Isolation and characterization of feruloylated oligosaccharides from Phyllostachys acuta and in vitro antioxidant activity.

Feruloylated oligosaccharides (FOs) generated by decomposing plant hemicellulose, offer a wide range of potential applications in both the food and biomedical areas. As a graminaceous plant, bamboo is rich in hemicellulose. However, the structural composition and activity studies of FOs from it were rarely reported. In this study, FOs from Phyllostachys acuta (pFOs) obtained by enzymatic hydrolysis were isolated by AmberliteXAD-2 and C18 SPE columns. Then, pFOs were qualitatively and quantitatively analyzed by UPLC-ESI-MS/MS after labeled by 3-Amino-9-ethyl-carbazole (AEC), and the chemical antioxidant activity of pFOs and effects of pFOs on H2O2-induced oxidative damage were investigated. Finally, 14 of pFOs isomers were distinguished and identified, of which 10 did not contain hexoses and 4 did, and the three most abundant pFO structures were 12 (Iso 7, F1A1X2H2-AEC, 29.04 %), 11 (Iso 6, F1A1X1H2-AEC, 17.96 %), and 4 (Iso 3-1, F1A1X3-AEC, 15.57 %). The results of antioxidant studies showed that pFOs possessed certain reducing power, scavenging DPPH radicals, scavenging superoxide anion radicals, and scavenging hydroxyl radicals. Among them, the ability to clear DPPH radicals was particularly significant. pFOs significantly reduced the viability of RAW264.7 cells after H2O2 induction, whereas pFOs had a significant protective effect (p < 0.001). pFOs increased the viability of T-AOC and SOD enzymes in oxidatively damaged cells, as well as had a significant inhibition effect on ROS elevation (p < 0.001). This study lays the foundation for the structural analysis and antioxidant activity evaluation of bamboo-derived feruloyl oligosaccharides for their application in food and pharmaceutical fields.

Transcytosis-Based Renal Tubular Reabsorption of Luminescent Gold Nanoparticles for Enhanced Tumor Imaging.

Transcytosis-based tubular reabsorption of endogenous proteins is a well-known energy-saving pathway that prevents nutrient loss. However, utilization of this well-known reabsorption pathway for the delivery of exogenous nanodrugs remains a challenge. In this study, using the surface mimic strategy of a specific PEPT1/2-targeted Gly-Sar peptide as a ligand, renal-clearable luminescent gold nanoparticles (P-AuNPs) were developed as protein mimics to investigate the transcytosis-based tubular reabsorption of exogenous substances. By regulating the influential factors (H+ content in tubular lumens and PEPT1/2 transporter counts in tubular cells) of Gly-Sar-mediated transcytosis, the specific and efficient interaction between P-AuNPs and renal tubular cells was demonstrated both in vitro and in vivo. Efficient transcellular transportation significantly guided the reabsorption of P-AuNPs back into the bloodstream, which enhanced the blood concentration and bioavailability of nanoparticles, contributing to high-contrast tumor imaging.

Luminescent Gold Nanoparticles with Discrete DNA Valences for Precisely Controlled Transport at the Subcellular Level.

Ultrasmall luminescent gold nanoparticles (AuNPs) with excellent capabilities to cross biological barriers offer great promise in designing intelligent model nanomedicines for investigating structure-property relationships at the subcellular level. However, the strict surface controllability of ultrasmall AuNPs is challenging because of their small size. Herein, we report a facile in situ method for precisely controlling DNA aptamer valences on the surface of luminescent AuNPs with emission in the second near-infrared window using a phosphorothioate-modified DNA aptamer, AS1411, as a template. The discrete DNA aptamer number of AS1411-functionalized AuNPs (AS1411-AuNPs, ≈1.8 nm) with emission at 1030 nm was controlled in one aptamer (V1), two aptamers (V2), and four aptamers (V4). It was then discovered that not only the tumor-targeting efficiencies but also the subcellular transport of AS1411-AuNPs were precisely dependent on valences. A slight increase in valence from V1 to V2 increased tumor-targeting efficiencies and resulted in higher nucleus accumulation, whereas a further increase in valence (e.g., V4) significantly increased tumor-targeting efficiencies and led to higher cytomembrane accumulation. These results provide a basis for the strict surface control of nanomedicines in the precise regulation of in vivo transport at the subcellular level and their translation into clinical practice in the future.

In Vivo Aggregation of Clearable Bimetallic Nanoparticles with Interlocked Surface Motifs for Cancer Therapeutics Amplification.

Although renal-clearable luminescent metal nanoparticles (NPs) have been widely developed, their application to efficient cancer therapy is still limited due to low reactive oxygen species (ROS) production. Here, a novel system of clearable mercaptosuccinic acid (MSA) coated Au-Ag bimetallic NPs is designed to enhance ROS production. The results show that the strong COO-Ag coordination bonds between the carboxylic acid groups of MSA and Ag atoms on the Au-Ag bimetallic NPs could construct high-rigidity interlocked surface motifs to restrict the intrananoparticle motions for enhanced ROS generation. Moreover, bimetallic NPs exhibit pH-responsive self-assembly capability under the acidic environment inside lysosomes of cancer cells at both in vitro and in vivo, restricting the internanoparticle motions to further boost ROS production. The well-designed bimetallic NPs show high tumor targeting efficiency, fast elimination from the body through rapid liver biotransformation, and extensive destruction to cancer cells, resulting in good security and prominent therapeutic performance.

Glutathione-Activated Emission of Ultrasmall Gold Nanoparticles in the Second Near-Infrared Window for Imaging of Early Kidney Injury.

Biomarker-activatable luminescent probes with high sensitivity and specificity show great promise in advanced bioimaging applications. However, the lack of stable biomarkers at an early stage is currently a major obstacle for sensitive early disease imaging. Herein, we develop a facile in vivo ligand exchange strategy to achieve renal-clearable activatable luminescent gold nanoparticles (AuNPs), which are independent of biomarkers for sensitive and long-time imaging of early kidney injury. Significantly activated emission in the second near-infrared region (∼1026 nm) is realized from the ligand exchange of triphenylphosphine-3,3',3″-trisulfonic acid (TPPTS)-coated AuNPs (∼1.4 nm, TPPTS-AuNPs) with quantitative amounts of glutathione (GSH). The abundant GSH in cells, particularly in liver sinusoids, is then demonstrated successfully to activate the emission of TPPTS-AuNPs with an extremely low background for both cell imaging and in vivo visualization of visceral organs (e.g., liver and kidneys). In addition, the in vivo GSH-exchanged TPPTS-AuNPs show enhanced interactions with acidic renal tubular epithelial cells, resulting in sensitive (contrast index, ∼3.9) and long-time (>6.5 h) noninvasive monitoring of acidosis-induced early kidney injury. This facile ligand exchange strategy opens new possibilities for designing activatable luminescent probes independent of biomarkers for earlier disease diagnosis and treatment.

Rapid Biotransformation of Luminescent Bimetallic Nanoparticles in Hepatic Sinusoids.

Liver sequestration, mainly resulting from the phagocytosis of mononuclear phagocyte system (MPS) cells, is a long-standing barrier in nanoparticle delivery, which severely decreases the disease-targeting ability, leads to nanotoxicity, and inhibits clinical translation. To avoid long-term liver sequestration, we elaborately designed luminescent gold-silver bimetallic nanoparticles that could be rapidly transformed by the hepatic sinusoidal microenvironment rich in glutathione and oxygen, significantly different from monometallic gold nanoparticles that were rapidly sequestrated by Kupffer cells due to the much slower biotransformation. We found that the rapid sinusoidal biotransformation induced by the synergistic reactions of glutathione and oxygen with the reactive silver atoms could help bimetallic nanoparticles to avoid MPS phagocytosis, promote fast release from the liver, prolong blood circulation, enhance renal clearance, and increase disease targeting. With the fast biotransformation in sinusoids, liver sequestration could be turned into a beneficial storage mechanism for nanomedicines to maximize targeting.

Noninvasive photoacoustic computed tomography/ultrasound imaging to identify high-risk atherosclerotic plaques.

PURPOSE: Noninvasive detection of high-risk plaques is still challenging. In this study, we aimed to noninvasively assess αvß3-integrin expression using a customed photoacoustic (PA) computed tomography (PACT)/ultrasound (US) system in atherosclerotic lesions of varying degrees of severity and to explore its potential value for detecting high-risk plaques. METHODS: We constructed αvß3-integrin-targeted ultrasmall gold nanorods (AuNRs) with cyclo Arg-Gly-Asp (cRGD) and tested their properties. Employing C57BL/6 J (wild-type, WT) mice and apolipoprotein E gene knockout (ApoE-/-) mice fed either a chow diet or a high-fat/high-cholesterol diet (HFHCD), we established varying degrees of lesion severity. In vivo PACT/US imaging was performed to assess αvß3-integrin expression in the 4 groups by cRGD-AuNRs. Further histopathologic examination was conducted to evaluate the plaque vulnerability indicators. RESULTS: The data showed that cRGD-AuNRs exhibited excellent photothermal conversion capacity, stability, targeting ability, and biocompatibility. The immunohistochemical results indicated that αvß3-integrin was upregulated with increasing aggravation of the lesions. In vivo PACT/US imaging showed good consistency with αvß3-integrin expression. Notably, ApoE-/- mice fed a HFHCD showed an abrupt PA intensity increase compared with the other groups. The histopathologic examination verified that the atherosclerotic plaques of ApoE-/- mice fed the HFHCD developed unstable phenotypes. Correlation analysis showed that PA intensity was mainly related to inflammation and angiogenesis among all of the indicators. CONCLUSION: Our data indicated that αvß3-integrin is an effective indicator of plaque instability, and noninvasive PACT/US molecular imaging assessment of αvß3-integrin holds promise in detecting high-risk plaques.

Surface-regulated injection dose response of ultrasmall luminescent gold nanoparticles.

With the rapid growth of the use of renal-clearable nanomedicines in disease targeting and therapy, a fundamental understanding of their injection dose responses is of great importance for future translation to clinical settings. Using glutathione-coated gold nanoparticles (GS-AuNPs) as a renal-clearable nanomedicine model for the construction of ultrasmall AuNPs with different serum protein binding abilities, we discover that the concentration-dependent serum protein binding capabilities endow GS-AuNPs with a more sensitive response to injection dose than NPs resistant to serum protein binding, resulting in greatly improved tumor-targeting efficiencies during both single and repeated low-dose injections; the performance is also distinct from nonrenal-clearable AuNPs coated with serum protein, which show decreased tumor-targeting efficiency with a decrease in the injection dose.

Ultrasmall Luminescent Metal Nanoparticles: Surface Engineering Strategies for Biological Targeting and Imaging.

In the past decade, ultrasmall luminescent metal nanoparticles (ULMNPs, d < 3 nm) have achieved rapid progress in addressing many challenges in the healthcare field because of their excellent physicochemical properties and biological behaviors. With the sharp shrinking size of large plasmonic metal nanoparticles (PMNPs), the contributions from the surface characteristics increase significantly, which brings both opportunities and challenges in the application-driven surface engineering of ULMNPs toward advanced biological applications. Here, the systematic advancements in the biological applications of ULMNPs from bioimaging to theranostics are summarized with emphasis on the versatile surface engineering strategies in the regulation of biological targeting and imaging performance. The efforts in the surface functionalization strategies of ULMNPs for enhanced disease targeting abilities are first discussed. Thereafter, self-assembly strategies of ULMNPs for fabricating multifunctional nanostructures for multimodal imaging and nanomedicine are discussed. Further, surface engineering strategies of ratiometric ULMNPs to enhance the imaging stability to address the imaging challenges in complicated bioenvironments are summarized. Finally, the phototoxicity of ULMNPs and future perspectives are also reviewed, which are expected to provide a fundamental understanding of the physicochemical properties and biological behaviors of ULMNPs to accelerate their future clinical applications in healthcare.

Weak Anchoring Sites of Thiolate-Protected Luminescent Gold Nanoparticles.

Surface functionalization of gold nanoparticles (AuNPs) with thiolate ligands is a successful strategy for controlling their stability, nanotoxicity, circulation, and interaction with biological environments as leading nanomedicines. However, the effects of the weak anchoring groups of NH2 and COOH have been long-term ignored because of the well-recognized strong anchoring site of S-Au. Herein, the authors achieve controllable weak anchoring sites of the luminescent AuNPs using a typical thiolate peptide such as glutathione with anchoring groups of SH, COOH, and NH2 . Additionally, they establish that not only the strong anchoring site of S-Au, but also the weak anchoring sites from N-Au and COO-Au are critical to the behavior of AuNPs at both in vitro and in vivo levels. These results open up new possibilities for the fundamental understanding of the significance of the weak anchoring sites in the future surface functionalization of nanomedicines toward advanced theranostics.

[Determination of atmospheric organochlorine pesticides using isotope dilution high-resolution gas chromatography/high-resolution mass spectrometry].

A method for the determination of 25 organochlorine pesticides (OCPs) in the atmosphere using isotope dilution high-resolution gas chromatography/high-resolution mass spectrometry (ID-HRGC/HRMS) was developed. Sample extraction was performed using an accelerated solvent extractor (ASE). The extraction parameters were as follows: the extraction solvent was 50% (v/v) hexane in dichloromethane, the extraction temperature was 100 ℃, the static time was 8 min, the cell was rinsed with 60% cell volume using the aforementioned extraction solvent, the purging time was 180 s with N2 gas, and the extraction proceeded through three cycles. The eluting solutions of common cartridges such as florisil, graphitized carbon black, alumina, and silica were determined via cartridge elution tests. Use of the aforementioned cartridges alone cannot remove the pigments in the air sample solution. Subsequently, all possible pairwise combinations of the four cartridges were used for sample cleaning, and only the combination of florisil and graphitized carbon black was found to completely remove the pigments. Thus, the combination of florisil and graphitized carbon black cartridges using 10 mL toluene for elution was determined as the final cleaning method in this study. A high-resolution mass spectrometer equipped with a gas chromatograph was used for quantification. A fused-silica capillary column (Rtx-CL Pesticides2, 30 m×0.25 mm×0.2 µm) was used to separate the target compounds. Injection was performed in the splitless mode at 250 ℃. The flow rate of nitrogen gas was maintained constant at 1 mL/min. The oven temperature was 110 ℃ (1 min), 20 ℃/min up to 210 ℃, 1.5 ℃/min up to 218 ℃ (1 min), and 2 ℃/min up to 260 ℃ (1 min). HRMS was conducted at >8000 resolution, the source temperature was 280 ℃ in the electron impact mode using ionization energy of 35 eV, and measurements were performed in the selective ion monitoring (SIM) mode. Twenty-five OCPs were identified by comparing their GC retention times with those of the corresponding labeled compounds, and the actual ion abundance ratios of two exact m/z values with the corresponding theoretical values. The 25 OCPs were quantified by average relative response factors (RRFs), and the relative standard deviations (RSDs) of the RRFs with six calibration solutions were no more than 20%. The linear range of this method was 0.4 to 800 µg/L, and the correlation coefficients (R2) were higher than 0.992. To validate the method, clean materials (one quartz fiber filter (QFF) and two polyurethane foam (PUF) plugs) were spiked with 100 pg, 400 pg, and 15 ng native OCP standards, respectively; the RSDs of the 25 OCPs for each spiked level ranged from 0.64% to 16%. The spiking recoveries of the native OCPs ranged from 67.2% to 135%. Penetration experiments were conducted by sampling various volumes of air (15-1000 m3) using a filter-PUF/PUF high-volume active sampler. The breakthrough volume was sampled when the amount of OCPs collected in the PUF of the non-sampling end reached 5% of the total amount collected by both PUFs. When a high-volume active sampler with filter-PUF/PUF was used as an adsorbent for sampling atmospheric OCPs, a serious breakthrough of pentachlorobenzene (PeCB) occurred. The effective sampling volume of hexachlorobenzene (HCB) was very low, and was no more than 30 m3 under the standard conditions (101.325 kPa, 273 K). The effective sampling volumes of other OCP compounds should be no more than 1200 m3. This will necessitate the use of high-adsorption-capacity adsorbents such as the PUF-XAD (a styrene-divinylbenzene copolymer) sandwich used for sampling air PeCB and HCB. Calculation with the effective sampling volumes from the penetration experiment revealed that the limits of detection of the 25 OCPs were in the range of 0.002 to 0.7 pg/m3. Thus, the detection levels of OCPs in this study were reduced to at least 2% of the current monitoring standards. Analysis of air samples in Beijing showed that all the target compounds except for trans-heptachlor epoxide, endrin, cis-nonachlor and 4,4'-DDD were 100% detected in the air samples. The concentrations of HCB (in volumes of 15-30 m3) ranged from 514 to 563 pg/m3, while those of the other OCPs (in a volume of 600 m3) ranged from 0.01 to 18.9 pg/m3. The recoveries of surrogate standards in this sample analysis were in the range of 33.9% to 155%, which satisfied the requirements of EPA Method 1699. Because of the very high detection limits, the current related monitoring standards cannot meet the requirements of atmospheric OCP analysis, especially at the ultra-trace level. In addition, highly sensitive monitoring standard methods are urgently needed. This method is suitable for analyzing most atmospheric OCPs, even at the ultra-trace level. It also lays the foundation for a new standard method formulation and provides strong support for the implementation of relevant international conventions.

Concentration-Dependent Subcellular Distribution of Ultrasmall Near-Infrared-Emitting Gold Nanoparticles.

The ability to accurately control the subcellular distribution of nanomedicines provides unique advantages on understanding of cellular biology and disease theranostics. The nanomedicine concentration is a key factor to affect the theranostic efficiency and systematic toxicity. Herein, we unravel a concentration-dependent subcellular distribution of near-infrared-emitting gold nanoparticles (AuNPs) co-coated with glutathione and a cell-penetrating peptide CR8 (CR-AuNPs), which shows a strong membrane-binding at high concentration but more endocytosis for mitochondria targeting at the low concentration region. Attributing to high content of AuI and microsecond luminescent lifetimes, these AuNPs can catalyze dissolved oxygen to generate singlet oxygen (1 O2 ) efficiently. Combining with the concentration-dependent subcellular distribution, the luminescent AuNPs show photocytotoxicity in the relative low concentration region. These findings facilitate the fundamental understanding of the biological behaviors and potential cytotoxicity of ultrasmall luminescent AuNPs toward future theranostics.

Precisely Regulated Luminescent Gold Nanoparticles for Identification of Cancer Metastases.

The nanoprobes for identification of cancer metastases in the mononuclear phagocyte system (MPS) organs are of significant importance but are limited due to the long-standing challenge of low tumor-targeting specificity with inadequate targeting efficiency and high nonspecific accumulation. Here, we report a surface regulation strategy that integrates the tumor-acidity-activated charge-reversal behavior and precise control in both hydrodynamic diameter (HD) and surface charge on ultrasmall luminescent gold nanoparticles (AuNPs) to achieve significantly high tumor-targeting specificity. The precise regulation of AuNPs to a rational HD and surface charge could rapidly and selectively recognize small metastatic tumors (∼1 mm) in liver and lung with high signal-to-noise ratios of 4.6 and 4.5, respectively. These results help further understand the in vivo transport of nanoprobes and provide guidance for design of translatable nanosized nanomedicines in cancer metastasis theranostics.

Strict DNA Valence Control in Ultrasmall Thiolate-Protected Near-Infrared-Emitting Gold Nanoparticles.

Realizing robust DNA functionalization with strict valence control in the sub-2-nm thiolate-protected luminescent gold nanoparticles (AuNPs) is highly demanded but remains unsolved due to their unique Au(0) core and Au(I)-S shell structures. Herein, we report a facile strategy using phosphorothioates (ps)-modified DNA (psDNA) as a template for in situ growth of near-infrared (NIR)-emitting AuNPs with precisely controlled DNA valence. In addition, the particle size could be finely tuned in ultrasmall ranges from 1.3 to 2.6 nm with regulation of the ps length of psDNA. The ultrasmall NIR-emitting AuNPs bearing strict DNA valence are also demonstrated to be as powerful building block for well-organized one-dimensional assembly and optical probe for targeted cellular imaging. Such a facile strategy in decoration of luminescent AuNPs with strict DNA valence provides a new pathway for development of surface-functionalizable ultrasmall metal nanoplatforms toward various downstream applications.

Facile in situ synthesis of ultrasmall near-infrared-emitting gold glyconanoparticles with enhanced cellular uptake and tumor targeting.

The simultaneous possession of high tumor-targeting efficiency, long blood circulation, and low normal-tissue retention is critical for future clinically translatable nanomedicines. Herein, we reported a facile in situ glycoconjugation strategy for the synthesis of near-infrared (NIR)-emitting gold glyconanoparticles (AuGNPs, ∼2.4 nm) using 1-thio-ß-d-glucose as both the surface ligand and the reducing agent in the presence of a gold precursor. The ultrasmall AuGNPs showed similar low healthy organ retention to that of the renal-clearable ultrasmall nonglyconanoparticles, but ∼10 and 2.5 times higher in vitro and in vivo tumor-targeting efficiencies, respectively, were observed. This facile glycoconjugation strategy of ultrasmall AuGNPs was found to show activity towards glucose transporters in the cancer cells and prolonged blood circulation with both renal and hepatobiliary clearance pathways, which synergistically enhanced the tumor targeting of the ultrasmall AuGNPs. This discovery provides a smart strategy for the improvement in tumor targeting by ultrasmall NPs and further strengthens our understanding of glycoconjugation in designing future clinically translatable nanomedicines.

Effect of Hydrophobicity on Nano-Bio Interactions of Zwitterionic Luminescent Gold Nanoparticles at the Cellular Level.

Fundamental understanding of how the hydrophobicity impacts cellular interactions of engineered nanoparticles is critical to their future success in healthcare. Herein, we report that inserting hydrophobic octanethiol onto the surface of zwitterionic luminescent glutathione coated gold nanoparticles (GS-AuNPs) of 2 nm enhanced their affinity to the cellular membrane and increased cellular uptake kinetics by more than one order of magnitude, rather than inducing the accumulation of the AuNPs in the bilayer core or enhancing their passive diffusion. These studies highlight the diversity and heterogeneity in the hydrophobicity-induced nano-bio interactions at the cellular level and offer a new pathway to expediting cellular uptake of engineered nanoparticles. In addition, the amphiphilic luminescent AuNPs with high affinity to cell membrane and rapid endocytosis potentially serve as dual-modality imaging probes to correlate fluorescence and electron microscopies at the cellular level.

Transformation from gold nanoclusters to plasmonic nanoparticles: A general strategy towards selective detection of organophosphorothioate pesticides.

Luminescent gold nanoclusters (AuNCs) synthesized using non-thiolate DNA ligands were reported to show both optical and structure responses toward diethyposphorthioate (DEP) derived from the hydrolysis of chlorpyrifos (CP). After incubation of AuNCs with DEP, the non-thiolate DNA ligands were immediately replaced and the tiny AuNCs with ultrasmall size transformed gradually to plasmonic nanoparticles, which resulted in significant luminescence quenching of the AuNCs, offering a new possibility to selectively detect organophosphorothioate pesticides that could be easily hydrolyzed to form the special structures such as DEP containing two binding sites (e.g. S and O atoms). Therefore, selecting CP as a model analyte, we here developed a general strategy for the construction of a novel chemosensor for the determination of CP using the non-thiolate DNA coated AuNCs as an optical probe. Based on aggregation-induced luminescence quenching, this strategy exhibited highly sensitive and selective responses towards CP with a limit of detection (LOD) of 0.50µM, and was applied successfully to the analysis of CP in real sample. More interestingly, this facile strategy could easily distinguish CP from other thiol reagents through solution color change in spite of the existence of the coordination between Au and S atom for both of them, and the response mechanisms for them were studied in detail. In additional, it could be extended to detect the other organophosphorothioate pesticides with the similar structure as CP, which exploits a new platform for the construction of chemosensor and application.

Bioapplications of renal-clearable luminescent metal nanoparticles.

Renal-clearable inorganic nanoparticles (NPs) that hold great potential in the future clinic translations are considered as the next generation of nanomedicine. In the past decade, enormous efforts have been dedicated to the development of renal-clearable NPs with fascinating optical properties, selective disease-targeting capabilities and low nanotoxicities. A further understanding of the design of renal-clearable luminescent metal NPs and their metabolic behavior in the body is important to achieve their clinical transition and extend their bioapplications in disease theranostics. In this review, we discuss the recent synthetic strategies of renal-clearable metal NPs in terms of the considerations of size and composition, surface chemistry and emission wavelength. We also summarize the current disease-related applications of these renal-clearable luminescent metal NPs in tumor targeting, kidney disease and antimicrobial investigations after a discussion of their biological behavior including the pharmacokinetics and biodistribution. Finally, we provide perspectives on the current challenges and upcoming chances for renal-clearable luminescent metal NPs.

pH-Guided Self-Assembly of Copper Nanoclusters with Aggregation-Induced Emission.

We here report a facile pH-guided strategy for the fabrication of water-soluble protein/copper nanoclusters (CuNCs) hybrid nanostructures with stable and bright luminescence resulted from aggregation-induced emission. Using l-cysteine as both the reducing and capping agents, the synthesized CuNCs showed a good reversible pH-responsive aggregation and dispersion in the solution. The CuNCs formed insoluble macroscopic aggregates with stable red-colored emission (620 nm) at pH 3.0 but became soluble with weak luminescence at pH <1.5 or pH >4.0. The highly reversible pH-responsive properties of the CuNCs made it feasible to achieve water-soluble protein/CuNCs hybrid nanostructures in the presence of protein without any external forces (e.g., sonication). The weak luminescent CuNCs were first mixed with protein under neutral condition (e.g., pH 7.0), followed by tuning of the pH to acidic conditions (e.g., pH 3.0) to form luminescent protein/CuNCs hybrid nanostructures, the sizes of which were much smaller than those of the protein-free macroscopic CuNC aggregates. This strategy was easily applicable to other dispersing agents (e.g., glucose oxidase), opening a new pathway for the construction of many other smart water-soluble luminescent biomolecule/nanocluster hybrid nanostructures with various applications.