Luminescence Nanoprobes for Drug Screening, Pharmaceutical Analysis, and Therapeutic Evaluations

Luminescence nanoprobes have been extensively investigated for many biomedical applications, ranging from cell/tissue imaging, real‐time monitoring pathophysiological changes, drug discovery, bioassays, image‐guided surgery, and therapeutic evaluations, mainly due to their excellent physicochemical properties such as tailorable size, controlled morphology, and regulatable luminescence performances. In particular, rationally designed luminescence nanoprobes with reasonable functional integration enable pharmacological effect‐based drug screening, quantification of biological/pharmaceutical agents, and monitoring therapeutic responses of different drugs. This review provides an overview of the applications of different types of luminescence nanoprobes in drug discovery and development, involving various nanoprobes for high‐throughput and high‐content drug screening, pharmaceutical analysis, and therapeutic evaluations in cancers, cardiovascular diseases, and other typical inflammatory diseases. Moreover, challenges and future perspectives regarding future applications of luminescence nanoprobes in the drug discovery field are also discussed.


Introduction
Over the past decades, tremendous advances have been achieved in the development of various types of luminescence nanoprobes DOI: 10.1002/adsr.202200103 for numerous biomedical applications, such as early diagnosis, [1] real-time monitoring pathophysiological changes, discovery of therapeutic targets, [2] drug screening, pharmaceutical analysis, image-guided surgery, [3] and therapeutic evaluations. [4,5]Owing to their unique physicochemical properties, such as a large surface area to volume ratio, tailorable size, controlled structure/morphology, and broadly regulatable luminescence performances, luminescence nanoprobes derived from nanoparticles (NPs) or other nanomaterials have received much more attention in pharmaceutical sciences.For example, nanoprobes with sizes comparable to antibodies own approximately 1500 potential sites for modification, which is notably more than those on antibodies. [6,7]The high specific surface area affords great surface functionality, which can be used to tune nanoprobes for in vitro and in vivo tissue or cell targeting, sensing, and imaging. [8,9]As well documented, NPs with appropriate biophysicochemical properties, such as size, morphology, and surface chemistry, can efficiently accumulate in specific tissues and/or diseases sites, cells, and subcellular organelles.These outstanding features have enabled luminescence nanoprobes to monitor specific biological processes, study targeting capability of nanomedicines, determine interactions between drugs and targets, discover novel therapeutic targets, perform pharmacological effect-based drug screening, and evaluate drug efficacies.
Besides their effects on tissue and cellular distribution, physicochemical characters (such as size and morphology) of NPs can affect the luminescence ability of some nanoprobes, such as quantum dots (QDs) and plasmonic NPs. [10,11]For example, the emission wavelength of QDs can be precisely regulated by their particle size, with the large size generating long-wavelength fluorescence.In addition, the optical and catalytic properties, as well as surface-enhanced Raman scattering (SERS) or plasmon resonance energy transfer signals can be adjusted by the morphology of nanoprobes. [12]Therefore, luminescence characters of different types of nanoprobes can be tailored, according to their specific biomedical applications, thus affording precision readout for in vitro/vivo imaging, biosensing, and bioassays.[15][16][17] For example, chemiluminescence NPs can be coupled with traditional

Luminescence nanoprobes Applications References
Terbium ion-carbon dots Quantitative monitoring of ATP [202]   Molecularly imprinted polymer-carbon dots Evaluation of AChE activity and screening its inhibitors [203]   CdSeTe QDs-fibrils nanocrystals Screening drugs for modulating the prion-like transmission [204]   CuFeS 2 QDs Detection of cyclin D1 for screening anticancer drugs [205]  popular explanation of DS is to investigate the interactions of candidate drug molecules with pharmacological targets and corresponding biological effects, by using specific models and testing strategies. [30,31]The emphasis of DS lies in the evaluation of biological activity, pharmacological detection, and medicinal value with proper DS methods.In this regard, drug discovery has been accelerated by the rapid development of high-throughput screening (HTS) technologies for rapid candidate molecule screening, new pharmacological target exploration, virtual screening to understand structure-activity relationships, and pharmaceutical applications of Omics approaches to seek health and disease-related targets.In the past decades, noninvasive optical imaging has played an important role in drug discovery, especially the screening platform based on luminescence nanoprobes.As a powerful tool for HTS, luminescence nanoprobes can provide abundant and accurate biological information, including the interaction between candidate drugs and targets, in vivo drug distribution and metabolism, drug efficacies, and possible toxicities based on the variations of absorption/emission spectra, luminescent intensities, and other imaging parameters.Furthermore, luminescence nanoprobes can provide HTS with desirable rapidity and sensitivity for analytes detection. [32]In this section, different strategies and corresponding examples of luminescent probes, mainly including QDs, upconversion nanophosphors, dye-doped luminescent nanoprobes, and fluorescent gold nanoclusters, are summarized and discussed (Table 1).

Quantum Dots (QDs)
QDs, i.e., semiconductor nanocrystals, are commonly synthesized from binary mixtures of III-V or II-VI semiconductor materials, with unique quantum-confined photonic and electronic properties resulting from their sizes being smaller than the exciton Bohr radius of the bulk constituent materials. [33]Owing to the specific structure, QDs possess excellent photophysical properties, such as size-dependent and symmetric photoluminescence spectra ranging from ultraviolet (UV) to near-infrared (NIR), strong resistance to photobleaching and chemical degradation, high quantum yield, and large Stokes shift, which permit the development of novel bioimaging strategies. [34]The synthesis of nanoprobes based on QDs generally involves two steps.First, QDs are prepared by organic phase or aqueous phase methods, affording QDs with specific physicochemical characteristics.Similarly, glowing graphene QDs and CDs can be synthesized using top-down and bottom-up approaches. [35]The former involves cleaving or breaking down carbonaceous materials, while the latter is based on pyrolysis, carbonization, or a step-wise chemical fusion of small aromatic molecules. [36]Second, surface engineering is commonly performed to improve the quantum yield and other functionalities of QDs, in which controlled oxidation, polymer passivation, and conjugation of different functional moieties Figure 1.Schematic of the fabrication and application of a ratiometric fluorescent nanoprobe for imaging of singlet oxygen generation and intracellular survivin mRNA.Reproduced with permission. [38]Copyright 2020.Royal Society of Chemistry.are frequently involved. [37]Herein we mainly focus on the applications of QD-based nanoprobes for DS.The superior photophysical characteristics of QDs combined with advances in microscopy notably simplify technical requirements for a cell-based drug discovery process, thus properly meeting the demand for HTS.
To date, considerable efforts have been made toward the establishment of QD-based screening systems.For example, Lin et al. designed and synthesized a nanoprobe for photodynamic therapy (PDT) of tumors as well as cancer cell identification and antitumor drug screening based on ratio-metric fluorescence imaging of intracellular survivin mRNA (Figure 1). [38]The engineered nanoprobe can detect the high expression of survivin, a strong apoptosis suppressor protein, on the tumor cell surface.Based on the combination of biomass QDs and DyLight680-labeled singlestranded DNA, this nanoprobe enabled screening of survivin inhibitors as potential anti-cancer drugs.As a proof of concept, sepantronium bromide (YM-155), an antitumor drug, was used to establish the screening model to test the developed nanoprobe.Comparison of the control and YM-155 groups indicated that the fluorescence intensity of nanoprobe-labeled cells at 670 nm notably decreased, since YM-155 significantly inhibited the survivin expression level in tumor cells.This suggested that the proposed ratiometric fluorescence imaging method can be used to screen antitumor drugs.Chang et al. reported a new antagonistconjugated QD nanoprobe for screening antidepressant medications by monitoring single serotonin (5-HT) transporter proteins on the surface of serotonergic cells (Figure 2a). [39]Typically, the potential of QDs surface functionalization was fully utilized in this study.Liu et al. developed -cyclodextrin-coated QDs to detect the -glucosidase activity and screen related inhibitors in a sensitive manner, since p-nitrophenol, a hydrolysis product of the glucosidase reaction, can quench fluorescence of -cyclodextrincoated QDs via an electron transfer process (Figure 2b). [40]Notably, the discovery of antivirals for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a hot area of research since 2019. [41]The major limitations for relevant studies lie in the strict requirement of containment facilities.One way to solve this problem is to establish appropriate SARS-CoV-2 models.For example, Gorshkov et al. reported a new screen model of SARS-CoV-2 based on QDs conjugated with a recombinant Spike receptor binding domain.This approach allowed to monitor the binding event in a solution due to fluorescence quenching generated by the energy transfer between the nanoprobe with angiotensin-converting enzyme (ACE) 2-conjugated gold NPs, resulting in an efficient and facile biosensor for biochemical and cell-based assays. [42]ollectively, owing to beneficial photophysical characteristics and the versatility of surface functionalization, the utilization of QDs has considerably promoted the development of HTS.Copyright 2012.Society for Neuroscience.b) Schematic illustration of the -glucosidase inhibitor screening mechanism.Reproduced with permission. [40]Copyright 2015.Wiley.

Upconversion Nanophosphors (UCNPs)
UCNPs are a type of rare-earth metal-doped NPs with NIRto-visible luminescence generated by a non-linear and anti-Stokes process, whereby low-energy photons are converted to high-energy photons. [32]Depending on their size, structure, morphology, crystalline phase, and purity, UCNPs exhibit various upconversion photoluminescence efficiencies and emission wavelengths. [43,44]To date, numerous synthesis techniques have been employed to produce UCNPs, including thermal decomposition, coprecipitation, solvo-hydrothermal synthesis, solgel, microemulsion, electrospinning, mechanochemical preparation, and microwave synthesis. [45]Compared with QDs and traditional organic dyes, UCNPs have unique characteristics of minimizing the background interference due to autofluorescence of bio-samples and enhancing tissue penetration.This indicates that UCNPs can be used for establishment of DS platforms with strong anti-jamming capability.Lanthanide (Ln 3+ )doped luminescent inorganic nanocrystals (NCs) are representative UCNPs, which possess high chemical stability and superior optical features such as long-lived luminescence (from several to tens of milliseconds), large antenna-generated Stokes or anti-Stokes shifts, narrow emission bands, and excellent photostability. [46]These desirable optical properties enable broad applications of Ln 3+ -doped NCs in biodetection, particularly in bioassays of model analytes, nucleic acids, ions, and diseaserelated biomarkers. [47,48]Nevertheless, the coordination environment plays an important role in fluorescence properties of the Ln 3+ -based sensing system, therefore appropriate confinement conditions are necessary to promote the maximal fluorescence signal. [49]In this aspect, Liang et al. developed a nanoprobe to monitor the activity of deubiquitinating enzymes (DUBs), a group of proteases capable of catalyzing the cleavage of proteinubiquitin bonds that are closely related to various diseases (Figure 3). [50]Specifically, mesoporous silica NPs (MSNs) were used to load terbium (Tb), and the confinement effect obviously enhanced the fluorescence emission property of Tb.Furthermore, the combination of rhodamine B-labeled ubiquitin (UBR) and MSN-Tb afforded fluorescence resonance energy transfer (FRET), in which fluorescence emission would be transferred from Tb to rhodamine B and this effect will be attenuated in the presence of DUB inhibitors.Overall, this research established a close relationship between the FRET signal of the constructed nanoprobe with DUBs or DUB inhibitors, thereby allowing the discovery and evaluation of potential inhibitors for DUBs.
Due to their unique and excellent properties, UCNPs show great potential in DS with a high sensitivity and high signal-tonoise ratio.Liu et al. developed a mix-and-read luminescence resonance energy transfer assay by integrating the outstanding features of rare-earth-doped UCNPs for highly selective recognition of protein kinases (PKs)-induced phosphopeptides. [51]By using UCNPs themselves as affinity materials to selectively capture phosphopeptides catalyzed by PKs, this strategy effectively improved the signal-to-noise ratio and greatly simplified the detection procedures.In other words, this type of nanoprobes can screen potential kinase-targeted drugs in a highly efficient manner.

Dye-Doped Luminescent Nanoprobes (DDLNs)
DDLNs are commonly described as certain NPs doped with small-molecular fluorescent dyes.In past decades, the convenience and easy to detection of fluorescence signals have drawn Engineering of a terbium-based nanoarchitecture probe and schematic illustration of detection of the deubiquitinating enzyme activity.Reproduced with permission. [50]Copyright 2020.Royal Society of Chemistry.
broad attention to traditional fluorescence dyes in DS.However, many limitations of conventional luminophores hinder their practical applications, such as poor photostability, low quantum yield, and aggregation under physiological conditions. [52]Recently, the increased numbers of pharmacological targets have promoted the efficiency and simplification of screening technologies.To overcome the drawbacks of traditional fluorophores, new efficient nanoplatforms have been developed to achieve good biocompatibility, weak autofluorescence, and higher penetration depth. [53,54]To obtain high-performance DDLNs, the chemical structure of the chromophore skeleton should be first optimized, such as introducing the donor-acceptor structure to realize redshift absorption and emission.Second, water solubility of organic dyes need to be increased by chemical modification with hydrophilic functional groups or polymers.In addition, fluorescent dyes should be doped into nanomaterials to improve their performance. [55]Importantly, the chromophore skeleton plays a crucial role for fluorescence properties of the used organic dyes.58] Dye-doped silica NPs (DDSNs) are regarded as successful examples, which exhibit synthetic versatility and all the required features to obtain high effective tools for bioimaging and DS.On the one hand, silica-coated materials have ideal physical properties, including stability, biocompatibility, and the capacity of binding with biomolecules.On the other hand, silica-coated materials can efficiently enhance the quantum yield and photostability of the coated dyes as well as improve their endocytosis by targeting cells and concomitantly preventing the coated dyes from capturing by tissues.Moreover, due to facile and versatile surface functionalization via either physical absorption or covalent conjugation, DDSNs enable efficient screening of candidate molecules and exploration of new therapeutic targets.
With the development of novel fluorescence dyes, DDLNs have played an important role in DS.For instance, Li et al. reported a two-photon fluorescence silica NP-based FRET nanoprobe platform, which showed ratiometric fluorescence responses for adenosine triphosphate (ATP) detection with high sensitivity and high selectivity in vivo. [59]By doping a two-photon fluorescent dye into silica NPs, the obtained nanoprobes possess unique advantages of two-photon dyes such as anti-Stokes shift, less fluorescence background, low light scattering, and high tissue penetration depth under NIR excitation.Meanwhile, this approach can overcome the disadvantages like high biological toxicity and instability of the fluorescence efficiency. [60]These excellent performances of the mentioned nanoprobe can effectively achieve DS assay for ATP-related diseases.In another study, Juan et al. reported a mucin 1-specific dye-doped NIR emitting mesoporous silica nanoprobe for monitoring the efficacy of anti-cancer drugs, based on the close correlation between mucin 1 and cancer progression. [61]This nanoprobe showed high targeting for mucin 1 and favorable permeability to the tumor tissue.

Fluorescent Gold Nanoclusters
Fluorescent gold nanoclusters (AuNCs) generally consist of several to a few hundreds of gold atoms. [62,63]Due to the unique structure, AuNCs possess many valuable characteristics, such as the high quantum yield, stability, biocompatibility, and particularly size-dependent fluorescence capacities. [64]Similar to CDs, both top-down and bottom-up approaches can be applied to synthesize AuNCs. [65]Currently employed synthetic techniques for AuNCs include chemical reduction, photoreduction, and chemical etching, among others. [66]Different factors, such as the types and concentrations of ligands and templates, Au 3+ concentration, and pH level, can affect the size, oxidation state, optical properties, and other key features of AuNCs. [67]igure 4. Drug detection using a nanoprobe AuNCs.a) Schematic of AuNCs formed by using HSA and HSA-warfarin as a template.b) Photoemission spectra of HSA-Au NCs prepared at 60 °C in the presence of different drugs.A control experiment was carried out in the absence of drugs.Reproduced with permission. [68]Copyright 2021.Royal Society of Chemistry.
AuNCs have been widely investigated in the field of DS, such as evaluation of the binding strength between candidate molecules and the targets.Yu et al. established a facile drug screening method by evaluating the relative affinity of various proteinbinding drugs by monitoring the formation kinetics of fluorescent AuNCs inside the protein-drug templates (Figure 4a). [68]pecifically, the authors prepared AuNCs using ligand-bound proteins under heat treatment and ingeniously built a relationship between the fluorescence intensity and binding affinity of a drug due to the size-dependent fluorescence capacities of AuNCs (Figure 4b).It was also found that the preparation conditions have remarkable effects on physicochemical properties and fluorescence performances of resulting AuNCs.Liu et al. reported a ratiometric fluorescence approach for rapid and facile screening of drug-induced acute kidney injury using chromophoremodified AuNCs. [69]Notably, AuNCs examined in this study were prepared using thiolate ligands such as glutathione (GSH), leading to the recruitment of GSH on AuNCs surface.Thus obtained GSH-coated AuNCs showed outstanding optical performance, high stability, low toxicity, and reactive oxygen species (ROS)responsiveness.In this way, drug-induced nephrotoxins were screened by monitoring the ROS level in kidneys.Further, aristolochic acid, tanshinone IIA, and geniposidic acid were chosen as model drugs, and their efficacies for drug-induced early renal injury were demonstrated using the developed nanoprobe.Also, the result implicated that the nanoprobe can accurately monitor the renal lesions caused by nephrotoxic triptolide, indicating that the examined nanoprobe has the potential to screen drugs for relevant diseases.Consequently, screening platforms based on AuNCs considerably enhance the screening efficiency for different candidate molecules mainly due to the simplified procedures, thus deserving further development of AuNC-derived systems for HTS.

Luminescence Nanoprobe-Mediated Pharmaceutical Analysis
In recent years, the pharmaceutical analysis field not only aims to develop innovative technologies for quantifying drug compo-nents and their biological effects, but also attempts to solve critical scientific issues encountered in pharmacology.In particular, there is much more emphasis on the collaborative innovation by integrating pharmaceutical analysis, analytical chemistry, biology, medicine, and other disciplines.Recent advances in fluorescent nanomaterial-based biosensoring platforms have enabled promising approaches for analyzing different therapeutic agents, varying from small-molecule drugs, biotechnological drugs, nanotherapies, and cellular therapies (Table 2).

Plasmonic Nanoparticles for Pharmaceutical Analysis
Plasmonic NPs are a special type of luminescence nanoprobes, and their unique optical properties mainly originate from surface plasmon resonance (SPR). [70,71]The past few years have witnessed broad explorations and applications of plasmonic NPs in the multidisciplinary fields ranging from photocatalysis to chemical and bio-sensing. [72,73]Interestingly, the optical, chemical, and catalytic properties of plasmonic NPs are closely correlated to their morphology and electron density. [74][77] Basically, chemical or physical stimuli from the local environment can be converted efficiently into optical signals with suitable plasmonic NPs, such as Au and Ag NPs that represent the most commonly employed plasmonic NPs because of their superior ability to support plasmons.Moreover, owing to excellent biocompatibility, tunable optical properties, good conductivity, and large surface areas for attachment with biomolecules, plasmonic NPs enable a broad range of biological sensing and imaging applications.Especially, as excellent optical reporters, plasmonic NPs are extremely sensitive to chemical composition, surface chemistry, and the surrounding medium, thus serving as diverse optical techniques for nucleic acid detection, protein determination, measurement of cellular redox substances, and cell imaging. [12]The following section mainly focuses on plasmonic nanoprobes for bioassay.

Quantification of Small-Molecule Drugs
As a powerful technique, SERS spectroscopy has numerous advantages, [78] thus enabling detection of different biomolecules at ultralow concentrations and with high sensitivity and specificity.By combining plasmonic NPs (such as Au and Ag NPs) and Raman reporter molecules, numerous SERS nanoprobes have been implied for quantification of drugs.Theoretically, local electromagnetic field enhancement of NPs can effectively improve the Raman scattering signal.Based on this issue, Adam and coworkers developed an extremely sensitive detection platform for kanamycin with a 2-mercaptobenzothiazole (MBT)-labeled Au-core@Ag-shell nanoprobe serving as a SERS substrate and anti-kanamycin functionalized hybrid magnetic NPs (Fe 3 O 4 ) functioning as a recognition substrate. [79]After separating kanamycin from the sample matrix with a recognition substrate, the SERS and recognition substrates were bound each other via kanamycin molecules (Figure 5a).When the captured kanamycin molecules were labeled with the Au/Ag nanoprobe, relatively sharp peaks at 1248, 1394, and 1578 cm −1 appeared because of the MBT Raman signal (Figure 5b).According to the results, the detection limit was 2 pg mL −1 , which was about 3-7 times more sensitive than that of previously reported probes.Furthermore, the method was also tested for the kanamycin specificity, by comparing with neomycin, gentamycin, and streptomycin.
In a separate study, Kang et al. developed a simple and sensitive system based on SERS competitive immunoassay for chloramphenicol (CAP) detection. [80]The method can overcome the drawback of conventional SERS immunoassay that is unsuitable for small molecule detection.Briefly, functionalized Au NPs were labeled with a Raman reporter molecule 4,4'-dipyridyl (4,4'-DP) and a chloramphenicol-bovine serum albumin conjugate (CAP-BSA).Meanwhile, magnetic NPs were modified with CAP antibody as a separation tool in this system.With the addition of free CAP, a competitive immune reaction was initiated between CAP and AuNPs conjugated with CAP-BSA (Figure 5c).After separation by a magnet, the quantity of SERS units in the supernatant would decrease.The higher the concentration of free CAP, the less the SERS signal associated with CAP antibody-modified magnetic beads would be.Thus, the SERS luminescence unit in the supernatant would be higher and the SERS signal obtained from the supernatant would be more intense.The underlying relationship between the SERS intensity and CAP concentration enabled the quantification of CAP levels.

Protein Quantification
To develop immunoassay methods with high sensitivity and selectivity for quantification of different drugs, the surface and morphology of plasmonic NPs can be modified with a variety of ) SERS spectra of MBT-labeled-Au@Ag NPs.Reproduced with permission. [79]Copyright 2014.Elsevier.c) SERS-based magnetic quantification for CAP.Reproduced with permission. [80]Copyright 2016.Elsevier.
antibodies to specifically bind with the corresponding antigens. [81]Lu et al. developed a fiber optic-SPR platform using plasmonic NPs for sensitive detection of infliximab (IFX), a therapeutic monoclonal antibody of tumor necrosis factor (TNF)-.In this study, gold coated optical fibers were functionalized with a carboxyl self-assembling monolayer (SAM), onto which the IFX specific monoclonal antibody, i.e., MA-IFX20G2, was conjugated as a capture antibody.In order to reach clinically relevant sensitivity for detecting serum IFX of patients, the SPR signal was amplified by employing Au NPs functionalized with other IFX specific antibodies, such as MA-IFX3D5 and MA-IFX6B7.The constructed optic fiber probes functionalized with the capture antibody were used to quantify IFX either directly or through a sandwich method with detection antibody.Notably, the performance of this IFX detection platform was validated using five IFX-treated patient samples, demonstrating an excellent intraclass correlation coefficient.Moreover, the assay time was significantly reduced, as compared to enzyme-linked immunosorbent assay (ELISA).
In other findings, a simple yet robust method has been established to quantify three different cancer-related proteins, i.e., carcinoembryonic antigen, prostate-specific antigen (PSA), and -fetoproten, by detecting scattering intensities of single NPs. [82]lasmon resonance energy transfer (PRET), an optical phenomenon arising between plasmonic NPs and molecular acceptors, similar to chemiluminescence resonance energy transfer (CRET) or FRET, will cause decrease of the Rayleigh scattering intensity of plasmonic NPs, thus providing special signal changes in immunoassays. [83,84]By utilizing Au NPs and QDs as donoracceptor pairs, Liu and coworkers developed separation-free homogeneous immunoassays for PSA quantification.To this end, detection antibody-modified QDs and capture antibody-modified Au NPs were indiscriminately linked to PSA, in which PRET occurred between AuNPs and QDs.The quenching efficiency of PRET can be adjusted by changing the distance between the donor and acceptor pairs.The authors found that the PRET efficiency was closely correlated with the PAS concentration, thus realizing PAS quantification. [85]Besides immunoassays, enzymes can be indirectly determined using plasmonic NPs.Because DNA conformations and peptide/protein structures can be regulated by diverse enzymes, the strategies capable of sensing or imaging nucleic acids are also suitable for measuring the activity of enzymes.For instance, a core-satellite assembled nanostructure of Au 50 @Au 13 has been constructed with a nicked hairpin DNA containing a telomerase substrate primer as the linker to connect the core (Au 50 ) and the satellite (Au 13 ).In the presence of telomerase, the nicked hairpin DNA formed a rigid hairpin structure, thereby leading to disassembly of Au 50 @Au 13 and notable decrease of the plasmon coupling effect.The engineered core-satellite NPs have been proved suitable for in situ monitoring the variation of telomerase activity in vivo and distinguishing cancer cells from normal cells. [86]

Nucleic Acid Detection
During the past decades, many elegant studies have demonstrated the outstanding performance of plasmonic NPs for nucleic acid detection.In 2002, a universal method named single particle counting for single nucleotide analysis was proposed by Oldenburg and coworkers. [87]Briefly, the level of single stranded DNA can be easily quantified according to the counts of light spots scattered by a single plasmonic nanoprobe under darkfield microscopy.The number of luminescence spots generated by target-bound nanoprobes (such as Au NPs) is highly correlated with the level of single stranded DNA, thus allowing quantification of gene expression with the sensitivity approximately 60-fold higher than that of traditional fluorescence intensity measurements. [88]In practice, however, the light spots still can be scattered by small defects, impurities, and other interfering substances.This issue considerably limits the application of plasmonic nanoprobe-based DNA detection, although some positive results shows excellent sensitivity. [89]o overcome the above mentioned limitations, another improved technique termed as plasmon coupling was proposed. [90,91]When the distance between two plasmonic NPs is 2.5 times shorter than the particle diameter, their plasmonic oscillations can be coupled together, and this phenomenon is called as plasmon coupling. [92]The formation of structure similar to the sandwich can vividly demonstrate the key process of this DNA detection technology.Also, plasmonic NPs can be modified by conjugating separately with two different nucleic acid probes and complementarily linked to the target DNA by sandwich hybridization.Therefore, the luminescence colors of individual nanoprobes and the dimers are naturally different from each other.The amount of formed sandwich luminescence particles represents the level of DNA.Using this technique, Xiao and coworkers successfully detected target DNA at a single molecule level. [93]In addition, two types of probes adopted guarantee specific counting of positive luminescence events, thereby circumventing the limitation of single particle counting. [94]urthermore, similar to the change in colors, the spectrum position of the hybridized plasmonic nanoprobes shifts either.Based on the spectrum peak shifts resulting from hybridization of Au NPs and mRNA, target mRNA splice variants in living cells can be tracked and quantified in single-copy sensitivity. [95]ue to the adjustability and improvability of the plasmon coupling method, its performance in nucleic acid detection has been optimized via many strategies.In this aspect, Hwu et al. designed a new device for high throughput micro-RNAs measurement on dark-field microwells, which can simultaneously quantify several nucleic acids. [96]However, it is difficult to distinguish the aggregation state of plasmonic NPs.The addition of target nu-cleic acids can result in the formation of both plasmonic NP dimmers and clusters containing more than 2 single particles.Identification of dimmers is generally relied on experience.To overcome this drawback, Liu et al. established a colorimetrybased algorithm to distinguish the aggregation states of plasmonic NPs. [97]In addition, uniform NPs derived from coupling of plasmonic NPs are suitable SERS substrates to produce desirable SERS signals with high efficiency and reproducibility.Accordingly, Qi et al. designed a uniform sunflower-like gold nanostructure, which was utilized to dynamically monitor DNA damage in apoptotic cancer cells at a DNA base level. [98]Another promising type of nanoprobes employed for nucleic acid detection by the SERS technique involves anisotropic plasmonic NPs, such as gold nanostars and nanorods.In this case, target nucleic acids were successfully detected with gold nanostar probes implanted in the pig skin by Wang and colleagues. [99]

Chemiluminescence Nanoparticle-Coupled HPLC/CE Analysis
Chemiluminescence (CL) is the emission of electromagnetic radiation, including ultraviolet, visible, or infrared light, by electronically excited intermediates produced via chemical reactions.Since no external light source is required for CL, it shows relatively low background signals.As a result, CL-based bioassays have been examined in clinical laboratory science, pharmaceutical analysis, and food safety studies. [100,101]It has been proved that diverse metal NPs can function as nanoprobes or nanointerfaces to initiate various liquid-phase CL reactions. [102]Studies by Zhang et al. indicated that the CL system of luminol-H 2 O 2 can be amplified by Au NPs. [103]Other metal NPs, such as Ag NPs, Pt NPs, and Au/Ag alloy NPs have also been found able to catalyze CL reactions, [104,105] particularly for luminol-based systems.Depending on the size and morphology, Au NPs can facilitate the radical generation and electron transfer processes on their surface, thereby affecting the catalytic activity.Of note, it has been demonstrated that Au NPs of 38-nm diameter exhibit the strongest catalysis activity, owing to the desirable electronic structure, large specific surface area, and high electron density.Inspired by these advantages of Au NPs, irregularly shaped NPs and triangular NPs have also been designed and applied in the CL reaction. [106]The catalytic efficiency of irregularly shaped or triangular NPs was notably higher than that of spherical NPs.In addition, it was found that a variety of organic compounds containing hydroxyl, amino, and mercapto groups can inhibit the CL signals in the AuNP-luminol-H 2 O 2 system, which makes it possible to apply this system for quantification of catechol, epinephrine, norepinephrine, dopamine, and cysteine. [103]By virtue of the AuNP-catalyzed luminol-H 2 O 2 CL system, the concentrations of fluoroquinolone derivatives and estrogens could be determined. [107]espite the high sensitivity of the metal NP-luminol-H 2 O 2 reaction system, its applications in complicated biological samples are still limited due to the poor selectivity.To address this issue, two powerful separation methods, i.e., HPLC and CE, were introduced to couple with the CL detection. [108]For instance, triangular Au NPs were used as a novel postcolumn CL reagent to establish a simple HPLC-CL method for quantifying low molecular weight aminothils, including cysteine, glutathione, cysteinylglycine, and glutamylcysteine. [109]Zhang and coworkers established a novel online catalyzed CL detection platform in tandem with HPLC, in which Au NPs were produced by online reaction of H 2 O 2 , NaHCO 3 -Na 2 CO 3 , and HAuCl 4 .The NP-catalyzed CL detector satisfied quantitative requirements of eight different phenolic compounds, such as caffeic acid, dihydroxybenzoic acid, gallic acid, and protocatechuic acid. [110]CE is another important complement separation technique for HPLC owing to the minimal sample volume requirement, short analysis time, and high efficient separation.The metal NP-involved system coupled with CE has been considered as a promising technology in CLbased bioassays.For instance, uric acid in serum was determined with an AuNP-enhanced CE-CL detection platform. [111]Uric acid eluted from CE showed inhibitory effects on the AuNP-mediated CL reaction between luminol and H 2 O 2 .According to the correlation between the suppressed CL intensity and serum concentrations of uric acid, uric acid was quantified with high sensitivity.Furthermore, Zhao et al. also introduced AuNPs to enhance the sensitivity of luminol-H 2 O 2 CE-CL detection system for quantification of epinephrine and norfloxacin. [112]

Luminescence Nanoprobe-Mediated Chiral Discrimination
In the nature, many important biological species are chiral, such as amino acids, proteins, enzymes, and nucleic acids.Although enantiomers of chiral molecules exhibit similar physicochemical properties, they generally display notably different biological activities, metabolic mechanisms, pharmacological functions, and toxicities.Therefore, chiral discrimination is a critical issue in the research areas of biochemistry, biotechnology, asymmetric catalysts, and pharmaceutical industries.Traditionally, electrochemical, [113,114] spectroscopic, and chiroptical methods have been used for chiral identification.In recent years, NPbased probes for chiral discrimination and quantification have attracted more attentions, due to their outstanding physicochemical properties and fine-tuning of surface properties.In particular, luminescent QDs and CDs are mainly used nanoprobes in this field.

QDs
As a popular type of fluorescent nanoprobes, QDs have been extensively used for the chiral recognition of drugs, amino acids, and other biomolecules because of their exceptional advantages, including excellent chemical stability, broad absorption spectra, and narrow emission bands.Meanwhile, cyclodextrins, a type of cyclic oligosaccharide composed of glucopyranosyl units linked via -1,4-glycosidic bonds, are special chiral selectors originating from their intrinsic chirality and guest-inclusion capacity.Therefore, cyclodextrin-capped QDs are commonly employed for identification of chiral compounds, taking advantage of changes of the fluorescent intensity of nanoprobes in the presence of host-guest interactions between cyclodextrins and target enantiomers.For instance, -cyclodextrin and -cyclodextrin with different cavity sizes were used as surface coating units to modify CdSe/ZnS QDs for chiral recognition of methionine and tyrosine. [115,116]The addition of L-methionine or L-tyrosine to cyclodextrin-QD pairs restricted the cyclodextrin conformation and induced a uniform arrangement.Concomitantly, the luminescent intensity of probes was enhanced by suppressing the quenching path to the medium attributing to the ordered orientation and conformational rigidity of the surface.In addition, -cyclodextrin-modified CdSe/ZnS QDs have been used for the recognition of D-penicillamine (PA), a toxic enantiomer of penicillamine. [117]Selective identification of L-PA and D-PA was accomplished by virtue of host-guest recognition between PAs and -cyclodextrin pockets on the QDs surface.In the presence of L-PA, the fluorescence intensity of modified QDs decreased, while it was increased with D-PA.Notably, the fluorescence intensity was closely correlated with the D-PA concentration, showing a good linear relationship, which was used for D-PA analysis in pharmaceutical formulations.Furthermore, based on the FRET mechanism, other strategies have been proposed for chiroselective sensing of enantiomers using cyclodextrin-functionalized CdSe/ZnS QDs. [19]In this case, FRET between QDs and rhodamine B incorporated in the -cyclodextrin receptor was used for optical discrimination of D/L-phenylalanine and D/L-tyrosine through competitive analysis.The competitive displacement of rhodamine B by the chiral target resulted in the decreased intensity of FRET emission and enhancement in luminescence of QDs (Figure 6a).
Besides cyclodextrins, porphyrins, chiral amino acids, and chiral drugs have been used to decorate QDs for accomplishing chiral discrimination based on different mechanisms.Particularly, as fluorescence quenchers of QDs, porphyrins have received considerable attention in chiral recognition.As a typical example, an "On-Off-On" fluorescent platform has been established by chiral self-assembly of porphyrin (ZnTPyP) and Nacetyl-L-cysteine-capped CdTe QDs for chiral discrimination of amino acids. [118]Competitive binding of ZnTPyP with D-proline, D-lysine, and L-serine increased the distance between ZnTPyP and QDs.Thus the fluorescence signals of CdTe QDs were switched between quenching by chiral ZnTPyP and then restoring in the presence of enantiomer amino acids.Furthermore, chiral amino acid-functionalized QDs have received much attention in chiral recognition, because the homochiral interaction in such systems is weaker than that of heterochiral ones.For instance, chiral cysteine-capped CdSe/ZnS QDs were adopted to determine carnitine enantiomers.In this case, L-and Denantiomers of carnitine selectively quenched the fluorescence intensity of D-and L-cysteine-capped QDs owing to the homochiral and heterochiral interactions on the surface of QDs. [119]In other cases, chiral drugs have been employed for surface modification of QDs, which were utilized for recognition of chiral drugs and amino acids.Delgado-Perez and coworkers designed a chiral nanoprobe using CdSe/ZnS core-shell QDs capped with N-acetyl-L-cysteine methyl ester for analyzing enantiomers of drugs, especially aryl propionic acid analogues, such as ketoprofen and flurbiprofen. [120]The enantiomers of tested drugs quenched the QDs fluorescence in a concentration-dependent manner, due to the reorganization of chiral ligands on the surface of QDs.This fluorescence quenching system demonstrated extraordinary capacity for chiral discrimination in model drugs.Reproduced with permission. [19]Copyright 2009.American Chemical Society.b) The recognition mechanism of D/L-glucose achieved through the formation of CDs@AuNPs complexes triggered by a stereoselective enzymatic reaction.Reproduced with permission. [123]Copyright 2018.Royal Society of Chemistry.c) Nanopaper-based enantioselective recognition of D/L-lysine (L-Lys) using L-cystine capped CDs, and the nanoprobe exhibited dosedependent fluorescence enhancement upon the addition of L-lysine.Reproduced with permission. [124]Copyright 2018.Elsevier.

CDs
CDs, as the newest member of carbon-based nanomaterials, have aroused great interest in various fields in most recent years, due to their unique optical properties and abundant functional groups.The application of CDs in stereospecific molecular recognitions has opened a new window for the design of chiroptical nanoprobes. [121,122]For identification of enantiomers with CDs, surface modification with free chiroptical sensors is generally Glucose oxidase, a natural chiral ligand selector, was first introduced for chiral analysis by Zhou and coworkers. [123]Briefly, a novel dual-mode chiral responsive platform using a CDs@AuNPs complex for the recognition of glucose enantiomers was developed.Based on the stereoselective enzymatic reaction, D-glucose was selectively catalyzed by glucose oxidase, which induced the production of H 2 O 2 and led to the accelerated formation of CDs@AuNPs.Due to the conjugation of CDs with Au NPs, the fluorescence intensity of CDs decreased via energy transfer (Figure 6b).
Among different molecules used for surface modification of CDs, amino acids can provide ideal chiral selectors.Copur et al. used L-cystine to produce enantioselective CDs for nanopaperbased sensing of L-lysine. [124]With the addition of L-lysine, the fluorescence intensity of nanoprobes was enhanced, while no noticeable response was observed in the presence of D-lysine (Figure 6c).In the presence of lysine enantiomers, changes in the fluorescence intensity of CDs are attributed to the restriction of intra-molecular vibration and rotation of functional units as a result of the interaction between lysine and CDs, leading to the signal enhancement for visual chiral discrimination of D/L-lysine.Interestingly, porous organic cages (POCs) with chiral frameworks can also be linked to CDs to afford chiroselective characteristics.In a previous study, Lu et al. prepared a novel CD-decorated chiral POC hybrid nanocomposite (CD@RCC3) for enantio-recognition of nitrophenol isomers as well as phenylalaninol and phenylethanol enantiomers. [125]The authors supposed that the formation of strong complexes is due to binding of the acidic -OH proton of target phenolic compounds to the basic amino unit of CD@RCC3.The chiral recognition ability of the complex toward target alcohols was considered to be mainly caused by the numerous NH active sites and chiral pockets with high affinity to chiral molecules.

Luminescence Nanoprobe-Mediated Therapeutic Evaluations
As described above, many luminescence nanoprobes possess unique properties, which enable them as a fundamental tool for many biomedical applications, such as drug screening and pharmaceutical analysis.In addition, nanoprobes provide new tools for precise monitoring of therapeutic responses of different diseases, such as cancer, [126,127] cardiovascular diseases, [128,129] and other inflammation-associated diseases. [130,131]Luminescence nanoprobes can be rationally designed to provide detailed information regarding structures of biomolecules and relevant biological processes at the subcellular level, thus benefiting both fundamental and translation studies.] Here we highlight recent advances in luminescence nanoprobe-mediated therapeutic evaluations in several diseases.

Cancer
Despite great achievements in cancer diagnosis and treatment over the past decades, cancer remains the leading cause of global mortality.The development and application of high spatiotemporal resolution luminescence nanoprobes can simultaneously achieve specific tumor biomarker detection and real-time monitoring of therapeutic effects, making them promising candidates precision medicines for tumors. [134,135]urrently, a large number of luminescence nanoprobes are available for cancer diagnosis and therapeutic evaluation by different imaging strategies. [136,137][140] Several diagnostic and therapeutic NPs have been developed based on the strong luminescence of metal nanoclusters (MNCs) in combination with multiple treatment methods, which have promising development prospects in cancer therapeutic evaluation. [141,142]In addition to strong luminescence, MNCs have the ability to produce ROS and photothermal effects under excited light, thereby showing PDT capacity. [143]Also, CDs can act as a luminescence imaging and synergistic anticancer agent by combining with photothermal therapy, PDT, and chemodynamic therapy. [144,145]According to the latest development, Ag 2 S QDs can function as a new type of nanoprobes for fluorescent imaging in NIR and second near-infrared (NIR-II) regions due to their dual-modality imaging properties and photothermal effect. [146]Moreover, Ag 2 S QDs exhibit better biocompatibility and a versatile surface for modifications with specific ligands or biocompatible molecules for their different biomedical applications, as compared to traditional QDs.In a decent study, Ag 2 S QDs were covalently conjugated with a CXCR4 antagonist, i.e., AMD3100, for accurate detection of CXCR4 expression in tumors. [147]Particularly, the obtained theranostic nanosystem (QD-AMD) can accurately distinguish highly metastatic breast cancer cells from normal cells, therefore it is promising for identifying, tracking, predicting, and inhibiting the metastatic spread of breast tumor in vivo.Notably, correlative analysis showed that the fluorescence intensity of QD-AMD in the lung decreased with the number of lung metastatic 4T1 tumor cells.Therefore, the information revealed by this theranostic nanosystem can be used to evaluate therapeutic effects of different treatments in mice with breast cancer lung metastasis.
As well recognized, pathophysiological features of the tumor microenvironment, including slightly acidic pH, hypoxia, overexpressed enzymes, and high levels of ROS and GSH, are closely associated with the development of cancers. [148]Therefore, measurement of these tumor-associated parameters is essential for tumor detection, therapeutic evaluation, and treatment regimen regulation.Pathologically activatable luminescence nanoprobes coupled with noninvasive imaging technologies provide a feasible approach for tumor-associated microenvironment imaging and specific therapeutic evaluation. [149,150]The bioresponsive luminescence nanoprobes, such as O 2 -sensitive nanoprobes, [151] pH-sensitive nanoprobes, [152,153] enzymesensitive nanoprobes, [154,155] redox-sensitive nanoprobes, [156] and luminescence nanothermometers, [157] can be used to track biological processes and assess disease progression directly with high resolution.For instance, a multifunctional pH-activatable indocyanine green-encoded nanoprobe (PINS) was designed for high sensitive and specific detection of head/neck and breast cancer by using existing clinical cameras. [158]Most recently, our group developed self-illuminating NPs by self-assembly of an amphiphilic copolymer CLP comprised of chlorin e6 (Ce6) simultaneously conjugated with luminol and a hydrophilic chain of polyethylene glycol (PEG) (Figure 7). [159,160]Upon activation by myeloperoxidase (MPO) and ROS, CLP NPs can produce bioluminescence, thus enabling imaging of neutrophil infiltration and oxidative stress in the tumor microenvironment before and after therapeutic interventions.On the other hand, since the bioluminescence intensity of CLP NPs is proportional to the count of high ROS-expressing tumor cells, this self-illuminating nanoprobe enables real-time evaluation of therapeutic responses by monitoring tumor size and/or tumor cell numbers.In addition, our study indicated that this type of luminescence nanoprobes was able to generate 1 O 2 for in situ PDT upon triggering by different levels of ROS, thereby exhibiting antitumor activity in H 2 O 2 -high tumors. [160]dditionally, recent studies have suggested that tumorassociated neutrophils or tumor-associated macrophages may serve as alternative prognostic and diagnostic indicators for cancer patients. [161,162]According to these findings, our group recently developed high-performance luminescence nanoprobes (LAD NPs) to detect lung metastasis of breast cancer by realtime monitoring infiltrated neutrophils in the lungs. [163]In this aspect, a biocompatible and neutrophil-triggerable selfilluminating cyclodextrin-derived material and an aggregationinduced emission (AIE) agent PPV were employed to build the nanoprobe LAD NPs (Figure 8).It was found that the in vivo luminescence intensity of neutrophils was linearly correlated with 4T1 tumor cell counts in the lungs.We also demonstrated that tumor growth and the lung metastasis process could be monitored by luminescence imaging using the newly engineered nanoprobe.Moreover, luminescence imaging capacity of this nanoprobe can be further amplified by surface functionalizing with a neutrophiltargeting peptide.Consequently, this novel nanoprobe can be utilized for evaluation of therapeutic effects of drugs against breast cancer lung metastasis.
reduce the mortality and morbidity rate of CVDs. [129]Luminescence nanoprobes hold great potential for diagnosis, evaluation of disease progression, and monitoring therapeutic effects in CVDs. [128,168]171] As well known, AS is a major pathological cause of many CVDs, such as MI and stroke.In view of a crucial role of proinflammatory macrophages in the pathogenesis of AS, a variety of functional luminescence nanoprobes have been developed for imaging and recruitment and dynamics of macrophages in aortic plaques. [172]Kim's group developed a novel macrophage-targeted theranostic system named MMR-Lobe-Cy, which was constructed with mannose-PEG (MMR-PEG), chitosan-deoxycholic acid-cyanine 7, and lobeglitazone (Figure 9). [173]MMR-PEG was utilized as a common target unit for macrophages, while Cy7 served as a fluorescence nanoprobe.Thus obtained MMR-Lobe-Cy nanoplatform not only displayed robust anti-inflammatory effects and reversed plaques to a stable phenotype, but also showed good optical coherence tomography to macrophages in the inflamed plaques region, thus enabling evaluation of the inflammatory degree after different treatments.
Similar to AS, integration of deep-tissue luminescence imaging modalities with emerging therapeutic strategies can afford considerable opportunities for diagnosis and therapeutic monitoring of ischemic stroke.It has been well-documented that oxidative stress represents a reliable biomarker in determining the severity of ischemic stroke. [174]Thus, a ROS-responsive ratiometric NIR-II nanoprobe (IR-LnNPs) was successfully applied for distinguishing the salvageable ischemic tissue from the infarcted stroke core by visualizing the ROS level and the enrichment degree of the nanoprobe at the lesion site, offering a viable tool for real-time and dynamically assessing ischemic stroke. [175]Compared with MRI, IR-LnNPs allowed more rapid detection of the ischemia area at 30 min after cerebral ischemia.Moreover, luminescence nanoprobes can also be combined with other imaging modalities to precisely monitor the progression of cardiovascular and cerebrovascular diseases.As an example, Yang et al. constructed an activatable luminescent nanoprobe for X-ray-excited luminescence (XEL) imaging. [176]This probe can be integrated to certify the imaging sensitivity and credibility as well as monitor thrombosis progression.Due to the extensive clinical application of X-rays and their ability to penetrate deeply into tissues without notable limitation, X-ray-activated luminescence imaging provides a novel prospect for detection and diagnosis of deep-tissue diseases, with great potential for clinical translation.

Inflammatory Diseases
Inflammation is closely related to a wide range of acute and chronic diseases, varying from inflammatory bowel disease analyses of the luminescence intensity and the number of neutrophils in BALF f), the MPO level in the lungs g), or the 4T1-GFP cell counts in the lungs h).Reproduced according to the terms of the CC-BY license. [163]Copyright 2022.The authors, published by BioMed Central.
[179] As a straightforward and versatile approach, visualization of in vivo inflammation by luminescence nanoprobes can provide new insight into the pathogenesis of inflammatory diseases and directly evaluate therapeutic responses to different anti-inflammatory treatments. [180]ven the presence of inflammatory cells (such as neutrophils and macrophages), excessive ROS, and overexpressed specific enzymes (like MPO), different bio-responsive or inflammationactivatable luminescence nanoprobes have been developed for real-time and dynamic imaging of acute and chronic inflammatory diseases. [181,182]Reproduced with permission. [173]Copyright 2021.Ivyspring International Publisher.

Fluorescence Nanoprobes
Fluorescence nanoprobe-based imaging is the most commonly examined technique in basic studies on acute and chronic inflammatory diseases. [34]Oxidative stress characterized by excessive production of ROS and reactive nitrogen species (RNS) are important indicators for assessing the severity of many inflammatory diseases.In particular, ROS-responsive fluorescence nanoprobes have been utilized to visualize the oxidative stress level for evaluating inflammation in different animal models. [182,183]For instance, Tang group developed a peroxynitrite-responsive fluorescence nanoprobe with AIE characteristics for selective detection of elevated ONOO − generation and visualization of inflammation in vivo. [184]The engineered fluorescence nanoprobe, abbreviated as TPE-IPB-PEG, was composed of an AIEgen unit of tetraphenylethene (TPE) derivative, a peroxynitrite-responsive moiety phenylboronate, and a lipid-PEG matrix.The fluorescence of TPE-IPB-PEG can be switched on specifically in the bacterial-infected skin region of inflammationbearing mice with elevated generation of ONOO − .Further ex vivo imaging of different tissues not only confirmed the excellent selectivity and high efficiency of TPE-IPB-PEG in inflammation imaging, but also demonstrated that this nanoprobe can provide real-time feedback on in vivo efficacies.
Recently, there has been increasing interest in the development of fluorescence nanoprobes decorated with natural cell membranes or their analogues to monitor inflammatory diseases. [185]Beatriz Salinas's group reported goat milk exo-somes covalently labeled with fluorophore sulfo-cyanine 5 and BODIPY-FL, which allowed fluorescence imaging of peritonitis progression. [186]This approach can be potentially employed for real-time inflammation stratification and prediction of therapeutic effects efficacy.In another study, a platelet membrane-coated nanotheranostic agent (defined as TMSN@PM) was designed to target inflammatory sites and scavenge excessive ROS. [187]In addition to its therapeutic function, TMSN@PM was engineered as a diagnostic tool, thus providing information about the degree of inflammation in acute liver failure and pancreatitis.By combining both therapeutic and diagnostic capabilities, TMSN@PM is promising for treating and monitoring inflammatory diseases.

Chemiluminescence Nanoprobes
Besides fluorescence nanoprobes, various types of chemiluminescence nanoprobes have been examined for imaging inflamed sites, since their luminescence is triggered by some biochemical signals associated with inflammation, while no external radiation is required for excitation. [188,189]Especially, chemiluminescence or bioluminescence imaging has prominent advantages of the extremely high signal-to-noise ratio and ultrahigh sensitivity, thereby permitting imaging and therapeutic evaluation of deep-seated diseases. [56,190,191] Copyright 2017.Elsevier.
Compared with other bioluminescence agents, luminol-based nanoprobes exhibit more desirable imaging capacity, because their luminescence signals are closely related to the specific reaction with abnormally increased MPO and ROS at the inflammatory site.To overcome relatively week luminescence of luminol and its small-molecule derivatives due to nonspecific distribution and poor retention at inflamed sites, our group constructed a type of biodegradable and biocompatible chemiluminescence nanoprobes based on luminol-conjugated cyclodextrins (Figure 10). [195]Luminescence of these nanoprobes can be specifically triggered by overproduced ROS and MPO in the inflammatory microenvironment.Of note, the Lu-bCD nanoprobe, i.e., NPs based on luminol-conjugated -cyclodextrin, can selectively illuminate activated neutrophils during inflammatory responses.It was also demonstrated that the high and sustainable luminescent signals were positively correlated with neutrophil counts.In vivo studies in diverse mouse models of typical inflammatory diseases, including peritonitis, paw edema, colitis, and acute lung injury, indicated that these NPs derived from luminol-conjugated cyclodextrin materials can efficiently monitor the dynamical change of neutrophils in both superficial and deep tissues.In line with luminescence associated with the count and activation of neutrophils, treatment with anti-inflammatory and antioxidant drugs (such as Tempol and 4-ABAH) significantly attenuated luminescence signals.Consequently, therapeutic effects of different treatments can be directly evaluated by the magnitude of bioluminescence intensities.Notably, imaging performance of luminol-based nanoprobes can be further improved by chemiluminescence resonance energy transfer (CRET).The emission wavelength of luminol (≈440 nm) can be red-shifted through CRET with a fluorescent acceptor Ce6, which was achieved by conjugating both luminol and PEG onto Ce6. [159]The obtained amphiphilic conjugate CLP can assemble into core-shell structured NPs.CLP NPs enabled precise tracking of the development of inflammatory disorders.c) Schematic illustration of ROS-triggered luminescence imaging for peritonitis, acute liver injury, acute lung injury, and tumors by OVE-derived nanoprobes.Reproduced with permission. [196]Copyright 2021.Wiley.
Peroxalate compound-based nanomaterials containing fluorescent dyes represent another classical type of chemiluminescence nanoprobes for in vivo visualization and evaluation of oxidative stress and inflammation.In a recent study, our group engineered a ROS-responsive luminescence nanoplatform based on a vitamin E-derived peroxalate compound (OVE) (Figure 11). [196]By incorporating different fluorescent molecules into OVE NPs, the obtained nanoprobes could achieve long persistent chemiluminescence with broad emission wavelengths ranging from 432 to 855 nm, upon triggering by abnormally elevated H 2 O 2 under inflammatory conditions.These self-luminescent nanoprobes were found to be highly effective for real-time monitoring inflammation progression in mice with peritonitis, alcoholic liver injury, and acute lung injury.In a study by Zhang et al., a new type of NIR-II chemiluminescence sensors based on bis[3,4,6trichloro-2-(pentyloxycarbonyl)phenyl]oxalate (CPPO) and two donor-acceptor-donor fluorescent dyes (BTD540 and BBTD700) were used for detecting inflammatory arthritis. [197]The chemi-luminescence intensity and the higher signal-to-noise ratio at the arthritis site were well-correlated with time-dependent changing profiles of H 2 O 2 levels.Similarly, inflammatory processes were evaluated by chemiluminescent semiconducting polymer NPs (SPNs) containing peroxalate bis(2,4,6trichlorophenyl) oxalate (TCPO) in mouse models of peritonitis and neuroinflammation. [198]Moreover, both in vitro and in vivo results suggested that these peroxalate-based chemiluminescence nanoprobes exhibit good safety profiles, highlighting their great potential for evaluation of therapy responses to different treatments.
Overall, luminescence nanoprobes hold great promise for diagnosis and therapeutic evaluations in a variety of diseases, with respect to both fundamental research and clinical applications.In particular, luminescence nanoprobes have demonstrated their multiple advantages in molecular imaging for tumor detection and fluorescence imaging-guided cancer surgery.Currently, clinical trials of luminescence nanoprobes mainly involve the utilization of fluorescent dyes conjugated with antibodies, peptides, and proteases. [199,200]As a typical example, cyclic arginine-glycineaspartic acid-tyrosine (cRGDY)-decorated ultrasmall core-shell fluorescent silica NPs labeled with Cy5.5 have been tested in a phase II clinical trial (NCT02106598) for image-guided sentinel lymph node biopsy in patients with head and neck cancer. [201]On the other hand, although nanoprobe-based luminescence imaging shows great promise in preclinical studies, many obstacles remain to be overcome before available nanoprobes can be used for evaluating therapeutic effectiveness of different drugs or drug candidates in clinical settings.In addition to optimizing luminescence properties and elucidating safety issues of different types of nanoprobes, patient-friendly optical imaging instruments with high sensitivity need to be developed, with the detectors enabling collection of luminescent signals simply by scanning.

Challenge and Future Perspective
Despite recent advances in luminescence nanoprobes for drug screening, pharmaceutical analysis, and therapeutic evaluation at molecular, cellular, and animal levels, the development of luminescence nanoprobes for practical applications still encounters many challenges.For high-throughput drug screening, the necessity of repeatability, accuracy, and efficiency put forward high requirements for the preparation of different types of nanoprobes.Currently, few available nanoprobes fully meet these demands, since the majority of these nanoprobes are not intentionally synthesized for drug screening applications.Notably, the functionality of nanoprobes can be considerably affected by complicated biological samples, thus most likely resulting in the inaccurate detection and false positives.Moreover, the limited range of selective targets for nanoprobes cause challenges for extensive screening of candidate compounds, since only some specific biological molecules can be detected.Additionally, many approaches utilized for the nanoprobe production are highly complicate, necessitating further design innovations to reduce costs and simultaneously enhance both colloidal and luminescent stability for long-term storage.Importantly, in vivo distribution and metabolism profiles of many nanoprobes are inadequately explored, which arouses potential risks such as acute and chronic side effects.In terms of pharmaceutical analysis, the synthesis of uniform and reproducible nanoprobes is necessary to guarantee the development of advanced quantification techniques with high sensitivity, accuracy, and repeatability.Particularly, limited imaging accuracy compromises the capacity of nanoprobes in tracking the absorption, distribution, metabolism, and excretion of drugs.The imaging signals obtained from different instruments or laboratories cannot be directly compared by a unified standard, resulting in the sluggish application of luminescence nanoprobes in pharmaceutical analysis.As for therapeutic evaluation, although nanoprobes can be equipped with outstanding capacities for loading different diagnostic agents and presenting effective targeting moieties, useful luminescence signals of nanoprobes are susceptible to in vivo physiological and pathological microenvironments.As a result, the obtained imaging data may deviate, potentially compromising the reliability of drug efficacy evaluation.Insufficient imaging resolution and lack of advanced technologies are additional challenges limiting the accuracy and reliability of luminescence nanoprobes for clinical applications.
To address the above mentioned issues, engineering strategies or synthesis procedures need to be further improved to ensure the large-scale production of nanoprobes in facile, efficient, economical, and reproducible manners.Therefore, most existing luminescence nanoprobes should be further verified and considerably optimized, with respect to their physicochemical properties, stability, and luminescence performance.Besides, the discovery and applications of novel nanomaterials and combination of different available nanoprobes have been regarded as two actionable strategies to optimize the nanoprobe-based DS platforms in the future.In this context, nanoprobes with more sensitive and discriminative luminescence readout capability in response to pathological cues remain to be developed.In particular, nanoprobes with high specificity and selectivity to clinically useful biomarkers and/or well-recognized therapeutic indicators are highly desirable.To this end, novel nanoprobes need to be rationally designed and discovered according to new biomarkers.Importantly, comprehensive and intensive studies should be performed for existing nanoprobes to establish reliable correlations between luminescent signals and biomarkers according to the pathogenesis of specific diseases.In all these cases, machine learning-assisted design, synthesis, and optimization can be applied for the development of highly efficient nanoprobes.
Furthermore, multifunctional nanoprobe-based multimodality imaging techniques, such as the combination of MRI, computed tomography, ultrasound, and luminescence, represent intriguing strategies.By employing diverse imaging modalities, multi-dimensional detection and screening can be possibly achieved to enhance both efficiency and precision for drug screening.The capacity for visualization and real-time monitoring can also be strengthened to enable the observation of drug distribution and metabolism in vivo, thus providing additional insight into pharmacodynamics and mechanisms of drugs.
Finally, it is necessary to carefully address safety profiles of some luminescence nanoprobes that will be used for in vivo DS and therapeutic evaluations.At least, nanoprobes themselves should not cause considerable effects on drug efficacies and toxicities, to avoid false conclusions.

Figure 3 .
Figure3.Engineering of a terbium-based nanoarchitecture probe and schematic illustration of detection of the deubiquitinating enzyme activity.Reproduced with permission.[50]Copyright 2020.Royal Society of Chemistry.

Figure 6 .
Figure 6.Luminescence imaging-based analysis of chiral molecules.a) Schematic of competitive FRET assay using CdSe/ZnS QDs with rhodamine B.Reproduced with permission.[19]Copyright 2009.American Chemical Society.b) The recognition mechanism of D/L-glucose achieved through the formation of CDs@AuNPs complexes triggered by a stereoselective enzymatic reaction.Reproduced with permission.[123]Copyright 2018.Royal Society of Chemistry.c) Nanopaper-based enantioselective recognition of D/L-lysine (L-Lys) using L-cystine capped CDs, and the nanoprobe exhibited dosedependent fluorescence enhancement upon the addition of L-lysine.Reproduced with permission.[124]Copyright 2018.Elsevier.

Figure 7 .
Figure 7.A luminescence nanoprobe for tumor imaging and therapeutic evaluation.a) Design and synthesis of an amphiphilic copolymer CLP for detecting and treating solid tumors expressing high H 2 O 2 .b) Typical time-lapse in vivo images and quantitative analysis of luminescence intensities in mice bearing 4T1 tumors after local administration of CLP nanoparticles.Reproduced with permission.[160]Copyright 2020.American Chemical Society.

Figure 8 .
Figure 8. Early imaging of pulmonary micrometastasis by a self-luminescence nanoprobe.a) Engineering of a neutrophil-dependent luminescence nanoprobe LAD NPs for early imaging of lung metastasis of breast cancer.b) In vivo luminescence images showing pulmonary metastasis in mice.c) Quantitative analysis of pulmonary luminescence intensities in mice at various time points.d) Percentages of neutrophils in bronchoalveolar lavage fluid (BALF) of mice after intravenous inoculation of 4T1-GFP cells for different time periods.e) Quantified MPO levels in the lungs.f-h) Correlationanalyses of the luminescence intensity and the number of neutrophils in BALF f), the MPO level in the lungs g), or the 4T1-GFP cell counts in the lungs h).Reproduced according to the terms of the CC-BY license.[163]Copyright 2022.The authors, published by BioMed Central.

Figure 9 .
Figure 9. Schematic illustration of evaluation of the inflammatory degree of plaques in the coronary artery by a macrophage-targeted theranostic system MMR-Lobe-Cy.a) The synthetic route of MMR-Lobe-Cy.b) Ex vivo NIR fluorescence reflectance imaging (FRI) (top), brightfield (middle), and merged images (bottom) of arteries.Scale bars, 4 mm.c) Representative in vivo optical coherence tomography (OCT)-NIR fluorescence (NIRF) images of different groups corresponding to the fluorescence microscopy images (MMR-derived NIR fluorescence, red; autofluorescence, green).Scale bars, 500 m.Reproduced with permission.[173]Copyright 2021.Ivyspring International Publisher.

Figure 10 .
Figure 10.A ROS/MPO-responsive, biodegradable, and biocompatible chemiluminescence nanoprobe (Lu-bCD NP) for visualizing diseases associated with inflammation and oxidative stress.a) Schematic of the structure and composition of the luminescence material Lu-bCD and hydrolysis mechanism.b) Representative in vivo luminescence images and quantitative data of Lu-bCD NP in peritonitis mice in the presence or absence of Tempol or 4-ABAH.c) Luminescence imaging of mice with acute lung injury at different time points after intravenous administration of Lu-bCD NP.Reproduced with permission.[195]Copyright 2017.Elsevier.

Figure 11 .
Figure 11.Peroxalate compound-based chemiluminescence nanoprobes for real-time monitoring of different inflammatory diseases.a) Design and construction of broad-wavelength luminescent nanoprobes.b) Preparation of OVE-based chemiluminescence nanoprobes by a nanoprecipitation/selfassembly method.c)Schematic illustration of ROS-triggered luminescence imaging for peritonitis, acute liver injury, acute lung injury, and tumors by OVE-derived nanoprobes.Reproduced with permission.[196]Copyright 2021.Wiley.

Table 1 .
Applications of typical luminescence nanoprobes in drug screening.

Table 2 .
Applications of typical luminescence nanoprobes in pharmaceutical analysis.