Highly sensitive and accurate detection of C-reactive protein by CdSe/ZnS quantum dot-based fluorescence-linked immunosorbent assay

Abstract

Background: The conventional and widely used enzyme-linked immunosorbent assays (ELISA), due to specificity and high-sensitivity, were suitable in vitro diagnosis. But enzymes are vulnerable to the external conditions, and the complex operation steps limit its application. Semiconductor quantum dots have been successfully used in biological and medical research due to the high photoluminescence and high resistance to photobleaching. In this study, we have developed a novel quantum dot-labeled immunosorbent assay for rapid disease detection of C-reactive protein (CRP).

Results: The assay for the detection of CRP can provide a wide analytical range of 1.56-400 ng/mL with the limit of detection (LOD) = 0.46 ng/mL and the limit of quantification = 1.53 ng/mL. The precision of the assay has been confirmed for low coefficient of variation, less than 10% (intra-assay) and less than 15% (inter-assay). The accuracy of assay meets the requirements with the recoveries of 95.4-105.7%. Furthermore, clinical samples have been collected and used for correlation analysis between this FLISA and gold standard Roche immunoturbidimetry. It shows excellent accurate concordance and the correlation coefficient value (R) is as high as 0.989 (n = 34).

Conclusions: This in vitro quantum dot-based detection method offers a lower LOD and a wide liner detection range than ELISA. The total reaction time is only 50 min, which is much shorter than the commercialization ELISA (about 120 min). All of the results show that a convenient, sensitive, and accurate fluorescence-linked immunosorbent assay method has been well established for the detection of CRP samples. Therefore, this method has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.

Keywords: C-reactive protein; Fluorescence-linked immunosorbent assay; Fluorescent probe; Quantum dots.

Scheme 1

A schematic illustration of FLISA procedure

Scheme 2

Formation of QD labeled antibody

Fig. 1

Fluorescence spectra (a), dynamic light scattering (b), and agarose gel electrophoresis imaging (c) of the aqueous CdSe/ZnS QDs and QDs-mAb; and PL intensity of FLISA with different antigen concentrations (d)

Fig. 2

The PL intensity of QDs-mAb in various pH (a) and in different buffers (b). Fluorescence spectra of QDs-mAb in Tris buffer solutions with different ionic strengths (c). The PL intensity of QDs-mAb probes versus different storage time (d) in Tris buffer solution

Fig. 3

Evolution of PL intensity in different fluorescence developing time (a) and optimal fluorescence developing time (b)

Fig. 4

Photoluminescence spectra from FLISA for determination of CRP antigen (a) and standard curves (b) (n = 3)

Fig. 5

Cross-reactionof FLISA (a), and comparison of results for CRP concentrations by FLISA and Roche immunoturbidimetry (b)