Whole mount zebrafish antibody staining (ABS) is a common staining technique used to localize protein information in a zebrafish embryo or larva. Like most biological assays, the whole mount zebrafish ABS is still largely conducted manually through labor intensive and time-consuming steps which affect both consistency and throughput of the assay. In this work, we develop a milli fluidic device that can automatically trap and immobilize the fixed chorion-less zebrafish embryos for the whole mount ABS. With just a single loading step, the zebrafish embryos can be trapped by the milli fluidic device through a chaotic hydrodynamic trapping process. Moreover, a consistent body orientation (i.e., head point inward) for the trapped zebrafish embryos can be achieved without additional orientation adjustment device. Furthermore, we employed a consumer-grade SLA 3D printer assisted method for device prototyping which is ideal for labs with limited budgets. Notably, the milli fluidic device has enabled the optimization and successful implementation of whole mount zebrafish Caspase-3 ABS. We demonstrated our device can accelerate the overall procedure by reducing at least 50% of washing time in the standard well-plate-based manual procedure. Also, the consistency is improved, and manual steps are reduced using the milli fluidic device. This work fills the gap in the milli fluidic application for whole mount zebrafish immunohistochemistry. We hope the device can be accepted by the zebrafish community and be used for other types of whole mount zebrafish ABS procedures or expanded to more complicated in situ hybridization (ISH) procedure.Graphical

Sickle cell disease is characterized by painful vaso-occlusive crises, in which poorly deformable sickle cells play an important role in the complex vascular obstruction process. Existing techniques are mainly based on optical microscopy and video processing of sickle blood flow under normoxic condition, for measuring vaso-occlusion by a small fraction of dense sickle cells of intrinsic rigidity but not the vaso-occlusion by the rigid, sickled cells due to deoxygenation. Thus, these techniques are not suitable for rapid, point-of-care testing. Here, we integrate electrical impedance sensing and Polydimethylsiloxane-microvascular mimics with controlled oxygen level into a single microfluidic chip, for quantification of vaso-occlusion by rigid, sickled cells within 1min. Electrical impedance measurements provided a label-free, real-time detection of different sickle cell flow behaviors, including steady flow, vaso-occlusion, and flow recovery in response to the deoxygenation-reoxygenation process that are validated by microscopic videos. Sensitivity of the real part and imaginary part of the impedance signals to the blood flow conditions in both natural sickle cell blood and simulants at four electrical frequencies (10, 50, 100, and 500kHz) are compared. The results show that the sensitivity of the sensor in detection of vaso-occlusion decreases as electrical frequency increases, while the higher frequencies are preferable in measurement of steady flow behavior. Additional testing using sickle cell simulants, chemically crosslinked normal red blood cells, shows same high sensitivity in detection of vaso-occlusion as sickle cell vaso-occlusion under deoxygenation. This work enables point-of-care testing potentials in rapid, accurate detection of steady flow and sickle cell vaso-occlusion from microliter volume blood specimens. Quantification of sickle cell rheology in response to hypoxia, may provide useful indications for not only the kinetics of cell sickling, but also the altered hemodynamics as obseved at the microcirculatory level.

Contact behaviors of medical devices, such as guidewires and catheters, are critical in endovascular surgeries. In this work, a new method to predict adhesive contact force between catheter and vascular artery is presented. Multi-asperity adhesion on the surface of vascular artery, deformation of asperity and deformation of vascular substrate are all considered. The single asperity behavior is described with Johnson-Kendall-Roberts (JKR) contact model. The multi-asperity behavior is based on Greenwood-Williamson (GW) asperity model. Vascular substrate is considered as elastic bulk substrate and its deformation is determined with Hertzian pressure from asperity on a circular region on the elastic half space. The model shows that the deformation of vascular substrate accounts for the majority of the total contact deformation and significantly affects the predicted contact force. The model is verified with published experimental data. The comparison shows that the model produces very accurate prediction of contact force between catheter and vascular artery when the contact force is compressive. Parametric analysis based on asperity topography is carried out. The analysis shows that the diameter of the circular region of the interface between asperity and vascular substrate has more significant effect on the estimation of contact force than the radius of asperity. Further validation of prediction accuracy of the model under experiment is needed.

Lung cancer is the leading cause of cancer death in the United States. It has the lowest 5-year survival rate among the most common cancers and therefore, early diagnosis is critical to improve the survival rate. In this paper, a new handheld electronic device is proposed to detect nine lung cancer biomarkers in the exhaled breath. An electrochemical gas sensor was produced through deposition of a thin layer of graphene and Prussian blue on a chromium-modified silicon substrate. Selective binding of the analyte was formed by molecular imprinting polymer (MIP). Subsequent polymerization and removal of the analyte yielded a layer of a conductive polymer on top of the sensor containing molecularly imprinted cavities selective for the target molecule. The sensors were tested over 1-20 parts per trillion (ppt) level of concentration while the sensor resistance has been monitored as the sensors react to the analyte by resistance change. Pentane sensor was also tested for selectivity. A printed circuit board was designed to measure the resistance of each sensor and send the data to a developed application in smartphone through Bluetooth. This handheld device has the potential to be used as a diagnostic method in the near future.

Improving biosensor performance which utilize impedance cytometry is a highly interested research topic for many clinical and diagnostic settings. During development, a sensor's design and external factors are rigorously optimized, but improvements in signal quality and interpretation are usually still necessary to produce a sensitive and accurate product. A common solution involves digital signal processing after sample analysis, but these methods frequently fall short in providing meaningful signal outcome changes. This shortcoming may arise from a lack of investigative research into selecting and using signal processing functions, as many choices in current sensors are based on either theoretical results or estimated hypotheses. While a ubiquitous condition set is improbable across diverse impedance cytometry designs, there lies a need for a streamlined and rapid analytical method for discovering those conditions for unique sensors. Herein, we present a comprehensive dissemination of digital filtering parameters applied on experimental impedance cytometry data for determining the limits of signal processing on signal quality improvements. Various filter orders, cutoff frequencies, and filter types are applied after data collection for highest achievable noise reduction. After designing and fabricating a microfluidic impedance cytometer, 9µm polystyrene particles were measured under flow and signal quality improved by6.09dB when implementing digital filtering. This approached was then translated to isolated human neutrophils, where similarly, signal quality improved by7.50dB compared to its unfiltered original data. By sweeping all filtering conditions and devising a system to evaluate filtering performance both by signal quality and object counting accuracy, this may serve as a framework for future systems to determine their appropriately optimized filtering configuration.

We previously reported a single-cell adhesion micro tensile tester (SCAμTT) fabricated from IP-S photoresin with two-photon polymerization (TPP) for investigating the mechanics of a single cell-cell junction under defined tensile loading. A major limitation of the platform is the autofluorescence of IP-S, the photoresin for TPP fabrication, which significantly increases background signal and makes fluorescent imaging of stretched cells difficult. In this study, we report the design and fabrication of a new SCAμTT platform that mitigates autofluorescence and demonstrate its capability in imaging a single cell pair as its mutual junction is stretched. By employing a two-material design using IP-S and IP-Visio, a photoresin with reduced autofluorescence, we show a significant reduction in autofluorescence of the platform. Further, by integrating apertures onto the substrate with a gold coating, the influence of autofluorescence on imaging is almost completely mitigated. With this new platform, we demonstrate the ability to image a pair of epithelial cells as they are stretched up to 250% strain, allowing us to observe junction rupture and F-actin retraction while simultaneously recording the accumulation of over 800 kPa of stress in the junction. The platform and methodology presented here can potentially enable detailed investigation of the mechanics of and mechanotransduction in cell-cell junctions and improve the design of other TPP platforms in mechanobiology applications.

Neuropeptide Y (NPY) occurs in G-protein-coupled receptors and offers targeted effects at the active sites for therapeutic action in various conditions like depression, stress, obesity and cancer. Immune stimulating complexes (ISCOMs) associate peptides with the lipid systems for enhancing antigen targeting to provide site-specific action and B-cell response. The present study focused on the encapsulation of NPY in ISCOMs to comprise dual action in the form of immunity modulation and management of breast cancer by arresting G0/G1 phase. The colloidal ISCOMs were prepared by coupling method and further optimized by Box-Behnken design of Design of Experiment (DoE) software. The NPY-loaded ISCOMs (formulation ISCN) were characterized by various parameters with higher % encapsulation efficiency of 87.99 ± 1.87% and in-vitro release of 84.16±3.2% of NPY for 24h. The study of MTT assay on MCF-7 cell line for formulation ISCN exhibited a significant decrease in the cell growth of 66.41±4.7% at 10µg/mL compared to plain NPY (52.21±0.04%). The MCF-7 cells showed a significant reduction in cytokine levels in the presence of formulation ISCN wherein TH1(TNF-α) and TH2(IL-10) levels were found to be 25.12±3.11 pg/mL and 35.76±4.23 pg/mL, respectively. The cell cycle study demonstrated that significant cells were blocked in the G0/G1 phase with 57.8±3.02% of cell apoptosis using formulation ISCN. The formulation ISCN was found to prolong t1/2 and increase AUC than plain NPY via intravenous administration due to complex formation with phospholipid. Hence, ISCOMs-based NPY system will be a promising approach for dual action as immunomodulation and anticancer effects by controlling the release of NPY.

In this study, we developed a microfluidic device for a dual work of protein preconcentration and subsequent capture by an immunoassay system. The fabrication of nano-interstices (nanochannels) to generate ion concentration polarization effect (ICP) for the preconcentration was simply performed by exploiting the loose association of glass-on-modified AuNPs to the polydimethylsiloxane (PDMS) channel pads as well as spaces between the substrate and the PDMS pad caused by these AuNPs. As a result, 65-fold concentration enhancement was achieved when performed on a sample of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA). Furthermore, a modification procedure of antibodies responsible for capturing target proteins was performed on gold electrodes integrated into the proposed chip. After preconcentration, the immunoassay system was worked, and showed a good performance in capturing targets. Through this study, we demonstrated that the device can work efficiently for the dual purpose, has the potential to apply widely for the analysis and capture of various targets.

Isolation of exosome from culture medium in an effective way is desired for a less time consuming, cost saving technology in running the diagnostic test on cancer. In this study, we aim to develop an inertial microfluidic channel to separate the nano-size exosome from C666-1 cell culture medium as a selective sample. Simulation was carried out to obtain the optimum flow rate for determining the dimension of the channels for the exosome separation from the medium. The optimal dimension was then brought forward for the actual microfluidic channel fabrication, which consisted of the stages of mask printing, SU8 mould fabrication and ended with PDMS microchannel curing process. The prototype was then used to verify the optimum flow rate with polystyrene particles for its capabilities in actual task on particle separation as a control outcome. Next, the microchip was employed to separate the selected samples, exosome from the culture medium and compared the outcome from the conventional exosome extraction kit to study the level of effectiveness of the prototype. The exosome outcome from both the prototype and extraction kits were characterized through zetasizer, western blot and Transmission electron microscopy (TEM). The microfluidic chip designed in this study obtained a successful separation of exosome from the culture medium. Besides, the extra benefit from this microfluidic channels in particle separation brought an evenly distributed exosome upon collection while the exosomes separated through extraction kit was found clustered together. Therefore, this work has shown the microfluidic channel is suitable for continuous separation of exosome from the culture medium for a clinical study in the future.

The poses of robotic endoscopic capsules are indispensable for further follow-up examinations, potential targeted drug delivery, and closed-loop controlling of active locomotion. A novel tracking method using the multiple magnetic excitations with frequency division has been investigated. The multiple excitation coils can simultaneously work at different frequency to improve real-time tracking. A novel model between the magnetic flux density and the capsule's pose has been derived, which shows a nonlinear equation group with multiple local extremum. Then, a Back-Propagation (BP) neural network algorithm combined with the mother wavelet is investigated to solve the pose. To reduce the volume and power consumption, the wireless magnetic sensing module uses digital signal processing as the core framework, which is beneficial to be miniaturized to integrate with the capsule. The functional prototype of the tracking system has been developed, which consists of a wireless magnetic sensing module mounted in the capsule, a magnetic excitation module with frequency division, a wireless receiver and data interface, an excitation coil array and a platform for pose solving. The experimental results show that the mean errors are 0.0098m in x-component, 0.0122m in y-component, 0.0077m in z-component, 0.187rad in α-component and 0.161rad in β-component, respectively. The real-time performance of the tracking system is improved.