While imaging tests and tissue biopsies are the most common methods for diagnosing cancer, serum tests can also help doctors identify the cancer by the usage of certain indicators, the antigens.
Via measuring the levels of antigens, our APOllO E.Cotector Plus can help doctors to suggest the diagnosis as the reference for prescription of target therapy[1].
In our APOllO E.Cotector Plus design, we engineered the E.coli to display dual display system. One of the system displayed scFv by the transmembrane protein Lpp-OmpA. Meanwhile, the other system expressed transmembrane protein FadL fused with gold binding polypeptide (GBP) for the purpose of specifically binding on the gold chip, the material commonly used in biosensors[2].
By processing our E.Cotector Plus, with gold binding polypeptide connecting to the gold chip, we simplified the procedures of the self-assemble modification (SAM), the technique to immobilize antibodies on the gold surface[3]. We skipped the procedure of the gold surface decoration with sulfur bond and omitted the carbon chains attaching to antibody, which may occasionally block the binding sites of scFv,
further increasing the efficiency of antibody-antigen interactions.
Combined with the idea of the biosensor, we deemed our E.cotectorplus played the role of biological recognition part and the gold chip acted as the transducer part.
Therefore, our E.cotectorplus, which is able to attach on gold chip, can be regarded as the platform for precise physicochemical nanoscale detectors, such as Quartz crystal microbalance, Surface plasmon resonance Spectroscopy, Dual-polarization interferometry, ellipsometry, etcetera.
With our powerful APOllO E.Cotector Plus , we solved the time-consuming and sophisticated process of gold surface modification. What's more, by coupling our E.Cotector Plus to precise measurement instruments, we can achieve a huge boost to provide more sensitive and specific scFv detecting techniques options,
and give more reliable diagnosis for doctors to apply monoclonal antibody target drugs.
Figure 3. APOllO E.Cotector Plus contains dual display system.
One system displayed the transmembrane protein Lpp-OmpA fused with the sequence of scFv, which can bind with targeted antigens.
The other system expressed transmembrane protein FadL fused with gold binding polypeptide (GBP) for the purpose of specifically binding on the gold chip.
Figure 4. (a) When targeted antigens in patients' serum attach to scFv on APOllO E.Cotector Plus, the signal change of mass on the gold surface will be caught by the machines, such as Quartz crystal microbalance (QCM), Surface plasmon resonance Spectroscopy (SPR).Therefore, we can quantize and evaluate the level of binding antigens, and give the more accurate diagnosis for doctors.
(b)A biosensor is an analytical device, which can be divided into three elements including the sensitive biological element, the transducer and the detector with data evaluation device.
The introduction of gold binding polypeptide
The gold binding polypeptide, abbreviated as GBP, is the three-repeated of following 14 aminoacid sequences: [MHGKTQATSGTIQS], which was developed in an E. coli cell-surface display system[4].
According to the paper, the binding sequence of GBP does not contain cysteine which can form a covalent thiol linkage with gold, the linkage to the gold surface in Self-Assembled Monolayers (SAMs)[5].
The mechanism of the connection between GBP and gold metal plane remains unknown. By using Molecular Dynamics (MD), it indicates that GBP, with an antiparallel β-sheet structure, can recognize gold surface via OH-binding.
It is likely that the hydroxyl, together with amine, ligands on GBP recognize the atomic lattice of gold, aligning the molecule along the variants of a six-fold axis on the Au (111) surface[6].
The utilize of gold chip in biosensor
The gold is the best choice for our biosensor substrate because of its advantages of stability to external environment, the excellent capability of transducing electronic signals, the sensitive physicochemical properties
and, most important of all, the specific interaction with gold binding polypeptide.
Summary
This year, our hit project, E.Cotector is to assist the medical practitioners to choose the appropriate targeted drug therapies for various conditions of patients. Before doctors prescribing the targeted drugs for cancer patients, E.Cotector can mark the tumor cells or test the antigens in the serum by part of monoclonal antibodies (scFv) which is a kind of targeted drug directly binding with antigens. APOllO organization provided an advanced method in selecting personalized therapy for every particular patient.
E.Cotectors marked the tumor cells by displaying scFv on its outer membranes and fluorescence proteins:
- Simultaneously marked multiple kinds of overexpressed unique antigens on the cells.
- Amplified the signal by E. coli expressing fluorescence proteins.
E.Cotectors Plus detected the antigens in the serum by dual-displaying scFv and gold binding peptides on their outer membranes:
- An innovative indicator to combine synthetic biology and numerous precision measurement technology.
- Achieve the extraordinary degree of precision in detecting concentration of antigens in the serum.
- Enhance the process yield in immobilization of antibodies on the medium gold surface.
Want to see more, please see Achievements page.
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NCTU_Formosa APOllO
Engineering Building 6 EF455, 1001 University Road, Hsinchu 300, Taiwan, ROC.
