Accurate detection of tumor markers at low concentration, has significant promise for early diagnosis and therapeutic monitoring (1–3). Vascular endothelial growth factor (VEGF) is a significant regulator of both pathologic and physiologic angiogenesis by activating VEGF-receptor tyrosine kinases in all endothelial cells (4–7). VEGF is a signaling protein that has been applied as an important serum biomarker for a number of human diseases, such as cancer (8–11), rheumatoid arthritis (12), psoriasis (13) and proliferating retinopathy (14). The abnormally rapid growth and division of tumors make it stimulated the overexpression of VEGF due to the supply of more nutrients and oxygen, leading to in tumor lymphatic vessels induction and cancer metastasis (15). It is estimated that up to 60% of human cancer cells overexpress VEGF to create the essential vascular network to support tumor growth and metastasis (16). A high concentration of VEGF in the tumor tissue and serum of patients who suffer brain tumor was previously reported (17). Increased VEGF concentration is also highly correlated with tumor progression and survival in patients with malignant melanoma (18). According to previous researches, the VEGF level in healthy people is usually less than 100 pg mL− 1 and clearly increased by the progression of clinical stage (198 pg mL− 1 in stage I–II; 955 pg mL− 1 in stage III–IV) in ovarian cancer. Moreover, similar VEGF values were previously reported, indicating overexpression of VEGF in patients with brain tumor (19, 20). Hence, VEGF serum level has important effects as a biomarker on some diseases and subsequent monitoring of treatment.
Avastin, a humanized anti-VEGF antibody, is a potential candidate for the development of antiangiogenic drugs against VEGF (21, 22). Therefore, due to its specificity for VEGF, it is known to be an extremely potent angiogenesis inhibitor (23). It is a challenging and critical task to develop selective and sensitive detection of VEGF in the patients’ whole blood or serum for early diagnosis of disease and efficient therapeutic monitoring strategies.
VEGF165 is the most potent pro-angiogenic isoform and is usually overexpressed in a variety of human tumors (24, 25). Therefore, the receptor binding domain (RBD) of VEGF165 is selected as the target protein in this work. Tumor markers can be detected by different methods such as radioimmunoassay (26), enzyme-linked immunosorbent assays (ELISA) (27), electrophoretic immunoassay (28), and mass spectrometry-based proteomics (29). ELISA is one of the most common immunoassay methods that is widely used for the detection of different analytes due to its simplicity, rapidity, low cost, robustness, and high-throughput (30). However, the conventional colorimetric ELISA based on horseradish peroxidase (HRP) displays low sensitivity and fails to meet requirements for higher sensitivity application.
Here we improved the detection sensitivity of ELISA by three strategies. At first, catalase (CAT) was used instead of HRP due to its excellent catalytic efficiency. CAT turnover number exceeds any other enzymatic reaction and is estimated to be approximately 4 × 107 (31–34), which is almost 240-fold higher than the one for HRP (35). Secondly, increasing the ratio between the enzyme molecule and target analyte at each binding event which can amplify the detection signal and consequently improves the sensitivity of ELISA. Negatively because of steric reasons in conventional ELISA, a ratio of 1:1 for enzyme and detection antibody is usually used (29, 36). We use dextran that can address the problems described above as these molecules exhibit three-dimensional and flexible construction with high loading capacity. Meanwhile, to maintain a high CAT activity of CAT-VEGF conjugate to enhance the detection sensitivity, we proposed the use of the dextran system to achieve an indirect conjugation of the CAT and VEGF. In addition, due to relatively low color intensity, especially at low analyte concentrations, the signal-generation mechanism based on enzymes that catalyze chromogenic substrates (e.g., tetramethylbenzidine) is not suitable. To enhance the detection sensitivity of conventional ELISA, chromogenic substrates may be replaced by more sensitive signal-generation transducers to convert molecular recognition events into detectable outputs, including chemiluminescent substrates, as the third strategy.
Chemiluminescent ELISA (CL-ELISA) is a good alternative method for samples screening with the significant merits of high sensitivity, low noise, no interference from background scattering light, broader linearity, reduced assay time, free of radioactive reagents, simple instrument and ease of use. These advantages of CL-ELISA make it a useful detection system. Up to now, several chemiluminescent immunoassay methods have been established in the clinical detection of tumor markers, such as alpha-fetoprotein (AFP), prostate-specific antigen (PSA), carbohydrate antigen 125 (CA125), and neuron-specific enolase (NSH) (37–40).
Quantum dots (QDs) as a new family of versatile nanoparticle, have shown attractive prospects due to their unique optical properties and effects associated with quantum confinement (41–43). One considerable property of TGA-CdTe QDs is being sensitive to hydrogen peroxide (H2O2), leading to the QDs CL. We have recently developed a catalase assay method based on the finding that the CL of the TGA-CdTe/H2O2 system is reduced owing to the consumption of H2O2 by the catalytic action of CAT (44). In the present study, a novel CL based ELISA was developed for the sensitive detection of VEGF, in which H2O2-sensitive TGA capped CdTe QDs were introduced as a CL signal output, avastin (anti-VEGF monoclonal antibody) and CAT-VEGF conjugate as the coating antibody and competitive antigen, respectively. The analytical performances of our proposed method which is assessed on the basis of validation process using real sample demonstrated that the CL-ELISA can be applied for selective detection of VEGF molecules in real samples accurately.