Characterizing a Silver Nanoparticle-Based Electrochemical Biosensor for Shiga Toxin Detection.

Shiga toxins (1, 2) regularly cause outbreaks and food recalls and pose a significant health risk to the infected population. Therefore, new reliable tools are needed to rapidly detect Shiga toxin cost-effectively in food, water, and wastewater before human consumption. Enzyme immunoassay and polymerase chain reaction approaches are the gold standard detection methods for the Shiga toxin. However, these methods require expensive instruments along with expensive reagents, which makes them hard to convert into point-of-use and low-cost systems. This study introduces an electrochemical biosensing method that utilizes silver nanoparticles (AgNPs) as electrochemical tags and commercially available low-cost screen-printed carbon electrodes for detection. This study introduces the modification of reference electrodes on commercially available screen-printed carbon electrodes to detect AgNPs dissolved in nitric acid. This biosensor achieved a 2 ng/mL lowest measured concentration for Shiga toxin-1 in less than 3 h. These biosensor results also showed that the AgNP-based sensor has better linearity (for graph between peak current vs concentration) and lower standard deviation compared to gold nanoparticles (AuNP)-based electrochemical biosensors.

Transgenic expression in zebrafish embryos with an intact chorion by electroporation and microinjection.

Electroporation is regularly used to deliver agents into cells, including transgenic materials, but it is not used for mutating zebrafish embryos due to the lack of suitable systems, information on appropriate operating parameters, and the challenges posed by the protective chorion. Here, a novel method for gene delivery in zebrafish embryos was developed by combining microinjection into the space between the chorion and the embryo followed by electroporation. This method eliminates the need for chorion removal and injecting into the space between the chorion and embryo eliminates the need for finding and identifying key cell locations before performing an injection, making the process much simpler and more automatable. We also developed a microfluidic electroporation system and optimized electric pulse parameters for transgenesis of embryos. The study provided a novel method for gene delivery in zebrafish embryos that can be potentially implemented in a high throughput transgenesis or mutagenesis system.

Utilizing ChatGPT to assist CAD design for microfluidic devices.

ChatGPT is a generative AI model that has garnered tremendous public interest due to its ability to solve diverse problems through high-level reasoning and analysis. Among its features is an ability to create and debug code. While this capability has been explored with conventional programming languages such as Python, it has yet to be applied to computer-aided design (CAD). In this work, we utilized GPT-4 to create functional microfluidic components using OpenSCAD, an open-source CAD software package. Through an iterative dialogue, GPT-4 created functional designs for a helix/spiral, a valve, a t-junction, and a serpentine channel. This concept could facilitate CAD in the future for both technical and non-technical users and can be reasonably extended to other fields.

Insight into the formation and biological effects of natural organic matter corona on silver nanoparticles in water environment using biased cyclical electrical field-flow fractionation.

Natural organic matter (NOM) readily interacts with nanoparticles, leading to the formation of NOM corona structures on their surface. NOM corona formation is closely related to the surface coatings and bioavailability of nanoparticles. However, the mechanism underlying NOM corona formation on silver nanoparticles (AgNPs) remains largely unknown due to the lack of effective analytical methods for identifying the changes in the AgNP surface. Herein, the separation ability of biased cyclical electrical field-flow fractionation (BCyElFFF) for same-sized polyvinyl pyrrolidone-coated and poly(ethylene glycol)-coated silver nanoparticles (AgNPs) with different electrophoretic mobilities was evaluated under various electrical conditions. Then, the mechanism behind the NOM corona formation on these AgNP surfaces was elucidated based on the changes in the elution time and off-line characterization of the collected fractions during their elution time in a BCyElFFF run. Finally, the survival rates of E. coli exposed to polyvinyl pyrrolidone-coated and poly(ethylene glycol)-coated AgNPs with or without NOM collected during repeated BCyElFFF runs were observed to increase with increasing NOM concentration, clearly demonstrating the negative effect of NOM corona structures on the bioavailability of AgNPs. These findings highlight the powerful separation and isolation ability of BCyElFFF in studying the transformation and fate of nanoparticles in aqueous environments.

Using Electroporation to Improve and Accelerate Zebrafish Embryo Toxicity Testing.

Zebrafish have emerged as a useful model for biomedical research and have been used in environmental toxicology studies. However, the presence of the chorion during the embryo stage limits cellular exposure to toxic elements and creates the possibility of a false-negative or reduced sensitivity in fish embryo toxicity testing (FET). This paper presents the use of electroporation as a technique to improve the delivery of toxic elements inside the chorion, increasing the exposure level of the toxins at an early embryo stage (<3 h post-fertilization). A custom-made electroporation device with the required electrical circuitry has been developed to position embryos between electrodes that provide electrical pulses to expedite the entry of molecules inside the chorion. The optimized parameters facilitate material entering into the chorion without affecting the survival rate of the embryos. The effectiveness of the electroporation system is demonstrated using Trypan blue dye and gold nanoparticles (AuNPs, 20-40 nm). Our results demonstrate the feasibility of controlling the concentration of dye and nanoparticles delivered inside the chorion by optimizing the electrical parameters, including pulse width, pulse number, and amplitude. Next, we tested silver nanoparticles (AgNPs, 10 nm), a commonly used toxin that can lower mortality, affect heart rate, and cause phenotypic defects. We found that electroporation of AgNPs reduces the exposure time required for toxicity testing from 4 days to hours. Electroporation for FET can provide rapid entry of potential toxins into zebrafish embryos, reducing the time required for toxicity testing and drug delivery experiments.

Multiple-Streams Focusing-Based Cell Separation in High Viscoelasticity Flow.

Viscoelastic flow has been widely used in microfluidic particle separation processes, in which particles get focused on the channel center in diluted viscoelastic flow. In this paper, the transition from single-stream focusing to multiple-streams focusing (MSF) in high viscoelastic flow is observed, which is applied for cell separation processes. Particle focusing stream bifurcation is caused by the balance between elastic force and viscoelastic secondary flow drag force. The influence of cell physical properties, such as cell dimension, shape, and deformability, on the formation of multiple-streams focusing is studied in detail. Particle separation is realized utilizing different separation criteria. The size-based separation of red (RBC) and white (WBC) blood cells is demonstrated in which cells get focused in different streams based on their dimension difference. Cells with different deformabilities get stretched in the viscoelastic flow, leading to the change of focusing streams, and this property is harnessed to separate red blood cells infected with the malaria parasite, Plasmodium falciparum. The achieved results promote our understanding of particle movement in the high viscoelastic flow and enable new particle manipulation and separation processes for sample treatment in biofluids.

Separation of U87 glioblastoma cell-derived small and medium extracellular vesicles using elasto-inertial flow focusing (a spiral channel).

Nanoscale and microscale cell-derived extracellular vesicle types and subtypes are of significant interest to researchers in biology and medicine. Extracellular vesicles (EVs) have diagnostic and therapeutic potential in terms of biomarker and nanomedicine applications. To enable such applications, EVs must be isolated from biological fluids or separated from other EV types. Developing methods to fractionate EVs is of great importance to EV researchers. Our goal was to begin to develop a device that would separate medium EVs (mEVs, traditionally termed microvesicles or shedding vesicles) and small EVs (sEVs, traditionally termed exosomes) by elasto-inertial effect. We sought to develop a miniaturized technology that works similar to and provides the benefits of differential ultracentrifugation but is more suitable for EV-based microfluidic applications. The aim of this study was to determine whether we could use elasto-inertial focusing to re-isolate and recover U87 mEVs and sEVs from a mixture of mEVs and sEVs isolated initially by one round of differential ultracentrifugation. The studied spiral channel device can continuously process 5 ml of sample fluid per hour. Using the channel, sEVs and mEVs were recovered and re-isolated from a mixture of U87 glioma cell-derived mEVs and sEVs pre-isolated by one round of differential ultracentrifugation. Following two passes through the spiral channel, approximately 55% of sEVs were recovered with 6% contamination by mEVs (the recovered sEVs contained 6% of the total mEVs). In contrast, recovery of U87 mEVs and sEVs re-isolated using a typical second centrifugation wash step was only 8% and 53%, respectively. The spiral channel also performed similar to differential ultracentrifugation in reisolating sEVs while significantly improving mEV reisolation from a mixture of U87 sEVs and mEVs. Ultimately this technology can also be coupled to other microfluidic EV isolation methods in series and/or parallel to improve isolation and minimize loss of EV subtypes.

Viscoelastic Particle Focusing and Separation in a Spiral Channel.

As one type of non-Newtonian fluid, viscoelastic fluids exhibit unique properties that contribute to particle lateral migration in confined microfluidic channels, leading to opportunities for particle manipulation and separation. In this paper, particle focusing in viscoelastic flow is studied in a wide range of polyethylene glycol (PEO) concentrations in aqueous solutions. Polystyrene beads with diameters from 3 to 20 µm are tested, and the variation of particle focusing position is explained by the coeffects of inertial flow, viscoelastic flow, and Dean flow. We showed that particle focusing position can be predicted by analyzing the force balance in the microchannel, and that particle separation resolution can be improved in viscoelastic flows.

Compression of the vascular wall to create a friction fit in a vascular anastomotic coupler.

A previously reported microvascular coupler was shown to effectively create vascular anastomoses, but was too large for practical clinical use. To safely reduce coupler size, certain failure modes needed to be better understood. The coupler functions, in part, by compressing the vessel wall between two concentric rings, creating a friction fit that anchors the device to the vessel. This work investigates the relationship between vessel wall compression and resulting friction fit strength to ensure reducing coupler size will not unduly increase the risk that this friction fit might fail. Vascular walls were compressed to a specified strain and the tensile force required to overcome the resulting friction was measured. Experiments were conducted with various vessel types (Porcine common carotid artery, splenic artery, and jugular vein), across a range of compressive strains (55-95%), and by using either PEEK or HDPE to compress the vessel. Tensile force was increased at a rate of 5 g/min or held constant for 24 h. For experiments with incrementally increasing force, the force at failure varied with compressive strain via a power function. At 70% compression, PEEK produced 4.6 times stronger friction fits than HDPE, and common carotid arteries and splenic arteries produced 1.8 and 1.3 times stronger fits than jugular veins respectively. For experiments where tensile force was applied for 24 h, much lower forces were required to overcome friction. These results were compared to friction fit failure in a coupler prototype and it was found that the prototypes failed at just 30% of the force required to cause vessel slip under the other test conditions. These results were used to develop a model that predicts the probability of device failure via vessel slipping (one design, smaller than previously reported, was estimated to fail at maximum in vivo axial stress once in 500 anastomoses, a potentially safe level of risk).

High efficiency rare sperm separation from biopsy samples in an inertial focusing device.

Immotile and rare sperm isolation from a complex cell background is an essential process for infertility treatment. The traditional sperm collection process from a biopsy sample requires long, tedious searches, yet still results in low sperm retrieval. In this work, a high recovery, high throughput sperm separation process is proposed for the clinical biopsy sperm retrieval process. It is found that sperm have different focusing positions compared with non-sperm cells in the inertial flow, which is explained by a sperm alignment phenomenon. Separation in the spiral channel device results in a 95.6% sperm recovery in which 87.4% of non-sperm cells get removed. Rare sperm isolation from a clinical biopsy sample is performed with the current approach. The chance of finding sperm is shown to increase 8.2 fold in the treated samples. The achieved results highly support this method being used for the development of a rapid biopsy sperm sorting process. In addition, the mechanism was proposed and can be applied for the high-efficiency separation of non-spherical particles in general.

Development and Testing of a Continuous Flow-Electrical-Split-Flow Lateral Transport Thin Separation System (Fl-El-SPLITT).

In this work, a new high-volume, continuous particle separation device that separates based upon size and charge is described. Two continuous flow-electrical-split-flow lateral transport thin (Fl-El-SPLITT) device architectures (a platinum electrode on a porous membrane and a porous graphite electrode under a membrane) were developed and shown to improve particle separations over a purely electrical-SPLITT device. The graphite FL-El-SPLITT device architecture achieved the best separation of approximately 60% of small (28 nm) vs large (1000 nm) polystyrene particles. Fl-El-SPLITT (platinum) achieved a 75% separation on a single pass using these same particles. Fl-El-SPLITT (platinum) achieved a moderate 26% continuous separation of U87 glioma cell-derived small extracellular vesicles (EVs) from medium EVs. Control parameter testing showed that El-SPLITT continuously directed particle motility within a channel to exit a selected port based upon the applied voltage using either direct current or alternating current. The transition from one port to the other was dependent upon the voltage applied. Both large and small polystyrene particles transitioned together rather than separating at each of the applied voltages. These data present the first ever validation of El-SPLITT in continuous versus batch format. The Fl-El-SPLITT device architecture, monitoring, and electrical and fluid interfacing systems are described in detail for the first time. Capabilities afforded to the system by the flow addition include enhanced particle separation as well as the ability to filter out small particles or desalinate fluids. High-throughput continuous separations based upon electrophoretic mobility will be streamlined by this new technique that combines electrical and flow fields into a single device.

SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations.

The unprecedented global pandemic known as SARS-CoV-2 has exercised to its limits nearly all aspects of modern viral diagnostics. In doing so, it has illuminated both the advantages and limitations of current technologies. Tremendous effort has been put forth to expand our capacity to diagnose this deadly virus. In this work, we put forth key observations in the functionality of current methods for SARS-CoV-2 diagnostic testing. These methods include nucleic acid amplification-, CRISPR-, sequencing-, antigen-, and antibody-based detection methods. Additionally, we include analysis of equally critical aspects of COVID-19 diagnostics, including sample collection and preparation, testing models, and commercial response. We emphasize the integrated nature of assays, wherein issues in sample collection and preparation could impact the overall performance in a clinical setting.

Optimization of a microfluidic spiral channel used to separate sperm from blood cells.

Assisted reproductive technology includes medical procedures that confront the problem of infertility. In some cases of male infertility, blood cells are present in the sperm containing samples and must be removed. Spiral-channel devices have been developed to perform this task, but there is a strong need to increase their throughput. In this work, the theory behind the separation is employed to optimize the device for increased throughput. An existing device that is known to separate sperm and blood cells with a rectangular cross section of 600 × 100 µm2 was used as the baseline. Using its physics, theoretical models were generated to explore theoretical performances of larger-size channels. The models suggested that a channel of size 800 × 133 µm2 would likely work. This geometry enabled the throughput to be increased by 50%, from 2 ml/min in the case of the baseline-size to 3 ml/min in the designed device. Experiments using the larger device resulted in a recovery of more than 90% of sperm cells while removing 89% of red blood cells (RBCs). In comparison, the reference device results in a 90% recovery of sperm cells while removing 74% of white blood cells (WBCs). The length of the channel was also reduced to reduce the pressure required to operate the chip. Literature has shown the removal of WBCs to be higher than that of RBCs due to their larger size, spherical shape, and comparatively low deformability, suggesting that the revised chip would be faster and better for the separation of sperm and all blood cells.

Characterization of Human Glioblastoma versus Normal Plasma-Derived Extracellular Vesicles Preisolated by Differential Centrifugation Using Cyclical Electrical Field-Flow Fractionation.

Although many properties for small extracellular vesicles (sEVs, formerly termed "exosomes") isolated at ∼100 000g are known, a wide range of values are reported for their electrophoretic mobility (EM) measurements. This paper reports for the first time the effect of dilution on the EM of U87 glioblastoma cell-derived and plasma-derived sEVs and medium size EVs (mEVs, commonly termed "oncosomes") preisolated by differential centrifugation. Furthermore, the effect of resalting on the EM of sEVs and mEVs was evaluated. The EM of U87 sEVs and U87 mEVs showed an increase as the salt concentration decreased to 0.005% of the initial salt concentration. However, for the plasma sEVs and plasma mEVs, the electrophoretic mobility increased as the salt concentration decreased to 0.01% of the initial salt concentration and then increased to its initial value when the salt concentration decreased to 0.005% of the initial salt concentration. For both U87 and plasma sEVs and mEVs, the EM remained almost constant when the concentration of the particles changed and the salt concentration was kept the same as its initial value. This indicates that the EM of EVs is only a function of the salt concentration of the buffer and is independent of the concentration of the particles. The sEVs and mEVs were separated with cyclical ElFFF for the first time. The results indicate that ElFFF was able to fractionate the EVs, and a crescent-shaped trend was found for the retention time when the applied AC voltage was altered (increased).

Towards a better testicular sperm extraction: novel sperm sorting technologies for non-motile sperm extracted by microdissection TESE.

Non-obstructive azoospermia (NOA) is the most severe form of male factor infertility. It is characterized by a lack of spermatogenesis in the seminiferous tubules. Microdissection testicular sperm extraction (microTESE) has significantly improved testicular sperm retrieval rates compared to conventional techniques for NOA. Following testicular biopsy, the sperm is usually non-motile and contained within seminiferous tubules requiring extensive laboratory processing to find individual sperm sufficient for artificial reproductive technologies (ART). Current techniques include mechanical and enzymatic processing which is time-consuming and often damaging to sperm. We review novel techniques that may help improve sperm retrieval rates after microTESE including microfluidics (dielectrophoretic cell sorting, spiral channel sorting, and pinched flow fractionation), fluorescence-activated cell sorting (FACS), and magnetic-activated cell sorting (MACS).

Microfluidic System for Rapid Isolation of Sperm From Microdissection TESE Specimens.

OBJECTIVES: To demonstrate a novel prototype microfluidic system for rapid isolation of sperm from real and simulated microdissection testicular sperm extraction samples. METHODS: The novel microfluidic system was tested using minced testicular biopsies from patients with nonobstructive azoospermia. The samples were split into 2 portions, conventional processing vs microfluidic. The embryologists were blinded to the processing protocol and searched the specimens for sperm after processing. We recorded the number of sperm found and the time to sperm identification and compared the sperm retrieval rates. RESULTS: When compared to conventional methods, samples processed through the microfluidic system were cleaner (decreased somatic cells/debris), with the average number of sperm identified per minute improving from 1.52 sperm per minute for the control and 13.5 sperm per minute with the device yielding an 8.88 fold improvement in the sperm found per minute for the device as compared to the control. Preliminary viability and morphology tests show a minimal impact on sperm processed through the microfluidic system. CONCLUSION: The presented microfluidic system can facilitate rapid and efficient isolation of sperm from microdissection testicular sperm extraction samples. A prospective clinical trial to verify these results is needed to confirm this preliminary data.

Characterization and differential retention of Q beta bacteriophage virus-like particles using cyclical electrical field-flow fractionation and asymmetrical flow field-flow fractionation.

Virus-like particles (VLPs) are widely used in medicine, but can be difficult to characterize and isolate from aggregates. In this research, primarily cyclical electrical field-flow fractionation (CyElFFF) coupled with multi-angle light scattering (MALS), and dynamic light scattering (DLS) detectors, was used for the first time to perform size and electrical characterization of three different types of Q beta bacteriophage virus-like particles (VLPs): a blank Q beta bacteriophage which is denoted as VLP and two conjugated ones with different peptides. The CyElFFF results were verified with transmission electron microscopy (TEM). Asymmetrical flow field-flow fractionation (AF4) coupled with MALS was also applied using conditions similar to those used in the CyElFFF experiments, and the results of the two techniques were compared to each other. Using these techniques, the size and electrophoretic characteristics of the fractionated VLPs in CyElFFF were obtained. The results indicate that CyElFFF can be used to obtain a clear distribution of electrophoretic mobilities for each type of VLP. Accordingly, CyElFFF was able to differentially retain and isolate VLPs with high surface electric charge/electrophoretic mobility from the ones with low electric charge/electrophoretic mobility. Regarding the size characterization, the size distribution of the eluted VLPs was obtained using both techniques. CyElFFF was able to identify subpopulations that did not appear in the AF4 results by generating a shoulder peak, whereas AF4 produced a single peak. Different size characteristics of the VLPs appearing in the shoulder peak and the main peak indicate that CyElFFF was able to isolate aggregated VLPs from the monomers partially. Graphical abstract.

A Tunable Microfluidic Device Enables Cargo Encapsulation by Cell- or Organelle-Sized Lipid Vesicles Comprising Asymmetric Lipid Bilayers.

Cellular membranes play host to a wide variety of morphologically and chemically complex processes. Although model membranes, like liposomes, are already widely used to reconstitute and study these processes, better tools are needed for making model bilayers that faithfully mimic cellular membranes. Existing methods for fabricating cell-sized (µm) or organelle-sized (tens to hundreds of nanometers) lipid vesicles have distinctly different requirements. Of particular note for biology, it remains challenging for any technique to efficiently encapsulate fragile cargo molecules or to generate liposomes with stable, asymmetric lipid leaflets within the bilayer. Here a tunable microfluidic device and protocol for fabricating liposomes with desired diameters ranging from ≈10 µm to ≈100 nm are described. Lipid vesicle size is templated by the simple inclusion of a polycarbonate filter within the microfluidic system and tuned with flow rate. It is shown that the vesicles made with this device are stable, unilamellar, lipid asymmetric, and capable of supporting transmembrane protein assembly, peripheral membrane protein binding, as well as soluble cargo encapsulation (including designer nanocages for biotechnology applications). These fabricated vesicles provide a new platform for studying the biophysically rich processes found within lipid-lipid and lipid-protein systems typically associated with cellular membranes.

Hydrodynamic cavitation for the rapid separation and electrochemical detection of Cryptosporidium parvum and Escherichia coli O157:H7 in ground beef.

Foodborne illnesses are a major contributor to misery and health challenges in both rich and poor nations. Illnesses from pathogens such as Escherichia coli and Cryptosporidium parvum oocysts account for most of the cases of diarrhea in the world. Many standard methods exist for detecting these pathogens in water. However, these standard methods do not readily translate to the detection of the same pathogens in food. Detection techniques for pathogens in food are often inadequate, due to their inability to completely separate pathogens from food matrices. In this paper, we present a technique to separate and detect both Escherichia coli cells and Cryptosporidium parvum oocysts that have been embedded in ground meat. We achieve this objective by combining enzymatic digestion of the meat, hydrodynamic cavitation to disassemble pathogens from the meat, immunomagnetic separation to purify meat samples and indirect electrochemical detection of the target pathogens. Our use of hydrodynamic cavitation to separate pathogens is compared against an industry standard separation technique. Results indicate that the use of hydrodynamic cavitation amplifies the detection capabilities of our sensing technique and is overall comparable to or better than conventional stomacher sample preparation.