We present a laser-fabricated platform integrating a laminated capillary-driven microfluidic system with a laser-induced graphene (LIG) heater for on-chip loop-mediated isothermal amplification (LAMP) targeting human papillomavirus type 16 (HPV16) DNA. Both the microfluidic channels and LIG heater were patterned using a CO2 laser system, providing a rapid and mask-free fabrication strategy. The laminated microfluidic architecture enables passive capillary-driven fluid transport without external pumps and incorporates a push valve to retain the reaction mixture during amplification and release the amplified products into the downstream detection zone. LAMP reagents and a dicationic pyridine fluorescent dye (PY2+) were preloaded onto glass fiber pads within the channel, reducing manual reagent addition. The LIG heater, positioned directly beneath the amplification chamber, provided localized Joule heating for on-chip LAMP operation. Following LAMP, valve activation released the amplified products into the detection zone, where binding of PY2+ to double-stranded DNA generated a visible fluorescence. The platform was evaluated using HPV16 plasmid DNA concentrations from 0.01 pg/μL to 0.01 μg/μL, with calculated LOD and LOQ values of 0.019 pg/μL and 0.37 pg/μL, respectively. Assay specificity was supported by the absence of cross-reactivity toward the tested nontarget genital tract pathogens. By integrating capillary-driven fluid handling, reagent pre-storage, localized heating, and downstream fluorescence detection, the platform provides a simplified approach to integrated nucleic acid amplification and detection and a foundation for future portable sample-to-result analysis.
Portable nucleic acid amplification platforms commonly use an external thermostat clamped to the reaction cartridge, which introduces interfacial thermal resistance, constrains heater geometry, and reduces the reaction volume maintained within the target temperature range. This work reports a battery-powered lab-on-a-chip biosensing platform that addresses this limitation by directly laminating a flexible silver–polyimide microheater beneath a micro-milled poly(methyl methacrylate) (PMMA) microreactor array and regulating temperature through the resistance of the heater track. The heater functions as the transduction-enabling element because amplification performance depends on maintaining the enzymatic reaction volume within the active temperature window. The eight-chamber PMMA chip was fabricated by benchtop micro-milling, with spindle speed and feed rate optimized using a Taguchi design, reducing mean channel-floor roughness from 0.42 μm to 0.12 μm while maintaining channel width within 2.5% of the design value. The heater was fabricated by cleanroom-free surface-modification ion exchange and doctor-blade deposition of an Ag–poly(vinyl alcohol) composite, producing a temperature coefficient of resistance of 0.0019 °C-1 and resistance change below 1% after 5000 bending cycles. Integrated into a handheld closed-loop device, the cartridge maintained the 35–65 °C isothermal window with a worst-case steady-state error of 0.61 °C. At 40 °C, it reached set point within 3 min, drew less than 0.6 W, and limited spatial variation across eight chambers to ±0.2 °C. Finite-element modelling showed that more than 92% of the 20 μL reaction volume remained within ±1 °C. The chip amplified Trypanosoma evansi DNA with results concordant with micro-PCR and benchtop PCR.
The management of airway disease based on phenotypic characteristics is limited by the absence of diagnostic tools capable of concurrently addressing multiple inflammatory pathways. Traditional assessments provide isolated measurements, which fail to capture intricate interactions among early immune activation, systemic dissemination, neutrophil recruitment, and viral immune response. These are four critical mechanisms that influence disease progression and therapeutic outcomes. In this study, we introduce a non-faradaic electrochemical impedance spectroscopy biosensor designed for a label-free parallel measurement of C-reactive protein (CRP), IP-10 (CXCL10), interleukin-6 (IL-6), and interleukin-8 (IL-8). The platform uses frequency-specific impedance analysis to detect binding events in capacitance at the electrode-electrolyte interface, allowing for direct concentration-dependent detection. It offers wide dynamic ranges covering both physiological and pathological concentrations for all four analytes. Analytical validation showed a strong correlation between spiked and recovered concentrations (R2 > 0.95), with percent recovery within the 80-120% accuracy range and cross-reactivity below 20% under multiplexed conditions, confirming the platform's selectivity and quantitative reliability. Both intra- and inter-assay reproducibility met analytical standards. By incorporating four distinct inflammatory biomarkers into a single impedance framework, this platform moves airway diagnostics beyond isolated measurements to a multi-dimensional phenotype resolution, laying the groundwork for decentralized risk assessment and therapy-guided inflammatory monitoring.
Extracellular vesicles (EVs) are membrane-bound nanoparticles that serve as minimally invasive biomarkers, yet their clinical translation is hampered by limitations in existing isolation and detection strategies. Here, we systematically benchmarked EVs secreted from HeLa cell cultures using ultracentrifugation, microfluidic ultrafiltration, and magnetic bead–based capture, the latter implemented both conventionally and on OpenDrop, an off-the-shelf digital microfluidic (DMF) platform. This comparative evaluation encompassed key performance metrics, including yield, purity, workflow complexity, and operational efficiency. Nanoparticle tracking analysis and scanning electron microscopy revealed that EVs isolated from DMF exhibited recovery rates and vesicle morphology on par with those obtained through conventional bead-based workflows, while requiring markedly reduced reagent volumes. Western blotting and flow cytometry confirmed robust expression of tetraspanin EV markers (CD63 and CD81), across a dynamic concentration range. On-chip ELISA performed directly on the DMF device demonstrated strong linearity across EV concentrations ranging from 5 × 106 to 1 × 109 particles/mL (R2 = 0.8935), in close agreement with conventional benchtop measurements (R2 = 0.8388). Together, these results establish the DMF platform used in this study as a versatile, and cost-efficient solution capable of integrating EV isolation and detection within a single, unmodified device. By uniting conventional laboratory workflows with miniaturized, digitally controlled operations, this work underscores the potential of accessible DMF technologies to advance portable point-of-care EV diagnostics in the future.
Organ-on-a-chip (OoC) platforms are a key subclass of microphysiological systems (MPS), designed to mimic the dynamic and physiological properties of native human tissue and organs. These systems have attracted significant attention, particularly following the Food and Drug Administration (FDA) approval of their use in the pharmaceutical and biomedical industries as alternatives to traditional models. However, their physiological relevance is often limited due to the lack of functional immune compartments, which are responsible for maintaining homeostasis and to modulate disease progression in the human body. In this review, we present a critical overview of immune-integrated OoC platforms, highlighting their capacity to emulate complex immunological processes such as immune cell trafficking, extravasation, and cytokine-mediated inflammation. We further explore recent advances in microfluidic design and on-chip biosensor integrations to perform real-time monitoring of immune dynamics. Despite their immense potential for drug development and personalized medicine, we highlight that their clinical translation remains challenged by biological and microarchitectural limitations, the lack of systemic multi-organ networks, hurdles in on-chip biosensor integration, and operational drawbacks. Overall, we outline a strategic roadmap for the standardization of immunocompetent OoC platforms to generate the robust and reproducible datasets required for artificial intelligence (AI) models and subsequent FDA qualification.
