Hypoxia-targeted and spatial-selective tumor suppression by near infrared nanoantenna sensitized engineered bacteria.

It is an active research area in the development of engineered bacteria to address the bottleneck issue of hypoxic tumors, which otherwisely possess resistance to chemotherapies, radiotherapies, and photodynamic therapies. Here we report a new method to ablate hypoxic tumors with NIR-nanoantenna sensitized engineered bacteria (NASEB) in a highly effective and dual selective manner. It features engineered E. coli MG1655 (EB) with coatings of lanthanide upconversion nanoparticles (UCNPs) as external antennas on bacterial surface (MG1655/HlyE-sfGFP@UCNP@PEG), enabling NIR laser-switchable generation/secretion of HlyE perforin to kill cancer cells. We have demonstrated that NASEB enrichment on hypoxic tumor sites via their innate chemotactic tendency, in assistance of localized NIR laser irradiation, can suppress tumors with improved efficacy and selectivity, thus minimizing potential side effects in cancer treatment. The NIR-responsive nanoantenna sensitized switching in engineering bacteria is distinct from the previous reports, promising conceptually new development of therapeutics against hypoxic tumors. STATEMENT OF SIGNIFICANCE: Tumor hypoxia exacerbates tumor progression, but also reduces the efficacy of conventional chemotherapies, radiotherapies, or photodynamic therapies. Here we develop near infrared Nano Antenna Sensitized Engineered Bacteria (NASEB) to treat hypoxic tumors. NASEB can accumulate and proliferate on hypoxic tumor sites via their innate chemotactic tendency. After receiving NIR laser signals, the upconversion nanoparticles on NASEB surface as antennas can transduce them to blue light for activation of HlyE perforin in the protein factory of EB. Our method features dual selectivity on the tumor sites, contributed by hypoxic tumor homing of anaerobic bacteria and spatial confinement through selective NIR laser irradiation. The concept of NASEB promises to address the challenges of tumor hypoxia for cancer therapies.

Single-Cell Enzymatic Screening for Epithelial Mesenchymal Transition with an Ultrasensitive Superwetting Droplet-Array Microchip.

The phenotypic changes of circulating tumor cells (CTCs) during the epithelial-mesenchymal transition (EMT) have been a hot topic in tumor biology and cancer therapeutic development. Here, an integrated platform of single-cell fluorescent enzymatic assays with superwetting droplet-array microchips (SDAM) for ultrasensitive functional screening of epithelial-mesenchymal sub-phenotypes of CTCs is reported. The SDAM can generate high-density, volume well-defined droplet (0.66 nL per droplet) arrays isolating single tumor cells via a discontinuous dewetting effect. It enables sensitive detection of MMP9 enzyme activities secreted by single tumor cells, correlating to their epithelial-mesenchymal sub-phenotypes. In the pilot clinical double-blind tests, the authors have demonstrated that SDAM assays allow for rapid identification and functional screening of CTCs with different epithelial-mesenchymal properties. The consistency with the clinical outcomes validates the usefulness of single-cell secreted MMP9 as a biomarker for selective CTC screening and tumor metastasis monitoring. Convenient addressing and recovery of individual CTCs from SDAM have been demonstrated for gene mutation sequencing, immunostaining, and transcriptome analysis, revealing new understandings of the signaling pathways between MMP9 secretion and the EMT regulation of CTCs. The SDAM approach combined with sequencing technologies promises to explore the dynamic EMT plasticity of tumors at the single-cell level.

Integrating Cycled Enzymatic DNA Amplification and Surface-Enhanced Raman Scattering for Sensitive Detection of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) represents an emerging biomarker of liquid biopsies for the development of precision cancer diagnostics and therapeutics. However, sensitive detection of ctDNA remains challenging, due to their short half-life and low concentrations in blood samples. In this study, we report a new method to address this challenge by integrating cycled enzymatic DNA amplification technique and Au nanoparticle@silicon-assisted surface-enhanced Raman scattering (SERS) technique. We have demonstrated a reproducible identification of a single-base-mutated ctDNA sequence of diffuse intrinsic pontine gliomas (DIPGs), with the limit of detection (LOD) as low as 9.1 fM in the spiked blood samples. This approach can be used to analyze trace amounts of ctDNA in translational medicine for early diagnosis, therapeutic effect monitoring, and prognosis of patients with cancer.

Aptamer Conformation-Cooperated Enzyme-Assisted Surface-Enhanced Raman Scattering Enabling Ultrasensitive Detection of Cell Surface Protein Biomarkers in Blood Samples.

Developing novel strategies for sensitive and specific detection of protein biomarkers is a field of active research. Here, we report an ultrasensitive biosensor to detect protein tyrosine kinase-7 (PTK7), an important protein biomarker on the cell surface, by aptamer conformation-cooperated enzyme-assisted surface-enhanced Raman scattering (SERS) (ACCESS) technology. Our approach features a synergistic combination of the conformational alteration of the anglerfish aptamer triggered by the recognition of the membrane protein (PTK7) and Exo III enzyme-assisted nucleic acid amplification. It transduces the specific binding events between the aptamer and PTK7 protein into dramatically improved SERS signals. Sensitive and specific detection of PTK7 protein has been demonstrated both in the solution and directly on the surface of live CCRF-CEM cells, with a limit of detection better than the commercial enzyme-linked immunosorbent assay method by nearly 5 orders of magnitude. As a flexible, ultrasensitive, and specific approach, ACCESS promises important applications in clinical diagnostics, where only a very limited amount of the biological sample is available.

Sensitive detection of antibiotics using aptamer conformation cooperated enzyme-assisted SERS technology.

Serious healthcare concerns have been raised on the issue of antibiotic residues after overuse, especially by accumulation in the human body through food webs. Here, we report a methodological development for sensitive detection of antibiotics with aptamer conformation cooperated enzyme-assisted SERS (ACCESS) technology. We design and integrate a set of nucleic acid oligos, realizing specific recognition of chloramphenicol (CAP) and efficient exonuclease III-assisted DNA amplification. It features a "signal-on" analysis of CAP with the limit of detection (15 fM), the lowest concentration detectable in the literature. Our method exhibits a high selectivity on the target analyte, free of interference of other potential antibiotic contaminants. The ACCESS assay promises an ultrasensitive and specific detection tool for trace amounts of antibiotic residues in samples of our daily life.