Digital microfluidics-based digital counting of single-cell copy number variation (dd-scCNV Seq).

Single-cell copy number variations (CNVs), major dynamic changes in humans, result in differential levels of gene expression and account for adaptive traits or underlying disease. Single-cell sequencing is needed to reveal these CNVs but has been hindered by single-cell whole-genome amplification (scWGA) bias, leading to inaccurate gene copy number counting. In addition, most of the current scWGA methods are labor intensive, time-consuming, and expensive with limited wide application. Here, we report a unique single-cell whole-genome library preparation approach based on digital microfluidics for digital counting of single-cell Copy Number Variation (dd-scCNV Seq). dd-scCNV Seq directly fragments the original single-cell DNA and uses these fragments as templates for amplification. These reduplicative fragments can be filtered computationally to generate the original partitioned unique identified fragments, thereby enabling digital counting of copy number variation. dd-scCNV Seq showed an increase in uniformity in the single-molecule data, leading to more accurate CNV patterns compared to other methods with low-depth sequencing. Benefiting from digital microfluidics, dd-scCNV Seq allows automated liquid handling, precise single-cell isolation, and high-efficiency and low-cost genome library preparation. dd-scCNV Seq will accelerate biological discovery by enabling accurate profiling of copy number variations at single-cell resolution.

ARMOR: Auto-Assembled Resilient Biomimetic Calcified Ornaments for Selective Cell Protection by Dual-Aptamer-Driven Hybridization Chain Reaction.

Unlike plant and microbial cells having cell walls, the outermost layer of mammalian cell is a delicate, two-layered structure of phospholipids with proteins embedded, which is susceptible to environmental changes. It is necessary to create an "armor" on cell surface to protect cell integrity. Here, we propose an Auto-assembled Resilient bioMimetic calcified ORnaments (ARMOR) strategy driven by dual-aptamer-based hybridization chain reaction (HCR) and Ca2+ assisted calcification for selective cell protection. This co-recognition design enhances the selectivity and leverages robust in situ signal amplification by HCR to improve the sensitivity. The calcified shell is cogenerated by crosslinking the alginate-HCR product with Ca2+ ion. ARMOR has high efficiency for shielding cells from environmental assaults, which can be applied to circulating tumor cell (CTC) protection, isolation, and identification, maintaining the native state and intact genetic information for downstream analysis.

DNA-Programmed Orientation-Ordered Multivalent Microfluidic Interface for Liquid Biopsy.

Aptamer-functionalized microfluidic interfaces hold great potential for liquid biopsies owing to their programmable nature. However, most previous studies have focused on development of multivalent aptamers to improve binding affinity, while ignoring aptamer orientation on microfluidic interfaces, resulting in suboptimal accessibility and affinity. Herein, we report a Cubic DNA Nanostructure (CDN)-programmed strategy to precisely control the orientation and valency of the Aptamer on a microfluidic interface (CDN-Apt-Chip) for enhancing the capture and release of circulating tumor cells (CTCs). We demonstrate that the ordered orientation and multivalent configuration can synergistically increase the binding affinity of aptamers toward CTCs. By using CDN-Apt-Chip, we successfully isolated CTCs from the peripheral blood of T-cell leukemia patients and discriminated T-cell leukemia patients from healthy volunteers. Furthermore, the captured CTCs were nondestructively released via nuclease treatment. We then performed T-cell receptor sequencing on the released cells to demonstrate the compatibility with downstream analysis. Overall, this study provides a new paradigm for interface regulation of functional microfluidic chips and advances the clinical translation of aptamer-based liquid biopsy.

Single-Cell Digital Microfluidic Mass Spectrometry Platform for Efficient and Multiplex Genotyping of Circulating Tumor Cells.

Gene mutation profiling of heterogeneous circulating tumor cells (CTCs) offers comprehensive and real-time molecular information of tumors for targeted therapy guidance, but the lack of efficient and multiplex genotyping techniques for single-CTC analysis greatly hinders its development and clinical application. This paper reports a single-CTC mass spectrometry analysis method for efficient and multiplex mutation profiling based on digital microfluidics. Digital microfluidics affords integrated single-CTC manipulation, from single-CTC isolation to high-performance whole genome amplification, via nanoliter droplet-based wettability trapping and hydrodynamic adjustment of cell distribution. Coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, multiplex mutation information of individual CTCs can be efficiently and accurately identified by the inherent mass differences of different DNA sequences. This platform achieves Kirsten rat sarcoma viral oncogene mutation profiling of heterogeneous CTCs at the single-cell level from cancer patient samples, offering new avenues for genotype profiling of single CTCs and cancer therapy guidance.

HUNTER-Chip: Bioinspired Hierarchically Aptamer Structure-Based Circulating Fetal Cell Isolation for Non-Invasive Prenatal Testing.

Isolation and genetic analysis of circulating fetal cells from billions of maternal cells in peripheral blood are the cornerstone of fetal cell-based non-invasive prenatal testing. Inspired by the hierarchically multivalent architecture for enhanced capture of nature, an aptamer-based Hierarchically mUltivalent aNTibody mimic intERface (HUNTER) was designed with a tremendous avidity effect for highly efficient capture and non-destructive release of fetal cells. It was engineered by grafting Y-shaped DNA nanostructures to a linear polymer chain, creating a flexible polymer chain with bivalent aptamer side chains. This hierarchical arrangement of the aptamer ensures morphological complementarity, collective multiple-site interaction, and multivalent recognition between the aptamer and target cells. In combination with a deterministic lateral displacement (DLD)-patterned microdevice named as HUNTER-Chip, it achieves a binding affinity over 65-fold and a capture efficiency over 260%-fold due to the combination of hierarchically designed aptamers and frequent cell-ligand collision created by DLD. Moreover, a nuclease-assisted cell release strategy facilitates the release of fetal cells for gene analysis, such as fluorescence in situ hybridization. With the advantages of high affinity, excellent capture efficiency, and compatible downstream analysis, the HUNTER-Chip holds great potential for non-invasive prenatal diagnosis.

Rice Stripe Mosaic Virus-Encoded P4 Is a Weak Suppressor of Viral RNA Silencing and Is Required for Disease Symptom Development.

Viral suppressors of RNA silencing (VSRs) are a cluster of viral proteins that have evolved to counteract eukaryotic antiviral RNA silencing pathways, thereby contributing to viral pathogenicity. In this study, we revealed that the matrix protein P4 encoded by rice stripe mosaic virus (RSMV), which is an emerging cytoplasmic rhabdovirus, is a weak RNA silencing suppressor. By conducting yeast two-hybrid, bimolecular fluorescence complementation, and subcellular colocalization assays, we proved that P4 interacts with the rice endogenous suppressor of gene silencing 3 (OsSGS3). We also determined that P4 overexpression has no effect on OsSGS3 transcription. However, P4 can promote the degradation of OsSGS3 via ubiquitination and autophagy. Additionally, a potato virus X-based expression system was used to confirm that P4 enhances the development of mosaic symptoms on Nicotiana benthamiana leaves by promoting hydrogen peroxide accumulation but not cell death. To verify whether P4 is a pathogenicity factor in host plants, we generated transgenic P4-overexpressing rice plants that exhibited disease-related developmental defects including decreased plant height and excessive tillering. Our data suggest that RSMV-encoded P4 serves as a weak VSR that inhibits antiviral RNA silencing by targeting OsSGS3.

Stimuli-Responsive Microfluidic Interface Enables Highly Efficient Capture and Release of Circulating Fetal Cells for Non-Invasive Prenatal Testing.

Circulating fetal nucleated cells (CFCs) carrying whole genomic coding of the fetus in maternal blood have been pursued as ideal biomarkers for noninvasive prenatal testing (NIPT). However, a significant limitation is the need to enrich sufficient cells in quantity and purity for fetal genetic disorder diagnosis. This study for the first time demonstrates a stimuli-responsive ligand enabling interface on array patterned microfluidic chip (NIPT-Chip) for high efficient isolation and release of CFCs in untreated whole blood. Deterministic lateral displacement (DLD)-array was patterned in the chip to increase collision frequency between CFCs and surface-anchored antibody to achieve high efficient cell capture. More importantly, the stimuli-responsive interface enables gentle release of captured CFCs through a thiol exchange reaction for downstream gene analysis of NIPT. With the advantages of simple processing, efficient isolation, and gentle release, NIPT-Chip offers great potential for clinical translation of circulating fetal cell-based NIPT.

A green route for synthesizing pure silica zeolites with six-membered rings.

Green routes for synthesizing pure silica zeolites are attractive but still challenging. Herein, we for the first time report a green route for synthesizing pure silica zeolites with six-membered rings (6MRs) by a combined strategy of ethanol filling and zeolite seeding. As a result, pure silica zeolites with 6MRs, such as SOD, MTN, and NON, could be successfully synthesized.

Fluoride-free synthesis of beta zeolite with enrichment of polymorph B from a solvent-free route.

Beta zeolite with enrichment of polymorph B is successfully synthesized in the absence of fluorine species under solvent-free conditions. The phase composition of polymorph B in the sample is about 70%.

Stimulus-Responsive Microfluidic Interface Enables Efficient Enrichment and Cytogenetic Profiling of Circulating Myeloma Cells.

Minimal residual disease (MRD) provides an independent prognostic factor for multiple myeloma (MM) patients. However, clinical MRD assays suffer from highly invasive sampling, insufficient detection sensitivity, and high cost. Herein, a stiMulus-Responsive ligand-Decorated microfluidic chip (MRD-Chip) was developed for efficient capture and controlled release of circulating myeloma cells (CMCs) in the peripheral blood for noninvasive myeloma evaluation. The CD138 antibody-decorated herringbone chip with a disulfide linker was designed to enhance the collision probability between blood cells and capture antibodies, leading to high capture efficiency of CMCs. More importantly, the captured CMCs can be nondestructively released via a thiol-exchange reaction, allowing them to be used for subsequent cellular and molecular analysis. By fluorescence in situ hybridization assay, we successfully identified the cytogenetic abnormalities (chromosome 1q21 amplification and p53 deletion) of CMCs in clinical samples. Overall, with the merits of noninvasive sampling, high capture efficiency (70.93%), high throughput (1.5 mL/h), and nondestructive release of target cells (over 90% viability) for downstream analysis, our strategy provides new opportunities for myeloma evaluation, such as prognosis assessment, efficacy monitoring, and mechanism research of disease relapse and drug resistance.

Ultrasensitive and Reversible Nanoplatform of Urinary Exosomes for Prostate Cancer Diagnosis.

Prostate cancer cell-derived exosomes in urine have been extensively studied recently and regarded as novel biomarkers for cancer diagnosis and prognosis, which presents wide prospects in clinical applications. Sensitive detection and specific capture methods are essential for exosomes analysis. Herein, a dual functional platform composed of superparamagnetic conjunctions and molecular beacons (SMC-MB) is reported. The SMC-MB platform is designed based on aptamer immunoaffinity with ultrasensitive detection efficiency and reversible isolation capacity, which, respectively, profit from nonenzymatic amplification methods and magnetic separation along with restriction cleavage. It is noteworthy that exosomes quantification was exactly amplified and transformed into single strand DNA detection. Correlated measurements evidence that the limit of detection of SMC-MB is as low as ∼100 particles/µL in urine, and a linear relationship meets between the logarithmic concentration of exosomes and fluorescence intensity of the molecular beacon. Furthermore, employing prostate specific membrane antigen (PSMA) aptamer, the platform adapted to detect and capture PMSA-positive exosomes from urine samples provides excellent diagnostic efficiency for prostate cancer (PCa). The expression of typical biomarkers of PCa, i.e., PSA and PCA3 mRNA, is significantly higher in PSMA-positive exosomes. Altogether, the platform and strategy described in this paper are promising in urinary exosomes analysis and prostate cancer detection.