Spatially confined dual-emission nanoprobes assembled from silicon nanoparticles and gold nanoclusters for ratiometric biosensing.

Gold nanoclusters (AuNCs) possess weak intrinsic fluorescence, limiting their sensitivity in biosensing applications. This study addresses these limitations by developing a spatially confined dual-emission nanoprobe composed of silicon nanoparticles (SiNPs) and AuNCs. This amplified and stabilized fluorescence mechanism overcomes the limitations associated with using AuNCs alone, achieving superior sensitivity in the sensing platform. The nanoprobe was successfully employed for ratiometric detection of bleomycin (BLM) in serum samples, operating at an excitation wavelength of 365 nm, with emission wavelengths at 480 nm and 580 nm. The analytical performance of the system is distinguished by a linear detection range of 0-3.5 µM, an impressive limit of detection (LOD) of 35.27 nM, and exceptional recoveries ranging from 96.80 to 105.9%. This innovative approach significantly enhances the applicability and reliability of AuNC-based biosensing in complex biological media, highlighting its superior analytical capabilities.

An ATMND/SGI based three-way junction ratiometric fluorescent probe for rapid and sensitive detection of bleomycin.

In this work, we developed a rapid and sensitive label-free ratiometric fluorescent (FL) probe for the detection of bleomycin (BLM). The probe consists of a DNA sequence (D6) and two fluorophore groups, 2-amino-5,6,7-trimethyl-1,8-naphthalene (ATMND) and SYBR Green I (SGI). The D6 sequence could be folded into a three-way junction structure containing a C-C mismatch position in the junction pocket. The unique "Y" structure not only could entrap ATMND in the mismatch pocket with high affinity, leading to FL quenching at 408 nm, but also embed SGI in the grooves of the double-stranded portion, resulting in FL enhancement at 530 nm. In the presence of BLM-Fe(II), the "Y" structure of D6 was destroyed due to the specific cleavage of the BLM recognition site, the 5'-GT-3' site in D6. This caused the release of ATMND and SGI and thus the ratiometric signal change of FL enhancement by ATMND and FL quenching by SGI. Under optimal conditions, the ratiometric probe exhibited a linear correlation between the intensity ratio of F408/F530 and the concentration of BLM in the range of 0.5-1000 nM, with a detection limit of 0.2 nM. In addition, the probe was applied to detect BLM in human serum samples with satisfactory results, indicating its good clinical application potential.

Ratiometric fluorescence determination of alkaline phosphatase activity based on carbon dots and Ce3+-crosslinked copper nanoclusters.

A new ratiometric fluorescent probe for efficient determination of ALP was developed. The probe was constructed by combining Ce3+-crosslinked copper nanoclusters (Ce3+-CuNCs) which exhibit the aggregation-induced emission (AIE) feature with carbon dots (CDs). The introduction of phosphate (Pi) induced the generation of CePO4 precipitation, resulting in significant decrease of fluorescence emission of CuNCs at 634 nm. At the same time, the fluorescence of CDs at 455 nm was obviously enhanced, thus generating ratiometric fluorescence response. Based on the fact that the hydrolysis of pyrophosphate (PPi) by ALP can produce Pi, the CD/Ce3+-CuNCs ratiometric probe was successfully used to determine ALP. A good linear relationship between the ratiometric value of F455/F634 and ALP concentrations ranging from 0.2 to 80 U·L- 1 was obtained, with a low detection limit of 0.1 U·L- 1. The ratiometric responses of the probe resulted in the visible fluorescence color change from orange red to blue with the increase of ALP concentration. The smartphone-based RGB recognition of the fluorescent sample images was used for ALP quantitative determination. A novel ratiometric fluorescent system based on Ce3+-CuNCs with AIE feature and CDs were constructed for efficient detection of ALP.

Activatable Near-Infrared Fluorescent and Photoacoustic Dual-Modal Probe for Highly Sensitive Imaging of Sulfatase In Vivo.

Sulfatase is an important biomarker closely associated with various diseases. However, the state-of-the-art sulfatase probes are plagued with a short absorption/emission wavelength and limited sensitivity. Developing highly sensitive fluorescent probes for in vivo imaging of sulfatase remains a grand challenge. Herein, for the first time, an activatable near-infrared fluorescence/photoacoustic (NIRF/PA) dual-modal probe (Hcy-SA) for visualizing sulfatase activity in living cells and animals is developed. Hcy-SA is composed of a sulfate ester moiety as the recognition unit and a NIR fluorophore hemicyanine (Hcy-OH) as the NIRF/PA reporter. The designed probe exhibits a rapid response, excellent sensitivity, and high specificity for sulfatase detection in vitro. More importantly, cells and in vivo experiments confirm that Hcy-SA can be successfully applied for PA/NIRF dual-modal imaging of sulfatase activity in living sulfatase-overexpressed tumor cells and tumor-bearing animals. This probe can serve as a promising tool for sulfatase-related pathological research and cancer diagnosis.

Enzyme-free nucleic acid dual-amplification strategy combined with mimic enzyme catalytic precipitation reaction for the photoelectrochemical detection of microRNA-21.

A novel photoelectrochemical (PEC) biosensor based on an enzyme-free nucleic acid dual-amplification strategy combined with a mimic enzyme to catalyze the deposition of a quencher is reported for the ultrasensitive detection of miRNA-21. A limited amount of target miRNA-21 can trigger the formation of long DNA duplexes on the electrode, owing to the synergistic effect of the enzyme-free nucleic acid dual-amplification strategy of entropy-driven strand displacement reaction (ESDR) amplification and hybridization chain reaction (HCR) amplification. The embedded manganese porphyrin (MnPP) in the long DNA duplexes acts as a horseradish peroxidase (HRP)-mimicking enzyme to catalyze the transformation of benzo-4-chlorohexadienone on the electrode surface, resulting in a significant reduction in photocurrent intensity. As a photosensitive material, BiOCl-BiOI is used as a tag to provide strong initial PEC signals. Based on the cascade integration of the enzyme-free nucleic acid dual-amplification strategy and the mimic enzyme-catalyzed precipitation reaction, the current PEC biosensor exhibits outstanding performance for miRNA-21 detection with an ultralow detection limit (33 aM) and a wide quantification range (from 100 aM to 1 nM). This work provides a new avenue toward the ultrasensitive detection of miRNAs, and is expected to be used for clinical and biochemical samples. A unique PEC biosensor with the BiOCl-BiOI composite, as the photosensitive material, has been developed for ultrasensitive miRNA-21 determination based on the combination of an enzyme-free nucleic acid dual-amplification strategy and mimic enzyme catalytic precipitation reaction.

Clicking of organelle-enriched probes for fluorogenic imaging of autophagic and endocytic fluxes.

Autophagy and endocytosis are essential in regulating cellular homeostasis and cancer immunotherapeutic responses. Existing methods for autophagy and endocytosis imaging are susceptible to cellular micro-environmental changes, and direct fluorogenic visualization of their fluxes remains challenging. We develop a novel strategy via clicking of organelle-enriched probes (COP), which comprises a pair of trans-cyclooctenol (TCO) and tetrazine probes separately enriched in lysosomes and mitochondria (in autophagy) or plasma membrane (in endocytosis). These paired probes are merged and boost a fluorogenic click reaction in response to autophagic or endocytic flux that ultimately fuses mitochondria or plasma membrane into lysosomes. We demonstrate that this strategy enables direct visualization of autophagic and endocytic fluxes, and confer insight into correlation of autophagic or endocytic flux to cell surface expression of immunotherapeutic targets such as MHC-I and PD-L1. The COP strategy provides a new paradigm for imaging autophagic and endocytic fluxes, and affords potential for improved cancer immunotherapy using autophagy or endocytosis inhibitors.

Fluorescent and colorimetric determination of glutathione based on the inner filter effect between silica nanoparticle-gold nanocluster nanocomposites and oxidized 3,3',5,5'-tetramethylbenzidine.

Determination of glutathione (GSH) is closely related to the clinical diagnosis of many diseases. Thus, a fluorescent and colorimetric dual-readout strategy for the sensitive determination of glutathione was proposed. The mesoporous silica nanoparticle-gold nanocluster (MSN-AuNC) nanocomposites with significantly enhanced emission and effectively improved photostability characteristics were used as fluorescent probes. Based on the inner filter effect (IFE), the fluorescence of MSN-AuNCs at 570 nm can be effectively quenched by oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB) with absorption in the wavelength ranges of 330-470 nm and 500-750 nm. However, the addition of GSH could cause the reduction of blue oxTMB to colorless TMB, resulting in the inhibition of IFE and the recovery of the fluorescence of MSN-AuNCs. Therefore, using oxTMB as both quencher and color indicator, a dual-readout oxTMB/MSN-AuNC sensing system for the sensitive determination of GSH was constructed. As signal amplification is caused by the fluorescence enhancement of MSN-AuNCs, the detection limits as low as 0.12 µM and 0.34 µM can be obtained for fluorescent and colorimetric assay, respectively. This method may not only offer a new idea for the sensitive and effective determination of GSH, but also broaden the applications of AuNCs in fluorescent and colorimetric dual-readout bioanalysis.

In-situ synthesis of 3D Cu2O@Cu-based MOF nanobelt arrays with improved conductivity for sensitive photoelectrochemical detection of vascular endothelial growth factor 165.

Construction of novel photoelectrochemical (PEC) materials with unique structures can effectively improve the photoelectric conversion efficiency. Here, a self-supported Cu2O@Cu-MOF/copper mesh (CM) nanobelt arrays with high specific surface area, high orientation, and high photoelectric conversion performance is obtained by in-situ grown strategy. Such PEC aptasensor is constructed based on the Cu2O@Cu-MOF/CM combined with rolling circle amplification and enzymatic biocatalytic precipitation for vascular endothelial growth factor 165 analysis. This strategy achieves excellent cooperative signal amplification, which greatly improves the detection sensitivity. The PEC aptasensor exhibited a wide calibration ranged from 10 to 1 × 108 fM with a detection limit down to 2.3 fM (S/N = 3). The construction of semiconductor@MOFs has developed the potential application of MOFs in photoelectrochemical and found a reliable path for ultrasensitive detection of biomarkers.

A tumour mRNA-triggered nanoassembly for enhanced fluorescence imaging-guided photodynamic therapy.

A multifunctional theranostic nanoplatform, which integrates diagnostic and therapeutic functions in a single nanosystem, holds great promise for guiding disease treatment and improving the corresponding therapy efficacy. We report the development of a novel g-C3N4 nanosheet-based theranostic nanoassembly for both enhanced imaging of cancer-relevant mRNA in living cells and imaging-guided on-demand photodynamic therapy (PDT) for tumors. The nanoassembly was constructed by using highly fluorescent and water-dispersible g-C3N4 nanosheets which act as nanocarriers, enabling efficient and self-tracking transfection of the DNA hairpin probes. The presence of intracellular mRNA will initiate the DNA hairpin probes, ultimately resulting in an amplified fluorescence signal via hybridization and displacement with mRNA. Moreover, enhanced fluorescence imaging-guided precise PDT for tumors in living cells was also demonstrated, allowing the selective ablation of tumors without any obvious side effects. Therefore, the developed theranostic approach can provide a promising platform for low-abundance biomarker discovery and early treatment of related diseases.

Mitochondrion-Targeting Fluorescence Probe via Reduction Induced Charge Transfer for Fast Methionine Sulfoxide Reductases Imaging.

Methionine sulfoxide reductases (Msrs) play essential roles in maintaining mitochondrial function and are recognized as potential therapeutic targets. However, current probes for Msrs fail to target mitochondria and exhibit a relatively slow response and limited sensitivity. Here we develop a novel turn-on fluorescence probe that facilitates imaging of mitochondrial Msrs in living cells. The probe is constructed by conjugating a methyl phenyl sulfoxide, a mimic Msrs substrate, to an electron-withdrawing hydrophobic cation, methylpyridinium. The probe of acceptor-acceptor structure is initially nonemissive. Msrs catalyzed reduction of sulfoxide to sulfide generated a fluorophore of distinct donor-acceptor structure. The probe is demonstrated to exhibit high sensitivity, fast response, and high selectivity toward MsrA in vitro. Furthermore, the probe is successfully introduced to detect and image Msrs in living cells with excellent mitochondrial-targeting capability. Moreover, the probe also reveals decreased Msrs activity in a cellular Parkinson's disease model. Our probe affords a powerful tool for detecting and visualizing mitochondrial Msrs in living cells.

Proximity-induced hybridization chain assembly with small-molecule linked DNA for single-step amplified detection of antibodies.

A novel and versatile platform for single-step amplified fluorescence detection of antibodies via specific proximity-induced hybridization chain assembly is developed.

Programmable Self-Assembly of Protein-Scaffolded DNA Nanohydrogels for Tumor-Targeted Imaging and Therapy.

DNA hydrogels are biocompatible and are suitable for many biomedical applications. However, to be useful imaging probes or drug carriers, the ordinary bulk size of DNA hydrogels must be overcome. Here we put forward a new strategy for fabricating a novel and simple protein-scaffolded DNA nanohydrogel, constructed through a direct DNA self-assembly using three types of streptavidin (SA)-based DNA tetrad for the activation of imaging and targeting therapy of cancer cells. The DNA nanohydrogels are easily prepared, and we show that by varying the initial concentration of DNA tetrad, it is possible to finely control their size within nanoscale range, which are favorable as carriers for intracellular imaging and transport. By further incorporating therapeutic agents and tumor-targeting MUC1 aptamer, these multifunctionalized SA-scaffolded DNA nanohydrogels (SDH) can specifically target cancer cells and selectively release the preloaded therapeutic agents via a structure switching when in an ATP-rich intracellular environment, leading to the activation of the fluorescence and efficient treatment of cancer cells. With the advantages of facile modular design and assembly, effective cellular uptake, and excellent biocompatibility, the method reported here has the potential for the development of new tunable DNA nanohydrogels with multiple synergistic functionalities for biological and biomedical applications.

Development of large Stokes shift, near-infrared fluorescence probe for rapid and bioorthogonal imaging of nitroxyl (HNO) in living cells.

Nitroxyl (HNO), as an electron reduced and protonated form of nitric oxide, is emerging as a potential diagnostic and therapeutic biomarker. It is still of great interest to develop probes of desirable properties to study its biological functions. Here we develop a near infrared fluorescence probe for detecting and visualizing exogenous and endogenous HNO in living cells. The probe is designed by coupling a HNO-responsive moiety, diphenylphosphinobenzoyl group, with a near infrared fluorophore with large of Stokes shift via an ester linker. The probe was initially nonfluorescent. HNO-catalyzed oxidation reaction generates an aza-ylide, which intramolecularly attacks the carbonyl carbon, liberating the initial fluorophore with activated fluorescence signals. The probe is proportional to the concentrations of HNO in the range of 2.0-80 µM with a limit of detection of 0.05 µM. Furthermore, the probe also exhibits high selectivity and fast response (reaching plateau within 600 s) towards HNO in vitro. Moreover, imaging studies reveal that the probe is capable of detecting exogenous HNO with dose-dependent fluorescence signals. Its ability to image endogenous HNO without or with induction is also demonstrated in living cells. This turn-on fluorescence probe provides a useful tool for studying HNO in living cells.

DNA Polymer Nanoparticles Programmed via Supersandwich Hybridization for Imaging and Therapy of Cancer Cells.

Spherical nucleic acid (SNA) constructs are promising new single entity materials, which possess significant advantages in biological applications. Current SNA-based drug delivery system typically employed single-layered ss- or ds-DNA as the drug carriers, resulting in limited drug payload capacity and disease treatment. To advance corresponding applications, we developed a novel DNA-programmed polymeric SNA, a long concatamer DNA polymer that is uniformly distributed on gold nanoparticles (AuNPs), by self-assembling from two short alternating DNA building blocks upon initiation of immobilized capture probes on AuNPs, through a supersandwich hybridization reaction. The long DNA concatamer of polymeric SNA enables to allow high-capacity loading of bioimaging and therapeutics agents. We demonstrated that both of the fluorescence signals and therapeutic efficacy were effectively inhibited in resultant polymeric SNA. By further modifying with the nucleolin-targeting aptamer AS1411, this polymeric SNA could be specifically internalized into the tumor cells through nucleolin-mediated endocytosis and then interact with endogenous ATP to cause the release of therapeutics agents from long DNA concatamer via a structure switching, leading to the activation of the fluorescence and selective synergistic chemotherapy and photodynamic therapy. This nanostructure can afford a promising targeted drug transport platform for activatable cancer theranostics.

Activatable Fluorescence Probe via Self-Immolative Intramolecular Cyclization for Histone Deacetylase Imaging in Live Cells and Tissues.

Histone deacetylases (HDACs) play essential roles in transcription regulation and are valuable theranostic targets. However, there are no activatable fluorescent probes for imaging of HDAC activity in live cells. Here, we develop for the first time a novel activatable two-photon fluorescence probe that enables in situ imaging of HDAC activity in living cells and tissues. The probe is designed by conjugating an acetyl-lysine mimic substrate to a masked aldehyde-containing fluorophore via a cyanoester linker. Upon deacetylation by HDAC, the probe undergoes a rapid self-immolative intramolecular cyclization reaction, producing a cyanohydrin intermediate that is spontaneously rapidly decomposed into the highly fluorescent aldehyde-containing two-photon fluorophore. The probe is shown to exhibit high sensitivity, high specificity, and fast response for HDAC detection in vitro. Imaging studies reveal that the probe is able to directly visualize and monitor HDAC activity in living cells. Moreover, the probe is demonstrated to have the capability of two-photon imaging of HDAC activity in deep tissue slices up to 130 µm. This activatable fluorescent probe affords a useful tool for evaluating HDAC activity and screening HDAC-targeting drugs in both live cell and tissue assays.

A fluorescent graphitic carbon nitride nanosheet biosensor for highly sensitive, label-free detection of alkaline phosphatase.

Graphitic C3N4 (g-C3N4) nanosheets provide an attractive option for bioprobes and bioimaging applications. Utilizing highly fluorescent and water-dispersible ultrathin g-C3N4 nanosheets, a highly sensitive, selective and label-free biosensor has been developed for ALP detection for the first time. The developed approach utilizes a natural substrate of ALP in biological systems and thus affords very high catalytic efficiency. This novel biosensor is demonstrated to enable quantitative analysis of ALP in a wide range from 0.1 to 1000 U L(-1) with a low detection limit of 0.08 U L(-1), which is among the most sensitive assays for ALP. It is expected that the developed method may provide a low-cost, convenient, rapid and highly sensitive platform for ALP-based clinical diagnostics and biomedical applications.