Gold Nanoparticles-Based Electrochemical Method For The Assay Of Tumor Markers

Objective:Accurate detection methods are needed in each process of cancer prevention, diagnosis, treatment and prognosis, such as genetic susceptibility screening, auxiliary and differential diagnosis, treatment monitoring and prognosis evaluation. To develop novel analytical methods with promoted sensitivity and specificity will benefit the growing tumor population. This study is to establish a novel electrochemical system for the assay of tumor-related DNA, microRNA and protein. By taking the detection of breast cancer gene BRCA1, microRNA-21(miR-21) and vascular endothelial growth factor (VEGF), we have also proved the feasibility and versatility of our new method. This study may provide a new principle for clinical detection of tumor markers.Methods:Based on the ultra-high electron transfer efficiency of gold nanoparticles (GNPs), and the interaction between GNPs and single-stranded DNA (ssDNA) modified on gold electrode surface, we have constructed a novel electrochemical detection system. Due to the interaction between the gold and the nitrogenous base, GNPs can be adsorbed to a gold electrode surface that is modified with ssDNA. So, electrochemical communication between the negatively charged electrochemical species [Fe(CN)6]3-/4-and the electrode can be greatly facilitated through the adsorbed GNPs, and the charge transfer resistance (Rct) decreases. However, in the presence of target DNA, microRNA, or protein, ssDNA will form a secondary structure which cannot adsorb GNPs. With the exposure of the negatively charged phosphate backbone of DNA, a large Rct can be observed. Since the intensity of Rct is directly related to the amount of target molecules, quantitative relationship between the target molecules and the electrochemical signals can be established.Results:1、Establishment of the detection method(1) Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are selected as the primary means for detection in this experiment. Peak separation of the redox potential is reduced to50mV after the electrode is incubated with10nM GNPs for30min, and the EIS signal is decreased for93.8%. However, the change cannot be observed in the presence of the detection target.(2) It is found that ssDNA modified electrode has a better electron transfer ability than the bare electrode after incubation with GNPs. In the CV test, the relationship between scanning velocity (v) and peak current is followed:y=36.57x+1.04(R2=0.997) and y=-39.86x-0.65(R2=0.999), where y is the peak current, x is v1/2.(3) In the optimal experiments, we have found that even the shortest DNA fragment (5T) can effectively capture the GNPs and greatly reduce the impedance. In addition, the peak current still remains basically unchanged in continuous scan for20cycles, indicating a strong interaction between the GNPs and ssDNA.(4) By comparing the intensities of impedance of the electrodes prepared with different densities ssDNA modification, we have found that the modified electrode prepared with0.5μM ssDNA has the maximum efficiency. This may be attributed to the suitable distance between the probes, thus GNPs can be better adsorbed to the gold surface and accelerate the charge-transfer between the electrochemical species and the electrode.2、Application of the detection(1) Detection of breast cancer susceptibility gene BRCA1:the linear relation between the logarithm of BRCA1concentration (1pM-500nM) and Rct value can be elaborated by the equation:y=511.7x+1913.2(R2=0.986), where y is Rct, x is the logarithm of BRCA1concentration. Detection limit is as low as1pM. The detection of single base mismatches will be stronger around the melting temperature.(2) Detection of miR-21in lung cancer and adjacent tissues:miR-21expressions in five cases of squamous cell lung cancer and adjacent normal tissues have also been analyzed. The experimental results have confirmed that only100ng total RNA is enough to get a preferably result. Compared to normal tissues, miR-21in cancer tissues have raised2.5times.(3) Detection of VEGF:The linear equation between VEGF concentration and Rct value is y=11.3x+291.2(R2=0.989), where y is Rct, x is the concentration of VEGF (5-40pM), with the detected limit of5pM.Conclusion:We have constructed a novel electrochemical biosensor, which processes many features such as simplicity, sensitivity, low-cost and versatility. This will provide new opportunities for clinical detection of tumor-associated DNA, microRNA and protein.