Advances in FRET‐based biosensors from donor‐acceptor design to applications

Fluorogenic biosensors are essential tools widely used in biomedicine, chemical biology, environmental protection and food safety. Fluorescence resonance energy transfer (FRET) is a crucial technique for developing fluorogenic biosensors that provide mechanistic insight into bioprocesses through time‐spatial bioimaging in living cells and organisms. Although extensive FRET‐based sensors have been developed for detecting or imaging analytes of interest over the past decade, few comprehensive reviews have summarized the recent studies from the fundamental chemical angle about the design and application. In this work, the recent advance in the discovery of FRET biosensors using donor‐acceptor dye combinations is described and they are classified based on different types of analytes, such as mall molecules, proteins, enzymes, nucleic acids and metal ions. This review provides molecular‐level inspiration for the design of FRET‐based biosensors, aiding in their application in biosensing and bioimaging.


INTRODUCTION
Fluorescence sensing and imaging offer a unique and valuable approach for detecting specific analytes in biological environments.Consequently, fluorescence sensors have been used widely in biomedical research, environmental assessment, and materials science, since they have the benefits of low cost, non-invasiveness and high temporal-spatial resolution. [1][4] However, several key factors, such as the natural autofluorescence, the change of microenvironment and excitation intensity, directly affect the sensitivity of vast fluorescence sensors, leading to the failure in preclinical studies or implantation in humans. [5]herefore, the design and synthesis of ratiometric fluorescent probes, aimed at reducing these limitations and enhancing the signal-to-noise ratio, have attracted more attention in fluorescent biosensing and bioimaging. [6]Fluorescent sensors were mainly designed based on the following common mechanisms such as intramolecular charge transfer (ICT), fluorescence resonance energy transfer (FRET), The general application of FRET-based biosensors.
aggregation-induced emission (AIE), photoinduced electron transfer (PET) and other sensing mechanisms. [7]Among them, only ICT and FRET demonstrate dual-emission ratiometric sensing characteristic to reduce noise signal.FRET, in particular, allows for a substantial emission wavelength shift to avoid/reduce interference from the background, resulting in high sensitivity.0][11][12] FRET fluorescent sensors typically contain two fluorescent chromophores: a donor and an acceptor.When the donor and acceptor are at an appropriate distance apart (typically 2-10 nm), non-radiative energy transfer occurs between them, resulting in a decrease in the strength of donor fluorescence and enhance in the fluorescence intensity of acceptor. [13]ince FRET only occurs when donor-acceptor distance is less than 10 nm, it is a highly useful technology that has been commonly used in biological sensing applications, particularly to observe the conformational changes of certain biological molecules.Therefore, a comprehensive review of the design and application of recent FRET fluorescent sensors would be highly beneficial for ratiometric biosensors and the exploration of various biomacromolecules and biological phenomena.
In this work, the advances in FRET-based biosensors from donor-acceptor design to application were systematically reviewed.Firstly, the design principles of the FRET-based biosensors were briefly described.Then, the advantages and limitations of the commonly used or recently developed FRET pairs, organic dyes and nanomaterials, were summarized.Finally, the detailed applications of FRET-based biosensors in various fields, were categorized according to different types of analytes of interest, including proteins, metal ions, small molecules and nucleic acids, was comprehensively discussed.The goal of this review is to provide The energy transfer of the FRET process is depicted in a Jablonski. [17]The ground and excited states of fluorophores are represented by S 0 and S 1 , respectively.molecular-level inspiration for the development of FRETbased sensors and to promote their application in biosensing and bioimaging areas.

DESIGN PRINCIPLES OF FRET-BASED BIOSENSOR
In 1948, Theodor Förster proposed an equation to quantify the efficiency of electronic excitation transfer from an energy donor to an acceptor. [14]The overall process of the energy transfer is illustrated below.In the first step, photons are excited from the ground state to the donor's excited state.In the second step, energy is transferred from the excited donor (named as D) to the acceptor molecule (named as A).D + hv → D * (1) The energy transfer of the overall FRET process is illustrated in Figure 2 using a Jablonski plot, which explains the excitation and emission of the donor and acceptor and their energy-coupled ground state. [15]To achieve a successful FRET operation, three critical criteria must be satisfied. [16]irst, the donor's the emission dipole and the acceptor's absorption dipole must be oriented to each other.Second, there must be an overlap between the acceptor's excitation spectrum and the donor's emission spectrum.Third, the distance of donor and acceptor must be within 10 nm.FRET efficiency is a central parameter that determines the selectivity and sensitivity of FRET-based biosensors.
To better understand the factors affecting FRET efficiency, we can summarize them into four intrinsic factors.The first factor is spectral overlap integration (J), which refers to the degree of overlap between the acceptor's excitation spectrum and the donor's emission spectrum (Figure 3). [18,19] The FRET efficiency is highly determined by the value of R, and thus a slight change in R can significantly affect the FRET signal.The third factor is the term of dipole-dipole interaction (κ), which is determined by both dipole orientation and scale free parameter.Finally, the fourth factor is that the acceptor fluorophore has a long enough fluorescence lifetime to allow FRET to occur between two dipoles.Since the donor-acceptor distance is so critical to the FRET efficiency, FRET is frequently utilized as a molecular/spectral scale approach to monitor conformational changes in specific biomolecules or intermolecular interaction between a biomolecule of interest and its binding partners. [18]Of note, the theoretical treatment on Coulombic coupling that determines the FRET efficiency mainly involves the follow two factors, ideal dipole approximation (IDA) and multipolar coupling and the associated distance dependence.When considering isotropic averaging and the molecular size and shape, the IDA may not be applicable for distances of 20 Å or smaller, as well as for distances in certain directions larger than 50 Å. [20]In addition, it was found that short range interactions (multipolar and orbital overlap) dominates the transfer process, whereas long range dipolar interactions also provides contribution, based on the fit of the experimental and simulated fluorescence decays. [21]he fundamental principle for designing a FRET-based biosensor is to achieve precise modulation of the intermolecular distance between a donor-acceptor pair to construct the FRET system.The changes of fluorescence are usually induced by the initiation or cessation of the FRET process when exposed to the analyte.Thus, this FRET system can be utilized to indicate the presence and amount of a specific analyte by observing variations in fluorescence signal.

DIFFERENT TYPES OF FRET PAIRS
Typical FRET sensors consist of three elements: a donor molecule, an acceptor molecule and the linker for their connection.The selection of fluorophores, donor and acceptor molecules is critically important in the design and functionality of FRET sensors.There are three major types of fluorophores used in the creation of donor-acceptor pair, small organic dyes (such as boron dipyrromethene, rhodamine, fluorescein and coumarin), fluorescent proteins (FPs) and nanomaterials (such as different types of quantum dots and graphene oxides).All of the fluorophores exhibit diverse characteristics that contribute to their versatility in particular applications in FRET-based biosensing. [22,23]

Small-molecule fluorophores
Organic dyes are commonly used in developing FRET probes due to their simple preparation and low cost (Figure 4).Additional chemical substituents can be introduced into different dyes to modify their electronic properties and tune their absorption and emission properties for FRET-based biosensing. [24,25]Typically, In FRET-based systems, dyes are often connected by non-conjugated spacers, and energy transfer happens across them.In this section, we classified FRET systems based on different fluorophore backbones, with an emphasis on the role of boron dipyrromethene (BODIPY), rhodamine, [26] fluorescein, [27] coumarin [28] and other fluorophores in detecting proteins, small molecules, metal ions and other analytes.

Boron dipyrromethene
BODIPY dyes and their derivatives have received extensive attention due to their exceptional characteristics, including a high absorption extinction coefficient, intense emission, elevated quantum yield, robust photophysical stability and solubility in a wide range of solvent systems.Structural modifications of BODIPY with different reactive groups such as aryl and alkyl substituents can extend emission wavelengths, which facilitate minimal photodamage to biological tissue. [29,30]BODIPY also exhibits minimal autoabsorption and autofluorescence from biomolecules and deep tissue infiltration, making it an ideal fluorescent probe and bioimaging marker in biological systems. [31]] Yu et al. developed a new ratiometric fluorescence probe (P1) with BODIPY-rhodamine pair for sensitive detection of Hg 2+ ion (Figure 5A). [30,34]The presence of Hg 2+ ions hydrolyzed the spirolactam ring opening of rhodamine, resulting to the occurrence of FRET between BODIPY and rhodamine.Moreover, the probe successfully achieved the paper strip-based detection and cellular imaging for Hg 2+ ions.Ooyama et al. [35] developed a BODIPY skeleton-based fluorescent sensor (P2) based on the mechanism of PET and FRET for measuring trace water in organic samples (Figure 5B).With similar strategy, Li et al. [36] developed two probes for detecting Fe 3+ , which showed high sensitivity and selectivity in living cells.Hypochlorite levels are critical to in human health.In moderation, hypochlorite is known to help the immune system; however, in excess, hypochlorite can cause disease in humans.Therefore, developing a fluorescent probe for detection of hypochlorite is extremely important.Liu et al. [37] developed a FRET fluorescent sensor (P3) based on BODIPY and rhodamine-thiohydrazide system, which enabling the excellent detection of HOCl in living cells (Figure 5C).

Rhodamine
Rhodamine dyes are widely used in detecting ions, biological small molecules, and enzymes because of their high quantum yield and easy structural change in aqueous solution.
The absorption and emission wavelength of rhodamine are greater than 530 nm, and molecular modification caused the shift of both absorption and emission spectra to move to the near-infrared region.In FRET systems, rhodamine dyes act as energy acceptors and exhibit excellent photophysical properties due to their absorption and emission spectra shifting to longer wavelengths (≈580 nm).Thus, rhodamine derivatives are a good building block for the construction of FRET sensing systems.Cao et al. [38] synthesized a new FRET-based ratio probe (P4), aimed to detect Hg 2+ with excellent in vitro optical properties and cellular imaging capability (Figure 6A).The probe, utilizing imidazo [1,5] pyridine as the donor, piperazine as the linker and rhodamine as the acceptor, realized the detection of Hg 2+ in quite wide pH range (from 4 to 10) within 5 min.This overall limit of detection for this probe is 0.93 nM.Additionally, it has also been used suc-cessfully in bioimaging of Hg 2+ in living cells.Glutathione (GSH) serves as an important antioxidant in cells.Keitaro Umezawa et al. [39] designed fluorescent probe (P5) that allow real-time live cell imaging and quantitative analysis of GSH kinetics (Figure 6B).Measuring the acidity levels in tumor masses yields ample physiological insights.It is extremely important to detect small changes in pH of tumor masses as they have a significant impact on tumor growth, progression and treatment.Zhao et al. [40] developed a new FRET-based pH adjustable sensor (P6), which could monitor the pH in various tumor microenvironment (Figure 6C).With the appropriate selection of response regions in pH-adjustable sensors, it is possible to achieve reliable pH indication with P6 on the acidosis-driven tumor progression, providing valuable insights for tumor research and treatment.

Fluorescein
Fluorescein and its derivatives are another important organic dyes widely used as fluorescent probes in various fields of biology and medicine because of their good photostability, high quantum yield, high water solubility and easy chemical modification.As a derivative dye of xanthene, fluorescein exists in various forms such as cation, anion, neutral and dianion in aqueous solutions. [41]Based on its absorption properties, fluorescein has two conformations, lactone and quinone forms.The quinone form of fluorescein shows stronger absorption and fluorescence than that of the lactone form, and thus is more frequently adopted in the development of fluorescent sensors.The combination of fluorescein in FRET systems has proven to be a valuable tool in the study of biological systems, allowing for high sensitivity for detecting and imaging of molecular interactions and signaling events.Goswami et al. [42] developed a FRET-based ratio fluorescent probe (P7) using a fluorescein-Zn-naphthalene complex (NFHZn 2+ ) to detect CN − (Figure 7A).The detailed sensing mechanism for this probe is mainly based on the displacement reaction, where FRET was achieved by the mutual conversion of the complexing/decomplexing probes NFH and NFH/Zn 2+ through Zn 2+ /CN − modulation.This sensor has high selectivity and sensitivity to CN − , and the detection limit is about 0.509 μM.Yi et al. [43] synthesized a fluorescent probe (P8) that emits yellow-green light to detect quaternary ammonium salt structure (Figure 7B).The probe reached a linear detection range of 0-30 μM and the detection limit of 8.68 μM when sensing quaternary ammonium salt structure in aqueous solution.The probe was competent in detecting quaternary ammonium salt content in actual water samples with a recovery rates between 101% and 115%.These results suggest that the fluorescent probe holds great promise for the precise quantification of quaternary ammonium salt.Pakornsiri Sontisiri et al. [44] successfully developed a novel fluorescent probe (P9) utilizing dabsylfluorescein and exhibits super selectivity and specificity towards H 2 S (Figure 7C).This probe realized the direct visualization of H 2 S in living cells and has the potential to be applied in more complicated samples.

Coumarins
Coumarins are a class of fluorophores bearing a benzopyrone structure that are naturally found in many plants and essential oils.The coumarin fluorophore with relatively short emission usually acts as an energy donor in a FRET system.47][48] Shang et al. [45] designed and prepared an ICT-FRET fluorescent probe (P10) with a dual method for detecting H 2 O 2 in living cells (Figure 8A).The probe uses coumarin as a donor, naphthalimide as an acceptor, and a borate moiety as a recognition group in the FRET process.It was found that the borate as protecting group in this probe improved sensitivity towards H 2 O 2 in the assay.Additionally, the borate groups can be altered by H 2 O 2 to produce a hydroxyl product and cause an absorption red shift via the ICT process.The probe has good selectivity, enabled the successful application in detecting changes of exogenous and endogenous H 2 O 2 in living cells.The dual mechanism of the probe, with both FRET and ICT processes, enhances its sensitivity and selectivity, making it a valuable tool for studying H 2 O 2 dynamics in living systems.
As a vital cytoprotective enzyme regulating heme in human, the abnormal concentration of HO-1 is strongly linked to various diseases, such as neurodegenerative and cardiovascular diseases.Hence, quantitative determination of HO-1 is of great interest.Long et al. [49] reported the synthesis a coumarin-porphyrin based FRET probe (P11) and its ability to detect HO-1 activity.The probe was engineered to "break-apart" upon HO-1-catalyzed porphyrin degradation, providing an important tool for HO-1 related mechanistic study with live fluorescent imaging manner (Figure 8B).H 2 S n is an emerging signaling molecule with significant roles in various physiological processes.Thus, quantitative detection of H 2 S n is of great interest in biomedical and environmental applications.Liu et al. [50] reported a coumarin-naphthalimide derivative-based ratiometric fluorescent probe (P12) for sensing hydrogen polysulfides, integrating both mechanisms of ICT and FRET (Figure 8C).The probe has exceptional sensitivity, excellent selectivity and good biocompatibility for monitoring H 2 S n in living cells.

Fluorescent proteins
Fluorescent proteins (FPs) are a type of proteins that exhibit strong fluorescence when exposed to light of a specific wavelength.FPs are widely used as molecular biological tools for tracking various biological processes and interactions in living cells and organisms.[53][54][55] Compared to fluorescent small organic probes, FPs biosensors are genetically encoded and can be naturally introduced into living cells by transformation or transfection and become an integral part of the cellular system.In this regard, FPs biosensors are quite suitable for visible imaging in various biological samples, like cells, tissues, and even whole animals.Besides, FPs-based FRET sensors are highly valuable for long-term imaging because of their stability in vivo for a long time.Furthermore, the use of FPs as the donor or acceptor in FRET-based biosensors allows for multicolor imaging and simultaneously tracking of multiple biological processes simultaneously.
Ding et al. [56] successfully constructed a FRET system to detect sulfated chondroitin sulfate (OSCS) by using pressurized green FPs as donor and dye-labeled heparin (Hep) as acceptor (Figure 9A).Using this super-positively charged green fluorescent protein-based FRET sensing technology, OSCS with as little as 0.001% w/w in heparin was successfully detected.Most of FRET biosensors are made up of cyan and yellow fluorescent proteins (CFP and YFP), which limits their compatible integration with the optogenetic techniques that work with blue light.To solve this limitation, Kenta Terai et al. [57] discovered a new FRET biosensor with red-shifted emission by adjusting the arrangement of fluorescent proteins and modulatory domains (Figure 9B).The Booster biosensor can monitor the dynamic change of protein kinase A activity in living tissues of transgenic mice in realtime manner.Later, Robert E. Campbell et al. [58] reported a new FRET biosensor based on fluorescent proteins, using mScarlet-derived green fluorescent protein (mWatermelon) as donor and red fluorescent protein mScarlet-I as acceptor (Figure 9C).The functionality of this FRET pair was investigated by successful measurement of protease activity and common metal ions (Ca 2+ and K + ), separately.These new FPs based FRET pairs expand the range of available biosensors and provide researchers with more tools for studying complex biological processes and interactions.

Nanomaterials
Nanoparticles (NPs) bearing particular optical and electronic properties have been produced in recent decades for FRETbased applications, injecting new energy into the field of FRET and bringing about a rebirth of FRET in medical and biological applications.Compared to fluorescent proteins and organic dyes, NPs offer several advantages, including excellent energy transfer efficiencies, broad working distances and adjustable spectra to minimize crosstalk between the donor and acceptor.Common nanomaterials used in FRET applications include graphene oxide (GO), gold nanoparticles (AuNPs) and various quantum dots (QDs) (Figure 10).More specially, QDs are highly fluorescent and have a broad excitation spectrum, making them ideal for FRET systems.
GQDs have unique optical properties, including high water solubility, excellent photostability and low toxicity, making them ideal for biological imaging and sensing applications.UCNPs are highly suitable for deep-tissue imaging because they can convert near-infrared light to visible light.AuNPs have unique optical features, including surface plasmon resonance, making them suitable for biosensing applications.GO has excellent biocompatibility, high surface area and good water solubility, making it suitable for drug delivery applications.Recently, these NPs have been used in various biological and medicinal applications, such as nucleic acid analysis, immunobiometrics, diagnostics and drug delivery.

Semiconductor quantum dots
Semiconductor QDs are photoluminescent nanocrystals that have been increasingly used as fluorescent materials in a variety of applications.QDs can function as FRET donors or acceptors due to their size adjustability, large particle size, high extinction coefficient and wide absorption spectra.As FRET donors, QDs exhibit good spectral overlap with many different acceptors, allowing for efficient and selective excitation without stimulating receptors.Furthermore, the large surface area of QDs allows for the attachment of several donors, increasing the probability of FRET sensitization without affecting FRET efficiency.61] Due to their special optical characteristics, semi-conductor QDs have been widely employed in bioanalytical analysis, including DNA hybridization assays, protein binding assays and enzyme activity assays. [62,63]Many of the above assays use FRET as a detection protocol, allowing for a measurable QD emission signal that is specific to "on" (FRET sensitization) or "off" (FRET quenching) upon the recognition of target event, such as biomolecule binding or conformational changes.Tricia B.C. Forbes et al. [64] developed a simple and unique FRET-based fluorescence sensor using the core-shell structure of GSH-CdSe/ZnS QDs for the nanomolar detection of triclosan (TCS) in water samples.The fluorescence intensity of GSH-CdSe/ZnS QDs was remarkably increased by the addition of TCS, which may be attributed to the transfer of fluorescence resonance energy from TCS to QD, allowing it to be used as an "on" fluorescent probe for the detection and determination of TCS (Figure 11A).The use of QD-based FRET probes in bioanalytical assays shows great potential for achieving high sensitivity and specificity, enabling the F I G U R E 1 1 (A) Diagram of detecting TCS with FRET-based GSH-CdSe/ZnS QDs fluorescence sensor.Reproduced with permission. [65]Copyright 2015, Elsevier.(B) Illustration of DNA sensing platforms with FRET systems of GQD-GO pair.Reproduced with permission. [68]Copyright 2018, Elsevier.(C) UCNPs-SYBR Green I pair.Reproduced with permission. [68]Copyright 2018, Elsevier.(D) Sensing mechanism of a fluorometric and colorimetric dual-mode assay for DNA measurement with FAM-Apt.Reproduced with permission. [66]Copyright 2018, Elsevier.detection and quantification of various biological molecules and targets.The probe is used to the measurement of triclosan both in tap and river water, with about 94%-117.5% recovery.

Graphene quantum dots
Graphene is a 2D planar carbon atom sheet bonded to a honeycomb lattice in sp 2 hybridization, while GQD are welldefined new graphene nanoparticles with a diameter of less than 20 nm, which are small enough to trigger significant quantum confinement effects and edge effects.The synthesis of GQD can be divided into a "top-down" splitting method and a "bottom-up" organic method. [67]GQDs not only retain good properties of QDs, such as high brightness, tunable optical properties, superior light stability, but also display advantages of non-toxicity and excellent biocompatibility.In this scenario, GQDs are excellent candidates for building FRET systems for biomedical applications. [68]Further, as a carbon-based luminescent nanomaterial, GQD has an advantage of being easy to manufacture and low in cost compared with an organic fluorescent dye and a fluorescent protein.Therefore, GQDs have become very promising optoelectronic materials.Feng et al. [69] established a novel and efficient fluorescence detection DNA platform by regulating FRET between graphene oxide (GO) and GQDs (Figure 11B).These sensors achieved effective discrimination between complementary and non-complementary nucleic acids in good reproducibility.According to the calibration curve, F I G U R E 1 2 Sensing mechanism of GST based on the QDs@GSH-GO FRET system. [78]e DNA detection probe has an LOD of 0.075 nM and can quantify DNA in a broad linear range from 6.7 to 46.0 nM.Both sensors distinguish complementary nucleic acids from the mismatched nucleic acids with excellent sensitivity and specificity, suggesting a useful platform for in vivo detection.

Upconversion nanoparticles
Upconversion is the process of conversion of low-energy light (near-infrared (NIR)) to higher energy (ultraviolet, infrared) by gradual absorption and energy transfer of lanthanide ions. [70][73] As is well known, NIR light with strong penetrating power should produce less injury to biological system.In contrast, ultraviolet light can cause photo damage to biological samples such as tissues.Therefore, UCNP is considered to be an excellent substitute for traditional fluorophores and has attracted considerable attention in the field of biology.Wang et al. [74] developed a novel DNA probe (Figure 11C) that responds to NIR light with efficient FRET from UCNP to nucleic acid staining SG.This probe offers several benefits for detecting DNA, including easy preparation, minimized autofluorescence background, a strong FRET signal, high sensitivity and selectivity.

Gold nanoparticles (AuNPs)
Gold nanoparticles (AuNPs) are extensively used in biosensing applications as fluorescent quenchers due to their exceptional characteristics such as surface plasmon resonance, high electrical conductivity and facile surface modification. [75,76]he high surface area to volume ratio of AuNP also helps to increase FRET efficiency by providing a multi-donor single acceptor configuration.Several effective FRET systems were established based on the AuNPs for sensing environmental pollutants, disease markers, as well as biological substances.Yang et al. [66] reported a dual-mode assay for ATP determination, which was designed based on FRET between FAM-Apt and cDNAs-AuNPs (Figure 11D).The binding of ATP to the aptamers resulted in the displacement of the cDNAs-AuNPs, leading to fluorescence enhancement of FAM.Aptamers labeled with FAM (6-carboxyfluorescein) were used as probes to detect biologically relevant molecules, metal ions and proteins.

Graphene oxide
GO is a modified form of graphene with an epoxy hydroxyl and carboxyl groups on the surface, which results in the coexistence of different carbon clusters like the π state and the sp 2 matrix.This unique molecular structure enables that GO can work as a super nano-quencher for generalpurpose fluorophores.Thus, GO provides longer working distances and higher FRET efficiencies than traditional FRET acceptors.More interestingly, GO demonstrates the broad absorption spectra ranging from 200 to 800 nm, which is beneficial for FRET occurrence without strictly limiting spectral overlap. [77]Therefore, GO has been commonly used as a receptor in developing FRET biosensing system.Chen et al. [78] reported a novel off-on fluorescence sensor for glutathione S-transferase (GST) assay on the basis of FRET system constructed with GSH functionalized Mndoped ZnS QD and GO.GSH was introduced on the surface of QDs to manipulate the FRET process for the specific recognition towards GST (Figure 12).The developed sensor successfully achieved the quantitative determination of the GST in both living cells and human urine, with good recovery of 80%-90% and no complicated pretreatment.This sensor has an abundance of potential for the non-toxic, sensitive chemical/biological testing in the biomedical field.

Heavy metal ions in environment
Metal ions play a vital role in the environment, chemistry, medicine and biology, and their detection has received significant attention in recent years, leading to considerable achievements.FRET is one of the desirable approaches for measuring metal ions due to its reliability, rapid 80 nM [126]   performance and high sensitivity. [103]In last decade, extensive studies were reported on the detection of metal ions with particular FRET system.The detailed information about the donors-acceptors pairs are summarized in Table 1.

Macro metal ions
Macro metals, which account for more than 0.01% of the weight in an organism, are essential elements for life processes, such as Na + , Ca 2+ , K + and Mg 2+ .In physiological and pathological conditions, all of the above metal ions play essential roles in human body.Hence, exploring new FRETbased biosensors for detecting macro metal ions is of great interest.Tian et al. [127] developed a new nanosensor based on FRET between CDs (as a donor) and AuNPs (as an acceptor) (Figure 13A) to detect Ca 2+ and Mg 2+ .The hybrid nanosensor composed of CDs and hexametaphosphatecapped AuNPs, displaying dual characteristic emission peaks at 439 nm (CDs) and 608 nm (AuNPs), was synthesized and characterized accordingly.Upon the addition of Ca 2+ or Mg 2+ into the hybrid nanosensor, their negative charge was reduced, inducing the aggregation of AuNPs, which subsequently caused the enhancement of fluorescence intensity at 608 nm and the reduction of fluorescence intensity at 439 nm.The detection limits of Ca 2+ and Mg 2+ were about 16 μM and 70 μM, respectively.The nanosensor is capable of detecting fluoride in aqueous solution with good sensitivity (LOD ≈ 21 μM), and a fabricated paper-based sensor was developed accordingly for visualizing fluoride in real water sample.
Lee et al. [103] constructed a new K + sensor based on FRET system, using 15-crown-5-ether capped CdSe/ZnS quantum dots (QDCE) as a donor and 15-crown-5-ether attached rhodamine B (RBCE) as an acceptor.With the presence of K + , K + and two crown ethers (from QDCE and RBCE, respectively) formed a sandwich structure (Figure 13B), resulting in an effective FRET process by promoting the proximity of QDCE moiety with RBCE moiety.In this case, the resultant LOD is about 4.3 μM, while the liner range was 0-50 µM.However, the probe has no excellent selectivity for potassium ions because of the interferences caused by other metal ions, which limits its further application.

Trace elements
Trace elements, which accounts for less than 0.01% of weight in an organism, are indispensable for the proper functioning of the human body.The essential trace elements, such as Cr 3+ , Fe 3+ , Cu 2+ , Co 2+ and Zn 2+ , play critical roles in various physiological processes.For instance, Fe 3+ is involved in oxygen transport, electron transport, and oxidoreductase catalysis, among other fundamental functions. [128]Iron Detection principle of a hybrid ratiometric fluorescent nanosensor for Ca 2+ . [127](B) Detection principle of QDCE and RBCE based FRET sensor for K + . [103]I G U R E 1 4 Proposed mechanism for chemosensor + Fe 3+ complex.[131] deficiency, one of the leading causes to the global burden of diseases, can lead to many human problems, such as anemia, decreased immunity and low blood pressure.[129,130] Therefore, in recent years, the development of fluorescent chemosensors has emerged as a powerful tool for detecting trace elements for health indication.Piao et al. successfully developed a new fluorescent chemosensor for Fe 3+ analysis based on FRET system employing a dansyl moiety as donor and rhodamine as acceptor (Figure 14).The probe was prepared by a simple condensation reaction, using rhodamine dye and a dansyl group. When 3+ was added into the probe solution, it caused the spirolactam ring opening of rhodamine, facilitating the intramolecular FRET for the designed probe.[131] The detection limit was 1.05 μM, while the liner range was 10-150 μM.This probe has good potential for further application, because of the straightforward synthesis, uncomplicated structure and remarkable sensing capabilities.

Toxic metal ions
Despite the beneficial effects of the aforementioned metal ions on the human body, some others, including Hg 2+ , Al 3+ , Ag + and Pb 2+ , are toxic and can cause health problems such as neurotoxicity, Parkinson's disease, cancer and Alzheimer's disease. [132,133]Excessive exposure to mercury, for example, also lead to permanent damage of DNA and the central nervous system. [134]As a result, the development of FRETbased biosensors with excellent selectivity and sensitivity for the detection of toxic metal ions is in urgent demand.Zuo et al. [135] constructed an effective dual-color fluorescent sensing platform based on FRET between FAM/TAMRA (as a donor) and WS 2 nanosheets (as an acceptor) for homogeneous detection of Hg 2+ and Ag + (Figure 15A).The mixing probe, containing mercuryspecific DNA probes labeled with FAM and silver-specific DNA probes labeled with TAMRA, was absorbed by WS 2 nanosheets via van der Waals force.The two dyes were quenched by WS 2 nanosheets due to the occurrence of FRET.However, with the presence of Hg 2+ or Ag + , FAM-labeled probe and TAMRA-labeled probe would form Hg 2+ -mediated base pairs (T-Hg 2+ -T) and Ag + -mediated base pairs (C-Hg 2+ -C) respectively.This weakens the interaction formed by dsDNA and WS 2 nanosheets, causing the switched FAM-labeled probe and TAMRA-labeled probe move away from WS 2 nanosheets and resulting in the F I G U R E 1 5 (A) Illustration of WS 2 nanosheet-based sensing platform for fluorescent detection of Hg 2+ and Ag + .Reproduced with permission. [135]opyright 2016, Elsevier.(B) Fluorescent TCQ for Al 3+ detection based on FRET. [136]ecific emission of one dye was recovered while the other remains quenched.The detection limits for Hg 2+ and Ag + were about 3.3 nM with the liner range from 6-650 nM and 1.2 nM with the liner range from 5-1000 nM, respectively.The dual-color fluorescent biosensor realized the detection of Hg 2+ and Ag + in the complicated biological samples, like serum and cell lysates.
Similarly, the extended exposure to Al 3+ can pose a significant risk to human health, and the detection of Al 3+ is of great interest. [137]Zhu et al. [136] developed a ratiometric fluorescence sensor based on the FRET between quinoline moiety (as a donor) and coumarin (as an acceptor) for the detection of Al 3+ (Figure 15B).Before the addition of Al 3+ , the probe only showed the maximum emission of quinolone at 390 nm.After the addition of Al 3+ , the chelation of nitrogen and oxygen atoms with Al 3+ induced the occurrence of FRET from quinoline moiety to coumarin, leading to the decline of fluorescence intensity of quinoline and the enhancement of fluorescence intensity of coumarin.The detection limit was 0.024 μM while the liner range was 0.2-10 μM.In addition, this probe was utilized for fluorescent imaging of Al 3+ in living cells.

Small molecules
Small molecules, including inorganic ions and organic compounds, are crucial for various physiological processes and play a vital role in our daily lives.Monitoring and detecting these molecules is essential for understanding their functions and roles in different diseases.To accomplish this goal, FRET-based biosensors have become a potent tool for biosensing and bioimaging of small molecules of interest, because of high sensitivity and ratiometric characteristic.A series of FRET-based biosensors have been designed to detect various compounds, such as glucose, neurotransmitters like dopamine and acetylcholine, and metal ions like copper and zinc.These biosensors offer a wide dynamic range and provide valuable information about the content and distribution of small molecules in biological systems, which can aid in the diagnosis and treatment of diseases.Table 2 lists different donor-acceptor pairs for detecting and monitoring these small molecules.

Inorganic molecules and ions
As we all know, inorganic molecules (HOCl, NH 3 , H 2 O 2 , H 2 S etc.) and ions (HSO 3 ) play significant roles in many physiological and pathological processes, and thus their levels are highly associated with human health and food safety.[169] Wu et al. [163] developed a FRET sensor for the colorimetric and ratiometric detection of HSO 3 − using coumarin-piperazine-TCF (CPT) conjugate platform (Figure 16A).Without HSO 3 − , the FRET between coumarin (as a donor) and TCF moiety (as an acceptor) of CPT was "on".After incubating the probe with HSO 3 − , the FRET was terminated because of the interruption of energy transfer from the coumarin to the TCF.The detection limit of HSO 3 − was 45 nM with the linear range of 0-7 μM.The sensor demonstrated typical ratiometric fluorescent property when imaging HSO 3 − in living cells.F I G U R E 1 6 (A) Proposed sensing mechanism of probe CPT for HSO 3 − . [163](B) Illustration of the ARS-FPBA based FRET probe for ratiometric sensing of H 2 O 2 .Reproduced with permission. [175]Copyright 2017, American Chemical Society.
F I G U R E 1 7 Mechanism of TNT detection based on FRET system conjugated with gold nanoparticles and Quantum dots.Reproduced with permission. [149]Copyright 2017, American Chemical Society.
[177][178][179][180] To detect H 2 O 2 , Feng et al. [175] developed a dual-emission FRET sensor with 7-hydroxycoumarin (HC) and alizarin red S (ARS) were as donor and acceptor, respectively, using single wavelength excitation (Figure 16B).When ARS was not present, the polymeric nanoprobe displayed blue-colored fluorescence, characterized by an emission peak at 450 nm.On the contrary, with the presence of ARS, FRET between ARS and HC produced a new emission peak at the wavelength of 600 nm.Upon the addition of H 2 O 2 , ARS would dissociate from the nanoprobe surface, leading to the fluorescence reduction at 600 nm and fluorescence enhancement at 450 nm, respectively.The detection limit of H 2 O 2 was 0.76 μM with the linear range of 0-500 μM.The probe exhibits several benefits, including easy preparation, excellent selectivity and favorable fluorescence characteristics.

Hazardous organic compounds
Apart from inorganic molecules and ions, certain organic compounds such as explosives (TNT and TNP), pesticides (paraoxon), and toxic chemicals (thiourea and bisphenol A) can also be harmful to humans because of their broad distribution.For instance, TNT is widely used as an explosive in military and industrial applications, but prolonged exposure can cause anemia, abnormal liver function and affect male fertility.To address this issue, Sarita Devi et al. [149] developed a FRET-based biosensor for the detection of TNT using CdSe@MPA QDs as a donor and AuNPs@MEA NPs as an acceptor (Figure 17).The QD-MPA-AuNPs composition resulted in FRET and a decrease in the fluorescence intensity of CdSe@MPA QDs.However, upon the addition of TNT, the amine group on AuNPs@MEA NPs reacted with TNT, causing the separation of AuNPs@MEA NPs from QD-MPA-AuNPs and leading to the recovery of the fluorescence intensity of CdSe@MPA QDs.The detection limit of TNT was 21.9 nM with a linear range of 0-500 nM.FRETbased biosensors like this one have the potential to detect harmful organic compounds in various applications, helping to prevent their negative effects on human health and the environment.

Other biologically active small molecules
In addition to inorganic ions and molecules, several biological small molecules play crucial roles in human health.These molecules include ascorbic acid (AA), adenosine triphosphate (ATP), dopamine (DA), cysteine (Cys), glucose and adenosine.Therefore, accurate determination of the above biological small molecules is essential for disease diagnosis, food safety and pharmaceutical applications.
AA, also known as vitamin C, is an important reactive biomolecule in the human body.It has been shown to improve immunity, prevent and cure many diseases. [181]o address the need for accurate detection of AA, Cen et al. [182] developed a FRET-based biosensor for the detection of AA.The probe used a cobalt oxyhydroxide (CoOOH)-modified upconversion nanosystem (Table 2).The nanosystem was constructed by using UCNP NaYF 4 :30%Yb, 0.5%Tm@NaYF 4 as donor and CoOOH modified UCNPs as an acceptor (Figure 18).Initially, the fluorescence of UCNP was quenched by CoOOH nanoflakes.However, in the presence of AA, CoOOH was reduced to Co 2+ , and CoOOH was decomposed from the surface of UCNP, resulting in the recovery of UCNP fluorescence.The detection limit of AA was 0.2 μM with a linear range of 0-60 μM.The nanosystem utilizing UCNPs enables highly sensitive and selective detection of AA activity while minimizing background interference.In addition to AA, FRET-based biosensors have been developed for the detection of various other biological small molecules, such as dopamine, Cys, TNT, NH 3 , HOCl, H 2 S, trinitrotoluene, glucose, vitamin B 2, H 2 S, 17βestradiol (Table 2).These sensors have the potential to aid in the early diagnosis and treatment of the related diseases by accurately detecting and quantifying biological small molecules.

Proteins
Proteins are typical biomacromolecules that serve as the fundamental material basis of life and the function players of life activities.In recent years, FRET has emerged as a highly accurate and sensitive method for detecting proteins, making it a valuable tool for various applications.FRET-based biosensors have been created to detect a variety of proteins, including enzymes, antibodies and receptors.These biosensors typically use a donor-acceptor pair to specific quantification of the protein of interest.
The following is a summary of FRET-based biosensors for protein detection, including examples of donors, acceptors and analytes (Table 3).
In living organisms, a variety of proteins commonly present and essentially required for regular life activities, such as calmodulin, GSH (glutathione), PSA (anti-prostate specific antigen), PDGF-BB (platelet-derived growth factor), neurotoxin, EpCAM (epithelial cell adhesion molecule) and others.212][213][214][215] Shi et al. [73] designed and prepared a dual-mode nanosensor capable of discriminating GSH from Cys/Hcy.This nanosensor has both colorimetric and fluorometric readouts.The nanosensor used CQDs as a fluorometric donor and AuNPs as a fluorometric acceptor and colorimetric reporter.In the absence of GSH, AuNPs quenched the fluorescence of CQDs due to the aggregation of AuNPs and CQDs by controlling their surface properties, leading to a color change of AuNPs from red to blue and quenching of CQD fluorescence.However, in the presence of GSH, GSH preferentially encapsulated AuNPs because of its stronger affinity for AuNPs.This protected AuNPs from aggregation and increased the distance of CQD-AuNPs, resulting in a visible color change and recovery of fluorescent signal.The linear range of detection of GSH was 0.1-0.6 μM, with a detection limit of 50 nM.This nanosensor has the advantages of high sensitivity by fluorescence method and convenient visual detection.The nanosensor demonstrates high sensitivity for detecting GSH in aqueous solutions and human plasma.

Enzymes
Enzymes are a crucial class of biocatalysts, and FRET-based biosensors have been devised to detect various enzymes, including thrombin, adenosine deaminase (ADA), phospholipase D (PLD), alkaline/acid phosphatase, trypsin and human neutrophil elastase (HNE) (Table 3).Among them, HNE is a typical serine protease that is highly involved in lung cancer and pulmonary inflammatory diseases.HNE is a typical therapeutic target for COPD and is an important biomarker of pulmonary disease progression.Thus, development of FRET-based HNE-targeting probes are of great interest.
In 2020, we [216] achieved in vitro sensing and in vivo imaging of HNE by using a FRET nanoprobe, termed QDP, which incorporated QDs together with organic dyes (Figure 19).The QDP nanoprobe was able to measure HNE both endogenous and exogenous HNE in living cells, with the ultrasensitivity of 7.15 pM.Furthermore, it realized the temporal and spatial imaging of HNE in mouse models of lung cancer and acute lung injury.These findings demonstrate the potential of FRET-based biosensors for the detection of enzymes in various applications, including disease diagnosis and treatment monitoring.The creation of highly sensitive and biocompatible probes may pave the way for the in vivo detection and imaging of enzymes and other biological targets in living organisms.

Nucleic acids and other analytes
In addition to metal ions, small molecules, and proteins, FRET can also be used to detect other analytes related to the human body, such as DNA, RNA, HIV-1 gene, Listeria monocytogenes, ochratoxin A, norovirus, T-2 toxin, vitamin B2 and other biomarkers (Table 4).While traditional methods for DNA detection, such as polymerase chain reaction (PCR) and gel electrophoresis, have limitations in their detection limit, FRET-based probes have been developed to overcome this problem.For instance, Kudr, Jiri et al. [217] study was conducted to investigate the interaction between C-dots and DNA fragments, which revealed a strong affinity of C-dots towards DNA.The absorption and fluorescence spectra of C-dots were affected by DNA, and this finding was supported by changes in the DNA electrophoretic F I G U R E 1 9 FRET-based sensing mechanism for sensing and imaging HNE.The blue point in the peptide represents the cleave site for HNE catalysis.Reproduced with permission. [216]Copyright 2020, American Chemical Society.
mobility and a competitive binding assay (Figure 20A).The study also demonstrated that C-dots reduce DNA's ability to accept EtBr, which prevents solution quenching.However, Cdots transfer some of their excitation energy to EtBr, resulting in FRET.The fluorescence of EtBr was sensitive to the existing state of DNA and could efficiently distinguish between native DNA and DNA damaged by UV radiation or free radicals.These findings suggest that this mechanism could be used as part of biosensors to monitor the threat posed by increased UV radiation levels.Another example is related to ochratoxin A (OTA) detection.OTA, which is one of the most toxic and widespread naturally occurring mycotoxins, has been discovered in various food, such as oats, wheat, coffee beans, corn, cereals, and others. [218]OTA demonstrates numerous pharmacological activities, such as nephrotoxic, immunotoxic, F I G U R E 2 0 (A) A diagrammatic illustration of the proposed DNA sensing approach that utilizes GQD as a nanoquencher is presented.Reproduced with permission. [213]Copyright 2017, Elsevier B.V. (B) The operational mechanism of the aptasensor for OTA detection is based on FRET-based ratiometric fluorescence. [65]rcinogenic and teratogenic effects. [218,219]Qian et al. [65] discovered a new ratiometric FRET sensor for OTA detection through dual-mode of fluorescent sensing and direct visual screening.In this FRET system, green-emitting CdTe QDs (gQDs) served as donor, AuNPs as acceptor.As shown in Figure 20B, thiolated complementary DNA (cDNA) was attached to AuNPs to form AuNPs-cDNA, which then formed rQDs@SiO2@AuNPs-cDNA.When the hybridization reaction occurred between aptamer and cDNA, gQDs were close enough to AuNPs, leading to FRET and quenching of gQDs fluorescence.After adding OTA, OTA specifically bound to the aptamer, causing the gQDs-aptamer/OTA complex to move away from rQDs@SiO2@AuNPs-cDNA, followed by the recovery of gQDs fluorescence.The sensor was able to detect OTA in linear range of concentrations from 5 pg/mL to 10 ng/mL and a low detection limit of 1.67 pg/mL.Hence, it was used for rapid detection of OTA through dual-mode fluorescence sensing and high-throughput format.

CONCLUSIONS AND PROSPECT
In conclusion, the exploration of FRET-based biosensors has received increasing interest for various implications due to their unique advantages, such as ratiometric property and visible imaging capability.The ability to regulate the distance between the donor and acceptor makes FRET-based biosensors less prone to biological interference, enabling non-invasive and in situ biosensing and bioimaging of specific analytes of interest.In this review, we discussed the design principles of FRET-based biosensors, their classification based on energy donor-acceptor dye combinations, as well as the molecular mechanisms underlying their recognition of different analytes, such as proteins, small molecules, enzymes, metal ions and nucleic acids.We also summarized recent progress in the extensive application of FRET-based biosensors in biosensing and bioimaging.FRET is widely utilized for disease diagnosis and treatment, [231][232][233] as well as drug screening and delivery. [234,235]However, like other analytical techniques, there are some challenges in the development of FRET-based biosensor.(1) Better sensitivity.There are various ways to improve the limit of detection for low concentrations of the target molecules in biosystems, such as the optimization of fluorescent labels, minimization of background signals, optimization of the optical system, calibration and standardization, optimization of experimental conditions, and appropriate data analysis and processing.(2) Better selectivity.High selectivity is always indispensable for practical application.In this case, it would be valuable to use highly specific labeling agents to minimize interference from other factors, mitigation of optical interference and autofluorescence, leading to the reduction of false-positive results.(3) Better biocompatibility.It is very important to develop new materials with excellent biodegradability and long-term biocompatibility to realize the widespread adoption and clinical translation.Looking ahead, the development of FRET-based biosensors is expected to continue to grow, driven by advancements in nanotechnology, molecular biology and material science.For instance, the integration of FRET-based biosensors with novel materials, such as 2D materials and metal-organic frameworks, may lead to the development of highly sensitive and selective biosensors.Moreover, the use of FRET-based biosensors in clinical settings for disease diagnosis and treatment monitoring holds great promise.Overall, FRET-based biosensors offer tremendous potential for advancing biosensing and bioimaging, and we expect to see continued progress in their development and application in the years to come.

F I G U R E 3
The absorption and fluorescence spectra of a regular donor-acceptor pair.Brown region refers to the spectral overlap of donor-acceptor pair's the fluorescence spectrum.

F
I G U R E 4 Small-molecule fluorophores for developing FRET system.

F I G U R E 5
Structures and sensing mechanisms of BODIPY-based fluorogenic FRET sensors against (A) Hg 2+ , (B) H 2 O and (C) HOCl.

F I G U R E 6
Structures and sensing mechanisms of rhodamine-based fluorogenic FRET probes against (A) Hg 2+ , (B) GSH and (C) pH.

F I G U R E 7
Structures and sensing mechanisms of fluorescein-based fluorogenic FRET probes against (A) Zn 2+ , (B) NH 4 + and (C) HS − .F I G U R E 8 Structures and sensing mechanisms of coumarin-based fluorogenic FRET probes against (A) H 2 O 2 , (B) HO − and (C) H 2 S 2 .F I G U R E 9 (A) The ScGFP and Hep-RF1 based FRET FP biosensor for OSCS detection.[56](B) The FRET biosensors based on CFP and YFP are named Booster.(C) Construction of mWatermelon and mScarlet-I based GFP-RFP FRET system, and mWatermelon and mScarlet-I based FRET system for the detection of protease activity, Ca 2+ and K + .

F I G U R E 1 0
Diagrammatic representation of FRET biosensor based on nanoparticles and respond to various analytes.

F I G U R E 1 8
Design and principle of AA sensing with CoOOH modified UCNP nanosystem.
List of applications of FRET-based sensors for heavy metal ions.
TA B L E 1 FRET-based sensors for different small molecules.
TA B L E 2 FRET-based biosensors for various proteins.
TA B L E 3 TA B L E 4 FRET-based biosensors for nucleic acids and other analytes.