Research

The Laboratory for Biosensing conducts inter-disciplinary research. We are to exploit biomaterials with specific structures and functionalities by microbial surface display and protein engineering strategies. Further, we develop novel nano-bio interfaces, aiming at exploring biofuel cells, analytical methods and integrated intelligent sensing devices for bioenergy process, environment, industrial control, medical care, clinical diagnostics and food safety. Specifically, our research covers the following fields:

1. Protein engineering and microbial surface display

Functional proteins including enzymes play key roles in synthetic biology, efficient enzyme preparations, biofuel cells and biosensing. We express stable enzymes with high activity in vitro by conventional protein engineering, and explore whole-cell biocatalysts based on bacterial surface display. (Bioresource Technology 2013, 147, 492–498; Analytical Chemistry 2012, 84, 275–282)

Phage display library or yeast library based screening systems are also under development in our laboratory, aiming at selecting facile molecular probes and/or functional ligands with high affinity and specificity, exploring antibody substitutes and imitation of antibody functions, studying protein/peptide-target interactions. (Journal of Molecular Biology 2012, 417, 129–143; Analytical Chemistry 2014, 86, 2767–2774; Scientific Reports 2014, 4, 6808; Antiviral Research 2014, 109, 68–71.)


Figure 1
. Scheme for design and construction of biomacromolecule. A), protein engineering. B), phage display library-based affinity peptide screening.

2. Electrochemical biosensors

Electrochemical biosensors are developed by creating novel nano-bio interface.

For example, we constructed a novel Nafion/bacteria-displaying xylose dehydrogenase (XDH-bacteria)/multi-walled carbon nanotubes (MWNTs) composite film-modified electrode (Nafion/XDH-bacteria/MWNTs/GCE) (Figure 2A). D-xylose could be catalytically oxidized to D-xylonolactone in the presence of XDH-bacteria with NAD+ as its cofactor, which is reduced to NADH, the latter is further oxidized on the electrode surface (Figure 2B). The NADH oxidation emerges at a high positive potential of 0.70 V (vs. Ag/AgCl) at the Nafion/XDH-bacteria/GCE, however, which is lowered to 0.50 V at Nafion/XDH-bacteria/MWNTs/GCE. And the current response of Nafion/XDH-bacteria/MWNTs/GCE is much higher than that for Nafion/XDH-bacteria/GCE, suggesting the electrocatalytic role of MWNTs. The current-time curve was obtained with Nafion/XDH-bacteria/MWNTs/GCE by using amperometry at an applied potential of +0.5 V (Figure 2C). The oxidation current increased after addition of D-xylose and reached at 95% steady-state value within 5 s. The current linearly increased with the increasing concentration of D-xylose over the concentration of 0.6-100 μM. Compared with the current for 10 μM D-xylose, there is no interference in the presence of 300-fold excess of D-maltose, D-galactose, D-mannose, D-glucose, D-sucrose, D-fructose, D-xylitol and D-cellbiose, as well as 60-fold excess of L-arabinose (Figure 2D). The limit of detection of this biosensor by using the Nafion/XDH-bacteria/MWNTs composite-based biosensor is much lower than other methods reported so far. (Biosensors & Bioelectronics 2012, 33, 100–105)


Figure 2
. Xylose electrochemical biosensor based on xylose-dehydrogenase-displayed E. coli and MWNTs modified electrode. (A), Construction of modified electrode. (B), Proposed electrocatalytic oxidation of xylose by XDH-bacteria. (C), Current-time curve obtained for the Nafion/XDH-bacteria/MWNTs/GCE on the successive addition of xylose in 0.1 M PBS (pH 7.4) containing 2 mM NAD+.(D),Current-time curve obtained for the Nafion/XDH-bacteria/MWNTs/GCE on the successive addition of D-xylose (a-c) and a series of saccharides (d-l).

We also developed a novel glucose biosensor based on glucose oxidase (GOx) yeast surface displayed system. GOx was displayed on yeast cell surface using a-agglutinin as an anchor motif. Both the immunochemical analysis and enzymatic assay showed that active GOx was efficiently expressed and translocated on the cell surface (Figure3A, B). Compared with secreted expressed GOx, the cell surface displayed GOx (GOx-yeast) demonstrated excellent enzyme properties, such as good stability within a wide pH range (pH 3.5-11.5), good thermostability (retaining 84.2% enzyme activity at 56 °C) and high D-glucose specificity. In addition, direct electrochemistry was achieved at the GOx-yeast/MWNTs modified electrode, suggesting that the host cell of yeast did not have any adverse effect on the electrocatalytic property of the recombinant GOx. Thus, a novel electrochemical glucose biosensor based on this GOx-yeast and MWBTs was developed. The biosensor is stable, specific, reproducible, simple, and cost-effective, which can be applicable for real sample detection (Figure. 3C). (Analytical Chemistry 2013, 85, 6107–6112)


Figure 3
. (A), Photograph of (a) yeast cell control and (b) GOx surface-displayed yeast. (B), GOx activity of the surface-displayed yeast cells. (C), CVs of Nafion/GOx-yeast/MWNTs/GCE in PBS buffer (pH 7.4) containing different concentrations of glucose: 0.0 mM (a), 0.1 mM (b), 0.5 mM (c), 2.0 mM (d), 8.0 mM (e), and 12.0 mM (f). Inset shows a typical calibration graph of the glucose biosensor.

3. Biofuel cells

By integrating bioelectrochemistry, nanoscience and surface modification, novel modified electrodes are developed to immobilize electricigens for fast direct electron transfer. Another aim is to solve the key problems in the development of microbial fuel cells, for instance, we explore novel microbial electrolysis cells and get electric power benefiting from treatment of industrial and agricultural wastes. Meanwhile, strategies are established to improve the microbial conversion efficiency and output power density to develop portable, efficient and long-life microbial fuel cells.

For example, we developed one-compartment biofuel cell containing XDH-bacteria/poly (brilliant cresyl blue) (PBCB) /MWNTs modified glassy carbon electrode (GCE) (XDH-bacteria/PBCB/MWNTs/GCE) as the bioanode, and bilirubin-oxidase /MWNTs /GCE as the biocathode. The as-assembled BFC showed a maximum power density of 63 μWcm-2 at 0.44 V with an open circuit potential of 0.58V (Figure 5A, curve e), which is much better than other bioanodes including free-XDH/PBCB/MWNTs/GCE (Figure 5A, curve d). (Biosensors & Bioelectronics 2013, 44, 160–163)


Figure. 4
 (A) Dependence of the power density on the cell voltage varying bioanodes: XDH/MWNTs/GCE in 10 mM NAD+ (a); XDH/MWNTs/GCE in the presence of 10 mM NAD+ +30 mM xylose (b); XDH/PBCB/MWNTs/GCE in 10 mM NAD+ (c); XDH/PBCB/MWNTs/GCE in presence of 10 mM NAD+ and 30 mM xylose (d); and XDH-bacteria/PBCB/MWNTs/GCE in 10 mM NAD+ + 30 mM xylose (e). (B) Dependence of the power density on xylose concentration in the cell containing 10 mM NAD+ where the bioanode was XDH-bacteria/PBCB/MWNTs/GCE. Supporting electrolyte, 0.1 M PBS quiescent solution under O2-saturated condition.

A highly uniform three-dimensional (3D) macroporous gold (MP-Au) film was prepared by heating the gold “supraspheres”, which were synthesized by a bottom-up protein-templating approach. Laccase was covalently immobilized to the MP-Au/FTO electrode, which exhibited the optimal orientation for direct electron transfer. The as-prepared laccase/MP-Au biocathode exihibited an onset potential of 0.62 V versus saturated calomel electrode toward O2 reduction. On the other hand, mutated glucose dehydrogenase (GDH) surface displayed bacteria (GDH-bacteria) were used to improve the stability of the glucose oxidation at the bioanode. Then the membraneless glucose/O2 fuel cell was assembled, which showed a high power output of 55.8 μW•cm-2 and open circuit potential of 0.80 V (Figure 5). Moreover, the biofuel cell retained 84% of its maximal power density even after continuous operation for 55 h, indicating a favorably stable power output process. (Analytical Chemistry 2014, 86, 6057–6063)


Figure 5
. (A), Schematic representation of the working principle of the glucose/O2 BFC composing of a laccase-based 3D macroporous gold film biocathode and a bacterial surface displaying GDH mutant-based bioanode. (B),The polarization curves and power outputs of the glucose BFCs: GDH-bacteria/PMB/MWNTs/GCE bioanode vs laccase/MP-Au/FTO (a) or laccase/planar Au biocathode (b) in 50 mM acetate buffer (pH 5.5) containing 10 mM NAD+ and 10 mM glucose under an O2-saturated condition.

4. Controllable self-assembly of nano-bio structures

Multi-functional bio-nano structure is constructed by self-assembly in order to understand biological function, biomimicking as well as applications in biosensing, biofuel cell and nanomedicine. (Advanced Materials 2009, 21, 100–105; Analytical Chemistry 2014, 86, 2767–2774; Scientific Reports 2014, 4, 6808.)


Figure 6
. TEM images of negatively stained individual tetraArg–M13 phages A) and gold-nanoparticle self-assembled individual tetraArg–M13 phages B). (A. Liu, et al., Advanced Materials 2009, 21, 1001–1005.)

5. Protein microarray and glycan microarray

In post-genomics era, both proteomics and functional glycomics have received increasing attention in biology and bio-medicine. By developing novel probes which have specific binding sites for proteins or carbohydrates, novel protein microarrays and oligosaccharide microarrays can be constructed and assay kits are developed. Especially, we develop microarrays to monitor the bioenergy process, for example, to mointor the bio-conversion of lignocellulose to fuel ethanol in real time.

The cellulytic enzyme endoglucanase I (EG I) was used as a model for selection of its specific peptide ligands from the f8/8 landscape library. Three phage monoclones were selected and identified by the specificity array, of which one phage monoclone displaying the fusion peptide EGSDPRMV (phage EGSDPRMV) could bind EG I specifically with highest affinity. Subsequently, the phage EGSDPRMV was used directly to construct peptide microarray. For comparison, major coat protein pVIII fused EG I specific peptide EGSDPRMV (pVIII-fused EGSDPRMV) which was isolated from phage EGSDPRMV was also immobilized by traditional method to fabricate peptide microarray. The fluorescent signal of the phage EGSDPRMV-mediated peptide microarray was more reproducible and about four times higher than the value for pVIII-fused EGSDPRMV-based microarray (Figure 7), suggesting the high efficiency of the proposed phage EGSDPRMV-mediated peptide immobilization method. Further, the phage EGSDPRMV based microarray not only simplified the procedure of microarray construction but also exhibited significantly enhanced sensitivity due to the symmetrical carrier landscape phage, which dramatically increased the density and sterical regularity of immobilized peptides in each spot. Thus, given the high flexibility and wide application of the phage display platform, this peptide immobilization strategy can be of unsurpassed utility in the field of peptide microarrays. (Analytical Chemistry 2014, 86, 5844–5850)


Figure 7
. (A), Pattern of fabricated microarray, in which 1 indicated the pVIII-fused EGSDPRMV and 2 indicated phage EGSDPRMV. (B), Image of peptide microarray incubated with 100 nM EG I-Cy3. (C), Quantitative results of image B.