Flow-Based Immunosensing Using the Pore Channel of a Porous Membrane As a Reaction Space

Abstract

This Letter describes a new rapid and sensitive immunosensing device using the pore space of a porous membrane as the reaction space. A track-etched membrane with uniform cylindrical pores is used as the base substrate of this device. The capture antibodies are covalently and densely immobilized inside the membrane pores by the uniform introduction of poly­(acrylic acid) (PAAc) via the plasma graft polymerization technique, followed by the active ester method. This membrane shows excellent antibody retention by covalent binding. The detection test was carried out via a sandwich-type assay, and all reaction steps from the antigen–antibody reaction to the enzyme reaction were conducted by permeating each solution into the pores. The detection test showed a signal comparable to that of the conventional enzyme-linked immunosorbent assay, although the detection time required in the test was shortened to 35 min. The reason for achieving both high sensitivity and short detection time is that the antibody accumulated pore space with high selectivity and promoted contact between the reactants by solution permeation. This report is expected to aid the design of systems for membrane-based devices, which currently have problems associated with sensitivity, rapidity, selectivity, or amount of sample. We further expect that this system could be applied to various diagnostic areas, including point-of-care testing.

Numerical Modeling for Sensitive and Rapid Molecular Detection by Membrane-Based Immunosensors

“…The experimental result for the membrane-based biosensor was derived from the combined results of the BSA detection test and the detection test with contaminant human serum albumin. 16 Figure 3a,b shows that the signals for the model and experiment are similar in both the membrane-based sensor and ELISA without any fitting parameter. Although each experimental signal in our sensor is slightly higher than the calculation values, this may be due to the experimental factor, including remaining contact between the permeated solution and the membrane before the signal is emitted.…”

“…To investigate the molecular-recognition behavior of the first step of the sensing system, we calculated the distribution of the captured Ag relative to the membrane thickness direction when the C Ag = 10 ng/mL Ag (BSA) solution permeated the membrane under the experimental conditions, as previously reported. 16 The red line in Figure 4a shows that most of the Ags were quickly captured at the entrance of the pore; therefore, the amount of captured Ags at the exit was almost zero. In fact, according to our calculation of the total amount of accumulated Ags, most of the permeated Ags (>97%) were captured in the pore.…”

“…A detailed description of the sensor fabrication and experimental data used in this study can be found in a previous report. 16 In brief, a track-etched polyethylene terephthalate (PET) membrane with uniform cylindrical pores (porosity 19.6%, maximum pore diameter 1 μm, and thickness 22 μm) was modified with poly(acrylic acid) by plasma graft polymerization. 17 After converting the carboxy group of the grafted polymer into the active ester group, the capture antibody was immobilized into the pores by immersing the PET membrane in the capture antibody solution (100 μL, 100 μg/mL) at 4 °C overnight (Figure 2b).…”

“…We experimentally used bovine serum albumin (BSA) as the Ag, anti-BSA polyclonal antibody as the Ab 1 , HRP-labeled anti-BSA polyclonal antibody as the Ab 2 , and 3,3′,5,5′-tetramethylbenzidine (TMB) as the substrate. 16 Therefore, the parameters used in this model were in accordance with these conditions.…”

“…Recently, we succeeded in developing a membrane-based immunosensor that is rapid and sensitive (Figure 1a). 16 This sensor is based on a porous track-etched membrane, and nanometer-to micrometer-sized cylindrical pores are used as molecular-recognition channels. To achieve a high-reactivity space, the captured antibodies were uniformly and densely immobilized within the small-sized pores using a combination of the plasma-induced graft polymerization technique 17−19 and the active ester method.…”