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

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the etiological agent of coronavirus disease 2019 (COVID-19), has infected more than 340 million people since the outbreak of the pandemic in 2019, resulting in approximately 55 million deaths. The rapid and effective diagnosis of COVID-19 patients is vital to prevent the spread of the disease. In a previous study, we reported a novel temperature-responsive liposome-linked immunosorbent assay (TLip-LISA) using biotinylated-TLip that exhibited high detection sensitivity for the prostate-specific antigen. Herein, we used immunoglobulin-TLip (IgG-TLip), in which the antibodies were directly conjugated to the liposomal surface to simplify pretreatment procedures and reduce the detection time for SARS-CoV-2. The results indicated that TLip-LISA could detect the recombinant nucleocapsid protein and the nucleocapsid protein in inactivated virus with 20 min incubation time in total, and the limit of detection was calculated to be 2.2 and 1.0 pg/mL, respectively. Therefore, TLip-LISA has high potential to be used in clinic for rapid diagnosis and disease control.

Figure 1

Conceptual schematic of TLip-LISA using IgG-TLip. After incubation of IgG-TLip, the plate is heated with a hot plate and a fluorescence detector is used to monitor and record the fluorescence intensity change with time.

Figure 2

N protein detection by TLip-LISA using IgG-TLip. (a) Representative profiles of the change in fluorescence intensity of the positive sample (500 pg/mL) and the negative control. The arrow indicates the inflection time point in each group. (b) Representative profiles of the change in the increase rate of fluorescence intensity of the positive sample (500 pg/mL) and the negative control. The inflection time point was defined as the increase rate reached the highest magnitude as indicated by the arrow.

Figure 3

N protein detection using IgG-TLip. n = 4 for positive samples. (a) Inflection time points for different concentrations. (b) Linear regression between 0.5 and 5 pg/mL. The orange dot line indicates the corresponding level of LOD, and the orange dot indicates the LOD. NC, negative control. Rapid detection of N protein and the inactivated virus.

Figure 4

Rapid detection of N protein. n = 4 for positive samples and n = 10 for the negative control. Inflection times for different N protein concentrations with linear regression between 1000 and 0.1 pg/mL. The orange dot line indicates the corresponding level of LOD, and the orange dot indicates the LOD. The detection time for the negative control was 31.9 ± 1.72 s.

Figure 5

Rapid detection of the inactivated virus. n = 4 for positive samples, and the negative control was the same as that for the N protein. Linear regression curve was obtained between 1000 and 0.1 pg/mL. The orange dot line indicates the corresponding level of LOD, and the orange dot indicates the LOD.