Ultra-low intensity light pulses for large cargo delivery into hard-to-transfect cells using an rGO mixed PDMS microtip device.

Nanoparticle-mediated photoporation has arisen as a universal intracellular delivery tool; however, the direct interaction of nanoparticles and cells hampers its clinical translation. Here, we report a uniform contactless intracellular delivery that transfects a large number of cells within a minute and avoids direct contact of nanoparticles and cells, thereby improving the cell viability. Our platform consists of an array of polydimethylsiloxane (PDMS) mixed reduced graphene oxide (rGO) nanoflakes on pyramidal microtips, uniformly distributed at the apex of the tip. The extraordinary optoelectronic properties of rGO were combined with micro-pyramidal cavities to entrap light in micro-cavities and efficiently convert it into heat through multiple reflections and absorptions. As a result, ultralow infra-red laser pulse irradiation could create cavitation bubbles followed by cell membrane deformation and biomolecular delivery. Using this delivery platform, we have achieved the delivery of small to large cargo (668 Da to 465 kDa) in various mammalian cells, including hard-to-transfect H9C2 cardiomyocytes. The best results were achieved for enzyme (465 kDa) delivery with a transfection efficiency and cell viability of 95% and 98%, respectively, in SiHa cells. The highly efficient cargo delivery tool demonstrated a safe and effective approach for cell therapy and diagnostics.

A sequential liquid dispensing method in a centrifugal microfluidic device operating at a constant rotational speed for the multiplexed genetic detection of foodborne pathogens.

This study proposes a sequential liquid dispensing method using a centrifugal microfluidic device operating at a constant rotational speed for the multiplexed genetic detection of nucleic acid targets across multiple samples in a single operation. A pair of passive valves integrated into each microchamber enabled the liquid to fill towards the center of rotation against the centrifugal force, facilitating the complete removal of air inside the microchamber. Liquid manipulation can be achievable without any surface coating of the device by exploiting the inherent hydrophobicity of the polymer. Furthermore, design guidelines for the optimization of microfluidic devices are clarified. Consequently, our proposed method allows direct liquid dispensing into the reaction chambers without cross-contamination while simultaneously metering the sample/reagent volume for the colorimetric loop-mediated isothermal amplification (LAMP) reaction. In addition, we demonstrated the simultaneous detection of four foodborne pathogens (Salmonella spp., Vibrio parahaemolyticus, Campylobacter spp., and norovirus genogroup II (GII)) across four samples in a centrifugal microfluidic device within 60 min. Furthermore, the device exhibited high quantitation (R 2 > 0.98) of the DNA concentration in the sample. Our proposed method enables a more compact design by eliminating the need for metering chambers and offers a point-of-care testing platform with high simplicity as it operates at a constant rotational speed.

A microfluidic-based quantitative analysis system for the multiplexed genetic diagnosis of human viral infections using colorimetric loop-mediated isothermal amplification.

In this study, a microfluidic-based system utilizing colorimetric loop-mediated isothermal amplification (LAMP) is introduced for the quantitative analysis of nucleic acid targets. This system offers a user-friendly and cost-effective platform for the multiplexed genetic diagnosis of various infectious diseases across multiple samples. It includes time-lapse imaging equipment for capturing images of the microfluidic device during the LAMP assay and a hue-based quantitative analysis software to analyze the LAMP reaction, streamlining diagnostic procedures. An electric pipette was used to simplify the loading of samples and LAMP reagents into the device, allowing easy operation even by untrained individuals. The hue-based analysis software employs efficient image processing and post-processing techniques to calculate DNA amplification curves based on color changes in multiple reaction chambers. This software automates several tasks, such as identifying reaction chamber areas from time-lapse images, quantifying color information within each chamber, correcting baselines of DNA amplification curves, fitting experimental data to theoretical curves, and determining the threshold time for each curve. To validate the developed system, conventional off-chip LAMP assays were conducted with a 25 µL reaction mixture in 0.2 mL polymerase chain reaction (PCR) tubes using a real-time turbidimeter. The results indicated that the threshold time obtained using the colorimetric LAMP assay in the developed system is comparable to that obtained with real-time turbidity measurements in PCR tubes, demonstrating the system's capability for quantitative analysis of target nucleic acids, including those from human herpesviruses.

Automatic microdispenser-integrated multiplex enzyme-linked immunosorbent assay device with autonomously driven centrifugal microfluidic system.

In this study, we established the control and design theory of an autonomously driven dispenser at a steady rotation speed and proposed a dispenser-integrated multiplex enzyme-linked immunosorbent assay (ELISA) device. In establishing the theory of the dispenser, we estimated the flow rate in the dispenser and the applied pressure onto the passive valves, so that the suitable burst pressure of the valves and flow rate could be designed. The dispenser-integrated multiplex ELISA device has the potential to perform flow control for executing an ELISA of 6 samples/standards per chip or 18 samples/standards per compact disk by just steadily rotating a chip. In the immunoassay evaluation of the device using mouse IgG detection, it was confirmed that the device could assay 5 µL of several standards in just 30 min without nonspecific reactions, and although this system has a high limit of detection (LOD, 63.4-164 pg mL-1) it is equal to that of manual assay with a titer plate. The device can be fabricated by transferring the microchannel pattern from a mold without complex assembly or alignment, and it can control the liquid operation by just steadily rotating. Thus, the device system developed will contribute to reducing the cost of fabricating chips and control equipment for ELISA systems. Consequently, a compact, portable, and low-cost ELISA system for point-of-care testing is expected to be realized.

Microfluidic device-fabricated spiky nano-burflower shape gold nanomaterials facilitate large biomolecule delivery into cells using infrared light pulses.

Photothermal nanoparticle-sensitised photoporation is an emerging approach, which is considered an efficient tool for the intracellular delivery of biomolecules. Nevertheless, using this method to achieve high transfection efficiency generally compromises cell viability and uneven distribution of nanoparticles results in non-uniform delivery. Here, we show that high aspect ratio gold nano-burflowers, synthesised in a microfluidic device, facilitate highly efficient small to very-large cargo delivery uniformly using infrared light pulses without sacrificing cell viability. By precisely controlling the flow rates of shaping reagent and reducing agent, high-density (24 numbers) sharply branched spikes (∼80 nm tip-to-tip length) of higher aspect ratios (∼6.5) with a small core diameter (∼45 nm) were synthesised. As produced gold burflower-shape nanoparticles are biocompatible, colloidally stable (large surface zeta potential value), and uniform in morphology with a higher plasmonic peak (max. 890 nm). Theoretical analysis revealed that spikes on the nanoparticles generate a higher electromagnetic field enhancement upon interaction with light pulses. It induces plasmonic nanobubbles in the vicinity of the cells, followed by pore formation on the membrane leading to diverse biomolecular delivery into cells. Our platform has been successfully implemented for uniform delivery of small to very large biomolecules, including siRNA (20-24 bp), plasmid DNA expressing green fluorescent protein (6.2 kbp), Cas-9 plasmid (9.3 kbp), and ß-galactosidase enzyme (465 kDa) into diverse mammalian cells with high transfection efficiency and cell viability. For very large biomolecules such as enzymes, the best results were achieved as ∼100% transfection efficiency and ∼100% cell viability in SiHa cells. Together, our findings demonstrate that the spiky gold nano-burflower shape nanoparticles manufactured in a microfluidic system exhibited excellent plasmonic behaviour and could serve as an effective tool in manipulating cell physiology.

Ultrathin SU-8 membrane for highly efficient tunable cell patterning and massively parallel large biomolecular delivery.

Cell patterning is a powerful technique for the precise control and arrangement of cells, enabling detailed single-cell analysis with broad applications in therapeutics, diagnostics, and regenerative medicine. This study presents a novel and efficient technique that enables massively parallel high throughput cell patterning and precise delivery of small to large biomolecules into patterned cells. The innovative cell patterning device proposed in this study is a standalone, ultrathin 3D SU-8 micro-stencil membrane, with a thickness of 10 µm. It features an array of micro-holes ranging from 40 µm to 80 µm, spaced apart by 50 µm to 150 µm. By culturing cells on top of this SU-8 membrane, the technique achieves highly efficient cell patterns varying from single-cell to cell clusters on a Petri dish. Utilizing this technique, we have achieved a remarkable reproducible patterning efficiency for mouse fibroblast L929 (80.5%), human cervical SiHa (81%), and human neuroblastoma IMR32 (89.6%) with less than 1% defects in undesired areas. Single-cell patterning efficiency was observed to be highest at 75.8% for L929 cells. Additionally, we have demonstrated massively parallel high throughput uniform transfection of large biomolecules into live patterned cells by employing an array of titanium micro-rings (10 µm outer diameter, 3 µm inner diameter) activated through infrared light pulses. Successful delivery of a wide range of small to very large biomolecules, including propidium iodide (PI) dye (668.4 Da), dextran (3 kDa), siRNA (13.3 kDa), and ß-galactosidase enzyme (465 kDa), was accomplished in cell patterns for various cancer cells. Notably, our platform achieved exceptional delivery efficiencies of 97% for small molecules like PI dye and 84% for the enzyme, with corresponding high cell viability of 100% and 90%, respectively. Furthermore, the compact and reusable SU-8-based membrane device facilitates highly efficient cell patterning, transfection, and cell viability, making it a promising tool for diagnostics and therapeutic applications.

Massively Parallel High-Throughput Single-Cell Patterning and Large Biomolecular Delivery in Mammalian Cells Using Light Pulses.

The recent advancements of single-cell analysis have significantly enhanced the ability to understand cellular physiology when compared to bulk cellular analysis. Here a massively parallel single-cell patterning and very large biomolecular delivery is reported. Micro-pillar polydimethyl siloxane stamp with different diameters (40-100 µm with 1 cm × 1 cm patterning area) is fabricated and then imprint distinct proteins and finally pattern single-cell to small clusters of cells depending on the micro-pillar diameters. The maximum patterning efficiency is achieved 99.7% for SiHa, 96.75% for L929, and 98.6% for MG63 cells, for the 100 µm micro-pillar stamp. For intracellular delivery of biomolecules into the patterned cells, a titanium micro-dish device is aligned on top of the cells and exposed by infrared light pulses. The platform successfully delivers small to very large biomolecules such as PI dyes (668 Da), dextran 3000 Da, siRNA (20-24 bp), and large size enzymes (464 KDa) in SiHa, L929 and MG63 cells. The delivery efficiency for PI dye, Dextran 3000, siRNA, and enzyme for patterned cells are ≈95 ± 3%, 97 ± 1%, 96 ± 1% and 94 ± 3%, with cell viability of 98 ± 1%. Thus, the platform is compact, robust, easy for printing, and potentially applicable for single-cell therapy and diagnostics.

Visible Pulsed Laser-Assisted Selective Killing of Cancer Cells with PVP-Capped Plasmonic Gold Nanostars.

A new generation of nanoscale photosensitizer agents has improved photothermal capabilities, which has increased the impact of photothermal treatments (PTTs) in cancer therapy. Gold nanostars (GNS) are promising for more efficient and less invasive PTTs than gold nanoparticles. However, the combination of GNS and visible pulsed lasers remains unexplored. This article reports the use of a 532 nm nanosecond pulse laser and polyvinylpyrrolidone (PVP)-capped GNS to kill cancer cells with location-specific exposure. Biocompatible GNS were synthesized via a simple method and were characterized under FESEM, UV-visible spectroscopy, XRD analysis, and particle size analysis. GNS were incubated over a layer of cancer cells that were grown in a glass Petri dish. A nanosecond pulsed laser was irradiated on the cell layer, and cell death was verified via propidium iodide (PI) staining. We assessed the effectiveness of single-pulse spot irradiation and multiple-pulse laser scanning irradiation in inducing cell death. Since the site of cell killing can be accurately chosen with a nanosecond pulse laser, this technique will help minimize damage to the cells around the target cells.

Metallic micro-ring device for highly efficient large cargo delivery in mammalian cells using infrared light pulses.

Uniform transfection of biomolecules into live cells with high delivery efficiency and cell viability is an immensely important area of biological research and has many biomedical applications. In the present study, we report highly efficient, uniform parallel intracellular delivery of small to very large biomolecules into diverse cell types using a titanium micro-ring (TMR) device activated by infrared (IR) light pulse. A TMR array device (2 cm × 2 cm) consists of a 10 µm outer diameter and 3 µm inner diameter for each micro-ring, and 10 µm interspacing between two micro-rings. Upon IR (1050 nm) pulse laser irradiation on the TMR device, photothermal cavitation bubbles are generated, disrupting the cell plasma membrane, and biomolecules are gently delivered into the cells by a simple diffusion process. This TMR device successfully delivered diverse types of small to very large biomolecules such as propidium iodide (PI; 668.4 Da) dye, dextran (3 kDa), small interfering RNA (13.3 kDa), enhanced green fluorescent protein expression plasmid DNA (6.2 kb), and ß-galactosidase enzyme (465 kDa) into human cervical (SiHa), mouse fibroblast (L929), and mouse neural crest-derived (N2a) cancer cells. For smaller molecules (PI dye), delivery efficiency and cell viability were achieved at ∼96% and ∼97%, respectively, with a laser fluence of 21 mJ cm-2 for 250 pulses. In contrast, ∼85% transfection efficiency and ∼90% cell viability were achieved for plasmid DNA with 45 mJ cm-2 laser fluence for 250 pulses in SiHa cells. Moreover, the intracellular delivery of ß-galactosidase enzyme was confirmed with confocal microscopy and flow cytometry analysis resulting in ∼83% co-staining of ß-galactosidase enzyme and calcein AM. Based on these efficient deliveries of diverse types of biomolecules in different cell types, the device has the potential for cellular diagnostic and therapeutic applications.

Combinatorial physical methods for cellular therapy: Towards the future of cellular analysis?

The physical energy activated techniques for cellular delivery and analysis is one of the most rapidly expanding research areas for a variety of biological and biomedical discoveries. These methods, such as electroporation, optoporation, sonoporation, mechanoporation, magnetoporation, etc., have been widely used in delivering different biomolecules into a range of primary and patient-derived cell types. However, the techniques when used individually have had limitations in delivery and co-delivery of diverse biomolecules in various cell types. In recent years, a number of studies have been performed by combining the different membrane disruption techniques, either sequentially or simultaneously, in a single study. The studies, referred to as combinatorial, or hybrid techniques, have demonstrated enhanced transfection, such as efficient macromolecular and gene delivery and co-delivery, at lower delivery parameters and with high cell viability. Such studies can open up new and exciting avenues for understanding the subcellular structure and consequently facilitate the development of novel therapeutic strategies. This review consequently aims at summarising the different developments in hybrid therapeutic techniques. The different methods discussed include mechano-electroporation, electro-sonoporation, magneto-mechanoporation, magnetic nanoparticles enhanced electroporation, and magnetic hyperthermia studies. We discuss the clinical status of the different methods and conclude with a discussion on the future prospects of the combinatorial techniques for cellular therapy and diagnostics.

Automated detection of patterned single-cells within hydrogel using deep learning.

Single-cell analysis has been widely used in various biomedical engineering applications, ranging from cancer diagnostics, and immune response monitoring to drug screening. Single-cell isolation is fundamental for observing single-cell activities and an automatic finding method of accurate and reliable cell detection with few possible human errors is also essential. This paper reports trapping single cells into photo patternable hydrogel microwell arrays and isolating them. Additionally, we present an object detection-based DL algorithm that detects single cells in microwell arrays and predicts the presence of cells in resource-limited environments at the highest possible mAP (mean average precision) of 0.989 with an average inference time of 0.06 s. This algorithm leads to the enhancement of the high-throughput single-cell analysis, establishing high detection precision and reduced experimentation time.

Mixing Performance of a Planar Asymmetric Contraction-and-Expansion Micromixer.

Micromixers are one of the critical components in microfluidic devices. They significantly affect the efficiency and sensitivity of microfluidics-based lab-on-a-chip systems. This study introduces an efficient micromixer with a simple geometrical feature that enables easy incorporation in a microchannel network without compromising the original design of microfluidic devices. The study proposes a newly designed planar passive micromixer, termed a planar asymmetric contraction-and-expansion (P-ACE) micromixer, with asymmetric vertical obstacle structures. Numerical simulation and experimental investigation revealed that the optimally designed P-ACE micromixer exhibited a high mixing efficiency of 80% or more within a microchannel length of 10 mm over a wide range of Reynolds numbers (0.13 ≤ Re ≤ 13), eventually attaining approximately 90% mixing efficiency within a 20 mm microchannel length. The highly asymmetric geometric features of the P-ACE micromixers enhance mixing because of their synergistic effects. The flow velocities and directions of the two fluids change differently while alternately crossing the longitudinal centerline of the microchannel, with the obstacle structures asymmetrically arranged on both sidewalls of the rectangular microchannel. This flow behavior increases the interfacial contact area between the two fluids, thus promoting effective mixing in the P-ACE micromixer. Further, the pressure drops in the P-ACE micromixers were experimentally investigated and compared with those in a serpentine micromixer with a perfectly symmetric mixing unit.

A microfluidic diagnostic device with air plug-in valves for the simultaneous genetic detection of various food allergens.

The identification of accidental allergen contamination in processed foods is crucial for risk management strategies in the food processing industry to effectively prevent food allergy incidents. Here, we propose a newly designed passive stop valve with high pressure resistance performance termed an "air plug-in valve" to further improve microfluidic devices for the detection of target nucleic acids. By implementing the air plug-in valve as a permanent stop valve, a maximal allowable flow rate of 70 µL/min could be achieved for sequential liquid dispensing into an array of 10 microchambers, which is 14 times higher than that achieved with the previous valve arrangement using single-faced stop valves. Additionally, we demonstrate the simultaneous detection of multiple food allergens (wheat, buckwheat, and peanut) based on the colorimetric loop-mediated isothermal amplification assay using our diagnostic device with 10 microchambers compactly arranged in a 20-mm-diameter circle. After running the assays at 60 °C for 60 min, any combination of the three types of food allergens and tea plant, which were used as positive and negative control samples, respectively, yielded correct test results, without any cross-contamination among the microchambers. Thus, our diagnostic device will provide a rapid and easy sample-to-answer platform for ensuring food safety and security.

Microfluidic platforms for single neuron analysis.

Single-neuron actions are the basis of brain function, as clinical sequelae, neuronal dysfunction or failure for most of the central nervous system (CNS) diseases and injuries can be identified via tracing single-neurons. The bulk analysis methods tend to miscue critical information by assessing the population-averaged outcomes. However, its primary requisite in neuroscience to analyze single-neurons and to understand dynamic interplay of neurons and their environment. Microfluidic systems enable precise control over nano-to femto-liter volumes via adjusting device geometry, surface characteristics, and flow-dynamics, thus facilitating a well-defined micro-environment with spatio-temporal control for single-neuron analysis. The microfluidic platform not only offers a comprehensive landscape to study brain cell diversity at the level of transcriptome, genome, and/or epigenome of individual cells but also has a substantial role in deciphering complex dynamics of brain development and brain-related disorders. In this review, we highlight recent advances of microfluidic devices for single-neuron analysis, i.e., single-neuron trapping, single-neuron dynamics, single-neuron proteomics, single-neuron transcriptomics, drug delivery at the single-neuron level, single axon guidance, and single-neuron differentiation. Moreover, we also emphasize limitations and future challenges of single-neuron analysis by focusing on key performances of throughput and multiparametric activity analysis on microfluidic platforms.

Microfluidic mechanoporation for cellular delivery and analysis.

Highly efficient intracellular delivery strategies are essential for developing therapeutic, diagnostic, biological, and various biomedical applications. The recent advancement of micro/nanotechnology has focused numerous researches towards developing microfluidic device-based strategies due to the associated high throughput delivery, cost-effectiveness, robustness, and biocompatible nature. The delivery strategies can be carrier-mediated or membrane disruption-based, where membrane disruption methods find popularity due to reduced toxicity, enhanced delivery efficiency, and cell viability. Among all of the membrane disruption techniques, the mechanoporation strategies are advantageous because of no external energy source required for membrane deformation, thereby achieving high delivery efficiencies and increased cell viability into different cell types with negligible toxicity. The past two decades have consequently seen a tremendous boost in mechanoporation-based research for intracellular delivery and cellular analysis. This article provides a brief review of the most recent developments on microfluidic-based mechanoporation strategies such as microinjection, nanoneedle arrays, cell-squeezing, and hydroporation techniques with their working principle, device fabrication, cellular delivery, and analysis. Moreover, a brief discussion of the different mechanoporation strategies integrated with other delivery methods has also been provided. Finally, the advantages, limitations, and future prospects of this technique are discussed compared to other intracellular delivery techniques.

Microfluidic nanomaterials: From synthesis to biomedical applications.

Microfluidic platforms gain popularity in biomedical research due to their attractive inherent features, especially in nanomaterials synthesis. This review critically evaluates the current state of the controlled synthesis of nanomaterials using microfluidic devices. We describe nanomaterials' screening in microfluidics, which is very relevant for automating the synthesis process for biomedical applications. We discuss the latest microfluidics trends to achieve noble metal, silica, biopolymer, quantum dots, iron oxide, carbon-based, rare-earth-based, and other nanomaterials with a specific size, composition, surface modification, and morphology required for particular biomedical application. Screening nanomaterials has become an essential tool to synthesize desired nanomaterials using more automated processes with high speed and repeatability, which can't be neglected in today's microfluidic technology. Moreover, we emphasize biomedical applications of nanomaterials, including imaging, targeting, therapy, and sensing. Before clinical use, nanomaterials have to be evaluated under physiological conditions, which is possible in the microfluidic system as it stimulates chemical gradients, fluid flows, and the ability to control microenvironment and partitioning multi-organs. In this review, we emphasize the clinical evaluation of nanomaterials using microfluidics which was not covered by any other reviews. In the future, the growth of new materials or modification in existing materials using microfluidics platforms and applications in a diversity of biomedical fields by utilizing all the features of microfluidic technology is expected.

A method of sequential liquid dispensing for the multiplexed genetic diagnosis of viral infections in a microfluidic device.

In this study, we introduce polydimethylsiloxane (PDMS)-based microfluidic devices capable of sequential dispensing of samples into multiple reaction microchambers in a single operation to provide a fast and easy sample-to-answer platform for multiplexed genetic diagnosis of multiple viral infectious diseases. This approach utilizes the loop-mediated isothermal amplification (LAMP) method to amplify and detect specific nucleic acid (DNA/RNA) targets. We present a microfluidic flow control theory for sequential liquid dispensing phenomena, which provides design guidelines for device optimization. The device specifications, such as the possible dispensing number and maximal allowable flow rate, can be theoretically designed by optimizing the geometric dimensions of the microchannels and a pair of passive stop valves integrated into each microchamber together with the water contact angles of the materials used to fabricate the microfluidic devices. In addition, a passive stop valve with a vertical-type phaseguide structure was designed to improve device performance. We could simultaneously diagnose coronavirus disease 2019 (COVID-19) and other infectious diseases, such as severe acute respiratory syndrome (SARS), seasonal influenza A, and pandemic influenza A (H1N1) 2009. The colorimetric reverse transcription LAMP (RT-LAMP) assay suggests that the four viral infectious diseases can be detected within 30 min using a hue-based quantitative analysis, and the naked eye using our microfluidic devices.

Fabrication of TiO2 microspikes for highly efficient intracellular delivery by pulse laser-assisted photoporation.

The introduction of foreign cargo into living cells with high delivery efficiency and cell viability is a challenge in cell biology and biomedical research. Here, we demonstrate a nanosecond pulse laser-activated photoporation for highly efficient intracellular delivery using titanium dioxide (TiO2) microspikes as a substratum. The TiO2 microspikes were formed on titanium (Ti) substrate using an electrochemical anodization process. Cells were cultured on top of the TiO2 microspikes as a monolayer, and the biomolecule was added. Due to pulse laser exposure of the TiO2 microspike-cell membrane interface, the microspikes heat up and induce cavitation bubbles, which rapidly grow, coalesce and collapse to induce explosion, resulting in very strong fluid flow at the cell membrane surface. Thus, the cell plasma membrane disrupts and creates transient nanopores, allowing delivery of biomolecules into cells by a simple diffusion process. By this technique, we successfully delivered propidium iodide (PI) dye in HeLa cells with high delivery efficiency (93%) and high cell viability (98%) using 7 mJ pulse energy at 650 nm wavelength. Thus, our TiO2 microspike-based platform is compact, easy to use, and potentially applicable for therapeutic and diagnostic purposes.