Capillary-driven microfluidic device with integrated nanoporous microbeads for ultrarapid biosensing assays

I. Introduction Advances in wearable electronics, functional nanocomposite polymers, flexible microfluidics, and commercial polymer microfluidics are reported on a daily basis. However, much of this research is field-specific, despite the enormous potential that the convergence of these research areas has for the future of health care, worker safety, food safety, consumer devices, and commercial microfluidics. We present an innovative approach of powerful, general-purpose frameworks that facilitate interfacing of nanocomposite polymer-based, microfluidic, and electronic devices on flexible polymer, textile, or industrial polymer platforms. Such approaches offer practical solutions for areas such as safety lighting and health monitors for safety vests; heart monitors and perspiration monitors for athletic clothing; and other applications in real-time wearable bioelectric and biochemical monitoring. II. Wearable Microfluidic and Electronic Frameworks A. Wearable bioelectric sensors Intense research into wearable electronics has resulted in many innovative devices and systems, e.g.: flexible polyimide or Kapton printed circuit boards (PCBs) [1]; roll-to-roll foil and other printed devices [2]; printing and weaving of textiles [3]; and special geometries for flexible interconnect between rigid components [4]. We have investigated conductive nanocomposite polymers and metal transfer processes for wearable bioelectric sensors [5, 6]. These technologies can be employed in, for example, conformable and wearable systems for electrocardiogram (ECG) [5], tissue impedance [7] and pressure [8] sensors. Unlike many other techniques, the technology and materials employed for our devices are highly compatible with clothing-based textiles. B. Wearable microfluidics Much less research has been performed in the emerging area of wearable microfluidics. Traditionally, microfluidic devices are fabricated in rigid substrates or flexible materials such as polydimethylsiloxane (PDMS) that are bonded to rigid substrates. Free-standing PDMS devices are developed for, e.g., perspiration sensors [9]; however, such processes typically require long fabrication times, equipment in a cleanroom facility, and difficulty with integration onto textiles. Other devices employ porous materials or microneedles to deliver biofluids, e.g., perspiration or interstitial fluid, by capillary forces [10]; however, such devices are limited in fluid collection or are too invasive. Wearable textile-based devices have been recently demonstrated that use the textile itself as a fluidic “channel” [11]; however, such devices are dependent on the fluidic characteristics of the fabric. To overcome these limitations and facilitate the development of fully flexible, wearable, and durable microfluidic devices that can be used for a wider variety of applications, we have developed a printing-based fabrication process that employs screen printable plastisol ink [12]. This process is demonstrated for simple multi-level microfluidic devices toward the goal of fully wearable microfluidic systems. III. Summary Many methods have been developed for microelectronic and microfluidic devices in rigid or flexible packages (e.g., [13, 14, 15]). However, such technologies suffer from difficult integration and/or limited flexibility, limited geometries, limited microfluidic sample size, and/or difficult integration, especially on textile-based substrates. The technologies we present offer simple and compatible printing-based and transfer processes for microelectronic and microfluidic frameworks that facilitate cost-effective development of durable wearable biomedical systems. References Y. Chuo, et. al., IEEE Transactions Biomedical Circuits Systems 4(5):281-94 (2010).K. Jain, et. al., Proceedings of the IEEE 93(8):15000-10 (2005).F. Carpi, et. al., IEEE Transactions Information Technology Biomedicine 9(3):295-318 (2005).M. Gonzalez, et. al., Microelectronics Reliability 51:1069-76 (2011).D. Chung, et. al. Proc. SPIE 9060, (2014); doi:10.1117/12.2046548.D. Hilbich, et. al., Proc. SPIE 98020R (2016); doi:10.1117/12.2219284.D. Chung, et. al., J Electrochemical Society 161(2):B3071-76 (2014).A. Rahbar, et. al., Proc. SPIE 90600O (2014); doi:10.1117/12.2044342.J. Choi, et. al., Adv Healthcare Mater 6:1601355 (2017).F. Benito-Lopez, et. al., Procedia Chemistry 1(1):1103-1106 (2009).A. Nilghaz, et. al., Lab Chip 12:209-218 (2012).D. Chung & B.L. Gray, J Micromech Microeng, 27(11), (2017).S. Cheng., et. al., Advanced Functional Materials 21(12):2282-90 (2011).M. Scholles, et. al., Proc. of SPIE 7593:75930C-2 (2013).A. Wu, et. al., Lab Chip 10:519-521 (2010).

In this study, we successfully synthesized and crystallized metal complexes and functional proteins in a simple microfluidic device. The fabricated microfluidic device is dedicated to liquid and solid syntheses, including crystallization. A feature of this device is that the lid of the outlet synthesis chamber can be attached and removed. In the case of solid products such as protein crystals, removing the lid of the outlet portion and easily removing the solid products from the synthesis chamber is possible. Furthermore, partition walls were provided in the reaction (crystallization) chamber at 100 to 300 <tex xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">$\mu \mathrm{m}$</tex> intervals. The partition contributes to efficiently mixing the reaction solution and optimizing the crystallization conditions. Applying this device shortened the synthesis of metal complexes (liquid) from 6 hours to 10 seconds. Furthermore, we succeeded in synthesizing, crystallizing, and extracting Zn(II) complex proteins, which are difficult to synthesize.

A simple sheath flow microfluidic device is used to fabricate polymer micro/nanofibers that have precisely controlled shapes and sizes. Poly(methylmethacrylate) (PMMA) was used as the model polymer for these experiments. The sheath-flow device uses straight diagonal and chevron-shaped grooves integrated in the top and bottom walls of the flow channel to move sheath fluid completely around the polymer stream. Portions of the sheath stream are deflected in such a way as to define the cross-sectional shape of the polymer core. The flow-rate ratio between the sheath and core solution determines the fiber diameter. Round PMMA fibers with a diameter as small as 300 nm and flattened fibers with a submicron thickness are demonstrated.

Continuous fabrication of core-shell aerogel microparticles using microfluidic flows

This work focuses on the fabrication of polyimide aerogel microparticles of diameter 200-1000 μm from a surfactant-free, two-phase, silicone oil/dimethylformamide (DMF) oil-in-oil (O/O) system using a simple microfluidic device. The polyimide sol prepared in DMF is turned into droplets suspended in silicone oil in the microfluidic device. The droplets are guided to a heated silicone oil bath to accelerate sol-gel transition and imidization reactions, thereby yielding spherical, discrete gel microparticles that do not undergo coalescence. The discrete gel microparticles are isolated and supercritically dried to obtain aerogel microparticles. The microparticle size distribution shows dependence on dispersed and continuous phase flowrates in the microfluidic channels. The microparticle surface morphology shows dependence on the silicone oil bath temperature.

Polymeric porous particles are currently used for various applications in biotechnology, tissue engineering and pharmaceutical science, e.g., floating drug delivery systems and inhaled formulations. Particle shape and size depend on variable parameters; among them, polymer type and concentration, stirring speed, pH and type of solvent. In this study, porous poly(lactic-co-glycolic) acid (PLGA) and poly(d,l-lactide) (PLA) microspheres (MPs), with varying sizes and morphologies, were synthesized and optimized using both batch formulation and a flow-focusing microfluidic device. A well-established method of preparation utilizing solvent evaporation and the double emulsion technique was performed. Similar to other batch encapsulation methods, this technique is time and reagent consuming and consists of several steps. Hence, although porous structures provide tremendous opportunity in the design of new applications for tissue engineering and as improved controlled-release carriers, the synthesis of these particles with predefined properties remains challenging. We demonstrated the fabrication of porous MPs using a simple microfluidic device, compared to batch synthesis fabrication; and the effect of solvent, polymer concentration and type, post-hydrolysis treatment, on porosity degree. Moreover, a kinetic release study of fluorescent molecule was conducted for non-porous in comparison to porous particles. An overview of future prospects and the potential of these porous beads in this scientific area are discussed.

Microfluidic extraction based on a co-laminar flow of aqueous two-phase system is used to separate bovine serum albumin (BSA). Mass transfer between the continuous two-phase flows is demonstrated by the extraction of BSA in a microfluidic device. The protein concentrations of the BSA samples were determined using the Bradford method. Polyethylene glycol 4000 and ammonium sulfate ((NH4)2SO4) served as model aqueous two-phase solutions. The appropriate flow rates of the aqueous two phases were thus determined. We can flexibly control the mass transfer area and time by simply adjusting the flow rate. It takes only 3.6 s for three extraction cycles in a coaxial microfluidic device to achieve a BSA recovery yield of 71.1 %, which is superior to the traditional beaker aqueous two-phase extraction process. In this study, co-laminar flow-based continuous microextraction is demonstrated and its mass transfer is analyzed by solving the diffusion model, based on a large specific interfacial area and surface renewal.

The past few decades has witnessed an intensive drive in the development of microfluidic technologies and devices that have tremendous applications in diverse sectors from defence to healthcare. Entire analytical protocols, including sample pretreatment, sample/reagent manipulation, separation, reaction, and detection can be performed significantly quicker on these miniaturised and compact devices. A broad range of existing and new materials from silicon, glass, polymers, and paper have been demonstrated as viable compatible materials for creating advanced and low-cost microfluidic devices, with embedded micrometre-sized elements, to provide unique and often combined functionalities for microfluidic processing. Based on the category of materials, fabrication techniques and examples of applications will be discussed in this chapter. Factors influencing the choice of material, cost of processing, and suitability of specific applications are included. The integration of microfluidic devices and detection technologies suggests a solid understanding of fabrication procedures and their limitations is essential to the development of commercial microfluidic devices.

Drugs play an indispensable role in the fields of medicine， agriculture， and animal husbandry. However， their long-term and improper use may lead to drug residues in food， the environment and organisms， posing a potentially serious threat to human health and the ecological environment. For instance， antibiotic residues may induce bacterial resistance， pesticide residues may cause neurotoxicity， and hormone drugs may interfere with the endocrine system. Therefore， developing sensitive and accurate detection methods for drug residues has become an important prerequisite and current hot topic in drug research. Meanwhile， the complicated matrices and low contents of the residues make it necessary for the widely used chromatography/mass spectrometry （MS） determination technologies to be coupled with efficient sample pretreatment procedures. Molecularly imprinted solid-phase microextraction （MI-SPME） technology combines the rapidity， high efficiency and solvent-free characteristics of SPME， and the specific recognition and selective adsorption capabilities of molecularly imprinted polymers （MIPs）， and shows significant advantages in the highly selective separation and enrichment of drug residues in complex samples. In recent years， the MI-SPME technology has become a research hotspot in the field of drug residue detection.This work systematically reviews the research progress since 2019 on the application of MI-SPME coupled with chromatography/MS in drug residue detection across food safety， environmental monitoring and biomedical fields. First， this work introduces in detail on the working principle and operation process of SPME technology. SPME achieves efficient enrichment of target analytes through the selective adsorption of the stationary phase-coated fibers， offering simplicity， speed， minimal solvent use， and compatibility with analytical instruments such as chromatography/MS.Next， the review focuses on elaborating the preparation methods and new technologies and strategies of MIPs. The traditional methods for preparing MIPs mainly include free radical polymerization， in-situ polymerization and sol-gel methods. However， traditional MIPs have defects such as template leakage risk， limited binding ability， and irregular material morphology， which restrict the application range. To this end， researchers have developed a series of novel preparation technologies and strategies， such as surface imprinting， nanoimprinting， dummy template imprinting， multi-template imprinting， multifunctional monomer imprinting and stimulus-response imprinting. These technologies and strategies have significantly enhanced the recognition and enrichment ability of MIPs for trace drug residues in complex samples by optimizing their structures and performances.To meet the requirements of different sample types and analytical instruments， MI-SPME media need to be designed into specific technical configurations through chemical or physical methods. This review summarizes six different MI-SPME device modes： MIPs-coated fiber SPME， MIPs in-tube SPME （IT-SPME）， MIPs stir bar sorptive extraction （SBSE）， MIPs dispersive SPME （DSPME）， MIPs thin-film SPME （TFME）， and MIPs in-tip SPME. Each mode offers unique advantages for the separation， enrichment and determination of drug residues in real samples. For example， the coated fiber SPME is simple to operate and suitable for direct immersion or headspace extraction of liquid samples； IT-SPME features miniaturization and automation， with excellent compatibility with chromatographic and mass spectrometric systems； DSPME achieves efficient separation and enrichment by dispersing adsorbents directly into sample solutions.Then， the applications of MI-SPME in the fields of food safety， environmental monitoring and biological medicine are summarized， highlighting typical research examples. In the field of food safety， MI-SPME can be used to detect pesticide residues， veterinary drug residues， and drugs for human use in fruits， vegetables， animal meats and dairy products. In environmental monitoring， it can be used for the detection of drug residues in aqueous environments and soil. In the field of biological medicine， it can be used for the analysis of drug residues in biological samples such as plasma， urine， and serum.Although the MI-SPME technology has shown great potential in drug residue detection， it still faces some challenges. For example， the preparation process of MIPs needs to be further optimized to improve their selectivity and stability； the development and application of new materials （such as graphene， metal-organic frameworks） for composite MIPs still need to solve problems such as high cost and complex processes； the integration of MI-SPME technology and automated equipment is also a bottleneck and important direction for future development. Looking ahead， with the advancement of green chemistry principles and point-of-care testing technologies， MI-SPME is expected to play an even greater role in drug residue detection. It will provide more efficient and precise technical support for food safety， environmental monitoring， and biomedical research.

A target-responsive aptamer-cross-linked hydrogel was designed and synthesized for portable and visual quantitative detection of the toxin Ochratoxin A (OTA), which occurs in food and beverages. The hydrogel network forms by hybridization between one designed DNA strand containing the OTA aptamer and two complementary DNA strands grafting on linear polyacrylamide chains. Upon the introduction of OTA, the aptamer binds with OTA, leading to the dissociation of the hydrogel, followed by release of the preloaded gold nanoparticles (AuNPs), which can be observed by the naked eye. To enable sensitive visual and quantitative detection, we encapsulated Au@Pt core-shell nanoparticles (Au@PtNPs) in the hydrogel to generate quantitative readout in a volumetric bar-chart chip (V-Chip). In the V-Chip, Au@PtNPs catalyzes the oxidation of H2O2 to generate O2, which induces movement of an ink bar to a concentration-dependent distance for visual quantitative readout. Furthermore, to improve the detection limit in complex real samples, we introduced an immunoaffinity column (IAC) of OTA to enrich OTA from beer. After the enrichment, as low as 1.27 nM (0.51 ppb) OTA can be detected by the V-Chip, which satisfies the test requirement (2.0 ppb) by the European Commission. The integration of a target-responsive hydrogel with portable enrichment by IAC, as well as signal amplification and quantitative readout by a simple microfluidic device, offers a new method for portable detection of food safety hazard toxin OTA.

Wearable microfluidic detection devices for noninvasive sweat analysis are highly desirable for human health monitoring. Herein, we present a bioenzyme-free microfluidic colorimetric detection device for simultaneous detection of creatinine (Cre), urea, and Mg2+ in sweat, enabling comprehensive kidney function and mental stress assessment. The device leverages a ternary catalytic system composed of H2MoO5 nanomaterial, Cu2+, and Cre, which exhibits significantly enhanced oxidase-like activity. This system directly catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate a blue product (ox-TMB), providing a robust platform for Cre detection without the need for bioenzymes. For urea and Mg2+ detection, 4-dimethylaminobenzaldehyde and chromium black T were employed as effective chromogenic reagents, respectively. A flexible multichannel microfluidic chip was fabricated, incorporating specific colorimetric reagent-modified paper chips into the corresponding reaction reservoirs. This design allows for the simultaneous detection of Cre, urea, and Mg2+ in sweat with linear ranges of 10-100 μM, 10-70 mM, and 0.5-8 mM, respectively. The device's performance was validated using actual sweat samples, with detection results analyzed via a smartphone application, demonstrating high accuracy and reliability. This work introduces a simple, low-cost, and stable bioenzyme-free microfluidic colorimetric detection device, offering significant potential for in situ and intelligent sweat analysis. The integration of smartphone technology further enhances its practicality, making it a promising tool for real-time health monitoring and point-of-care testing.

A low-cost microfluidic paper-based analytical device (µPAD) with column chromatography preconcentration for the determination of paraquat in vegetable samples

Image-based evaluations of distribution and cytotoxicity of Irinotecan (CPT-11) in a multi-compartment micro-cell coculture device

We developed a novel strategy for fabrication of microfluidic paper-based analytical devices (μPADs) by selective wet etching of hydrophobic filter paper using a paper mask having a specific design. The fabrication process consists of two steps. First, the hydrophilic filter paper was patterned hydrophobic by using trimethoxyoctadecylsilane (TMOS) solution as the patterning agent. Next, a paper mask penetrated with NaOH solution (containing 30% glycerol) was aligned onto the hydrophobic filter paper, allowing the etching of the silanized filter paper by the etching reagent. The masked region turned highly hydrophilic whereas the unmasked region remains highly hydrophobic. Thus, hydrophilic channels, reservoirs, and detection zones were generated and delimited by the hydrophobic barriers. The effects of some factors including TMOS concentration, etching temperature, etching time, and NaOH concentration on fabrication of μPAD were studied. Being free of any expensive equipment, metal mask and expensive reagents, this rapid, simple, and cost-effective method could be used to fabricate μPAD by untrained personnel with minimum cost. A flower-shaped μPAD fabricated by this presented method was applied to the glucose assay in artificial urine samples with good performance, indicating its feasibility as a quantitative analysis device. We believe that this method would be very attractive to the development of simple microfluidic devices for point-of-care applications in clinical diagnostics, food safety, and environmental protection.

Single-cell analysis commonly requires the confinement of cell suspensions in an analysis chamber or the precise positioning of single cells in small channels. Hydrodynamic flow focusing has been broadly utilized to achieve stream confinement in microchannels for such applications. As imaging flow cytometry gains popularity, the need for imaging-compatible microfluidic devices that allow for precise confinement of single cells in small volumes becomes increasingly important. At the same time, high-throughput single-cell imaging of cell populations produces vast amounts of complex data, which gives rise to the need for versatile algorithms for image analysis. In this work, we present a microfluidics-based platform for single-cell imaging in-flow and subsequent image analysis using variational autoencoders for unsupervised characterization of cellular mixtures. We use simple and robust Y-shaped microfluidic devices and demonstrate precise 3D particle confinement towards the microscope slide for high-resolution imaging. To demonstrate applicability, we use these devices to confine heterogeneous mixtures of yeast species, brightfield-image them in-flow and demonstrate fully unsupervised, as well as few-shot classification of single-cell images with 88% accuracy.