(Invited) Development of Fibronectin-Modified Gold Egfet Sensor for the Detection of S. Epidermidis Biofilm and Staphylococcal 16S rRNA

Prolonged catheter implantation and placement expose patients to the risk of microbial infection and biofilm formation could lead to extensive hospitalization and mortality increase. Among frequent isolated pathogens (gram-positive bacteria (45-65%), gram-negative bacteria (25-40%) and fungi (3-6%), Staphylococcus epidermidis is the most common microbial agents, while Staphylococcus aureus is associated with more severe cases, a higher risk of hospitalization, catheter removal, and finally, death [1].  The pathogenesis of bacteria is mainly driven by their ability to colonize and form a thick multilayer biofilm on the polymer surface (plastics) leading to mild to sepsis that typically binds human matrix proteins such as fibrinogen or fibronectin [2]. Biofilm mostly consists of partially de-acetylated polymer of beta-1-6-linked N-acetylglucosamine polysaccharide intercellular adhesion (PIA) that acts  as glue to stick and wrap negatively charged bacterial surface. Substantial quantity of extracellular DNA from the lysed cells is also reported to play a major role in biofilm formation [3]. 

Within various developed biosensor, field effect transistor (FET) sensor may offer several benefits, in particular EGFET structure with its easy fabrication, small influence of optical illumination and operation temperature, and disposable gate. The application of a commercial metal oxide semiconductor field effect transistor (MOSFET) also reduces the complexity and cost in its fabrication processes [4].  In this opportunity, we would like to demonstrate a novel protocol for bacterial biofilm detection determined by fibronectin immobilization onto Au-EGFET sensing membrane, altogether with the measurement of cellular responses toward antibiotic treatment. Besides, the detection of electronic signals from 16S rRNA of S. epidermidis and S. aureus on Au-were conducted using new distinctive designed probe. Charge characteristics were shown by a responsive shift of Au-EGFET gate voltage.  Not only effective for biofilm detection, we have also discovered that the whole gate surface modification on Au-EGFET is a powerful tool to detect specific bacteria species involved in the infection. In biofilm test, layer upon layer had been built to provide a robust adhesion of biofilm molecules to Au surface. The immersion of Au-electrode in SAMs solution and the graft of fibronectin afterwards successfully increased the surface roughness of bare Au. Fibronectin mostly hosts the adhesion of bacterial cells in human body.

The structure of Au-SAMs-fibronectin gradually improved the surface topology of Au as expressed in the root mean square roughness (Rq) value at 3.22 nm, 3.82 nm and 6.32 nm for bare Au, with SAMs and with fibronectin, respectively. Subsequently, Staphylococcus epidermidis biofilm was attached onto Self Assembled Monolayers (SAMs) coated Au substrate of an extended-gate field-effect-transistor (EGFET) sensor with immobilized extracellular matrix protein, fibronectin. Biofilm mostly composed of positively charged Polysaccharide Intercellular Adhesin (PIA) significantly contributed threshold voltage (V_TH) shift from -6.67 to -94 mV for biofilm yielded by 3.8x10⁶ to 3.8x10⁸ CFU/ml S. epidermidis and obtained Limit of Detection (LOD) of 9×10⁵  CFU/ml S. epidermidis. SAMs-Au surface functionalization and its properties after fibronectin immobilization showed an optimal surface roughness from atomic force microscope (AFM) view [5]. The presence of biofilm on the surface was validated by (Scanning Electron Microscope) SEM screening after 1 hour incubation at 37^oC. Cellular response to antibiotic treatment against biofilm achieved as contaminated dialysate was treated with and without Vancomycin.  The hybridization of a highly conserved gene, 16S rRNA of S. epidermidis and S. aureus, the most leading pathogenic bacteria in peritoneal dialysis patients, was also successfully recorded.

This finding does not only provide insights into aetiologies of infectious disease but also may guide the clinicians in prescribing antibiotics and determining the treatment duration and infection control procedures when pathogenic species is well identified. In the future work, the functionalities of other SAMs and nanoparticles can be applied to improve the Debye-length issue and uniformity control of biomolecules density onto the surface. Overall, Au-EGFET sensor with its advantages including its simple passivation, packaging and fabrication, and flexibility of extended area gate, opens a new niche in bacterial biochip technology providing a comprehensive biofilm and pathogenic bacteria species identification.

References

[1] M. Otto, Staphylococcal biofilms., Curr. Top. Microbiol. Immunol. 322 (2008) 207–228.

[2] J.M. Patti, B.L. Allen, M.J. McGavin, M. Höök, MSCRAMM-mediated adherence of microorganisms to host tissues., Annu. Rev. Microbiol. 48 (1994) 585–617.

[3] C.B. Whitchurch, T. Tolker-Nielsen, P.C. Ragas, J.S. Mattick, Extracellular DNA required for bacterial biofilm formation., Science. 295 (2002) 1487. doi:10.1126/science.295.5559.1487.

[4] T. Goda, Y. Miyahara, Langmuir ACS J. Surfaces Colloids. (2012).doi:10.1021/la302977s.

[5] A. Purwidyantri, H.-C. Lai, S.-H. Tsai, J.-D. Luo, C.-C. Chiou, Y.-C. Tian, et al., Sensors Actuators B Chem. In Press (2014).