RESUMEN
Multiplex detection of low-abundance protein biomarkers in biofluids can contribute to diverse biomedical fields such as early diagnosis and precision medicine. However, conventional techniques such as digital ELISA, microarray, and hydrogel-based assay still face limitations in terms of efficient protein detection due to issues with multiplexing capability, sensitivity, or complicated assay procedures. In this study, we present the degassed micromold-based particle isolation technique for highly sensitive and multiplex immunoassay with enzymatic signal amplification. Using degassing treatment of nanoporous polydimethylsiloxane (PDMS) micromold, the encoded particles are isolated in the mold within 5 min absorbing trapped air bubbles into the mold by air suction capability. Through 10 min of signal amplification in the isolated spaces by fluorogenic substrate and horseradish peroxidase labeled in the particle, the assay signal is amplified with one order of magnitude compared to that of the standard hydrogel-based assay. Using the signal amplification assay, vascular endothelial growth factor (VEGF) and chorionic gonadotropin beta (CG beta), the preeclampsia-related protein biomarkers, are quantitatively detected with a limit of detection (LoD) of 249 fg/mL and 476 fg/mL in phosphate buffer saline. The multiplex immunoassay is conducted to validate negligible non-specific detection signals and robust recovery rates in the multiplex assay. Finally, the VEGF and CG beta in real urine samples are simultaneously and quantitatively detected by the developed assay. Given the high sensitivity, multiplexing capability, and process simplicity, the presented particle isolation-based signal amplification assay holds significant potential in biomedical and proteomic fields.
Asunto(s)
Técnicas Biosensibles , Límite de Detección , Factor A de Crecimiento Endotelial Vascular , Humanos , Técnicas Biosensibles/métodos , Inmunoensayo/métodos , Factor A de Crecimiento Endotelial Vascular/orina , Factor A de Crecimiento Endotelial Vascular/aislamiento & purificación , Factor A de Crecimiento Endotelial Vascular/análisis , Dimetilpolisiloxanos/química , Gonadotropina Coriónica Humana de Subunidad beta/orina , Gonadotropina Coriónica Humana de Subunidad beta/aislamiento & purificación , Gonadotropina Coriónica Humana de Subunidad beta/sangre , Gonadotropina Coriónica Humana de Subunidad beta/análisis , Biomarcadores/orina , Femenino , Embarazo , Diseño de EquipoRESUMEN
Less than optimal particle isolation techniques have impeded analysis of orthopaedic wear debris in vivo. The purpose of this research was to develop and test an improved method for particle isolation from tissue. A volume of 0.018â¯mm3 of clinically relevant CoCrMo, Ti-6Al-4V or Si3N4 particles was injected into rat stifle joints for seven days of in vivo exposure. Following sacrifice, particles were located within tissues using histology. The particles were recovered by enzymatic digestion of periarticular tissue with papain and proteinase K, followed by ultracentrifugation using a sodium polytungstate density gradient. Particles were recovered from all samples, observed using SEM and the particle composition was verified using EDX, which demonstrated that all isolated particles were free from contamination. Particle size, aspect ratio and circularity were measured using image analysis software. There were no significant changes to the measured parameters of CoCrMo or Si3N4 particles before and after the recovery process (KS tests, pâ¯>â¯0.05). Titanium particles were too few before and after isolation to analyse statistically, though size and morphologies were similar. Overall the method demonstrated a significant improvement to current particle isolation methods from tissue in terms of sensitivity and efficacy at removal of protein, and has the potential to be used for the isolation of ultra-low wearing total joint replacement materials from periprosthetic tissues. STATEMENT OF SIGNIFICANCE: This research presents a novel method for the isolation of wear particles from tissue. Methodology outlined in this work would be a valuable resource for future researchers wishing to isolate particles from tissues, either as part of preclinical testing, or from explants from patients for diagnostic purposes. It is increasingly recognised that analysis of wear particles is critical to evaluating the safety of an orthopaedic device.
Asunto(s)
Aleaciones , Procesamiento de Imagen Asistido por Computador , Articulación de la Rodilla/metabolismo , Prótesis de la Rodilla/efectos adversos , Programas Informáticos , Rodilla de Cuadrúpedos/metabolismo , Aleaciones/administración & dosificación , Aleaciones/química , Aleaciones/farmacocinética , Aleaciones/farmacología , Animales , Artroplastia de Reemplazo de Rodilla , Articulación de la Rodilla/patología , Masculino , Ratas , Ratas Wistar , Rodilla de Cuadrúpedos/patologíaRESUMEN
Budesonide (BDS) is a potent active pharmaceutical ingredient, often administered using respiratory devices such as metered dose inhalers, nebulizers, and dry powder inhalers. Inhalable drug particles are conventionally produced by crystallization followed by milling. This approach tends to generate partially amorphous materials that require post-processing to improve the formulations' stability. Other methods involve homogenization or precipitation and often require the use of stabilizers, mostly surfactants. The purpose of this study was therefore to develop a novel method for preparation of fine BDS particles using a microfluidic reactor coupled with ultrasonic spray freeze drying, and hence avoiding the need of additional homogenization or stabilizer use. A T-junction microfluidic reactor was employed to produce particle suspension (using an ethanol-water, methanol-water, and an acetone-water system), which was directly fed into an ultrasonic atomization probe, followed by direct feeding to liquid nitrogen. Freeze drying was the final preparation step. The result was fine crystalline BDS powders which, when blended with lactose and dispersed in an Aerolizer at 100 L/min, generated fine particle fraction in the range 47.6% ± 2.8% to 54.9% ± 1.8%, thus exhibiting a good aerosol performance. Subsequent sample analysis confirmed the suitability of the developed method to produce inhalable drug particles without additional homogenization or stabilizers. The developed method provides a viable solution for particle isolation in microfluidics in general.