The white arrow indicates the flow direction in the LFI. The three spots located on the front part of the strip, shown in Figure 3B-iCiii, presented well-developed circular fluorescent spots, indicating that the expected immunoaffinity binding was complete. 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 g/mL, covering the required clinical detection range, and the limit of detection (LOD) value was calculated to be 15.5 g/mL. Thus, the SQ-LFI system provides an avenue for the realization of an efficient quantification diagnostic device in resource-limited conditions. strong class=”kwd-title” Keywords: lateral flow immunoassay, renal failure, microalbuminuria, instrument-free quantitative analysis 1. Introduction The development of an accurate and user-friendly diagnostic device is one of the most significant objectives in the clinical field. In the academic and industrial sectors, extensive research has proceeded to develop efficient diagnostic tools, such as point-of-care testing (POCT) biosensors [1,2,3,4,5,6,7]. To develop an efficient POCT-type immunosensor, researchers have focused on the lateral flow immunoassay (LFI) method, which is an immunochromatographic test using a nitrocellulose (NC) membrane as a substrate [8,9,10,11,12,13,14,15]. In traditional LFI, an analyte forms an immunocomplex with detection antibody-conjugated gold nanoparticles (Ab-AuNPs), then flows through the membrane and is captured on the test and control lines by capture antibodies that are immobilized on the lines. Since the operation of LFI is automatically accomplished by capillary force and the result can be verified with the naked eye, LFI has merits such as low cost of operation, easy handling, and operation without external instruments. However, despite the merits of LFI, traditional colorimetric AuNP-based LFI is still used in limited tests that only require simple negative or positive results, Gabapentin Hydrochloride such as pregnancy and sexually transmitted disease tests, because of the drawbacks associated with signal evaluation [16,17]. Typically, the result of an LFI test is presented as a single colored line on the strip that only indicates the existence of the target analyte. If quantification is desired, the color signal in the test line must be converted to Gabapentin Hydrochloride optical density or color intensity values by an optical transducing device [18,19,20,21]. To overcome this weakness, barcode-type LFI strips that can easily quantify the analytes using LFI have been developed. However, this approach has limitations in the detailed quantitative analysis and requires an additional optical transducing device for further detailed quantitative analysis [22,23,24]. It also eliminates the inherent merits of LFI, such as its reasonable cost and equipment-free analysis. To develop a quantitative immunoassay using LFI while maintaining its inherent merits, the involvement of external Gabapentin Hydrochloride transducing instruments should be avoided. With the aforementioned goal in mind, we developed a semi-quantitative LFI (SQ-LFI) method that does not require any external optical instrument to quantify the analytes (Figure 1A). For the simple quantitative analysis of analytes using SQ-LFI, the concept of battery charging animation was employed. Because the animation indicates the charging status of electronic devices as several small boxes, a user simply counts the number of stacked boxes to estimate the charging status (Figure 1B). By adopting this concept, we designed a new SQ-LFI system, in which the single test line of the conventional LFI was converted to several countable test spots. The developed SQ-LFI can provide information on the antigen concentration as several colored spots and, similar to concept animation, a user simply counts the number of Gabapentin Hydrochloride colored spots to estimate the concentration of the antigen. In the sandwich-type immunoassay-based SQ-LFI, the numbers of colored spots that appear on the strip are proportional to the concentration of antigens in the samples. Because SQ-LFI proceeds automatically IgG2a/IgG2b antibody (FITC/PE) by capillary force and signal analysis can be conducted by the naked eye, quantitative analysis can be performed without any external instruments. In addition, despite the innovation of a new LFI test strip, the rest of the materials and procedures were the same as those used in conventional LFI. Therefore, the developed SQ-LFI can be produced using conventional methods with minimum procedural modifications. Open in a separate window 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 ideas of the SQ-LFI system. To validate the applicability of SQ-LFI, a practical immunoanalysis for Gabapentin Hydrochloride renal failure was.