A Low-Cost, Disposable and Portable Inkjet-Printed Biochip for the Developing World.

Electrowetting on dielectric-based digital microfluidic platforms (EWOD-DMF) have a potential to impact point-of-care diagnostics. Conventionally, EWOD-DMF platforms are manufactured in cleanrooms by expert technicians using costly and time consuming micro-nanofabrication processes such as optical lithography, depositions and etching. However, such high-end microfabrication facilities are extremely challenging to establish in resource-poor and low-income countries, due to their high capital investment and operating costs. This makes the fabrication of EWOD-DMF platforms extremely challenging in low-income countries, where such platforms are most needed for many applications such as point-of-care testing applications. To address this challenge, we present a low-cost and simple fabrication procedure for EWOD-DMF electrode arrays, which can be performed anywhere with a commercial office inkjet printer without the need of expensive cleanroom facilities. We demonstrate the utility of our platform to move and mix droplets of different reagents and physiologically conductive buffers, thereby showing its capability to potentially perform a variety of biochemical assays. By combining our low-cost, inkjet-printed EWOD-DMF platform with smartphone imaging technology and a compact control system for droplet manipulation, we also demonstrate a portable and hand-held device which can be programmed to potentially perform a variety of biochemical assays.

Personalized Drug Efficacy Monitoring Chip.

Cancer drug resistance mechanisms such as tumor heterogeneity and adaptable feedback loops are prevalent issues facing cancer therapy development. Drug resistance can be unique to a cancer type and, most importantly, to each individual cancer patient. Consequently, testing different dosages and therapeutics directly on each individual patient sample (i.e., tumor and cancer cells) has compelling advantages compared to large scale in vitro drug testing and is a step toward personalized drug selection and effective treatment development. Recently, microfluidic-based chemo-sensitivity assays on patient biopsies have been proposed. Despite their novelty, these platforms usually rely on optical labels, optical equipment, or complex microfabricated channel geometries and structures. In this work, we proposed a novel lab on a chip platform capable of real-time and continuous screening of drug efficacy on (cancer) cell subpopulations without the need of labels or bulky readout optical equipment. In this platform, several label-free and rapid techniques have been implemented for the precise capturing of cells of interest in parallel with the real-time measurement and characterization of the effectiveness of candidate therapeutic agents. To demonstrate the utility of the platform, the effect of an apoptotic inducer, topoisomerase I inhibitor, 7-ethyl-10-hydrocamptothecin (SN38) on human colorectal carcinoma cancer cells (HCT 116) was used as a study model. Additionally, electrical results were optically verified to examine the continuous measurements of the biological mechanisms, specifically, apoptosis and necrosis, during therapeutic agent characterizations. The proposed device is a versatile platform which can also be easily redesigned for the automated and arrayed analysis of cell-drug interaction down to the single cell level. Our platform is another step toward enabling the personalized screening of drug efficacy on individual patients' samples that potentially leads to a better understanding of drug resistance and the optimization of patients' treatments.

An orbital shear platform for real-time, in vitro endothelium characterization.

Electrical impedance techniques have been used to characterize endothelium morphology, permeability, and motility in vitro. However, these impedance platforms have been limited to either static endothelium studies and/or induced laminar fluid flow at a constant, single shear stress value. In this work, we present a microfabricated impedance sensor for real-time, in vitro characterization of human umbilical vein endothelial cells (HUVECs) undergoing oscillatory hydrodynamic shear. Oscillatory shear was applied with an orbital shaker and the electrical impedance was measured by a microfabricated impedance chip with discrete electrodes positioned at radial locations of 0, 2.5, 5.0, 7.5, 10.0, and 12.5 mm from the center of the chip. Depending on their radial position within the circular orbital platform, HUVECs were exposed to shear values ranging between 0.6 and 6.71 dyne/cm(2) (according to numerical simulations) for 22 h. Impedance spectra were fit to an equivalent circuit model and the trans-endothelial resistance and monolayer's capacitance were extracted. Results demonstrated that, compared to measurements acquired before the onset of shear, cells at the center of the platform that experienced low steady shear stress (∼2.2 dyne/cm(2) ) had an average change in trans-endothelial resistance of 6.99 ± 4.06% and 1.78 ± 2.40% change in cell capacitance after 22 hours of shear exposure; cells near the periphery of the well (r = 12.5 mm) experienced transient shears (2.5-6.7 dyne/cm(2) ) and exhibited a greater change in trans-endothelial resistance (24.2 ± 10.8%) and cell capacitance (4.57 ± 5.39%). This study, demonstrates that the orbital shear platform provides a simple system that can capture and quantify the real-time cellular morphology as a result of induced shear stress. The orbital shear platform presented in this work, compared to traditional laminar platforms, subjects cells to more physiologically relevant oscillatory shear as well as exposes the sample to several shear values simultaneously. Biotechnol. Bioeng. 2016;113: 1336-1344. © 2015 Wiley Periodicals, Inc.

Quantitative trait loci mapping of innate fear behavior in day-old F2 chickens of Japanese Oh-Shamo and White Leghorn breeds using restriction site-associated DNA sequencing.

Understanding the genetic mechanisms that underlie innate fear behavior is essential for improving the management and performance of the poultry industry. This study aimed to map QTL associated with innate fear responses in open field (OF) and tonic immobility (TI) tests, using an F2 chicken intercross population between 2 behaviorally distinct breeds: the aggressive Japanese Oh-Shamo (OSM) and the docile White Leghorn T-line (WL-T). Genome-wide QTL analysis for the OF and TI traits was conducted using 2,109 single nucleotide polymorphism (SNP) markers obtained through restriction site-associated DNA sequencing (RAD-seq). While several suggestive QTL were identified for TI and OF traits at genome-wide 20% significance threshold levels, the analysis revealed 2 significant QTL for 2 OF traits (total distance and maximum speed) at genome-wide 5% significance threshold levels. These significant QTL were located between 12.34 and 30.49 megabase (Mb) on chromosome 1 and between 40.02 and 63.38 Mb on chromosome 2, explaining 6.75 to 7.40% of the total variances. These findings provide valuable insights for the poultry industry, particularly in refining chicken management strategies and informing targeted breeding efforts.

Factors associated with unquantifiable total HIV-1 DNA in peripheral blood in persons living with HIV: An observational study.

Background: The human immunodeficiency virus type 1 (HIV-1) cannot be eradicated even with suppressive antiretroviral therapy because its retrotranscribed genome integrates into the DNA of host cells, creating a long-term reservoir. Quantification of total HIV-1 DNA in peripheral blood is a biomarker of this reservoir that can predict progression of the infection, treatment response, and HIV-1-related complications. A deeper understanding of the reservoir may help develop a cures. Objective: This study aimed to characterize persons living with HIV-1 (PLWH) with unquantifiable total HIV-1 DNA in blood (below the quantification threshold) and identify associated factors. Methods: We have conducted a retrospective observational study. During the study period, all PLWH who had total leukocyte-associated HIV-1 DNA measured by quantitative PCR were included. We have isolated a population of participants with HIV-1 DNA levels below the quantification threshold (40 copies/106 leukocytes). Results: Out of 1094 patients analysed, 62 had unquantifiable and 1032 quantifiable HIV-1 DNA levels in blood. We have found that those with unquantifiable HIV-1 DNA had a higher CD4 T cell nadir (p = 0.006) and a lower viral load zenith (p < 0.001). Multivariate analyses showed that initiation of treatment in primary infection was the only protective factor against HIV-1 DNA quantifiability, the odds of HIV-1 DNA quantifiability decreased by 82% in those treated within 30 days of infection, after controlling for other factors. Conclusion: Our research highlights the importance of an early start of anti-retroviral therapy to limit the size of the HIV-1 reservoir, as receiving treatment during primary infection was found as the only protective factor against quantifiability of HIV-1 DNA in blood.

Electrokinetic concentration and patterning of colloids with a scanning laser.

Optically-based lab-on-a-chip systems have the distinct advantage of being dynamically controlled in real time, providing reconfigurable operations that can be tuned to perform a variety of tasks. This manuscript demonstrates the concentration of liquid-suspended microparticles using a focused near-infrared laser (980 nm) and a parallel-plate electrode system. The parallel-plate electrodes consisted of an indium tin oxide-coated coverslip and a gold-coated glass substrate. When the laser was applied at 36 mW, the indium tin oxide surface is locally heated creating sharp temperature gradients on the order of 0.07(o) C/µm. When an AC field was applied, electrothermal hydrodynamic forces generated microfluidic vortices. At an AC frequency of 40 kHz, the optically controlled electro-hydrodynamics aggregated colloids at the center of fluid motion on the surface of the indium tin oxide coverslip. The nature of colloid aggregation, translation, and patterning was explored when the translational velocity of the laser spot was varied. This manuscript describes the design of the laser scanning system using commercially available components and the fabrication of the parallel-plate chip. The effect that the laser scanning rate has on the heat transfer, fluid velocity, and colloid aggregation is discussed.

A microfluidic impedance platform for real-time, in vitro characterization of endothelial cells undergoing fluid shear stress.

We introduce a microfluidic impedance platform to electrically monitor in real-time, endothelium monolayers undergoing fluid shear stress. Our platform incorporates sensing electrodes (SEs) that measure cell behavior and cell-free control electrodes that measure cell culture media resistance simultaneously but independently from SEs. We evaluated three different cellular subpopulations sizes through 50, 100, and 200 µm diameter SEs. We tested their utility in measuring the response of human umbilical vein endothelial cells (HUVECs) at static, constant (17.6 dyne per cm2), and stepped (23.7-35-58.1 dyne per cm2) shear stress conditions. For 14 hours, we collected the impedance spectra (100 Hz-1 MHz) of sheared cells. Using equivalent circuit models, we extracted monolayer permeability (RTER), cell membrane capacitance, and cell culture media resistance. Platform evaluation concluded that: (1) 50 µm SEs (∼2 cells) suffered interfacial capacitance and reduced cell measurement sensitivity, (2) 100 µm SEs (∼6 cells) was limited to measuring cell behavior only and cannot measure cell culture media resistance, and (3) 200 µm SEs (∼20 cells) detected cell behavior with accurate prediction of cell culture media resistance. Platform-based shear stress studies indicated a shear magnitude dependent increase in RTER at the onset of acute flow. Consecutive stepped shear conditions did not alter RTER in the same magnitude after shear has been applied. Finally, endpoint staining of VE-cadherin on the actual SEs and endpoint RTER measurements were greater for 23.7-35-58.1 dyne per cm2 than 17.6 dyne per cm2 shear conditions.

Microtechnology-based methods for organoid models.

Innovations in biomaterials and stem cell technology have allowed for the emergence of novel three-dimensional (3D) tissue-like structures known as organoids and spheroids. As a result, compared to conventional 2D cell culture and animal models, these complex 3D structures have improved the accuracy and facilitated in vitro investigations of human diseases, human development, and personalized medical treatment. Due to the rapid progress of this field, numerous spheroid and organoid production methodologies have been published. However, many of the current spheroid and organoid production techniques are limited by complexity, throughput, and reproducibility. Microfabricated and microscale platforms (e.g., microfluidics and microprinting) have shown promise to address some of the current limitations in both organoid and spheroid generation. Microfabricated and microfluidic devices have been shown to improve nutrient delivery and exchange and have allowed for the arrayed production of size-controlled culture areas that yield more uniform organoids and spheroids for a higher throughput at a lower cost. In this review, we discuss the most recent production methods, challenges currently faced in organoid and spheroid production, and microfabricated and microfluidic applications for improving spheroid and organoid generation. Specifically, we focus on how microfabrication methods and devices such as lithography, microcontact printing, and microfluidic delivery systems can advance organoid and spheroid applications in medicine.

3D-bioprinted all-inclusive bioanalytical platforms for cell studies.

Innovative drug screening platforms should improve the discovery of novel and personalized cancer treatment. Common models such as animals and 2D cell cultures lack the proper recapitulation of organ structure and environment. Thus, a new generation of platforms must consist of cell models that accurately mimic the cells' microenvironment, along with flexibly prototyped cell handling structures that represent the human environment. Here, we adapted the 3D-bioprinting technology to develop multiple all-inclusive high throughputs and customized organ-on-a-chip-like platforms along with printed 3D-cell structures. Such platforms are potentially capable of performing 3D cell model analysis and cell-therapeutic response studies. We illustrated spherical and rectangular geometries of bio-printed 3D human colon cancer cell constructs. We also demonstrated the utility of directly 3D-bioprinting and rapidly prototyping of PDMS-based microfluidic cell handling arrays in different geometries. Besides, we successfully monitored the post-viability of the 3D-cell constructs for seven days. Furthermore, to mimic the human environment more closely, we integrated a 3D-bioprinted perfused drug screening microfluidics platform. Platform's channels subject cell constructs to physiological fluid flow, while its concave well array hold and perfused 3D-cell constructs. The bio-applicability of PDMS-based arrays was also demonstrated by performing cancer cell-therapeutic response studies.

A Machine Learning-Assisted Nanoparticle-Printed Biochip for Real-Time Single Cancer Cell Analysis.

Cancers are a complex conglomerate of heterogeneous cell populations with varying genotypes and phenotypes. The intercellular heterogeneity within the same tumor and intratumor heterogeneity within various tumors are the leading causes of resistance to cancer therapies and varied outcomes in different patients. Therefore, performing single-cell analysis is essential to identify and classify cancer cell types and study cellular heterogeneity. Here, the development of a machine learning-assisted nanoparticle-printed biochip for single-cell analysis is reported. The biochip is integrated by combining powerful machine learning techniques with easily accessible inkjet printing and microfluidics technology. The biochip is easily prototype-able, miniaturized, and cost-effective, potentially capable of differentiating a variety of cell types in a label-free manner. n-feature classifiers are established and their performance metrics are evaluated. The biochip's utility to discriminate noncancerous cells from cancerous cells at the single-cell level is demonstrated. The biochip's utility in classifying cancer sub-type cells is also demonstrated. It is envisioned that such a chip has potential applications in single-cell studies, tumor heterogeneity studies, and perhaps in point-of-care cancer diagnostics-especially in developing countries where the cost, limited infrastructures, and limited access to medical technologies are of the utmost importance.

Dynamic optoelectric trapping and deposition of multiwalled carbon nanotubes.

In the path toward the realization of carbon nanotube (CNT)-driven electronics and sensors, the ability to precisely position CNTs at well-defined locations remains a significant roadblock. Highly complex CNT-based bottom-up structures can be synthesized if there is a method to accurately trap and place these nanotubes. In this study, we demonstrate that the rapid electrokinetic patterning (REP) technique can accomplish these tasks. By using laser-induced alternating current (AC) electrothermal flow and particle-electrode forces, REP can collect and maneuver a wide range of vertically aligned multiwalled CNTs (from a single nanotube to over 100 nanotubes) on an electrode surface. In addition, these trapped nanotubes can be electrophoretically deposited at any desired location onto the electrode surface. Apart from active control of the position of these deposited nanotubes, the number of CNTs in a REP trap can also be dynamically tuned by changing the AC frequency or by adjusting the concentration of the dispersed nanotubes. On the basis of a calculation of the stiffness of the REP trap, we found an upper limit of the manipulation speed, beyond which CNTs fall out of the REP trap. This peak manipulation speed is found to be dependent on the electrothermal flow velocity, which can be varied by changing the strength of the AC electric field.

Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm.

Carbon-based nanomaterials such as single-walled carbon nanotubes and reduced graphene oxide are currently being evaluated for biomedical applications including in vivo drug delivery and tumor imaging. Several reports have studied the toxicity of carbon nanomaterials, but their effects on human male reproduction have not been fully examined. Additionally, it is not clear whether the nanomaterial exposure has any effect on sperm sorting procedures used in clinical settings. Here, we show that the presence of functionalized single walled carbon nanotubes (SWCNT-COOH) and reduced graphene oxide at concentrations of 1-25 µg/mL do not affect sperm viability. However, SWCNT-COOH generate significant reactive superoxide species at a higher concentration (25 µg/mL), while reduced graphene oxide does not initiate reactive species in human sperm. Further, we demonstrate that exposure to these nanomaterials does not hinder the sperm sorting process, and microfluidic sorting systems can select the sperm that show low oxidative stress post-exposure.

Selection of functional human sperm with higher DNA integrity and fewer reactive oxygen species.

Fertilization and reproduction are central to the survival and propagation of a species. Couples who cannot reproduce naturally have to undergo in vitro clinical procedures. An integral part of these clinical procedures includes isolation of healthy sperm from raw semen. Existing sperm sorting methods are not efficient and isolate sperm having high DNA fragmentation and reactive oxygen species (ROS), and suffer from multiple manual steps and variations between operators. Inspired by in vivo natural sperm sorting mechanisms where vaginal mucus becomes less viscous to form microchannels to guide sperm towards egg, a chip is presented that efficiently sorts healthy, motile and morphologically normal sperm without centrifugation. Higher percentage of sorted sperm show significantly lesser ROS and DNA fragmentation than the conventional swim-up method. The presented chip is an easy-to-use high-throughput sperm sorter that provides standardized sperm sorting assay with less reliance on operators's skills, facilitating reliable operational steps.

Electrokinetic concentration, patterning, and sorting of colloids with thin film heaters.

Reliable and simple techniques for rapid assembly and patterning of colloid architectures advance the discovery and implementation of such nanomaterials. This work demonstrates rapid electrokinetic two-dimensional assembly of colloidal structures guided by the geometry of thin film heaters within a parallel-plate device. This system is designed to enable either independently addressable or massively parallel colloidal assembly. A combination of electrothermal hydrodynamics, particle-electrode, and particle-particle electrokinetic interactions governs their assembly. Concentration and patterning of structures are shown with 1.0 µm polystyrene particles and sorting between 1.0 µm and 2.0 µm particles is demonstrated.

Parásitos intestinales y bacterias enteropatógenas en niños de edad escolar de Maracaibo, Venezuela / Intestinal parasites and enteropathogenic bacteria in school age children of Maracaibo, Venezuela

Intestinal pathogens infection represents a global public health problem, and are associated with morbidity and mortality high rates, particularly in children; we determined the relative frequency of intestinal parasites and diarrheagenic bacteria in 22 children of the Basic State School "Comandante Remigio Negron", Maracaibo, Venezuela. Most of the children showed elevated parasites (72.73%) and polyparasitism (22.73%). The highest frequency corresponded to the protozoa and chromist (95.65%), finding a high frequency of Blastocystis sp. (52.17%); while Giardia intestinalis was detected only in 8.70%, in contrast to global data that indicate it is the most frequent protozoan species in children. In addition, the complex E. histolytica/E. dispar / E. moshkovskii (13.04%) was detected and also Hymenolepis nana (4.35%). Non-typhoidal Salmonella (4.54%) was also detected, the most common bacterial pathogen causing foodborne infection globally and the main stool-isolated bacteria in pediatric patients of the region. These results demonstrate the high relative frequency of intestinal parasites in the studied children, with predominance of protozoa and chromist, as well as the presence of Salmonella sp.; highlighting the need to promote the hygiene and environmental sanitation, to reduce the relative frequency of intestinal pathogens and their consequences to health and school performance.

Las infecciones por patógenos intestinales representan un problema de salud pública mundial y se encuentran asociadas con elevadas tasas de morbilidad y mortalidad, especialmente en niños; por lo cual se determinó la frecuencia relativa de parásitos intestinales y bacterias enteropatógenas entre 22 niños de la Escuela Básica Estadal "Comandante Remigio Negrón", Maracaibo, Venezuela. La mayoría de los niños presentaron parásitos (72,73%) y poliparasitismo elevado (22,73%). La frecuencia más alta correspondió a los protozoarios y cromistas (95,65%), encontrando una elevada frecuencia de Blastocystis sp. (52,17%); mientras que G. intestinalis fue detectada sólo en 8,70%, a diferencia de datos mundiales que indican es la especie de protozoario más frecuente en niños. Asimismo, se detectó el Complejo E. histolytica/E. dispar/E. moshkovskii (13,04%) e Hymenolepis nana (4,35%). Se detectó Salmonella no tifoidea (4,54%), que constituye el principal patógeno bacteriano que ocasiona infecciones transmitidas por alimentos a nivel mundial y la principal bacteria aislada de heces en pacientes pediátricos de la región. Estos resultados demuestran la elevada frecuencia relativa de parásitos intestinales en la población infantil estudiada, con predominio de protozoos y cromistas, así como la presencia de Salmonella sp.; señalando la necesidad de fomentar los hábitos higiénicos y el saneamiento ambiental, para disminuir la frecuencia relativa de patógenos intestinales y sus secuelas en el estado de salud y rendimiento escolar.

Micro- and nanotechnology approaches for capturing circulating tumor cells.

Circulating tumor cells (CTC) are cells that have detached from primary tumors and circulate in the bloodstream where they are carried to other organs, leading to seeding of new tumors and metastases. CTC have been known to exist in the bloodstream for more than a century. With recent progress in the area of micro- and nanotechnology, it has been possible to adopt new approaches in CTC research. Microscale and nanoscale studies can throw some light on the time course of CTC appearance in blood and CTC overexpression profiles for cancer-related markers and galvanize the development of drugs to block metastases. CTC counts could serve as endpoint biomarkers and as prognostic markers for patients with a metastatic disease. This paper reviews some of the recent researches on using micro- and nanotechnology to capture and profile CTC.