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  • Open access
  • 217 Reads
3D polymer structures for the identification of optimal dimensions for cellular growth for 3D lung alveolar models

Organ-on-chips and scaffolds for tissue engineering are vital assay tools for pre-clinical testing and prediction of human response to drugs and toxins, while providing an ethical sound replacement of animal testing and a low-cost alternative to expensive clinical studies. An important success criteria for these models is the ability to have structural parameters for optimized performance.

In this study we show how the two-photon polymerization fabrication method can be used to create 3D test platforms made for analysing optimal scaffold parameters for cell growth. We design and fabricate a 3D grid structure, designed as a set of wall structures with niches of various dimensions for probing the optimal niche for cell attachment. The 3D grid structures are fabricated from bio-compatible polymer SZ2080 and subsequently seeded with A549 lung epithelia cells. The seeded structures are incubated and imaged with multi-colour spectral confocal microscopy at several time points, to determine the volume of cell material present in the different niches of the grid structure. Spectral imaging with linear unmixing is used to separate the auto-fluorescence contribution from the scaffold from the fluorescence of the cells and use it to determine the volume of cell material present in the different sections of the grid structure. The variation in structural parameters influences the incubated A549 cells distribution and morphology. In the future this kind of differentiated 3D growth platform, could be applied for optimized culture growth, cell differentiation and advanced cell therapies.

  • Open access
  • 77 Reads
Sensitivity Analysis of a Portable Wireless PCB-MEMS Permittivity Sensor Node for Non-Invasive Liquid Recognition

Dielectric characteristics are useful to determine crucial properties of liquids and to differentiate between liquid samples with similar physical characteristics. Liquid recognition has found applications in a broad variety of fields, including healthcare, food science, and quality inspection, among others. This work demonstrates the fabrication, instrumentation, and functionality of a portable wireless sensor node for permittivity measurement of liquids that require characterization and differentiation. The node incorporates an interdigitated microelectrode array as transducer, and a microcontroller unit with radio communication electronics for data processing and transmission, which enables a wide variety of stand-alone applications. A laser-ablation-based microfabrication technique is applied to fabricate the microelectromechanical systems (MEMS) transducer on a printed circuit board (PCB) substrate. The surface of the transducer is covered with a thin layer of SU-8 polymer by spin coating, which prevents from direct contact between the Cu electrodes and the liquid sample. This helps to enhance durability, avoid electrode corrosion and contamination of the liquid sample, and to prevent undesirable electrochemical reactions to arise. The transducer’s impedance was modeled as a Randles cell, having resistive and reactive components determined analytically using a square wave as stimuli, and a resistor as a current-to-voltage converter. To characterize the node sensitivity under different conditions, three different transducer designs were fabricated and tested for four different fluids—i.e., air, isopropanol, glycerin, and distilled water—achieving a sensitivity of 1.6965 +/- 0.2028 εr/pF. Experimental results agree with theoretical calculations within relative percentage errors of 4% and 27%. The use of laser ablation allowed the reduction of the transducer footprint while maintaining its sensitivity within an adequate value for the targeted applications.

  • Open access
  • 73 Reads
Stable Porous Silicon Membranes for Fast Bacterial Detection

The rapid detection of hazardous bacteria is important for healthcare situations where such identification can lead to substantial gains for patient treatment and recovery, and a reduced usage of broad-spectrum antibiotics. Potential biosensors must be able to provide a fast, sensitive and selective response with as little sample preparation as possible. Indeed, some of these pathogens such as Staphylococcus aureus can be yet harmful at very low concentrations in the blood stream, e.g. below 10 colony forming units per mL (CFU/mL). These stringent requirements limit the number of candidates, especially for point-of-care applications.

Amongst several biosensing techniques, optical sensing using porous silicon (PSi) substrate has been widely suggested in recent years thanks to unique features such as a large surface area, tunable optical characteristics, and above all relatively easy and affordable fabrication techniques. In most configurations, PSi optical biosensors are close-ended porous layers that limits their sensitivity and responsiveness due to diffusion-limited infiltration of the analytes in the porous layer. Also, PSi is a reactive material, its oxidation in buffer solutions results in time-varying shifts. Despite its attractive properties, several challenges must still be overcome in order to reach practical applications.

Our work addresses three main improvement points. The first one is the stability over time in saline solutions helped by atomic layer deposition of metal oxides inside the pores. Besides a better stability, our solution is helping with an increase of the optical signal to noise ratio thus reducing the limit of detection. The second one is to perform the lysis of the bacteria prior to its exposure to the sensor, such that the selective detection is based upon the percolation of bacterial residues inside the pores rather than the bacteria themselves. The third one is to remove the bulk silicon below a PSi layer to create a membrane, that allows for flow-through of the analytes thus enhancing the interactions between the lysate and the sensor’s surface. This approach allows us to avoid the step of surface functionalization used in classical biosensors. We tested thanks to these improvements the selective detection of Bacillus cereus lysate with concentrations between 103 to 105 CFU/mL.

Future works are dedicated to further improvements, including optical signal enhancement techniques and dielectrophoretic assisted percolation in the porous silicon membrane.

  • Open access
  • 85 Reads
Separation microfluidic device fabricated by micromilling techniques

The diagnostic of several diseases can be performed by analyzing the blood plasma of the patient. Despite the extensive research work, there is still the need to improve the current low-cost fabrication techniques and devices for the separation of the plasma from the blood cells. Microfluidic biomedical devices have a great potential for that process. Hence, a microfluidic device made by micromilling and sealed with the oxygen plasma technique was tested by means of two different blood analogue fluids. The device has four microchannels having similar geometries but with different channel depths. A high-speed video microscopy system was used for the visualization and acquisition of the flow of the analogue fluids throughout the microchannels of the device. Then, the separation of particles and plasma was evaluated using the software ImageJ by measuring and comparing the grey values at the entrance and at the exit of the channel. The device has shown a significant reduction of the amount of cells between the entrance and the exit of the microchannels. No major influences were found from the depth of the channels and size of the particles on the separation process. However, it was found that flow rate has affected the separation results where the best results were obtained for a flow rate of 100 μL/min. Although these results are promising, further analysis and optimizations of the microfluidic devices will be conducted in future works, as well as, the comparison between devices sealed using different methods, such as, the solvent bonding technique.

  • Open access
  • 111 Reads
Rapid lipid content screening in Neochloris Oleoabundans by carbon-based dielectrophoresis

The use of microalgae as a biomass source for biofuels production has drawn attention of many scientists due to several associated environmental advantages over conventional terrestrial crops, including microalgae growing using wastewaters, and higher CO2 fixation rate, contributing to reduction of atmospheric concentration. Consequently, a reliable cytoplasmic lipid screening process in microalgae is a valuable asset for harvesting optimization in mass production processes. In this study, heterogeneous cytoplasmic lipid content of Neochloris Oleoabundans was dielectrophoretically assorted in a microfluidic device using castellated carbon microelectrodes. Experiments carried out over a wide frequency window (100 kHz to 30 MHz) at a fixed amplitude of 7 VPP, showed a significant contrast between the dielectrophoretic behavior of high-lipid content and low-lipid content cells at the low frequency range (100-800 kHz). A weak response for the mid and high frequency ranges (1-30 MHz) was also identified for high and low lipid content samples, allowing to establish an electrokinetic footprint of the studied strain.

These results suggest that development of a reliable screening process for harvesting optimization is possible through a fast and straightforward mechanism such as dielectrophoresis, employing glassy carbon, a low-cost and easy-to-machine material.

Experimental setup in this study involved in-vitro culturing of nitrogen-replete (N+) and a nitrogen-deplete (N-) cell suspensions to promote low and high lipid production in cells, respectively. Cell populations were monitored using spectrophotometry, and the resulting lipid development among cells was quantified by Nile red fluorescence.

  • Open access
  • 250 Reads
Magneto-Catalytic Janus Micromotors for Selective Inactivation of Bacteria Biofilms

Janus micromotors are a unique class of materials whose surfaces have two or more distinct physical properties, allowing thus for two types of chemistry to occur simultaneously. Judicious design of the micromotor structure allows to incorporate different functionalities in a single unit to adapt the propulsion behaviour along with the incorporation of specific receptors for a myriad of applications. Herein we report the preparation of graphene oxide (GO)/PtNPs/Fe2O3 Janus micromotors for highly selective capture/inactivation of gram-positive bacteria units and biofilms. The strategy is based on the combination of a lanbiotic (Nisin) with Janus micromotors. Lanbiotics are peptides composed of methyllanthionine residues with a highly selective antimicrobial activity towards multidrug resistant bacteria. Nisin is a natural compound normally used for food preservation, which display specific antimicrobial activity towards gram-positive bacteria. Such peptide can bind to lipid II unit of the bacteria membranes, damaging its morphology and releasing its contents. The coating of micromotors with GO impart them with a Janus structure for the subsequent asymmetric assembly of catalytic (PtNPs) and magnetic (Fe2O3) engines and results in an active rough layer for a higher loading of Nisin via covalent interactions. The micromotors possess adaptative propulsion mechanisms, including catalytic mode (PtNPs) in peroxide solutions or magnetic actuation (fuel free) by the action of an external magnetic field. The enhanced movement and localized delivery of the micromotors (both in catalytic and magnetic actuated mode) results in a 2-fold increase of the capture/killing ability towards Staphylococcus Aureus bacteria in raw media (juice, serum and tap water samples), as compared with free Nisin and static counterparts. The micromotor strategy display also high selectivity towards such bacteria, as illustrated by the dramatically lower capture/killing ability towards gram-negative Escherichia Coli. Unlike previous micromotors based strategies, this approach displays higher selectivity towards a type of bacteria along with enhanced stability, prolonged use and adaptative propulsion modes, holding considerable promise to treat methicillin resistant antibiotic infections, for environmental remediation or food safety, among others applications.

  • Open access
  • 56 Reads
Geometric understanding of local fluctuation distribution of conduction time in lined-up cardiomyocyte network in agarose-microfabrication multi-electrode measurement assay

We examined characteristics of the propagation of conduction in width-controlled cardiomyocyte cell networks for understanding the contribution of the geometrical arrangement of cardiomyocytes for their local fluctuation distribution. We tracked a series of extracellular field potentials of linearly lined-up mouse primary cardiomyocytes and human embryonic stem cell-derived cardiomyocytes with 100 kHz sampling intervals of multi-electrodes signal acquisitions and an agarose microfabrication technology to localize the cardiomyocyte geometries in the lined-up cell networks with 100-300 µm wide agarose microstructures. Conduction time between two neighbor microelectrodes showed Gaussian distribution, which indicates this conduction propagation in a unit length was a stochastic firing phenomenon. However, the distributions of conduction time were not expanded but maintained within an identical range of distribution regardless of their propagation distances from a unit microelectrode distance, 0.3 mm, to five units of the distance, 1.5 mm, which is against the expected distance-dependent enlarging of the distribution based on the faster firing regulation. In contrast, when Quinidine was applied to the cardiomyocytes, the distributions of conduction time were expanded as propagation distance increased as predicted by the conduction propagation model of faster firing regulation. The results indicate the “faster firing regulation” is not sufficient to explain this conservation of the propagation time distribution in cardiomyocyte networks, and suggest the existence of some cooperative conduction propagation regulation, which is disappeared by the sodium channel blocking. In this meeting, we discuss the possible interpretation of this synchronous behavior more in detail and the influence of this phenomenon for diagnostics.

  • Open access
  • 87 Reads
On-Chip Multiple Particle Velocity and Size Measurement Using Single-Shot Two-Wavelength Differential Image Analysis

Precise and quick measurement of samples' flow velocities is essential for cell sorting timing control and reconstruction of acquired image-analyzed data. We have developed a simple technique for single-shot measurement of flow velocities of particles simultaneously in a microfluidic pathway. The speed was calculated from the difference in the particles' elongation in an acquired image that appeared when two wavelengths of light with different irradiation times were applied. We ran microparticles through an imaging flow cytometer and irradiated two wavelengths of light with different irradiation times simultaneously to those particles. The mixture of two wavelengths transmitted lights was divided into two wavelengths, and the images of the same microparticles for each wavelength were acquired in a single shot. We estimated the velocity from the difference of its elongation divided by the difference of irradiation time by comparing these two images. The distribution of polystyrene beads' velocity was parabolic and highest at the center of the flow channel, consistent with the expected velocity distribution of the laminar flow. Applying the calculated velocity, we also restored the accurate shapes and cross-sectional areas of particles in the images, indicating the capability of this simple method for improvement of imaging flow cytometry and cell sorter for diagnostic screening of circulating tumor cells. In this meeting, we describe the data more in detail and discuss the further application of this method.

  • Open access
  • 122 Reads
Nanoscopic Biosensors in Microfluidics

Synergy between, physics, material sciences and biotechnology during last decade has led to a tremendous scientific progress in the fields of biodetection and nanomedicine. This tight interaction led to the emergence of a new class of bioinspired systems that enables to bring the area of biosensorics e.g. for cell or molecular diagnostics and analytics to the new level. The advances are expected in terms of (i) possibility of early diagnostics of diseases due to the increased sensitivity of the detectors, (ii) real time and high throughput analysis offered by combination of integrated electronics and microfluidic approach, and (iii) establishing the new functional formats for the bioassays. One of the most promising candidates for the future diagnostics are the electronic nanobiosensors that have attracted great attention in the last decades since they provide rich quantitative information for medical and biotechnological assays without pre-treatment and specific optical labelling of the detected species.

At the same time, to bring state‐of‐the‐art biomedical diagnostic devices to the hands of the people, it is important to reduce the price of the devices and allow for their high‐volume delivery in a cost‐efficient manner, e.g., container transportation. For the latter, a crucial aspect is to reduce the weight of the device. This can be achieved by replacing the conventional rigid substrates, like Si or glass by light weight and large area polymeric foils.

Here I will focus on two flexible diagnostic platforms for the analysis at the micro- and nanoscale, represented by (a) silicon nanowires based field effect transistors and (b) 2D materials based on molybdenum disulfide.

  • [1] Karnaushenko, et al., Adv. Healthc. Mater. 4(10), 1517 (2015).
  • [2] Zhang, et al., Small 15 (23), 1901265 (2019).
  • [3] Baraban, et al. Advanced Science 6 (15), 1900522 (2019).
  • Open access
  • 76 Reads
The Effect of Micromixer Geometry on The Diameters of Emulsion Droplets: NIR Spectroscopy and Artificial Neural Networks Modeling

In this work, teardrop micromixer and swirl micromixer were used for preparation of oil-in-water (O/W) emulsions with Tween 20 and PEG 2000 as emulsifiers (concentrations: 2 % and 4 %) at different total flow rates (20 - 280 µL/min). Stability of the prepared O/W emulsions was evaluated based on the droplet size of the dispersed phase. For determination of the droplet size, the average Feret diameter was used. Furthermore, near infrared (NIR) spectra of all prepared samples were collected. Obtained results showed that the change in the droplet size followed the same trend for both micromixers used in the experiment. At higher total flow rates, emulsification resulted in smaller values of the average Feret diameter. Values of the average Feret diameter were higher for emulsions prepared in the swirl micromixer, compared to the teardrop micromixer. Artificial Neural Network (ANNs) models, based on the recorded NIR spectra of emulsions, were developed to predict the droplet size of the dispersed phase. The obtained ANN models have high values of R2 for training, test, and validation, with small error values and show that NIR spectroscopy, in combination with ANNs, could be efficiently used for evaluation of the stability of oil-in-water emulsions.