Fluorescent Probes for Disease Diagnosis.

The identification and detection of disease-related biomarkers is essential for early clinical diagnosis, evaluating disease progression, and for the development of therapeutics. Possessing the advantages of high sensitivity and selectivity, fluorescent probes have become effective tools for monitoring disease-related active molecules at the cellular level and in vivo. In this review, we describe current fluorescent probes designed for the detection and quantification of key bioactive molecules associated with common diseases, such as organ damage, inflammation, cancers, cardiovascular diseases, and brain disorders. We emphasize the strategies behind the design of fluorescent probes capable of disease biomarker detection and diagnosis and cover some aspects of combined diagnostic/therapeutic strategies based on regulating disease-related molecules. This review concludes with a discussion of the challenges and outlook for fluorescent probes, highlighting future avenues of research that should enable these probes to achieve accurate detection and identification of disease-related biomarkers for biomedical research and clinical applications.

A programmable topological photonic chip.

Controlling topological phases of light allows the observation of abundant topological phenomena and the development of robust photonic devices. The prospect of more sophisticated control with topological photonic devices for practical implementations requires high-level programmability. Here we demonstrate a fully programmable topological photonic chip with large-scale integration of silicon photonic nanocircuits and microresonators. Photonic artificial atoms and their interactions in our compound system can be individually addressed and controlled, allowing the arbitrary adjustment of structural parameters and geometrical configurations for the observation of dynamic topological phase transitions and diverse photonic topological insulators. Individual programming of artificial atoms on the generic chip enables the comprehensive statistical characterization of topological robustness against relatively weak disorders, and counterintuitive topological Anderson phase transitions induced by strong disorders. This generic topological photonic chip can be rapidly reprogrammed to implement multifunctionalities, providing a flexible and versatile platform for applications across fundamental science and topological technologies.

Reversible Fluorescent Probes for Dynamic Imaging of Liver Ischemia-Reperfusion Injury.

ConspectusHepatic ischemia-reperfusion injury (HIRI) is an inevitable complication of clinical surgeries such as liver resection or transplantation, often resulting in postoperative liver dysfunction, hepatic failure in up to 13% of postresection patients, and early graft failure in 11-18% of liver transplantation patients. HIRI involves a series of biochemical events triggered by abnormal alterations in multiple biomarkers, characterized by short lifespans, dynamic changes, subcellular regional distribution, and multicollaborative regulation. However, traditional diagnosis, including serology, imaging, and liver puncture biopsy, suffers from low sensitivity, poor resolution, and hysteresis, which hinder effective monitoring of HIRI markers. Thus, to address the unique properties of HIRI markers, there is a pressing demand for developing novel detection strategies that are highly selective, transiently responsive, dynamically reversible, subcellular organelle-targeted, and capable of simultaneous multicomponent analysis.Optical probe-based fluorescence imaging is a powerful tool for real-time monitoring of biomarkers with the advantages of high sensitivity, noninvasiveness, rapid analysis, and high-fidelity acquisition of spatiotemporal information on signaling molecules compared with conventional methods. Moreover, with the growing demand for continuous monitoring of biomarkers, probes with reversible detection features are receiving more and more attention. Importantly, reversible probes can not only monitor fluctuations in marker concentrations but also distinguish between transient bursts of markers during physiological events and long-term sustained increases in pathological marker levels. This can effectively avoid false-positive test results, and in addition, reversible probes can be reutilized with green and economical features. Therefore, our team has employed various effective methods to design reversible optical probes for HIRI. We proposed reversible recognition strategies based on specific reactions or interactions to detect dynamic changes in markers. Given the biomarkers' unique signaling in subcellular organelles and the synergistic regulatory properties of multiple markers for HIRI, bifunctional reversible detection strategies are exploited, including organelle-targeted reversible and multicomponent simultaneous detection. With these strategies, we have tailored a variety of high-fidelity fluorescent probes for a series of HIRI markers, including reactive oxygen/nitrogen species (O2•- and ONOO-), ATP, protein (Keap1), mitochondrial DNA, etc. Utilizing the probes, the in situ dynamic imaging detection of the HIRI markers was successfully achieved. While performing the precise examination of the earlier occurrence of HIRI disease and visualizing the real-time monitoring of the disease process, we have also further elucidated the HIRI-associated signaling pathways. It is envisioned that our summarized work will inspire the design of future reversible fluorescent probes and help to improve the clinical diagnosis and therapeutic efficiency of these diseases.

Blockade of CCR5 and CXCR3 attenuates murine acute graft-versus-host disease through modulating donor-derived T-cell distribution and function.

Lymphocyte trafficking via chemokine receptors such as C-C chemokine receptor 5 (CCR5) and CXCR3 plays a critical role in the pathogenesis of acute graft-versus-host disease (aGVHD). Our previous studies showed that the addition of CCR5 or CXCR3 antagonists could only slightly alleviate the development of aGVHD. Given the specificity of T lymphocytes bearing CXCR3 and CCR5, we investigated whether combined CCR5 and CXCR3 blockade could further attenuate murine aGVHD. A mouse model of aGVHD was established to assess the efficacy of CCR5 and/or CXCR3 blockade on the development of aGVHD. The distribution of lymphocytes was calculated by quantification of immunostaining cells. The immunomodulatory effect on T cells was assessed by evaluating T-cell proliferation, viability, and differentiation. Using the murine allogeneic hematopoietic stem cell transplantation model, we demonstrated that blockade of both CCR5 and CXCR3 could efficiently alleviate the development of aGVHD. Further investigation on the immune mechanisms for this prophylactic effect showed that more T cells were detained into secondary lymphoid organs (SLOs), which may lead to reduced infiltration of T cells into GVHD target organs. Our study also showed that T cells detained in SLOs dampened the activation, suppressed the polarization toward T helper type 1 (Th1) and T cytotoxic type 1 (Tc1) cells, and induced the production of Treg cells. These data suggest that concurrent blockade of CCR5 and CXCR3 attenuates murine aGVHD through modulating donor-derived T-cell distribution and function, and this might be applicable for aGVHD prophylaxis in clinical settings.

Identification of DNase I hypersensitive sites in the human genome by multiple sequence descriptors.

DNase I hypersensitive sites (DHSs) are chromatin regions highly sensitive to DNase I enzymes. Studying DHSs is crucial for understanding complex transcriptional regulation mechanisms and localizing cis-regulatory elements (CREs). Numerous studies have indicated that disease-related loci are often enriched in DHSs regions, underscoring the importance of identifying DHSs. Although wet experiments exist for DHSs identification, they are often labor-intensive. Therefore, there is a strong need to develop computational methods for this purpose. In this study, we used experimental data to construct a benchmark dataset. Seven feature extraction methods were employed to capture information about human DHSs. The F-score was applied to filter the features. By comparing the prediction performance of various classification algorithms through five-fold cross-validation, random forest was proposed to perform the final model construction. The model could produce an overall prediction accuracy of 0.859 with an AUC value of 0.837. We hope that this model can assist scholars conducting DNase research in identifying these sites.

Fluorescent small molecule donors.

Small molecule donors (SMDs) play subtle roles in the signaling mechanism and disease treatments. While many excellent SMDs have been developed, dosage control, targeted delivery, spatiotemporal feedback, as well as the efficiency evaluation of small molecules are still key challenges. Accordingly, fluorescent small molecule donors (FSMDs) have emerged to meet these challenges. FSMDs enable controllable release and non-invasive real-time monitoring, providing significant advantages for drug development and clinical diagnosis. Integration of FSMDs with chemotherapeutic, photodynamic or photothermal properties can take full advantage of each mode to enhance therapeutic efficacy. Given the remarkable properties and the thriving development of FSMDs, we believe a review is needed to summarize the design, triggering strategies and tracking mechanisms of FSMDs. With this review, we compiled FSMDs for most small molecules (nitric oxide, carbon monoxide, hydrogen sulfide, sulfur dioxide, reactive oxygen species and formaldehyde), and discuss recent progress concerning their molecular design, structural classification, mechanisms of generation, triggered release, structure-activity relationships, and the fluorescence response mechanism. Firstly, from the large number of fluorescent small molecular donors available, we have organized the common structures for producing different types of small molecules, providing a general strategy for the development of FSMDs. Secondly, we have classified FSMDs in terms of the respective donor types and fluorophore structures. Thirdly, we discuss the mechanisms and factors associated with the controlled release of small molecules and the regulation of the fluorescence responses, from which universal guidelines for optical properties and structure rearrangement were established, mainly involving light-controlled, enzyme-activated, reactive oxygen species-triggered, biothiol-triggered, single-electron reduction, click chemistry, and other triggering mechanisms. Fourthly, representative applications of FSMDs for trackable release, and evaluation monitoring, as well as for visible in vivo treatment are outlined, to illustrate the potential of FSMDs in drug screening and precision medicine. Finally, we discuss the opportunities and remaining challenges for the development of FSMDs for practical and clinical applications, which we anticipate will stimulate the attention of researchers in the diverse fields of chemistry, pharmacology, chemical biology and clinical chemistry. With this review, we hope to impart new understanding thereby enabling the rapid development of the next generation of FSMDs.

Reprogramming Tumor-Associated Macrophages with a Se-Based Core-Satellite Nanoassembly to Enhance Cancer Immunotherapy.

Tumor-associated macrophages (TAMs), as the most prevalent immune cells in the tumor microenvironment, play a pivotal role in promoting tumor development through various signaling pathways. Herein, we have engineered a Se@ZIF-8 core-satellite nanoassembly to reprogram TAMs, thereby enhancing immunotherapy outcomes. When the nanoassembly reaches the tumor tissue, selenium nanoparticles and Zn2+ are released in response to the acidic tumor microenvironment, resulting in a collaborative effort to promote the production of reactive oxygen species (ROS). The generated ROS, in turn, activate the nuclear factor κB (NF-κB) signaling pathway, driving the repolarization of TAMs from M2-type to M1-type, effectively eliminating cancer cells. Moreover, the nanoassembly can induce the immunogenic death of cancer cells through excess ROS to expose calreticulin and boost macrophage phagocytosis. The Se@ZIF-8 core-satellite nanoassembly provides a potential paradigm for cancer immunotherapy by reversing the immunosuppressive microenvironment.

Nonvolatile Electro-optic Response of Graphene Driven by Ferroelectric Polarization.

Two-dimensional materials (2DMs) have exhibited remarkably tunable optical characteristics, which have been applied for significant applications in communications, sensing, and computing. However, the reported tunable optical properties of 2DMs are almost volatile, impeding them in the applications of multifarious emerging frameworks such as programmable operation and neuromorphic computing. In this work, nonvolatile electro-optic response is developed by the graphene-Al2O3-In2Se3 heterostructure integrating with microring resonators (MRRs). In such compact devices, the optical absorption coefficient of graphene is substantially tuned by the out-of-plane ferroelectric polarization in α-In2Se3, resulting in a nonvolatile optical transmission in MRRs. This work demonstrates that integrating graphene with ferroelectric materials paves the way to develop nonvolatile devices in photonic circuits for emerging applications such as optical neural networks.

Posttranslational regulation of liver kinase B1 in human cancer.

Liver kinase B1 (LKB1) is a serine-threonine kinase that participates in multiple cellular and biological processes, including energy metabolism, cell polarity, cell proliferation, cell migration, and many others. LKB1 is initially identified as a germline-mutated causative gene in Peutz-Jeghers syndrome and is commonly regarded as a tumor suppressor due to frequent inactivation in a variety of cancers. LKB1 directly binds and activates its downstream kinases including the AMP-activated protein kinase (AMPK) and AMPK-related kinases by phosphorylation, which has been intensively investigated for the past decades. An increasing number of studies have uncovered the posttranslational modifications (PTMs) of LKB1 and consequent changes in its localization, activity, and interaction with substrates. The alteration in LKB1 function as a consequence of genetic mutations and aberrant upstream signaling regulation leads to tumor development and progression. Here, we review current knowledge about the mechanism of LKB1 in cancer and the contributions of PTMs, such as phosphorylation, ubiquitination, SUMOylation, acetylation, prenylation, and others, to the regulation of LKB1 function, offering new insights into the therapeutic strategies in cancer.

Mechanistic Insights into N2O5-Halide Ions Chemistry at the Air-Water Interface.

The activation of halogens (X = Cl, Br, I) by N2O5 is linked to NOx sources, ozone concentrations, NO3 reactivity, and the chemistry of halide-containing aerosol particles. However, a detailed chemical mechanism is still lacking. Herein, we explored the chemistry of the N2O5···X- systems at the air-water interface. Two different reaction pathways were identified for the reaction of N2O5 with X- at the air-water interface: the formation of XNO2 or XONO, along with NO3-. In the case of the Cl- system, the ClNO2 generation pathway is more favorable, while for the Br- and I- systems, the formation of BrONO and IONO is barrierless, making them the predominant products. Furthermore, the mechanisms of formation of X2 from XNO2 and XONO were also investigated. The high energy barriers of reactions and the high free energies of the products compared to those of the reactants indicate that ClNO2 is stable at the air-water interface. Contrary to the widely held belief regarding X2 producing from the reaction of XNO2 with X-, our calculations demonstrate that BrONO and IONO initially form stable BrONO···Br- and IONO···I- complexes, which then subsequently react with Br- and I- to form Br3- and I3-, respectively. Finally, Br3- and I3- decompose to form Br2 and I2. These findings have significant implications for experimental interpretation and offer new insights into halogen cycling in the atmosphere.

Glowing Octopus-Inspired Nanomachine: A Versatile Aptasensor for Efficient Capture, Imaging, Separation, and NIR-Triggered Release of Cancer Cells.

The separation and analysis of circulating tumor cells (CTCs) in liquid biopsy significantly facilitated clinical cancer diagnosis and personalized therapy. However, current methods face challenges in simultaneous efficient capturing, separation, and imaging of CTCs, and most of the devices cannot be reused/regenerated. We present here an innovative glowing octopus-inspired nanomachine (GOIN), capable of capturing, imaging, separating, and controlling the release of cancer cells from whole blood and normal cells. The GOIN comprises an aptamer-decorated magnetic fluorescent covalent organic framework (COF), which exhibits a strong affinity for nucleolin-overexpressed cancer cells through a multivalent binding effect. The captured cancer cells can be directly imaged using the intrinsic stable fluorescence of the COF layer in the GOIN. Employing magnet and NIR laser assistance enables easy separation and mild photothermal release of CTCs from the normal cells and the nanomachine without compromising cell viability. Moreover, the GOIN demonstrates a reusing capability, as the NIR-triggered CTC release is mild and nondestructive, allowing the GOIN to be reused at least three times.

A Multimode Optical Sensor for Selective and Sensitive Detection of Harmful Heavy Metal Cr(VI) in Fresh Water and Sea Water.

Water pollution originating from heavy metals has shown great impacts on the ecological environment and human health due to their extremely low biodegradability. Hexavalent chromium Cr(VI), as one harmful heavy metal with strong oxidation, high biological permeability, and high carcinogenicity, is becoming an increasingly serious threat to human health. Therefore, conveniently but accurately, monitoring the Cr(VI) level in water to maintain its normal level and ensuring the stability of the ecosystem and human health become very valuable. However, most of these heavy metal sensors reported are turn-off type single-emission sensors. In this work, a ratiometric fluorescence/colorimetry/smartphone triple-mode turn-on optical sensor for Cr(VI) was developed based on a multifunctional metal-organic framework platform. The detection limits for these three mutual verification modes were only 1.28, 4.89, and 68.4 nM, respectively. Additionally, the color changes of the detection system under sunlight can also be observed directly by the naked eye. The accuracy and practicability of this multimode sensor were further proved by the detection of Cr(VI) in actual water and seawater samples, and the recovery rate ranged from 97.308 to 104.041%.

Advancing Nonsmall Cell Lung Cancer Diagnosis Accuracy via Dual Detection Fluorescent Nanoprobes.

Among the primary threats to human health worldwide, nonsmall cell lung cancer (NSCLC) remains a significant factor and is a leading cause of cancer-related deaths. Due to subtle early symptoms, NSCLC patients are diagnosed at advanced stages, resulting in low survival rates. Herein, novel Au-Se bond nanoprobes (NPs) designed for the specific detection of Calpain-2 (CAPN2) and Human Neutrophil Elastase (HNE), pivotal biomarkers in NSCLC, were developed. The NPs demonstrated exceptional specificity and sensitivity toward CAPN2 and HNE, enabling dual-color fluorescence imaging to distinguish between NSCLC cells and normal lung cells effectively. The NPs' performance was consistent across a wide pH range (6.2 to 8.0), and it exhibited remarkable resistance to biological thiol interference, indicating its robustness in complex physiological environments. These findings suggest the nanoprobe is a promising tool for early NSCLC diagnosis, offering a novel approach for enhancing the accuracy of cancer detection.

Synergistically Activated Aggregation-Induced Emission Probe for Precise In Situ Staining of Lipids in Atherosclerotic Plaques.

Visualizing the localization and distribution of lipids within arteries is crucial for studying atherosclerosis. However, existing lipid-specific probes face challenges such as strong hydrophobicity and nonspecific staining of lipophilic organelles or tissues, making them impractical for the precise identification of atherosclerotic plaques. To address this issue, we design a synergistically activated probe, Cbz-Lys-Lys-TPEB, which responds to cathepsin B (CTB) and H2O2 for the in situ generation of aggregation-induced emission luminogens (AIEgens). This enables specific staining of lipids within arteries and precise imaging of atherosclerotic plaques. The probe combines a tetraphenylethene building block with a hydrophilic peptide sequence (Cbz-Lys-Lys) and phenylboric acid module, providing excellent water solubility and fluorescence quenching in a molecular dispersion state. Upon interaction with H2O2 and CTB within plaques, the hydrophilic Cbz-Lys-Lys-TPEB probe is specifically cleaved and converted into hydrophobic AIEgens, leading to rapid aggregation and significant fluorescence enhancement. Interestingly, the in situ-liberated AIEgens display distinct lipid binding ability, effectively tracking the location and distribution of lipids in plaques. This synergistic target-activated AIEgen liberation strategy demonstrates significant feasibility for the reliable and accurate identification of atherosclerotic plaques, holding tremendous potential for clinical diagnosis and risk stratification of atherosclerosis.

Thermophoretic Aggregation Imaging of Tumor-Derived Exosomes by CRISPR-Cas13a-Based Nanoprobes.

The inherent heterogeneity of tumor-derived exosomes holds great promise for enhancing the precision of cancer diagnostics. MicroRNAs (miRNAs) encapsulated in tumor-associated exosomes have emerged as valuable biomarkers for the early detection of cancers. Nevertheless, the flexible structure and inherent instability of RNA limit its application in biological diagnostics. The CRISPR-Cas13a system, distinguished by its target-responsive "collateral effect", represents a powerful tool for advancing cancer diagnostics. In this study, we harness the CRISPR-Cas13a system as an innovative signal amplification tool to image cancer-related exosomal miRNA in situ. Furthermore, we capitalize on the thermophoretic aggregation effect exhibited by gold nanoparticles (Au NPs) to consolidate the fluorescent signals generated by the CRISPR-Cas13a system. Subsequently, the developed nanoprobe is applied to detect lung cancer-related exosomal miRNA from human serum, enabling the aggregated visualization of low-abundance cancer exosomes in individuals with lung cancer compared with healthy individuals. This sensitive thermophoretic aggregation assay provides a diagnostic tool for lung cancer in the clinic.

Filter Membrane-Based Colorimetric Approach for Point-of-Care Detection of Biomarkers Using CRISPR-Cas12a.

CRISPR-Cas-based point-of-care testing (POCT) strategies have been widely explored for the detection of diverse biomarkers. However, these methods often require complicated operations, such as careful solution transfer steps, to achieve high sensitivity and accuracy. In this study, we combine a filter membrane-based POCT method with CRISPR-Cas12a for colorimetric detection of biomarkers. For the nucleic acid target, the trans-cleavage activity of CRISPR-Cas12a is directly triggered, cutting the single-stranded DNA linkers on glucose oxidase (GOx)-modified polymer nanoparticles. Due to the size difference between GOx and the polymer nanoparticles, GOx can be separated using a filter membrane. The filtrate containing GOx reacts with the substrate to generate a colorimetric signal. For the non-nucleic acid target, the non-nucleic acid signal is converted into a nucleic acid signal that activates CRISPR-Cas12a, resulting in a colorimetric signal. The entire operation is easy to perform, and the signal can be directly observed via the naked eye, which circumvents the use of costly instruments. The developed strategy holds great promise for accurate and accessible POCT detection of disease biomarkers in resource-limited settings.

Simultaneous Detection of Aldehyde Metabolites by Light-Assisted Ambient Ionization Mass Spectrometry.

In the clinic, small-molecule metabolites (SMMs) in blood are highly convincing indicators for disease diagnosis, such as cancer. However, challenges still exist for detection of SMMs due to their low concentration and complicated components in blood. In this work, we report the design of a novel "selenium signature" nanoprobe (Se nanoprobe) for efficient identification of multiple aldehyde metabolites in blood. This Se nanoprobe consists of magnetic nanoparticles that can enrich aldehyde metabolites from a complex environment, functionalized with photosensitive "selenium signature" hydrazide molecules that can react with aldehyde metabolites. Upon irradiation with UV, the aldehyde derivatives can be released from the Se nanoprobe and further sprayed by mass spectrometry through ambient ionization (AIMS). By quantifying the selenium isotope distribution (MS/MS) from the derivatization product, accurate detection of several aldehyde metabolites, including valeraldehyde (Val), heptaldehyde (Hep), 2-furaldehyde (2-Fur), 10-undecenal aldehyde (10-Und), and benzaldehyde (Ben), is realized. This strategy reveals a new solution for quick and accurate cancer diagnosis in the clinic.

A Sequentially Activated Probe for Imaging of Superoxide Anion and Peroxynitrite in PC12 Cells under Oxidative Stress.

Superoxide anion (O2·-) and peroxynitrite (ONOO-), two important oxidants under oxidative stress, coexist in complex cell and organism systems, playing crucial roles in various physiological and pathological processes, particularly in neurodegenerative diseases. Despite the absence of robust molecular tools capable of simultaneously visualizing O2·- and ONOO- in biosystems, the relationship between these two species remains understudied. Herein, we present sequentially activated fluorescent probe, DHX-SP, which exhibits exceptional selectivity and sensitivity toward O2·- and ONOO-. This probe enables precise imaging of these species in living PC12 cells under oxidative stress conditions using distinct fluorescence signal combinations. Furthermore, the probe DHX-SP has the ability to visualize changes in O2·- and ONOO- levels during ferroptosis of PC12 cells and in the Parkinson's disease model. These findings establish a connection between the crosstalk of the phosphorus group of O2·- and ONOO- in PC12 cells under oxidative stress.

In Situ Imaging of GGT and HOBr-Triggered Atherosclerotic Plaque Rupture via Activating the RunX2/Col IV Signaling Pathway.

Calcification and abnormal collagen deposition within blood vessels constitute causative factors for atherosclerotic plaque rupture, and their occurrence is intimately linked with γ-glutamyltranspeptidase (GGT) and hypobromous acid (HOBr). However, the underlying regulatory mechanisms of GGT and HOBr in plaque rupture remain unclear. Hence, we developed a dual-responsive near-infrared (NIR) fluorescent probe (BOC-H) that effectively avoids spectral crosstalk for the in situ visualization of the fluctuations in GGT and HOBr levels during atherosclerotic plaque rupture. We found that both GGT and HOBr contents increase significantly in the calcification models of cells and animals. The overexpressed GGT participated in intracellular oxygen-promoting behavior, which obviously upregulated the expression of RunX2 and Col IV by facilitating H2O2 and HOBr secretion. This process triggered calcification and abnormal collagen deposition within the plaque, which raised the risk of plaque rupture. PM2.5-induced arteriosclerotic calcification models further verified the results that GGT and HOBr accelerate plaque rupture via activation of the RunX2/Col IV signaling pathway. Moreover, the assessment of GGT and HOBr in serum samples from patients with acute myocardial infarction further confirmed the co-regulation of GGT and HOBr in the plaque rupture. Together, our studies highlight the involvement of GGT and HOBr in driving plaque rupture and offer new targets for the prevention and treatment of acute cardiovascular disease.

Layer-By-Layer Assembly of Atomically Precise Alloy Nanoclusters Photosystems for Solar Water Oxidation.

Atomically precise metal nanoclusters (NCs) have garnered tremendous attention as light-harvesting antennas in heterogeneous photocatalysis due to unique atomic stacking mode, quantum confinement effect, and enriched active sites. However, metal NCs as photosensitizers suffer from extremely short carrier lifetime, poor photostability, and difficulty in carrier migration, which hinder the wide-spread utilization of metal NCs in solar energy conversion. To solve these problems, herein, Ag-doped glutathione (GSH)-capped gold NCs, i.e., alloy Au1- x Agx @GSH NCs and non-conjugated insulating polymer of poly(diallyl-dimethylammonium chloride) (PDDA) are utilized as the building blocks for layer-by-layer assembly of spatially multilayered alloy NCs/metal oxide (MO) photosystems. The alternately deposited ultrathin PDDA layer in-between Au1- x Agx @GSH NCs on the MO substrate functions as an efficient charge flow mediator to relay the directional photoelectron transfer over Au1- x Agx @GSH NCs, giving rise to the cascade charge transfer chain. This peculiar carrier migration mode endowed by exquisite interface configuration design significantly boosts the unidirectional electron migration from the Au1- x Agx @GSH NCs to the MO substrate, substantially improving the visible-light-driven photoelectrochemical water oxidation performances of MO/(PDDA-Au1- x Agx )n multilayer heterostructured photoanodes. The work will inspire the rational construction of alloy metal NCs-based photosystems for modulating spatially controllable charge transfer pathway for solar energy conversion.