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Our purpose in this study was to determine the feasibility of assessing children’s prewriting with a new tablet tool, the Quantitative Assessment of Prewriting Skills (QAPS), while determining the validity of the QAPS for identifying visual motor skill differences. We recruited 10 children who were receiving occupational therapy (OT) services for visual motor deficits from a local OT clinic and 10 age-matched typically developing (TD) children from the local community. The QAPS assesses the accuracy of copying patterns on a tablet that records the child’s finger position on the tablet, and the data are then analyzed for different dimensions of pattern copying. We found a large effect size difference in the QAPS total score between our two participant groups, with the OT group showing poorer performance than TD children; and, among nine assessment dimensions, roundness of a drawn circle showed the largest effect size difference between groups. The QAPS appears to be a promising tool for assessing visual motor skills, and it warrants additional testing in larger participant samples.

Antibody therapy has been one of the most successful therapies for a wide range of diseases, including cancer. One way of expediting antibody therapy development is through phage display technology. Here, by screening thousands of randomly assembled peptide sequences, it is possible to identify potential therapeutic candidates. Conventional screening technologies do not accommodate perfusion through the system, as is the case of standard plate-based cultures. This leads to a poor translation of the experimental results obtained

A hydrogenated amorphous silicon p-i-n photodiode was optimized for enhanced sensitivity in the 200–300 nm wavelength range, in particular at 215 nm, 260 nm and 280 nm, which are absorbance peaks for key biomolecules. The UV-optimized photodiode was characterized in terms of its dark current density-voltage characteristics; its external quantum efficiency as a function of incident light wavelength; photocurrent density as a function of incident photon flux and wavelength; and the noise equivalent power at the identified key UV wavelengths. A dark current of $1\times 10^{-10}$ Acm−2 was obtained at 0 V. An external quantum efficiency of 6%, 7% and 8% and noise equivalent power of $7.49\times 10^{-13}\text{W}$ , $5.33 \times 10^{-13}\text{W}$ and $5 \!\times \!10^{-13}\text{W}$ were obtained at 215 nm, 260 nm, and 280 nm, respectively. The UV absorbance spectrum between 205–355 nm was determined for thiolated single-stranded DNA (SH-ssDNA), bovine serum albumin (BSA) and ascorbic acid with the UV-optimized photodiode coupled with a microfluidic structure. Limits of detection of $9.97~\mu \text{M}$ and $0.18~\mu \text{M}$ were obtained for BSA and SH-ssDNA, respectively. The absorbance of ascorbic acid at 215 nm was successfully detected.

Biocatalytic processes are amongst the most cost efficient and sustainable methods of production of high-value biological compounds, as well as several chemical commodities. These enzyme-based processes require lengthy optimization studies that can become extremly costly due to the amount of the catalyst required for a thorough testing of a wide array of working conditions. Microfluidics technology, which enables the manipulation of liquids in the range from a few nL to μL, presents itself as an interesting alternative to traditional optimization sequences. By using microfluidics, one can drastically reduce the amount of enzyme used per working condition tested, which will drive down the cost of optimization. Besides this, microfluidics technology allows for a high degree of parallelization which enables the testing of a wide variety of process conditions in a short time frame when compared to traditional approaches such as well plate technology. In this review article, we aim to clarify some basic definitions concerning the use of microfluidics technology, while providing examples of multiple approaches, with varying degrees of complexity, being used throughout the world. It is our intent to demonstrate not only the usefulness of these devices for the improvement of industrial processing, but also to show our colleagues the advantages of implementing this type of technology in academic research labs across the globe.

Trophoblast stem cells (TSCs) have recently been derived from human embryos and early-first-trimester placenta; however, aside from ethical challenges, the unknown disease potential of these cells limits their scientific utility. We have previously established a bone morphogetic protein 4 (BMP4)-based two-step protocol for differentiation of primed human pluripotent stem cells (hPSCs) into functional trophoblasts; however, those trophoblasts could not be maintained in a self-renewing TSC-like state. Here, we use the first step from this protocol, followed by a switch to newly developed TSC medium, to derive bona fide TSCs. We show that these cells resemble placenta- and naive hPSC-derived TSCs, based on their transcriptome as well as their invitro and invivo differentiation potential. We conclude that primed hPSCs can be used to generate functional TSCs through a simple protocol, which can be applied to a widely available set of existing hPSCs, including induced pluripotent stem cells, derived from patients with known birth outcomes.

Idiopathic toe-walking (ITW) refers to persistent walking without heel contact for unknown reasons. An underexplored area is the relationship of sensory processing to ITW. This study presents methods to assess sensory differences in individuals with ITW and summarizes results from a pilot testing of the measures. This pilot study included nine children and one young adult with ITW. Ten age-matched controls were recruited to provide a comparison group when norms were not available in the literature. The measures included in this study were as follows: sensory questionnaires; electrodermal activity response to sensory stimuli; monofilaments; biothesiometer; gait on different surfaces; NeuroCom® SMART Balance Master® Sensory Organization Test and Adaptation Test; and ankle position matching. All study procedures were completed in about 3 hours. Children as young as 4 years were able to complete the measures. We observed overall differences in sensory processing, specifically, higher Sensory Processing Measure scores (p = .011), higher resting electrodermal activity (p = .012), increases in heel-toe walking on novel surfaces (p = .034), and more falls with balance perturbation (p = .007) in individuals with ITW. A subset of individuals also showed tactile hyposensitivity (5 out of 10 in the ITW group) and poor equilibrium scores in the Sensory Organization Test (4 out of 9 in the ITW group, 1 unable to complete the test). Our results confirmed the heterogeneity in the etiology of ITW. We propose that further testing in sensory modulation, tactile processing, and vestibular processing is needed to fully explore the impact of sensory processing on children with ITW.

The development of a portable, Point-of-Need device to measure plant biomarkers in the field is reported. The system consists of a microcontroller-based hand-held device capable of reading photosensors at low signal levels (pA-nA) and driving light sources or pump actuators that couples to a microfluidic chip for performing colorimetric assays. The user interface is through a touchscreen and data is stored on-board in a memory card. Although robust and well-established bench-top instrumentation is available for these types of assays, many applications in the Agro-Food industry require portable systems that can make fast and inexpensive measurements in the field. We apply this system to the detection of Azelaic Acid (AzA), a known biomarker for plant health, in the nM regime. The developed system is versatile and can be applied to different types of ELISA, DNA and enzymatic based biosensing assays at the point-of-need, for plant, animal and human health applications.

Early diagnosis of fungal infections, which have seen an increase due to different environmental factors, is essential to an appropriate treatment of the plant by avoiding proliferation of the pathogen without excessive fungicide applications. In this work, we propose a microfluidic based approach to a multiplexed, point-of-need detection system capable of identifying infected grape cultivars. The system relies on the simultaneous detection of three plant hormones: salicylic, azelaic and jasmonic acids with a total assay time under 7 minutes, with LODs of 15 μM, 10 μM and 4.4 nM respectively. The three detection assays are based on optical transduction, with the detection of salicylic and azelaic acids using transmission measurements, while the detection of jasmonic acid is a fluorescence-based assay. The molecular recognition event for each metabolite is different: nanoparticle conjugation for salicylic acid, enzymatic reaction for azelaic acid and antibody-antigen recognition for jasmonic acid. In this work, two cultivars, Trincadeira and Carignan, presented infections with two fungal pathogens, Botrytis cinerea and Erysiphe necator. The grapes were tested using the microfluidic system alongside the benchmark techniques such as, high-performance liquid chromatography and enzyme-linked immunosorbent assay. The microfluidic system was not only capable of distinguishing infected from healthy samples, but also capable of distinguishing between different infection types.

Bloodstream infections (BSIs) are triggered by the existence of pathogens in blood and are considered a major health burden worldwide, especially when they result in sepsis and septic shock. Common diagnostic methods are time-consuming, present low specificity, or suffer from interference of blood components, which hampers a timely and effective treatment of BSIs. In this work, a novel microfluidic assay was developed combining a bead-based chip and bacteriophage receptor binding proteins (RBPs) as extremely specific and sensitive recognition molecules for the multiplex concentration and detection of Escherichia coli and Pseudomonas aeruginosa, which are highly prevalent bacteria in BSIs. The device comprises a microcolumn in which antibody-functionalized agarose beads were packed allowing the entrapment of the target bacterium from blood, providing its concentration and separation. For bacterial detection, two recombinant RBPs (Gp54 and Gp17) were fused with different fluorescent proteins and used for the identification of P. aeruginosa and E. coli by the measurement of the distinct fluorescent signals obtained. The developed microfluidic-based assay enabled a fast (70min) and highly specific multiplex detection of both pathogens in whole blood, achieving a detection limit of around 103 CFU, without requiring any time-consuming bacterial pre-enrichment step. Furthermore, it provided a quantitative assessment of bacterial loads present in blood. Noteworthy, this miniaturized and inexpensive device presents simple fabrication and operation, showing great potential to be fully automated, demonstrating to be ideal in point-of-care settings., The authors acknowledge the funding from the Portuguese Foundation for Science and Technology (FCT) under the scope of the project “Phages‐on‐chip” PTDC/BTM‐SAL/32442/2017 (POCI‐01-0145‐FEDER‐032442) and the strategic funding of the research units CEB (UIDB/04469/2020) and INESC MN (UID/05367/2020) through the pluriannual BASE and PROGRAMATICO financing and BioTecNorte operation (NORTE‐01-0145‐FEDER‐000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. S.P.C. and C.R.F.C. acknowledge the FCT for the grants SFRH/BD/130098/2017 and PD/BD/135274/2017, respectively., info:eu-repo/semantics/publishedVersion

A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.

Label-free approaches to biomolecular detection in microfluidics avoid potential labeling interference, are suitable for the screening of analytes that are not easily tagged, and simplify assay development toward point-of-use applications. In this paper, multimodal label-free detection of biomolecules was performed in a microfluidic system: 1) optically, with an integrated hydrogenated amorphous silicon thin-film photodiode and 2) electrically, using integrated electrochemical impedance interdigitated microelectrodes. Bovine serum albumin, single-stranded DNA, and a mixture of human immunoglobulins G were selected as model biomolecules. The sensitivities of the biomolecular detection were first evaluated in a bare microfluidic channel. The lower limit of detection obtained, for each transducing technique, was 10 nM for the impedimetric detection of ssDNA and 80 nM for the optical detection of a mixture of antibodies. These sensitivities were considered fit-for-purpose when applied to microfluidic systems aimed at monitoring molecular adsorption to different immobilized ligands. To validate the proposed multimodal detection, the model biomolecules were detected in a microfluidic structure packed with ion exchange chromatography agarose microbeads and the target biomolecules were monitored in real time both at bead level and downstream of the packed beads. The application of these label-free sensors ranges from bioprocess optimization to molecular purification and potentially the development of rapid analytics, with the advantages of device compactness, cost effectiveness, high speed of analysis, and multiplexed signal acquisition.

Microelectromechanical sensors have extreme mass sensitivities, making them promising for mass sensing, gas sensing, and biosensing applications. Despite the numerous examples of MEMS/NEMS mass sensors in the literature, the characterization of the resonator or oscillator system is often incomplete. Resonant mass sensor studies should include phase noise (or Allan deviation), as the use of these metrics allows for the identification of the noise mechanisms, the optimization of the measurement and actuation parameters, the prediction of the limit of detection, and the comparison of different types of resonators or vibration modes. In this paper, we summarize the metrics we consider to be the most important in the characterization of dynamic mass sensors, and then apply them to low-cost, hydrogenated amorphous silicon (a-Si:H) thin-film MEMS cantilever resonators. The effects of the actuation voltage on the phase noise were studied. The mass sensitivities of these resonators were calibrated using patterned thin-films localized at the tip of the cantilevers as well as uniform ultra-thin films of SiO2. The experimental mass sensitivities in vacuum ranged −98 Hz/pg to −4.6 kHz/pg. The limits of detection were calculated to be 100-833 fg in vacuum and 37-846 pg at atmospheric pressure. [2019-0006]

The demand for portable, rapid, sensitive and low-cost DNA detection systems that could be used for rapid diagnostic of disease and infection has greatly motivated the development of new DNA detection platforms in recent years. However, these platforms are often highly complex, requiring an excessively long time of analysis and/or suffer from inadequate sensitivity. Here we report a rapid and simple bead-based microfluidic platform to detect a specific 22-mer DNA sequence via hybridization, serving as a model of a miRNA biomarker for tumor and cardiovascular diseases. Commercial nanoporous chromatography beads were used as solid support for probe DNA immobilization, using either a single-mode electrostatic interaction or multi-mode interactions (electrostatic and hydrophobic). Using a mass balance approach, a probe density of 2.4 × 10 13 ± 18 % ( ± RSD%) molecules/cm2 was quantified in optimized conditions, which was found to provide hybridization efficiencies above 95 % and a hybridization dissociation constant below 1 nM. Comparing targets with different optical labels, namely Atto 430LS, quantum dots and horseradish peroxidase, the lowest limit of detection of 9.5 ± 1.1 pM was achieved with an assay time of 10 min using a quantum dot label coupled with a multi-mode immobilization. These results highlight the potential of the system to be applied as a simple and highly sensitive DNA hybridization platform, achieving low pM sensitivities without the need of a DNA amplification procedure.

Bacterial, fungal and viral infections in plant systems are on the rise, most of which tend to spread quickly amongst crops. These pathogens are also gaining resistance to known treatments, which makes their early detection a priority to avoid extensive loss of crops and the spreading of disease to animal systems. In this work, we propose a microfluidic platform coupled with integrated thin-film silicon photosensors for the detection of pathogen infections in grapes. This detection was achieved by monitoring the concentration of Azelaic Acid (AzA). This small organic acid plays a significant role in the defense mechanism in plant systems. In this platform, the enzyme tyrosinase was immobilized on microbeads inside a microfluidic system. By colorimetric monitoring of the inhibitory effect of AzA on the enzyme tyrosinase in real time, it was possible, in under 10 minutes, to detect different concentrations of AzA in both buffer and spiked solutions of grape juice, in both cases with limits of detection in the 5-10 nM range. In addition, with this microfluidic device, it was possible to clearly distinguish infected from healthy grape samples at three different grape maturation points. Healthy grape samples showed AzA concentrations in the range of 10-20 nM (post-dilution) while infected samples have an estimated increase of AzA of 10-30×, results which were confirmed using HPLC. In both juice and grape samples an integrated sample preparation stage that decreases the phenol content of the solutions was required to achieve fit-for-purpose sensitivities to AzA.

Microfluidic strategies combined with transduction and electronic integration have the promise of enabling miniaturized, combinatorial assays at higher speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In this chapter we introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively evaluate the transient calcium response of the endogenous Muscarinic type 1 receptor (M1) in HEK 293 T cells. The microfluidic fabrication protocol is described as well as a methodology to monitor the cell response in real time, after stimulation with M1 agonists (e.g., carbachol) and antagonists (e.g., Pirenzepine).

The detection of pathogens in primary care settings is critical, not only for virus detection but also for the detection of antibiotic resistant bacteria. Microfluidic devices can be used to develop point-of-care detection systems, but are usually insufficiently sensitive for the application. An on-chip isothermal amplification technique, such as the padlock probing-based rolling circle amplification technique, could potentially achieve the desired specificity and sensitivity, while keeping the complexity of the microfluidic device low. In this work, we describe an innovative microfluidic rolling circle amplification performed on microbeads using a continuous flow of solutions with the potential to achieve the required clinical demands in fast and sensitive fashion. Moreover, the integration with amorphous-hydrogenated silicon thin film p-i-n photodiode and a high-pass interference filter to allow the acquisition of the fluorescence signal from the amplification product on-chip, demonstrates the capability of performing the full assay on-chip along with the portability potential of the device.

Microfluidic strategies combined with transduction and electronic integration have the promise of enabling miniaturized, combinatorial assays at higher speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In this chapter we introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively evaluate the transient calcium response of the endogenous Muscarinic type 1 receptor (M1) in HEK 293 T cells. The microfluidic fabrication protocol is described as well as a methodology to monitor the cell response in real time, after stimulation with M1 agonists (e.g., carbachol) and antagonists (e.g., Pirenzepine).

Prostate cancer (PCa) diagnosis methods lack specificity and sensitivity or are extremely invasive. Thus, the fast, simple, reliable and accurate detection of low-grade PCa biomarkers could have a great clinical impact, saving lives and reducing patient uncertainty. Studies reported the overexpression of the protein nucleolin (NCL) on the cell surface of PCa cells and can be considered as a cancer-selective target. NCL binds specifically G-rich sequences, namely DNA G-quadruplex (G4) aptamers like AS1411, which can be modified and act as a molecular beacon (MB) for detection. In the present work, we have designed a microfluidic platform for the detection of NCL using a modified AS1411 aptamer beacon (AS1411-N5). When the MB recognizes NCL, it is prompted to undergo a conformational change, separating the fluorophore from the quencher and restoring fluorescence. Firstly, we started to characterize structurally the MB. Circular dichroism and Nuclear Magnetic Resonance spectroscopies indicated that the MB forms a parallel G4 structure. The affinity with NCL was determined fluorometric titration. Localization of MB was assessed in the PCa cell line (PC-3), further demonstrating the ability of the MB to recognize cell-surface NCL. To assess the clinically relevant NCL concentration of human PCa, ELISA assay was performed using peripheral blood mononuclear cells of PCa patients. Finally, the microfluidic assays showed the capacity of the microfluidic device in recognizing NCL in complexed samples like spiked human plasma. Concluding, it is here demonstrated that the microfluidic platform based on the AS1411-N5 could be used as a specific and selective approach to detect NCL in biological samples and contribute to the early-stage diagnosis of PCa.

Colorectal cancer (CRC) is responsible for millions of deaths every year. The staggering numbers call for the development of efficient diagnostic and treatment strategies, since the survival chances increase with earlier detection, in addition to the interpatient variability of treatment efficiency. Phage display emerges as a promising technique that allows the screening of new therapeutics for a specific target. In the present work, a microfluidic platform to culture CRC cells from the HCT-116 cell line for at least 48 h was developed. The cultured cells were then used as targets for BODIPY-FL labelled M13 phage capture and the signals were monitored through fluorescence microscopy imaging. Furthermore, a microfabricated a-Si:H photodiode and a custom-designed high-pass interference filter were developed and incorporated in the acquisition setup to allow the acquisition of fluorescence signal from the labelled M13 phages without the need of a fluorescence microscope.