Team:BioMarvel/Project/Description

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Project Description

Heart failure (HF) is a major public health problem, with a prevalence of over 5.8 million in the USA, and over 40 million worldwide (Lancet 388:1545-1602, 2016). Heart failure is when the heart is unable to pump sufficiently to maintain blood flow to meet the body's needs. Common causes of heart failure include coronary artery disease including a previous myocardial infarction (heart attack), high blood pressure, atrial fibrillation, excess alcohol use, infection, and cardiomyopathy of an unknown cause. Heart failure is a common, costly, and potentially fatal condition. (Lancet 365:1877–89, 2005). Early diagnosis of heart failure is essential for the successful medical treatment of the disease. However, current diagnosis of heart failure patients takes considerable time from initial sampling to detection results due to sample transportation time and operating costs. Also, there is no convenient and affordable service system for continuous monitoring of patient status.

Therefore, biosensors that are portable and permit on-site analysis of samples are very useful for effective diagnosis and monitoring of heart failure patients. Among them, electrochemical immunosensors have several advantages of simplicity, inexpensive cost, accuracy, and high sensitivity. In the immunosensors, biological recognition events between antigens and antibodies immobilized on a sensing platform generate.

It is necessary to produce effective, high sensitivity, and low-cost immunosensors. Effective immobilization of antibodies on a sensing platform is crucial in designing immunosensors. Self-assembled monolayers (SAMs) method is widely used to immobilize antibodies on gold surfaces of a sensing area via covalent binding in biosensors, but the chemical method is very complex and takes a long time. Therefore, it is necessary to develop a simple method of antibody immobilization on gold sensing surfaces. To overcome this problem, we will present a synthetic biology method by producing a novel fusion protein constructed by genetically fusing gold binding polypeptides (GBP) to protein G (ProG) as a crosslinker for effective immobilization of antibodies on gold sensing surfaces. The resulting GBP-ProG fusion protein will directly be self-immobilized onto gold surfaces via the GBP portion, followed by the oriented binding of antibodies onto the ProG domain targeting the Fc region of antibodies. As a result, we expect that the GBP-ProG protein can be a valuable crosslinker for simple and oriented immobilization of antibodies onto gold sensing surfaces without any surface chemical modification.

We created a vector capable of producing the fusion protein which includes GBP and ProG. PCR was conducted using plasmid pTGE (Park et al., Anal.Chem., 78:7197, 2006) and protein G gene of Streptocococcus G418 as a template. Eventually, 6His-GBP and Protein G was obtained as the combined form of gene products. The DNA obtained by PCR was separated about 700 bps of DNA with electrophoresis. These were cut with two restrict enzymes, NdeI and BamHI, then cloned to plasmid pET-22b (+) cut with the same restriction enzyme to create recombinant plasmid pET-6HisGBP-ProG.

Then parts EcoR1 and PstI in the GBP-ProG sequence will be removed through DNA synthesis. Then PCR process for GBP-ProG sequence will be performed as a manufactured primer. These will be cut with two restricted enzymes, EcoRI and PstI, then clone to plasmid pSB1C3 cut with the same restriction enzyme to create recombinant plasmid pSB1C3-GBP-ProG. DH5α E. coli strain will be transformed by The vector produced above and then screened from LB plates with chloramphenicol. After the induction expression, the entire culture medium centrifugation and discard the upper layer. The remaining cells will be suspended in the solution and the cells will be crushed at 4 ℃ using an ultrasonic thresher. The crushed solution centrifugation at 4 ℃ for 30 minutes at 13,000 rpm to obtain the supernatant. Supernatant (total protein) will be refined using the Talon resin column and then quantified using the Bradford protein quantification method and dilute to 1mg/ml with a fixed buffer solution (PBS, pH 7.4).

Fig 1. Schematic diagram of the process of producing fusion proteins

This finally purified fusion protein will be used as an effective crosslinker for antibody immobilization for fabrication of an electrochemical immunosensor which can monitor heart failure patients. A sensitive and rapid electrochemical immunosensor will be fabricated by an interdigitated array (IDA) gold electrode. This will be developed for the detection of human B-type natriuretic peptide (BNP) in the early diagnosis of heart failure. The GBP-ProG fusion protein will be especially used as a crosslinker for effective immobilization of anti-proBNP antibodies on the gold electrode surfaces. The detection experiments will be performed with successive injection of BNP, alkaline phosphatase (AP)-labeled anti-BNP, and p-aminophenylphosphate. Then, cyclic voltammograms will be obtained by the oxidation peak current proportionally to the concentration of enzymatic product, p-aminophenol.

After the gold binding protein immobilized to the gold electrode surface, we will confirm effective binding of antibodies using anti-proBNP antibodies. The results of the meaningful generation of electrochemical detection signals will be obtained and the usefulness of fusion proteins will be proved.

This electrochemical immunosensor shown in this study can attribute to the efficient treatment and monitoring of heart failure cases, which have increased in number worldwide, to reduce the time loss and mortality rates of individuals.

Fig 2. Schematic diagram of the electrochemical biosensor using GBP-ProG

Reference

  1. McMurray JJ, Pfeffer MA (2005). "Heart failure". Lancet. 365 (9474): 1877–89.
  2. GBD 2015 Disease and Injury Incidence and Prevalence, Collaborators (2016). Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990–2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 388 (10053): 1545–1602.
  3. Dickstein K, Cohen-Solal A, Filippatos G, et al. (2008). ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: the Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM). Eur. Heart J. 29 (19): 2388–442
  4. Park TJ, Ko S et al. (2006). Protein Nanopatterns and Biosensors Using Gold Binding Polypeptide as a Fusion Partner. Anal.Chem. 78:7197-205

Lab.

  • open bench 1
  • open bench 2
  • open bench 3
  • clean bench