Near‐Infrared Optical Sensing of Biomacromolecules with Upconversion Nanoplatforms

Optical sensing and imaging have perceived massive success in biomedical analysis and disease diagnosis in terms of minimal invasiveness, good sensitivity, high accuracy, and time‐/cost‐effectiveness. Upconversion nanoparticles (UCNPs) as a kind of promising luminescent material hold many merits like unique frequency‐converting capability, emission fine‐tuning, low auto‐fluorescence interference, good tissue‐penetration ability, high photostability, and excellent biocompatibility, which are widely applied for optical sensing of diverse chemically or biologically derived analytes. Extensive efforts are dedicated to the rational fabrication of reliable upconversion nanoplatforms (UCNs) through ingenious modulation of the luminescent energy transfer process for various optical biosensing applications. Herein, the advancement of biomacromolecules (e.g., nucleic acids, proteins, and enzymes) detection using multiplex UCNs from on‐paper platforms to in‐solution as well in living systems is specifically focussed. Detailed summarizations of the probe design strategy, responsive mechanisms, and sensing performance have been presented. In addition, based on the current research achievements, the challenges and future perspectives are emphasized to facilitate further clinical sensing utilizations with upconversion photonic technology.

DOI: 10.1002/adpr.202200175 Optical sensing and imaging have perceived massive success in biomedical analysis and disease diagnosis in terms of minimal invasiveness, good sensitivity, high accuracy, and time-/cost-effectiveness. Upconversion nanoparticles (UCNPs) as a kind of promising luminescent material hold many merits like unique frequency-converting capability, emission fine-tuning, low auto-fluorescence interference, good tissue-penetration ability, high photostability, and excellent biocompatibility, which are widely applied for optical sensing of diverse chemically or biologically derived analytes. Extensive efforts are dedicated to the rational fabrication of reliable upconversion nanoplatforms (UCNs) through ingenious modulation of the luminescent energy transfer process for various optical biosensing applications. Herein, the advancement of biomacromolecules (e.g., nucleic acids, proteins, and enzymes) detection using multiplex UCNs from on-paper platforms to in-solution as well in living systems is specifically focussed. Detailed summarizations of the probe design strategy, responsive mechanisms, and sensing performance have been presented. In addition, based on the current research achievements, the challenges and future perspectives are emphasized to facilitate further clinical sensing utilizations with upconversion photonic technology.
been fabricated as unique optical nanotransducers for optogenetic regulation, such a NIR photoactivation paradigm allows deep brain stimulations and other types of deep-tissue optogenetic therapeutics. [15] Among the diverse biomedical utilizations, UCNs-based techniques for macromolecules sensing have witnessed significant development in the past few years. Biomacromolecules like nucleic acids (DNA, RNA), proteins, and enzymes are involved in the fundamental biological functions of living organisms, and their expression levels, structural changes, and activities disturbance are closely related to the occurrence and progression of diseases. [16] Compared to other approaches for biomacromolecules detection through chromatographic, colorimetric, and electrometric assays, [17] UCNsbased NIR luminescent sensing holds obvious advantages including low background interference, high sensitivity, and cost-/time-effectiveness. Moreover, recent research advances have boosted UCNs applications for macromolecule sensing from the molecular level to living animals, showing the potential for clinical translations.
Hence, in this review we specifically focus on the advances of UCNs-based biomacromolecules detection (e.g., DNA, RNA, proteins, and enzymes) through on-paper platforms, in-solution as well in living systems ( Figure 1). The detailed summarizations of design strategies, responsive mechanisms, and sensing performances of upconversion probes have been presented. In addition, the challenges and future perspectives in line with current achievements are emphasized to facilitate further clinical utilizations of upconversion biosensing technology.

Design of Diverse UCNs
UCNPs contain three main components; sensitizer, emitter, and host matrix. Yb 3þ ion is usually introduced to sensitize the photon upconversion process due to its relatively large absorption cross-section and suitable energy levels for energy transfer. [18] The trivalent-state ions Tm 3þ , Er 3þ , Ho 3þ , etc. are usually used as emitters in UCNPs with versatile energy level structures covering a wide-ranging spectrum, enabling the multicolor upconversion luminescence (UCL) ranging from the ultraviolet (UV), visible to NIR optical window (Figure 2a). [19] Notably, recent studies of the upconversion sensitizers have expanded to Ce 3þ , Gd 3þ , Tb 3þ , Dy 3þ , Eu 3þ , and Sm 3þ ions in core-shelldesigned upconversion nanocrystals, and some transition metal ions Mn 2þ and Cu 2þ , metal ion Pb 2þ and even the lanthanide divalent ion Sm 2þ have also proven to be potent activators for multiplex UCL tuning. [20] Generally, lanthanide-based upconversion processes can be categorized into six classes: excited-state absorption (ESA), energy transfer upconversion (ETU), cooperative upconversion (CUC), cross-relaxation (CR), photon avalanche (PA), and energy migration-mediated upconversion (EMU). [21] In the upconversion process, UCNPs are excited by multiple low-energy photons with a long wavelength (e.g., 980 nm), and emit a single high-energy photon with a shorter wavelength through the multiphoton energy transfer process. The excitation wavelength can be shifted to 808 nm by doping Nd 3þ ions, where Yb 3þ ions act as an energy transfer bridge that traps directly sensitized Nd 3þ energy and then transfer it to the emitters ( Figure 2b). [22] Besides, dye-sensitized UCNPs have also been proved to be an alternative strategy to achieve both 800 nm excitable and efficient luminescence enhancing performances. [23] Compared with 980 nm excited UCNPs, such a shift of the excitation wavelength significantly reduces the laser-induced photothermal effect during the operation of bioimaging and phototherapy. [24] Since bare UCNPs are not capable of responding to the analytes, thus necessary modification and functionalization of UCNPs as sensitive biosensors are crucial for practical sensing applications. Similar to other molecular probes, stimuliresponsive upconversion nanoprobes can be rationally designed through either affinity ligands installation or manipulation of the UCL energy transfer. [27] Generally, UCNPs for specific biosensing can be divided into two types, termed targeted and activatable probes in line with the responsive principles. As illustrated in Figure 3, targeted upconversion probes for biomacromolecules  sensing rely on the specific luminescent signal accumulation upon interaction with the target of interest (e.g., proteins, tumors), such as immunofluorescence assay and ligand-directed biological imaging. [28] Based on this concept, the surface of luminescent UCNPs is modified by tumor-targeting antibodies or other specific ligands that allow in vitro tumor cells or biomarkers detection, and even in-vivo tumor theranostics upon selective accumulation and uptake of the nanoparticles. [29] However, targeted optical sensing usually suffers from nonspecific signal interference and a lack of quantitative capability. In contrast, activatable upconversion probes with the stimuliresponsive signal variations are capable of correlating the luminescence intensity for sensitive quantification of biomacromolecules. Moreover, activatable UCNPs-based biosensing exhibit Reproduced with permission. [25] Copyright 2008, American Chemical Society. b) Representative UCNPs energy level structures and energy transfer diagram in the photon upconversion process. Reproduced with permission. [26] Copyright 2013, Wiley-VCH. a higher signal-to-noise ratio (SNR) and lower limit of detection (LOD) in comparison with that of targeted approaches. [30] There are two major routes to design activatable upconversion probes, which involve different mechanisms including luminescence resonance energy transfer (LRET) or fluorescence resonance energy transfer (FRET) and the inner filter effect (IFE). [12b,31] In the LRET process, the nonradiative photon energy from the luminescence donor UCNPs transfers to the nearby chemically modified energy receptor (e.g., dyes or other photon absorbers) as indicated in Figure 3, leading to the UCL quenching effect or consequently lights up the receptor fluorescent molecules. Alternatively, the UCL photon reabsorbed by the surrounding acceptor molecules after the response of the analyte is termed the IFE. It should be noted that the IFE efficiency mainly depends on the absorption spectral tuning of appropriate chromophores, while the LRET effect is also affected by the distance (<10 nm) between donor UCNPs and acceptor molecules. Following these strategies, various kinds of upconversion nanoprobes have been fabricated for sensitive detection of diverse biomacromolecules in recent years, the detailed applications are discussed in the following. Table 1 summarizes currently reported studies of different types of UCNs and their applications for optical sensing of diverse biomacromolecules, including DNA, RNA, proteins, and enzymes. The probe structures, optical properties, and responsive mechanisms of representative UCNs are also highlighted.

DNA
The trace amount of DNA/RNA analysis is very important in molecular medicine and clinical diagnosis. In recent decades, extensive efforts have been devoted to developing effective sensing strategies, in which the classical DNA hybridization-based detection is widely implemented. [75] A lot of sensitive UCNs have been reported for DNA/RNA imaging and sensing in the buffer solutions or living subjects.
In 2006, Wheeler et al. reported a sandwich-type hybridizationbased nucleotide upconversion nanosensor that uses two shorter oligonucleotides with a specific sequence to capture the longer target oligonucleotide (Figure 4a). [61a] Of which, one short oligonucleotide is covalently bound to UCNPs, while another one is labeled with an organic fluorophore that the excitation spectrum overlaps with the UCL spectrum. In the presence of the target DNA, the LRET effect takes place due to a closed distance between UCNPs and the fluorophore, leading to the decrease of UCL and lighting up the fluorophore. Thanks to the merits of high sensitivity (LOD, 1.3 nM), low background, and photobleaching, such nucleotide upconversion sensors are expected to be effective for applications in both DNA/RNA detection and protein-DNA/RNA interaction studies. By following the same concept, Huang et al. prepared carboxylic acid ligands-functionalized UCNPs and further conjugated them with reporter-DNA through the streptavidin-biotin interaction (Figure 5a). [61b] Consequently, these non-covalent streptavidin-functionalized UCNPs can also be used for sensitive DNA detection on the basis of the LRET mechanism.
Different from the aforementioned dye-labeled reporter DNA facilitated UCNs, Zhang et al. reported another detection system based on the upconversion LRET effect, which is very simple with only one capturing DNA probe on the UCNPs surface, without the fluorophore-labeled reporter DNA, showing higher sensitivity (LOD, 20 fM) and specificity (Figure 4b). [62] Since then, several research studies have reported focusing on the design of novel upconversion DNA sensors and diversifying their sensing applications. One typical direction is to extend the scope of LRET acceptors by introducing other optical materials, such as graphene oxide (GO), polymers, QDs, AuNPs, and so forth. [12b,76] Compared with commonly used organic dyes, water-soluble GO has been validated to be an ultra-highly efficient quencher toward UCNPs. [77] On this basis, Kanaras et al. developed a DNA sensor platform using the LRET pair formed by UCNPs as the luminescence donor and GO as the quencher (Figure 5b). [39] The single-strand DNA (ssDNA) on the UCNPs surface shows a strong affinity towards GO due to the π-π interactions between the aromatic nucleobases and the unsaturated structure of GO, resulting in a significant UCL quenching.
In the presence of the complementary DNA strand (cDNA), the hybridization process leads to double-stranded dsDNA that destructs the LRET effect and turns on the UCL. Such UCNP-GO nanoprobes are highly sensitive to detecting cDNA, and LOD is low to the picomolar level. Alternatively, poly-mphenylenediamine (PMPD) nanospheres have also been used as the energy acceptor in upconversion DNA sensing platforms (Figure 5c), which is reported by Liu et al. in 2012. [40] This UCNP-PMPD biosensor linearly responds to the target DNA concentration with the LOD at 0.03 nM, and it has the applicability to complicated sample matrix (e.g., human serum). In light of high extinction coefficients and broadband absorbing features of AuNPs, Zourob et al. reported an LRET-based pair of UCNP-AuNP nanohybrid platforms for "turn on" sensing of ssDNA ( Figure 5d). [41] After the complementary DNA response, the separation of UCNPs and the quencher (AuNPs) leads to a measurable increase in the UCL intensity. Such a system is sensitive to detecting the target DNA concentration down to the picomolar.
Another research direction in UCNPs-based DNA sensing platforms is to further extend their practical applications in the biomedical field. For instance, Zhang  In vitro telomerase activity sensing [55] www.advancedsciencenews.com www.adpr-journal.com sensitive and straightforward upconversion LRET system to evaluate the intermolecular fates of small interference RNA (siRNA) during their cellular delivery ( Figure 6a). [44] The dynamic variation of the LRET efficiency between UCNPs and the BOBO-3 dye provides real-time evidence for cellular uptake, release, and biostability of siRNA molecules in live cells, which further shows the possibility for in vitro and even in vivo detection of other biological macromolecules, such as nucleic acids and proteins. Xu et al. constructed a chiral nanoscale assembly with the UCNP-AuNP LRET pair and applied it for ultrasensitive endogenous microRNA (miRNA) sensing in living cells (Figure 6b,c). [47] When miRNA is present, it will be complementary to the recognition sequence on each side of Au-UCNP pyramids, which leads to the complete dissociation of the DNA frame and destructs the LRET quenching effect. This platform enables both the luminescent and chiroptical dual-mode responses for abnormal cellular miRNA in situ quantitation. Besides, Rubio-Retama et al. developed a graphene quantum dots (GQDs) and ssDNA-UCNP-based LRET sensor that has the capacity of complementary miRNA sensing through π-π stacking  [78] ; In addition, the ratiometric sensing of thymidine kinase 1 (TK1) mRNA using an upconversion nanobeacon (UCNPs-MB/Dox) has been reported by Xing et al., [64] which shows the potential as a precise theranostic agent for tumor biomarker imaging in living cells; To further improve the sensing performance, Kuang et al. introduced an AuNR@Pt-UCNP satellite assembly based LRET sensor for mRNA analysis. [79] Such a method achieves effective in situ imaging and quantifiable detection of TK1 mRNA with a LOD of 0.67 fmol/10 μg RNA.
Recently, Zhu et al. carried out a study using UCNP-AuNPbased LRET system to detect tumor-related noncoding RNA (ncRNA), meanwhile an exonuclease III (Exo III)-assisted cycling amplification strategy introduced further improves the sensing sensitivity and performance (Figure 6d). [46] Briefly, AuNPs bearing one hairpin DNA (Hp) are conjugated to the linker DNA-modified UCNPs by DNA hybridization, leading to UCL quenching through LRET. A signal DNA (SDNA) sequence is designed to open Hp, recovering the UCL. To improve the universality and high sensitivity, an Exo III-assisted cycling amplification strategy is introduced, in which a multifunctional hairpin DNA (mHp) containing ncRNA recognition sequence and SDNA sequence is designed to recognize ncRNA and trigger Exo III as a biocatalyst to stepwise disintegrate itself, releasing both ncRNA and SDNA. The released ncRNA can be reused to release more SDNA thereby achieving the signal amplification. By changing the recognition portion of mHp, various ncRNA can be detected. This study provides new thinking to develop highly sensitive ncRNA sensing platforms for cancer diagnosis.

Proteins
The application of UCNs for protein analysis also shows great promise in recent years. As early as 2005, Li et al. reported an LRET biosensor with bioconjugated UCNPs and AuNPs for the detection of trace amounts of avidin (Figure 7a). [48] In which, the biotin-avidin interaction mediated LRET quenching effects are specifically and linearly varied with the concentration of avidin in the nanomolar range. With a similar concept, Xu et al. then applied such techniques for immunoassay of goat antihuman immunoglobulin G (igG) antibody (Figure 7b). [56] The human IgG-modified UCNPs and rabbit antigoat IgG modified AuNPs based LRET system is worked upon the addition of goat antihuman IgG, which initiates sandwich-type immunoreaction and in turn shortens the distance between the donors and the acceptors for LRET occurrence. This sensing platform has good sensitivity (LOD, 0.88 μg mL À1 ) and precision (relative standard deviation, RSD, 1.3%). In light of the specific antibody-antigen interaction, Sirkka et al. used UCNPs as reporters in the highly sensitive heterogeneous immunoassay of cardiac troponin I [38] ; Kim et al. also reported an immunosensor based on LRET quenching effects to detect homogeneous glycated hemoglobin (HbA1c) in blood samples (Figure 7c) [57] ; Very recent, Wang, Li and Xiao et al. further used this sandwichtype immunoassay design based single-particle enumeration (SPE) technology to detect prostate-specific antigen (PSA), a typical prostate cancer biomarker with the LOD at 2.3 pM in the serum sample. [58] Besides, Pang et al. constructed an LRET-based biosensor (UCNPs-CNPs) for thrombin detection through protein-aptamer recognition (Figure 7d). [50] As aforementioned, UCNPs tagged with the thrombin aptamer can bound to carbon nanoparticles (CNPs) surface via π-π stacking, which induces obvious LRET quenching; The UCL signal can be restored in the presence of thrombin through destructing the π-π interaction in a concentration-dependent manner. In regard to improving the sensitivity of such a system, Wang et al. developed a thrombin aptamer-based LRET UCNPs-AuNRs sensor, which is capable of monitoring thrombin concentrations in aqueous buffer and human blood samples with the LOD as low as 0.118 and 0.129 nM, respectively. [80] Such versatile protein-aptamer-based strategies could be ready to develop other UCNs for various biomacromolecules by linking different aptamers or ligands to UCNPs. For instance, Zhang and Wang et al. reported a mycotoxins aptasensor based on multiplexed LRET between ochratoxin A (OTA)-, fumonisin B1 (FB1)-aptamers immobilized UCNPs and GO. [49] In addition to the fabrication of directly linked donor-acceptor pairs, other types of LRET-based upconversion platforms have been developed for protein detection. Wang et al. proposed a  Reproduced with permission. [39] Copyright 2015, American Chemical Society. c) The upconversion LRET DNA sensor with PMPD as the energy acceptor. Reproduced with permission. [40] Copyright 2013, American Chemical Society. d) Illustration of the upconversion LRET-based detection of ssDNA using UCNP-decorated PSA/SiO 2 nanohybrids and cyclometalated Ir(III)-AuNPs as the energy receptor. Reproduced with permission. [41] Copyright 2015, American Chemical Society. non-complemented part.) Reproduced with permission. [47] Copyright 2015, American Chemical Society. d) Fabrication of the LRET-based ncRNA sensing nanoplatform using UCNPs-AuNPs as the energy donor-acceptor pair and the Exo III-assisted cycling amplification strategy. Reproduced with permission. [46] Copyright 2018, Wiley-VCH.
www.advancedsciencenews.com www.adpr-journal.com method combining molecular imprinting technology (MIP) with UCNPs for sensing of Cytochrome c (Cyt c). [59] The molecularly imprinted material-coated UCNPs (UCNPs@MIP) can be prepared by in situ coating Cyt c imprinted materials to the carboxyl-modified UCNPs through the sol-gel technique. Then its fluorescence intensity is sensitively reduced with the recognition of Cyt c. Zhang et al. developed a label-free UCNP-AuNPbased LRET nanosensor for ultrasensitive detection of protamine and heparin. [81] The LRET pair is formed by the electrostatic adsorption of AuNPs on UCNPs in the solution. The LRET system can be destructed upon protamine-induced AuNPs aggregation, while the addition of both protamine and heparin leads to an enhanced LRET effect owing to the stronger interaction between heparin and protamine than that with AuNPs. Such upconversion LRET platforms allow sensitive and simultaneous detection of protamine and heparin with low LOD (6.7 and 0.7 ng mL À1 , respectively). Another study reported by Chen and You et al.
introduced a novel sensing strategy by hybridized time-resolved (TR) upconversion LRET with the indicator displacement assay (IDA) concept (Figure 7e). [66] The TR-LRET-IDA system can be further used for sensitive and quantitative detection of anions, glyphosate, and relevant proteins. Other than the advances in quantitative protein detection, UCNPs-based LRET platforms have been engineered as nanoscopic reporters for protein-protein interaction analysis in living cells by Piehler and Haase et al. (Figure 7f ). [67] Such LRET systems are formed by anti-GFP nanobody (aGFP) functionalized UCNPs that can site-specifically target a bait protein fused to EGFP, as well as an acceptor-tagged prey protein. Upon cellular proteins interaction, the upconversion LRET happens that can be specifically imaged with high fidelity. This study provides opportunities to design novel bio-analytical applications by integrating upconversion LRET with commonly used fluorophores, such as fluorescence proteins (FPs) and dyes.  [48] Copyright 2010, Wiley-VCH. b) Design of LRET-based UCNP/AuNP platform for goat antihuman immunoglobulin G (IgG) antibody sensing. Reproduced with permission. [56] Copyright 2009, American Chemical Society. c) LRET-based homogeneous immunosensor for hemoglobin detection using antibody-conjugated UCNPs. Reproduced with permission. [57] Copyright 2011, American Chemical Society. d) LRET-based thrombin sensor by specific aptamer-modified UCNPs and carbon nanoparticles (CNPs). Reproduced with permission. [50] Copyright 2011, American Chemical Society. e) Upconversion LRET-based indicator-displacement assay (IDA) for the detection and patterning of biologically relevant glyphosate and proteins. Reproduced with permission. [66] Copyright 2015, American Chemical Society. f ) LRET reporter systems for the investigation of protein-protein interaction. UCNPs functionalized with an anti-GFP nanobody (aGFP) are site-specifically targeted to a bait protein fused to EGFP. Adapted with permission. [67] Copyright 2016, Wiley-VCH.

Enzymes
As another kind of highly complex biomacromolecules, enzymes (also called catalytic proteins) play an increasingly important role in biochemical reactions and basic biological functions in microorganisms, plants, and animals. [82] Therefore, sensitive and accurate enzyme-activity assays are crucial for the investigation of disease biology and the development of novel drugs. With regard to the enzyme assay scope, upconversion nanotechnology-based optical sensing has shown advantageous features in this field. In 2008, Rantanen et al. creatively designed an LRET-based upconversion probe for the measurement of benzonase endonuclease that efficiently degrades the oligonucleotide to short fragments ( Figure 8a). [54] Compared with the traditional method by measuring the organic dye AF680 directly with the excitation wavelength at 655 nm, the upconversion-based system using 980 nm excitation could detect benzonase activities at around 0.01 U with higher signal-to-background ratios. Such a design concept is also applicable for measuring other hydrolyzing enzyme activity by rationally changing the internally quenched fluorescent substrate.
LRET-based UCNs can be further used for sensing proteases, for example, matrix metalloproteinases (MMPs), a family of zincdependent endopeptidases that participate in the degradation of extracellular matrix (ECM), playing vital roles in physiological and pathological processes. [83] In 2012, Liu et al. reported an LRET biosensor based on upconversion phosphors and CNPs for MMP-2 determination in blood samples. [51] This nanoplatform contains a specific MMP-2 substrate domain and a π-electron-rich region, the π-π stacking between the probe peptide and CNPs induces the FRET process between UCNPs and the energy receptor. Such a NIR optical sensing method largely eliminates the background interference in complex matrixes and enables facile, highly selective detection of MMP-2 in blood. Then Min et al. strategically designed a similar LRET system using UCNPs and graphene oxide (GO) as the energy donor-acceptor pair for highly sensitive and specific detection of MMP-9 in living tumor cells, the LOD is low to 12 ng mL À1 and the detection time (3 h) is significantly reduced compared to the conventional zymography technique (16 h). [84] Notably, Ma et al. further developed a multichannel optical sensor based on UCNPs for multiplex ratiometric sensing of proteolytic activities of both MMP-2 and MMP-7 (Figure 8b). [68] This probe was constructed via a facile  [54] Copyright 2008, Wiley-VCH. b) Illustration of LRET-based sensing of MMP-2 and MMP-7 activities, representative luminescence spectra and calibration curve of UCNPs treated with various concentrations of MMP-2 or MMP-9. Reproduced with permission. [68] Copyright 2018, American Chemical Society. c) Illustration of apoptosis-associated signal events for caspase-3 activation and subsequent cleavage of UCNP probes for apoptosis imaging. Reproduced with permission. [52] Copyright 2015, American Chemical Society. d) The design of Rhod-Lipo-UCNP nanoprobe and its sensing principle to phospholipase D. Reproduced with permission. [70] Copyright 2014, American Chemical Society.
www.advancedsciencenews.com www.adpr-journal.com phase transfer modification of multi-emissive UCNPs using two polyhistidine-containing peptides conjugated with different fluorophores (FITC and TAMRA). The blue and green emissions can be specifically activated by MMP-7 and MMP-2, respectively, and the red emission could serve as an internal reference for ratiometric sensing. Moreover, such a system allows high specificity and sensitivity for multiplex detection with little signal crosstalk and cross-reactivity. UCNPs-based optical probes have also been used for sensing caspase enzymes, a family of proteases that are closely associated with cell death and disease. [85] In 2013, Xing et al. reported the NIR light-controlled tumor apoptosis with upconversion platforms and simultaneously sensing of activated caspase-3 through cleaving the peptide probe with a DEVD sequence on the UCNPs surface, thus turning on the quenched NIR fluorescence of Cy5. [86] Following this caspase-responsive concept, Ma et al. prepared LRET-based UCNP probes with optimal bioresponsivity for sensitive detection of caspase-3 and imaging of apoptosis in living mice for chemotherapy efficacy evaluation (Figure 8c). [52] Besides, upconversion-based caspase-3 sensing has been proved to be highly sensitive with a LOD of 0.01 pg mL À1 . [87] By using a different peptide containing a specific motif of LEHD, Song et al. fabricated upconversion sensing platforms for the detection of caspase-9 activity in vitro and in vivo. [69] Overall, these caspasesensitive UCNs serve as powerful tools for biomedical applications, such as monitoring of the apoptotic process, the discovery of apoptosis-targeted drugs, and the evaluation of anti-cancer drug efficacy.
Efforts have also been made to develop UCNs for optical sensing of other kinds of enzymes. For example, Chu et al. reported a phospholipid-modified upconversion nanoprobe for ratiometric sensing and bioimaging of phospholipase D (PLD) activity both in cell lysates and in living cells (Figure 8d). [70] Liu et al. developed a sensitive and generic upconversion assay for protein kinase, which is in a simple mix-and-read format without the requirement of surface modification, substrate immobilization, separation, or washing steps, showing the potential in protein kinases-related clinical diagnosis and drug discovery ( Figure 9a). [71] Guo et al. constructed an LRET-based UCNPpeptide-AuNP nanosensor for trypsin detection, and further potentially be used to diagnose trypsin-related diseases or screen trypsin inhibitors. [88] Ma et al. designed hyaluronic acid (HA)bearing upconversion fluorescence nanoparticles (HA-UCNPs) and successfully used them to determine hyaluronidase in human serum samples from both colorectal cancer patients and healthy people (Figure 9b). [53] Chen et al. applied the UCNs for tyramine and tyrosinase (TYR) activity sensing, the responsive mechanism is based on the effective luminescence quenching caused by TYR catalytic oxidation of tyramine to the melanin-like polymers product (Figure 9c). [60] Tang et al. introduced a novel fluorescent sensing platform for telomerase activity assay by coupling a 3D DNA walker on UCNP-MnO 2 -based LRET system (Figure 9d). [55] Wang et al. fabricated an LRET-based upconversion system to detect glutathione S-transferase in human serum samples at a sub-nanomolar level. [89] Among the research for alkaline phosphatase (ALP) activity sensing, Xian et al. used label-free UCNP-based fluorescent probes for sequential detection of Cu 2þ , pyrophosphate, and ALP (Figure 9e), [73] Zhang et al. modified UCNPs as upconversion ratiometric fluorescence and colorimetric dualreadout nanosensors for ALP activity monitoring. [72] To improve the sensitivity, the same research group further provided a highly sensitive ALP quantifying strategy based on enzyme-triggered cascade signals amplification (ECSAm) with rapid fusing reaction. [90] The final example reported the ultrasensitive DNA methyltransferase activity with UCNs by Wu et al., such a nanosensor demonstrated high testing and screening capability for enzyme inhibitors' evaluation ( Figure 9f ). [74] In addition to the aforementioned studies, recent advances in UCN-based biosensing have extended to other types of bioanalytes. For instance, Du et al. reported a NIR-excited nongenetic voltage nanosensor that combined membrane-anchored UCNPs and the membrane-embedded dipicrylamine (DPA), which achieves stable recording of neuronal membrane potential in intact animals. [91] Shi et al. introduced a highly sensitive and selective upconversion nanosensor for NIR imaging of K þ in living cells and mouse brain. [92] Although the aforementioned studies are not in the category of biomacromolecules sensing, these well-designed UCNs are capable of indirectly evaluating the ion channel proteins' status.

Fabrication of Different Sensing Platforms
As we know from the above, the diverse optical sensing purposes for various biomacromolecules require respective upconversion platforms. Thus, the following sections will discuss the typical biomacromolecules sensing studies using multiplex UCNs from on-paper platforms to in-solution as well in living systems.

Sensing on Paper
As early as 2001, Tanke et al. creatively applied upconverting phosphor technology (UPT) for nucleic acid detection using paper-based microarrays with 980 nm light illumination. [93] Compared with the dye (Cy5) molecule-based model system, UPT reporter technology was proved to be superior in sensitivity and good image contrast for spotted cDNA sensing (Figure 10a). This study significantly promotes the use of such a unique UPTfacilitated microarray analysis for biomedical applications, owing to that the combination of upconversion nanotechnology with paper-based platforms allows low-cost and scalable fabrication of the optical sensing devices. [94] Since then, similar sensing paradigms by paper-based methods using NIR-excited UCNs have been widely developed for multiple bioanalytes, such as DNA, [32] proteins, [33,37] exosomes, [35] tumor biomarkers, [36] in which different design strategies are presented to either enable the sensing specificity or improve the sensitivity.
As shown in Figure 10b, Krull et al. prepared LiYF 4 UCNPs with intense and tunable UC emissions, followed by the combination of single-stranded DNA oligonucleotides that allows for selectively detecting the trace amounts of target DNA via a sandwich hybridization assay on a paper-based platform.
[32a] Such a study demonstrates high sensitivity with the limit of detection (LOD) of about 3.6 fmol for a clinically used genetic marker. Moreover, it also shows the capacity to determine the presence of a single-base mismatch. In another study, Xu et al. developed a sensitive and quantitative household prognosis platform www.advancedsciencenews.com www.adpr-journal.com UC-LFS, integrating a smartphone-based reader with multiplexed upconversion fluorescent lateral flow strip (LFS), which can simultaneously quantify two target antigens including brain natriuretic peptide (BNP) and suppression of tumorigenicity 2 (ST2) associated with heart failure. [34] In addition to paper-based "always on" luminescent sensors, Liu et al. constructed a paper-supported LRET aptasensor using UCNPs as the energy donor while carbon nanoparticles (CNPs) as acceptors (Figure 10c). [33] The π-π stacking between the aptamer and CNPs induces the LRET process on the paper surface and thus led to the luminescence quenching of UCNPs. Upon IgE interaction, the energy transfer can be inhibited and thereby turning on the UCL in a concentration-dependent manner. This aptasensor can be further used to quantify IgE concentration from 0.5 to 80 ng mL À1 in either buffer solution or human serum samples.
Meanwhile, some paper-based detections of other types of proteins and even tumor biomarkers have been developed in recent years. For example, Song et al. fabricated a normal filter papersupported device that UCNPs tagged with specific antibodies are printed to the test zones (Figure 10d). [36] After binding with FITC-labeled biomarkers (e.g., carcinoembryonic antigen, CEA) through antigen-to-antibody reactions, the LRET-mediated luminescence changes enable sensitive and ratiometric sensing. Moreover, the multichannel test paper can be easily engineered for simultaneous detection of multiple cancer biomarkers, showing high anti-interfere, stable, and reproducible features with low LOD down to 0.89 ng mL À1 . Qu et al. have also developed a Figure 9. Representative UCNs for enzyme detection (part II). a) Design principle of the UCNPs-based LRET assay for the detection of protein kinases activity. Reproduced with permission. [71] Copyright 2014, American Chemical Society. b) Fluorescence response of the upconversion nanoprobe for hyaluronidase detection. Reproduced with permission. [53] Copyright 2015, American Chemical Society. c) Illustration of quenching UCNPs for tyrosinase activity detection. Reproduced with permission. [60] Copyright 2019, Elsevier. d) 3D DNA walker amplified FRET sensing platform for telomerase activity assay based on the MnO 2 -UCNP system. Reproduced with permission. [55] Copyright 2019, The Royal Society of Chemistry. e) The fluorescence "off-onoff" switch for sequential detection of Cu 2þ , pyrophosphate, and alkaline phosphatase with the upconversion sensor. Reproduced with permission. Copyright 2017, Elsevier. f ) Ultrasensitive DNA methyltransferase activity sensing and inhibitor evaluation with UCNP-AuNP-based LRET transducer. Reproduced with permission. [74] Copyright 2021, Springer Nature.
www.advancedsciencenews.com www.adpr-journal.com convenient and sensitive visual method for the detection of tumor-associated telomerase activity on cellulose paper with functionalized UCNPs (Figure 10e). [37a] The telomerase substrate (TS) primer and dNTP mixtures-immobilized paper has the potential of fulfilling telomerase-mediated polymerase chain reactions (PCRs), then UCNPs can conjugate to the telomerase reaction products (TRP) through sequence-specific hybridization. This study further demonstrates the reliability of the analysis of telomerase activities in different cell lines, and the initial screening of telomerase inhibitors. Apart from the earlier studies, Liu et al. have also reported a portable UCNPs-based paper device for roadside field testing of the drug (cocaine) abuse (Figure 10f ). [95] Upon cocaine recognition by the aptamer fragments, the UCL intensity of UCNPsloaded paper is quenched by AuNPs due to the LRET effect. This platform can sensitively give quantitative results using only a smartphone as the apparatus, which is applicable in human saliva and blood samples. Chen et al. reported a similar paper-supported aptasensor based on the LRET principle from UCNPs to gold nanorods (AuNRs) for the accessible determination of exosomes. [35] When exosomes are present, modified aptamers can combine with the CD63 protein on the surface of exosomes, forming a conjugation to close the distance between UCNPs and AuNRs, which initiates the LRET and promotes UCL Figure 10. Spotted microarray and paper-based bio-analysis using UCNs. a) The experiment designed to compare the detection sensitivity of nucleic acid microarrays provided by conventional Cy5 fluorescence and upconverting phosphor particles. Reproduced with permission. [93] Copyright 2001, Nature Portfolio. b) Scheme of sandwich paper-based DNA assay using UCNs. Reproduced with permission. [32a] Copyright 2014, Wiley-VCH. c) The papersupported aptasensor for IgE detection using UCNPs and CNPs as LRET energy donor-acceptor pair. Reproduced with permission. [33] Copyright 2016, Elsevier. d) Paper-based upconversion LRET biosensor for sensitive detection of multiple cancer biomarkers. Reproduced with permission. [36] Copyright 2016, Nature Portfolio. e) Tumor-associated telomerase activity sensing on UCNPs functionalized cellulose paper. Reproduced with permission. [37a] Copyright 2015, The Royal Society of Chemistry. f ) UCNPs-based paper device for the on-site detection of drug (cocaine) abuse. Reproduced with permission. [95] Copyright 2016, American Chemical Society.
Overall, the earlier studies validate the merits of reduced optical background interference, the superb photostability of UCNs, the inexpensive NIR laser, the simple paper strip, and the portable detection apparatus (e.g., eye or a cell phone camera). The development of practical bio-analytical systems using UCNs in paper-based platforms is of great promise for biomedical and point-of-care applications.

Sensing in Solution, in Cells and Animals
Other than the advances in paper-based solid-phase sensing, most of UCNs have been developed to image and detect a wide range of biomacromolecules from buffer solutions to biological systems, such as cells, disease tissues (e.g., tumor, lymph node, liver, and blood vessel) and living animals. Thus, in this part, we will provide synoptic descriptions of the initial UCNs utilization and their consecutive evolutions for different biomacromolecules sensing, meanwhile, the significance and inadequacies for further real-world diagnostic translations are also emphasized.
Different from the design of paper-based upconversion sensing platforms, there is no need to use paper substrates to load UCNs for biomacromolecules detection in the solution. Similar to the conventional bioassay technologies, such as the PCR and enzyme-linked immunosorbent assay (ELISA), UCNs can be used as sensitive luminescent reporters for nucleic acids or proteins sensing from different sample sources. For example, Chen et al. introduced a novel upconversion LRET sensor that can be used for quantitative and sensitive detection of anions, glyphosate, and relevant proteins in a pure aqueous solution. [66] Kanaras et al. successfully developed an LRET-based GO-UCNP sensor that can specifically detect messenger RNA (mRNA) in complex biological fluids (Figure 11a). [45a] This sensing platform also shows the capability to quantify mRNA biomarkers present in Alzheimer's disease and prostate cancer in human blood serum; In addition, Liu et al. reported a UCNP-based LRET biosensor for MMP-2 determination in blood samples. [51] Figure 11. Representative UCNs for biomacromolecules sensing in solution, in cells and animals. a) Graphene oxide (GO)-UCNPs-based optical sensors for targeted detection of mRNA biomarkers present in Alzheimer's disease and prostate cancer. Adapted with permission. [45a] Copyright 2016, American Chemical Society. b) Confocal images of MCF-7, HeLa, PCS-460-010 cells with Au-UCNP pyramids nanobiosensor for miR-21 cellular sensing. Scale bar ¼ 20 μm. Reproduced with permission. [47] Copyright 2015, American Chemical Society. c) Upconversion nanoprobes for sensitive detection of miR-122 and in vivo optical imaging of a nude mouse with human liver cancer. Reproduced with permission. [65] Copyright 2015, American Chemical Society.
www.advancedsciencenews.com www.adpr-journal.com To further enable UCNs for in vitro and in vivo utilizations, upconversion nanoprobes are normally modified with specific coatings or ligands to improve the biocompatibility and tissuetargeting efficiency, which is different from that on paper and in solution sensing platforms. There has been a very detailed discussion in aforementioned Section 3 about the biomacromolecule sensing in cells and animals. Among them, Xu et al. used a chiral UCNP-AuNP-based LRET system and applied it for ultrasensitive miRNA sensing in living cells (Figure 11b). [47] Endogenous miRNA can be complementary to the recognition sequence of Au-UCNP pyramids, leading to the structural dissociation of the DNA frame and turning on the UCL. This nanoplatform allows the chiroptical and luminescent dual-mode quantitation of abnormal cellular miRNA. Na et al. fabricated a luminescence-enhanced LRET-based upconversion nanoprobe to detect the liver-specific miRNA 122 (miR-122) via the sequence complementary hybridization, this method can be successfully used for the imaging of both exogenous and endogenous miR-122 in cells and in living mice (Figure 11c). These studies provide the potential for future diagnostic applications.

Summary and Perspective
In the past decades, continuous efforts have been dedicated to develop intelligent UCNs for optical sensing of diverse biomacromolecules including DNA, RNA, proteins and enzymes. The main design principles of these sensitive UCNs involve the following factors: 1) UCNPs with desired luminescence; 2) the recognition moieties for effective interaction with bioanalytes; 3) luminescent energy acceptors with appropriate spectral overlap and spatial distance; 4) responsive mechanisms (LRET or IFE); 5) sensing platforms (test paper, array, nanoprobe, etc.); 6) biocompatible modifications and functionalization for in vitro and in vivo uses. Advanced studies of biomacromolecules sensing with UCNs have extended their applicability from basic biological analysis to disease diagnosis and pathological profiling. Compared with conventional fluorescent dye molecules and chromophores-based biosensing systems, UCNs show obvious merits such as good photostability, low background interference, high sensitivity, and satisfied biocompatibility. Despite great achievements that have been made, there remain several critical concerns that need to be addressed before UCNs can be translated from the bench studies to practical applications.
First, the relatively low upconversion quantum yield of UCNPs is the major obstacle that always limits their biomedical applications, especially for in vivo models. Some strategies demonstrated the feasibility to improve the photon-upconverting efficacy through either manipulating the local environment or enhancing the energy transfer, which includes surface passivation with core-shell/multi-shell structures, and surface modifications with sensitizers/activators as well as engineering of the excitation source. [96] Although these efforts are of value for further refining UCNs as reliable biosensors, it is still a long way for improvement toward practical uses.
Second, the low energy transfer efficiency in UCNPs-based detection systems is another issue that affects the sensing performance. In the LRET process, besides the optimum spectra overlap between upconversion luminescence and the absorption band of energy acceptors, the distance (<10 nm) between luminescent UCNPs and energy acceptors are key parameters for sensitive luminescence response in biosensing [97] ; IFE-based upconversion sensing depends on the tuning of appropriate chromophores for maximum luminescence reabsorption. However, most of the reported UCNs with either inadequate spectral overlap or long spatial distance between luminescent centers and energy acceptors that are unable to match the theoretical radius for effective energy transfer.
Last but not the least, so far the development of UCNs for biomacromolecules sensing is still at the basic research stage, many big or small obstacles need to be well addressed for future daily clinical uses. For example, the biosafety issues owing to the nanotoxicity or photodamage are mostly encountered in in vivo studies; UCNs for specific sensing of tumor biomarkers in living conditions are limited to passive targeting through conjugating tumor-targeted FA and RGD peptides, which always leads to the low tumor accumulating efficacy and nonspecific interference, thus actively targeted biosensing based on UCNPs is highly demanded in future studies; the complex protocols for UCNs construction limit the prospect of industrial preparation and commercial transformation; there are lack of unified standards in the field of upconversion biosensing, which leads to unsatisfied reproducibility and quantitative accuracy; most of the optical detectors used in upconversion biosensing are custom built-in biological research labs, commercially available instrumentations that are compatible with the existing fluorescence microscope, microarray scanner or readers are highly demanded.
Although the aforementioned potential problems limit the biosensing applications of UCNs in real life, there are still some future research directions and development prospects of upconversion biophotonics worth mentioning. In particular, upconversion optogenetics is gradually moving towards a broader field of biological regulation and optical therapeutics; NIR-II upconversion or downconversion technology shows the merits for deeptissue bioimaging and phototheranostics; UCNPs open the emerging frontiers of super-resolution imaging based on the unique optical mechanisms, such as the photon avalanche and nonlinearity. Overall, future innovative studies focusing on both technical refinements and application extensions of UCNs are still required, it is expected that the continued efforts will offer upconversion optical sensing great promise for practical biomedical analysis and diagnostic utilizations.