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Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia, characterized by the accumulation of amyloid beta (Aß) peptides in the brain. Here, we present a simple, rapid, and affordable CRISPR-powered aptasensor for the quantitative detection of Aß40 and Aß42 biomarkers in cerebrospinal fluid (CSF) samples, enabling early and accurate diagnostics of AD patients. The aptasensor couples the high specificity of aptamers for Aß biomarkers with CRISPR-Cas12a-based fluorescence detection. The CRISPR-powered aptasensor enables us to detect Aß40 and Aß42 in CSF samples within 60 min, achieving a detection sensitivity of 1 pg/mL and 0.1 pg/mL, respectively. To validate its clinical utility, we quantitatively detected Aß40 and Aß42 biomarkers in clinical CSF samples. Furthermore, by combining CSF Aß42 levels with the c(Aß42)/c(Aß40) ratio, we achieved an accurate diagnostic classification of AD patients and healthy individuals, showing superior performance over the conventional ELISA method. We believe that our innovative aptasensor approach holds promise for the early diagnostic classification of AD patients.

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Alpha-fetoprotein (AFP) is an important protein biomarker of liver cancer, as its serum levels are highly correlated with the progression of disease. Conventional immunoassays for AFP detection rely on enzyme-linked immunosorbent assay analyses with expensive and bulky equipment. Here, we developed a simple, affordable, and portable CRISPR-powered personal glucose meter biosensing platform for quantitative detection of the AFP biomarker in serum samples. The biosensor takes advantage of the excellent affinity of aptamer to AFP and the collateral cleavage activity of CRISPR-Cas12a, enabling sensitive and specific CRISPR-powered protein biomarker detection. To enable point-of-care testing, we coupled invertase-catalyzed glucose production with the glucose biosensing technology to quantify AFP. Using the developed biosensing platform, we quantitatively detected AFP biomarker in spiked human serum samples with a detection sensitivity of down to 10 ng/mL. Further, we successfully applied the biosensor to detect AFP in clinical serum samples from patients with liver cancer, achieving comparable performance to the conventional assay. Therefore, this novel CRISPR-powered personal glucose meter biosensor provides a simple yet powerful alternative for detecting AFP and potentially other tumor biomarkers at the point of care.

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Nanoparticulate formulations are being developed toward enhancing the bioavailability of orally administrated biologics. However, the processes mediating particulate carriers' intestinal uptake and transport remains to be fully elucidated. Herein, an optical clearing-based whole tissue mount/imaging strategy was developed to enable high quality microscopic imaging of intestinal specimens. It enabled the distribution of nanoparticles within intestinal villi to be quantitatively analyzed at a cellular level. Two-hundred and fifty nm fluorescent polystyrene nanoparticles were modified with polyethylene glycol (PEG), Concanavalin A (ConA), and pectin to yield mucopenetrating, enterocyte targeting, and mucoadhesive model nanocarriers, respectively. Introducing ConA on the PEGylated nanoparticles significantly increased their uptake in the intestinal epithelium (∼4.16 fold for 200 nm nanoparticle and ∼2.88 fold for 50 nm nanoparticles at 2 h). Moreover, enterocyte targeting mediated the trans-epithelial translocation of 50 nm nanoparticles more efficiently than that of the 200 nm nanoparticles. This new approach provides an efficient methodology to obtain detailed insight into the transcytotic activity of enterocytes as well as the barrier function of the constitutive intestinal mucus. It can be applied to guide the rational design of particulate formulations for more efficient oral biologics delivery.

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Understanding the intestinal transport of particles is critical in several fields ranging from optimizing drug delivery systems to capturing health risks from the increased presence of nano- and micro-sized particles in human environment. While Caco-2 cell monolayers grown on permeable supports are the traditional in vitro model used to probe intestinal absorption of dissolved molecules, they fail to recapitulate the transcytotic activity of polarized enterocytes. Here, an intestine-on-chip model is combined with in silico modeling to demonstrate that the rate of particle transcytosis is ≈350× higher across Caco-2 cell monolayers exposed to fluid shear stress compared to Caco-2 cells in standard "static" configuration. This relates to profound phenotypical alterations and highly polarized state of cells grown under mechanical stimulation and it is shown that transcytosis in the microphysiological model is energy-dependent and involves both clathrin and macropinocytosis mediated endocytic pathways. Finally, it is demonstrated that the increased rate of transcytosis through cells exposed to flow is explained by a higher rate of internal particle transport (i.e., vesicular cellular trafficking and basolateral exocytosis), rather than a change in apical uptake (i.e., binding and endocytosis). Taken together, the findings have important implications for addressing research questions concerning intestinal transport of engineered and environmental particles.

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The mucus covering of epithelial tissues presents one significant biological barrier to the uptake and absorption of particulate carriers. Improved understanding of the mechanisms mediating the transport of nanoparticles across such mucus layers would accelerate their development as optimised mucosal drug delivery formulations (e.g. via oral and rectal routes). Herein, an in vitro mucus model ("Mucus-on-Chip") was developed to enable the interaction and transport of functionalised nanoparticles and reconstituted mucus to be quantitatively investigated in real-time. We verified that the diffusion of nanoparticles into mucus is highly dependent on their biointerfacial properties. Muco-inert modification (PEGylation) significantly enhanced the mucopenetration of 50 nm and 200 nm nanoparticles, whereas limited mucopenetration was observed for pectin coated mucoadhesive nanoparticles. Furthermore, this model can be easily adapted to mimic specific physiological mucus environments. Mucus pre-treated with a mucolytic agent displayed reduced barrier function and therefore significantly accelerated mucopenetration of nanoparticles, which was independent of their size and biointerfacial properties. This new "Mucus-on-Chip" methodology provides detailed insight into the dynamics of nanoparticle-mucus interaction, which can be applied to refine the design of particulate formulations for more efficient mucosal drug delivery.

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Diverse nanoparticulate systems have been engineered as vehicles towards enhancing the bioavailability of orally administrated vaccines. Substantial evidence suggests that targeting microfold cells (M cells) within Peyer's patches (PPs) is a prerequisite for vaccine-loaded nanocarriers to induce an effective antigen-specific immune response. Improved understanding of the contribution of M cells to sampling luminal nanoparticles into the underlying gut associated lymphoid tissues would accelerate the development of oral vaccine formulations. Herein, a novel clearing-based whole tissue mount/imaging technique was developed to enable the specific distribution of nanoparticles within ex vivo murine PPs to be quantitatively determined at the cellular level. This revealed that 200 nm nanoparticles modified with M cell targeting ligands (lectin Ulex europaeus agglutinin-1, UEA-1) were translocated into subepithelial domes 7.6 and 16.3 times greater than the non-targeted ones at 60 min and 120 min, respectively. This approach provides a new methodology to quantitatively investigate the transcytotic activity of M cells for particulate formulations, which may aid in the design of improved oral vaccines.

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Developing ultrasensitive contrast agents for the early detection of malignant tumors in liver is highly demanded. Constructing hepatic tumors specific targeting probes could provide more sensitive imaging information but still faces great challenges. Here we report a novel approach for the synthesis of ultra-small Fe3O4 nanoparticles conjugated with c(RGDyK) and their applications as active-target T1-weighted magnetic resonance imaging (MRI) contrast agent (T1-Fe3O4) for imaging tiny hepatic tumors in vivo. RGD-modified T1-Fe3O4 nanoprobes exhibited high r1 of 7.74 mM(-1)s(-1) and ultralow r2/r1 of 2.8 at 3 T, reflecting their excellent T1 contrast effect at clinically relevant magnetic field. High targeting specificity together with favorable biocompatibility and strong ability to resist against non-specific uptake were evaluated through in vitro studies. Owing to the outstanding properties of tumor angiogenesis targeting with little phagocytosis in liver parenchyma, hepatic tumor as small as 2.2 mm was successfully detected via the T1 contrast enhancement of RGD-modified T1-Fe3O4. It is emphasized that this is the first report on active-target T1 imaging of hepatic tumors, which could not only significantly improve diagnostic sensitivity, but also provide post therapeutic assessments for patients with liver cancer.

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