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Digital droplet PCR (ddPCR) is a powerful amplification technique for absolute quantification of viral nucleic acids. Although commercial ddPCR devices are effective in the lab bench tests, they cannot meet current urgent requirements for on-site and rapid screening for patients. Here, we have developed a portable and fully integrated lab-on-a-disc (LOAD) device for quantitively screening infectious disease agents. Our designed LOAD device has integrated (i) microfluidics chips, (ii) a transparent circulating oil-based heat exchanger, and (iii) an on-disc transmitted-light fluorescent imaging system into one compact and portable box. Thus, droplet generation, PCR thermocycling, and analysis can be achieved in a single LOAD device. This feature is a significant attribute for the current clinical application of disease screening. For this custom-built ddPCR setup, we have first demonstrated the loading and ddPCR amplification ability by using influenza A virus-specific DNA fragments with different concentrations (diluted from the original concentration to 107 times), followed by analyzing the droplets with an external fluorescence microscope as a standard calibration test. The measured DNA concentration is linearly related to the gradient–dilution factor, which validated the precise quantification for the samples. In addition to the calibration tests using DNA fragments, we also employed this ddPCR-LOAD device for clinical samples with different viruses. Infectious samples containing five different viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), varicella zoster virus (VZV), Zika virus (ZIKV), and adenovirus (ADV), were injected into the device, followed by analyzing the droplets with an external fluorescence microscope with the lowest detected concentration of 20.24 copies/µL. Finally, we demonstrated the proof-of-concept detection of clinical samples of IAV using the on-disc fluorescence imaging system in our fully integrated device, which proves the capability of this device in clinical sample detection. We anticipate that this integrated ddPCR-LOAD device will become a flexible tool for on-site disease detection. [ABSTRACT FROM AUTHOR]

An innovative modular approach for facile design and construction of flexible microfluidic biosensor platforms based on a dry manufacturing "craft-and-stick" approach is developed. The design and fabrication of the flexible graphene paper electrode (GPE) unit and polyethylene tetraphthalate sheet (PET)6/adhesive fluidic unit are completed by an economic and generic xurographic craft approach. The GPE widths and the microfluidic channels can be constructed down to 300 μm and 200 μm, respectively. Both units were assembled by simple double-sided adhesive tapes into a microfluidic integrated GPE (MF-iGPE) that are flexible, thin (<0.5 mm), and lightweight (0.4 g). We further functionalized the iGPE with Prussian blue and glucose oxidase for the fabrication of MF-iGPE glucose biosensors. With a closed-channel PET fluidic pattern, the MF-iGPE glucose biosensors were packaged and sealed to protect the integrated device from moisture for storage and could easily open with scissors for sample loading. Our glucose biosensors showed 2 linear dynamic regions of 0.05–1.0 and 1.0–5.5 mmol L−1 glucose. The MF-iGPE showed good reproducibility for glucose detection (RSD < 6.1%, n = 6) and required only 10 μL of the analyte. This modular craft-and-stick manufacturing approach could potentially further develop along the concept of paper-crafted model assembly kits suitable for low-resource laboratories or classroom settings. [ABSTRACT FROM AUTHOR]

We present a low-cost, accessible, and rapid fabrication process for electrochemical microfluidic sensors. This work leverages the accessibility of consumer-grade electronic craft cutters as the primary tool for patterning of sensor electrodes and microfluidic circuits, while commodity materials such as gold leaf, silver ink pen, double-sided tape, plastic transparency films, and fabric adhesives are used as its base structural materials. The device consists of three layers, the silver reference electrode layer at the top, the PET fluidic circuits in the middle and the gold sensing electrodes at the bottom. Separation of the silver reference electrode from the gold sensing electrodes reduces the possibility of cross-contamination during surface modification. A novel approach in mesoscale patterning of gold leaf electrodes can produce generic designs with dimensions as small as 250 μm. Silver electrodes with dimensions as small as 385 μm were drawn using a plotter and a silver ink pen, and fluid microchannels as small as 300 μm were fabricated using a sandwich of iron-on adhesives and PET. Device layers are then fused together using an office laminator. The integrated microfluidic electrochemical platform has electrode kinetics/performance of ΔEp = 91.3 mV, Ipa/Ipc = 0.905, characterized by cyclic voltammetry using a standard ferrocyanide redox probe, and this was compared against a commercial screen-printed gold electrode (ΔEp = 68.9 mV, Ipa/Ipc = 0.984). To validate the performance of the integrated microfluidic electrochemical platform, a catalytic hydrogen peroxide sensor and enzyme-coupled glucose biosensors were developed as demonstrators. Hydrogen peroxide quantitation achieves a limit of detection of 0.713 mM and sensitivity of 78.37 μA mM−1 cm−2, while glucose has a limit of detection of 0.111 mM and sensitivity of 12.68 μA mM−1 cm−2. This rapid process allows an iterative design–build–test cycle in under 2 hours. The upfront cost to set up the system is less than USD 520, with each device costing less than USD 0.12, making this manufacturing process suitable for low-resource laboratories or classroom settings. [ABSTRACT FROM AUTHOR]

A new approach to perform electrochemical immunoassay based on the utilization of encapsulated microcrystal was developed. The microcrystal labels create a 'supernova effect' upon exposure to a desired releasing agent. The microcrystal cores dissolve, and large amounts of signal-generating molecules diffuse across the capsule wall into the outer environment. Layer-by-Layer (LbL) technology was employed for the encapsulation of electrochemical signal-generating microcrystals (ferrocene microcrystals). The encapsulated microcrystals were conjugated with antibody molecules through the adsorption process. The biofunctionalized microcrystals were utilized as a probe for immunoassays. The microcrystal-based label system provided a high-signal molecule to antibody (S/P) ratio of [10.sup.4]-[10.sup.5]. Microcrystal biolabels with different antibody surface coverage (1.60-5.05 mg [m.sup.-2]) were subjected to a solid-phase immunoassay for the detection of mouse immunoglobulin G (M-IgG) molecules. The microcrystal-based immunoassay for the detection of M-IgG performed with microcrystals having antibody surface coverage of 5.05 mg [m.sup.-2] showed a sensitivity of 3.93 nA [micro][g.sup.-1] [L.sup.-1] with a detection limit of 2.82 [micro]g [L.sup.-1].

Extracellular matrix (ECM) components govern a range of cell functions, such as migration, proliferation, maintenance of stemness, and differentiation. Cell niches that harbor stem‐/progenitor cells, with matching ECM, have been shown in a range of organs, although their presence in the heart is still under debate. Determining niches depends on a range of in vitro and in vivo models and techniques, where animal models are powerful tools for studying cell‐ECM dynamics; however, they are costly and time‐consuming to use. In vitro models based on recombinant ECM proteins lack the complexity of the in vivo ECM. To address these issues, we present the spatiotemporal extracellular matrix model for studies of cell‐ECM dynamics, such as cell niches. This model combines gentle decellularization and sectioning of cardiac tissue, allowing retention of a complex ECM, with recellularization and subsequent image processing using image stitching, segmentation, automatic binning, and generation of cluster maps. We have thereby developed an in situ representation of the cardiac ECM that is useful for assessment of repopulation dynamics and to study the effect of local ECM composition on phenotype preservation of reseeded mesenchymal progenitor cells. This model provides a platform for studies of organ‐specific cell‐ECM dynamics and identification of potential cell niches. [ABSTRACT FROM AUTHOR]

The rapidly emerging field of organic bioelectronics has witnessed the wide use of conducting polymers (CPs) to fabricate advanced chemically modified electrodes (CMEs) for biosensors and biomedical devices. The electrochemical performance of the CPs in such devices is closely related to the quality and physiochemical nature of the dopants. A bi-functional graphene oxide derivative with high reduction degree and negatively-charged sulphonate functionality, i.e. sulphonate-coupled reduced graphene oxide (S-RGO), was developed and used as an efficient dopant for a CP with enhanced electrochemical performance. The S-RGO was synthesised via a facile one-pot hydrothermal reaction using 4-hydrazinobenzosulphonic acid (4-HBS) as reductant and sulphonate precursor simultaneously. The resulting S-RGO possesses high aqueous dispersion stability (more than 6 months), high electrical conductivity (1493.0 S m−1) and sulphonate functionality. Due to these specific properties, S-RGO demonstrated improved electropolymerisation efficiency for poly(3,4-ethylenedioxythiophene) (PEDOT) proving an effective dopant for the preparation of a PEDOT:S-RGO film (5 mC) with faster polymerisation time (37 s) compared to the conventional 2D dopants GO (PEDOT:GO, 129 s) and RGO (PEDOT:RGO, 66 s). The resulting PEDOT:S-RGO appeared as a homogenous film with uniformly distributed S-RGO dopant, low equivalent series resistance and low charge transfer resistance. Moreover, the electrochemical transduction performance of the PEDOT:S-RGO interface was evaluated with 4 different analytes, including ferric/ferrocyanide redox probe, dopamine, nicotinamide adenine dinucleotide and hydrogen peroxide. As a result of the synergistic effect of S-RGO and PEDOT, the PEDOT:S-RGO demonstrated enhanced electrochemical performance with respect to faster electrode kinetics (smaller ΔEp), ∼2 and ∼4 times increased current responses, and lower peak potentials compared to PEDOT:GO and PEDOT:RGO. This bi-functional S-RGO dopant combined the advantages of conventional GO and RGO to deliver sulphonate functionality and high conductivity for the preparation of advanced PEDOT interface with improved electrochemical performance, that could potentially be applied for applications in electrochemical sensors, biosensors and bioelectronic devices. [ABSTRACT FROM AUTHOR]

Microparticles have attracted much attention for medical, analytical and biological applications. Calcium carbonate (CaCO3) templating method with the advantages of having narrow size distribution, controlled morphology and good biocompatibility that has been widely used for the synthesis of various protein-based microparticles. Despite CaCO3 template is biocompatible, most of the conventional methods to create stable protein microparticles are mainly driven by chemical crosslink reagents which may induce potential harmful effect and remains undesirable especially for biomedical or clinical applications. In this article, we demonstrate the fabrication of protein microparticles and microcapsules with an innovative method using biomolecular tools such as enzymes and affinity molecules to trigger the assembling of protein molecules within a porous CaCO3 template followed by a template removal step. We demonstrated the enzyme-assisted fabrication of collagen microparticles triggered by transglutaminase, as well as the affinity-assisted fabrication of BSA-biotin avidin microcapsules triggered by biotin-avidin affinity interaction, respectively. Based on the different protein assemble mechanisms, the collagen microparticles appeared as a solid-structured particles, while the BSA-biotin avidin microcapsules appeared as hollow-structured morphology. The fabrication procedures are simple and robust that allows producing protein microparticles or microcapsules under mild conditions at physiological pH and temperature. In addition, the microparticle morphologies, protein compositions and the assemble mechanisms were studied. Our technology provides a facile approach to design and fabricate protein microparticles and microcapsules that are useful in the area of biomaterials, pharmaceuticals and analytical chemistry. [ABSTRACT FROM AUTHOR]

A new approach for the facile fabrication of electrochemical biosensors using a biohybrid conducting polymer was demonstrated using glucose oxidase (GOx) and poly (3, 4-ethylenedioxythiophene) (PEDOT) as a model. The biohybrid conducting polymer was prepared based on a template-assisted chemical polymerisation leading to the formation of PEDOT microspheres (PEDOT-MSs), followed by in-situ deposition of platinum nanoparticles (PtNPs) and electrostatic immobilisation of glucose oxidase (GOx) to form water processable GOx-PtNPs-PEDOT-MSs. The morphology, chemical composition and electrochemical performance of the GOx-PtNPs-PEDOT-MS-based glucose biosensor were characterised using scanning electron microscopy (SEM), energy-dispersive X-ray spectrometry (EDS), Fourier transform infrared (FTIR) spectroscopy, zeta potential and electrochemical measurements, respectively. The biosensor delivered a linear response for glucose over the range 0.1–10 mM (R 2 = 0.9855) with a sensitivity of 116.25 µA mM −1 cm −2 , and limit of detection of 1.55 µM (3×SD/sensitivity). The sensitivity of the developed PEDOT-MS based biosensor is significantly higher (2.7 times) than the best reported PEDOT-based glucose biosensor in the literature. The apparent Michaelis–Menten constant (K m app ) of the GOx-PtNPs-PEDOT-MS-based biosensors was calculated as 7.3 mM. Moreover, the biosensor exhibited good storage stability, retaining 97% of its sensitivity after 12 days storage. This new bio-hybrid conducting polymer combines the advantages of micro-structured morphology, compatibility with large-scale manufacturing processes, and intrinsic biocatalytic activity and conductivity, thus demonstrating its potential as a convenient material for printed bioelectronics and sensors. [ABSTRACT FROM AUTHOR]

Microstructured materials are becoming important for high performance electrochemical device especially for energy storage due to their advantageous diffusion and flux properties. Utilizing a rationally designed hollow structured polypyrrole microparticles (PPyMPs) with controllable wall thicknesses of ∼110 to 340 nm, we observed a significant morphological effect on electrocapacitive kinetics of the PPyMPs modulated by the voltammetric potential window and scan rate. The thin-hollow architecture of PPyMPs revealed significant enhancement of charge storage performance (up to 447 %), high retention at high scan rate and faster charge/discharge kinetics compared to the thick-hollow PPyMPs due to the larger accessible surface area and decrease of diffusion length. These findings demonstrated the electrocapacitive kinetics performance of microstructured soft materials related to morphological effect modulated by operational conditions. Our study provides new insight on electrochemistry of soft electrode materials with controlled nanostructured morphology for understanding the mechanism of charge insertion and mass diffusion for the future development of high performance porous electrode material. [ABSTRACT FROM AUTHOR]