RESUMEN
Paper-based analytical device (PAD) has received more and more attention in the field of point-of-care test (POCT) due to its low cost, portability and simple operation. Sensitivity and selectivity are two important aspects in clinical diagnostic analysis. However, low sensitivity of a PAD limits its wider application for POCT. Here we introduced a PAD that can clean and enrich the protein content from salty media with both electric field (E) and pH gradient (double E/pH gradients), with which 100-fold enrichment effect could be achieved within 70 s as demonstrated by fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) from artificial urine media. With post staining of the protein stacking band with bromophenol blue (BPB), selective colorimetric detection of human serum albumin (HSA) was achieved simply with smartphone camera in the clinically significant range of 10-300 mgâ§L-1 (R2 = 0.99) with a limit of detection (LOD) of 4.9 mgâ§L-1. Detection of microalbuminuria (MAU) of diabetic patients was demonstrated with this method without difference (É = 0.01) to that by the clinical method (immunoturbidimetry). This work demonstrated the potential of this PAD method in online sample pretreatment and detection of target component from complex physiological samples.
Asunto(s)
Técnicas Analíticas Microfluídicas , Papel , Albúmina Sérica Bovina/aislamiento & purificación , Albúmina Sérica Humana/aislamiento & purificación , Teléfono Inteligente , Animales , Bovinos , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/orina , Campos Electromagnéticos , Humanos , Concentración de Iones de Hidrógeno , Técnicas Analíticas Microfluídicas/instrumentación , Sales (Química)/química , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/orina , Albúmina Sérica Humana/química , Albúmina Sérica Humana/orinaRESUMEN
Field-amplified stacking (FAS) is a commonly used method for enhancing the sensitivity of charged species from low conductive media in capillary electrophoresis. FAS also showed significant sensitivity enhancement effect on a uniform paper fluidic channel by proper design of the electrolyte. In this paper, a novel method of introducing electric field gradient is proposed by geometry design of a 2D paper fluidic channel, and field amplification effect was successfully demonstrated with reduced requirement on the sample's conductivity. Sensitive colorimetric detection of microalbuminuria (MAU) from urine samples was demonstrated by mobile phone camera. Experimental results showed that, with active electric field motivation, up to 93.5% of the loaded protein probe could be effectively transferred and stacked into a narrow band on the newly designed paper fluidic channel. A limit of detection (LOD) of 6.5â¯mgâ§L-1 HSA was achieved with a dynamic range of 10-300â¯mgâ§L-1 (linear in the range of 10-100â¯mgâ§L-1, R2â¯=â¯0.991). Combined with selective staining of albumin with bromophenol blue (BPB), the established method was applied to the detection of MAU from clinical urine samples, and consistent results with that of the clinical method were obtained. With this paper-based analytical device (PAD), MAU from highly conductive urine samples can be directly loaded and detected without any pretreatment. This method provides a way to develop highly sensitive point-of-care test (POCT) for rapid screening of some diseases.
Asunto(s)
Albuminuria/diagnóstico , Colorimetría/métodos , Electroforesis Capilar/métodos , Papel , Albúmina Sérica Humana/orina , Azul de Bromofenol/química , Colorimetría/instrumentación , Colorantes/química , Electroforesis Capilar/instrumentación , Humanos , Límite de DetecciónRESUMEN
In this work, fast isoelectric focusing (IEF) was successfully implemented on an open paper fluidic channel for simultaneous concentration and separation of proteins from complex matrix. With this simple device, IEF can be finished in 10 min with a resolution of 0.03 pH units and concentration factor of 10, as estimated by color model proteins by smartphone-based colorimetric detection. Fast detection of albumin from human serum and glycated hemoglobin (HBA1c) from blood cell was demonstrated. In addition, off-line identification of the model proteins from the IEF fractions with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was also shown. This PAD IEF is potentially useful either for point of care test (POCT) or biomarker analysis as a cost-effective sample pretreatment method.
Asunto(s)
Hemoglobina Glucada/análisis , Focalización Isoeléctrica/instrumentación , Dispositivos Laboratorio en un Chip , Pruebas en el Punto de Atención , Albúmina Sérica Humana/análisis , Diseño de Equipo , Hemoglobina Glucada/aislamiento & purificación , Humanos , Focalización Isoeléctrica/economía , Dispositivos Laboratorio en un Chip/economía , Papel , Pruebas en el Punto de Atención/economía , Albúmina Sérica Humana/aislamiento & purificación , Factores de TiempoRESUMEN
We present a novel electrokinetic stacking (ES) method based on field amplification on a simple paper device for sample preconcentration. With voltage application, charged probe ions in a solution of lower conductivity stack and form a narrow band at the boundary between the sample and the background electrolyte of higher conductivity. The stacking band appears quickly and stabilizes in a few minutes. With this ES method, three orders of magnitude signal improvement was successfully achieved for both a fluorescein probe and a double-stranded DNA within 300 s. This enhanced stacking efficiency is attributed to a focusing effect due to the balance between electromigration and counter electroosmotic flow. We also applied this ES method to other low-cost fiber substrates such as cloth and thread. Such a simple and highly efficient ES method will find wide applications in the development of sensitive paper-based analytical devices (PADs), especially for low-cost point-of-care testing (POCT).