Critical considerations in determining the surface charge of small extracellular vesicles.

Small extracellular vesicles (EVs) have emerged as a focal point of EV research due to their significant role in a wide range of physiological and pathological processes within living systems. However, uncertainties about the nature of these vesicles have added considerable complexity to the already difficult task of developing EV-based diagnostics and therapeutics. Whereas small EVs have been shown to be negatively charged, their surface charge has not yet been properly quantified. This gap in knowledge has made it challenging to fully understand the nature of these particles and the way they interact with one another, and with other biological structures like cells. Most published studies have evaluated EV charge by focusing on zeta potential calculated using classical theoretical approaches. However, these approaches tend to underestimate zeta potential at the nanoscale. Moreover, zeta potential alone cannot provide a complete picture of the electrical properties of small EVs since it ignores the effect of ions that bind tightly to the surface of these particles. The absence of validated methods to accurately estimate the actual surface charge (electrical valence) and determine the zeta potential of EVs is a significant knowledge gap, as it limits the development of effective label-free methods for EV isolation and detection. In this study, for the first time, we show how the electrical charge of small EVs can be more accurately determined by accounting for the impact of tightly bound ions. This was accomplished by measuring the electrophoretic mobility of EVs, and then analytically correlating the measured values to their charge in the form of zeta potential and electrical valence. In contrast to the currently used theoretical expressions, the employed analytical method in this study enabled a more accurate estimation of EV surface charge, which will facilitate the development of EV-based diagnostic and therapeutic applications.

CoVSense: Ultrasensitive Nucleocapsid Antigen Immunosensor for Rapid Clinical Detection of Wildtype and Variant SARS-CoV-2.

The widespread accessibility of commercial/clinically-viable electrochemical diagnostic systems for rapid quantification of viral proteins demands translational/preclinical investigations. Here, Covid-Sense (CoVSense) antigen testing platform; an all-in-one electrochemical nano-immunosensor for sample-to-result, self-validated, and accurate quantification of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N)-proteins in clinical examinations is developed. The platform's sensing strips benefit from a highly-sensitive, nanostructured surface, created through the incorporation of carboxyl-functionalized graphene nanosheets, and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) conductive polymers, enhancing the overall conductivity of the system. The nanoengineered surface chemistry allows for compatible direct assembly of bioreceptor molecules. CoVSense offers an inexpensive (<$2 kit) and fast/digital response (<10 min), measured using a customized hand-held reader (<$25), enabling data-driven outbreak management. The sensor shows 95% clinical sensitivity and 100% specificity (Ct<25), and overall sensitivity of 91% for combined symptomatic/asymptomatic cohort with wildtype SARS-CoV-2 or B.1.1.7 variant (N = 105, nasal/throat samples). The sensor correlates the N-protein levels to viral load, detecting high Ct values of ≈35, with no sample preparation steps, while outperforming the commercial rapid antigen tests. The current translational technology fills the gap in the workflow of rapid, point-of-care, and accurate diagnosis of COVID-19.

Capillary-Assisted Molecular Pendulum Bioanalysis.

The development of robust biosensing strategies that can be easily implemented in everyday life remains a challenge for the future of modern biosensor research. While several reagentless approaches have attempted to address this challenge, they often achieve user-friendliness through sacrificing sensitivity or universality. While acceptable for certain applications, these trade-offs hinder the widespread adoption of reagentless biosensing technologies. Here, we report a novel approach to reagentless biosensing that achieves high sensitivity, rapid detection, and universality using the SARS-CoV-2 virus as a model target. Universality is achieved by using nanoscale molecular pendulums, which enables reagentless electrochemical biosensing through a variable antibody recognition element. Enhanced sensitivity and rapid detection are accomplished by incorporating the coffee-ring phenomenon into the sensing scheme, allowing for target preconcentration on a ring-shaped electrode. Using this approach, we obtained limits of detection of 1 fg/mL and 20 copies/mL for the SARS-CoV-2 nucleoproteins and viral particles, respectively. In addition, clinical sample analysis showed excellent agreement with Ct values from PCR-positive SARS-CoV-2 patients.

Immuno-affinity Potent Strip with Pre-Embedded Intermixed PEDOT:PSS Conductive Polymers and Graphene Nanosheets for Bio-Ready Electrochemical Biosensing of Central Nervous System Injury Biomarkers.

Future point-of-care (PoC) and wearable electrochemical biosensors explore new technology solutions to eliminate the need for multistep electrode modification and functionalization, overcome the limited reproducibility, and automate the sensing steps. In this work, a new screen-printed immuno-biosensor strip is engineered and characterized using a hybrid graphene nanosheet intermixed with the conductive poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) polymers, all embedded within the base carbon matrix (GiPEC) of the screen-printing ink. This intermixed nanocomposite ink is chemically designed for self-containing the "carboxyl" functional groups as the most specific chemical moiety for protein immobilization on the electrodes. The GiPEC ink enables capturing the target antibodies on the electrode without any need for extra surface preparation. As a proof of concept, the performance of the non-functionalized ready-to-immobilize strips was assessed for the detection of glial fibrillary acidic protein (GFAP) as a known central nervous system injury blood biomarker. This immuno-biosensor exhibits the limit of detection of 281.7 fg mL-1 (3 signal-to-noise ratio) and the sensitivity of 322.6 Ω mL pg-1 mm-2 within the clinically relevant linear detection range from 1 pg mL-1 to 10 ng mL-1. To showcase its potential PoC application, the bio-ready strip is embedded inside a capillary microfluidic device and automates electrochemical quantification of GFAP spiked in phosphate-buffered saline and the human serum. This new electrochemical biosensing platform can be further adapted for the detection of various protein biomarkers with the application in realizing on-chip immunoassays.

µDrop: Multi-analyte portable electrochemical-sensing device for blood-based detection of cleaved tau and neuron filament light in traumatic brain injury patients.

Over 27 million individuals are affected every year worldwide with central nervous system (CNS) injuries. These injuries include but are not limited to traumatic brain injury (TBI) and spinal cord injury (SCI). CNS injuries remain a significant public health concern which demands reliable tools for rapid, on-sight, on-field, and point-of-care diagnostic (POC) solutions. To address these challenges, we developed a low-cost, open-source, hand-held, portable, and POC detection technology, termed as MicroDrop (µDrop), which can simultaneously detect up to eight target biomolecules and display results in both analog and digital formats. The data acquired is stored wirelessly in a cloud server for further investigation and statistical analysis. Multiplexing capability of µDrop and immuno-biosensors detects and quantifies Cleaved-Tau Protein (C-Tau) and Neuron-Filament (NFL) proteins in the blood of TBI patients. Immuno-biosensors rapidly sense the two target proteins in less than 30 min, with µDrop and a conventional potentiostat. C-Tau and NFL were selectively detected with µDrop within the dynamic range of 10 pg/mL - 100 ng/mL and the sensitivity range of 47 µA/pg mm2 - 65 µA/pg mm2. Comparing the biosensing performance with enzyme-linked immunosorbent assays (ELISA) shows that the immuno-biosensors combined with µDrop could successfully differentiate between clinical controls and injured patients.

Role of temperature on bio-printability of gelatin methacryloyl bioink in two-step cross-linking strategy for tissue engineering applications.

Additive manufacturing has shown promising results in reconstructing three-dimensional (3D) living tissues for various applications, including tissue engineering, regenerative medicine, drug discovery, and high-throughput drug screening. In extrusion-based bioprinters, stable formation of filaments and high-fidelity deposition of bioinks are the primary challenges in fabrication of physiologically relevant tissue constructs. Among various bioinks, gelatin methacryloyl (GelMA) is known as a photocurable and physicochemically tunable hydrogel with a demonstrated biocompatibility and tunable biodegradation properties. The two-step crosslinking of GelMA (reversible thermal gelation and permanent photo-crosslinking) has attracted researchers to make complex tissue constructs. Despite promising results in filament formation and printability of this hydrogel, the effect of temperature on physicochemical properties, cytocompatibility, and biodegradation of the hydrogel are to be investigated. This work studies the effect of thermoreversible, physical crosslinking on printability of GelMA. The results of 3D printing of GelMA at different temperatures followed by irreversible chemical photo-crosslinking show that the decrease in temperature improves the filament formation and shape fidelity of the deposited hydrogel, particularly at the temperatures around 15 °C. Time dependant mechanical testing of the printed samples revealed that decreasing the extruding temperature increases the elastic properties of the extruded filaments. Furthermore, our novel approach in minimizing the slippage effect during rheological study enabled to measure changes in linear and non-linear viscoelastic properties of the printed samples at different temperatures. A considerable increase in storage modulus of the extruded samples printed at lower temperatures confirms their higher solid behavior. Scanning electron microscopy revealed a remarkable decrease in porosity of the extruded hydrogels by decreasing the temperature. Chemical analysis by Fourier-transform infrared spectroscopy and circular dichroism showed a direct relationship between the coil-helix transition in hydrogel macromers and its physical alterations. Finally, biodegradation and cytocompatibility of the extruded hydrogels decreased at lower extruding temperatures.

Effect of cell imprinting on viability and drug susceptibility of breast cancer cells to doxorubicin.

This study demonstrates the effect of substrate's geometrical cues on viability and the efficacy of an anti-cancer drug, doxorubicin (DOX), on breast cancer cells. It is hypothesized that the surface topographical properties can mediate the cellular drug intake. Pseudo-three dimensional (3D) platforms were fabricated using imprinting technique from polydimethylsiloxane (PDMS) and gelatin methacryloyl (GelMA) hydrogel to recapitulate topography of cells' membranes. The cells exhibited higher viability on the cell-imprinted platforms for both PDMS and GelMA materials compared to the plain/flat counterparts. For instance, MCF7 cells showed a higher metabolic activity (11.9%) on MCF7-imprinted PDMS substrate than plain PDMS. The increased metabolic activity for the imprinted GelMA was about 44.2% compared to plain hydrogel. The DOX response of cells was monitored for 24 h. Although imprinted substrates demonstrated enhanced biocompatibility, the cultured cells were more susceptible to the drug compared to the plain substrates. In particular, MCF7 cells on imprinted PDMS and GelMA substrates showed 37% and 50% higher in cell death compared to the corresponding plain PDMS and GelMA, respectively. Interestingly, the drug susceptibility of the cells on the imprinted hydrogel was about 70% higher than the cells cultured on imprinted PDMS substrates. Having MCF7 cell-imprinted substrates, DOX responses of two other breast cancer cell lines, SKBR3 and ZR-75-1, were also evaluated. The results support that cell membrane curvature developed by multiscale topography is able to mediate intracellular signaling and drug intake. STATEMENT OF SIGNIFICANCE: Research in biological sciences and drug discovery mostly rely on two dimensional (2D) cell culture techniques which cannot provide a reliable physiologically relevant environment. Lack of extracellular matrix and a large shift in physicochemical properties of conventional 2D substrates can induce aberrant cellular behaviors. While chemical composition, topographical, and mechanical properties of substrates have remarkable impacts on drug susceptibility, gene expression, and protein synthesis, the most cell culture plates are from rigid and plain substrates. A number of (bio)polymeric 3D-platforms have been introduced to resemble innate cell microenvironment. However, their intricate culture protocols restrain their applications in demanding high-throughput drug screening. To address the above concerns, in the present study, a hydrogel-based pseudo-3D substrate with imprinted cell features has been introduced.

Dynamics of temperature-actuated droplets within microfluidics.

Characterizing the thermal behavior of dispersed droplets within microfluidic channels is crucial for different applications in lab-on-a-chip. In this paper, the physics of droplets volume during their transport over a heater is studied experimentally and numerically. The response of droplets to external heating is examined at temperature ranges of 25-90 °C and at different flow rates of the dispersed phase respect to the continuous flow. The results present a reliable prediction of the droplet volume and stability when heating is applied to the droplets at the downstream channel in a quite far distance from the droplets' ejection orifice. Increasing the ratio of flow rate resulted in larger droplets; for instance, the flow ratio of 0.25 produced drops with 40% larger diameter than the flow rate of 0.1. For every 10 °C increase in temperature of the droplets, the droplet diameter increased by about 5.7% and 4.2% for pure oil and oil with a surfactant, respectively. Also, the droplets showed a degree of instability during their transport over the heater at higher temperatures. Adding SPAN 20 surfactant improved the stability of the droplets at temperatures higher than 60 °C. The experimentally validated numerical model helped for systemic analysis of the influence of key temperature-dependence parameters (e.g. surface tension, density and viscosity of both phases) on controlling the volume and stability of droplets. Our findings supported to develop highly functional systems with a predetermined droplets performance under high temperatures up to 90 °C. This report provides a preliminary basis for enhancing the performance of droplet microfluidic systems for digital droplet polymerase chain reaction (ddPCR), continuous flow digital loop-mediated isothermal PCR (LAMP), and droplet-based antibiotic susceptibility testing.

Filter-based isolation, enrichment, and characterization of circulating tumor cells.

Isolation of circulating tumor cells (CTCs) from blood has long been a challenge due to the rarity and heterogeneity of these cells. Detection technologies have predominantly focused on different molecular or physical properties of CTCs. Size-based isolation approach using microfilters have been widely used to capture CTCs because of the difference in size and stiffness of the cells compared to other hemocytes. Isolation of rare cells based on their size was the original CTC enrichment technique and it demonstrated a simple yet rapid method that enhanced the recovery of cells with high throughput. In this review, we highlight key technical aspects of filter-based isolation, detection, and characterization of CTCs, and compare the clinical performance of filter-based devices with the approved platforms and immunoassays used for the analysis of CTCs. We have also discussed future prospective and incorporation of advances in immunochemistry technique into the filter-based platforms for enhancing the utility in clinical settings.

A microfluidic optical platform for real-time monitoring of pH and oxygen in microfluidic bioreactors and organ-on-chip devices.

There is a growing interest to develop microfluidic bioreactors and organ-on-chip platforms with integrated sensors to monitor their physicochemical properties and to maintain a well-controlled microenvironment for cultured organoids. Conventional sensing devices cannot be easily integrated with microfluidic organ-on-chip systems with low-volume bioreactors for continual monitoring. This paper reports on the development of a multi-analyte optical sensing module for dynamic measurements of pH and dissolved oxygen levels in the culture medium. The sensing system was constructed using low-cost electro-optics including light-emitting diodes and silicon photodiodes. The sensing module includes an optically transparent window for measuring light intensity, and the module could be connected directly to a perfusion bioreactor without any specific modifications to the microfluidic device design. A compact, user-friendly, and low-cost electronic interface was developed to control the optical transducer and signal acquisition from photodiodes. The platform enabled convenient integration of the optical sensing module with a microfluidic bioreactor. Human dermal fibroblasts were cultivated in the bioreactor, and the values of pH and dissolved oxygen levels in the flowing culture medium were measured continuously for up to 3 days. Our integrated microfluidic system provides a new analytical platform with ease of fabrication and operation, which can be adapted for applications in various microfluidic cell culture and organ-on-chip devices.

Humidified microcontact printing of proteins: universal patterning of proteins on both low and high energy surfaces.

Microcontact printing (µCP) of proteins is widely used for biosensors and cell biology but is constrained to printing proteins adsorbed to a low free energy, hydrophobic surface to a high free energy, hydrophilic surface. This strongly limits µCP as harsh chemical treatments are required to form a high energy surface. Here, we introduce humidified µCP (HµCP) of proteins which enables universal printing of protein on any smooth surface. We found that by flowing water in proximity to proteins adsorbed on a hydrophilized stamp, the water vapor diffusing through the stamp enables the printing of proteins on both low and high energy surfaces. Indeed, when proteins are printed using stamps with increasing spacing between water-filled microchannels, only proteins adjacent to the channels are transferred. The vapor transport through the stamp was modeled, and by comparing the humidity profiles with the protein patterns, 88% relative humidity in the stamp was identified as the threshold for HµCP. The molecular forces occurring between PDMS, peptides, and glass during printing were modeled ab initio to confirm the critical role water plays in the transfer. Using HµCP, we introduce straightforward protocols to pattern multiple proteins side-by-side down to nanometer resolution without the need for expensive mask aligners, but instead exploiting self-alignment effects derived from the stamp geometry. Finally, we introduce vascularized HµCP stamps with embedded microchannels that allow printing proteins as arbitrary, large areas patterns with nanometer resolution. This work introduces the general concept of water-assisted µCP and opens new possibilities for "solvent-assisted" printing of proteins and of other nanoparticles.

Future of portable devices for plant pathogen diagnosis.

The demand for rapid and accurate diagnosis of plant diseases has risen in the last decade. On-site diagnosis of single or multiple pathogens using portable devices is the first step in this endeavour. Despite extensive attempts to develop portable devices for pathogen detection, current technologies are still restricted to detecting known pathogens with limited detection accuracy. Developing new detection techniques for rapid and accurate detection of multiple plant pathogens and their associated variants is essential. Recent single DNA sequencing technologies are a promising new avenue for developing future portable devices for plant pathogen detection. In this review, we detail the current progress in portable devices and technologies used for detecting plant pathogens, the current position of emerging sequencing technologies for analysis of plant genomics, and the future of portable devices for rapid pathogen diagnosis.

Microfluidic positioning of pollen grains in lab-on-a-chip for single cell analysis.

A lab-on-a-chip device with a knot shaped microfluidic network is presented to enable trapping of single pollen grains at the entrances of a series of microchannels. This set-up serves to create identical growth conditions for serially arranged tip growing plant cells such as pollen tubes. The design consists of an inlet to introduce the pollen suspension into the chip, three outlets to evacuate excess medium or cells, a distribution chamber to guide the pollen grains toward the growth microchannels and a serial arrangement of microchannels with different geometries connected to the distribution chamber. These microchannels are to harbor the individual pollen tubes. Two different criteria were established to assess the efficiency and optimize the device: trapping probability and uniformity of fluid flow conditions within the microchannels. The performance of different geometries of the microfluidic network was numerically analyzed and experimentally tested.

Applications of microfluidics for studying growth mechanisms of tip growing pollen tubes.

Pollen tube, the fastest tip growing plant cell, plays essential role in life cycle of flowering plants. It is extremely sensitive to external cues and this makes it as a suitable cellular model for characterizing the cell response to the influence of various signals involved in cellular growth metabolism. For in-vitro study of pollen tube growth, it is essential to provide an environment the mimics the internal microenvironment of pollen tube in flower. In this context, microfluidic platforms take advantage of miniaturization for handling small volume of liquids, providing a closed environment for in-vitro single cell analysis, and characterization of cell response to external cues. These platforms have shown their ability for high-throughput cellular analysis with increased accuracy of experiments, and reduced cost and experimental times. Here, we review the recent applications of microfluidic devices for investigating several aspects of biology of pollen tube elongation.

In vitro study of oscillatory growth dynamics of Camellia pollen tubes in microfluidic environment.

A hallmark of tip-growing cells such as pollen tubes and fungal hyphae is their oscillatory growth dynamics. The multiple aspects of this behavior have been studied to identify the regulatory mechanisms that drive the growth in walled cells. However, the limited temporal and spatial resolution of data acquisition has hitherto prevented more detailed analysis of this growth behavior. To meet this challenge, we employed a microfluidic device that is able to trap pollen grains and to direct the growth of pollen tubes along microchannels filled with liquid growth medium. This enabled us to observe the growth behavior of Camellia pollen tubes without the use of the stabilizer agarose and without risking displacement of the cell during time lapse imaging. Using an acquisition interval of 0.5 s, we demonstrate the existence of primary and secondary peak frequencies in the growth dynamics. The effect of sucrose concentration on the growth dynamics was studied through the shift in these peak frequencies indicating the pollen tube's ability to modulate its growth activity.

Quantification of the Young's modulus of the primary plant cell wall using Bending-Lab-On-Chip (BLOC).

Biomechanical and mathematical modeling of plant developmental processes requires quantitative information about the structural and mechanical properties of living cells, tissues and cellular components. A crucial mechanical property of plant cells is the mechanical stiffness or Young's modulus of its cell wall. Measuring this property in situ at single cell wall level is technically challenging. Here, a bending test is implemented in a chip, called Bending-Lab-On-a-Chip (BLOC), to quantify this biomechanical property for a widely investigated cellular model system, the pollen tube. Pollen along with culture medium is introduced into a microfluidic chip and the growing pollen tube is exposed to a bending force created through fluid loading. The flexural rigidity of the pollen tube and the Young's modulus of the cell wall are estimated through finite element modeling of the observed fluid-structure interaction. An average value of 350 MPa was experimentally estimated for the Young's modulus in longitudinal direction of the cell wall of Camellia pollen tubes. This value is in agreement with the result of an independent method based on cellular shrinkage after plasmolysis and with the mechanical properties of in vitro reconstituted cellulose-callose material.