Recent progress in sensing application of metal nanoarchitecture-enhanced fluorescence

Fluorescence analytical methods, as real time and in situ analytical approaches to target analytes, can offer advantages of high sensitivity/selectivity, great versatility, non-invasive measurement and easy transmission over long distances. However, the conventional fluorescence assay still suffers from low specificity, insufficient sensitivity, poor reliability and false-positive responses. By exploiting various metal nanoarchitectures to manipulate fluorescence, both increased fluorescence quantum yield and improved photostability can be realized. This metal nanoarchitecture-enhanced fluorescence (MEF) phenomenon has been extensively studied and used in various sensors over the past years, which greatly improved their sensing performance. Thus in this review, we primarily give a general overview of MEF based sensors from mechanisms to state-of-the-art applications in environmental assays, biological/medical analysis and diagnosis areas. Finally, their pros and cons as well as further development directions are also discussed.


Introduction
Fluorescence-based techniques are simple and universal analytical methods extensively used in trace detection, biomedical imaging, diagnosis, optoelectronics and forensics. [1][2][3][4][5][6][7] As a real time and in situ analytical approach to target analytes, uorescence-based sensors can offer advantages of high sensitivity, high selectivity, non-invasive measurements and easy transmission over long distances. [7][8][9] However, they still suffer from shortcomings, such as low uorescence quantum efficiency, environment-dependent intensity and photobleaching, which severely hinder their wider application.
Localized surface plasmons (LSPs) refer to free electron collective oscillations induced by external electromagnetic waves, in conned metal nanostructures such as nanoparticles (see Fig. 1a). Resonance may occur when the light frequency matches that of the conduction electron oscillation. Under this condition, the electric eld near the nanoparticle surface is largely enhanced and the metal optical extinction is greatly increased. 10,11 Thus LSPs offer unique absorption and scattering properties to metallic nanoparticles. 12,13 It should be noted that absorption (corresponding to nonradiative decays) induces uorescence quenching of nearby uorophores, while scattering contributes to uorescence enhancement. And the proportion of scattering in total extinction spectra greatly affects the degree of uorescence enhancement induced by these metallic nanoparticles. Silver and gold nanoparticles with larger sizes are preferably used for uorescence enhancement, as for smaller ones, absorption loss tends to dominate. 14 Based on the above theory, we summarized that metal nanoarchitecture-enhanced uorescence (MEF, rstly reported in the 1960s 15,16 and rstly used for biosensors in 1991) arises from resonant interactions between emission dipoles and LSPs of metal nanoarchitectures (see Fig. 1a). The enhanced electric eld experienced by uorophores gives rise to extra increased excitation rates E m , while the resultant coupling between uorophores and nearby metal nanoparticles induces extra radiative rates G m . This process can be more easily understood using the Jablonski diagram shown in Fig. 1b. Thus MEF can effectively enhance the uorescence quantum efficiency of uorophores by increasing both excitation and radiative decay rates. 14,[17][18][19] To Meiling Wang is an associate professor in the School of Physics and Materials Science, Anhui University, and she received her PhD degree in Material Physics and Chemistry from the University of Chinese Academy of Sciences (CAS) in 2012. Her research interests include nanomaterials and uorescence sensors.
obtain effective MEF, there should be overlaps between excitation/emission spectra and the metal plasmon bands. It should be noted that, as a result of enhanced radiative decay rates, shortened effective uorescence lifetimes can be obtained, which greatly improved the photostability of uorophores. 2,14,20 Thus MEF can solve the inherent shortcomings of uorophores by improving both quantum efficiency and stability.
As reported, various kinds of metals have been used for MEF, such as gold, 21,22,34,48,49,75,100,[107][108][109][110][111][112][113] silver, 30,31,35,45,69,110,112,[114][115][116][117][118][119][120] aluminum, 121,122 copper, 123-125 nickel, 125,126 chromium, 125,127 zinc 125 and so on. So far, MEF across the ultra-violet to the second near infrared wavelength range has been reported. 128 Considering their visible and near infrared plasmon transitions, low damping associated with inter-and intra-band transitions, 129 as well as easy fabrication and functionalization, gold and silver are most commonly used for MEF in practical applications. 130 Gold and silver nanoparticles show plasmon frequencies decreasing with increased particle size, and thus their plasmon bands can be tuned over the entire visible and near infrared spectrum. Besides composition and size, plasmon transition frequencies can also be affected by shape and local dielectric environments of metallic nanostructures. Silver shows a much higher scattering efficiency than gold in the wavelength range of l < 600 nm, 129,131 while gold is known for its better biocompatibility and chemical stability.
MEF induced uorescence intensity is principally determined by the following two factors: the degree of spectral overlap between uorophore excitation and plasmon transitions, and the distance between the uorophore and the metal surface. 14,132,133 For uorophores in close proximity (less than 5 nm) or even directly attached to the metal surface, quenching may occur as a result of the excited electron transfer to the metal via nonradiative decay ways. 134 However, as the enhanced electric eld decays nearly exponentially with distance, the uorophore should not be too far from the plasmonic nanostructure. The efficiency of plasmon induced MEF is highest when they are separated by a few nanometers, 14 and thus distance control is important.
To achieve a superior MEF effect, a core/shell structure or a sandwich structure is commonly used, which consists of a metal core/lm, a dielectric shell/lm as a spacer layer and a uorophore layer. 10,135 The spacer layer is usually a dielectric layer with precisely/atomically controlled thickness, and used to separate and connect the metal core/lm and the uorophore layer. 26,50,56,58,74,136 Both the defect-free quality and the dielectric properties of the spacer can affect the plasmonic enhancement. 137 In addition, an ideal spacer layer should play roles in protecting the metal from oxidation. Based on these theories, various spacer layers such as silica, 18 144 have been reported. It should be pointed out that each kind of spacer layer has its advantages and disadvantages, 17,136 and we can choose the most suitable one according to requirements.
To produce the integrated core/shell or sandwich plasmonic nanostructure, commonly the following three processes should be considered: preparation of the metallic nanostructure, coating of the spacer layer and immobilization of the uorophore layer. 27,[59][60][61][62][63][64]147 Various geometrical nanostructures for MEF have been studied so far, and they can be categorized into two main groups: solution-based colloidal nanoparticles (NPs) 27,31,48,52,54,92,145,150 and periodical/non-periodical plasmonic and presence (right) of a metal nanostructure. Eexcitation rate, Gradiation rate, K nrnonradiation decay rate, E mmetal-enhanced excitation rate, and G mmetal enhanced-radiation rate.  Fig. 2. Design, preparation and performance study of these different geometrical plasmonic nanostructures are well summarized in previous reviews. 10,31,108,112,125,136,139,153 In this review, aer elaborating the related MEF theory and platform assembling method, in the following sections, we will review research advances and the implications/limitations of these integrated MEF plasmon nanostructures in uorescence sensing applications. Finally, we will present our views on future directions and further perspectives in this area. We hope that this review can provide a comprehensive coverage of current development achievements in this eld and put forward new perspectives for the future development of improved sensing performance.
2 MEF-based fluorescence sensing for environmental analysis

Detection of heavy metal ions
So far, most of the MEF sensing is performed using solutionbased colloidal NPs, where plasmonic NPs are dispersed into analyte-containing solutions, as they are attractive for biological detection, bioimaging and nanophotonic applications. 136 Among various types of spacer layers, silica is highly regarded for its ease of preparation, thickness controllability, light transparency, stability and biocompatibility. 154 So far, such

Cy3
Not mentioned Pb 2+ Ratiometric uorescence analysis in the range of 10 À12 to 3 Â 10 À6 M; LOD of 3 Â 10 À13 M; good recyclability and selectivity; real-time visual detection silica-coated core-shell NPs have been taken as the most successful class of hybrid plasmonic colloids. Various functions of silica shells are summarized in ref. 139. In this section, we will give a detailed introduction to Ag/Au@SiO 2 core-shell colloidal NPs used in environmental analysis areas. Heavy metal ions, such as Cu 2+ , Hg 2+ and Pb + , are considered as persistent toxic pollutants, which mainly come from uncontrolled battery manufacturing, metal melting, automobile exhaust and old ship demolition. 155 Once discharged into the environment, these heavy metals show high biotoxicity, degradation-resistance and bioaccumulation. Thus their trace detection in aqueous solutions is urgent and important.
Therefore, MEF-based uorescence sensors for these heavy metal ions have been developed in virtue of their improved emission properties (see Table 2). For example, Kim and his coworkers fabricated a new kind of Ag@SiO 2 core-shell NP, with silica chosen as the rigid spacer to adjust the distance between the Ag core and uorophores, and Au 25 nanoclusters modied on its surface acting as the uorescence indicator. 45 MEF of Au 25 nanoclusters was studied with varied core sizes, shell thicknesses and excitations. And an enhancement factor of 7.4 was obtained under optimal conditions. Furthermore, the Au 25adsorbed Ag@SiO 2 NPs were used for highly sensitive and selective 'turn-off' sensing of Cu 2+ , and it was proved that the turn-off ratio is 3.3 times larger than that of free Au 25 nanoclusters under optimal conditions, indicating their much superior sensing ability for Cu 2+ in aqueous solutions.
Ag@SiO 2 based core-shell colloidal NPs of Ag@SiO 2 -AuNCs (AuNCs refer to Au nanoclusters) with good water dispersibility, high stability and good biocompatibility have been synthesized by Xu and his co-workers for multi-component detection of Cu 2+ , pyrophosphate (PPi) and pyrophosphatase (PPase) (Fig. 3). Interactions between silver cores and the outer Au NPs greatly enhanced the emission of Au NPs via improving their excitation efficiency, and the composite core-shell nanostructures were used for developing a sensing platform based on OFF-ON-OFF switching of the uorescence signal in the presence of Cu 2+ , PPi and PPase, and the detection limits were 39 nM, 78.7 nM and 0.976 mU respectively. These sensing nanostructures have also been applied for uorescence cellular imaging. 56 However, unfortunately, their uorescence imaging results can only be used for qualitative research of Cu 2+ , PPi or PPase, but not for quantitative research.
Sui and his co-workers reported an Ag@SiO 2 based coreshell nanoprobe for Hg 2+ , using a sensing strategy by combining MEF and hybridization chain reaction (HCR). As shown in Fig. 4, in the absence of Hg 2+ , HCR occurred, resulting in the formation of long DNA chains with uorescent indicator SYBR Green intercalated into the double DNA helix. Thereaer, positively charged Ag@SiO 2 NPs were added aer a magnetic separation process from the test solution, which will be electrostatically adsorbed onto negatively charged DNA chains, just as 'smart dust' to enhance the uorescence signal. In the presence of Hg 2+ , the uorescence signal gradually decreased with [Hg 2+ ], and a detection limit of 25 pM was obtained under current experimental conditions. 63 And this probe has already been used for selective detection of Hg 2+ in real water.
Moreover, Rajbongshi and his co-workers synthesized uorescein isothiocyanate (FITC) modied Ag/SiO 2 /SiO 2 core-shell NPs and nanorods (NRs), and uorescence quenching detection of Fe 3+ was achieved using these plasmon nanostructures of different geometries. Lower detection limits of 19.4 and 0.83 nM were obtained for NPs (highest uorescence enhancement factor of 2.63) and NRs (highest uorescence enhancement factor of 3.5) respectively. 52 As shown in Fig. 5A, a new MEF-based probe was designed using the distance-dependent uorescence quenchingenhancement effect. Hg 2+ was detected via the formation of  the thymine-mercuric-thymine structure, which can open the hairpin and induce distance changes between the uorophore and the Ag NPs. This process can realize uorescence dequenching and MEF induced by Ag NPs, and 1 nM Hg 2+ was detected. In this work, Ag NPs were functionalized as both the quencher to reduce blank signals and the enhancement substrate for MEF, which greatly improved detection sensitivity. And this design principle can be universal for MEF-based probes. 147 As Raman spectroscopy has attractive vibrational ngerprint features with a bandwidth of over 100 times smaller than uorescence, 156 by combining the high-level multiplexing and specicity of surface-enhanced Raman scattering (SERS) with the large area rapid read-out of uorescence signal, the dualmode optical analysis is emerging as a powerful sensing analytical tool especially in biological and biomedical applications. 156 Furthermore, as reported, much more pronounced photostability can be obtained for uorophores near to a mixed metal substrate than to a single one. 157 Thus Li and her coworkers fabricated a dual-channel biosensor by immobilizing versatile signal indicator Au@Ag/graphene upconversion nanohybrids (Au@Ag-GU) onto the surface of magnetic beads (MBs) through the complementary pairing reaction between the aptamer and the complementary DNA (cDNA) (denoted as MBsaptamer/cDNA-Au@Ag15-GU), with the conjugated aptamer used for specic capture Hg 2+ . And the obtained sensor can export dual channels of SERS and uorescence signals for simultaneous Hg 2+ detection. As shown in Fig. 5B, in the absence of Hg 2+ , the MBs-aptamer/cDNA-Au@Ag15-GU can be easily attracted to one side using an external magnet, and the resulting supernatant solution did not include Au@Ag15-GU, and thus no SERS or uorescence signal was observed. However, in the presence of Hg 2+ , specic binding of the aptamer with Hg 2+ occurred, resulted in liberation of some cDNA-Au@Ag15-GU into the supernatant, which revealed strong SERS and enhanced uorescence signals with increasing [Hg 2+ ]. The as-fabricated dual-channel biosensor showed excellent performances for Hg 2+ with detection limits of 0.33 and 1 ppb for SERS and uorescence mode respectively, under the optimized conditions. And it has also been used for quantify Hg 2+ in spiked tap water and milk samples. SERS can be used to achieve accurate results while the uorescence method gives a much wider linear range, cheaper instruments and good reproducibility. It should be noted that this strategy bridged the gap between uorescence sensing and SERS assays, which broadens future applications of MEF-based sensing.
And on this basis, the same research group synthesized a novel bimodal sensor based on rhodamine derivative (RhD) graed Au nanospheres@Ag core-shell nanocubes (denoted as CSN-RhD), which show both MEF and SERS dual signals for Hg 2+ detection (see Fig. 5C). Both the SERS and MEF intensity increased with [Hg 2+ ]. With an optimized Ag cubic shell thickness, this CSN-RhD showed wide linear ranges of 0.001-1000 ppm and 0.01-1000 ppm, and detection limits of 0.94 and 5.16 ppb for MEF and SERS mode, respectively. These excellent sensing performances for Hg 2+ can be attributed to effective signal enhancement ability of the CSN plasmon nanostructures. 55 Moreover, Liang and his co-workers developed another novel MEF ratiometric/naked eye bimodal biosensor for Pb 2+ , which was composed of a ZnFe 2 O 4 @Au-Ag core-shell bifunctional nanocomposite conjugated with double-stranded DNA (including the Pb 2+ -specic DNAzyme strand labeled with Cy3 and the corresponding substrate strand labeled with N,S-doped carbon dots (N,S-CDs)). The uorescence of N,S-CDs was signicantly quenched with the formation of double-stranded DNA, which brought N,S-CDs and the super quencher CeO 2 into close proximity, see Fig. 6. At the same time, Cy3 uorescence was enhanced by the MEF effect of Au-Ag core-shell hollow nanocubes. In the presence of Pb 2+ , the DNAzyme strand was activated, broken away from the substrate strand and cleaved at the cleavage site into two fragments (red dot in Fig. 6). This process resulted in dequenching of N,S-CDs emission, while the uorescence of the Cy3 labeled fragment was efficiently quenched as it can be easily adsorbed onto the CeO 2 . As shown in Fig. 6b, the disengaged DNA/CeO 2 complex could result in a color change aer adding H 2 O 2 as a result of CeO 2 autocatalysis, and thus real-time visual detection of Pb 2+ can be realized. At the same time, by using the good linear relationships between log(I 562 /I 424 ) and log[Pb 2+ ] in the range of 10 À12 to 3 Â 10 À6 M, ratiometric uorescence quantitative analysis of Pb 2+ can be realized. 158 Their work might provide potential applications for on-site and real-time Pb 2+ detection in real water systems.

Analysis of pH and organic pollutants
Furthermore, MEF-based uorescent sensors for pH and other organic pollutants were developed (as shown in Table 3). For example, a novel ratiometric sensor consisting of Ag@SiO 2 core-shell NPs and a kind of pH sensitive dye of HPTS is developed by Bai and his co-workers for the pH assay. 58 With a shell thickness of 8 nm, uorescence was enhanced by 4 and 9 fold with excitation of 405 and 455 nm respectively. And the emission ratio of 513 nm excited by 455 nm to that excited by 405 nm versus pH in the range of 5-9 was determined, showing its potential application for pH detection in environmental and biological samples.
Trace detection of 2-aminoanthracene (2-AA), an aromatic amine, is of great signicance for environmental monitoring. Recently, Jin and co-workers synthesized a kind of Ag@SiO 2 core-shell NP with an $40 nm Ag core and an $7 nm SiO 2 shell, which could efficiently increase 2-AA emission via MEF. Based on this theory, 2-AA was detected within a wide linear range of 1-800 nM, with outstanding selectivity over co-existing polycyclic aromatic hydrocarbons. 50 Besides Ag/Au@SiO 2 core-shell colloidal NPs, other MEFbased detection in environmental analysis using Ag/Au colloidal NPs with different spacers have been reported, which we will review in this section too.
Tetracycline (TC), as a most frequently used antibiotic, also causes water pollution and enter the human body through the food chain, posing hazards to both the ecological environment and human health. Thus ultrasensitive detection of TC residues in water environments is necessary. 25 Xu and his co-workers developed a smart silver-enhanced uorescence platform via a simple modication of the interior and external surfaces of natural halloysite nanotubes (HNT) with Ag nanoparticles and Cit-Eu nanoprobes (schematically shown in Fig. 7). The appropriate thickness of the HNT walls results in effective MEF of Cit-Eu. And this Ag@HNTs-Cit-Eu nanocomposite was used for ultra-sensitive detection of TC with a detection limit of 4.8 nM. 25 As compared to solution-based colloidal NPs, attaching NPs to a substrate to integrate a plasmonic nanochip can reduce the tedious washing steps, but usually very low and positiondependent enhancement factors are obtained using this method as a result of random NP distributions. Considering this aw, researchers have explored various ordered nanostructure arrays with much better plasmonic coupling between

QDs
Not mentioned Paraoxon LOD of 1.0 Â 10 À10 M; exhibiting sensitivities over three orders of magnitude higher than those without MEF effect adjacent nanostructures and more hot-spot generation in nanogaps. For example, Kim and his co-workers developed an enzymebased miniaturized biosensor of hydrogel microarray entrapping QD-Ag@Silica and AChE for detection of neurotoxic paraoxon. In this design, MEF of Ag NPs was adopted to improve sensing performance, and paraoxon was detected via amplied QD uorescence quenching once exposed to p-nitrophenol produced by the AChE-catalyzed hydrolytic reaction (see Fig. 8). 64 This biosensor exhibited sensitivities (1.0 Â 10 À10 M) over three orders of magnitude higher than those without MEF effect. Its successful integration with microuidic systems further demonstrated potential applications for micro-totalanalysis-systems.

MEF-based sensing for the bioassay
Today, to improve disease curative and survival rates, early diagnosis is crucially important. Very low concentrations of biomarkers must be detected with great efficiency and reliability at preliminary stages. As a result of improved optical properties of uorophores, MEF allows for much lower detection limits and much earlier diagnosis. Thus great efforts have been made by researchers in developing MEF-based sensing for bioassays.

Immunoassay
As biochips based on plasmonic nanoparticles exhibit robust, rapid, and convenient detection, their fabrications have attracted increasing study. And the MEF phenomenon has already been widely exploited for immunoassays (as summarized in Table 4). For example, Xu and his co-workers developed a Ag@SiO 2 @SiO 2 -RuBpy core-shell composite, and a strategy for the detection of prostate specic antigen (PSA) by combining MEF and the magnetic separation technique was developed (as shown in Fig. 9A), which showed a good linear relationship between uorescence intensity and the PSA concentration in the range of 0.1-100 ng mL À1 . 159 And it has also been successfully applied for the detection of PSA in human serum with high sensitivity and specicity, proving its potential in tumor diagnosis.
Inuenza viruses, which can cause u infections, pose major threats to human health, and thus their specic and sensitive diagnosis is urgently needed. Takemura and his co-workers developed a solution-based nanobiosensor based on MEF of alloyed quaternary CdSeTeS QDs (QD) induced by Au NPs for the detection of inuenza viruses. 43 The QDs were conjugated with anti-hemagglutinin (HA) antibodies (anti-HA Abs), while AuNPs were conjugated with anti-neuraminidase (NA) antibodies (anti-NA Abs). With the presence of inuenza viruses, antigen interactions occurred on their surface, which along with adjacent Au NPs triggered MEF of QDs, as shown in Fig. 9B. Detection limits of 0.03 and 0.4 pg mL À1 were obtained for inuenza H1N1 viruses in deionized water and human serum respectively. And the detection of the clinically isolated H3N2 was also accomplished with a detection limit of 10 PFU mL À1 .
As there may be more than one denitive biomarker that can reliably diagnose early stages of many diseases, simultaneous assays with high specicity and sensitivity are necessary. 128 Moreover, driven by demands for cost efficiency, there are also increasing needs to acquire more information from a single experiment. Multiplexed assays can be used to measure multiple target analytes in a single run of the assay, which includes protein and nucleic acid-based multiplexing. 161,162 Considering the perfect specicity of immunoassays, multiplex detection of different biomarkers can be realized by conjugating plasmonic NPs with different capture antibodies. Yuan and his co-workers reported a kind of size-encoded plasmonic magnetic microbead (PMMB)-based multiplexed suspension array for simultaneous detection of multiple biomarkers, see Fig. 10. These PMMBs realized 60-fold uorescence enhancement, and have improved the detection limit by 2-orders of magnitude toward 100 fM for biomarkers. 160 The multiplexing ability of the as-prepared PMMB platform is particularly attractive for simultaneous detection of multiple targets with high output efficiency in complex samples. And we predict that this MEF-based PMMB could be employed as the next generation probe for biomarker detection and disease diagnosis in a multiplexed manner.  As plasmon nanostructures can effectively enhance emission, the uorescence can be modulated via assembly of various plasmon structures. 75 Using the Ag NP-gold nano lm assembly via biorecognition binding, an over 800 fold dequenched uorescence signal was observed by Cao and his co-workers. 75 As shown in Fig. 11a, the sensing of target antigens can be conveniently conrmed by the intense uorescence signal. As this Ag NP-gold nano lm assembly works in an 'off-on' mode with a neglectable initial background signal, it may have limitless potential applications in biosensing. Their study may pave ways for plasmonic coupling assembly and application.
By incorporating immune hybridization chain reaction (HCR) and CD MEF, Xu and his co-workers constructed a dual amplication uorescence sensor for alpha fetal protein (AFP). 38 With the help of the capture-antibody coated plasmonic slide of gold island lms (Fig. 11b) and the detection antibodyconjugated oligonucleotide initiator, HCR was triggered aer the introduction of CD-tagged DNA hairpins, which are complemented to the oligonucleotide initiator. In this cleverly designed AFP sensor, CD emission was greatly enhanced by the gold nano island lm, HCR provided secondary uorescence amplication simultaneously, and the two together resulted in a 17.2-fold total signal amplication. A wide linear detection range of over 5 orders of magnitude between CD emission and AFP concentration (0.0005-5 ng mL À1 ) was obtained, with a low detection limit of 94.3 fg mL À1 . They also realized detection of real samples using this dual amplication uorescence assay method. 38 An immunosensor for immunoglobulins (IgGs), with the uorescence signal enhanced by patterned Au NPs and speci-city realized via biological functionalization (see Fig. 11c), was developed. 95 The as-prepared device can directly detect IgGs from a drop of a patient's urine without any pretreatments, and a detection limit of 10 mg L À1 was realized with a linear response range of 10-100 mg L À1 . Further experiments proved its excellent specicity rarely interfered by other biomolecules and reliable analysis results comparable with those obtained using standard techniques. Liu and his co-workers developed an immunouorescence probe by combining MEF with the surface acoustic wave (SAW) technique to lower the detection limit for the carcinoembryonic antigen (CEA), 61 which is a prognostic biomarker of colorectal cancer (see Fig. 11d). By incubating with 50 nm silver nanocubes (AgNCs) on a SAW device with an optimal surface density, emission was plasmon enhanced, which improved sensitivity to antigens by a factor of 6 and lowered the detection limit down to below 1 ng mL À1 . Moreover, nonspecically bound proteins were much more effectively removed and incubation times was   tremendously reduced by the introduction of acoustic streaming. Overall, clinical levels of colorectal cancer biomarkers can be detected using this AgNC-based MEF probe.
Deng and his co-workers constructed another core-shell Ag@SiO 2 @SiO 2 -RuBpy nanocomposite for the detection of prostate specic antigen (PSA) using the target-triggered MEF 'turn-on' strategy, with RuBpy acting as the donor and BHQ-2 as the acceptor. BHQ-2 was brought to close proximity to the surface of the RuBpy-doped silica shell aer hybridization occurred ('off' state). However, with the addition of target PSA, the BHQ-PSA aptamer could be dissociated from the RuBpydoped silica shell ('on' state). Thus PSA quantitative analysis can be realized by recording uorescence intensity, and a detection limit of 0.20 ng mL À1 was obtained under current experimental conditions. 28

RNA and DNA detection
MicroRNAs (miRNAs), as noncoding RNAs, play crucial roles in regulating diverse physiological functions. Expression levels of tumor-related miRNAs may predict tumor growth and invasiveness, and thus their accurate detection is important. However, as a result of their low expression levels, miRNA detection remains a challenge. And MEF has also been applied for RNA and DNA analysis for its much lower detection limit (see Table 5).
As reported, a stronger electromagnetic eld can be generated in the gap regions of two neighboring particles, which will result in a much more dramatic uorescence enhancement. 163 Thus a gold nanogap antenna through target-triggered assembly of Au NRs was constructed by Peng and her coworkers. Dual-amplication of the uorescence signal was realized via combining the MEF effect of nanogap antennas and a strand displacement amplication (SDA) reaction, see Fig. 12.
In the presence of target miRNA, uorophores can be settled into the gap region of the gradually formed end-to-end Au NR dimers. Thus quenched uorescence induced by the Au NRs exhibited a dramatic enhancement by the MEF of the nanogap antennas, and a 'turn on' uorescence signal was observed. The SDA reaction resulted in secondary uorescence amplication simultaneously. A low detection limit of 97.2 Â 10 À18 M miRNA was realized by combining this method with the single-molecule counting technique. 98 Moreover, this proposed method has potential in monitoring expression levels for lowabundance nucleic acid biomarkers via miRNA imaging.
Protein and DNA detections are important steps for biological and diagnostic assays. Thus Ji and her co-workers developed a Ag lm/Ag zigzag nanorod array multilayer lm for DNA detection (as shown in Fig. 13A). With a folding number of 7, a 14-fold uorescence enhancement of alex488 was obtained, and the detection limit was determined to be 0.01 pm. Moreover, another study by Li and his co-workers using Au NPenhanced QD emission for detection of DNA was developed, and a high sensitivity of 19.6 pg was established by controlling the distance between the QDs and Au NPs using different numbers of oligonucleotides. 118 Mei and his co-workers developed an innovative gold nanorod (GNR) array biochip with vertical standing arrays of ordered GNR on a glass surface, as shown in Fig. 13B. 100 As shown in Fig. 13B, the initial uorescence of the biochip is minimal when the hairpin-structured ssDNA probe is conjugated to the array, as a result of quenching induced by nearby GNRs. However, the hairpin loop could be opened up with the formation of duplex DNA via hybridization, which brought the uorophore away from the GNR array (45-nucleotide-long). And this process can induce  100 Fluorophore ssDNA A linear range from 10 pM to 10 nM ORA-enabled molecular beacons 152 CDs DNA LOD of 300 fM; with four orders of sensitivity improvement a dramatically intensied uorescence signal, which could be used for quantitative ssDNA detection with a linear range from 10 pM to 10 nM. Further, Kannegulla and his co-workers reported an Ag open-ring nanoarray (ORA)-enabled molecular beacon (MB) probe for DNA detection (see Fig. 13C), which yielded a detection limit of $300 fM (equivalent subattomoles), four orders of improvement compared with that using a plane Ag substrate. 152 The ultrahigh sensitivity resulted from both the intensied uorescence signal due to MEF of the ORA-enabled MB platform and the reduced background signal level.

Enzyme detection
Acetylcholinesterase (AChE) in human blood is considered to be a biomarker of neurotoxin exposure, which inhibits AChE. Thus its trace detection in human blood is important. So far, scientists have also developed various MEF-based sensors for enzyme detection (see Table 6). For example, Ma and his co-workers designed a new kind of Ag@SiO 2 core-shell NP based in situ probe for AChE by combining the AChE catalytic reaction with MEF. 94 The surface of Ag@SiO 2 NPs is negatively charged, while AChE can catalyze acetylthiocholine (ATCh) hydrolysis to positively charged thiocholine (TCh), and thus the negatively surface charge of the core-shell Ag@SiO 2 NPs can be reversed as a result of electrostatic adsorption of TCh. Then the negatively charged uorescent dye (8-hydroxypyrene-1,3,6-trisulfonic acid, HPTS) was conned to the Ag@SiO 2 NP surface, which resulted in an enhanced uorescence signal. A dynamic range of 0-0.005 U mL À1 AChE was detected via this mechanism, with a detection limit of 0.05 mU mL À1 . 94 This work provides a new way of design for MEF-based probes for biomolecules. Moreover, Xu and his co-workers developed a novel solutionbased nanoprobe for in situ uorescence visualized 'turn on' detection of telomerase in live cells, 92 which is one of the most common biomarkers for cancer diagnosis and pathogenesis. In this work, gold nanobipyramids (Au NBPs) were used as both the uorescence resonance energy-transfer (FRET) quencher and MEF signal enhancement substrates of Cy5.5. Aer being conjugated to the Au NBPs, as a result of FRET, the uorescence of Cy5.5 was totally quenched, while with the addition of deoxyribonucleotide triphosphates (dNTPs) and telomerase, the hairpin loop was opened, followed by the dequenching of Cy5.5. The best MEF factor can be obtained by adjusting the number of oligonucleotide bases. Thus this kind of nicked molecular beacon-functionalized Au NBP is a dual-functional substrate, and a low detection limit of 23 HeLa cells with a dynamic range of 40-1200 was obtained (Fig. 14A). In situ dynamic telomerase activity uorescence imaging in live HeLa cells was realized as a result of its outstanding biocompatibility, stability and specicity. And cancer cells were also successfully distinguished from co-cultured normal ones, proving its potential in clinical diagnosis. 92 This work undoubtedly demonstrated a new pathway for designing sensitive and specic MEF based probes for cancer-related biomolecules.
Proteases are recognized as biomarkers of various diseases. Lucas and his co-workers reported a MEF-based 'turn-off' assay for proteolytic enzymes using a nano-silvered 96-well plate. And the use of uorescein isothiocyanate-labeled YebF protein as a coating layer for the enzymatic activity assay using trypsin as the model enzyme was demonstrated (as shown in Fig. 14B). 96 A detection limit of 1.89 ng was achieved, corresponding to 10% uorescence quenching. However, the present work needed lengthy incubation times and washing steps, and they also put forward that the overall assay time might be shortened via a microwave-accelerated MEF technology in the future assay design.

ATP detection
Adenosine triphosphate (ATP), as a typical energy storage molecule, can transport chemical energy for cellular metabolism processes. It is an important biomarker for disease diagnosis. Compared to electrochemical and colorimetric methods, uorescent sensors exhibit the ability to measure ATP in real-time. However, lower detection limits of the present uorescence methods for ATP are still at ranges of millimolar to micromolar. Developing ATP uorescent sensors with high sensitivity and stability is highly desirable, which can be realized by combining MEF with uorescence methods (as summarized in Table 7). And we will review these advances in this part.
Song and his co-workers presented a label-free aptasensor by using the Ag@SiO 2 NPs core-shell structure and the common nucleic acid stain PicoGreen (PG) as a uorescent indicator, for highly sensitive and selective detection of ATP in aqueous solutions (see Fig. 15A). 120 A signicant uorescence reduction was observed in the presence of ATP, as a result of the aptamer release from the complementary DNA (cDNA)/aptamer duplexes conned on the Ag@SiO 2 NP surface. 120 And this aptasensor achieved a wide linear detection range for ATP with a detection limit of 14.2 nM, exhibiting its excellent assay performances. This work may provide new avenues for assembly of MEF-based label-free biosensors.
Near-infrared uorescence (NIR) is extensively used in biological elds for its high penetration, low photothermal damage and immunity from autouorescence. However, common NIR uorophores suffer from low quantum efficiency. As optical antennas with anisotropic nanostructures have been widely used in the eld of MEF, Yu and her co-workers developed a NIR based MEF platform of functionalized gold nanorods for the bioassay of ATP. 164 As shown in Fig. 15B, in the absence of ATP, the self-hybridized DNA sequence brought the IR dye 800CW close to the AuNR surface, which resulted in a 'uorescence off' state, while the presence of ATP could disrupt this selfhybridization and push IR dye 800CW away from the AuNR surface, as a result of its higher specic affinity with the aptamer moiety. A low detection limit of 0.634 nM was obtained for ATP as a result of the greatly enhanced quantum efficiency of IR dye 800CW from 0 to 0.61. The biosensor was also successfully applied to detect low levels of ATP in rat brain, demonstrating its applicability for monitoring intracellular ATP.
Aptamers are a kind of recognition molecule composed of single-stranded nucleic acid that folds into special 3-D structures, and can specically bind to targets with high affinity. 165 They have advantages of low molecular weight, high stability and ease of chemical synthesis compared to antibodies. Thus Song and his co-workers developed a label-free uorescence aptasensor for highly sensitive and selective detection of ATP and thrombin, with PicoGreen (PG) used as a signal molecule and silver island lms (SIFs) as MEF substrates for signal enhancers. 120 PG uorescence can be magnied by SIFs without ATP or thrombin, as shown in Fig. 15C. However, with the presence of ATP or thrombin, as the aptamers underwent structure switching, PG uorescence intensity was reduced. And detection limits of 1.3 nM and 0.073 nM were obtained for ATP and thrombin respectively using this method. ATP and thrombin could be linearly detected in ranges from 10 nM to 100 mM and 0.1 nM to 100 nM in the logarithmic scale, respectively. And this aptamer can also be reliably used for ATP measurements in biological samples.

Detection of other biomolecules
Besides applications in immunoassays, RNA/DNA, enzyme and ATP detection, uorescence detection of other biomolecules such as PPi, dopamine (DA) and riboavin (RF) using MEF has rapidly developed, and we will summarize these recent reports in this section (Table 8).
Based on a similar sensing mechanism for Cu 2+ , PPi and PPase previously referred to in ref. 56, another two Au@SiO 2 based core-shell nanostructures composed of Au nanorods (NRs) and elongated gold nanobipyramids (Au NBPs) encapsulated by SiO 2 were reported and used for NIR detection of PPi, 60,62 which is closely related to the DNA replication process and the genetic information expression. As shown in Fig. 16A, meso-tetra(4-carboxyphenyl)porphyrin (TCPP) molecules were covalently immobilized onto the outer shell surface of the AuNR@SiO 2 . 60 As a result of the strong affinity between Cu 2+ and PPi, the turn-off state of TCPP-Cu 2+ can be disassembled, and uorescence was recovered. Combined with the MEF imparted by AuNRs, a detection limit of 8.20 Â 10 À7 M PPi was realized. Cell imaging using this sensor was also realized, proving its potential applications in biological mechanism studies.
Both experiments and theoretical simulations indicate that Au NBPs can induce signal enhancement several times higher than that of Au NRs with similar longitudinal plasmon resonance wavelength. Thus Niu and her co-workers demonstrated a novel NIR MEF system composed of an elongated gold nanobipyramid (Au NBP) antenna core, a silica shell and a NIR dye (see Fig. 16B). 62 The largely enhanced uorescence could be quenched by Cu 2+ and further recovered by PPi, owing to the stronger affinity between Cu 2+ and PPi. And this provided a method for 'switch-on' detection of PPi with a limit of 80 nM in aqueous solutions.
Furthermore, the probe was used for microRNA detection with a low detection limit of 8.4 pM. 62 Dopamine (DA) acting as an important neurotransmitter has always been a bioresearch focus. Yang and his co-workers fabricated a novel zinc-salophen (ZnSa) complex nanowire (NW)-Ag NP 1D hybrid nanostructure. Narrow gaps between the Ag NPs, acting as optical antennas that can produce a largely enhanced electrical eld for signal amplication, are shown in Fig. 17a. Thus sensing occurring at these nanogaps can realize improved performance. The specic binding of DA with ZnSa NWs realized DA selective detection, while the introduction of Ag NPs induced a substantially improved performance for DA  164 IR dye 800CW ATP LOD of 10 and 0.634 nM; successfully applied to detect ATP in rat brain Silver island lms (SIFs) 151 PG ATP and thrombin LOD of 1.3 and 0.073 nM, and linear detection ranges from 10 nM to 100 mM and 0.1 nM to 100 nM in the logarithmic scale for ATP and thrombin respectively detection via remarkable uorescence enhancements. And a detection limit as low as 3 nM was obtained. Excellent selectivity and long-term stability of the hybrid nanostructure were also predicted in the article. 51 Molecularly imprinted polymers (MIPs) are a kind of biomimetic receptor, synthesized through polymerization reactions in the presence of a template molecule. They are tailor-made for target molecules, and have antibody-like specic binding properties. 166 Herein, He and his co-workers reported a molecularly imprinted core-shell Ag@SiO 2 NP for sensitive and specic MEF assay of riboavin (RF), 99 as shown in    Aggregation-induced emission (AIE) molecules, which are non-emissive in the dissolved state while highly uorescent in the aggregated state, have advantages of a large Stokes' shi, excellent photostability, and high signal-to-noise ratio. 167 Thus Cui and her co-workers developed a MEF sensor based on AIE molecules for monitoring protein conformational changes. 106 A water-miscible sulfonate salt of 9,10-bis(2-(6-sulfonaphthalen-2yl)vinyl)anthracene (BSNVA) with excellent AIE properties was introduced into the MEF system to prepare the MEF-AIE sensor, using a PrP aptamer as the bridge (as schematically shown in Fig. 18). When mixed with cellular prion protein (PrPc), the MEF-AIE sensor is almost non-emissive, while brightly uorescent when mixed with disease-associated prion protein (PrPSc). Thus protein conformational conversion can be monitored through this PEF-AIE sensor. And a detection limit of 10 pM lower than that of the traditional AIE probe has been achieved in human serum samples. 106 This work realized signal amplication without labeling, and also provided new insights for protein detection and conformational monitoring.

Conclusions and prospects
In this review, we summarized the recent key advances of MEFbased uorescence sensing, including design, synthesis, assembly and applications of both solution-based colloidal NPs and plasmonic chip platforms. So far, applications of MEFbased uorescence sensing are at varying degrees of progress. In general, both environmental detection and bioanalysis of special target analytes using plasmonic MEF have been extensively studied, and much lower detection limits and more reliable results have been achieved. Future work may focus on improving the selectivity and anti-interference performance of sensors, which could be realized by combining MEF-based uorescence with other techniques including SERS, colorimetric methods, molecular imprinting and even immunoassays or aptasensor techniques. In particular, especially for bioassays, as there are usually more than one biomarker that can diagnose early stages of special diseases, we forecast that multiplexed assays using protein and nucleic acid could be a promising development direction. Furthermore, by preparing heteronanocomposite plasmonic nanostructures, owing to the synergistically enhanced optical properties of individual components and new features arising from the integrated systems, unexpected or improved sensing performances may be realized. 168 Lastly, integration of plasmonic MEF uorescence sensing with portable platforms, such as wearable devices and microuidics has always been the goal of current research, which would greatly accelerate transformations from conventional benchtop assays to powerful point-of-care tests. However, challenges still remain for its implementation, and we expect that this goal can be achieved in the near future.

Conflicts of interest
There are no conicts to declare.