Recent advances in autofluorescence-free biosensing and bioimaging based on persistent luminescence nanoparticles

Persistent luminescence nanoparticles (PLNPs) refer to a series of luminescent nanomaterials that can swiftly store the excitation energy and emit persistently after ceasing the excitation. Due to the characteristics of quickly storing the excitation energy and slowly emitting luminescence for a long time after ceasing excitation, they can effectively diminish background fluorescence, and are ideal for fluorescent analysis, especially in autofluorescencefree biosensing and bioimaging.     

Development of a Ruthenium(II) Complex Based Luminescent Probe for Imaging Nitric Oxide Production in Living Cells

A unique ruthenium(II) complex, bis(2,2′‐bipyridine)(4‐(3,4‐diaminophenoxy)‐2,2′‐bipyridine)ruthenium(II) hexafluorophosphate ([(Ru(bpy)₂(dabpy)][PF₆]₂), has been designed and synthesized as a highly sensitive and selective luminescence probe for the imaging of nitric oxide (NO) production in living cells. The complex can specifically react with NO in aqueous buffers under aerobic conditions to yield its triazole derivative with a high reaction rate constant at the 10¹⁰ M ^?1 s^?1 level; this reaction is accompanied by a remarkable increase of the luminescence quantum yield from 0.13 to 2.2 %. Compared with organic probes, the new Ru^II complex probe shows the advantages of a large Stokes shift (>150 nm), water solubility, and a wide pH‐availability range (pH independent at pH>5). In addition, it was found that the new probe could be easily transferred into both living animal cells and plant cells by the coincubation method, whereas the triazole derivative was cell‐membrane impermeable. The probe was successfully used for luminescence‐imaging detection of the exogenous NO in mouse macrophage cells and endogenous NO in gardenia cells. The results demonstrated the efficacy and advantages of the new probe for NO detection in living cells.     

Recent advances in autofluorescence-free biosensing and bioimaging based on persistent luminescence nanoparticles

Persistent luminescence nanoparticles (PLNPs) are a series of emerging luminescent nanomaterials which can emit persistently after ceasing the external excitation. Due to the long decay time of persistent luminescence, the background autofluorescence in complex sample and tissues can be effectively eliminated, thus significantly improving the sensitivity of bioanalysis. Besides, such a long decay time of luminescence also makes PLNPs valuable for long-term bioimaging. Benefiting from these merits, PLNPs have been widely used for biomedical applications, especially biosensing and bioimaging. In this review, we conclude the progress in the application of PLNPs at biosensing and bioimaging in recent years, and also provide our understanding of the prospects.     

Immobilization of the Gas Signaling Molecule H2S by Radioisotopes: Detection,Quantification, and In Vivo Imaging

Hydrogen sulfide (H₂S) has multifunctional roles as a gas signaling molecule in living systems. However, the efficient detection and imaging of H₂S in live animals is very challenging. Herein, we report the first radioisotope‐based immobilization technique for the detection, quantification, and in vivo imaging of endogenous H₂S. Macrocyclic ⁶⁴Cu complexes that instantly reacted with gaseous H₂S to form insoluble ⁶⁴CuS in a highly sensitive and selective manner were prepared. The H₂S concentration in biological samples was measured by a thin‐layer radiochromatography method. When ⁶⁴Cu–cyclen was injected into mice, an elevated H₂S concentration in the inflamed paw was clearly visualized and quantified by Cerenkov luminescence and positron emission tomography (PET) imaging. PET imaging was also able to pinpoint increased H₂S levels in a millimeter‐sized infarcted lesion of the rat heart.     

Immobilization of the Gas Signaling Molecule H2S by Radioisotopes: Detection,Quantification, and In Vivo Imaging

Hydrogen sulfide (H₂S) has multifunctional roles as a gas signaling molecule in living systems. However, the efficient detection and imaging of H₂S in live animals is very challenging. Herein, we report the first radioisotope‐based immobilization technique for the detection, quantification, and in vivo imaging of endogenous H₂S. Macrocyclic ⁶⁴Cu complexes that instantly reacted with gaseous H₂S to form insoluble ⁶⁴CuS in a highly sensitive and selective manner were prepared. The H₂S concentration in biological samples was measured by a thin‐layer radiochromatography method. When ⁶⁴Cu–cyclen was injected into mice, an elevated H₂S concentration in the inflamed paw was clearly visualized and quantified by Cerenkov luminescence and positron emission tomography (PET) imaging. PET imaging was also able to pinpoint increased H₂S levels in a millimeter‐sized infarcted lesion of the rat heart.     

Controlling electron trap depth to enhance optical properties of persistent luminescence nanoparticles for in vivo imaging

Focusing on the use of nanophosphors for in vivo imaging and diagnosis applications, we used thermally stimulated luminescence (TSL) measurements to study the influence of trivalent lanthanide Ln(3+) (Ln = Dy, Pr, Ce, Nd) electron traps on the optical properties of Mn(2+)-doped diopside-based persistent luminescence nanoparticles. This work reveals that Pr(3+) is the most suitable Ln(3+) electron trap in the diopside lattice, providing optimal trap depth for room temperature afterglow and resulting in the most intense luminescence decay curve after X-ray irradiation. This luminescence dependency toward the electron trap is maintained through additional doping with Eu(2+), allowing UV-light excitation, critical for bioimaging applications in living animals. We finally identify a novel composition (CaMgSi(2)O(6):Eu(2+),Mn(2+),Pr(3+)) for in vivo imaging, displaying a strong near-infrared afterglow centered on 685 nm, and present evidence that intravenous injection of such persistent luminescence nanoparticles in mice allows not only improved but highly sensitive detection through living tissues.     

Iridium(III) Complex-Based Activatable Probe for Phosphorescent/Time-Gated Luminescent Sensing and Imaging of Cysteine in Mitochondria of Live Cells and Animals

This study reports an activatable iridium(III) complex probe for phosphorescence/time-gated luminescence detection of cysteine (Cys) in vitro and in vivo. The probe, [Ir(ppy)₂(NTY-bpy)](PF₆) [ppy: 2-phenylpyridine; NTY-bpy: 4-methyl-4′-(2-nitrovinyl)-2,2′-bipyridine], is developed by incorporating a strong electron-withdrawing group, nitroolefin, into a bipyridine ligand of the Ir^III complex. The luminescence of the probe is quenched owing to the intramolecular charge transfer (ICT) process, but switched on by a specific recognition reaction between the probe and Cys. [Ir(ppy)₂(NTY-bpy)](PF₆) shows high sensitivity and selectivity for Cys detection and good biocompatibility. The long-lived emission of [Ir(ppy)₂(NTY-bpy)](PF₆) allows time-gated luminescence analysis of Cys in cells and human sera. These properties make it convenient for the phosphorescence and time-gated luminescence imaging and flow cytometry analysis of Cys in live samples. The Cys images in cancer cells and inflamed macrophage cells reveal that [Ir(ppy)₂(NTY-bpy)](PF₆) is distributed in mitochondria after cellular internalization. Visualizations and flow cytometry analysis of mitochondrial Cys levels and Cys-mediated redox activities of live cells are achieved. By using [Ir(ppy)₂(NTY-bpy)](PF₆) as a probe, in vivo sensing and imaging of Cys in D. magna, zebrafish, and mice are then demonstrated.     

X-Ray-triggered Carbon Monoxide and Manganese Dioxide Generation based on Scintillating Nanoparticles for Cascade Cancer Radiosensitization

Carbon monoxide (CO) is an endogenous signaling molecule with broad therapeutic effects. Here, a multifunctional X-ray-triggered carbon monoxide (CO) and manganese dioxide (MnO₂) generation nanoplatform based on metal carbonyl and scintillating nanoparticles (SCNPs) is reported. Attributed to the radioluminescent characteristic of SCNPs, UV-responsive Mn₂(CO)₁₀ is not only indirectly activated to release CO by X-ray but can also be degraded into MnO₂. A high dose of CO can be used as a glycolytic inhibitor for tumor suppression; it will also sensitize tumor cells to radiotherapy. Meanwhile MnO₂, as the photolytic byproduct of Mn₂(CO)₁₀, has both glutathione (GSH) depletion and Fenton-like Mn²⁺ delivery properties to produce highly toxic hydroxyl radical (⋅OH) in tumors. Thus, this strategy can realize X-ray-activated CO release, GSH depletion, and ⋅OH generation for cascade cancer radiosensitization. Furthermore, X-ray-activated Mn²⁺ in vivo demonstrates an MRI contrast effect, making it a potential theranostic nanoplatform.     

A Smart DNAzyme–MnO2 Nanosystem for Efficient Gene Silencing

DNAzymes hold promise for gene‐silencing therapy, but the lack of sufficient cofactors in the cell cytoplasm, poor membrane permeability, and poor biostability have limited the use of DNAzymes in therapeutics. We report a DNAzyme–MnO₂ nanosystem for gene‐silencing therapy. MnO₂ nanosheets adsorb chlorin e6‐labelled DNAzymes (Ce6), protect them from enzymatic digestion, and efficiently deliver them into cells. The nanosystem can also inhibit ¹O₂ generation by Ce6 in the circulatory system. In the presence of intracellular glutathione (GSH), MnO₂ is reduced to Mn²⁺ ions, which serve as cofactors of 10–23 DNAzyme for gene silencing. The release of Ce6 generates ¹O₂ for more efficient photodynamic therapy. The Mn²⁺ ions also enhance magnetic resonance contrast, providing GSH‐activated magnetic resonance imaging (MRI) of tumor cells. The integration of fluorescence recovery and MRI activation provides fluorescence/MRI bimodality for monitoring the delivery of DNAzymes.     

Biodegradable nanoprobe based on MnO<Subscript>2</Subscript> nanoflowers and graphene quantum dots for near infrared fluorescence imaging of glutathione in living cells

Near infrared (NIR) emitting semiconductor quantum dots can be excellent fluorescent nanoprobes, but the poor biodegradability and potential toxicity limits their application. The authors describe a fluorescent system composed of graphene quantum dots (GQDs) as NIR emitters, and novel MnO₂ nanoflowers as the fluorescence quenchers. The system is shown to be an activatable and biodegradable fluorescent nanoprobe for the “turn-on” detection of intracellular glutathione (GSH). The MnO₂-GQDs nanoprobe is obtained by adsorbing GQDs onto the surface of MnO₂ nanoflowers through electrostatic interaction. This results in the quenching of the NIR fluorescence of the GQDs. In the presence of GSH, the MnO₂-GQDs nanoprobe is degraded and releases Mn²⁺ and free GQDs, respectively. This gives rise to increased fluorescence. The nanoprobe displays high sensitivity to GSH and with a 2.8 μM detection limit. It integrates the advantages of NIR fluorescence and biodegradability, selectivity, biocompatibility and membrane permeability. All this makes it a promising fluorescent nanoprobe for GSH and for cellular imaging of GSH as shown here for the case of MCF-7 cancer cells.

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Graphical abstract A biodegradable NIR fluorescence nanoprobe (MnO₂-GQDs) for the “turn-on” detection of GSH in living cell was established, with the NIR GQD as the fluorescence reporter and the MnO₂ nanoflower as the fluorescence quencher.

     

Direct low-temperature synthesis of ultralong persistent luminescence nanobelts based on a biphasic solution-chemical reaction

The ultralong Zn₂GeO₄:Mn²⁺ persistent luminescence nanobelts (PLNBs) were synthesized using a direct hydrothermal route. The persistent luminescence performance is fine-turned upon prolonging the hydrothermal time and controlling the doping ratio of Mn²⁺. This solid-statereaction-free chemical approach will promote the broad use of these unique nanostructured PLNBs in developing imaging device.     

Non‐Aqueous Sol–Gel Synthesis of Ultra Small Persistent Luminescence Nanoparticles for Near‐Infrared In Vivo Imaging

Ultra‐small ZnGa₂O₄:Cr³⁺ nanoparticles (6 nm) that exhibit near‐infrared (NIR) persistent luminescence properties are synthesized by using a non‐aqueous sol–gel method assisted by microwave irradiation. The nanoparticles are pegylated, leading to highly stable dispersions under physiological conditions. Preliminary in vivo studies show the high potential for these ultra‐small ZnGa₂O₄:Cr³⁺ nanoparticles to be used as in vivo optical nanotools as they emit without the need for in situ excitation and, thus, avoid the autofluorescence of tissues.     

用于活体和细胞内SO2衍生物检测的线粒体靶向双光子磷光铱(Ⅲ)配合物

暴露于高剂量的二氧化硫（SO₂）及其衍生物（SO₃^2-和HSO₃^-）会导致血管疾病甚至肺癌发生。双光子磷光成像显微术（TPPIM）和双光子磷光寿命成像显微术（TPPLIM）具有良好的时空分辨能力、抗光漂白、抗自体荧光以及较强组织穿透性等优点,可以实现SO₂衍生物在生物样品中实时检测。得益于铱配合物的长磷光寿命（~110 ns）和线粒体靶向特性,本文报道了首例基于TPPLIM技术的SO₂衍生物检测探针Ir-EA。Ir-EA对水溶液中的亚硫酸氢盐表现出高特异性和灵敏性的识别能力（69倍磷光增强,10倍亚硫酸氢盐）和较低的检测限（65 nmol·L^-1）。更为重要的是,Ir-EA对活细胞和斑马鱼的线粒体中SO₂衍生物表现出良好的成像效果。     

MnO2 nanosheets as a carrier and accelerator for improved live-cell biosensing application of CRISPR/Cas12a

Besides gene-editing, the CRISPR/Cas12a system has also been widely used in in vitro biosensing, but its applications in live-cell biosensing are rare. One reason is lacking appropriate carriers to synchronously deliver all components of the CRISPR/Cas12a system into living cells. Herein, we demonstrate that MnO₂ nanosheets are an excellent carrier of CRISPR/Cas12a due to the two important roles played by them. Through a simple mixing operation, all components of the CRISPR/Cas12a system can be loaded on MnO₂ nanosheets and thus synchronously delivered into cells. Intracellular glutathione (GSH)-induced decomposition of MnO₂ nanosheets not only results in the rapid release of the CRISPR/Cas12a system in cells but also provides Mn²⁺ as an accelerator to promote CRISPR/Cas12a-based biosensing of intracellular targets. Due to the merits of highly efficient delivery, rapid intracellular release, and the accelerated signal output reaction, MnO₂ nanosheets work better than commercial liposome carriers in live-cell biosensing analysis of survivin messenger RNA (mRNA), producing much brighter fluorescence images in a shorter time. The use of MnO₂ nanosheets might provide a good carrier for different CRISPR/Cas systems and achieve the rapid and sensitive live-cell biosensing analysis of different intracellular targets, thus paving a promising way to promote the applications of CRISPR/Cas systems in living cells.

Herein, we demonstrate that MnO₂ nanosheets are an excellent carrier of CRISPR/Cas12a due to the two important roles played by them.     

Simultaneous Fenton‐like Ion Delivery and Glutathione Depletion by MnO2‐Based Nanoagent to Enhance Chemodynamic Therapy

Chemodynamic therapy (CDT) utilizes iron‐initiated Fenton chemistry to destroy tumor cells by converting endogenous H₂O₂ into the highly toxic hydroxyl radical (^.OH). There is a paucity of Fenton‐like metal‐based CDT agents. Intracellular glutathione (GSH) with ^.OH scavenging ability greatly reduces CDT efficacy. A self‐reinforcing CDT nanoagent based on MnO₂ is reported that has both Fenton‐like Mn²⁺ delivery and GSH depletion properties. In the presence of HCO₃^?, which is abundant in the physiological medium, Mn²⁺ exerts Fenton‐like activity to generate ^.OH from H₂O₂. Upon uptake of MnO₂‐coated mesoporous silica nanoparticles (MS@MnO₂ NPs) by cancer cells, the MnO₂ shell undergoes a redox reaction with GSH to form glutathione disulfide and Mn²⁺, resulting in GSH depletion‐enhanced CDT. This, together with the GSH‐activated MRI contrast effect and dissociation of MnO₂, allows MS@MnO₂ NPs to achieve MRI‐monitored chemo–chemodynamic combination therapy.     

Quantitative Monitoring and Visualization of Hydrogen Sulfide In Vivo Using a Luminescent Probe Based on a Ruthenium(II) Complex

Development of novel bioanalytical methods for monitoring of H₂S is key toward understanding the physiological and pathological functions of this gasotransmitter in live organisms. A ruthenium(II)‐complex‐based luminescence probe, Ru‐MDB (MDB: 4’‐methyl‐[2,2’‐bipyridine]‐4‐yl)methyl 2‐((2,4‐dinitrophenyl)thio)benzoate), was developed by introducing a new H₂S responsive masking moiety to a red‐emitting Ru^II luminophore. Cleavage of this masking group by a H₂S‐triggered reaction leads to a luminescence “off–on” response. The long‐lived emissions of Ru‐MDB and its reaction product with H₂S allowed quantitative detection of H₂S in autofluorescence‐rich human sera and adult zebrafish organs using the time‐gated luminescence mode. Ru‐MDB exhibits red emission, a large Stokes shift, high specificity and sensitivity for H₂S detection, and low cytotoxicity, which enables imaging and flow cytometry analysis of lysosomal H₂S generation in live inflamed cells under drug stimulation. Monitoring of H₂S in live Daphnia magna, zebrafish embryos, adult zebrafish, and mice, was conducted by in vivo imaging using Ru‐MDB as a probe.     

Li5Zn8Ga5Ge9O36: Cr3+, Ti4+: A Long Persistent Phosphor Excited in a Wide Spectral Region from UV to Red Light for Reproducible Imaging through Biological Tissue

Near‐infrared (NIR) long‐persistent phosphors (LPPs) have emerged as a potential solution for bio‐imaging applications over the past few years. However, there are enormous challenges regarding their in situ application based on their dependence on short‐wavelength excitation. In this paper, we report a multi‐spectral excited NIR LPP, Li₅Zn₈Ga₅Ge₉O₃₆: 1.5 % Cr³⁺, 0.5 % Ti⁴⁺, which overcomes the limitations of functional processes in biological tissues and other complex systems. This LPP exhibits a high luminescent intensity and a long emission duration in the NIR region (700–800 nm). The applicability of this phosphor to tissue imaging is demonstrated experimentally. Its persistent luminescence (PersL) can easily penetrate approximately 2 mm of pork flesh. More importantly, this phosphor can be re‐charged in situ using a red LED or laser diode array to provide renewed NIR PersL for biological tissues, which is beneficial for long‐term biological tissue imaging applications with high signal‐to‐noise ratios. Systematic investigations of the nature of energy traps and PersL mechanisms are also reported in this paper.     

A covalent organic framework (COF)-MnO2 based dual signal sensing platform for sensitive alkaline phosphatase activity detection via dynamic regulating the mimicking oxidase content

It is of great significance to accurately monitor the alkaline phosphatase (ALP) level as it plays an important role in living body activities. Herein, we develop a COF- MnO₂ system for ALP activity detection via the dynamic regulating the MnO₂ nanosheets content. MnO₂ nanosheets with oxidase-mimicking property can oxide the colorless 3,3′,5,5′-Tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB). The hexagonal structure and ordered mesoporous channels of DMTP-TAPB COF provide excellent space to accommodate the product oxTMB. The confinement of the dye molecules into COF structure leads to enhance color change and obvious fluorescence quench of the sensing system. The fluorescence quenching and color change dependent on the ALP level as it can dynamic regulate the MnO₂ content via the enzymatic hydrolysis of ascorbate-2-phosphate. Therefore, a COF-MnO₂ based dual signal sensing platform is successfully constructed to detect ALP activity, giving detection limit of 0.11 U L^-1 and 0.23 U L^-1 for fluorescence and colorimetric procedures, respectively. The practical application of the designed sensing platform is verified through the detection of ALP activity in serum samples, and satisfactory results are obtained.     

Photoluminescent and electrochemiluminescent dual-signaling probe for bio-thiols based on a ruthenium(II) complex

Photoluminescence (PL) and electrochemiluminescence (ECL) detection techniques are highly sensitive and widely used methods for clinical diagnostics and analytical biotechnology. In this work, a unique ruthenium(II) complex, [Ru(bpy)₂(DNBSO-bpy)](PF₆)₂ (bpy: 2,2′-bipyridine; DNBSO-bpy: 2,4-dinitrobenzenesulfonate of 4-(4-hydroxyphenyl)-2,2′-bipyridine), has been designed and synthesized as a highly sensitive and selective PL and ECL dual-signaling probe for the recognition and detection of bio-thiols in aqueous media. As a thiol-responsive probe, the complex can specifically and rapidly react with bio-thiols in aqueous solutions to yield a bipyridine-Ru(II) complex derivative, [Ru(bpy)₂(HP-bpy)]²⁺ (HP-bpy: 4-(4-hydroxyphenyl)-2,2′-bipyridine), accompanied by the remarkable PL and ECL enhancements. The complex was used as a probe for the PL and ECL detections of cysteine (Cys) and glutathione (GSH) in aqueous solutions. The dose-dependent PL and ECL enhancements showed good linear relationships against the Cys/GSH concentrations with the detection limits at nano-molar concentration level. Moreover, the complex-loaded HeLa cells were prepared for PL imaging of the endogenous intracellular thiols. The results demonstrated the practical utility of the complex as a cell-membrane permeable probe for PL imaging detection of bio-thiols in living cells.     

尺寸可控Zn1.4Ga1.97-2xO4∶1.5%Cr，xIn近红外发光长余辉纳米颗粒的制备及其光学性质

采用一步水热法,通过In³⁺的掺杂,获得了尺寸可控的近红外(NIR)发光ZGO∶1.5%Cr,xIn(Zn_1.4Ga_1.97-2xO₄∶1.5%Cr,xIn,x=0%、0.1%、0.2%、0.3%、0.4%、0.5%)长余辉纳米颗粒(PLNPs),考察了In³⁺掺杂量对ZGO∶1.5%Cr,xIn PLNPs尺寸大小、余辉发光性能以及晶体结构的影响。In³⁺的掺杂不仅能有效控制ZGO∶1.5%Cr,xIn PLNPs尺寸,还可以增强发光和余辉时间。结果表明,当In³⁺掺杂量为0.2%时,ZGO∶1.5%Cr,0.2%In PLNPs平均粒径为13.79 nm,分布最为均匀,粒径最小,NIR发光最强,余辉时间超过5 d,可通过LED灯再激发。In³⁺的掺杂对ZGO∶1.5%Cr,xIn PLNPs的晶体结构无影响,均为纯相的尖晶石结构。