Preparation of a plasmid encoding GBP-ProG fusion protein

We created a vector capable of producing the fusion protein, including gold binding polypeptide (GBP) and Protein G (ProG). The GBP specifically bound against the structure of gold surface was genetically fused to the N-terminus of protein G, and a six-histidine (6His) tag was fused to the N-terminus of the GBP for easy purification of the fusion proteins. The DNA fragments encoding GBP-ProG also include anderson promotor (J23100), ribosome binding site (B0034), and double terminators (B0010, B0012). Whole amplifying procedures of the construct were carried out from Integrated DNA Technologies (IDT).

We conducted A-tailing process at the end of our gene fragment for TA cloning. A-tailing is one of enzymatic methods to add a non-templated nucleotide to the 3' end of a blunt, double-stranded DNA. Tailing is a general procedure to prepare a T-vector or to A-tail a PCR product amplified with high-fidelity polymerase for use in TA cloning.

The A-tailed gene fragment was ligated with the TA vector at room temperature for 2 hours. Then, 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 DNA.

We picked 4 white colonies from the plates the cells were grown in LB broth with 0.1mM of ampicillin at 37°C and 220 rpm. The expanded vectors were isolated through DNA mini prep kit. The TA-GBP-ProG vector was incubated with XbaI and SpeI at 37°C for 2 hours. The DNA fragment parts separated from the plasmid backbone were purified using gel electrophoresis (Fig. 1) and ligated with pSB1C3 vector. The reaction conditions for restriction enzymes and ligation is shown in table 1.

Table 1. Reaction conditions for restriction enzymes and ligation

Figure 1. Gel electrophoresis of XbaI and SpeI-digested plasmid from pSB1C3 and TA-GBP-ProG

The ten microliter of the ligation sample was transformed to 100 μl of E. coli DH5-alpha. Then, the overnight culture was perform in LB plate including chloramphenicol. We picked 10 colonies from the plate and an additional overnight culture were achieved at 37°C and 220 rpm. The amplified plasmid was purified through DNA mini prep kit. We checked appropriate insertion of the construct in the vector by gel electrophoresis after reaction with XbaI and SpeI to verify the exact band scale. Figure 2 showed that lane 1, 4, 5, 6 and 7 is proper orientation producing of pSB1C3-GBP-ProG.

Figure 2. Gel electrophoresis of XbaI and SpeI-digested plasmid from bacteria transformed with the ligation mixture to generate pSB1C3-GBP-ProG

Plasmid sequence was confirmed by BIOFACT™ sequencing analysis service. We have confirmed that whole sequence of the engineered parts were correct (Fig. 3). Primer sequence was as follows (Fig. 3).

VF2 (Forward): 5’-TGCCACCTGACGTCTAAGAA-3’

VR (Reverse): 5’-ATTACCGCCTTTGAGTGAGC-3’

Figure 3. Sequencing results from BIOFACT™ sequencing analysis service

Amplification and purification of GBP-ProG fusion Protein

After IPTG-induced expression, the entire culture was centrifuged and discarded the upper layer. The remaining cell pellet wassuspended in a resin binding bufferand the cells werelysed at 4 ℃ by sonication protocol. The lysed solution was centrifuged at 4 ℃ for 30 minutes at 13,000 rpm to obtain the supernatant which contains whole proteins from the bacterial cells. With the TALON metal affinity resin, the purified proteins were confirmed by SDS-PAGE, and then quantified using the Bradford protein assay to dilute to 1mg/ml with a phosphate buffered solution (PBS).

Figure 4. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of amplified recombinant GBP-ProG and total proteins. M, molecular weight marker; T,total protein; B,aliquot of resin binding residue; W1,aliquot of 1st washing residue; W2,aliquot of 2nd washing residue;W3,aliquot of 3rd washing residue;E,purified GBP-ProG

Function test of the GBP-ProG protein via an electrochemical microfluidic chip

To test the function of the purified GBP-ProG fusion protein, we first fabricated the electrochemical microfluidic chip. Interdigitated array (IDA) chip with three-electrode system of reference, counter, and working electrode, fabricated by standard photolithography and lift-off process. Chrome (50 Åm thick) and gold (100 Åm thick) were sequentially deposited on a cyclic olefin copolymer (COC) wafer by evaporation. The photoresist (PR) was spin-coated on the COC wafer at 4000 rpm, then baking at 90 °C for 90 s. PR exposed to UV light (2.0 s, 16 mW/cm2) via a photomask after, and then developed. Gold and chrome was removed by etchant solution. The PR was removed by washing with acetone. The resulting IDA is shown in Fig. 5.

Figure 5. IDA Chip fabrication Process

The electrochemical microfluidic chip was fabricated by assembling the IDA gold electrode with the bare COC wafer that inlet/outlet holes (1 mm in diameter) punched for interconnection of a channel. To stick two parts together, we used a double-sided tape (thickness: 50 μm) wounded at direction of the channel, followed by generation of 50 μm gap between the upper and lower wafer. The finally fabricated electrochemical microfluidic chip is demonstrated in Fig. 6.

Figure 6. The fabricated electrochemical microfluidic chip

Result

The GBP-ProG was applied to the gold surface of the electrode inside the microchannel, and then the alkaline phosphatase (AP)-labeled immunoglobulin G (IgG) was immobilized onto the GBP-ProG formed. As a control, AP-labeled IgG also was treated on the other bare gold surface inside the microchannel. Then PAPP was injected into both microchannels to obtain a cyclic voltammograms by its enzymatic reaction as a substrate. CV current values were 38.63 μA at conditions in which GBP-ProG exists, albeit no cyclic voltammetric peak responses were detected in without GBP-ProG. This result showed that GBP-ProG was directly 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, without any chemical treatments (Fig. 7).

Fig 7. Cyclic voltammograms for the function test of the GBP-ProG fusion protein

Application of the GBP-ProG based-electrochemical immunosensor in diagnosis and monitoring of heart failure disease

After fabricating the immunochip, cyclic voltammograms were measured with the injection of PBS as a background signal. Then, the GBP-ProG (1 mg/mL) was injected into the microfluidic channels of device to allowthe GBP-ProG to binds to the partially gold surface inside the channel through its GBP domain without any chemical modification. The microchannels were rinsed with PBS. The anti-NT-proBNP (100 μg/mL) was injected into the microchannels. The excess binding sites were blocked by passing BSA (1 mg/mL), followed by PBS washing. After washing any unbound anti-NT-ProBNP with PBS, AP-anti- NT-ProBNP (100 μg/mL) was injected into the GBP-ProG/ anti-NT-ProBNP layered microchannel followed by rinsing with PBS. The enzyme substrate solution (4 mM PAPP in Tris-HCl, pH 9.0) was injected to produce the enzymatic product, r-aminophenol (PAP). The PAP was detected on the surface of the gold electrode through cyclic voltammetry (CV) from the oxidation peak current, which was proportional to its concentration during electro-oxidation into easily reducible r-quinoneimine (PQI).

Two concentrations (10 ng/ml and 10 μg/ml) of the NT-proBNP were applied, and Figure 8 showed the results. The CV current values were 21.07 μA and 37.65 μA at the concentrations, respectively, albeit no cyclic voltammetric responses were detected in PBS buffer as a control. The result indicates that the successful immobilization of antibodies onto the gold surface of the electrochemical immunosensor via the GBP-ProG protein, which attributed to simple and rapid fabrication of a highly sensitive biosensor.

Fig 8. Cyclic voltammograms at different concentrations of NTproBNP

In this study, a new strategy for effective immobilization of antibodies and sensitivity enhancement in the development of an electrochemical biosensor is presented. The proposed strategy entails directly coupling antibodies onto gold chip surfaces and employing genetically engineered GBP-ProG fusion proteins as a novel crosslinker. Experimental results demonstrated that the GBP-ProG proteins were successfully direct-immobilized onto immunochip via the GBP domain, and simple and oriented binding of antibodies could be achieved through the ProG portion of the layered GBP-ProG without any additional chemical treatment. These results provide a simple and effective approach for the fabrication of sensitive immunosensor. The electrochemical biosensor developed herein also could be used for an effective diagnosis and monitoring of not only heart failure but also other disease such as various cancers.