Ultrasensitive Electrochemical Immunosensor for Multiplex Sandwich Bioassaying Based on the Functional Antibodies.

In vitro diagnostics (IVDs) based on electrochemical immunosensors are crucial for disease screening, diagnosis, prognosis, and treatment monitoring. However, label-free electrochemical immunosensors commonly suffer from poor specificity, leading to false positives. To address this issue, we propose a highly sensitive and precise electrochemical immunosensor for protein marker detection. This approach involves directly labeling the detection antibodies (Ab2) with thionine (Thi). The Ab2 labeled by Thi exhibits a distinct redox peak upon targeted voltage stimulation, enabling accurate quantification of protein biomarkers. Thi-modified antibodies provide significant advantages over traditional antibody modification methods, such as enhanced detection sensitivity, improved accuracy, and specificity in protein marker identification. The method is straightforward and efficient, ensuring specific analyte detection while minimizing interference from other substances in the sample. Additionally, a multielectrode detection method was employed, achieving remarkably low limits of detection (LoDs) for tumor necrosis factor-alpha (TNF-alpha), cardiac troponin I (cTnI), and interleukin-6 (IL-6), with LoDs of 9.38 fg/mL, 1.70 fg/mL, and 8.14 fg/mL, respectively. The proposed electrochemical immunosensor also exhibited high selectivity and repeatability, with relative standard deviations (RSD) of 6.39% for TNF-alpha, 2.42% for cTnI, and 2.72% for IL-6 (n = 5). Moreover, it demonstrated high sensitivity and was evaluated for serum detection using the standard addition method. The results highlight the great potential of the proposed electrochemical immunosensor for clinical applications, offering a novel approach for future utilization in clinical settings.

Automatic ultrasensitive lateral flow immunoassay based on a color-enhanced signal amplification strategy.

Lateral flow immunoassays (LFIAs) are an essential and widely used point-of-care test for medical diagnoses. However, commercial LFIAs still have low sensitivity and specificity. Therefore, we developed an automatic ultrasensitive dual-color enhanced LFIA (DCE-LFIA) by applying an enzyme-induced tyramide signal amplification method to a double-antibody sandwich LFIA for antigen detection. The DCE-LFIA first specifically captured horseradish peroxidase (HRP)-labeled colored microspheres at the Test line, and then deposited a large amount of tyramide-modified signals under the catalytic action of HRP to achieve the color superposition. A limit of detection (LOD) of 3.9 pg/mL and a naked-eye cut-off limit of 7.8 pg/mL were achieved for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein. Additionally, in the inactivated virus detections, LOD equivalent to chemiluminescence (0.018 TCID50/mL) was obtained, and it had excellent specificity under the interference of other respiratory viruses. High sensitivity has also been achieved for detection of influenza A, influenza B, cardiac troponin I, and human chorionic gonadotrophin using this DCE-LFIA, suggesting the assay is universally applicable. To ensure the convenience and stability in practical applications, we created an automatic device. It provides a new practical option for point-of-care test immunoassays, especially ultra trace detection and at-home testing.

Discovery of Trametinib as an orchestrator for cytoskeletal vimentin remodeling.

The dynamic remodeling of the cytoskeletal network of vimentin intermediate filaments network supports various cellular functions, including cell morphology, elasticity, migration, organelle localization, and resistance against mechanical or pathological stress. Currently available chemicals targeting vimentin predominantly induce network reorganization and shrinkage around the nucleus. Effective tools for long-term manipulation of vimentin network dispersion in living cells are still lacking, limiting in-depth studies on vimentin function and potential therapeutic applications. Here, we verified that a commercially available small molecule, Trametinib, is capable of inducing spatial spreading of the cellular vimentin network without affecting its transcriptional or translational regulation. Further evidence confirmed its low cytotoxicity and similar effects on different cell types. Importantly, Trametinib has no impact on the other two cytoskeletal systems, actin filaments and the microtubule network. Moreover, Trametinib regulates vimentin network dispersion rapidly and efficiently, with effects persisting for up to 48 h after drug withdrawal. We also ruled out the possibility that Trametinib directly affects the phosphorylation level of vimentin. In summary, we identified an unprecedented regulator, Trametinib, capable of spreading the vimentin network toward the cell periphery, and thus complemented the existing repertoire of vimentin remodeling drugs in the field of cytoskeletal research.

Rapid droplet-based mixing for single-molecule spectroscopy.

Probing non-equilibrium dynamics with single-molecule spectroscopy is important for dissecting biomolecular mechanisms. However, existing microfluidic rapid-mixing systems for this purpose are incompatible with surface-adhesive biomolecules, exhibit undesirable flow dispersion and are often demanding to fabricate. Here we introduce droplet-based microfluidic mixing for single-molecule spectroscopy to overcome these limitations in a wide range of applications. We demonstrate its robust functionality with binding kinetics of even very surface-adhesive proteins on the millisecond timescale.

Towards an active droplet-based microfluidic platform for programmable fluid handling.

Droplet-based microfluidic systems have emerged as powerful alternatives to conventional high throughput screening platforms, due to their operational flexibility, high-throughput nature and ability to efficiently process small fluid volumes. However, the challenges associated with performing bespoke operations on user-defined droplets often limit their utility in screening applications that involve complex workflows. To this end, the marriage of droplet- and valve-based microfluidic technologies offers the prospect of balancing the controllability of droplet manipulations and analytical throughput. In this spirit, we present a microfluidic platform that combines the capabilities of integrated microvalve technology with droplet-based sample compartmentalization to realize a highly adaptable programmable fluid handling functionality. The microfluidic device consists of a programmable formulator linked to an automated droplet generation device and storage array. The formulator leverages multiple inputs coupled to a mixing ring to produce combinatorial solution mixtures, with a peristaltic pump enabling titration of reagents into the ring with picoliter resolution. The platform allows for the execution of user-defined reaction protocols within an array of storage chambers by consecutively merging programmable sequences of pL-volume droplets containing specified reagents. The precision in formulating solutions with small differences in concentration is perfectly suited for the accurate estimation of kinetic parameters. The utility of our platform is showcased through the performance of enzymatic kinetic measurements of beta-galactosidase and horseradish peroxidase with fluorogenic substrates. The presented platform provides for a range of automated manipulations and paves the way for a more diverse range of droplet-based biological experiments.

Microfluid Switching-Induced Transient Refractive Interface.

The laminar flow interface (LFI) developed at low Reynolds numbers is one of the most prominent features of microscale flows and has been employed in a diverse range of optofluidic applications. The formation of LFIs usually requires the manipulation of multiple streams within a microchannel using a complex hydrodynamic pumping system. Herein, we present a new type of LFI that is generated by fluid switching within a three-dimensional (3D) microlens-incorporating microfluidic chip (3D-MIMC). Since Poiseuille flows exhibit a parabolic velocity profile, the LFI is cone-like in shape and acts as a transient refractive interface (TRI), which is sensitive to the refractive index (RI) and the Péclet number (Pe) of the switching fluids. In response to the TRI, the intensity of the transmitted light can be intensified or attenuated depending on the sequence of fluid switching operations. By incorporating three-dimensional (3D) microlenses and increasing the Pe values, the profile and amplitude of the intensity peak are both significantly improved. The limit of detection (LoD) for a sodium chloride (NaCl) solution at Pe = 1363 is as low as 0.001% (w/w), representing an improvement of 1-2 orders of magnitude when compared to existing optofluidic concentration sensors based on intensity modulation. Fluid switching of a variety of inorganic and organic sample fluids confirms that the specific optical response (Kor) correlates positively with both Pe and the specific RI (Knc), obeying a linear relationship. This model is further verified through cross-validations and used to estimate the molecular diffusion coefficient (D) of a range of species. Furthermore, by virtue of the TRI, we achieve a sensitive measurement of optical-equivalent total dissolved solids (OE-TDS) for environmental samples.

PDMS-PUA bi-directional replication technology and its applications.

Polydimethylsiloxane (PDMS) and polyurethane acrylate (PUA) are excellent pattern transfer materials. In this study, PDMS-PUA bi-directional replication technology is explored using the PDMS grating as a template, and relevant technical issues are discussed in detail. Special surface treatment and process optimization are applied to solve the problems of demolding, PDMS polymerization inhibition, and substrate flatness. Further experiments show that the technology can be employed to replicate nanoscale structures and has the potential value of prolonging the longevity of the original template. Additionally, utilizing the advantage of the high elasticity of PDMS materials, two applications of bi-directional replication technology are demonstrated. One is to increase the line-density of the grating by stretching, and the experimental results show that the line-density of the grating increased by 26.6%. The other one is to fabricate the convex grating. Compared with the original planar PDMS grating, the resolution of the first-order diffraction spectrum of the convex grating at the focal point has been greatly improved. Since this technology requires simple equipment, and PDMS and PUA are reusable, it has the advantages of low cost, simplicity, and rapid fabrication. The two application examples also indicate that the technology has good application value.

DropCRISPR: A LAMP-Cas12a based digital method for ultrasensitive detection of nucleic acid.

Since their discovery, CRISPR/Cas systems have been extensively exploited in nucleic acid biosensing. However, the vast majority of contemporary platforms offer only qualitative detection of nucleic acid, and fail to realize ultrasensitive quantitative detection. Herein, we report a digital droplet-based platform (DropCRISPR), which combines loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a to realize ultrasensitive and quantitative detection of nucleic acids. This is achieved through a novel two-step microfluidic system which combines droplet LAMP with a picoinjector capable of injecting the required CRISPR/Cas12a reagents into each droplet. This method circumvents the temperature incompatibilities of LAMP and CRISPR/Cas12a and avoids mutual interference between amplification reaction and CRISPR detection. Ultrasensitive detection (at fM level) was achieved for a model plasmid containing the invA gene of Salmonella typhimurium (St), with detection down to 102 cfu/mL being achieved in pure bacterial culture. Additionally, we demonstrate that the DropCRISPR platform is capable of detecting St in raw milk samples without additional nucleic acid extraction. The sensitivity and robustness of the DropCRISPR further demonstrates the potential of CRISPR/Cas-based diagnostic platforms, particularly when combined with state-of-the-art microfluidic architectures.

Multi-compartment supracapsules made from nano-containers towards programmable release.

The assembly of nanomaterials into suprastructures offers the possibility to fabricate larger scale functional materials, whose inner structure strongly influences their functionality for a vast range of applications. In spite of the many current strategies, achieving multi-compartment structures in a targeted and versatile way remains highly challenging. Here, we describe a controllable and straightforward route to create uniform suprastructured materials with a multi-compartmentalized architecture by confining primary nanocapsules into droplets using a cross-junction microfluidic device. Following solvent evaporation from the droplets, the nanocapsules spontaneously assemble into precisely sized multi-compartment particles, which we term supracapsules. Thanks to the process, each spatially separated nanocapsule unit retains its cargo and functionalities within the resulting supracapsules. However, new collective properties emerge, and, particularly, programmable release profiles that are distinct from those of single-compartment capsules. Finally, the suprastructures can be disassembled into single-compartment units by applying ultra-sonication, switching their release to a burst-release mode. These findings open up exciting opportunities to fabricate multi-compartment suprastructures incorporating diverse functionalities for materials with emerging properties.

Replicating the Cynandra opis Butterfly's Structural Color for Bioinspired Bigrating Color Filters.

Multilayer grating structures, such as those found on the wings of the butterfly Cynandra opis, are able to interact with light to generate structural coloration. When illuminated and viewed at defined angles, such structural color is characterized by exceptional purity and brightness. To provide further insight into the mechanism of structural coloration, two-photon laser lithography is used to fabricate bioinspired bigrating nanostructures, whose optical properties may be controlled by variation of the height and period of the grating features. Through the use of both spectral measurements and finite-element method simulations, herein specific feature dimensions are identified that due to the combined effects of multilayer interference and diffraction generate excellent spectral characteristics and high color purity over the entire visible range. Additionally, it is demonstrated that variation of feature period and/or height plays a central role in controlling both hue and purity. Importantly, such tuneable bigrating structures are of significant utility in color filtering applications.

Flexibility-Patterned Liquid-Repelling Surfaces.

Droplets impacting solid surfaces is ubiquitous in nature and of practical importance in numerous industrial applications. For liquid-repelling applications, rigidity-based asymmetric redistribution and flexibility-based structural oscillation strategies have been proven on artificial surfaces; however, these are limited by strict impacting positioning. Here, we show that the gap between these two strategies can be bridged by a flexibility-patterned design similar to a trampoline park. Such a flexibility-patterned design is realized by three-dimensional projection micro-stereolithography and is shown to enhance liquid repellency in terms of droplet impalement resistance and contact time reduction. This is the first demonstration of the synergistic effect obtained by a hybrid solution that exploits asymmetric redistribution and structural oscillation in liquid-repelling applications, paving the rigidity-flexibility cooperative way of wettability tuning. Also, the flexibility-patterned surface is applied to accelerate liquid evaporation.

Biomimetic Water-Repelling Surfaces with Robustly Flexible Structures.

Biomimetic liquid-repelling surfaces have been the subject of considerable scientific research and technological application. To design such surfaces, a flexibility-based oscillation strategy has been shown to resolve the problem of liquid-surface positioning encountered by the previous, rigidity-based asymmetry strategy; however, its usage is limited by weak mechanical robustness and confined repellency enhancement. Here, we design a flexible surface comprising mesoscale heads and microscale spring sets, in analogy to the mushroomlike geometry discovered on springtail cuticles, and then realize this through three-dimensional projection microstereolithography. Such a surface exhibits strong mechanical robustness against ubiquitous normal and shear compression and even endures tribological friction. Simultaneously, the surface elevates water repellency for impacting droplets by enhancing impalement resistance and reducing contact time, partially reaching an improvement of ∼80% via structural tilting movements. This is the first demonstration of flexible interfacial structures to robustly endure tribological friction as well as to promote water repellency, approaching real-world applications of water repelling. Also, a flexibility gradient is created on the surface to directionally manipulate droplets, paving the way for droplet transport.

Optical-Switch-Enabled Microfluidics for Sensitive Multichannel Colorimetric Analysis.

The implementation of colorimetric analysis within microfluidic environments engenders significant benefits with respect to reduced sample and reagent consumption, system miniaturization, and real-time measurement of flowing samples. That said, conventional approaches to colorimetric analysis within microfluidic channels are hampered by short optical pathlengths and single-channel configurations, which lead to poor detection sensitivities and low analytical throughputs. Although the use of multiplexed light source/photodetector modules allows for multichannel analysis, such configurations significantly increase both instrument complexity and cost. To address these issues, we present a four-channel colorimetric measurement scheme within an optical-switch-enabled microfluidic chip (OSEMC) fabricated by two-photon stereolithography. The integration of optical switches enables sequential signal readout from each detection channel, and thus, only a single light source and a photodetector are required for operation. Optical switches can be controlled in a bespoke manner by changing the medium in the switch channel between a "light-transmitting" fluid and a "light-blocking" fluid using pneumatic microvalves. Such optical switches are characterized by fast response times (approximately 200 ms), tunable switching frequencies (between 0.1 and 1.0 Hz studied), and excellent stability. Operational performance demonstrates both good sensitivity and reproducibility through the colorimetric analysis of nitrite and ammonium samples using four detection channels. Furthermore, the use of OSEMC for parallel and real-time analysis of flowing samples is investigated via characterization of the adsorption kinetics of tartrazine on activated charcoal and the catalytic reaction kinetics of horseradish peroxidase (HRP).

In Situ Visualization of the Structural Evolution and Alignment of Lyotropic Liquid Crystals in Confined Flow.

Self-assembled materials such as lyotropic liquid crystals offer a wide variety of structures and applications by tuning the composition. Understanding materials behavior under flow and the induced alignment is wanted in order to tailor structure related properties. A method to visualize the structure and anisotropy of ordered systems in situ under dynamic conditions is presented where flow-induced nanostructural alignment in microfluidic channels is observed by scanning small angle X-ray scattering in hexagonal and lamellar self-assembled phases. In the hexagonal phase, the material in regions with high extensional flow exhibits orientation perpendicular to the flow and is oriented in the flow direction only in regions with a high enough shear rate. For the lamellar phase, a flow-induced morphological transition occurs from aligned lamellae toward multilamellar vesicles. However, the vesicles do not withstand the mechanical forces and break in extended lamellae in regions with high shear rates. This evolution of nanostructure with different shear rates can be correlated with a shear thinning viscosity curve with different slopes. The results demonstrate new fundamental knowledge about the structuring of liquid crystals under flow. The methodology widens the quantitative investigation of complex structures and identifies important mechanisms of reorientation and structural changes.

Laminar Flow-Based Fiber Fabrication and Encoding via Two-Photon Lithography.

In recent years, flow photolithography (FL) has emerged as a powerful synthetic tool for the creation of barcoded microparticles with complex morphologies and chemical compositions which have been shown to be useful in a range of multiplexed bioassay applications. More specifically, FL has been highly successful in producing micron-sized, encoded particles of bespoke shape, size, and color. That said, to date, FL has been restricted to generating barcoded microparticles and has lacked the ability to produce hybrid fibers which are structurally and spectrally encoded. To this end, we herein present a method that combines a continuous flow microfluidic system with two-photon polymerization (2PP) to fabricate microscale-encoded fibers and Janus strips in a high-throughput manner. Specifically, two co-flow liquid streams containing a monomer and initiator are introduced through a Y-shape channel to form a stable interface in the center of a microfluidic channel. The flow containing the (fluorescently labeled) monomer is then patterned by scanning the voxel of the 2PP laser across the interface to selectively polymerize different regions of the forming fiber/particle. Such a process allows for rapid spectral encoding at the single fiber level, with the resulting structurally coded fibers having obvious application in the fields of security identification and anticounterfeiting.

Liquid repellency enhancement through flexible microstructures.

Artificial liquid-repellent surfaces have attracted substantial scientific and industrial attention with a focus on creating functional topological features; however, the role of the underlying structures has been overlooked. Recent developments in micro-nanofabrication allow us now to construct a skin-muscle type system combining interfacial liquid repellence atop a mechanically functional structure. Specifically, we design surfaces comprising bioinspired, mushroom-like repelling heads and spring-like flexible supports, which are realized by three-dimensional direct laser lithography. The flexible supports elevate liquid repellency by resisting droplet impalement and reducing contact time. This, previously unknown, use of spring-like flexible supports to enhance liquid repellency provides an excellent level of control over droplet manipulation. Moreover, this extends repellent microstructure research from statics to dynamics and is envisioned to yield functionalities and possibilities by linking functional surfaces and mechanical metamaterials.

Self-aligned 3D microlenses in a chip fabricated with two-photon stereolithography for highly sensitive absorbance measurement.

Absorbance measurement is a widely used method to quantify the concentration of an analyte. The integration of absorbance analysis in microfluidic chips could significantly reduce the sample consumption and contribute to the system miniaturization. However, the sensitivity and limit of detection (LoD) of analysis in microfluidic chips with conventional configuration need improvements due to the limited optical pathway and unregulated light propagation. In this work, a 3D-microlens-incorporating microfluidic chip (3D-MIMC) with a greatly extended detection channel was innovatively fabricated using two-photon stereolithography. The fabrication was optimized with a proposed hierarchical modular printing strategy. Due to the incorporation of 3D microlenses, the light coupling efficiency and the signal-to-noise ratio (SNR) were respectively improved approximately 9 and 4 times. An equivalent optical path length (EOL) of 62.9 mm was achieved in a 3.7 µl detection channel for testing tartrazine samples. As a result, the sensitivity and LoD of the 3D-MIMC assay were correspondingly improved by one order of magnitude, compared with those of the 96-well plate assay. Notably, the 3D-MIMC has the potential to be integrated into a general microanalysis platform for multiple applications.

A Counter Propagating Lens-Mirror System for Ultrahigh Throughput Single Droplet Detection.

Fluorescence-based detection schemes provide for multiparameter analysis in a broad range of applications in the chemical and biological sciences. Toward the realization of fully portable analysis systems, microfluidic devices integrating diverse functional components have been implemented in a range of out-of-lab environments. That said, there still exits an unmet and recognized need for miniaturized, low-cost, and sensitive optical detection systems, which provide not only for efficient molecular excitation, but also enhanced photon collection capabilities. To this end, an optofluidic platform that is adept at enhancing fluorescence light collection from microfluidic channels is presented. The central component of the detection module is a monolithic parabolic mirror located directly above the microfluidic channel, which acts to enhance the number of emitted photons reflected toward the detector. In addition, two-photon polymerization is used to print a microscale-lens below the microfluidic flow channel and directly opposite the mirror, to enhance the delivery of excitation radiation into the channel. Using such an approach, it is demonstrated that fluorescence signals can be enhanced by over two orders of magnitude, with component parallelization enabling the detection of pL-volume droplets at rates up to 40 000 droplets per second.

Growing and Shaping Metal-Organic Framework Single Crystals at the Millimeter Scale.

Controlling and understanding the mechanisms that harness crystallization processes is of utmost importance in contemporary materials science and, in particular, in the realm of reticular solids where it still remains a great challenge. In this work, we show that environments mimicking microgravity conditions can harness the size and shape of functional biogenic crystals such as peptide-based metal-organic frameworks (MOFs). In particular, we demonstrate formation of the largest single crystals with controlled nonequilibrium shapes of peptide-based MOFs reported to date (e.g., those featuring curved crystal habits), as opposed to the typical polyhedral microcrystals obtained under bulk crystallization conditions. Such unique nonequilibrium morphologies arise from the interplay between the diffusion-controlled supply of precursors in simulated microgravity environments and the physical constraints imposed during crystal growth. In fact, our method mimics two main strategies of morphogenesis in biomineralization, i.e., spatial and morphological control, both being largely unexplored in the field of self-assembled functional materials. The presented results may open new opportunities to study and understand fundamental questions of relevance to materials science, such as how the size and shape of artificial crystals can influence their properties and functions while providing a strategy to tailor the size and shape of peptide-based MOF single crystals to specific applications.

Self-Compensating Liquid-Repellent Surfaces with Stratified Morphology.

Artificial liquid-repellent surfaces have recently attracted vast scientific attention; however, achieving mechanical robustness remains a formidable challenge before industrialization can be realized. To this end, inspired by plateaus in geological landscapes, a self-compensating strategy is developed to pave the way for the synthesis of durable repellent surfaces. This self-compensating surface comprises tall hydrophobic structural elements, which can repel liquid droplets. When these elements are damaged, they expose shorter structural elements that also suspend the droplets and thus preserve interfacial repellency. An example of this plateau-inspired stratified surface was created by three-dimensional (3D) direct laser lithography micro-nano fabrication. Even after being subjected to serious frictional damage, it maintained static repellency to water with a contact angle above 147° and was simultaneously able to endure high pressures arising from droplet impacts. Extending the scope of nature-inspired functional surfaces from conventional biomimetics to geological landscapes, this work demonstrates that the plateau-inspired self-compensating strategy can provide an unprecedented level of robustness in terms of sustained liquid repellency.