Flow production of practical and quantitative capillary driven-flow immune sensing chip using a circumferentially-grooved island micro-surface
Abstract
Practical immunoassay chip devices are high-priority needs in point-of-care testing (POCT) for rapid diagnoses. Compared to conventional POCT microchip devices, our report describes a manufacturing process involving laser ablation, inkjet deposition, and cover film sealing that is superior to that used for practical mass production of microchip devices, and produces devices capable of sensitive and reproducible measurements using a capillary-flow-driven system at a practical point-of-care setting. To promote sensitivity and reproducibility, circular islands surrounded by 10-µmdeep grooves were prepared to provide uniformity of printed antibody spots on a capillary flow immunoassay chip. The island surface enabled dense antibody fixation due to droplet surface tension as well as antibody determination by enzyme linked immunosorbent assay (ELISA), which demonstrated greater sensitivity than that of a device using a non-island surface. The luminescence intensity of the spots of the carboxyterminal propeptide of type I procollagen (PICP) exhibited a good linear relation with PICP concentration in the range 0-600 ng·mL-1 , which is suitable for clinical estimation in blood.
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Introduction
Analysis of biomarkers when the patient is located is known as point-of-care testing (POCT), and needs to be a simple and rapid medical diagnostic method [1-3]. Most of the available POCT devices involve immunoassay systems, and enzymelinked immunosorbent assay (ELISA) has been utilized as a standard analytical system due to its sensitivity and specificity [4]. Because a practical and simple POCT device must be easily available to the end user in many environments, a capillarydriven flow system is a good candidate because it does not require electrical power and is easy to operate. Indeed, a capillarydriven, flow-based immuno chromatographic assay is considered the simplest commercially available POCT device in the diagnostic market. Therefore, a capillary-driven, flow-based immunoassay system was developed for an ELISA using a piezoelectric inkjet printing system [5-7].
Practical approaches for immobilizing antibodies on a microchannel surface include microfluidic patterning [8], photolithography [9], physical entrapment [10], micro-contact printing [11, 12], and inkjet printing [5-7]. Among these approaches, printing has many advantages, such as simplicity, flexibility, low cost, minimal consumption of reagents, and simultaneous patterning of multiple reagents [13]. Many important studies have been successfully reported by printing technologies, for example, electrohydrodynamic printing [14], water-based inkjet printing [15] and printed wiring board assembly [16]. These features allow the mass production of this device [17]. Although inkjet printing can precisely deposit a drop of antibody solution at the picoliter level on a microchannel surface, a high concentration of antibodies can destabilize the ejection spray from the nozzle head due to its high viscosity, which is a problem for the development of a diagnostic immunoassay system. Accurate ELISA measurements require printing spots of identical size and equivalent amounts of antibody immobilization, which ensures reproducible determination by chemical luminescence.
To overcome the viscosity problem, a solution with a high concentration of antibodies was continuously deposited and immobilized on a circumferentially-grooved island micro surface. The island microstructure enables formation of a spherical droplet by surface tension, producing a precise antibody spot and the same amount of antibody deposition. In addition, the droplet does not adhere to the wall of the microchannel surface and the antibody spots have a uniform size, even when the ejection spray may become uneven.
Conclusion
A novel and simple approach for developing a sandwich ELISA based on a capillary-driven flow system for sensitive and reproducible measurement of PICP was developed. The circumferentially-grooved island surface allowed uniformity in printing of antibody spots onto a microchannel surface, enabling concentrated antibody solution fixation due to the droplet’s surface tension. Evaluation of the precision of antibody spot printing demonstrated that the variation in the surface area of spots was 0.05% RSD on the island surface and 31.3% for spots on the non-island surface. The spots on the island surface maintained a spherical form even at 1000 shots of antibody inkjet deposition, and produced greater luminescence intensity than those on the non-island surface. In contrast, spots on the non-island surface lost their spherical form after 500 shots and had slightly reduced luminescence intensity. A concentrated antibody solution was deposited at 1000 shots on the island surface and the spot intensity in a sandwich ELISA was greater than that for the non-island surface. The intensity of 0-600 ngmL-1 PICP spots showed a concentration-dependent relation and were greater overall than that for spots on non-island surfaces.
This microfluidic ELISA system using capillary-driven flow possesses many advantages for POC situations, such as simple operation, minimal sample consumption, and rapid results. The antibody immobilization process was performed consecutively using laser processing, inkjet deposition, and cover film sealing. This process is driven by mechatronic operation, which provides high throughput. This report demonstrates the potential of practical POC chip fabrication in the future.