Team:BioMarvel/WET LAB/Experiments
Functional Fusion Protein-Based Biochip for Diagnosis and Monitoring of Heart Failure
The fundamental objective of this project was to produce a novel fusion protein, gold binding polypeptides (GBP)-protein G (ProG), to simply and rapidly fabricate a highly sensitive electrochemical biosensor as a crosslinker for effective antibody immobilization on gold surface without any chemical treatments. The electrochemical biosensor fabricated in this study will be used for early diagnosis and real-time monitoring of heart failure. A genetically modified vector encoding the GBP and ProG was constructed from Integrated DNA Technologies (IDT). The vector was delivered to E. coli DH5-alpha competent cell. The bacteria were grown in LB plate supplemented with X-gal for selection bacterial cells containing recombinant. The fusion protein was purified by TALON metal affinity resin after the amplification by IPTG-induced expression. The resultant GBP-ProG was immobilized onto the surface of gold electrode. Primary antibodies (anti-NT-proBNP) were bound to the GBP-ProG layer by interaction between the Fc region of antibody and ProG portion of the fusion protein. Then, antigen (NT-proBNP; one of the heart failure markers) and secondary antibody labeled by alkaline phosphatase (AP) to generate electrochemical signals were serially loaded inside the electrochemical microchannel. The results demonstrated that the genetically modified GBP-ProG protein was useful for effective fabrication of a highly sensitive electrochemical biosensor.
Immobilization of GBP-ProG onto the Gold Surfaces of the Heart Failure Assessing Practical Protein-Immunochip (HAPPI)
To verify the function of the GBP-ProG onto the gold surface of the channel, we injected primary antibody (anti-NT-proBNP), antigen (NT-proBNP) and alkaline phosphatase (AP) labeled-secondary antibody onto both bare and GBP-ProG layered gold surface inside microchannel of the chip, respectively. Figure 1 shows the results. The electrical signal peak was observed at the chip with GBP-ProG, but no signal was detected at the chip without GBP-ProG, indicating that the GBP-ProG fusion protein was simply and rapidly self-immobilized onto the gold surface of the chip without any chemical treatments and could successfully capture antibodies as we expected.
Figure 1. Cyclic voltammograms for evaluating the function of the GBP-ProG
Electrochemcial Immunoassay as a New Platform for Diagnosis and Monitoring Heart Failure
We applied two different concentrations of NT-proBNP to evaluate the sensitivity of the GBP-ProG fusion protein-based electrochemical biosensor for early diagnosis and potential real-time monitoring of heart failure. The resulting cyclic voltammograms (CV) were obtained (Figure 2). The current values were enhanced with increased concentration of NT-proBNP, suggesting that detection of NT-proBNP could be sensitively carried out in a dose dependent manner. This result indicates that oxidation peak currents are reliant on immunoreactions between NP-proBNP and its antibodies and interactions of AP enzyme and its substrate. Consequentially this result shows that the novel GBP-ProG fusion protein can attribute to simple and rapid fabrication of a highly sensitive electrochemical biosensors.
Figure. 2 Cyclic voltammograms at different concentrations of NT-proBNP
Electrochemical Biosensors in the Near Future
An integrated system of a minimized electrochemical biosensor and smartphones could be helpful for effective diagnosis and real time-monitoring heart failure patient, greatly contributing to our community and public health by reducing heart failure-mediated mortality and medical cost. NT-proBNP concentration in blood will be self-tested, and the data will be analyzed by individual’s mobile applications on their smartphones, followed by sending the data to their doctors. This will also allow efficient monitoring of the heart failure patients for doctors. We believe this so-called U-healthcare system might be magnified in the near future as one of new healthcare industries.
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