As a novel carrier for chemiluminescent immunoassays, the stability of the thin film microfluidic pouch reagents is directly related to the accuracy and reproducibility of test results. Due to the unique structure of the microfluidic pouch (film material, microchannel design) and the sensitive nature of the chemiluminescent reaction, the reagents are susceptible to environmental factors during storage, transportation, and use. Therefore, a multi-dimensional stability assurance system must be established, encompassing material selection, packaging processes, and control of reaction conditions.
The material properties of the thin film microfluidic pouch are fundamental to reagent stability. Traditional plastic films can cause reagent oxidation or water loss due to their oxygen and moisture permeability. However, new composite films, using multi-layer co-extrusion technology, combine barrier layers, adhesive layers, and functional layers to significantly reduce oxygen and water vapor permeability. For example, barrier layers containing aluminum foil or silicon oxide effectively isolate the reagents from environmental interference. Furthermore, the chemical inertness of the film is crucial, ensuring that it does not adsorb or react with reagent components, thereby preventing increased background signal or inactivation of active substances.
The sealing process of the microfluidic pouch directly impacts the long-term stability of the reagents. If there are even minor leaks during the packaging process, oxygen, carbon dioxide, or microorganisms in the air may enter the bag, causing degradation of the enzyme-labeled antibody and deterioration of the luminescent substrate. Currently, hot-melt sealing and laser welding are the mainstream technologies. The former forms a seal by melting the film edge at high temperatures, while the latter uses laser energy to locally melt the material. Both require strict control of temperature, pressure, and time parameters to ensure seal integrity. Furthermore, integrity testing, such as negative pressure testing or dye permeation testing, is necessary after packaging to eliminate potential leakage risks.
Reagent formulation optimization is a key step in improving stability. In chemiluminescent immunoassays, the stability of the enzyme-labeled antibody, luminescent substrate, and buffer are interrelated. For example, the addition of protective agents (such as bovine serum albumin and carbohydrates) can reduce aggregation and inactivation of the enzyme-labeled antibody; selecting a pH-stable buffer system (such as Tris-HCl and phosphate) can maintain a stable reaction environment; and the addition of antioxidants (such as vitamin C and sodium thiosulfate) can inhibit oxidation reactions from interfering with the luminescent signal. Furthermore, the use of solid-state reagents (such as lyophilized powders) can further extend shelf life, but homogeneity after reconstitution must be addressed.
Controlling the microenvironment within the microfluidic bag is a key technology. Due to the small volume and large surface area within the bag, temperature fluctuations or light exposure can accelerate reagent decomposition. Therefore, light-proof packaging materials (such as polyethylene with carbon black) must be used, and the storage temperature must be controlled (typically 2-8°C). During transportation, buffering materials (such as foam boxes and ice packs) can minimize the impact of temperature fluctuations on the reagents. Furthermore, the design of the microfluidic bag must consider the mixing efficiency of the reagents to avoid localized concentration variations due to dead spots in the flow path, which can affect reaction kinetics.
Pretreatment steps before use are also crucial. For example, when reconstituting lyophilized reagents, the diluent should be added dropwise slowly to avoid bubbles that could lead to loss of active substances. When mixing multiple reagents, the stirring speed and duration must be controlled to prevent mechanical shear forces from disrupting protein structure. Furthermore, the operating environment must meet cleanliness requirements to prevent dust or microbial contamination of the reagents within the bag.
Long-term stability monitoring is essential for quality control. Accelerated aging tests (such as those under high temperature and high humidity conditions) can be used to predict the actual shelf life of the reagents, allowing for adjustments to the formulation and process based on real-time stability studies. Furthermore, establishing a standardized stability assessment system (e.g., luminescence intensity decay rate and background signal change) can provide data support for product optimization.
Ensuring the stability of chemiluminescence immunoassay reagents in thin film microfluidic pouches is a systematic project, requiring coordinated efforts across the entire supply chain, from materials science and packaging technology to formulation design and usage specifications. In the future, with the development of intelligent packaging (e.g., humidity indicator cards and temperature recorders) and new stabilizers, reagent stability will be further enhanced, providing more reliable technical support for point-of-care testing and precision medicine.
