Enhanced CRISPR/Cas12a-based quantitative detection of nucleic acids using double emulsion droplets.

Pairing droplet microfluidics and CRISPR/Cas12a techniques creates a powerful solution for the detection and quantification of nucleic acids at the single-molecule level, due to its specificity, sensitivity, and simplicity. However, traditional water-in-oil (W/O) single emulsion (SE) droplets often present stability issues, affecting the accuracy and reproducibility of assay results. As an alternative, water-in-oil-in-water (W/O/W) double emulsion (DE) droplets offer superior stability and uniformity for droplet digital assays. Moreover, unlike SE droplets, DE droplets are compatible with commercially available flow cytometry instruments for high-throughput analysis. Despite these advantages, no study has demonstrated the use of DE droplets for CRISPR-based nucleic acid detection. In our study, we conducted a comparative analysis to assess the performance of SE and DE droplets in quantitative detection of human papillomavirus type 18 (HPV18) DNA based on CRISPR/Cas12a. We evaluated the stability of SEs and DEs by examining size variation, merging extent, and content interaction before and after incubation at different temperatures and time points. By integrating DE droplets with flow cytometry, we achieved high-throughput and high-accuracy CRISPR/Cas12a-based quantification of target HPV18 DNA. The DE platform, when paired with CRISPR/Cas12a and flow cytometry techniques, emerges as a reliable tool for absolute quantification of nucleic acid biomarkers.

Relaxed purifying selection pressure drives accelerated and dynamic gene rearrangements in thrips (Insecta: Thysanoptera) mitochondrial genomes.

Insect mitochondrial genomes (mitogenome) generally present a typical gene order, which is considered as the ancestral arrangement. All sequenced mitogenomes in the Thysanoptera display high levels of gene rearrangement. Due to limited number of thrips mitogenomes sequenced, how gene rearrangement may be shaped by evolution remain unclear. Here, we analyzed 33 thrips mitogenomes, including 14 newly sequenced. These mitogenomes were diverse in organization, nucleotides substitution and gene arrangements. We found 28 highly rearranged gene orders with the breakpoints of gene rearrangements from 25 to 33. Reconstruction of the ancestors mitochondrial gene arrangements states indicated that Tubulifera have more complex pathways than Terebrantia in the gene order evolution. Molecular calibration estimated that divergence of two suborders occurred in the middle Triassic while the radiation of thrips was associated with the arose and flourish of angiosperm. Our evolutionary hypothesis testing suggests that relaxation of selection pressure enabled the early phase of Thysanoptera evolution, followed by a stronger selective pressure fixed diversification. Our analyses found gene inversion increases the nonsynonymous substitution rates and provide an evolutionary hypothesis driving the diverse gene orders.

Tuneable Cell-Laden Double-Emulsion Droplets for Enhanced Signal Detection.

Water-in-oil-in-water (w/o/w) or double-emulsion (DE) droplets have been widely used for cellular assays at a single-cell level because of their stability and biocompatibility. The oil shell of w/o/w droplets plays the role of a semipermeable membrane that allows substances with low molecular weight (e.g., water) to travel through but restricts those with high molecular weight (e.g., fluorescent biomarkers). Therefore, the core of DEs can be manipulated using osmosis, resulting in the shrinking or swelling of the core. Water leaves the inner aqueous phase to the outer phase via the oil shell when the osmotic pressure of the outer phase is higher than that in the inner phase, causing the shrinkage of DEs and vice versa. These processes can be achieved by transferring the DEs to hypertonic or hypotonic solutions. Manipulation of the core size of DEs can be beneficial to cellular assays. First, due to the selectivity of the oil shell of DEs, the concentration of biomarkers in the core increases when the inner aqueous phase is shrunk, resulting in the enhancement of biosignals. We demonstrate this by encapsulating the Bgl3 enzyme-secreting yeast with a substrate that displays fluorescence after hydrolyzation. In a second application, a single GFP-tagged yeast cell was encapsulated in DEs. After swelling the core of DEs, we observe that the larger core of DEs promotes cell growth compared to those with the smaller cores, leading to more intracellular proteins (green-fluorescent protein) for screening. These osmotic manipulations provide new tools for droplet-based biochemistry.

Certification of visinin-like protein-1 (VILIP-1) certified reference material by amino acid-based and sulfur-based liquid chromatography isotope dilution mass spectrometry.

As an emerging neurodegenerative disease, Alzheimer's disease (AD) has become a leading cause of dementia in older adults. Visinin-like protein-1 (VILIP-1) is an increasingly used biomarker for AD besides the widely accepted Aß1-40, Aß1-42, and tau. However, significant variations exist in the commercial immuno-based assays for VILIP-1 quantification, underlining the necessity to establish a traceability chain. Certified reference materials (CRMs) located at the top of the traceability chain are traceability sources for relevant matrix standard materials. In this work, VILIP-1 solution CRM with a certified value and uncertainty of 39.82±1.52 µg·g-1 was developed and certified using amino acid-based isotope dilution mass spectrometry (AA-ID-MS) and sulfur-based isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS). Certified values from both strategies showed great consistency, with traceability to SI units. Moreover, the candidate VILIP-1 CRM shows excellent homogeneity and can be stable for at least 7 days at -20°C and 12 months at -70°C. The VILIP-1 CRM developed can be used in value assignment to secondary calibrators and clinical matrix CRMs, showing prospects in early diagnosis and disease monitoring for AD.

Computer-Aided Design of Lasso-like Self-Assembling Anticancer Peptides with Multiple Functions for Targeted Self-Delivery and Cancer Treatments.

Anticancer peptides are promising drug candidates for cancer treatment, but the short circulation time and low delivery efficiency limit their clinical applications. Herein, we designed several lasso-like self-assembling anticancer peptides (LASAPs) integrated with multiple functions by a computer-aided approach. Among these LASAPs, LASAP1 (CRGDKGPDCGKAFRRFLGALFKALSHLL, 1-9 disulfide bond) was determined to be superior to the others because it can self-assemble into homogeneous nanoparticles and exhibits improved stability in serum. Thus, LASAP1 was chosen for proving the design idea. LASAP1 can self-assemble into nanoparticles displaying iRGD on the surface because of its amphiphilic structure and accumulate to the tumor site after injection because of the EPR effect and iRGD targeting to αVß3 integrin. The nanoparticles could disassemble in the acidic microenvironment of the solid tumor, and cleaved by the overexpressed hK2, which was secreted by prostate tumor cells, to release the effector peptide PTP-7b (FLGALFKALSHLL), which was further activated by the acidic pH. Therefore, LASAP1 could target the orthotopic prostate tumor in the model mice after intraperitoneal injection and specifically inhibit tumor growth, with low systematic toxicity. Combining the multiple targeting functions, LASAP1 represents a promising design of self-delivery of peptide drugs for targeted cancer treatments.

Shape-based separation of drug-treated Escherichia coli using viscoelastic microfluidics.

Here, we achieve shape-based separation of drug-treated Escherichia coli (E. coli) by viscoelastic microfluidics. Since shape is critical for modulating biological functions of E. coli, the ability to prepare homogeneous E. coli populations adopting uniform shape or sort bacterial sub-population based on their shape has significant implications for a broad range of biological, biomedical and environmental applications. A proportion of E. coli treated with 1 µg mL-1 of the antibiotic mecillinam were found to exhibit changes in shape from rod to sphere, and the heterogeneous E. coli populations after drug treatment with various aspect ratios (ARs) ranging from 1.0 to 5.5 were used for experiment. We demonstrate that E. coli with a lower AR, i.e., spherical E. coli (AR ≤ 1.5), are directed toward the middle outlet, while rod-shaped E. coli with a higher AR (AR > 1.5) are driven to the side outlets. Further, we demonstrate that the separation performance of the viscoelastic microfluidic device is influenced by two main factors: sheath-to-sample flow rate ratio and the concentration of poly-ethylene-oxide (PEO). To the best of our knowledge, this is the first report on shape-based separation of a single species of cells smaller than 4 µm by microfluidics.

Recent progress of carbon dots in targeted bioimaging and cancer therapy.

Carbon dots (CDs), as one new class of carbon nanomaterials with various structure and extraordinary physicochemical properties, have attracted tremendous interest for their potential applications in tumor theranostics, especially in targeted bioimaging and therapy. In these areas, CDs and its derivatives have been employed as highly efficient imaging agent for photoluminescence bioimaging of tumors cells. With unique structure, optical and/or dose attention properties, CDs have been harnessed in various nanotheranostic strategies for diverse tumors through integrating with other functional nanoparticles or utilizing their inherent physical properties. Up to now, CDs have been approved as novel biomaterials by their excellent performances in precise targeted bioimaging and therapy for tumors. Herein, the latest progress in the development of CDs in targeted bioimaging and tumor therapy are reviewed. Meanwhile, the challenges and future prospects of the application of CDs in promising nanotheranostic strategies are discussed and proposed.

Length-based separation of Bacillus subtilis bacterial populations by viscoelastic microfluidics.

In this study, we demonstrated the label-free continuous separation and enrichment of Bacillus subtilis populations based on length using viscoelastic microfluidics. B. subtilis, a gram-positive, rod-shaped bacterium, has been widely used as a model organism and an industrial workhorse. B. subtilis can be arranged in different morphological forms, such as single rods, chains, and clumps, which reflect differences in cell types, phases of growth, genetic variation, and changing environmental factors. The ability to prepare B. subtilis populations with a uniform length is important for basic biological studies and efficient industrial applications. Here, we systematically investigated how flow rate ratio, poly(ethylene oxide) (PEO) concentration, and channel length affected the length-based separation of B. subtilis cells. The lateral positions of B. subtilis cells with varying morphologies in a straight rectangular microchannel were found to be dependent on cell length under the co-flow of viscoelastic and Newtonian fluids. Finally, we evaluated the ability of the viscoelastic microfluidic device to separate the two groups of B. subtilis cells by length (i.e., 1-5 µm and >5 µm) in terms of extraction purity (EP), extraction yield (EY), and enrichment factor (EF) and confirmed that the device could separate heterogeneous populations of bacteria using elasto-inertial effects.

Recent advances in magnetic digital microfluidic platforms.

Magnetic Digital microfluidics (DMF), which enables the manipulation of droplets containing different types of samples and reagents by permanent magnets or electromagnet arrays, has been used as a promising platform technology for bioanalytical and preparative assays. This is due to its unique advantages such as simple and "power free" operation, easy assembly, great compatibility with auto control systems, and dual functionality of magnetic particles (actuation and target attachment). Over the past decades, magnetic DMF technique has gained a widespread attention in many fields such as sample-to-answer molecular diagnostics, immunoassays, cell assays, on-demand chemical synthesis, and single-cell manipulation. In the first part of this review, we summarised features of magnetic DMF. Then, we introduced the actuation mechanisms and fabrication of magnetic DMF. Furthermore, we discussed five main applications of magnetic DMF, namely drug screening, protein assays, polymerase chain reaction (PCR), cell manipulation, and chemical analysis and synthesis. In the last part of the review, current challenges and limitations with magnetic DMF technique were discussed, such as biocompatibility, automation of microdroplet control systems, and microdroplet evaporation, with an eye on towards future development.

Rapid, Simple, and Inexpensive Spatial Patterning of Wettability in Microfluidic Devices for Double Emulsion Generation.

Water-in-oil-in-water (w/o/w) double emulsion (DE) encapsulation has been widely used as a promising platform technology for various applications in the fields of food, cosmetics, pharmacy, chemical engineering, materials science, and synthetic biology. Unfortunately, DEs formed by conventional emulsion generation approaches in most cases are highly polydisperse, making them less desirable for quantitative assays, controlled biomaterial synthesis, and entrapped ingredient release. Microfluidic devices can generate monodisperse DEs with controllable size, morphology, and production rate, but these generally require multistep fabrication processes and use of different solvents or bulky external instrumentation to pattern channel wettability. To overcome these limitations, we propose a rapid, simple, and inexpensive method to spatially pattern wettability in microfluidic devices for the continuous generation of monodisperse DEs. This is achieved by applying corona-plasma treatment to a select zone of the microchannel surface aided by a custom-designed corona resistance microchannel to strictly confine the plasma-treatment zone in a single polydimethylsiloxane (PDMS) microfluidic device. The properties of PDMS channel surfaces and key microchannel regions for DE generation are characterized under different levels of treatment. The size, shell thickness, and number of inner cores of generated DEs are shown to be highly controllable by tuning the phase flow rate ratios. Using DEs as templates, we successfully achieve a one-step generation and collection of gelatin microgels. Additionally, we demonstrate the biological capability of generated DEs by flow cytometric screening of the encapsulation and growth of yeast cells within DEs. We expect that the proposed approach will be widely used to create microfluidic devices with more complex wettability patterns.

Correction: Modular off-chip emulsion generator enabled by a revolving needle.

Correction for 'Modular off-chip emulsion generator enabled by a revolving needle' by Yuxin Zhang et al., Lab Chip, 2020, 20, 4592-4599, DOI: 10.1039/D0LC00939C.

Calcium-bisphosphonate Nanoparticle Platform as a Prolonged Nanodrug and Bone-Targeted Delivery System for Bone Diseases and Cancers.

Bone and bone-related diseases are the major cause of mobility hindrance and mortality in humans and there is no effective and safe treatment for most of them, especially, for bone and bone metastatic cancers. Bisphosphonates (BPs) are a group of small-molecule drugs for treating osteoporosis and bone cancers but have a very short half-life in circulation, requiring high doses and long-term repeat use that can cause severe side effects. Previous attempts of using nanoparticles to deliver BPs have issues of drug loading capacity and endosome escape/drug release. The present study reports the direct synthesis of BP nanoparticles by precipitating bone-favorable calcium ions and a third-generation BP, risedronate (Ca-RISNPs), to achieve high drug loading, endosomal release, and strong bone-targeting properties. The Ca-RISNPs are monodispersed with high stability at physiological pH but readily dissociate at endosomal pH conditions. They demonstrate strong penetration ability and uniform distribution in human bone and cartilage tissues and the superior drug and DNA (plasmid and oligo double strand DNA) delivery capacity in bone cells. These NPs also exhibit high specificity in killing tumor-associated macrophages (TAMs) and inhibit TAM-induced tumor cell migration. Collectively, our data indicate that this BP nanodrug platform has a great potential in managing bone-related diseases and cancers as a prolonged BP nanodrug and simultaneously as the bone-targeted drug delivery system.

Droplet flow cytometry for single-cell analysis.

The interrogation of single cells has revolutionised biology and medicine by providing crucial unparalleled insights into cell-to-cell heterogeneity. Flow cytometry (including fluorescence-activated cell sorting) is one of the most versatile and high-throughput approaches for single-cell analysis by detecting multiple fluorescence parameters of individual cells in aqueous suspension as they flow past through a focus of excitation lasers. However, this approach relies on the expression of cell surface and intracellular biomarkers, which inevitably lacks spatial and temporal phenotypes and activities of cells, such as secreted proteins, extracellular metabolite production, and proliferation. Droplet microfluidics has recently emerged as a powerful tool for the encapsulation and manipulation of thousands to millions of individual cells within pico-litre microdroplets. Integrating flow cytometry with microdroplet architectures surrounded by aqueous solutions (e.g., water-in-oil-in-water (W/O/W) double emulsion and hydrogel droplets) opens avenues for new cellular assays linking cell phenotypes to genotypes at the single-cell level. In this review, we discuss the capabilities and applications of droplet flow cytometry (DFC). This unique technique uses standard commercially available flow cytometry instruments to characterise or select individual microdroplets containing single cells of interest. We explore current challenges associated with DFC and present our visions for future development.

Separation and Enrichment of Yeast Saccharomyces cerevisiae by Shape Using Viscoelastic Microfluidics.

Yeast Saccharomyces cerevisiae (S. Cerevisiae) is one of the most attractive microbial species used for industrial production of value-added products and is an important model organism to understand the biology of the eukaryotic cells and humans. S. Cerevisiae has different shapes, such as spherical singlets, budded doublets, and clusters, corresponding to phases of the cell cycle, genetic, and environmental factors. The ability to obtain high-purity populations of uniform-shaped S. Cerevisiae cells is of significant importance for a wide range of applications in basic biological research and industrial processes. In this work, we demonstrate shape-based separation and enrichment of S. Cerevisiae using a coflow of viscoelastic and Newtonian fluids in a straight rectangular microchannel. Due to the combined effects of lift inertial and elastic forces, this label-free and continuous separation arises from shape-dependent migration of cells from the Newtonian to the non-Newtonian viscoelastic fluid. The lateral position of S. Cerevisiae cells with varying morphologies is found to be dependent on cell major axis. We also investigate the effects of sheath and sample flow rate, poly(ethylene oxide) (PEO) concentration and channel length on the performance of the viscoelastic microfluidic device for S. Cerevisiae enrichment and separation by shape. Moreover, the separation efficiency, cell extraction yield, and cell viability after sorting operations are studied.

Infliximab-based self-healing hydrogel composite scaffold enhances stem cell survival, engraftment, and function in rheumatoid arthritis treatment.

Rheumatoid arthritis (RA) is a severe inflammatory autoimmune disease, but its treatment has been very difficult. Recently, stem cell-based therapies have opened up possibilities for the treatment of RA. However, the hostile RA pathological conditions impede the survival and differentiation of transplanted cells, and it remains challenging to fabricate a suitable biomaterial for the improvement of stem cells survival, engraftment, and function. Here we construct an optimal scaffold for RA management through the integration of 3D printed porous metal scaffolds (3DPMS) and infliximab-based hydrogels. The presence of rigid 3DPMS is appropriate for repairing large-scale bone defects caused by RA, while the designed infliximab-based hydrogels are introduced because of their self-healable, anti-inflammatory, biocompatible, and biodegradable properties. We demonstrate that the bioengineered composite scaffolds support adipose-derived mesenchymal stem cells (ADSCs) proliferation, differentiation, and extracellular matrix production in vitro. The composite scaffolds, along with ADSCs, are then implanted into the critical-sized bone defect in the RA rabbit model. In vivo results prove that the bioengineered composite scaffolds are able to down-regulate inflammatory cytokines, rebuild damaged cartilage, as well as improve subchondral bone repair. To the best of the authors' knowledge, this is the first time that using the antirheumatic drug to construct hydrogels for stem cell-based therapies, and this inorganic-organic hybrid system has the potential to alter the landscape of RA study.

Microdroplet enabled cultivation of single yeast cells correlates with bulk growth and reveals subpopulation phenomena.

Yeast has been engineered for cost-effective organic acid production through metabolic engineering and synthetic biology techniques. However, cell growth assays in these processes were performed in bulk at the population level, thus obscuring the dynamics of rare single cells exhibiting beneficial traits. Here, we introduce the use of monodisperse picolitre droplets as bioreactors to cultivate yeast at the single-cell level. We investigated the effect of acid stress on growth and the effect of potassium ions on propionic acid tolerance for single yeast cells of different species, genotypes, and phenotypes. The results showed that the average growth of single yeast cells in microdroplets experiences the same trend to those of yeast populations grown in bulk, and microdroplet compartments do not significantly affect cell viability. This approach offers the prospect of detecting cell-to-cell variations in growth and physiology and is expected to be applied for the engineering of yeast to produce value-added bioproducts.

Modular off-chip emulsion generator enabled by a revolving needle.

Microfluidic chips have demonstrated unparalleled abilities in droplet generation, including precise control over droplet size and monodispersity. And yet, their rather complicated microfabrication process and operation can be a barrier for inexperienced researchers, which hinders microdroplets from unleashing their potential in broader fields of research. Here, we attempt to remove this barrier by developing an integrated and modular revolving needle emulsion generator (RNEG) to achieve high-throughput production of uniformly sized droplets in an off-chip manner. The RNEG works by driving a revolving needle to pinch the dispersed phase in a minicentrifuge tube. The system is constructed using modular components without involving any microfabrication, thereby enabling user-friendly operation. The RNEG is capable of producing microdroplets of various liquids with diameters ranging from tens to hundreds of micrometres. We further examine the principle of operation using numerical simulations and establish a simple model to predict the droplet size. Moreover, by integrating curing and centrifugation processes, the RNEG can produce hydrogel microparticles and transfer them from an oil phase into a water phase. Using this ability, we demonstrate the encapsulation and culture of single yeast cells within hydrogel microparticles. We envisage that the RNEG can become a versatile and powerful tool for high-throughput production of emulsions to facilitate diverse biological and chemical research.

Improving Single-Cell Encapsulation Efficiency and Reliability through Neutral Buoyancy of Suspension.

Single-cell analysis is of critical importance in revealing cell-to-cell heterogeneity by characterizing individual cells and identifying minority sub-populations of interest. Droplet-based microfluidics has been widely used in the past decade to achieve high-throughput single-cell analysis. However, to maximize the proportion of single-cell emulsification is challenging due to cell sedimentation and aggregation. The purpose of this study was to investigate the influence of single-cell encapsulation and incubation through the use of neutral buoyancy. As a proof of concept, OptiPrep™ was used to create neutrally buoyant cell suspensions of THP-1, a human monocytic leukemia cell line, for single-cell encapsulation and incubation. We found that using a neutrally buoyant suspension greatly increased the efficiency of single-cell encapsulation in microdroplets and eliminated unnecessary cell loss. Moreover, the presence of OptiPrep™ was shown to not affect cellular viability. This method significantly improved the effectiveness of single-cell study in a non-toxic environment and is expected to broadly facilitate single-cell analysis.

Molecular Dynamics of the Recruitment of Immunoreceptor Signaling Module DAP12 Homodimer to Lipid Raft Boundary Regulated by PIP2.

Lipid raft microdomain of the plasma membrane is implicated in various biological and pathological processes. The involvement of lipid raft in T cell receptor (TCR) signal transduction has been widely studied, whereas the role of these structures in immunoreceptor signaling by DAP12 in natural killing (NK) cells remains largely unknown. Here, we demonstrate that phosphatidylinositol 4,5-bisphosphate (PIP2) lipid localized to lipid raft boundary in our coarse-grained (CG) model raft-forming membrane, and this negatively charged lipid recruits DAP12 homodimer into lipid raft boundary through protein-lipid interaction between the basic-rich regions and signaling immunoreceptor tyrosine-based activation motifs (ITAMs) of DAP12 and PIP2. Furthermore, our results reveal that the protein-lipid interaction can be disrupted by Ca2+, which competitively binds to PIP2 instead of DAP12. As a result, the cytoplasmic region of DAP12 homodimer is dissociated from the membrane back to the nonraft domain, and the ITAMs are exposed to allow further downstream signaling. These findings provide fundamental insights to understand the mechanism of signal transduction in NK cells regulated by membrane microenvironment.

Multifunctional Flexible Sensor Based on Laser-Induced Graphene.

The paper presents the design and fabrication of a low-cost and easy-to-fabricate laser-induced graphene sensor together with its implementation for multi-sensing applications. Laser-irradiation of commercial polymer film was applied for photo-thermal generation of graphene. The graphene patterned in an interdigitated shape was transferred onto Kapton sticky tape to form the electrodes of a capacitive sensor. The functionality of the sensor was validated by employing them in electrochemical and strain-sensing scenarios. Impedance spectroscopy was applied to investigate the response of the sensor. For the electrochemical sensing, different concentrations of sodium sulfate were prepared, and the fabricated sensor was used to detect the concentration differences. For the strain sensing, the sensor was deployed for monitoring of human joint movements and tactile sensing. The promising sensing results validating the applicability of the fabricated sensor for multiple sensing purposes are presented.