RESUMO
We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 µm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL.
Assuntos
Imunoensaio/instrumentação , Dispositivos Lab-On-A-Chip , Rickettsia conorii/isolamento & purificação , Animais , Anticorpos Imobilizados/metabolismo , Automação Laboratorial , Células Imobilizadas , Desenho Assistido por Computador , Desenho de Equipamento , Processamento de Imagem Assistida por Computador , Imunoensaio/métodos , Separação Imunomagnética , Limite de Detecção , Fenômenos Magnéticos , Microscopia , Microscopia Eletrônica de Varredura , Microesferas , Microtecnologia/métodos , Polimetil Metacrilato/química , Estudo de Prova de Conceito , Reprodutibilidade dos Testes , Rickettsia conorii/crescimento & desenvolvimento , Rickettsia conorii/imunologia , Propriedades de SuperfícieRESUMO
Point-of-care detection of pathogens is medically valuable but poses challenging trade-offs between instrument complexity and clinical and analytical sensitivity. Here we introduce a diagnostic platform utilizing lithographically fabricated micron-scale forms of cubic retroreflectors, arguably one of the most optically detectable human artifacts, as reporter labels for use in sensitive immunoassays. We demonstrate the applicability of this novel optical label in a simple assay format in which retroreflector cubes are first mixed with the sample. The cubes are then allowed to settle onto an immuno-capture surface, followed by inversion for gravity-driven removal of nonspecifically bound cubes. Cubes bridged to the capture surface by the analyte are detected using inexpensive, low-numerical aperture optics. For model bacterial and viral pathogens, sensitivity in 10% human serum was found to be 10(4) bacterial cells/mL and 10(4) virus particles/mL, consistent with clinical utility.