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.

Enhancing the sensitivity and stability of electrochemical aptamer-based sensors by AuNPs@MXene nanocomposite for continuous monitoring of biomarkers.

Electrochemical aptamer-based (E-AB) sensors offer exciting potential for real-time tracking of various biomarkers, such as proteins and small molecules, due to their exceptional selectivity and adaptability. However, most E-AB sensors rely on planar gold structures, which inherently limit their sensitivity and operational stability for continuous monitoring of biomarkers. Although gold nanostructures have recently enhanced E-AB sensor performance, no studies have explored the combination of gold nanostructure with other types of nanomaterials for continuous molecular monitoring. To fill this gap, we employed gold nanoparticles and MXene Ti3C2 (AuNPs@MXene), a versatile nanocomposite, in designing an E-AB sensor targeted at vascular endothelial growth factor (VEGF), a crucial human signaling protein. Remarkably, the AuNPs@MXene nanocomposite achieved over thirty-fold and half-fold increases in active surface area compared to bare and AuNPs-modified gold electrodes, respectively, significantly elevating the analytical capabilities of E-AB sensors during continuous operation. After a systematic optimization and characterization process, the newly developed E-AB sensor, powered by AuNPs@MXene nanocomposite, demonstrated both enhanced stability and heightened sensitivity. Overall, our findings open new avenues for the incorporation of nanocomposites in E-AB sensor design, enabling the creation of more sensitive and durable real-time monitoring systems.

Microfluidic flow cytometry for blood-based biomarker analysis.

Flow cytometry has proven its capability for rapid and quantitative analysis of individual cells and the separation of targeted biological samples from others. The emerging microfluidics technology makes it possible to develop portable microfluidic diagnostic devices for point-of-care testing (POCT) applications. Microfluidic flow cytometry (MFCM), where flow cytometry and microfluidics are combined to achieve similar or even superior functionalities on microfluidic chips, provides a powerful single-cell characterisation and sorting tool for various biological samples. In recent years, researchers have made great progress in the development of the MFCM including focusing, detecting, and sorting subsystems, and its unique capabilities have been demonstrated in various biological applications. Moreover, liquid biopsy using blood can provide various physiological and pathological information. Thus, biomarkers from blood are regarded as meaningful circulating transporters of signal molecules or particles and have great potential to be used as non (or minimally)-invasive diagnostic tools. In this review, we summarise the recent progress of the key subsystems for MFCM and its achievements in blood-based biomarker analysis. Finally, foresight is offered to highlight the research challenges faced by MFCM in expanding into blood-based POCT applications, potentially yielding commercialisation opportunities.

Gallium-Based Liquid Metal Particles for Therapeutics.

Gallium (Ga) and Ga-based liquid metal (LM) alloys offer low toxicity, excellent electrical and thermal conductivities, and fluidity at or near room temperature. Ga-based LM particles (LMPs) synthesized from these LMs exhibit both fluidic and metallic properties and are suitable for versatile functionalization in therapeutics. Functionalized Ga-based LMPs can be actuated using physical or chemical stimuli for drug delivery, cancer treatment, bioimaging, and biosensing. However, many of the fundamentals of their unique characteristics for therapeutics remain underexplored. We present the most recent advances in Ga-based LMPs in therapeutics based on the underlying mechanisms of their design and implementation. We also highlight some future biotechnological opportunities for Ga-based LMPs based on their extraordinary advantages.

Biomedical Applications of Liquid Metal Nanoparticles: A Critical Review.

This review is focused on the basic properties, production, functionalization, cytotoxicity, and biomedical applications of liquid metal nanoparticles (LMNPs), with a focus on particles of the size ranging from tens to hundreds of nanometers. Applications, including cancer therapy, medical imaging, and pathogen treatment are discussed. LMNPs share similar properties to other metals, such as photothermal conversion ability and a propensity to form surface oxides. Compared to many other metals, especially mercury, the cytotoxicity of gallium is low and is considered by many reports to be safe when applied in vivo. Recent advances in exploring different grafting molecules are reported herein, as surface functionalization is essential to enhance photothermal therapeutic effects of LMNPs or to facilitate drug delivery. This review also outlines properties of LMNPs that can be exploited in making medical imaging contrast agents, ion channel regulators, and anti-pathogenic agents. Finally, a foresight is offered, exemplifying underexplored knowledge and highlighting the research challenges faced by LMNP science and technology in expanding into applications potentially yielding clinical advances.

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells.

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.