مقالات پذیرفته شده در نهمین کنگره بین المللی زیست پزشکی
Integrated LAMP-Microfluidic System for Diagnosis of Moraxella catarrhalis
Integrated LAMP-Microfluidic System for Diagnosis of Moraxella catarrhalis
Fereshteh Chitsazian,1,*Solamz Khalighfard,2
1. Biological Department, Research Center for Development of Advanced Technologies, Tehran, Iran 2. Biological Department, Research Center for Development of Advanced Technologies, Tehran, Iran
Introduction: Timely and accurate identification of respiratory pathogens is essential for effective clinical decision-making and public health surveillance. Moraxella catarrhalis, a Gram-negative diplococcus, is a significant human pathogen responsible for various respiratory tract infections, including otitis media, sinusitis, and exacerbations of chronic obstructive pulmonary disease (COPD). Its high prevalence among children and immunocompromised individuals emphasizes the need for rapid and reliable diagnostic tools to enable early treatment and limit transmission.
Traditional diagnostic methods such as microbial culturing and biochemical assays, while dependable, are often slow, labor-intensive, and require specialized laboratory infrastructure. In contrast, molecular techniques have transformed pathogen detection by offering faster, more sensitive, and specific alternatives. Among these, loop-mediated isothermal amplification (LAMP) has emerged as a powerful method due to its ability to amplify DNA at a constant temperature, eliminating the need for thermal cycling. LAMP is particularly suitable for point-of-care diagnostics because of its robustness against inhibitors and its capacity to deliver results within an hour.
To further enhance the portability and efficiency of molecular diagnostics, microfluidic systems have gained increasing attention. These platforms allow precise control of fluid movement at the microscale, enabling the miniaturization of laboratory processes and integration of multiple analytical steps into compact devices. Polymethyl methacrylate (PMMA), a transparent thermoplastic polymer, is widely used in microfluidic chip fabrication due to its excellent optical clarity, biocompatibility, ease of machining, and cost-effectiveness.
This study presents the development of a PMMA-based microfluidic chip integrated with LAMP for the rapid and sensitive detection of Moraxella catarrhalis. By combining the specificity of LAMP with the scalability and portability of microfluidics, the proposed system aims to provide a reliable, low-cost diagnostic tool suitable for clinical and field applications.
Methods: A three-layer microfluidic chip was fabricated from 2 mm-thick PMMA sheets using CO₂ laser engraving and thermal bonding at 110°C. The design, created in SolidWorks and optimized via COMSOL simulations, included reaction wells, microchannels, and inlet/outlet ports for efficient fluid control.
Genomic DNA of Moraxella catarrhalis (strain IBRC-M 10685) was extracted using the Favorgen kit and quantified with a NanoDrop spectrophotometer. LAMP primers targeting specific genomic regions were used to prepare a 25 µL reaction mixture containing necessary reagents and hydroxynaphthol blue (HNB) for colorimetric detection. One microliter of the prepared reaction mixture was loaded into microfluidic wells and incubated at 64°C for 60 minutes. Amplification was visually confirmed by a color change from violet to sky blue. Negative controls were included to verify specificity.
Results: The PMMA-based microfluidic chip successfully enabled on-chip LAMP amplification for the detection of Moraxella catarrhalis. The chip’s three-layer design provided efficient fluid handling and thermal stability. COMSOL simulations verified uniform fluid distribution across the reaction zones, supporting consistent amplification conditions throughout the chip.
Visual detection using HNB offered a straightforward and reliable readout. In a representative test, two wells were loaded: one with a negative control and one with DNA extracted from M. catarrhalis. After incubation, the control well retained its violet color, while the test well turned sky blue, indicating successful amplification. This clear colorimetric contrast allowed rapid interpretation without specialized equipment.
The integrated system demonstrated high sensitivity and specificity, with a fast turnaround time suitable for point-of-care respiratory diagnostics. Its simplicity and portability make it ideal for use in resource-limited or decentralized healthcare settings.
Conclusion: This study demonstrates the successful integration of LAMP with a PMMA-based microfluidic platform for rapid and specific detection of Moraxella catarrhalis. The chip’s optimized design ensured reliable fluid control, critical for consistent on-chip amplification. The use of HNB as a colorimetric indicator enabled easy visual differentiation between positive and negative samples, eliminating the need for complex instrumentation.
The platform offers a scalable, low-cost solution for early detection of respiratory pathogens, supporting timely clinical decisions and effective infection control. Its portability and ease of use make it a promising tool for field diagnostics and surveillance, particularly in settings with limited laboratory infrastructure.