Stereotactic Photodynamic Therapy Using a Two-Photon AIE Photosensitizer.

Two-photon photodynamic therapy (TP-PDT) is emerging as a powerful strategy for stereotactic targeting of diseased areas, but ideal photosensitizers (PSs) are currently lacking. This work reports a smart PS with aggregation-induced emission (AIE) feature, namely DPASP, for TP-PDT with excellent performances. DPASP exhibits high affinity to mitochondria, superior photostability, large two-photon absorption cross section as well as efficient reactive oxygen species generation, enabling it to achieve photosensitization both in vitro and in vivo under two-photon excitation. Moreover, its capability of stereotactic ablation of targeted cells with high-precision is also successfully demonstrated. All these merits make DPASP a promising TP-PDT candidate for accurate ablation of abnormal tissues with minimal damages to surrounding areas in the treatment of various diseases.

Quantification of placental extracellular vesicles in different pregnancy status via single particle analysis method.

BACKGROUND: The nano-sized, lipid bilayer-delimited placental extracellular vesicles (PEVs) released by the placenta are now regarded as important mediators involved in various physiological and pathological processes of pregnant women. The number and contents of PEVs are significantly altered in preeclampsia and are considered as potential biomarkers. However, the distribution pattern of PEVs in the maternal circulation in different pregnancy status is still unclear for the limitation of the traditional method with low sensitivity. METHODS: In this work, we recruited 561 pregnant women with different pregnancy status and investigated the distribution pattern of PEVs in the maternal circulation based on a single extracellular vesicle analysis method and placental alkaline phosphatase (PLAP), a placenta-specific marker. RESULTS: The concentration of PEVs in pregnant women increased with the progression of gestational age, while the ratio of PEVs decreased to about 10% in the third trimester. Surprisingly, the PLAP+ EVs also presented in the plasma of non-pregnant women and normal male about 5%. The change in the ratio of PEVs can reflect the pregnancy status and also had a better diagnostic value in severe preeclampsia (AUC = 0.7811). CONCLUSIONS: Our study not only reveals the distribution pattern of PEVs, but also identifies the diagnostic potential of PEVs as biomarkers.

In Situ Generation of Red-to-NIR Emissive Radical Cations in the Stomach for Gastrointestinal Imaging.

Red-to-near-infrared (NIR) fluorescent probes, with advantages such as high spatiotemporal resolution and in situ sensing abilities, are highly attractive for diagnosis of gastrointestinal diseases and targeted drug development. However, conventional red-to-NIR fluorophores with electron closed-shell structures require tedious synthetic procedures for preparation, and it is difficult to further decorate them with sensing groups. In this study, a series of easily prepared pyrroles with simple structures that can quickly be transformed into red-to-NIR emissive radical cations in acidic buffer solution and in vivo stomachs is developed. The in-situ-generated red-to-NIR emissive pyrrole radical cations in the stomach have excellent biocompatibility and stability and can be used not only for intravital gastrointestinal imaging with high spatiotemporal resolution, but also for dynamic monitoring of the gastric emptying process and assessment of anti-gastric-acid therapy. The acidity-induced generation of pyrrole radical cations is believed to provide a facile strategy for developing red-to-NIR fluorophores and studying gastrointestinal diseases.

A Blood-Responsive AIE Bioprobe for the Ultrasensitive Detection and Assessment of Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a severe subtype of stroke caused by the rupturing of blood vessels in the brain. The ability to accurately assess the degree of bleeding in an SAH model is crucial for understanding the brain-damage mechanisms and developing therapeutic strategies. However, current methods are unable to monitor microbleeding owing to their limited sensitivities. Herein, a new bleeding assessment system using a bioprobe TTVP with aggregation-induced emission (AIE) characteristics is demonstrated. TTVP is a water-soluble, small-molecule probe that specifically interacts with blood. Taking advantage of its AIE characteristics, cell membranes affinity, and albumin-targeting ability, TTVP fluoresces in bleeding areas and detects the presence of blood with a high signal-to-noise (S/N) ratio. The degree of SAH bleeding in an endovascular perforation model is clearly evaluated based on the intensity of the fluorescence observed in the brain, which enables the ultrasensitive detection of mirco-bleeding in the SAH model in a manner that outperforms the current imaging strategies. This method serves as a promising tool for the sensitive analysis of the degree of bleeding in SAHs and other hemorrhagic diseases.

Tetrahedral framework nucleic acids linked CRISPR/Cas13a signal amplification system for rare tumor cell detection.

The sensitive and accurate detection of rare tumor cells provides precise diagnosis and dynamic assessment information in various tumor spectrums. However, rare tumor cells assay is still a challenge due to the exceedingly rare presence in the blood. In this research, we develop a fluorescent approach for the identification of rare tumor cells based on a combination of immunosorbent capture and a three-step signal amplification strategy. First, rare tumor cells are captured by immunoadsorption on 96-well plates. Second, self-synthesized tetrahedral framework nucleic acids (tFNAs) spontaneously anchor into the lipid bilayer of rare tumor cells, resulting in a "one to more" amplification effect. Then, the double-stranded DNA (dsDNA) binds to the vertices of the tFNAs and generates a large amount of target RNA by T7 polymerase, which is the secondary signal amplification. Finally, the target RNA activates the collateral cleavage ability of CRISPR/Cas13a, and the reporter RNA is cleaved for third signal amplification. The detection limit of the proposed method is down to 1 cell mL-1. Furthermore, the tFNAs-Cas13a system is also shown to be capable of detecting rare tumor cells in spiked-in samples and clinical blood samples. This platform enables speedy detection of rare tumor cells with high sensitivity and good specificity, and shows great potential for tumor diagnosis.

Point-of-Care Urinalysis with One Drop of Sample Using an Aggregation-Induced Emission Luminogen under the Coffee-Ring Effect.

Development of a practical point-of-care test for urinalysis is crucial for early diagnosis and treatment of chronic kidney disease (CKD). However, the classical gold standard detection method depends on sophisticated instruments and complicated procedures, impeding them from being utilized in resource-limited settings and daily screening. Herein, we report a rapid point-of-care device for the simultaneous quantification of microalbuminuria and leukocyte using one drop of urine. A luminogen (TTVP) with an aggregation-induced emission property can selectively activate its near-infrared fluorescence in the presence of albumin and leukocyte via hydrophobic or electrostatic interactions. The fluorescence signals from urine albumin and leukocyte could be well-separated combined with the coffee-ring effect. Using a smartphone-based detection device, simultaneous quantification of urine albumin and leukocyte was successfully achieved, which only took 20 min and required one drop of urine. The performance of this system is also verified with 120 clinical samples, which might serve as a simple, low-cost, and rapid tool for CKD screening and disease monitoring at the point of care.

A novel nest hybridization chain reaction based electrochemical assay for sensitive detection of circulating tumor DNA.

As an ideal biomarker candidate, circulating tumor DNA (ctDNA) plays a vital role in noninvasive diagnosis of cancer. However, most traditional approaches for quantifying ctDNA are cumbersome and expensive. In the present work, a novel electrochemical biosensor based on nest hybridization chain reaction was proposed for the sensitive and specific detection of PIK3CA E545K ctDNA with a simple process. The nest hybridization chain reaction was initiated by the hybridization of two dumbbell-shaped DNA units which were assembled by two classes of well-designed DNA probes respectively, leading to the formation of a complex DNA structure. In the presence of target ctDNA, the amplified hybridization chain reaction products were captured by target ctDNA, resulting in a significant increase of electrochemical signal. Under the optimal conditions, the developed biosensor exhibited good analytical performance for the detection of target ctDNA with the linear range from 5 pM to 0.5 nM and the detection limit of 3 pM. Furthermore, this assay was successfully applied to the detection of ctDNA in spiked-in samples, pleural effusion and serum samples of malignant tumor patients. This simple and cost-effective sensing system holds great potentials for ctDNA detection and cancer diagnosis.

Isolation of circulating fetal trophoblasts by a four-stage inertial microfluidic device for single-cell analysis and noninvasive prenatal testing.

Noninvasive detection of circulating fetal cells carrying the entire fetal genome is a promising way for prenatal testing of genetic diseases. However, ideal approaches for efficient separation of these valuable cells are not available. Here, a novel inertial microfluidic chip (CelutriateChip 1) is developed for ultra-fast, label-free enrichment of circulating trophoblasts (CTBs) from the whole blood samples of pregnant women. The unique structural design of the four-stage curved channel in CelutriateChip 1 enables CTBs with larger size to be efficiently separated from the blood samples under the effect of inertial and Dean drag forces. The transition of the target cells among the stages enables CelutriateChip 1 to achieve one or two orders of magnitude higher throughput compared to single channel inertial microfluidic chips. After optimization of conditions, CTBs can be recovered from 2 mL of whole blood within 5 min with an average recovery efficiency ranging from 52.3% to 65.8% and high white blood cell depletion (99.95%). CTBs collected from the chip can be isolated at the single-cell level and used for downstream immunofluorescence staining and genetic genotyping. Clinical tests are performed on 30 pregnant women and the results demonstrate that CTBs are obtainable in 86.67% of pregnancy cases. A single-base variant in the HBB gene can be accurately detected by sequencing of rare CTBs. This simple, antibody-free and low-cost approach holds promise for obtaining rare CTBs for prenatal detection of various genetic diseases.

Nondestructive Identification of Rare Trophoblastic Cells by Endoplasmic Reticulum Staining for Noninvasive Prenatal Testing of Monogenic Diseases.

Noninvasive prenatal detection of monogenic diseases based on cell-free DNA is hampered by challenges in obtaining a sufficient fraction and adequate quality of fetal DNA. Analyzing rare trophoblastic cells from Papanicolaou smears carrying the entire fetal genome provides an alternative method for noninvasive detection of monogenic diseases. However, intracellular labeling for identification of target cells can affect the quality of DNA in varying degrees. Here, a new approach is developed for nondestructive identification of rare fetal cells from abundant maternal cells based on endoplasmic reticulum staining and linear discriminant analysis (ER-LDA). Compared with traditional methods, ER-LDA has little effect on cell quality, allowing trophoblastic cells to be analyzed on the single-cell level. Using ER-LDA, high-purity of trophoblastic cells can be identified and isolated at single cell resolution from 60 pregnancies between 4 and 38 weeks of gestation. Pathogenic variants, including -SEA/ deletion mutation and point mutations, in 11 fetuses at risk for α- or ß-thalassemia can be accurately detected by this test. The detection platform can also be extended to analyze the mutational profiles of other monogenic diseases. This simple, low-cost, and noninvasive test can provide valuable fetal cells for fetal genotyping and holds promise for prenatal detection of monogenic diseases.