Leveraging the future of diagnosis and management of diabetes: From old indexes to new technologies.

BACKGROUND: Diabetes is a heterogeneous and multifactorial disease. However, glycemia and glycated hemoglobin have been the focus of diabetes diagnosis and management for the last decades. As diabetes management goes far beyond glucose control, it has become clear that assessment of other biochemical parameters gives a much wider view of the metabolic state of each individual, enabling a precision medicine approach. METHODS: In this review, we summarize and discuss indexes that have been used in epidemiological studies and in the clinical practice. RESULTS: Indexes of insulin secretion, sensitivity/resistance and metabolism have been developed and validated over the years to account also with insulin, C-peptide, triglycerides or even anthropometric measures. Nevertheless, each one has their own objective and consequently, advantages and disadvantages for specific cases. Thus, we discuss how new technologies, namely new sensors but also new softwares/applications, can improve the diagnosis and management of diabetes, both for healthcare professionals but also for caretakers and, importantly, to promote the empowerment of people living with diabetes. CONCLUSIONS: In long-term, the solution for a better diabetes management would be a platform that allows to integrate all sorts of relevant information for the person with diabetes and for the healthcare practitioners, namely glucose, insulin and C-peptide or, in case of need, other parameters/indexes at home, sometimes more than once a day. This solution would allow a better and simpler disease management, more adequate therapeutics thereby improving patients' quality of life and reducing associated costs.

A simple polystyrene microfluidic device for sensitive and accurate SERS-based detection of infection by malaria parasites.

Early and accurate detection of infection by pathogenic microorganisms, such as Plasmodium, the causative agent of malaria, is critical for clinical diagnosis and ultimately determines the patient's outcome. We have combined a polystyrene-based microfluidic device with an immunoassay which utilises Surface-Enhanced Raman Spectroscopy (SERS) to detect malaria. The method can be easily translated to a point-of-care testing format and shows excellent sensitivity and specificity, when compared to the gold standard for laboratorial detection of Plasmodium infections. The device can be fabricated in less than 30 min by direct patterning on shrinkable polystyrene sheets of adaptable three-dimensional microfluidic chips. To validate the microfluidic system, samples of P. falciparum-infected red blood cell cultures were used. The SERS-based immunoassay enabled the detection of 0.0012 ± 0.0001% parasitaemia in a P. falciparum-infected red blood cell culture supernatant, an ∼7-fold higher sensitivity than that attained by most rapid diagnostic tests. Our approach successfully overcomes the main challenges of the current Plasmodium detection methods, including increased reproducibility, sensitivity, and specificity. Furthermore, our system can be easily adapted for detection of other pathogens and has excellent properties for early diagnosis of infectious diseases, a decisive step towards lowering their high burden on healthcare systems worldwide.

Hybrid Digital-Droplet Microfluidic Chip for Applications in Droplet Digital Nucleic Acid Amplification: Design, Fabrication and Characterization.

Microfluidic-based platforms have become a hallmark for chemical and biological assays, empowering micro- and nano-reaction vessels. The fusion of microfluidic technologies (digital microfluidics, continuous-flow microfluidics, and droplet microfluidics, just to name a few) presents great potential for overcoming the inherent limitations of each approach, while also elevating their respective strengths. This work exploits the combination of digital microfluidics (DMF) and droplet microfluidics (DrMF) on a single substrate, where DMF enables droplet mixing and further acts as a controlled liquid supplier for a high-throughput nano-liter droplet generator. Droplet generation is performed at a flow-focusing region, operating on dual pressure: negative pressure applied to the aqueous phase and positive pressure applied to the oil phase. We evaluate the droplets produced with our hybrid DMF-DrMF devices in terms of droplet volume, speed, and production frequency and further compare them with standalone DrMF devices. Both types of devices enable customizable droplet production (various volumes and circulation speeds), yet hybrid DMF-DrMF devices yield more controlled droplet production while achieving throughputs that are similar to standalone DrMF devices. These hybrid devices enable the production of up to four droplets per second, which reach a maximum circulation speed close to 1540 µm/s and volumes as low as 0.5 nL.

Stacking-Dependent Electrical Transport in a Colloidal CdSe Nanoplatelet Thin-Film Transistor.

Here, we report an exceptional feature of the one-dimensional threadlike assemblies of a four-monolayer colloidal CdSe nanoplatelet (NPL)-based thin-film transistor. A series of different lengths of threads (200-1200 nm) was used as an active n channel in thin-film transistors (TFTs) to understand the change in mobility with the length of the threads. The film with the longest threads shows the highest conductivity of ∼12 S/cm and electron mobility of ∼14.3 cm2 V-1 s-1 for an applied gate voltage of 2 V. The mobility trends with the length seem to be driven mostly by the lower defects in threads, where the loss of electron hopping is less. Furthermore, our results show the mobility trends in stacking-dependent CdSe NPL threads and provide a new insight into fabricating high-mobility TFTs with the use of colloidal CdSe NPLs.

Bandlike Transport in FaPbBr3 Quantum Dot Phototransistor with High Hole Mobility and Ultrahigh Photodetectivity.

Halide perovskites have been widely explored for numerous optoelectronic applications among which phototransistors have appeared as one of the most promising light signal detectors. However, it is still a great challenge to endow halide perovskites with both mobility and high photosensitivity because of their high sensitivity to moisture in ambient atmosphere. Here, we explore an FAPbBr3 perovskite quantum dot (QD) phototransistor with bandlike charge transport and measure a dark hole mobility of 14.2 cm2 V-1 s-1 at ambient atmosphere. Attaining both high mobility and good optical figures of merit, a detectivity of ∼1016 Jones is achieved, which is a record for halide perovskite nanocrystals. Simple A-site salt (FABr) treatments offer a mechanism for connecting between perovskite QDs for better charge transfer in high-quality devices. All of these important properties are superior to most advanced inorganic semiconductor phototransistors, indicating a promising future in optoelectronic applications.

Paper-based laser-induced graphene for sustainable and flexible microsupercapacitor applications.

Laser-induced graphene (LIG) is as a promising material for flexible microsupercapacitors (MSCs) due to its simple and cost-effective processing. However, LIG-MSC research and production has been centered on non-sustainable polymeric substrates, such as polyimide. In this work, it is presented a cost-effective, reproducible, and robust approach for the preparation of LIG structures via a one-step laser direct writing on chromatography paper. The developed strategy relies on soaking the paper in a 0.1 M sodium tetraborate solution (borax) prior to the laser processing. Borax acts as a fire-retardant agent, thus allowing the laser processing of sensitive substrates that other way would be easily destroyed under the high-energy beam. LIG on paper exhibiting low sheet resistance (30 Ω sq-1) and improved electrode/electrolyte interface was obtained by the proposed method. When used as microsupercapacitor electrodes, this laser-induced graphene resulted in specific capacitances of 4.6 mF cm-2 (0.015 mA cm-2). Furthermore, the devices exhibit excellent cycling stability (> 10,000 cycles at 0.5 mA cm-2) and good mechanical properties. By connecting the devices in series and parallel, it was also possible to control the voltage and energy delivered by the system. Thus, paper-based LIG-MSC can be used as energy storage devices for flexible, low-cost, and portable electronics. Additionally, due to their flexible design and architecture, they can be easily adapted to other circuits and applications with different power requirements.

Condensation and Protection of DNA by the Myxococcus xanthus Encapsulin: A Novel Function.

Encapsulins are protein nanocages capable of harboring smaller proteins (cargo proteins) within their cavity. The function of the encapsulin systems is related to the encapsulated cargo proteins. The Myxococcus xanthus encapsulin (EncA) naturally encapsulates ferritin-like proteins EncB and EncC as cargo, resulting in a large iron storage nanocompartment, able to accommodate up to 30,000 iron atoms per shell. In the present manuscript we describe the binding and protection of circular double stranded DNA (pUC19) by EncA using electrophoretic mobility shift assays (EMSA), atomic force microscopy (AFM), and DNase protection assays. EncA binds pUC19 with an apparent dissociation constant of 0.3 ± 0.1 µM and a Hill coefficient of 1.4 ± 0.1, while EncC alone showed no interaction with DNA. Accordingly, the EncAC complex displayed a similar DNA binding capacity as the EncA protein. The data suggest that initially, EncA converts the plasmid DNA from a supercoiled to a more relaxed form with a beads-on-a-string morphology. At higher concentrations, EncA self-aggregates, condensing the DNA. This process physically protects DNA from enzymatic digestion by DNase I. The secondary structure and thermal stability of EncA and the EncA-pUC19 complex were evaluated using synchrotron radiation circular dichroism (SRCD) spectroscopy. The overall secondary structure of EncA is maintained upon interaction with pUC19 while the melting temperature of the protein (Tm) slightly increased from 76 ± 1 °C to 79 ± 1 °C. Our work reports, for the first time, the in vitro capacity of an encapsulin shell to interact and protect plasmid DNA similarly to other protein nanocages that may be relevant in vivo.

Deacetylation and Desuccinylation of the Fucose-Rich Polysaccharide Fucopol: Impact on Biopolymer Physical and Chemical Properties.

FucoPol is an acylated polysaccharide with demonstrated valuable functional properties that include a shear thinning fluid behaviour, a film-forming capacity, and an emulsion forming and stabilizing capacity. In this study, the different conditions (concentration, temperature, and time) for alkaline treatment were investigated to deacylate FucoPol. Complete deacetylation and desuccinylation was achieved with 0.02 M NaOH, at 60 °C for 15 min, with no significant impact on the biopolymer's sugar composition, pyruvate content, and molecular mass distribution. FucoPol depyruvylation by acid hydrolysis was attempted, but it resulted in a very low polymer recovery. The effect of the ionic strength, pH, and temperature on the deacetylated/desuccinylated polysaccharide, d-FucoPol, was evaluated, as well as its emulsion and film-forming capacity. d-FucoPol aqueous solutions maintained the shear thinning behaviour characteristic of FucoPol, but the apparent viscosity decreased significantly. Moreover, contrary to FucoPol, whose solutions were not affected by the media's ionic strength, the d-FucoPol solutions had a significantly higher apparent viscosity for a higher ionic strength. On the other hand, the d-FucoPol solutions were not affected by the pH in the range of 3.6-11.5, while FucoPol had a decreased viscosity for acidic pH values and for a pH above 10.5. Although d-FucoPol displayed an emulsification activity for olive oil similar to that of FucoPol (98 ± 0%) for an oil-to-water ratio of 2:3, the emulsions were less viscous. The d-FucoPol films were flexible, with a higher Young's modulus (798 ± 152 MPa), a stress at the break (22.5 ± 2.5 MPa), and an elongation at the break (9.3 ± 0.7%) than FucoPol (458 ± 32 MPa, 15.5 ± 0.3 MPa and 8.1 ± 1.0%, respectively). Given these findings, d-FucoPol arises as a promising novel biopolymer, with distinctive properties that may render it useful for utilization as a suspending or emulsifier agent, and as a barrier in coatings and packaging films.

Solution Combustion Synthesis: Towards a Sustainable Approach for Metal Oxides.

Solution combustion synthesis (SCS) has been widely used to produce simple and complex oxides with a desired morphology (size and shape). SCS is valuable due to low cost, simplicity and energy efficient synthesis. To guarantee the best molecular-level mixing of reactants in an aqueous or solvent-based solution some parameters need to be controlled, such as fuel type, metal cations precursors, stoichiometry ratio (φ), pH effect, atmosphere and initiation type. These determine the final properties of the oxide materials, providing the potential to reach different morphologies, which are essential for their final applications. This Review article focuses on the crucial parameters in SCS and how these affect the overall materials properties from nanostructures to thin films. To finalize, special attention is given to the application of SCS to form metal oxide thin films at low temperature and their application in thin film transistors (TFTs).

Supramolecular protein polymers using mini-ferritin Dps as the building block.

A missense mutant of a Dps protein (DNA-binding protein from starved cells) from Marinobacter hydrocarbonoclasticus was used as a building block to develop a new supramolecular assembly complex which enhances the iron uptake, a physiological function of this mini-ferritin. The missense mutation was conducted in an exposed and flexible region of the N-terminal, wherein a threonine residue in position 10 was replaced by a cysteine residue (DpsT10C). This step enabled a click chemistry approach to the variant DpsT10C, where a thiol-ene coupling occurs. Two methods and two types of linker were used resulting in two different mini-ferritin supramolecular polymers, which have maintained secondary structure and native iron uptake physiological function. Electrophoretic assays and mass spectrometry were utilized to confirm that both functionalization and coupling reactions occured as predicted. The secondary structure has been investigated by circular dichroism and synchrotron radiation circular dichroism. Size and morphology were obtained by dynamic light scattering, size exclusion chromatography and atomic force microscopy, respectively. The iron uptake of the synthesized protein polymers was confirmed by UV-Vis spectroscopy loading assays.

Transduction Mechanisms, Micro-Structuring Techniques, and Applications of Electronic Skin Pressure Sensors: A Review of Recent Advances.

Electronic skin (e-skin), which is an electronic surrogate of human skin, aims to recreate the multifunctionality of skin by using sensing units to detect multiple stimuli, while keeping key features of skin such as low thickness, stretchability, flexibility, and conformability. One of the most important stimuli to be detected is pressure due to its relevance in a plethora of applications, from health monitoring to functional prosthesis, robotics, and human-machine-interfaces (HMI). The performance of these e-skin pressure sensors is tailored, typically through micro-structuring techniques (such as photolithography, unconventional molds, incorporation of naturally micro-structured materials, laser engraving, amongst others) to achieve high sensitivities (commonly above 1 kPa-1), which is mostly relevant for health monitoring applications, or to extend the linearity of the behavior over a larger pressure range (from few Pa to 100 kPa), an important feature for functional prosthesis. Hence, this review intends to give a generalized view over the most relevant highlights in the development and micro-structuring of e-skin pressure sensors, while contributing to update the field with the most recent research. A special emphasis is devoted to the most employed pressure transduction mechanisms, namely capacitance, piezoelectricity, piezoresistivity, and triboelectricity, as well as to materials and novel techniques more recently explored to innovate the field and bring it a step closer to general adoption by society.

Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers.

Microfluidic (MF) advancements have been leveraged toward the development of state-of-the-art platforms for molecular diagnostics, where isothermal amplification schemes allow for further simplification of DNA detection and quantification protocols. The MF integration with loop-mediated isothermal amplification (LAMP) is today the focus of a new generation of chip-based devices for molecular detection, aiming at fast and automated nucleic acid analysis. Here, we combined MF with droplet digital LAMP (ddLAMP) on an all-in-one device that allows for droplet generation, target amplification, and absolute quantification. This multilayer 3D chip was developed in less than 30 minutes by using a low-cost and extremely adaptable production process that exploits direct laser writing technology in "Shrinky-dinks" polystyrene sheets. ddLAMP and target quantification were performed directly on-chip, showing a high correlation between target concentration and positive droplet score. We validated this integrated chip via the amplification of targets ranging from five to 500,000 copies/reaction. Furthermore, on-chip amplification was performed in a 10 µL volume, attaining a limit of detection of five copies/µL under 60 min. This technology was applied to quantify a cancer biomarker, c-MYC, but it can be further extended to any other disease biomarker.

Biofilm development and computational screening for new putative inhibitors of a homolog of the regulatory protein BrpA in Streptococcus dysgalactiae subsp. dysgalactiae.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.

Antibody modified gold nanoparticles for fast colorimetric screening of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic joint inflammation and one of the main causes of chronic disability worldwide with high prevalence in the ageing population. RA is characterized by autoantibody production, synovial inflammation and bone destruction, and the most accepted biomarker is rheumatoid factor (RF) autoantibodies. In this work, we developed a low-cost approach for the detection and quantification of the RF marker. This colorimetric immunosensor is based on gold nanoprobe crosslinking that results in extensive aggregation in the presence of the pentameric IgM RF. Aggregation of the nanoconjugates yields a color change from red to purple that can be easily observed by the naked eye. The interaction between nanoconjugates and the specific target was confirmed via dynamic light scattering (DLS), Raman spectroscopy and atomic force microscopy (AFM) imaging. This conceptual system shows a LOD of 4.15 UA mL-1 IgM RF (clinical threshold is set for 20 IU mL-1). The one-step biosensor strategy herein proposed is much faster than conventional detection techniques, without the need for secondary antibodies, additional complex washing or signal amplification protocols. To the best of our knowledge this is the first report on target induced aggregation of gold nanoprobes for quantitative colorimetric autoantibody detection.

E-Skin Bimodal Sensors for Robotics and Prosthesis Using PDMS Molds Engraved by Laser.

Electronic skin (e-skin) is pursued as a key component in robotics and prosthesis to confer them sensing properties that mimic human skin. For pressure monitoring, a great emphasis on piezoresistive sensors was registered due to the simplicity of sensor design and readout mechanism. For higher sensitivity, films composing these sensors may be micro-structured, usually by expensive photolithography techniques or low-cost and low-customizable molds. Sensors commonly present different sensitivities in different pressure ranges, which should be avoided in robotics and prosthesis applications. The combination of pressure sensing and temperature is also relevant for the field and has room for improvement. This work proposes an alternative approach for film micro-structuration based on the production of highly customizable and low-cost molds through laser engraving. These bimodal e-skin piezoresistive and temperature sensors could achieve a stable sensitivity of -6.4 × 10-3 kPa-1 from 1.6 kPa to 100 kPa, with a very robust and reproducible performance over 27,500 cycles of objects grasping and releasing and an exceptionally high temperature coefficient of resistance (TCR) of 8.3%/°C. These results point toward the versatility and high benefit/cost ratio of the laser engraving technique to produce sensors with a suitable performance for robotics and functional prosthesis.

Critical role of a double-layer configuration in solution-based unipolar resistive switching memories.

Lately, resistive switching memories (ReRAM) have been attracting a lot of attention due to their possibilities of fast operation, lower power consumption and simple fabrication process and they can also be scaled to very small dimensions. However, most of these ReRAM are produced by physical methods and nowadays the industry demands more simplicity, typically associated with low cost manufacturing. As such, ReRAMs in this work are developed from a solution-based aluminum oxide (Al2O3) using a simple combustion synthesis process. The device performance is optimized by two-stage deposition of the Al2O3 film. The resistive switching properties of the bilayer devices are reproducible with a yield of 100%. The ReRAM devices show unipolar resistive switching behavior with good endurance and retention time up to 105 s at 85 °C. The devices can be programmed in a multi-level cell operation mode by application of different reset voltages. Temperature analysis of various resistance states reveals a filamentary nature based on the oxygen vacancies. The optimized film was stacked between ITO and indium zinc oxide, targeting a fully transparent device for applications on transparent system-on-panel technology.

Digital Microfluidics for Nucleic Acid Amplification.

Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

A Digital Microfluidics Platform for Loop-Mediated Isothermal Amplification Detection.

Digital microfluidics (DMF) arises as the next step in the fast-evolving field of operation platforms for molecular diagnostics. Moreover, isothermal schemes, such as loop-mediated isothermal amplification (LAMP), allow for further simplification of amplification protocols. Integrating DMF with LAMP will be at the core of a new generation of detection devices for effective molecular diagnostics at point-of-care (POC), providing simple, fast, and automated nucleic acid amplification with exceptional integration capabilities. Here, we demonstrate for the first time the role of coupling DMF and LAMP, in a dedicated device that allows straightforward mixing of LAMP reagents and target DNA, as well as optimum temperature control (reaction droplets undergo a temperature variation of just 0.3 °C, for 65 °C at the bottom plate). This device is produced using low-temperature and low-cost production processes, adaptable to disposable and flexible substrates. DMF-LAMP is performed with enhanced sensitivity without compromising reaction efficacy or losing reliability and efficiency, by LAMP-amplifying 0.5 ng/µL of target DNA in just 45 min. Moreover, on-chip LAMP was performed in 1.5 µL, a considerably lower volume than standard bench-top reactions.

Validating silicon polytrodes with paired juxtacellular recordings: method and dataset.

Cross-validating new methods for recording neural activity is necessary to accurately interpret and compare the signals they measure. Here we describe a procedure for precisely aligning two probes for in vivo "paired-recordings" such that the spiking activity of a single neuron is monitored with both a dense extracellular silicon polytrode and a juxtacellular micropipette. Our new method allows for efficient, reliable, and automated guidance of both probes to the same neural structure with micrometer resolution. We also describe a new dataset of paired-recordings, which is available online. We propose that our novel targeting system, and ever expanding cross-validation dataset, will be vital to the development of new algorithms for automatically detecting/sorting single-units, characterizing new electrode materials/designs, and resolving nagging questions regarding the origin and nature of extracellular neural signals.

Single nucleotide polymorphism detection using gold nanoprobes and bio-microfluidic platform with embedded microlenses.

The use of microfluidics platforms combined with the optimal optical properties of gold nanoparticles has found plenty of application in molecular biosensing. This paper describes a bio-microfluidic platform coupled to a non-cross-linking colorimetric gold nanoprobe assay to detect a single nucleotide polymorphism associated with increased risk of obesity fat-mass and obesity-associated (FTO) rs9939609 (Carlos et al., 2014). The system enabled significant discrimination between positive and negative assays using a target DNA concentration of 5 ng/µL below the limit of detection of the conventionally used microplate reader (i.e., 15 ng/µL) with 10 times lower solution volume (i.e., 3 µL). A set of optimization of our previously reported bio-microfluidic platform (Bernacka-Wojcik et al., 2013) resulted in a 160% improvement of colorimetric analysis results. Incorporation of planar microlenses increased 6 times signal-to-loss ratio reaching the output optical fiber improving by 34% the colorimetric analysis of gold nanoparticles, while the implementation of an optoelectronic acquisition system yielded increased accuracy and reduced noise. The microfluidic chip was also integrated with a miniature fiber spectrometer to analyze the assays' colorimetric changes and also the LEDs transmission spectra when illuminating through various solutions. Furthermore, by coupling an optical microscope to a digital camera with a long exposure time (30 s), we could visualise the different scatter intensities of gold nanoparticles within channels following salt addition. These intensities correlate well to the expected difference in aggregation between FTO positive (none to small aggregates) and negative samples (large aggregates).