Instrumentation-Free Semiquantitative Immunoanalysis Using a Specially Patterned Lateral Flow Assay Device

Abstract

In traditional colorimetric lateral flow immunoassay (LFI) using gold nanoparticles (AuNPs) as a probe, additional optical transducers are required to quantify the signal intensity of the test line because it presents as a single red-colored line. In order to eliminate external equipment, the LFI signal should be quantifiable by the naked eye without the involvement of optical instruments. Given this objective, the single line test zone of conventional LFI was converted to several spots that formed herringbone patterns. When the sandwich immunoassay was performed on a newly developed semi-quantitative (SQ)-LFI system using AuNPs as an optical probe, the spots were colorized and the number of colored spots increased proportionally with the analyte concentration. By counting the number of colored spots, the analyte concentration can be easily estimated with the naked eye. To demonstrate the applicability of the SQ-LFI system in practical immunoanalysis, microalbumin, which is a diagnostic marker for renal failure, was analyzed using microalbumin-spiked artificial urine samples. Using the SQ-LFI system, the calibration results for artificial urine-based microalbumin were studied, ranging from 0 to 500 μg/mL, covering the required clinical detection range, and the limit of detection (LOD) value was calculated to be 15.5 μg/mL. Thus, the SQ-LFI system provides an avenue for the realization of an efficient quantification diagnostic device in resource-limited conditions.

Keywords: instrument-free quantitative analysis; lateral flow immunoassay; microalbuminuria; renal failure.

Figure 1

Schematic illustrations of the semi-quantitative lateral flow immunoassay (SQ-LFI) configuration and quantification strategy. (A) Configuration of the SQ-LFI system. (B) Quantification concepts of the SQ-LFI system.

Figure 2

Examinations of various pattern types to select the most effective spot arrangement. (A) A linear pattern comprising nine linearly arranged spots. (B) A diagonal pattern comprising nine diagonally arranged spots. (C) A herringbone pattern comprising 15 spots arranged in a herringbone formation with five spots per unit.

Figure 3

Schematic illustration of the flow observation procedure and resulting images of the flow around the developed spot. (A) Schematic illustration of the flow observation procedure. (B) Fluorescence images of the developed spots. The white arrow indicates the flow direction in the LFI.

Figure 4

Optimization of capture antibody concentration in the test zone. (A) A graph representing the number of colored spots on strips using three different concentrations of capture antibody. (B) Original and threshold-adjusted images obtained from the immunoassay strip. The SD values are shown in parentheses.

Figure 5

Quantitative analysis of microalbumin-spiked PBS samples. (A) The calibration curve of the various concentrations of microalbumin in PBS samples. (B) Original and threshold-adjusted images obtained from the immunoassay strip. The SD values are shown in parentheses.

Figure 6

Quantitative analysis of microalbumin-spiked artificial urine and diabetes urine samples. (A) The calibration curve of microalbumin at various concentrations in the artificial urine samples. Original and threshold-adjusted images of the strip. (B) The calibration curve of microalbumin at various concentrations in the artificial diabetes urine samples. Original and threshold-adjusted images of the strip. The two calibration curves are overlapped in the inset graph. The SD values are shown in parentheses.