Nanoconfinement-Enhanced Electrochemiluminescence for <i>in Situ</i> Imaging of Single Biomolecules

Direct imaging of electrochemical reactions at the single-molecule level is of potential interest in materials, diagnostic, and catalysis applications. Electrochemiluminescence (ECL) offers the opportunity to convert redox events into photons. However, it is challenging to capture single photons emitted from a single-molecule ECL reaction at a specific location, thus limiting high-quality imaging applications. We developed the nanoreactors based on Ru(bpy)32+-doped nanoporous zeolite nanoparticles (Ru@zeolite) for direct visualization of nanoconfinement-enhanced ECL reactions. Each nanoreactor not only acts as a matrix to host Ru(bpy)32+ molecules but also provides a nanoconfined environment for the collision reactions of Ru(bpy)32+ and co-reactant radicals to realize efficient in situ ECL reactions. The nanoscale confinement resulted in enhanced ECL. Using such nanoreactors as ECL probes, a dual-signal sensing protocol for visual tracking of a single biomolecule was performed. High-resolution imaging of single membrane proteins on heterogeneous cells was effectively addressed with near-zero backgrounds. This could provide a more sensitive tool for imaging individual biomolecules and significantly advance ECL imaging in biological applications

Nanoconfinement-Enhanced Electrochemiluminescence for <i>in Situ</i> Imaging of Single Biomolecules

Direct imaging of electrochemical reactions at the single-molecule level is of potential interest in materials, diagnostic, and catalysis applications. Electrochemiluminescence (ECL) offers the opportunity to convert redox events into photons. However, it is challenging to capture single photons emitted from a single-molecule ECL reaction at a specific location, thus limiting high-quality imaging applications. We developed the nanoreactors based on Ru(bpy)32+-doped nanoporous zeolite nanoparticles (Ru@zeolite) for direct visualization of nanoconfinement-enhanced ECL reactions. Each nanoreactor not only acts as a matrix to host Ru(bpy)32+ molecules but also provides a nanoconfined environment for the collision reactions of Ru(bpy)32+ and co-reactant radicals to realize efficient in situ ECL reactions. The nanoscale confinement resulted in enhanced ECL. Using such nanoreactors as ECL probes, a dual-signal sensing protocol for visual tracking of a single biomolecule was performed. High-resolution imaging of single membrane proteins on heterogeneous cells was effectively addressed with near-zero backgrounds. This could provide a more sensitive tool for imaging individual biomolecules and significantly advance ECL imaging in biological applications

Nanoconfinement-Enhanced Electrochemiluminescence for <i>in Situ</i> Imaging of Single Biomolecules

Direct imaging of electrochemical reactions at the single-molecule level is of potential interest in materials, diagnostic, and catalysis applications. Electrochemiluminescence (ECL) offers the opportunity to convert redox events into photons. However, it is challenging to capture single photons emitted from a single-molecule ECL reaction at a specific location, thus limiting high-quality imaging applications. We developed the nanoreactors based on Ru(bpy)32+-doped nanoporous zeolite nanoparticles (Ru@zeolite) for direct visualization of nanoconfinement-enhanced ECL reactions. Each nanoreactor not only acts as a matrix to host Ru(bpy)32+ molecules but also provides a nanoconfined environment for the collision reactions of Ru(bpy)32+ and co-reactant radicals to realize efficient in situ ECL reactions. The nanoscale confinement resulted in enhanced ECL. Using such nanoreactors as ECL probes, a dual-signal sensing protocol for visual tracking of a single biomolecule was performed. High-resolution imaging of single membrane proteins on heterogeneous cells was effectively addressed with near-zero backgrounds. This could provide a more sensitive tool for imaging individual biomolecules and significantly advance ECL imaging in biological applications

Nanoconfinement-Enhanced Electrochemiluminescence for <i>in Situ</i> Imaging of Single Biomolecules

Direct imaging of electrochemical reactions at the single-molecule level is of potential interest in materials, diagnostic, and catalysis applications. Electrochemiluminescence (ECL) offers the opportunity to convert redox events into photons. However, it is challenging to capture single photons emitted from a single-molecule ECL reaction at a specific location, thus limiting high-quality imaging applications. We developed the nanoreactors based on Ru(bpy)32+-doped nanoporous zeolite nanoparticles (Ru@zeolite) for direct visualization of nanoconfinement-enhanced ECL reactions. Each nanoreactor not only acts as a matrix to host Ru(bpy)32+ molecules but also provides a nanoconfined environment for the collision reactions of Ru(bpy)32+ and co-reactant radicals to realize efficient in situ ECL reactions. The nanoscale confinement resulted in enhanced ECL. Using such nanoreactors as ECL probes, a dual-signal sensing protocol for visual tracking of a single biomolecule was performed. High-resolution imaging of single membrane proteins on heterogeneous cells was effectively addressed with near-zero backgrounds. This could provide a more sensitive tool for imaging individual biomolecules and significantly advance ECL imaging in biological applications

Table_2_Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Stimulated by Mycobacterium tuberculosis PPE57 Identifies Characteristic Genes Associated With Type I Interferon Signaling.xls

Proline-glutamic acid (PE)- and proline-proline-glutamic acid (PPE)-containing proteins are exclusive to Mycobacterium tuberculosis (MTB), the leading cause of tuberculosis (TB). In this study, we performed global transcriptome sequencing (RNA-Seq) on PPE57-stimulated peripheral blood mononuclear cells (PBMCs) and control samples to quantitatively measure the expression level of key transcripts of interest. A total of 1367 differentially expressed genes (DEGs) were observed in response to a 6 h exposure to PPE57, with 685 being up-regulated and 682 down-regulated. Immune-related gene functions and pathways associated with these genes were evaluated, revealing that the type I IFN signaling pathway was the most significantly enriched pathway in our RNA-seq dataset, with 14 DEGs identified therein including ISG15, MX2, IRF9, IFIT3, IFIT2, OAS3, IFIT1, IFI6, OAS2, OASL, RSAD2, OAS1, IRF7, and MX1. These PPE57-related transcriptomic profiles have implications for a better understanding of host global immune mechanisms underlying MTB infection outcomes. However, more studies regarding these DEGs and type I IFN signaling in this infectious context are necessary to more fully clarify the underlying mechanisms that arise in response to PPE57 during MTB infection.</p

Table_1_Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Stimulated by Mycobacterium tuberculosis PPE57 Identifies Characteristic Genes Associated With Type I Interferon Signaling.xlsx

Proline-glutamic acid (PE)- and proline-proline-glutamic acid (PPE)-containing proteins are exclusive to Mycobacterium tuberculosis (MTB), the leading cause of tuberculosis (TB). In this study, we performed global transcriptome sequencing (RNA-Seq) on PPE57-stimulated peripheral blood mononuclear cells (PBMCs) and control samples to quantitatively measure the expression level of key transcripts of interest. A total of 1367 differentially expressed genes (DEGs) were observed in response to a 6 h exposure to PPE57, with 685 being up-regulated and 682 down-regulated. Immune-related gene functions and pathways associated with these genes were evaluated, revealing that the type I IFN signaling pathway was the most significantly enriched pathway in our RNA-seq dataset, with 14 DEGs identified therein including ISG15, MX2, IRF9, IFIT3, IFIT2, OAS3, IFIT1, IFI6, OAS2, OASL, RSAD2, OAS1, IRF7, and MX1. These PPE57-related transcriptomic profiles have implications for a better understanding of host global immune mechanisms underlying MTB infection outcomes. However, more studies regarding these DEGs and type I IFN signaling in this infectious context are necessary to more fully clarify the underlying mechanisms that arise in response to PPE57 during MTB infection.</p

Nanoarray Enabled Size-Dependent Isolation and Proteomics Profiling of Small Extracellular Vesicle Subpopulations toward Accurate Cancer Diagnosis and Prognosis

Small extracellular vesicles (sEVs) have emerged as noninvasive biomarkers in liquid biopsy due to their significant function in pathology and physiology. However, the phenotypic heterogeneity of sEVs presents a significant challenge to their study and has significant implications for their applications in liquid biopsies. In this study, anodic aluminum oxide films with different pore sizes (AAO nanoarray) were introduced to enable size-based isolation and downstream proteomics profiling of sEV subpopulations. The adjustable pore size and abundant Al3+ on the framework of AAOs allowed size-dependent isolation of sEV subpopulations through nanoconfined effects and Lewis acid–base interaction between AAOs and sEVs. Benefiting from the strong concerted effect, the simple AAO nanoarray enabled specific isolation of three sEV subpopulations, termed “50”, “90”, and “150 nm” groups, from 10 μL of complex biological samples within 10 min with high capture efficiencies and purities. Moreover, the nanopores of AAOs also acted as nanoreactors for comprehensive proteomic profiling of the captured sEV subpopulations to reveal their heterogeneity. The AAO nanoarray was first investigated on sEVs from a cell culture medium, where sEV subpopulations could be clearly distinguished, and three traditional sEV-specific proteins (CD81, CD9, and FLOT1) could be identified by proteomic analysis. A total of 3946, 3951, and 3940 proteins were identified from 50, 90, and 150 nm sEV subpopulations, respectively, which is almost twice the number compared to those obtained from the conventional approach. The concept was further applied to complex real-case sample analysis from prostate cancer patients. Machine learning and gene ontology (GO) information analysis of the identified proteins indicate that different-sized sEV subpopulations contain unique protein cargos and have distinct cellular components and molecular functions. Further receiver operating characteristic curve (ROC) analysis of the top five differential proteins from the three sEV subpopulations demonstrated the high accuracy of the proposed approach toward prostate cancer diagnosis (AUC > 0.99). More importantly, several proteins involved in focal adhesion and antigen processing and presentation pathways were found to be upregulated in prostate cancer patients, which may serve as potential biomarkers of prostate cancer. These results suggest that the sEV subpopulation-based AAO nanoarray is of great value in facilitating the early diagnosis and prognosis of cancer and opens a new avenue for sEVs in liquid biopsy

Table_3_Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Stimulated by Mycobacterium tuberculosis PPE57 Identifies Characteristic Genes Associated With Type I Interferon Signaling.xls

Proline-glutamic acid (PE)- and proline-proline-glutamic acid (PPE)-containing proteins are exclusive to Mycobacterium tuberculosis (MTB), the leading cause of tuberculosis (TB). In this study, we performed global transcriptome sequencing (RNA-Seq) on PPE57-stimulated peripheral blood mononuclear cells (PBMCs) and control samples to quantitatively measure the expression level of key transcripts of interest. A total of 1367 differentially expressed genes (DEGs) were observed in response to a 6 h exposure to PPE57, with 685 being up-regulated and 682 down-regulated. Immune-related gene functions and pathways associated with these genes were evaluated, revealing that the type I IFN signaling pathway was the most significantly enriched pathway in our RNA-seq dataset, with 14 DEGs identified therein including ISG15, MX2, IRF9, IFIT3, IFIT2, OAS3, IFIT1, IFI6, OAS2, OASL, RSAD2, OAS1, IRF7, and MX1. These PPE57-related transcriptomic profiles have implications for a better understanding of host global immune mechanisms underlying MTB infection outcomes. However, more studies regarding these DEGs and type I IFN signaling in this infectious context are necessary to more fully clarify the underlying mechanisms that arise in response to PPE57 during MTB infection.</p