A paper-embedded thermoplastic microdevice integrating additive-enhanced allele-specific amplification and silver nanoparticle-based colorimetric detection for point-of-care testing.

This study introduces a thermoplastic microdevice integrated with additive-enhanced allele-specific amplification and hydrazine-induced silver nanoparticle-based detection of single nucleotide polymorphism (SNP) and opportunistic pathogens. For point-of-care testing of SNP, an allele-specific loop-mediated isothermal amplification reaction using nucleotide-mismatched primers and molecular additives was evaluated to discriminate single-nucleotide differences in the samples. The microdevice consists of purification and reaction units that enable DNA purification, amplification, and detection in a sequential manner. The purification unit enables the silica-based preparation of samples using an embedded glass fiber membrane. Hydrazine-induced silver nanoparticle formation was employed for endpoint colorimetric detection of amplicons within three min at room temperature. The versatile applicability of the microdevice was demonstrated by the successful identification of SNPs related to sickle cell anemia, genetically-induced hair loss, and Enterococcus faecium. The microdevice exhibited a detection limit of 103 copies per µL of SNP targets in serum and 102 CFU mL-1 of Enterococcus faecium in tap water within 70 min. The proposed microdevice is a promising and versatile platform for point-of-care nucleic acid testing of different samples in low-resource settings.

A point-of-care platform for hair loss-related single nucleotide polymorphism genotyping.

Rapid genotyping of single nucleotide polymorphism (SNP) is crucial for prognostics and disease management, enabling more rapid therapy selection and treatment determination. Here, we introduce a point-of-care platform for hair loss-related SNP genotyping based on allele-specific loop-mediated isothermal amplification (AS-LAMP) combined with naked-eye visualization. The specificity of the AS-LAMP assay was significantly enhanced by using mismatched allele-specific primers. AS-LAMP reaction and Schiff's reagent-based colorimetric detection were successfully performed using a thermoplastic genotyping chip. This strategy also showed potential for determining homozygotes and heterozygotes in a target sample. To assess SNP genotyping capacity, the genotyping chip was fabricated to visually detect rs6152 polymorphism of an androgen receptor gene associated with genetically induced hair loss. The genotyping platform rapidly identified the SNP within 40 min, and the detection limit was as low as 1 pg/µL of the target DNA contained in human serum. The introduced strategy showed high specificity and stability in discriminating low-abundance mutations, making it suitable as a portable and affordable point-of-care platform for rapid and accurate SNP discrimination applicable for bedside detection.

Chitosan: a green adhesive for surface functionalization and fabrication of thermoplastic biomedical microdevices.

Chitosan (CS) is a natural polymer that exhibits many biological properties and is used as a biomaterial for antibacterial coatings, tissue engineering, cell research, drug delivery, and negatively charged molecule capture. In our previous study, we used a CS-polydopamine mixture to realize UV-assisted bonding between poly(methyl methacrylate) (PMMA) substrates to fabricate microdevices for self-assembled stem cell spheroid cultures. Herein, we attained reliable adhesive bonding between PMMAs using CS at room temperature assisted by oxygen plasma. The bond strength of adhesion was as high as 2.1 MPa, which could be stable for over two months according to the leak test. The adhesive bonding and surface functionalization of the microchannels were simultaneously completed such that the microdevices could be directly used for mesenchymal stem cell culture for spheroid generation and DNA purification for point-of-care testing (POCT) devices. Surface characterization was performed by contact angle measurements, Fourier-transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy. The POCT device allows sequential on-chip DNA purification, amplification, and colorimetric detection of pathogenic bacteria. This method provides a convenient and reliable strategy for the fabrication of PMMA microdevices that can be directly implemented in biological studies and POCT applications without involving prior surface modification steps.

Bonding Strategies for Thermoplastics Applicable for Bioanalysis and Diagnostics.

Microfluidics is a multidisciplinary science that includes physics, chemistry, engineering, and biotechnology. Such microscale systems are receiving growing interest in applications such as analysis, diagnostics, and biomedical research. Thermoplastic polymers have emerged as one of the most attractive materials for microfluidic device fabrication owing to advantages such as being optically transparent, biocompatible, cost-effective, and mass producible. However, thermoplastic bonding is a key challenge for sealing microfluidic devices. Given the wide range of bonding methods, the appropriate bonding approach should be carefully selected depending on the thermoplastic material and functional requirements. In this review, we aim to provide a comprehensive overview of thermoplastic fabricating and bonding approaches, presenting their advantages and disadvantages, to assist in finding suitable microfluidic device bonding methods. In addition, we highlight current applications of thermoplastic microfluidics to analyses and diagnostics and introduce future perspectives on thermoplastic bonding strategies.

Microfluidic electrical cell lysis for high-throughput and continuous production of cell-free varicella-zoster virus.

Varicella-zoster virus (VZV), the causative agent of varicella and herpes zoster, is highly cell-associated and spreads via cell-to-cell contact in tissue culture. The lack of cell-free VZV hampers studies on VZV biology as well as antiviral and vaccine development. In the present study, a poly(methylmethacrylate) microfluidic device integrated with arrays of microelectrode was fabricated to continuously electrolyse VZV-infected cells to produce cell-free viruses. By designing multiple constrictions and microelectrode arrays, a high electric field is focused on the constricted region of the microchannel to disrupt large numbers of virus-infected cells with high-throughput on a microfluidic platform. Plaque assay and scanning electron microscopy were conducted to quantify and characterize cell-free VZV produced using the microfluidic continuous-flow electrical cell lysis device. The process of microfluidic electrical cell lysis followed by subsequent filtration and virus concentration process yielded a 1.4-2.1 × 104 plaque-forming units (PFUs) per mL of cell-free VZV from 7.0 × 106 VZV-infected human foreskin fibroblasts (HFF) cells. The high electric field formed inside a microfluidic channel combined with the continuous-flow of virus-infected cells within the microchannel enabled the rapid and efficient production of high-titer cell-free virus in large quantities with relatively low input of the voltage.

A paper-based colorimetric chemosensor for rapid and highly sensitive detection of sulfide for environmental monitoring.

In this study, we report on paper-based colorimetric detection of sulfide using a newly synthesized chemical acting as a chemosensor, based on the deprotonation mechanism. Paper strips were also fabricated and incorporated with the chemosensor for on-site monitoring. The presence of sulfide induced deprotonation of a hydroxyl group of the chemosensor, which eventually resulted in a distinct spectral change in the tube as well as a visible color change on a paper strip. The chemosensor showed a highly selective colorimetric response to sulfide by changing its color from colorless to yellow without any interference from a mixture containing other anions. Moreover, the chemosensor effectively differentiated sulfide from other thiols, including cysteine and glutathione. The chemosensor colorimetrically detected sulfide with a fast response time of 10 s under physiological conditions. Practically, the paper test strip enabled colorimetric visualization of as low as 30 µM sulfide and a good recovery in quantitative analysis in water samples. The introduced paper-based chemosensor is a promising colorimetric strategy with rapid, selective, and sensitive sensing abilities for sulfide monitoring in environmental water samples.

Rapid Fabrication of Poly(methyl methacrylate) Devices for Lab-on-a-Chip Applications Using Acetic Acid and UV Treatment.

In the present study, we introduce a new approach for rapid bonding of poly(methyl methacrylate) (PMMA)-based microdevices using an acetic acid solvent with the assistance of UV irradiation. For the anticipated mechanism, acetic acid and UV irradiation induced free radicals on the PMMA surfaces, and acrylate monomers subsequently formed cross-links to create a permanent bonding between the PMMA substrates. PMMA devices effectively bonded within 30 s at a low pressure using clamps, and a clogging-free microchannel was achieved with the optimized 50% acetic acid. For surface characterizations, contact angle measurements and bonding performance analyses were conducted using predetermined acetic acid concentrations to optimize bonding conditions. In addition, the highest bond strength of bonded PMMA was approximately 11.75 MPa, which has not been reported before in the bonding of PMMA. A leak test was performed over 180 h to assess the robustness of the proposed method. Moreover, to promote the applicability of this bonding method, we tested two kinds of microfluidic device applications, including a cell culture-based device and a metal microelectrode-integrated device. The results showed that the cell culture-based application was highly biocompatible with the PMMA microdevices fabricated using an acetic acid solvent. Moreover, the low pressure required during the bonding process supported the integration of metal microelectrodes with the PMMA microdevice without any damage to the metal films. This novel bonding method holds great potential in the ecofriendly and rapid fabrication of microfluidic devices using PMMA.