Microfluidic particle counter visualizing mucosal antibodies against SARS-CoV-2 in the upper respiratory tract for rapid evaluation of immune protection.

Mucosal antibodies in the upper respiratory tract are the earliest and most critical responders to prevent respiratory infections, providing an indication for the rapid evaluation of immune protection. Here, we report a microfluidic particle counter that directly visualizes mucosal antibody levels in nasal mucus. The mucosal anti-SARS-CoV-2 spike receptor binding domain (RBD) antibodies in nasal secretions first react with magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that are surface-modified to form a "MMPs-anti-spike RBD IgG-PMPs" complex when RBD is present. After magnetic separation and loading into the microfluidic particle counter, the free PMPs, which are reduced with increasing anti-spike RBD IgG antibody levels, are trapped by a microfluidic particle dam and accumulate in the trapping channel. A sensitive mode [limit of detection (LOD): 14.0 ng mL-1; sample-to-answer time: 70 min] and an equipment-free rapid mode (LOD: 37.4 ng mL-1; sample-to-answer time: 20 min) were achieved. Eighty-seven nasal secretion (NS) samples from vaccinees were analyzed using our microfluidic particle counter, and the results closely resemble those of the gold-standard enzyme-linked immunosorbent assay (ELISA). The analysis shows that higher antibody levels were found in convalescent volunteers compared to noninfected volunteers. Together, we demonstrate a rapid kit that directly indicates immune status, which can guide vaccine strategy for individuals and the government.

Theranostic DNA nanostructure based on phenotype-specific activation of antisense oligonucleotides.

Antisense oligonucleotide (ASO) is a powerful agent for gene therapy, designed to form complementary pairs with specific mRNA to inhibit gene expression. However, low specificity limits its potential. To overcome this challenge, we developed a Y-shape DNA nanostructure that enhances the specificity in ASO-based treatment by introducing a detection trigger. The design incorporates the phenotype-specific miR21 activation and the sequential release of Bcl2 ASO. As a result, our Y-shape DNA nanostructure downregulates >50 % Bcl2 mRNA expression and induces >60 % cell death in breast cancer cells. Meanwhile, this approach shows no obvious damage to the non-cancerous cells, indicating the therapeutic potential as a theranostics agent in precision medicine with the combination of biomarker sensing and treatment. Overall, our Y-shape DNA nanostructure serves as a promising strategy providing potential in customized conformation design with specific target sequences in gene therapy.

Scalable pattern formation of skeletal myotubes by synergizing microtopographic cues and chiral nematics of cells.

Topographical cues have been widely used to facilitate cell fusion in skeletal muscle formation. However, an unexpected yet consistent chiral orientation of myotubes deviating from the groove boundaries is commonly observed but has long been unattended. In this study, we report a method to guide the formation of skeletal myotubes into scalable and controlled patterns. By inducing C2C12 myoblasts onto grooved patterns with different widths (from 0.4 to 200µm), we observed an enhanced chiral orientation of cells developing on wide grooves (50 and 100µm width) since the first day of induction. Active chiral nematics of cells involving cell migration and chiral rotation of the cell nucleus subsequently led to a unified chiral orientation of the myotubes. Importantly, these chiral myotubes were formed with enhanced length, diameter, and contractility on wide grooves. Treatment of latrunculin A (Lat A) suppressed the chiral rotation and migration of cells as well as the myotube formation, suggesting the essence of chiral nematics of cells for myogenesis. Finally, by arranging wide grooved/striped patterns with corresponding compensation angles to synergize microtopographic cues and chiral nematics of cells, intricate and scalable patterns of myotubes were formed, providing a strategy for engineering skeletal muscle tissue formation.

Multimodal detection of flap endonuclease 1 activity through CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides.

Flap endonuclease 1 (FEN1) is an endonuclease that specially removes 5' single-stranded overhang of branched duplex DNA (5' flap). While FEN1 is essential in various DNA metabolism pathways for preventing the malignant transformation of cells, an unusual expression of FEN1 is often associated with tumor progression, making it a potential biomarker for cancer diagnosis and treatment. Here we report a multimodal detection of FEN1 activity based on CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides (ssDNA). A dumbbell DNA structure with a 5' flap was designed, which can be cleaved by the FEN1 and the dumbbell DNA is subsequently ligated by T4 DNA ligase. The resulting closed duplex DNA contains a specific protospacer adjacent motif (PAM) that activates trans-cleavage of ssDNA after binding to CRISPR/Cas12a-crRNA. The trans-cleavage is activated only once and is independent to length or sequence of the ssDNA, which allows efficient signal amplification and multimodal signals such as fluorescence or cleaved connection between magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that alters solution turbidity after magnetic separation. In addition, by loading the particle solution into a microfluidic chip, unconnected PMPs escaping from a magnetic separator are amassed at the particle dam, enabling a visible PMP accumulation length proportional to the FEN1 activity. This multimodal detection is selective to FEN1 and achieves a low limit of detection (LOD) with only 40 min of reaction time. Applying to cell lysates, higher FEN1 activity was detected in breast cancer cells, suggesting a great potential for cancer diagnosis.

Detection of intracellular sodium ions based on phenotype-specific activation of NaA43 DNAzyme.

The intracellular sodium ion is one of the crucial elements for regulating physiological functions such as action potential and muscle contractions. However, detecting sodium ions in live cells is challenging because false signals may arise from the abundant sodium ions in the extracellular environment when introducing the detection agents. To minimize it, we report a DNAzyme-based detection of sodium ions in live cells via activation by endogenous mRNA. The substrate strand of DNAzyme first hybridizes to a blocking strand that prevents undesired cleavage of DNAzyme when delivered. Once entering cells, an endogenous mRNA biomarker binds to the toehold region of the blocking strand and displaces it, allowing the proper formation of the DNAzyme, which specifically catalyzes the cleavage of the substrate strand in the presence of intracellular Na+ and produces fluorescence signals. Using differentiating skeletal muscle cells as the model system, we demonstrated the successful delivery and phenotype-specific detection of intracellular sodium ions only in differentiated myotubes with highly-expressed myosin heavy chain mRNA. Moreover, using a drug cocktail to increase the permeability of the cell membrane, elevated levels of intracellular sodium ion was observed. This platform offers a broad and promising strategy for detecting intracellular metal ions, suggesting a great potential for understanding its role in cell/tissue physiology.

Microfluidic particle dam for direct visualization of SARS-CoV-2 antibody levels in COVID-19 vaccinees.

Various COVID-19 vaccines are currently deployed, but their immunization varies and decays with time. Antibody level is a potent correlate to immune protection, but its quantitation relies on intensive laboratory techniques. Here, we report a decentralized, instrument-free microfluidic device that directly visualizes SARS-CoV-2 antibody levels. Magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) can bind to SARS-CoV-2 antibodies simultaneously. In a microfluidic chip, this binding reduces the incidence of free PMPs escaping from magnetic separation and shortens PMP accumulation length at a particle dam. This visual quantitative result enables use in either sensitive mode [limit of detection (LOD): 13.3 ng/ml; sample-to-answer time: 70 min] or rapid mode (LOD: 57.8 ng/ml; sample-to-answer time: 20 min) and closely agrees with the gold standard enzyme-linked immunosorbent assay. Trials on 91 vaccinees revealed higher antibody levels in mRNA vaccinees than in inactivated vaccinees and their decay in 45 days, demonstrating the need for point-of-care devices to monitor immune protection.

Portable microfluidic device with thermometer-like display for real-time visual quantitation of Cadmium(II) contamination in drinking water.

Cadmium (Cd2+) is a toxic metal ion widely existing in water, soil and food. Conventional water quality control heavily relies on expensive, bulky and sophisticated instrument such as spectrometry, which is time-consuming and incompatible with on-site, real-time detection. Here, a portable microfluidic device with thermometer-like visual readouts is developed for real-time quantitation of cadmium (II) contamination in drinking water. We use Cd2+-dependent DNAzyme (Cd16), which is cleaved when Cd2+ is present, creating a single strand DNA which triggers catalytic hairpin assembly (CHA) with two hairpins H1 and H2 as the building blocks. Plenty of H1H2 complex, the product after the Cd2+-mediated CHA, are generated, which can connect magnetic microparticles (MMPs) and polystyrene microparticles (PMPs), forming "MMPs-H1H2-PMPs" sandwich structure. To provide visual readout to quantitate the particle connection, the particle solution is loaded into a portable microfluidic chip. A magnetic separator first removes MMPs and the connected PMPs, while free PMPs can continue flowing until accumulating into a bar at the particle dam. Shown as a thermometer-like display, the accumulating length is inversely proportional to the concentration of Cd2+, enabling quantitative detection of Cd2+ by the naked eye. The proposed device exhibits a limit of detection of 11.3 nM of Cd2+, selectivity >200-fold against other metal ions, high tolerance to the interferents present in drinking water and high recovery rate in tap water. With high analytical performance without any sample preparation step, this portable device is highly promising in real-time monitoring in urban drinking water at sites.

Early Committed Clockwise Cell Chirality Upregulates Adipogenic Differentiation of Mesenchymal Stem Cells.

Cell chirality is observed with diverse forms and coordinates various left-right (LR) asymmetry in tissue morphogenesis. To give rise to such diversity, cell chirality may be coupled with cell differentiation. Here, using micropatterned human mesenchymal stem cells (hMSCs), an early committed clockwise (CW) cell chirality that can itself upregulate the adipogenic differentiation is reported. hMSC chirality enables a positively tilted chiral orientation on micropatterned stripes. When cultured as single cells on circular micropatterns, an anticlockwise (ACW)-biased nucleus rotation and swirling pattern of actin filament are observed. Interestingly, with adipogenic induction for 3-6 days, such chirality is reversed to negative chiral orientation and CW-biased rotation, which is earlier than the maturation of other differentiation markers, and consistently expressed in terminally differentiated adipocytes. Using latrunculin A (LatA), cytochalasin D (CD), and nocodazole (Noco) that forces a CW-biased actin filament and nucleus rotation resembling the early differentiated chirality upon adipogenic induction, an upregulation of adipogenic differentiation is found. The result demonstrates that the early differentiated chirality may serve as a mechanical precursor to engage the lineage commitment, suggesting a feedback mechanism of chiral actin in regulating cell differentiation and LR morphogenesis.

Microfluidic Particle Dam for Visual and Quantitative Detection of Lead Ions.

Lead contamination in drinking water is a primary concern in public health, but it is difficult to monitor by end-users. Here, we provide a rapid and power-free microfluidic particle dam which enables visual quantification of lead ions (Pb2+) by the naked eye. GR-5 DNAzyme with extended termini can connect magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) by DNA hybridization, forming "MMPs-GR-5-PMPs". When Pb2+ is present, GR-5 is cleaved, resulting in an increasing number of free PMPs. To visually count the free PMPs, the solution is loaded to a capillary-driven microfluidic device that consists of a magnetic separator to remove the MMPs-GR-5-PMPs, followed by a particle dam that traps and accumulates the free PMPs into a visual bar with growing length proportional to the concentration of lead. The device achieved a limit of detection at 2.12 nM (0.44 ppb), high selectivity (>20,000-fold) against other metal ions, high tolerance to different pH and water hardness, and is compatible with tap water with a high recovery rate, enabling visual quantification and user-friendly interface for rapid screening of water safety.

Microfluidic bead trap as a visual bar for quantitative detection of oligonucleotides.

We demonstrate a microfluidic bead trap capable of forming a dipstick-type bar visible to the naked eye for simple and quantitative detection of oligonucleotides. We use magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that are connected and form MMPs-targets-PMPs when target oligonucleotides are present, leaving free PMPs with a number inversely proportional to the amount of targets. Using a capillary flow-driven microfluidic circuitry consisting of a magnetic separator to remove the MMPs-targets-PMPs, the free PMPs can be trapped at the narrowing nozzle downstream, forming a visual bar quantifiable based on the length of PMP accumulation. Such a power-free and instrument-free platform enables a limit of detection at 13 fmol (0.65 nM in 20 µl, S/N = 3) of oligonucleotides and is compatible with single-nucleotide polymorphisms and operation in a complex bio-fluid. Moreover, using DNAzyme as the target oligonucleotide that catalyzes a specific hydrolytic cleavage in the presence of lead ions, we demonstrate a model application that detects lead ions with a limit of detection of 12.2 nM (2.5 µg l-1), providing quantitative and visual detection of lead contamination at resource-limited sites.

Enzyme-Free Amplification by Nano Sticky Balls for Visual Detection of ssDNA/RNA Oligonucleotides.

Visual detection of nucleic acids provides simple and rapid screening for infectious diseases or environmental pathogens. However, sensitivity is the current bottleneck, which may require enzymatic amplification for targets in low abundance and make them incompatible with detection at resource-limited sites. Here we report an enzyme-free amplification that provides a sensitive visual detection of ssDNA/RNA oligonucleotides on the basis of nano "sticky balls". When target oligonucleotides are present, magnetic microparticles (MMPs) and gold nanoparticles (AuNPs) were linked together, allowing the collection of AuNPs after magnetic attraction. Subsequently, the collected AuNPs, which carry many oligonucleotides, were used as the sticky balls to link a second pair of MMPs and polymer microparticles (PMPs). Thus, because the magnetic field can attract the MMPs as well as the linked PMPs to the sidewall, the reduction of suspended PMPs yields a change of light transmission visible by the naked eye. Our results demonstrate that the limit of detection is 10 amol for ssDNAs (228 fM in 45 µL) and 75 amol for ssRNAs (1.67 pM in 45 µL). This method is also compatible with the serum environment and detection of a microRNA, miR-155, derived from human breast cancer cells. With significantly improved sensitivity for visual detection, it provides great potential for point-of-care applications at resource-limited sites.