Gas Biosensor Arrays Based on Single-Stranded DNA-Functionalized Single-Walled Carbon Nanotubes for the Detection of Volatile Organic Compound Biomarkers Released by Huanglongbing Disease-Infected Citrus Trees.

Volatile organic compounds (VOCs) released by plants are closely associated with plant metabolism and can serve as biomarkers for disease diagnosis. Huanglongbing (HLB), also known as citrus greening or yellow shoot disease, is a lethal threat to the multi-billion-dollar citrus industry. Early detection of HLB is vital for removal of susceptible citrus trees and containment of the disease. Gas sensors are applied to monitor the air quality or toxic gases owing to their low-cost fabrication, smooth operation, and possible miniaturization. Here, we report on the development, characterization, and application of electrical biosensor arrays based on single-walled carbon nanotubes (SWNTs) decorated with single-stranded DNA (ssDNA) for the detection of four VOCs-ethylhexanol, linalool, tetradecene, and phenylacetaldehyde-that serve as secondary biomarkers for detection of infected citrus trees during the asymptomatic stage. SWNTs were noncovalently functionalized with ssDNA using π-π interaction between the nucleotide and sidewall of SWNTs. The resulting ssDNA-SWNT hybrid structure and device properties were investigated using Raman spectroscopy, ultraviolet (UV) spectroscopy, and electrical measurements. To monitor changes in the four VOCs, gas biosensor arrays consisting of bare SWNTs before and after being decorated with different ssDNA were employed to determine the different concentrations of the four VOCs. The data was processed using principal component analysis (PCA) and neural net fitting (NNF)

Versatile microbial surface-display for environmental remediation and biofuels production

Surface display is a powerful technique that utilizes natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production

A Pathogen Secreted Protein as a Detection Marker for Citrus Huanglongbing.

The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Candidatus Liberibacter asiaticus (CLas) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), CLas colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of CLas cells in infected trees and the long latency for disease symptom development makes sampling of trees for CLas detection challenging. Here, we report that a CLas secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from CLas cells can presumably move along the phloem, beyond the site of ACP inoculation and CLas colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect CLas infection. This work demonstrates that antibody-based diagnosis using a CLas secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs

Improvement in organophosphorus hydrolase activity of cell surface-engineered yeast strain using Flo1p anchor system

Organophosphorus hydrolase (OPH) hydrolyzes organophosphorus esters. We constructed the yeast-displayed OPH using Flo1p anchor system. In this system, the N-terminal region of the protein was fused to Flo1p and the fusion protein was displayed on the cell surface. Hydrolytic reactions with paraoxon were carried out during 24 h of incubation of OPH-displaying cells at 30°C. p-Nitrophenol produced in the reaction mixture was detected by HPLC. The strain with highest activity showed 8-fold greater OPH activity compared with cells engineered using glycosylphosphatidylinositol anchor system, and showed 20-fold greater activity than Escherichia coli using the ice nucleation protein anchor system. These results indicate that Flo1p anchor system is suitable for display of OPH in the cell surface-expression systems