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.

Chronoamperometric interrogation of an electrochemical aptamer-based sensor with tetrahedral DNA nanostructure pendulums for continuous biomarker measurements.

Tetrahedral DNA nanostructure (TDN) is highly promising in developing electrochemical aptamer-based (E-AB) sensors for biomolecular detection, owing to its inherit programmability, spatial orientation and structural robustness. However, current interrogation strategies applied for TDN-based E-AB sensors, including enzyme-based amperometry, voltammetry, and electrochemical impedance spectroscopy, either require complicated probe design or suffer from limited applicability or selectivity. In this study, a TDN pendulum-empowered E-AB sensor interrogated by chronoamperometry for reagent-free and continuous monitoring of a blood clotting enzyme, thrombin, was developed. TDN pendulums with extended aptamer sequences at three vertices were immobilized on a gold electrode via a thiolated double-stranded DNA (dsDNA) at the fourth vertex, and their motion is modulated by the bonding of target thrombin to aptamers. We observed a significantly amplified signalling output on our sensor based on the TDN pendulum compared to E-AB sensors modified with linear pendulums. Moreover, our sensor achieved highly selective and rapidly responsive measurement of thrombin in both PBS and artificial urine, with a wide dynamic range from 1 pM to 10 nM. This study shows chronoamperometry-enabled continuous biomarker monitoring on a sub-second timescale with a drift-free baseline, demonstrating a novel approach to accurately detect molecular dynamics in real time.

Passive microfluidic devices for cell separation.

The separation of specific cell populations is instrumental in gaining insights into cellular processes, elucidating disease mechanisms, and advancing applications in tissue engineering, regenerative medicine, diagnostics, and cell therapies. Microfluidic methods for cell separation have propelled the field forward, benefitting from miniaturization, advanced fabrication technologies, a profound understanding of fluid dynamics governing particle separation mechanisms, and a surge in interdisciplinary investigations focused on diverse applications. Cell separation methodologies can be categorized according to their underlying separation mechanisms. Passive microfluidic separation systems rely on channel structures and fluidic rheology, obviating the necessity for external force fields to facilitate label-free cell separation. These passive approaches offer a compelling combination of cost-effectiveness and scalability when compared to active methods that depend on external fields to manipulate cells. This review delves into the extensive utilization of passive microfluidic techniques for cell separation, encompassing various strategies such as filtration, sedimentation, adhesion-based techniques, pinched flow fractionation (PFF), deterministic lateral displacement (DLD), inertial microfluidics, hydrophoresis, viscoelastic microfluidics, and hybrid microfluidics. Besides, the review provides an in-depth discussion concerning cell types, separation markers, and the commercialization of these technologies. Subsequently, it outlines the current challenges faced in the field and presents a forward-looking perspective on potential future developments. This work hopes to aid in facilitating the dissemination of knowledge in cell separation, guiding future research, and informing practical applications across diverse scientific disciplines.

Machine learning implementation strategy in imaging and impedance flow cytometry.

Imaging and impedance flow cytometry is a label-free technique that has shown promise as a potential replacement for standard flow cytometry. This is due to its ability to provide rich information and archive high-throughput analysis. Recently, significant efforts have been made to leverage machine learning for processing the abundant data generated by those techniques, enabling rapid and accurate analysis. Harnessing the power of machine learning, imaging and impedance flow cytometry has demonstrated its capability to address various complex phenotyping scenarios. Herein, we present a comprehensive overview of the detailed strategies for implementing machine learning in imaging and impedance flow cytometry. We initiate the discussion by outlining the commonly employed setup to acquire the data (i.e., image or signal) from the cell. Subsequently, we delve into the necessary processes for extracting features from the acquired image or signal data. Finally, we discuss how these features can be utilized for cell phenotyping through the application of machine learning algorithms. Furthermore, we discuss the existing challenges and provide insights for future perspectives of intelligent imaging and impedance flow cytometry.

An optimized PDMS microfluidic device for ultra-fast and high-throughput imaging flow cytometry.

Imaging flow cytometry (IFC) is a powerful tool for cell detection and analysis due to its high throughput and compatibility in image acquisition. Optical time-stretch (OTS) imaging is considered as one of the most promising imaging techniques for IFC because it can realize cell imaging at a flow speed of around 60 m s-1. However, existing PDMS-based microchannels cannot function at flow velocities higher than 10 m s-1; thus the capability of OTS-based IFC is significantly limited. To overcome the velocity barrier for PDMS-based microchannels, we proposed an optimized design of PDMS-based microchannels with reduced hydraulic resistance and 3D hydrodynamic focusing capability, which can drive fluids at an ultra-high flow velocity (of up to 40 m s-1) by using common syringe pumps. To verify the feasibility of our design, we fabricated and installed the microchannel in an OTS IFC system. The experimental results first proved that the proposed microchannel can support a stable flow velocity of up to 40 m s-1 without any leakage or damage. Then, we demonstrated that the OTS IFC is capable of imaging cells at a velocity of up to 40 m s-1 with good quality. To the best of our knowledge, it is the first time that IFC has achieved such a high flow velocity just by using a PDMS-glass chip. Moreover, high velocity can enhance the focusing of cells on the optical focal plane, increasing the number of detected cells and the throughput. This work provides a promising solution for IFC to fully release its capability of advanced imaging techniques by operating at an extremely high screening throughput.

10 µm thick ultrathin glass sheet to realize a highly sensitive cantilever for precise cell stiffness measurement.

The micro-cantilever-based sensor platform has become a promising technique in the sensing area for physical, chemical and biological detection due to its portability, small size, label-free characteristics and good compatibility with "lab-on-a-chip" devices. However, traditional micro-cantilever methods are limited by their complicated fabrication, manipulation and detection, and low sensitivity. In this research, we proposed a 10 µm thick ultrathin, highly sensitive, and flexible glass cantilever integrated with a strain gauge sensor and presented its application for the measurement of single-cell mechanical properties. Compared to conventional methods, the proposed ultrathin glass sheet (UTGS)-based cantilever is easier to fabricate, has better physical and chemical properties, and shows a high linear relationship between resistance change and applied small force or displacement. The sensitivity of the cantilever is 15 µN µm-1 and the minimum detectable displacement at the current development stage is 500 nm, which is sufficient for cell stiffness measurement. The cantilever also possesses excellent optical transparency that supports real-time observation during measurement. We first calibrated the cantilever by measuring the Young's modulus of PDMS with known specific stiffness, and then we demonstrated the measurement of Xenopus oocytes and fertilized eggs in different statuses. By further optimizing the UTGS-based cantilever, we can extend its applicability to various measurements of different cells.

Inertial Separation of Particles Assisted by Symmetrical Sheath Flows in a Straight Microchannel.

Over the past two decades, inertial microfluidics, which works at an intermediate range of Reynolds number (∼1 < Re < ∼100), has been widely used for particle separation due to its high-throughput and label-free features. This work proposes a novel method for continuous separation of particles by size using inertial microfluidics, with the assistance of symmetrical sheath flows in a straight microchannel. Here, larger particles (>3 µm) are arranged close to the channel sidewalls, while smaller particles (<2 µm) remain flowing along the channel centerline. This conclusion is supported by experimental data with particles of different sizes ranging from 0.79 to 10.5 µm. Symmetrical Newtonian sheath flows are injected on both sides of particle mixtures into a straight rectangular microchannel with an aspect ratio (AR = height/width) of 2.5. Results show that the separation performance of the developed microfluidic device is affected by three main factors: channel length, total flow rate, and flow rate ratio of sheath to sample. Besides, separation of platelets from whole blood is demonstrated. The developed microfluidic platform owns the advantages of low fabrication cost, simple experiment setup, versatile selections of particle candidates, and stable operations. This systematic study provides a new perspective for particle separation, which is expected to find applications across various fields spanning physics, biology, biomedicine, and industry.

A review on intelligent impedance cytometry systems: Development, applications and advances.

Impedance cytometry is a well-established technique for counting and analyzing single cells, with several advantages, such as convenience, high throughput, and no labeling required. A typical experiment consists of the following steps: single-cell measurement, signal processing, data calibration, and particle subtype identification. At the beginning of this article, we compared commercial and self-developed options extensively and provided references for developing reliable detection systems, which are necessary for cell measurement. Then, a number of typical impedance metrics and their relationships to biophysical properties of cells were analyzed with respect to the impedance signal analysis. Given the rapid advances of intelligent impedance cytometry in the past decade, this article also discussed the development of representative machine learning-based approaches and systems, and their applications in data calibration and particle identification. Finally, the remaining challenges facing the field were summarized, and potential future directions for each step of impedance detection were discussed.

Microfluidic Separation and Enrichment of Escherichia coli by Size Using Viscoelastic Flows.

Here, we achieve the separation and enrichment of Escherichia coli clusters from its singlets in a viscoelastic microfluidic device. E. coli, an important prokaryotic model organism and a widely used microbial factory, can aggregate in clusters, leading to biofilm development that can be detrimental to human health and industrial processes. The ability to obtain high-purity populations of E. coli clusters is of significance for biological, biomedical, and industrial applications. In this study, polystyrene particles of two different sizes, 1 and 4.8 µm, are used to mimic E. coli singlets and clusters, respectively. Experimental results show that particles migrate toward the channel center in a size-dependent manner, due to the combined effects of inertial and elastic forces; 4.8 and 1 µm particles are found to have lateral equilibrium positions closer to the channel centerline and sidewalls, respectively. The size-dependent separation performance of the microdevice is demonstrated to be affected by three main factors: channel length, the ratio of sheath to sample flow rate, and poly(ethylene oxide) (PEO) concentration. Further, the separation of E. coli singlets and clusters is achieved at the outlets, and the separation efficiency is evaluated in terms of purity and enrichment factor.

Microsecond cell triple-sorting enabled by multiple pulse irradiation of femtosecond laser.

Femtosecond-laser-assisted cell manipulation, as one of the high throughput cell sorting techniques, is tailored for single-step multiple sorting based on controllable impulsive force. In this paper, femtosecond laser pulses are focused within a pocket structure and they induce an impulse force acting on the flowing objects. The impulsive force is shown to be controllable by a new method to adjust the femtosecond pulse properties. This allows precise streamline manipulation of objects having various physical qualities (e.g., weight and volume). The pulse energy, pulse number, and pulse interval of the femtosecond laser are altered to determine the impulsive force strength. The method is validated in single cell or bead triple-sorting experiments and its capability to perform streamline manipulation in as little as 10 µs is shown. The shift profiles of the beads acting under the impulsive force are studied in order to better understand the sorting mechanism. Additionally, beads and cells with different fluorescence intensities are successfully detected and directed into different microchannels, with maximum success rates of 90% and 64.5%, respectively. To sum up, all results suggest that this method has the potential to sort arbitrary subpopulations by altering the number of femtosecond pulses and that it takes the first step toward developing a single-step multi-selective system.

Microenvironmental Analysis and Control for Local Cells under Confluent Conditions via a Capillary-Based Microfluidic Device.

Sophisticated functions of biological tissues are supported by small biological units of cells that are localized within a region of 100 µm scale. The cells in these units secrete molecules to form their microenvironment to play a vital role in biological functions. Various microfluidic devices have been developed to analyze the microenvironment but were not designed for cells in a culture dish in a confluent condition, a typical setup for cell and tissue cultivation. This study presents a novel glass capillary-based microfluidic device for studying confluent cells in a culture dish. The multiple capillaries allow the device to confine the local flow in 100 µm or smaller scale to form two adjacent regions with different chemical properties; it can simultaneously perform local cell stimulation and collect secreted molecules from the stimulated cells. Cell removal was achieved upon trypsin stimulation from a limited area (3.8 × 10-3 ± 1.0 × 10-3 mm2), which corresponded to 7.6 ± 2.0 cells, using the mouse skeletal myoblast cell line (C2C12 cells) in a confluent condition. Microenvironmental analysis was demonstrated by measuring the secreted tumor necrosis factor alpha (TNF-α) collected from the microenvironment of the stimulated and unstimulated mouse leukemic monocyte cell line (RAW264 cells) to track temporal changes in the TNF-α production. The TNF-α secreted from stimulated cells was approximately four-fold higher than that from unstimulated cells in 90 min. This device enables local cell stimulation and the collection of secreted molecules for cells under confluent conditions, which contributes to the analysis of the cellular microenvironment.

Parallel Impedance Cytometry for Real-Time Screening of Bacterial Single Cells from Nano- to Microscale.

The benefits of impedance cytometry include high-throughput and label-free detection, while long-term calibration is required to remove the effects of the detection circuits. This study presents a novel impedance cytometry system, called parallel impedance cytometry, to simplify the calibration and analysis of the impedance signals. Furthermore, target objects can be detected even when benchmarked against similar objects. Parallel dual microchannels allow the simultaneous detection of reference and target particles in two separate microchannels, without the premixing of reference and target suspensions. The impedance pulses of both can appear separately on the opposite sides of the same time series, which have been verified via simulation and experimental results. Raw impedance signals can easily distinguish target particles from reference ones. Polystyrene beads with different sizes ranging from nano- to microscale (e.g., 500, 750 nm, 1, 2, 3, and 4.5 µm) confirm the nanosensitivity of the system. In addition, the detection of antibiotic-treated Escherichia coli cells demonstrates that our system can be used for the quantitative assessment of the dielectric properties of individual cells, as well as for the proportion of susceptible cells. Through benchmarking against untreated E. coli cells in the other channel, our method enables the discrimination of susceptible cells from others and the comparison of susceptible and insusceptible cells in the target suspension. Those findings indicate that the parallel impedance cytometry can greatly facilitate the measurement and calibration of the impedances of various particles or cells and provide a means to compare their dielectric properties.

Identification of Single Yeast Budding Using Impedance Cytometry with a Narrow Electrode Span.

Impedance cytometry is wildly used in single-cell detection, and its sensitivity is essential for determining the status of single cells. In this work, we focus on the effect of electrode gap on detection sensitivity. Through comparing the electrode span of 1 µm and 5 µm, our work shows that narrowing the electrode span could greatly improve detection sensitivity. The mechanism underlying the sensitivity improvement was analyzed via numerical simulation. The small electrode gap (1 µm) allows the electric field to concentrate near the detection area, resulting in a high sensitivity for tiny particles. This finding is also verified with the mixture suspension of 1 µm and 3 µm polystyrene beads. As a result, the electrodes with 1 µm gap can detect more 1 µm beads in the suspension than electrodes with 5 µm gap. Additionally, for single yeast cells analysis, it is found that impedance cytometry with 1 µm electrodes gap can easily distinguish budding yeast cells, which cannot be realized by the impedance cytometry with 5 µm electrodes gap. All experimental results support that narrowing the electrode gap is necessary for tiny particle detection, which is an important step in the development of submicron and nanoscale impedance cytometry.

A pressure driven electric energy generator exploiting a micro- to nano-scale glass porous filter with ion flow originating from water.

We demonstrated a pressure driven energy harvesting device using water and that features a glass filter with porous channels. We employed powder sintering to fabricate the glass filter (2 cm diameter, 3 mm thickness) by packing a powder of borosilicate glass particles into a carbon mold and then thermally fusing this at 700°C under pressure. In constant flow rate experiment, the optimum average pore radius of the filter for power generation was 12 µm. Using this filter, power of 3.8 mW (27 V, 0.14 mA, 0.021% energy efficiency) was generated at a water flow speed of 50 mm/s. In constant pressure experiment, a power generator was equipped with a foot press unit with a 60 kg weight (830 kPa) and 50 mL of water. The optimum average pore radius for power generation in this experiment was 12 µm and power of 4.8 mW (18 V, 0.26 mA, 0.017% energy efficiency) was generated with 1.7 s duration. This was enough power for direct LED lighting and the capacitors could store enough energy to rotate a fan and operate a wireless communicator. Our pressure driven device is suitable for energy harvesting from slow movements like certain human physiological functions, e.g. walking.

Anhydrobiotic chironomid larval motion-based multi-sensing microdevice for the exploration of survivable locations.

African chironomid (Polypedilum vanderplanki) larvae can suspend their metabolism by undergoing severe desiccation and then resume this activity by simple rehydration. We present a microdevice using interdigital comb electrodes to detect the larval motion using the natural surface charge of the living larvae in water. The larvae were most active 2 h after soaking them in water at 30°C; they exhibited motions with 2 Hz frequency. This was comparable to the signal obtained from the microdevice via fast Fourier transform (FFT) processing. The amplitude of the voltage and current were 0.11 mV and 730 nA, respectively. They would be enough to be detected by a low power consumption microcomputer. Temperature and pH sensing were demonstrated by detecting the vital motions of the revived larvae under different conditions. This multi-functional biosensor will be a useful microdevice to search for survivable locations under extreme environmental conditions like those on other planets.

Assessment of the electrical penetration of cell membranes using four-frequency impedance cytometry.

The electrical penetration of the cell membrane is vital for determining the cell interior via impedance cytometry. Herein, we propose a method for determining the conductivity of the cell membrane through the tilting levels of impedance pulses. When electrical penetration occurs, a high-frequency current freely passes through the cell membrane; thus, the intracellular distribution can directly act on the high-frequency impedance pulses. Numerical simulation shows that an uneven intracellular component distribution can affect the tilting levels of impedance pulses, and the tilting levels start increasing when the cell membrane is electrically penetrated. Experimental evidence shows that higher detection frequencies (>7 MHz) lead to a wider distribution of the tilting levels of impedance pulses when measuring cell populations with four-frequency impedance cytometry. This finding allows us to determine that a detection frequency of 7 MHz is able to pass through the membrane of Euglena gracilis (E. gracilis) cells. Additionally, we provide a possible application of four-frequency impedance cytometry in the biomass monitoring of single E. gracilis cells. High-frequency impedance (≥7 MHz) can be applied to monitor these biomass changes, and low-frequency impedance (<7 MHz) can be applied to track the corresponding biovolume changes. Overall, this work demonstrates an easy determination method for the electrical penetration of the cell membrane, and the proposed platform is applicable for the multiparameter assessment of the cell state during cultivation.

Bio-actuated microvalve in microfluidics using sensing and actuating function of Mimosa pudica.

Bio-actuators and sensors are increasingly employed in microscale devices for numerous applications. Unlike other artificial devices actuated by living cells or tissues, here we introduce a microvalve system actuated by the stimuli-responsive action plant, Mimosa pudica (sleepy plant). This system realizes the control of the valve to open and close by dropping and recovering responses of Mimosa pudica branch upon external physical stimulations. The results showed that one matured single uncut Mimosa pudica branch produced average force of 15.82 ± 0.7 mN. This force was sufficient for actuating and keeping the valve open for 8.46 ± 1.33 min in a stimulation-recovering cycle of 30 min. Additionally, two separately cut Mimosa pudica branches were able to keep the valve open for 2.28 ± 0.63 min in a stimulating-recovering cycle of 20min. The pressure resistance and the response time of the valve were 4.2 kPa and 1.4 s, respectively. This demonstration of plant-microfluidics integration encourages exploiting more applications of microfluidic platforms that involve plant science and plant energy harvesting.

Recent advances in microfluidic devices for single-cell cultivation: methods and applications.

Single-cell analysis is essential to improve our understanding of cell functionality from cellular and subcellular aspects for diagnosis and therapy. Single-cell cultivation is one of the most important processes in single-cell analysis, which allows the monitoring of actual information of individual cells and provides sufficient single-cell clones and cell-derived products for further analysis. The microfluidic device is a fast-rising system that offers efficient, effective, and sensitive single-cell cultivation and real-time single-cell analysis conducted either on-chip or off-chip. Here, we introduce the importance of single-cell cultivation from the aspects of cellular and subcellular studies. We highlight the materials and structures utilized in microfluidic devices for single-cell cultivation. We further discuss biological applications utilizing single-cell cultivation-based microfluidics, such as cellular phenotyping, cell-cell interactions, and omics profiling. Finally, present limitations and future prospects of microfluidics for single-cell cultivation are also discussed.

Dual-frequency impedance assays for intracellular components in microalgal cells.

Intracellular components (including organelles and biomolecules) at the submicron level are typically analyzed in situ by special preparation or expensive setups. Here, a label-free and cost-effective approach of screening microalgal single-cells at a subcellular resolution is available based on impedance cytometry. To the best of our knowledge, it is the first time that the relationships between impedance signals and submicron intracellular organelles and biomolecules are shown. Experiments were performed on Euglena gracilis (E. gracilis) cells incubated under different incubation conditions (i.e., aerobic and anaerobic) and 15 µm polystyrene beads (reference) at two distinct stimulation frequencies (i.e., 500 kHz and 6 MHz). Based on the impedance detection of tens of thousands of samples at a throughput of about 900 cells per second, three metrics were used to track the changes in biophysical properties of samples. As a result, the electrical diameters of cells showed a clear shrinkage in cell volume and intracellular components, as observed under a microscope. The morphology metric of impedance pulses (i.e., tilt index) successfully characterized the changes in cell shape and intracellular composition distribution. Besides, the electrical opacity showed a stable ratio of the intracellular components to cell volume under the cellular self-regulation. Additionally, simulations were used to support these findings and to elucidate how submicron intracellular components and cell morphology affect impedance signals, providing a basis for future improvements. This work opens up a label-free and high-throughput way to analyze single-cell intracellular components by impedance cytometry.

Specific capture and intact release of breast cancer cells using a twin-layer vein-shaped microchip with a self-assembled surface.

Breast cancer is the most fatal disease among female cancers yet its detection still relies on needle biopsy. The unique physical and immune characteristics of breast cancer cells different from blood cells make them suitable to be employed as excellent biomarkers in liquid biopsy, through which breast cancer cells are collected from peripheral blood for further cancer diagnosis, medical treatment monitoring, and drug screening. Although the separation and enrichment of breast cancer cells from peripheral blood have been studied for years, there are still two problems to be solved in these methods: the low efficiency of on-chip immunologic capture in the flow state and the influence of the conjugated antibodies for the following analyses during cell release. In this paper, a vein-shaped microchip with self-assembled surface was developed for the specific and robust capture (91.2%) of breast cancer cells in the flow state. A protein-recovery process was proposed, in which trypsin served as a mild release reagent, releasing 92% of cells with high viability (96%), normal adherent proliferation, and complete proteins on the cell membrane, avoiding disturbance of the conjugated chemical molecules in the following clinical study. The excellent performance demonstrated in isolating free breast cancer cells from real peripheral blood sample, originating from the orthotopic 4T1 breast cancer metastatic models, suggest the microchip could be utilized as a multiple circulating tumor cell capture and release platform that could allow providing more reliable information in liquid biopsies.