Detection of severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in human serum using a localized surface plasmon coupled fluorescence fiber-optic biosensor

Abstract

In order to enhance the sensitivity of conventional immunoassay technology for the detection of SARS coronavirus (SARS-CoV) nucleocapsid protein (N protein), we developed a localized surface plasmon coupled fluorescence (LSPCF) fiber-optic biosensor that combines sandwich immunoassay with the LSP technique. Experimentally, a linear relationship between the fluorescence signal and the concentration of recombinant SARS-CoV N (GST-N) protein in buffer solution could be observed from 0.1 pg/mL to 1 ng/mL. In addition, the concentration of GST-N protein in diluted serum across a similar range could also be measured. The correlation coefficients (linear scale) for these two measurements were 0.9469 and 0.9624, respectively. In comparison with conventional enzyme linked immunosorbent assay (ELISA), the detection limit of the LSPCF fiber-optic biosensor for the GST-N protein was improved at least 10(4)-fold using the same monoclonal antibodies. Therefore, the LSPCF fiber-optic biosensor shows an ability to detect very low concentration (approximately 1 pg/mL) of SARS-CoV N protein in serum. The biosensor should help with the early diagnosis of SARS infection.

Fig. 1

Detection of GST-N protein by conventional sandwich ELISA. (A) Serially diluted GST-N proteins in PBS were detected by sandwich ELISA. Different concentrations of GST-N protein were assayed on a microtiter plate coated with anti-N-1 and anti-N-2 monoclonal antibodies. The absorbance of OD450 in each well was observed after further incubation with polyclonal anti-GST-N antibody followed by peroxidase-conjugated anti-rabbit antibody. (B) GST-N protein was serially diluted in 10-fold diluted human serum and assayed by ELISA. Results shown are the mean of duplicates. Error bars indicate the standard deviation.

Fig. 2

Detection of different concentrations of GST-N protein in PBS with the LSPCF fiber-optic biosensor. (A) The binding processes of fluorescence probe interaction with different concentrations of GST-N protein in PBS [●: 0 pg/mL; ○: 0.1 pg/mL; ▾: 1 pg/mL; ▿: 10 pg/mL; ■: 100 pg/mL; □: 1000 pg/mL]. The uncertainty of the measurement is smaller than the size of data points in this figure. (B) The linear relationship [y = 0.0122 Ln(x) + 0.0206; R² = 0.9469] of GST-N protein concentration in PBS versus fluorescent signal. The error bar is smaller than the size of data points and equivalent to one standard derivation in this experiment.

Fig. 3

Detection of different concentrations of GST-N protein in 10-fold diluted human serum with LSPCF fiber-optic biosensor. (A) The binding processes of fluorescence probe interaction with different concentrations of GST-N protein in diluted serum [●: 0 pg/mL; ○: 0.1 pg/mL; ▾: 1 pg/mL; ▿: 10 pg/mL; ■: 100 pg/mL; □: 1000 pg/mL]. The uncertainty of the measurement is smaller than the size of data points in this figure. (B) The linear relationship [y = 0.0096 Ln(x) + 0.0337; R² = 0.9624] of GST-N protein concentration in diluted serum versus fluorescent signal. The error bar is smaller than the size of data points and equivalent to one standard derivation in this experiment.