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Recent key technological developments, such as super-resolution microscopy and microfabrication, enabled investigation of biological processes, including macroautophagy/autophagy, with unprecedented spatiotemporal resolution and control over experimental conditions. Such disruptive innovations deepened our capability to provide mechanistic understandings of the autophagic process and its causes. This addendum aims to expand the guidelines on autophagy in three key directions: optical methods enabling visualization of autophagic machinery beyond the diffraction-limited resolution; bioengineering enabling accurate designs and control over experimental conditions; and theoretical advances in mechanobiology connecting autophagy and mechanical processes of the cell. Abbreviation: 3D: three-dimensional; SIM: structured illumination microscopy; STORM: stochastic optical reconstruction microscopy.

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BACKGROUND: Developing disposable paper-based devices has positively impacted analytical science, particularly in developing countries. Some benefits of those devices include their versatility, affordability, environmentally friendly, and the possibility of being integrated with portable electrochemical or colorimetric detectors. Paper-based analytical devices (PADs) comprising circular zones and microfluidic networks have been successfully employed in the analytical chemistry reign. However, the combination of the stencil-printing method and alternative binder has not been satisfactorily explored for fabricating colorimetric paper devices. RESULTS: We developed PADs exploring the stencil printing approach and glass varnish as the hydrophobic chemical agent. As a proof-of-concept, the colorimetric assay of salivary α-amylase (sAA) was performed in saliva samples. Through the scanning electron microscopy measurements, it was possible to indicate satisfactory definitions between native fibers and barrier, and that the measured values for the channel width revealed suitable fidelity (R2 = 0.99) with the nominal widths (ranging from 400 to 5000 µm). The proposed hydrophobic barrier exhibited excellent chemical resistance. The analytical applicability for detecting sAA revealed linear behavior in the range from 2 to 12 U mL-1 (R2 = 0.99), limit of detection of 0.75 U mL-1, reproducibility (RSD ≤2.4%), recovery experiments ranged from 89 to 108% and AGREE response (0.86). In addition, the colorimetric analysis of sAA in four different saliva samples demonstrated levels ranging from 202 to 2080 U mL-1, which enabled monitoring the absence and presence of periodontitis. SIGNIFICANCE: This report has presented the first use of a self-adhesive mask and glass varnish for creating circular zones and microfluidic architectures on paper without using thermic or UV curing treatments. Also, the proposed analytical methodology for detecting sAA exhibited suitable ecological impact considering the AGREE tool. We believe the proposed fabrication of paper devices emerges as a novel, simple, high-fidelity microfluidic channel and portable analytical approach for colorimetric sensing.

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3D printers utilize cutting-edge technologies to create three-dimensional objects and are attractive tools for engineering compact microfluidic platforms with complex architectures for chemical and biochemical analyses. 3D printing's popularity is associated with the freedom of creating intricate designs using inexpensive instrumentation, and these tools can produce miniaturized platforms in minutes, facilitating fabrication scaleup. This work discusses key challenges in producing three-dimensional microfluidic structures using currently available 3D printers, addressing considerations about printer capabilities and software limitations encountered in the design and processing of new architectures. This article further communicates the benefits of using three-dimensional structures, including the ability to scalably produce miniaturized analytical systems and the possibility of combining them with multiple processes, such as mixing, pumping, pre-concentration, and detection. Besides increasing analytical applicability, such three-dimensional architectures are important in the eventual design of commercial devices since they can decrease user interferences and reduce the volume of reagents or samples required, making assays more reliable and rapid. Moreover, this manuscript provides insights into research directions involving 3D-printed microfluidic devices. Finally, this work offers an outlook for future developments to provide and take advantage of 3D microfluidic functionality in 3D printing. Graphical abstract Creating three-dimensional microfluidic structures using 3D printing will enable key advances and novel applications in (bio)chemical analysis.

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Microfluidics is an interdisciplinary field that encompasses both science and engineering, which aims to design and fabricate devices capable of manipulating extremely low volumes of fluids on a microscale level. The central objective of microfluidics is to provide high precision and accuracy while using minimal reagents and equipment. The benefits of this approach include greater control over experimental conditions, faster analysis, and improved experimental reproducibility. Microfluidic devices, also known as labs-on-a-chip (LOCs), have emerged as potential instruments for optimizing operations and decreasing costs in various of industries, including pharmaceutical, medical, food, and cosmetics. However, the high price of conventional prototypes for LOCs devices, generated in clean room facilities, has increased the demand for inexpensive alternatives. Polymers, paper, and hydrogels are some of the materials that can be utilized to create the inexpensive microfluidic devices covered in this article. In addition, we highlighted different manufacturing techniques, such as soft lithography, laser plotting, and 3D printing, that are suitable for creating LOCs. The selection of materials and fabrication techniques will depend on the specific requirements and applications of each individual LOC. This article aims to provide a comprehensive overview of the numerous alternatives for the development of low-cost LOCs to service industries such as pharmaceuticals, chemicals, food, and biomedicine.

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An important field of research is the miniaturization of analytical systems for laboratory applications and on-field analysis. In particular, gas chromatography (GC) has benefited from the recent advances in enabling technologies like photolithography, micromachining, hot embossing, and 3D-printing to improve sampling and sample preparation, microcolumn technologies, and detection. In this article, the developments and applications reported since 2015 were reviewed and summarized. Important applications using benchtop instruments, portable GCs, and micro-GCs (µGCs) were showcased to illustrate the current challenges associated with each miniaturized interfaces and systems. For instance, portable instruments need to be energy-efficient and ideally depend on renewable sources for carrier gas generation. Lastly, multidimensional separations were addressed using miniaturized systems to effectively improve the peak capacity of portable systems.

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The incorporation of sophisticated capabilities within microfluidic devices often requires the assembly of different layers in a correct arrangement. For example, when it is desired to include electrodes inside microfluidic channels or to create 3D microfluidic structures. However, the alignment between different substrates at the microscale requires expensive equipment not available for all research groups. In this work, we present an affordable, compact and portable aligner for assembling multilayered composite microfluidic chips. The instrument is composed of aluminum machined pieces combined with precision stages and includes a digital microscope with a LED illumination system for monitoring the alignment process. An interchangeable holder was created for substrate fixing, allowing the bonding of PDMS with other materials. Microscopic visualization is achieved through any device with internet access, avoiding the need of a computer attached to the aligner. To test the performance of the aligner, the center of an indium tin oxide microelectrode on a glass substrate was aligned with the center of a microchannel in a PDMS chip. The accuracy and precision of the instrument are suited for many microfluidic applications. The small and inexpensive design of the aligner makes it a cost-effective option for small groups working in microfluidics.

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In this article, we show an alternative low-cost fabrication method to obtain poly(dimethyl siloxane) (PDMS) microfluidic devices. The proposed method allows the inscription of micron resolution channels on polystyrene (PS) surfaces, used as a mold for the wanted microchip's production, by applying a high absorption coating film on the PS surface to ablate it with a focused low-power visible laser. The method allows for obtaining micro-resolution channels at powers between 2 and 10 mW and can realize any two-dimensional polymeric devices. The effect of the main processing parameters on the channel's geometry is presented.

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In recent years, controlled release of drugs has posed numerous challenges with the aim of optimizing parameters such as the release of the suitable quantity of drugs in the right site at the right time with the least invasiveness and the greatest possible automation. Some of the factors that challenge conventional drug release include long-term treatments, narrow therapeutic windows, complex dosing schedules, combined therapies, individual dosing regimens, and labile active substance administration. In this sense, the emergence of micro-devices that combine mechanical and electrical components, so called micro-electro-mechanical systems (MEMS) can offer solutions to these drawbacks. These devices can be fabricated using biocompatible materials, with great uniformity and reproducibility, similar to integrated circuits. They can be aseptically manufactured and hermetically sealed, while having mobile components that enable physical or analytical functions together with electrical components. In this review we present recent advances in the generation of MEMS drug delivery devices, in which various micro and nanometric structures such as contacts, connections, channels, reservoirs, pumps, valves, needles, and/or membranes can be included in their design and manufacture. Implantable single and multiple reservoir-based and transdermal-based MEMS devices are discussed in terms of fundamental mechanisms, fabrication, performance, and drug release applications.

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This study reports, for the first time, the possibility to manufacture analytical devices on polyester substrates using a cutting printer. The protocol involves the design of a layout in a graphical software, the cut into polyester films and the lamination against one or multiple polyester films coated with a thermosensitive layer. The feasibility of the proposed approach was demonstrated through the fabrication of 96-microwell plates, 3D microfluidic mixing and distance-based microfluidic devices. The printer has enabled cutting microchannels wider than 300 µm on polyester films and a thickness of 250 µm. Urea and glucose assays were performed on microwell plates aiming for their quantification in artificial urine and serum samples. The presented results revealed good agreement with the expected values. The complexation reaction between Fe2+ and o-phenanthroline was selected as model to investigate the feasibility of the 3D mixing device. Absorbance measurements were recorded for the reaction product performed in both on and off-chip modes. Considering the achieved data, the on-chip mixing exhibited similar behavior when compared to off-chip reaction, thus demonstrating to be efficient to perform mixtures due to the turbulence generated inside three-dimensional channels. Lastly, a distance-based device was designed to detect H2O2 based on the displacement of a dye plug promoted by the oxygen generation using a copper-modified paper sheet. The distance-based peroxymeter revealed a linear behavior in the concentration range between 1 and 5% (v/v) and a LOD equal to 0.5% (65.2 mM). Based on the results herein reported, the proposed method represents a simple and alternative protocol to produce microdevices, using affordable and inexpensive raw materials, within 10 min, and at a cost lower than US$ 0.10 per unit.

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Microfluidics has become a very promising technology in recent years, due to its great potential to revolutionize life-science solutions. Generic microfabrication processes have been progressively made available to academic laboratories thanks to cost-effective soft-lithography techniques and enabled important progress in applications like lab-on-chip platforms using rapid- prototyping. However, micron-sized features are required in most designs, especially in biomimetic cell culture platforms, imposing elevated costs of production associated with lithography and limiting the use of such devices. In most cases, however, only a small portion of the structures require high-resolution and cost may be decreased. In this work, we present a replica-molding method separating the fabrication steps of low (macro) and high (micro) resolutions and then merging the two scales in a single chip. The method consists of fabricating the largest possible area in inexpensive macromolds using simple techniques such as plastics micromilling, laser microfabrication, or even by shrinking printed polystyrene sheets. The microfeatures were made on a separated mold or onto existing macromolds using photolithography or 2-photon lithography. By limiting the expensive area to the essential, the time and cost of fabrication can be reduced. Polydimethylsiloxane (PDMS) microfluidic chips were successfully fabricated from the constructed molds and tested to validate our micro-macro method.