A review of the recent achievements and future trends on 3D printed microfluidic devices for bioanalytical applications.

3D printing has revolutionized the manufacturing process of microanalytical devices by enabling the automated production of customized objects. This technology promises to become a fundamental tool, accelerating investigations in critical areas of health, food, and environmental sciences. This microfabrication technology can be easily disseminated among users to produce further and provide analytical data to an interconnected network towards the Internet of Things, as 3D printers enable automated, reproducible, low-cost, and easy fabrication of microanalytical devices in a single step. New functional materials are being investigated for one-step fabrication of highly complex 3D printed parts using photocurable resins. However, they are not yet widely used to fabricate microfluidic devices. This is likely the critical step towards easy and automated fabrication of sophisticated, complex, and functional 3D-printed microchips. Accordingly, this review covers recent advances in the development of 3D-printed microfluidic devices for point-of-care (POC) or bioanalytical applications such as nucleic acid amplification assays, immunoassays, cell and biomarker analysis and organs-on-a-chip. Finally, we discuss the future implications of this technology and highlight the challenges in researching and developing appropriate materials and manufacturing techniques to enable the production of 3D-printed microfluidic analytical devices in a single step.

Sandpaper-based electrochemical devices assembled on a reusable 3D-printed holder to detect date rape drug in beverages.

This study describes the development of a new electrochemical paper-based analytical device (ePAD) on alumina sandpaper substrate through a pencil-drawing process for square wave voltammetry measurements of midazolam maleate used as a "date rape drug" in beverages. The proposed ePAD was assembled on a reusable 3D printed holder to delimit its geometric area and ensure better robustness. The ePAD was characterized by scanning electron microscopy, cyclic voltammetry, electrochemical impedance spectroscopy and Raman spectroscopy. The direct drawing of ePADs on sandpaper platforms through a graphite pencil has offered suitable repeatability (RSD = 1.0%) and reproducibility (RSD = 4.0%) using [Fe(CN)6]4- as redox probe. The proposed ePAD provided linear behaviour in the midazolam maleate concentration range between 2.5 and 150 mg L-1 and a limit of detection of 2.0 mg L-1. The feasibility of the ePAD for forensic application was successfully demonstrated through the detection of midazolam in different beverages (water, beer, liquor, and vodka). The intended application revealed low interference of other compounds present in beverages. Based on the achieved results, the proposed ePAD has offered great accuracy with no statistical difference at 95% confidence level from the data recorded by high performance liquid chromatography. The operational simplicity and the robustness ensured by the assembling on a reusable 3D printed holder make the ePAD drawn on sandpaper platform a powerful and promising analytical tool for the analysis of "date rape drugs" opening new possibilities for on-site forensic investigations.

Inexpensive and nonconventional fabrication of microfluidic devices in PMMA based on a soft-embossing protocol.

This study describes an inexpensive and nonconventional soft-embossing protocol to produce microfluidic devices in poly(methyl methacrylate) (PMMA). The desirable microfluidic structure was photo-patterned in a poly(vinyl acetate) (PVAc) film deposited on glass substrate to produce a low-relief master. Then, this template was used to generate a high-relief pattern in stiffened PDMS by increasing of curing agent /monomer ratio (1:5) followed by thermal aging in a laboratory oven (200°C for 24 h). The stiffened PDMS masters were used to replicate microfluidic devices in PMMA based on soft embossing at 220-230°C and thermal sealing at 140°C. Both embossing and sealing stages were performed by using binder clips. The proposed protocol has ensured the replication of microfluidic devices in PMMA with great fidelity (>94%). Examples of MCE devices, droplet generator devices and spot test array were successfully demonstrated. For testing MCE devices, a mixture containing inorganic cations was selected as model and the achieved analytical performance did not reveal significant difference from commercial PMMA devices. Water droplets were successfully generated in an oil phase at rate of ca. 60 droplets/min (fixing the continuous phase flow rate at 100 µL/h) with size of ca. 322 ± 6 µm. Glucose colorimetric assay was performed on spot test devices and good detectability level (5 µmol/L) was achieved. The obtained results for two artificial serum samples revealed good agreement with the certified concentrations. Based on the fabrication simplicity and great analytical performance, the proposed soft-embossing protocol may emerge as promising approach for manufacturing PMMA devices.

Fabrication of microwell plates and microfluidic devices in polyester films using a cutting printer.

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.

Environmentally Friendly Manufacturing of Flexible Graphite Electrodes for a Wearable Device Monitoring Zinc in Sweat.

Electrochemical sensors based on graphite and polymers have emerged as powerful analytical tools for bioanalytical applications. However, most of the fabrication processes are not environmentally friendly because they often involve the use of toxic reagents and generate waste. This study describes an alternative method to produce flexible electrodes in plastic substrates using graphite powder and thermal laminating sheets by solid-solid deposition through hot compression, without the use of hazardous chemical reagents. The electrodes developed through the proposed approach have successfully demonstrated flexibility, robustness, reproducibility (relative standard deviation around 6%), and versatility. The electrodes were thoroughly characterized by cyclic voltammetry, electrochemical impedance spectroscopy, Raman spectroscopy, and scanning electron microscopy. As a proof of concept, the electrode surfaces were modified with bismuth and used for zinc analysis in sweat. The modified electrodes presented linearity (R2 = 0.996) for a wide zinc concentration range (50-2000 ppb) and low detection limit (4.31 ppb). The proposed electrodes were tested using real sweat samples and the achieved zinc concentrations did not differ statistically from the data obtained by atomic absorption spectroscopy. To allow wearable applications, a 3D-printed device was fabricated, integrated with the proposed electrochemical system, and fixed at the abdomen by using an elastic tape to collect, store, and analyze the sweat sample. The matrix effect test was performed, spiking the real sample with different zinc levels, and the recovery values varied between 85 and 106%, thus demonstrating adequate accuracy and robustness of the flexible electrodes developed based on the proposed fabrication method.

Paper-Based Electrophoresis Microchip as a Powerful Tool for Bioanalytical Applications.

This chapter describes the development of paper-based microchip electrophoresis (pME) devices for the separation of clinically relevant compounds. pME were fabricated by laser cut and thermal lamination process using polyester pouches. In addition, hand-drawn pencil electrodes were integrated to the device to perform capacitively coupled contactless conductivity detection (C4D). Finished device costs less than US$ 0.10 and did not require either sophisticated instrumentation or clean room facilities. Furthermore, pME is lightweight, easy to handle, flexible, and robust. pME-C4D device revealed an excellent capacity to separate BSA and creatinine in less than 150 s with baseline resolution. The device proposed in this chapter has proven to be a good alternative as a platform for the diagnosis of diseases from renal disorders such as diabetes mellitus and heart disease.

Colorimetric analysis of the decomposition of S-nitrosothiols on paper-based microfluidic devices.

A disposable microfluidic paper-based analytical device (µPAD) was developed to easily analyse different S-nitrosothiols (RSNOs) through colorimetric measurements. RSNOs are carriers of nitric oxide (NO) that play several physiological and physiopathological roles. The quantification of RSNOs relies on their decomposition using several protocols and the colorimetric detection of the final product, NO or nitrite. µPADs were fabricated by wax printing technology in a geometry containing one central zone for the sample inlet and eight circular detection zones interconnected by microfluidic channels for decomposition and posterior detection of decayed products. Different decomposition protocols including mercuric ions and light (UV, visible, and infrared) were tested on µPADs. For this purpose, a 3D printed holder was coupled with µPADs to easily design a simultaneous decomposition procedure using different light sources. The Griess reagent was added to detect NO and nitrite produced by the different decomposition methods. µPADs were then scanned using a flat board scanner and calibration curves based on color intensity were plotted. The limit of detection (LOD) values achieved for nitrite (used as a reference compound) and S-nitrosoglutathione (GSNO) using mercuric decomposition were 3 and 4 µM, respectively. The LOD reported herein for nitrite is considered among the lowest LODs already reported for this compound using µPADs. The results also show that low-molecular-weight RSNO, namely S-nitrosocysteine, decomposes more easily than high-molecular-weight RSNOs with light. As a proof of concept, RSNOs in human plasma were successfully detected on µPADs. For this purpose, a preliminary treatment step was optimized and the presence of high-molecular-weight (HMW) RSNOs was evidenced in the available plasma samples. The concentrations of HMW-RSNOs and nitrite in the various samples ranged from 5 to 16 µM and from 37 to 58 µM, respectively.

Hand drawing of pencil electrodes on paper platforms for contactless conductivity detection of inorganic cations in human tear samples using electrophoresis chips.

This paper describes for the first time the fabrication of pencil drawn electrodes (PDE) on paper platforms for capacitively coupled contactless conductivity detection (C(4) D) on electrophoresis microchips. PDE-C(4) D devices were attached on PMMA electrophoresis chips and used for detection of K(+) and Na(+) in human tear samples. PDE-C(4) D devices were produced on office paper and chromatographic paper platforms and their performance were thoroughly investigated using a model mixture containing K(+) , Na(+) , and Li(+) . In comparison with chromatographic paper, PDE-C(4) D fabricated on office paper has exhibited better performance due to its higher electrical conductivity. Furthermore, the detector response was similar to that recorded with electrodes prepared with copper adhesive tape. The fabrication of PDE-C(4) D on office paper has offered great advantages including extremely low cost (< $ 0.004 per unit), reduced fabrication time (< 5 min), and minimal instrumentation (pencil and paper). The proposed electrodes demonstrated excellent analytical performance with good reproducibility. For an inter-PDE comparison (n = 7), the RSD values for migration time, peak area, and separation efficiency were lower than 2.5, 10.5, and 14%, respectively. The LOD's achieved for K(+) , Na(+) , and Li(+) were 4.9, 6.8, and 9.0 µM, respectively. The clinical feasibility of the proposed approach was successfully demonstrated with the quantitative analysis of K(+) and Na(+) in tear samples. The concentration levels found for K(+) and Na(+) were, respectively, 20.8 ± 0.1 mM and 101.2 ± 0.1 mM for sample #1, and 20.4 ± 0.1 mM and 111.4 ± 0.1 mM for sample #2.