P-Glycoprotein Detection for Multidrug Resistance of Leukemia Using SERS Immunoassay

Corresponding author: Baoan Chen, MD, PhD; E-mail: cba8888@hotmail.com Zhuyuan Wang, PhD; E-mail: wangzy@seu.edu.cn

Yiping Cui, PhD; E-mail: cyp@seu.edu.cn

Keywords: leukemia; multi-drug resistance; P-glycoprotein; surface-enhanced Raman scattering; SERS intensity.

Background

Acquisition of multidrug resistance (MDR) in the chemotherapy of leukemia could decrease the survival rate of refractory/relapsing patients. One of the best characterized mechanisms of MDR in leukemia is mediated by multidrug resistance protein-1 and its product, P-glycoprotein (P-gp). Thus, accurate detection of P-gp is necessary for MDR diagnosis. In the recent years, surface-enhanced Raman scattering (SERS) has emerged as a new detection technology of biological label for immunoassay with the advantages of ultrasensitive screening ability and extensive adaptability. However, few researches have focused on the application of SERS immunoassay in the diagnostics of leukemia MDR. The aim of our study is to investigate the expressions of P-gp on the cell surface of K562/ADM cells, and in the whole-blood samples of leukemia using a SERS-based immunoassay technique.

Methods

To simulate the MDR occurrence, we mixed the K562 and K562/ADM cells at different ratios. Besides, we built up the concentration gradient of K562/ADM cells for the quantitative analysis. We also divided 30 blood samples (AML n=14, ALL n=16; female n=12, male n=18; age<60 n=17, age≥60 n=13) into two groups (primary patients in Group A; relapsing patients with over-expressed P-gp in Group B) and compared their SERS-based results with those measured by FCM assay. After preparation of our targeted samples, we synthesized a sandwich immunocomplex, which comprised of magnetic nanobeads (MBs) decorated with anti-CD45, SERS nanoprobes (NPs) decorated with P-gp antibodies, and our targeted samples. Then, SERS measurements were performed on the sandwich immunocomplex. Briefly, the immunocomplex can be precipitated by magnet and the SERS signals could be detected in the precipitates due to the specific binding. Without target samples, only negligible SERS signals could be detected. In this way, the SERS immunoassay can be used to evaluate the presence or expression level of P-gp.

Results

There were positive and stable SERS signals of peak intensity at 1078 cm^-1 after suspended with target samples. First, the SERS intensity of K562/ADM was significantly higher than that of K562 (P <0.01). Second, the SERS intensities of different K562/ADM fractions showed a good linear response to the fractions of K562/ADM cells. Furthermore, the SERS intensities decreased with the decliningK562/ADM concentrations (from 5×10⁶ to 50 cells/mL) and the limit of detection (LOD) could reach 50 cells/ml, which was significantly lower than FCM. Furthermore, the SERS intensity of whole blood samples in Group B were about five folds more than those in Group A (P <0.01). It indicated great application potential and reliability of SERS for MDR assessment in clinic.

Conclusion

We have proposed a SERS-based immunoassay to evaluate the expression of P-gp, a product of MDR protein of leukemia. Qualitative and quantitative analysis of K562/ADM indicated excellent specificity, high sensitivity and detection limit, as well as great reproducibility of this immunoassay. It was also demonstrated that this immunoassay was with acceptable accuracy and detection reproducibility for clinical whole blood samples, which was of great importance and convenience for practical clinical application. These features have made SERS-based immunoassay a selective and convenient technique for the identification of leukemia MDR diagnosis.