NIR ‘‘water window’’.87 C-dot-based systems play a major role in industrial and technological advances. A lot of work has been devoted to the study of C-dots, especially on the energy and charge transfer research. However, only a few sensors based on FRET have been reported. Qu’s group reveal energy transfer from C-dots to graphene and consequently construct a fluorescencece resonance energy transfer (FRET) sensor, which could be used for measuring the concentration of potassiumions (K+) with high selectivity and tunable dynamic range.88 They design a model in which the donor (C-dots) and the acceptor (graphene) are brought into FRET proximity through specific cation−ligand complexation. C-dots are covalently aminated, and grapheme is noncovalently functionalized with 18-crown-6 ether (18C6E). The known tight binding of primary alkylammonium with 18C6E will bring C-dots and grapheme into appropriate proximity and hence induce energy transfer. Thereafter, the FRET process is inhibited because of competition between K+ and ammonium for 18C6E, which has high potassium selectivity. Wu’s group reported a carbondot-based ratiometric fluorescent sensor for detecting hydrogen sulfide in aqueous media and inside live cells.89 In this sensor, the C-dots not only serve as the energy donor but also as the anchoring site for the sensor, a naphthalimide azide derivative. The schematic illustration for the selective sensing for H2S by the C-dots-based sensor is shown in Figure 33.

Figure 33. Schematic illustration for the structure of the carbon-dotbased sensor and its ratiometric detection of H2S.

7. SUMMARY AND OUTLOOK The demand for highly sensitive nonisotopic bioanalysis systems for biotechnology applications, such as those needed in clinical diagnostics, food quality control, and drug delivery, has driven research in the use of nanomaterials for biomedical and biotechnological applications. The use of nontraditional combinations with materials other than the usual organic donor and acceptor dyes will expand the applicability of FRET analysis. Despite the numerous examples of FRET systems based on nanomaterials presented in this review, FRET remains an underused and underappreciated analytical tool. Researchers have made advances using systems based on FRET in bioimaging, labeling, separation, disease diagnosis, and therapy, and several obstacles and limitations still exist. First, large and bulky sized NPs may block some biological binding sites in close proximity or limit the use of NPs in certain applications, such as the FRET-based investigations. The cytotoxicity of nanomaterials must be fully investigated before considering the injection of NPs into diseased tissues and organs in the human body. Although significant challenges remain in current studies, the use of FRET nanosystem could serve as a practical tool for biological studies.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-411-84986307. E-mail: songfl@dlut.edu.cn. Tel.: +86-0411-84986306. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported financially by the NSF of China (21222605, 21006009, 21136002, 21076032, and 20923006), the Fundamental Research Funds for the Central Universities of China, 973 program of China (2009CB724700 and 2012CB733702), and 863 program of China (2011AA02A105).

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Fluorescent Nanosensors Based on Fluorescence Resonance Energy

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