A New Method For Simultaneous Detection Of Multiple Tumor Markers On The Surface Of Breast Cancer Cells Based On Aptamer Specificity

Cancer is the leading cause of death worldwide, several millions of deaths die of cancer diseases every year. The survival of cancer patients is strongly associated with the early diagnosis of tumor diseases. Simultaneous detection of multiple tumor-related proteins in cells is significant for accurate diagnosis of cancers and further understanding the roles of these proteins in cancer development because cancer is usually associated with various tumor-related proteins. Changes in expression of these proteins in cells are closely correlated with tumor burden and malignant progression. Moreover, simultaneous multiple-protein assay will greatly increase reliability of evaluation of pathological states due to its great improvement in detection sensitivity (true positive rate) and specificity (true negative rate) over the single-protein assay. This assay can be used for estimating multiple tumor-related proteins expression in breast cancer cells with good sensitivity and reliability, and can also discriminate the subtype of breast cancers. The method has potential application in cancer detection with tumor-related proteins. The main results are as follows:We report a novel strategy for high specific and simultaneous detection of multiple tumor-related proteins in living cancer cells based on recovered fluorescence signal, which is activated by specific-binding-triggered conformation alteration of the designed activatable aptamer probes. The activatable aptamer probe consists two fragments:a target-protein-recognized aptamer sequence for specifically recognizing the target protein on the surface of the cell, and an extending spacer making the aptamer in hairpin structure (before the probe binds with target protein) and facilitating fluorescent resonance energy transfer (FRET) between the fluorophore and quencher, which are labeled at 5’- and 3’-terminus, respectively, of the aptamer. Before interaction with cell, the fluorescence of activatable aptamer probe is quenched due to the close proximity of quencher to fluorophore. After interacted with cell, the fluorescence signal is activated due to specific binding of the probe with target protein on the cell surface, causing the conformation alteration and forcing the separation of fluorophore from the quencher. We achieve simultaneous detection of multiple tumor-related proteins in living cancer cells by designing the different activatable aptamer probes and selecting various fluorophore/quencher combinations. Moreover, it can also be used for highly sensitive detection of cancer cells at a very low abundance (-10 cells mL-1 for example MCF-7 cells) and for specific discrimination of the subtype of cancers. The advantage of this approach is that it has high detection sensitivity because of the significant suppression of background with use of the designed activatable aptamer probe. It has the ability of avoiding false signals arising due to the nonspecific adsorption of interferants because it operates via monitoring the activated fluorescence signals of the designed activatable aptamer probe. In addition, this assay can work in complicated environments, requiring no complicated washing processes. We believe that this approach promises a great potential of becoming a tool for diagnosis, treatment, and prognosis of cancers. Therefore, the activatable aptamer probes supply valuable information for biomedical research and early clinical diagnosis of cancer diseases.