Free Flow Ion Concentration Polarization Focusing (FF-ICPF).

Electrokinetic separation techniques in microfluidics are a powerful analytical chemistry tool, although an inherent limitation of microfluidics is their low sample throughput. In this article we report a free-flow variant of an electrokinetic focusing method, namely ion concentration polarization focusing (ICPF). The analytes flow continuously through the system via pressure driven flow while they separate and concentrate perpendicularly to the flow by ICPF. We demonstrate free flow ion concentration polarization focusing (FF-ICPF) in two operating modes, namely peak and plateau modes. Additionally, we showed the separation resolution could be improved by the use of an electrophoretic spacer. We report a concentration factor of 10 in human blood plasma in continuous flow at a flow rate of 15 µL min-1.

Flow-Free Microfluidic Device for Quantifying Chemotaxis in Spermatozoa.

Current male fertility diagnosis tests focus on assessing the quality of semen samples by studying the concentration, total volume, and motility of spermatozoa. However, other characteristics such as the chemotactic ability of a spermatozoon might influence the chance of fertilization. Here we describe a simple, easy to fabricate and handle, flow-free microfluidic chip to test the chemotactic response of spermatozoa made out of a hybrid hydrogel (8% gelatin/1% agarose). A chemotaxis experiment with 1 µM progesterone was performed that significantly demonstrated that boar spermatozoa are attracted by a progesterone gradient.

Mass Transport Determined Silica Nanowires Growth on Spherical Photonic Crystals with Nanostructure-Enabled Functionalities.

A robust and facile method has been developed to obtain directional growth of silica nanowires (SiO2 NWs) by regulating mass transport of silicon monoxide (SiO) vapor. SiO2 NWs are grown by vapor-liquid-solid (VLS) process on a surface of gold-covered spherical photonic crystals (SPCs) annealed at high temperature in an inert gas atmosphere in the vicinity of a SiO source. The SPCs are prepared from droplet confined colloidal self-assembly. SiO2 NW morphology is governed by diffusion-reaction process of SiO vapor, whereby directional growth of SiO2 NWs toward the low SiO concentration is obtained at locations with a high SiO concentration gradient, while random growth is observed at locations with a low SiO concentration gradient. Growth of NWs parallel to the supporting substrate surface is of great importance for various applications, and this is the first demonstration of surface-parallel growth by controlling mass transport. This controllable NW morphology enables production of SPCs covered with a large number of NWs, showing multilevel micro-nano feature and high specific surface area for potential applications in superwetting surfaces, oil/water separation, microreactors, and scaffolds. In addition, the controllable photonic stop band properties of this hybrid structure of SPCs enable the potential applications in photocatalysis, sensing, and light harvesting.

Increasing the Sensitivity of Electrochemical DNA Detection by a Micropillar-Structured Biosensing Surface.

The available active surface area and the density of probes immobilized on this surface are responsible for achieving high specificity and sensitivity in electrochemical biosensors that detect biologically relevant molecules, including DNA. Here, we report the design of gold-coated, silicon micropillar-structured electrodes functionalized with modified poly-l-lysine (PLL) as an adhesion layer to concomitantly assess the increase in sensitivity with the increase of the electrochemical area and control over the probe density. By systematically reducing the center-to-center distance between the pillars (pitch), denser micropillar arrays were formed at the electrode, resulting in a larger sensing area. Azido-modified peptide nucleic acid (PNA) probes were click-reacted onto the electrode interface, exploiting PLL with appended oligo(ethylene glycol) (OEG) and dibenzocyclooctyne (DBCO) moieties (PLL-OEG-DBCO) for antifouling and probe binding properties, respectively. The selective electrochemical sandwich assay formation, composed of consecutive hybridization steps of the target complementary DNA (cDNA) and reporter DNA modified with the electroactive ferrocene functionality (rDNA-Fc), was monitored by quartz crystal microbalance. The DNA detection performance of micropillared electrodes with different pitches was evaluated by quantifying the cyclic voltammetric response of the surface-confined rDNA-Fc. By decrease of the pitch of the pillar array, the area of the electrode was enhanced by up to a factor 10.6. A comparison of the electrochemical data with the geometrical area of the pillared electrodes confirmed the validity of the increased sensitivity of the DNA detection by the design of the micropillar array.

Point-of-care vertical flow allergen microarray assay: proof of concept.

BACKGROUND: Sophisticated equipment, lengthy protocols, and skilled operators are required to perform protein microarray-based affinity assays. Consequently, novel tools are needed to bring biomarkers and biomarker panels into clinical use in different settings. Here, we describe a novel paper-based vertical flow microarray (VFM) system with a multiplexing capacity of at least 1480 microspot binding sites, colorimetric readout, high sensitivity, and assay time of <10 min before imaging and data analysis. METHOD: Affinity binders were deposited on nitrocellulose membranes by conventional microarray printing. Buffers and reagents were applied vertically by use of a flow controlled syringe pump. As a clinical model system, we analyzed 31 precharacterized human serum samples using the array system with 10 allergen components to detect specific IgE reactivities. We detected bound analytes using gold nanoparticle conjugates with assay time of ≤10 min. Microarray images were captured by a consumer-grade flatbed scanner. RESULTS: A sensitivity of 1 ng/mL was demonstrated with the VFM assay with colorimetric readout. The reproducibility (CV) of the system was <14%. The observed concordance with a clinical assay, ImmunoCAP, was R(2) = 0.89 (n = 31). CONCLUSIONS: In this proof-of-concept study, we demonstrated that the VFM assay, which combines features from protein microarrays and paper-based colorimetric systems, could offer an interesting alternative for future highly multiplexed affinity point-of-care testing.

A lateral flow paper microarray for rapid allergy point of care diagnostics.

There is a growing need for multiplexed specific IgE tests that can accurately evaluate patient sensitization profiles. However, currently available commercial tests are either single/low-plexed or require sophisticated instrumentation at considerable cost per assay. Here, we present a novel convenient lateral flow microarray-based device that employs a novel dual labelled gold nanoparticle-strategy for rapid and sensitive detection of a panel of 15 specific IgE responses in 35 clinical serum samples. Each gold nanoparticle was conjugated to an optimized ratio of HRP and anti-IgE, allowing significant enzymatic amplification to improve the sensitivity of the assay as compared to commercially available detection reagents. The mean inter-assay variability of the developed LFM assay was 12% CV, and analysis of a cohort of clinical samples (n = 35) revealed good general agreement with ImmunoCAP, yet with a varying performance among allergens (AUC = [0.54-0.88], threshold 1 kU). Due to the rapid and simple procedure, inexpensive materials and read-out by means of a consumer flatbed scanner, the presented assay may provide an interesting low-cost alternative to existing multiplexed methods when thresholds >1 kU are acceptable.

Innovative electrode and chip designs for transendothelial electrical resistance measurements in organs-on-chips.

Many different epithelial and endothelial barriers in the human body ensure the proper functioning of our organs by controlling which substances can pass from one side to another. In recent years, organs-on-chips (OoC) have become a popular tool to study such barriers in vitro. To assess the proper functioning of these barriers, we can measure the transendothelial electrical resistance (TEER) which indicates how easily ions can cross the cell layer when a current is applied between electrodes on either side. TEER measurements are a convenient method to quantify the barrier properties since it is a non-invasive and label-free technique. Direct integration of electrodes for TEER measurements into OoC allows for continuous monitoring of the barrier, and fixed integration of the electrodes improves the reproducibility of the measurements. In this review, we will give an overview of different electrode and channel designs that have been used to measure the TEER in OoC. After giving some insight into why biological barriers are an important field of study, we will explain the theory and practice behind measuring the TEER in in vitro systems. Next, this review gives an overview of the state of the art in the field of integrated electrodes for TEER measurements in OoC, with a special focus on alternative chip and electrode designs. Finally, we outline some of the remaining challenges and provide some suggestions on how to overcome these challenges.

'Staying Hot': Investigating the influence of overnight conditions on the penile skin temperature during male sexual arousal-A novel methodology for nocturnal erection detection.

Objective: The objective of this study is to assess the impact of overnight environmental conditions on erectile penile temperature within a controlled setting, with the aim of investigating the feasibility of using temperature measurements for nocturnal erection detection in erectile dysfunction diagnostics. Subjects/patients and methods: We conducted a proof-of-concept study involving 10 healthy male participants aged 20 to 25. The study was carried out at the Department of Urology, St. Antonius Ziekenhuis, the Netherlands. Penile temperature thermistor measurements were taken during visually aroused erections of participants in naked state and in simulated overnight condition (underwear and blankets). Main outcome variables were peak and baseline temperature during erectile periods. To minimize the impact of differences in erectile strength and duration between consecutive measurements, we applied randomization to the order of the environmental conditions. Results: We observed a significant increase in penile temperature during erection in both the naked (p < 0.01) and simulated overnight condition (p < 0.01). The mean temperature increase was 1.70 and 0.67°C, respectively. While penile temperature returned to baseline immediately after naked erections, the 'Staying Hot effect' was noted in the simulated overnight condition measurements, where the temperature remained elevated at peak temperature for the entire 30-min period following the erection. Conclusions: The findings from this study indicate that the penile temperature not only significantly increases during naked sexual arousal but is also detectable under simulated overnight conditions. This underscores the potential of using temperature measurements for nocturnal erection detection, representing a crucial initial step in developing a modernized, non-invasive sensor system for ambulatory erectile dysfunction diagnostics. Further research, including an overnight study, is needed to gain insights into the feasibility of utilizing penile temperature measurements for nocturnal erection detection and to assess the impact of the 'Staying Hot effect' on subsequent erection detection.

Label-free, high-throughput, electrical detection of cells in droplets.

Today, droplet based microfluidics has become a standard platform for high-throughput single cell experimentation and analysis. However, until now no label-free, integrated single cell detection and discrimination method in droplets is available. We present here a microfluidic chip for fast (>100 Hz) and label-free electrical impedance based detection of cells in droplets. The microfluidic glass-PDMS device consists of two main components, the droplet generator and the impedance sensor. The planar electrode pair in the main channel allows the detection of only cells and cell containing droplets passing the electrodes using electrical impedance measurements. At a measurement frequency of 100 kHz non-viable cells, in low-conducting (LC) buffer, show an increase in impedance, due to the resistive effect of the membrane. The opposite effect, an impedance decrease, was observed when a viable cell passed the electrode pair, caused by the presence of the conducting cytoplasm. Moreover, we found that the presence of a viable cell in a droplet also decreased the measured electrical impedance. This impedance change was not visible when a droplet containing a non-viable cell or an empty droplet passed the electrode pair. A non-viable cell in a droplet and an empty droplet were equally classified. Hence, droplets containing (viable) cells can be discriminated from empty droplets. In conclusion, these results provide us with a valuable method to label-free detect and select viable cells in droplets. Furthermore, the proposed method provides the first step towards additional information regarding the encapsulated cells (e.g., size, number, morphology). Moreover, this all-electric approach allows for all-integrated Lab on a Chip (LOC) devices for cell applications using droplet-based platforms.

An optical aptasensor for real-time quantification of endotoxin: From ensemble to single-molecule resolution.

Endotoxin is a deadly pyrogen, rendering it crucial to monitor with high accuracy and efficiency. However, current endotoxin detection relies on multistep processes that are labor-intensive, time-consuming, and unsustainable. Here, we report an aptamer-based biosensor for the real-time optical detection of endotoxin. The endotoxin sensor exploits the distance-dependent scattering of gold nanoparticles (AuNPs) coupled to a gold nanofilm. This is enabled by the conformational changes of an endotoxin-specific aptamer upon target binding. The sensor can be used in an ensemble mode and single-particle mode under dark-field illumination. In the ensemble mode, the sensor is coupled with a microspectrometer and exhibits high specificity, reliability (i.e., linear concentration to signal profile in logarithmic scale), and reusability for repeated endotoxin measurements. Individual endotoxins can be detected by monitoring the color of single AuNPs via a color camera, achieving single-molecule resolution. This platform can potentially advance endotoxin detection to safeguard medical, food, and pharmaceutical products.

Automated assessment of human engineered heart tissues using deep learning and template matching for segmentation and tracking.

The high rate of drug withdrawal from the market due to cardiovascular toxicity or lack of efficacy, the economic burden, and extremely long time before a compound reaches the market, have increased the relevance of human in vitro models like human (patient-derived) pluripotent stem cell (hPSC)-derived engineered heart tissues (EHTs) for the evaluation of the efficacy and toxicity of compounds at the early phase in the drug development pipeline. Consequently, the EHT contractile properties are highly relevant parameters for the analysis of cardiotoxicity, disease phenotype, and longitudinal measurements of cardiac function over time. In this study, we developed and validated the software HAARTA (Highly Accurate, Automatic and Robust Tracking Algorithm), which automatically analyzes contractile properties of EHTs by segmenting and tracking brightfield videos, using deep learning and template matching with sub-pixel precision. We demonstrate the robustness, accuracy, and computational efficiency of the software by comparing it to the state-of-the-art method (MUSCLEMOTION), and by testing it with a data set of EHTs from three different hPSC lines. HAARTA will facilitate standardized analysis of contractile properties of EHTs, which will be beneficial for in vitro drug screening and longitudinal measurements of cardiac function.

Density Control over MBD2 Receptor-Coated Surfaces Provides Superselective Binding of Hypermethylated DNA.

Using the biomarker hypermethylated DNA (hmDNA) for cancer detection requires a pretreatment to isolate or concentrate hmDNA from nonmethylated DNA. Affinity chromatography using a methyl binding domain-2 (MBD2) protein can be used, but the relatively low enrichment selectivity of MBD2 limits its clinical applicability. Here, we developed a superselective, multivalent, MBD2-coated platform to improve the selectivity of hmDNA enrichment. The multivalent platform employs control over the MBD2 surface receptor density, which is shown to strongly affect the binding of DNA with varying degrees of methylation, improving both the selectivity and the affinity of DNAs with higher numbers of methylation sites. Histidine-10-tagged MBD2 was immobilized on gold surfaces with receptor density control by tuning the amount of nickel nitrilotriacetic acid (NiNTA)-functionalized thiols in a thiol-based self-assembled monolayer. The required MBD2 surface receptor densities for DNA surface binding decreases for DNA with higher degrees of methylation. Both higher degrees of superselectivity and surface coverages were observed upon DNA binding at increasing methylation levels. Adopting the findings of this study into hmDNA enrichment of clinical samples has the potential to become more selective and sensitive than current MBD2-based methods and, therefore, to improve cancer diagnostics.

Systematic characterization of cleanroom-free fabricated macrovalves, demonstrating pumps and mixers for automated fluid handling tuned for organ-on-chip applications.

Integrated valves enable automated control in microfluidic systems, as they can be applied for mixing, pumping and compartmentalization purposes. Such automation would be highly valuable for applications in organ-on-chip (OoC) systems. However, OoC systems typically have channel dimensions in the range of hundreds of micrometers, which is an order of magnitude larger than those of typical microfluidic valves. The most-used fabrication process for integrated, normally open polydimethylsiloxane (PDMS) valves requires a reflow photoresist that limits the achievable channel height. In addition, the low stroke volumes of these valves make it challenging to achieve flow rates of microliters per minute, which are typically required in OoC systems. Herein, we present a mechanical 'macrovalve' fabricated by multilayer soft lithography using micromilled direct molds. We demonstrate that these valves can close off rounded channels of up to 700 µm high and 1000 µm wide. Furthermore, we used these macrovalves to create a peristaltic pump with a pumping rate of up to 48 µL/min and a mixing and metering device that can achieve the complete mixing of a volume of 6.4 µL within only 17 s. An initial cell culture experiment demonstrated that a device with integrated macrovalves is biocompatible and allows the cell culture of endothelial cells over multiple days under continuous perfusion and automated medium refreshment.

Label-Free Microfluidic Impedance Cytometry for Acrosome Integrity Assessment of Boar Spermatozoa.

Microfluidics and lab-on-chip technologies have been used in a wide range of biomedical applications. They are known as versatile, rapid, and low-cost alternatives for expensive equipment and time-intensive processing. The veterinary industry and human fertility clinics could greatly benefit from label-free and standardized methods for semen analysis. We developed a tool to determine the acrosome integrity of spermatozoa using microfluidic impedance cytometry. Spermatozoa from boars were treated with the calcium ionophore A23187 to induce acrosome reaction. The magnitude, phase and opacity of individual treated and non-treated (control) spermatozoa were analyzed and compared to conventional staining for acrosome integrity. The results show that the opacity at 19 MHz over 0.5 MHz is associated with acrosome integrity with a cut-off threshold at 0.86 (sensitivity 98%, specificity 97%). In short, we have demonstrated that acrosome integrity can be determined using opacity, illustrating that microfluidic impedance cytometers have the potential to become a versatile and efficient alternative in semen analysis and for fertility treatments in the veterinary industry and human fertility clinics.

Contractility analysis of human engineered 3D heart tissues by an automatic tracking technique using a standalone application.

The use of Engineered Heart Tissues (EHT) as in vitro model for disease modeling and drug screening has increased, as they provide important insight into the genetic mechanisms, cardiac toxicity or drug responses. Consequently, this has highlighted the need for a standardized, unbiased, robust and automatic way to analyze hallmark physiological features of EHTs. In this study we described and validated a standalone application to analyze physiological features of EHTs in an automatic, robust, and unbiased way, using low computational time. The standalone application "EHT Analysis" contains two analysis modes (automatic and manual) to analyzes the contractile properties and the contraction kinetics of EHTs from high speed bright field videos. As output data, the graphs of displacement, contraction force and contraction kinetics per file will be generated together with the raw data. Additionally, it also generates a summary file containing all the data from the analyzed files, which facilitates and speeds up the post analysis. From our study we highlight the importance of analyzing the axial stress which is the force per surface area (µN/mm2). This allows to have a readout overtime of tissue compaction, axial stress and leave the option to calculate at the end point of an experiment the physiological cross-section area (PSCA). We demonstrated the utility of this tool by analyzing contractile properties and compaction over time of EHTs made out of a double reporter human pluripotent stem cell (hPSC) line (NKX2.5EGFP/+-COUP-TFIImCherry/+) and different ratios of human adult cardiac fibroblasts (HCF). Our standalone application "EHT Analysis" can be applied for different studies where the physiological features of EHTs needs to be analyzed under the effect of a drug compound or in a disease model.

A New Versatile Platform for Assessment of Improved Cardiac Performance in Human-Engineered Heart Tissues.

Cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) hold a great potential as human in vitro models for studying heart disease and for drug safety screening. Nevertheless, their associated immaturity relative to the adult myocardium limits their utility in cardiac research. In this study, we describe the development of a platform for generating three-dimensional engineered heart tissues (EHTs) from hPSC-CMs for the measurement of force while under mechanical and electrical stimulation. The modular and versatile EHT platform presented here allows for the formation of three tissues per well in a 12-well plate format, resulting in 36 tissues per plate. We compared the functional performance of EHTs and their histology in three different media and demonstrated that tissues cultured and maintained in maturation medium, containing triiodothyronine (T3), dexamethasone, and insulin-like growth factor-1 (TDI), resulted in a higher force of contraction, sarcomeric organization and alignment, and a higher and lower inotropic response to isoproterenol and nifedipine, respectively. Moreover, in this study, we highlight the importance of integrating a serum-free maturation medium in the EHT platform, making it a suitable tool for cardiovascular research, disease modeling, and preclinical drug testing.

A CRISPR/Cas12a-assisted in vitro diagnostic tool for identification and quantification of single CpG methylation sites.

The excellent specificity and selectivity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) is determined by CRISPR RNA's (crRNA's) interchangeable spacer sequence, as well as the position and number of mismatches between target sequence and the crRNA sequence. Some diseases are characterized by epigenetic alterations rather than nucleotide changes, and are therefore unsuitable for CRISPR-assisted sensing methods. Here we demonstrate an in vitro diagnostic tool to discriminate single CpG site methylation in DNA by the use of methylation-sensitive restriction enzymes (MSREs) followed by Cas12a-assisted sensing. Non-methylated sequences are digested by MSREs, resulting in fragmentation of the target sequence that influences the R-loop formation between crRNA and target DNA. We show that fragment size, fragmentation position and number of fragments influence the subsequent collateral trans-cleavage activity towards single stranded DNA (ssDNA), enabling deducting the methylation position from the cleavage activity. Utilizing MSREs in combination with Cas12a, single CpG site methylation levels of a cancer gene are determined. The modularity of both Cas12a and MSREs provides a high level of versatility to the Cas12a-MSRE combined sensing method, which opens the possibility to easily and rapidly study single CpG methylation sites for disease detection.

Bottom-Up Assembled Photonic Crystals for Structure-Enabled Label-Free Sensing.

Photonic crystals (PhCs) display photonic stop bands (PSBs) and at the edges of these PSBs transport light with reduced velocity, enabling the PhCs to confine and manipulate incident light with enhanced light-matter interaction. Intense research has been devoted to leveraging the optical properties of PhCs for the development of optical sensors for bioassays, diagnosis, and environmental monitoring. These applications have furthermore benefited from the inherently large surface area of PhCs, giving rise to high analyte adsorption and the wide range of options for structural variations of the PhCs leading to enhanced light-matter interaction. Here, we focus on bottom-up assembled PhCs and review the significant advances that have been made in their use as label-free sensors. We describe their potential for point-of-care devices and in the review include their structural design, constituent materials, fabrication strategy, and sensing working principles. We thereby classify them according to five sensing principles: sensing of refractive index variations, sensing by lattice spacing variations, enhanced fluorescence spectroscopy, surface-enhanced Raman spectroscopy, and configuration transitions.

Measuring barrier function in organ-on-chips with cleanroom-free integration of multiplexable electrodes.

Transepithelial/transendothelial electrical resistance (TEER) measurements can be applied in organ-on-chips (OoCs) to estimate the barrier properties of a tissue or cell layer in a continuous, non-invasive, and label-free manner. Assessing the barrier integrity in in vitro models is valuable for studying and developing barrier targeting drugs. Several systems for measuring the TEER have been shown, but each of them having their own drawbacks. This article presents a cleanroom-free fabrication method for the integration of platinum electrodes in a polydimethylsiloxane OoC, allowing the real-time assessment of the barrier function by employing impedance spectroscopy. The proposed method and electrode arrangement allow visual inspection of the cells cultured in the device at the site of the electrodes, and multiplexing of both the electrodes in one OoC and the number of OoCs in one device. The effectiveness of our system is demonstrated by lining the OoC with intestinal epithelial cells, creating a gut-on-chip, where we monitored the formation, as well as the disruption and recovery of the cell barrier during a 21 day culture period. The application is further expanded by creating a blood-brain-barrier, to show that the proposed fabrication method can be applied to monitor the barrier formation in the OoC for different types of biological barriers.

On-chip determination of spermatozoa concentration using electrical impedance measurements.

In this article we describe the development of a microfluidic chip to determine the concentration of spermatozoa in semen, which is a main quality parameter for the fertility of a man. A microfluidic glass-glass chip is used, consisting of a microchannel with a planar electrode pair that allows the detection of spermatozoa passing the electrodes using electrical impedance measurements. Cells other than spermatozoa in semen also cause a change in impedance when passing the electrodes, interfering with the spermatozoa count. We demonstrate that the change in electrical impedance is related to the size of cells passing the electrodes, allowing to distinguish between spermatozoa and HL-60 cells suspended in washing medium. In the same way we are able to distinguish between polystyrene beads and spermatozoa. Thus, by adding a known concentration of polystyrene beads to a boar semen sample, the spermatozoa concentrations of seven mixtures are measured and show a good correlation with the actual concentration (R(2)-value = 0.97). To our knowledge this is the first time that the concentration of spermatozoa has been determined on chip using electrical impedance measurements without a need to know the actual flow speed. The proposed method to determine the concentration can be easily applied to other cells. The described on-chip determination of the spermatozoa concentration is a first step towards a microfluidic system for a complete quality analysis of semen.