A rapid and highly sensitive biomarker detection platform based on a temperature-responsive liposome-linked immunosorbent assay

Abstract

The enzyme-linked immunosorbent assay (ELISA) is widely used in various fields to detect specific biomarkers. However, ELISA tests have limited detection sensitivity (≥ 1 pM), which is insufficiently sensitive for the detection of small amounts of biomarkers in the early stages of disease or infection. Herein, a method for the rapid and highly sensitive detection of specific antigens, using temperature-responsive liposomes (TLip) containing a squaraine dye that exhibits fluorescence at the phase transition temperature of the liposomes, was developed. A proof-of-concept study using biotinylated TLip and a streptavidin-immobilized microwell plate showed that the TLip bound to the plate via specific molecular recognition could be distinguished from unbound TLip within 1 min because of the difference in the heating time required for the fluorescence emission of TLip. This system could be used to detect prostate specific antigen (PSA) based on a sandwich immunosorbent assay using detection and capture antibodies, in which the limit of detection was as low as 27.6 ag/mL in a 100-μL PSA solution, 0.97 aM in terms of molar concentration. The present temperature-responsive liposome-linked immunosorbent assay provides an advanced platform for the rapid and highly sensitive detection of biomarkers for use in diagnosis and biological inspections.

Temperature-Responsive Liposome-Linked Immunosorbent Assay for the Rapid Detection of SARS-CoV-2 Using Immunoliposomes

“…The method used to determine the LOD for the TLip-LISA was based on previous studies. 25 A linear fitting line for the N protein concentration and inflection time points was obtained between 0.5 and 5 pg/mL in the regular incubation time experiment. Then, the LOD was determined as the N protein concentration that corresponded to an inflection time equal to the average inflection time of the negative control minus three times the negative control standard deviation.…”

“…In a previous study, TLip-LISA exhibited high sensitivity for detecting prostate-specific antigen with a good limit of detection (LOD) of 0.97 aM. 25 In this study, instead of using biotinylated-TLip, we performed immunoglobulin conjugation to TLip to reduce the total number of incubation steps and the assay time ( Figure 1 ), thereby enabling rapid detection and subsequent diagnosis, while also developing a point-of-care test that can be used in the field (i.e., in homes, workplaces, and airports) instead of requiring a specialized clinical laboratory. The reduced number of incubation steps also permitted rapid detection of SARS-CoV-2, which may be useful in the event of community outbreaks.…”

Modified ELISA for Ultrasensitive Diagnosis

“…Using this method, human-prostate-specific antigen, which is a prostate cancer biomarker, was detected at 0.8 pg/mL, a level lower than that detected by conventional ELISA. Furthermore, a temperature-responsive liposome-linked immunosorbent assay was reported [ 58 ]. This technique uses temperature-responsive liposomes containing a squaraine dye that exhibits fluorescence at the phase transition temperature of the liposomes.…”

Liposome-based Biosensors and Diagnosis Imaging Agents

“…Different types of assays have employed liposomes for biosensing by simply mixing the liposomes with test samples, such as immunoassays [liposome agglutination, liposome immunoaggregation, liposome immunoblotting, immunochromatographic liposome, and liposome-linked immunosorbent assays, (30,31) ]. Liposomes have also been employed as biosensors to detect various analytes (e.g., toxins, pesticides, enzyme, and viruses) (Table 1).…”