Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 2020 - Engineering | 100% |
The multi-billion dollars citrus industry in U.S. and Worldwide is at grave risk of irrepairable damage from citrus greening disease, also called Huanglongbing (HLB). This deadly disease is caused by the bacterial pathogen Candidatus Liberibacter for which currently there is no cure and management is heavily dependent on early detection. Current methods of HLB detection, which are based on disease symptoms and nucleic acid assay, are not only inaccurate but also unsuitable for field surveys due to variable latent time and sporadic distribution of the pathogen in infected trees. The objective of the proposed research is to develop a point-of-use immunosensor for highly sensitive and selective, facile, rapid, cost-effective and quantitative detection of a HLB biomarker in tree phloem extract. We will target proteins secreted by Ca. Liberibacter as candidate biomarkers as these proteins can systemically distribute in the infected trees, therefore providing direct, selective and reliable detections of HLB. To detect the HLB biomarker we will develop a novel electrical nanobiosensor that will detect the antigen based on change in electrical conductivity resulting from bridging of a nanogap by the trapping of nanoparticle. This nanosensor will address critical and urgent need of the multi-billion dollars citrus industry and food security issues by providing a highly sensitive and selective analytical tool for simple, rapid and low cost detection of citrus greening disease.
Generate high affinity antibodies against secreted proteins of Ca. Liberibacter and select one secreted protein as the biomarker for HLB. Fabricate a pair of graphene electrodes separated by nanometer gap distance. Perform site specific immobilization of one antibody against the selected HLB biomarker in the nanogap between the graphene electrodes. Synthesize magnetic core-gold shell nanoparticles and functionalizing them with second antibody against the selected HLB biomarker for isolation and concentration. Perform analytical characterization of nanogap electrode conductivity sensor for HLB biomarker. Test and validate the sensor using HLB-infected citrus samples.
Target Audience
During the present reporting period the research supported by this grantwas disseminated to graduate students, citrus growers and peer-researchers.
Changes / Problems
Nothing Reported
Training & Professional Development
This project has provided excellent training opportunities to two Ph.D students, one in Bioengineering and one (female) in Microbiology, in the two participating laboratories at UC Riverside. These students/trainees have been exposed to designing and conducting experiments using interdisciplinary approaches that involved cloning of antigen gene in bacteria for expression,antibody generation,purification and characterization of poolyclonal antibodies against specific targets, develop Enzyme-labeled immunosorbentassays using antibodies, fabircation of nanomaterials (such as carbon nanotubes) based nano-field-effect transistor immunosensorand applying it for immunosensor,synthesis of single layer graphene, making nanogap electrode and applying it for nanogap-basedbiosensor. Additionally theygained experience on grant writing, manuscript preparation, and research presentation.
Dissemination Streams
The results have also been disseminated through peer-reviewed publications and oral and poster presentations bythe PD/co-PD and the participating researchers in conferences, universities and research institutions in the US and abroad. The co-PD also talked to the public about the HLB in general as well as the current research efforts through interviews by local media and national broadcast venues. Overthe duration of the grant the researchers published 12 peer-reviewed journal articles, 1 patent and made16 oral and poster presentations at conferences, universities and research institutions.
Next Reporting Steps
Nothing Reported
Target Audience
Research supported by this project was disseminated tograduate students, citrus growers andpeer-researchers during the reporting period.
Changes / Problems
Nothing Reported
Training & Professional Development
Two Ph.D. students, one from Bioengineering and other (female) from Microbiology, received training in antibody generation, purification and characterization of poolyclonal antibodies against specific targets, develop Enzyme-labeled immunosorbent assays using antibodies, synthesis of single layer graphene, making nanogap electrode and applying it for nanogap-based biosensor.
Dissemination Streams
Research supported by this project was disseminated to citrus growers in the following two presentations. 1. Effectors as detection markers for HLB. Oral presentation by Wenbo Ma (invited talk). California Asian Citrus Psyllid and Huanglongbing Research and Extension Summit. Riverside, CA 2016. 2. Effectors as detection markers for HLB.Oral presentation by Kelley Clark (invited talk). California Citrus Nursery Society Annual Meeting. Auburn, CA. 2016
Next Reporting Steps
In the next reporting period, we will continue to provide purified antibodies and the biomarker proteins for the development of the nanosensor. We will provide HLB-infected and healthy citrus tissues for evaluation of the nano-device by comparing to the current standard HLB detection method based on quantitative PCR. To develop a dual antibody-based platform for the nanogap nanosensor, we are generating monoclonal antibodies against the biomarker protein. Two monoclonal antibody projects were initiated simultaneously to increase the possibilities of finding a mAb that can be used as a pair or pair with our current pAbs. The mAbs will be evaluated using ELISA and then used to develop nanogap nanosensors. Per the design of our biosensor platform, we will continue to characterize new monoclonal antibodies to obtain a pair of antibodies for performing a sandwich-assay for the CLas biomarker. We will continue optimizing the nanogap biosensor system for detection of CLas biomarkers. Meanwhile, per objective 4, we will synthesize magnetic core-gold shell nanoparticles functionalized with the second antibody against the selected HLB biomarker for isolation and concentration. Once we have achieved a sensitive and robust working nanogap sensor for detecting purified HLB biomarkers, we will proceed to testing the biosensor system with healthy plant samples artificially spiked with the HLB biomarkers. Concurrently, for the chemiresistive SWNT-based biosensor platform, we will continue to test and optimize our biosensors to detect CLas biomarkers in phloem extracts. We will explore various strategies for blocking our biosensor from nonspecific binding of biomolecules from complex phloem extracts. <br><br>
<br>What was accomplished under these goals? In order to obtain antibodies with minimal cross-reactivity to plant sample matrix, we proceed with our plan to generate the biomarker proteins using a different expression system. The biomarker was cloned into the SUMO-RSF-Duet vector, which allowed the purification of the fusion protein using the HIS tag and the subsequent cleavage of the tag to generate tag-free proteins. The tag-free proteins were then used in the affinity purification of the polyclonal antisera to obtain purified antibodies. The purified antibodies were validated for biomarker binding using western blotting, and the binding affinity was determined by ELISA. The antibodies were also verified for their ability to bind the biomarker in citrus samples, including healthy citrus extract spiked with purified biomarker proteins and HLB-infected tissues, using competitive ELISA. After these validation experiments, the antibodies have been used to develop nanosensors. Using electron beam lithography (EBL), we have achieved fabrication of our graphene nanogap electrode width ranging 65 nm and 100 nm. We have tested biosensing using a model biotin-avidin system. Results showed that increased concentration of gold-conjugated avidin bound to the biotin-functionalized nanogap led to increased conductance in the biosensor. We have also begun integrating antibodies against the CLas biomarker (anti-5315F pAbs) into the graphene nanogap biosensor. We specifically immobilized the first sandwich antibody to the nanogap region followed by synthesis of gold nanoparticles conjugated with the second sandwich antibody to perform preliminary testing. Initial testing showed detection of CLas biomarkers in phosphate buffer. Concurrently, we have also integrated these anti-5315F pAbs on our existing nano-FET/chemiresistor platform using single-walled carbon nanotubes. We have shown label-free nanomolar detection of the biomarker in simple phosphate buffer. Using our chemiresistor platform, we have also begun for detection of the CLas biomarker spiked citrus phoem extracts. The project has provided training to two graduate students in antibodies generation, purification and characterization and in the fabrication of nanosensor based on graphene nanopgap electrodes. <br><br><b>Publications</b><br>
Target Audience
Research supported by this project was disseminated to citrus growers and researchers during the reporting period. The PIs presented their findings in scientific and commodity conferences including International Research Conference on Huanglongbing (Orlando FL, 2015), Gordon Research Conference (GRC) on "Nanoscale Science and Engineering for Agriculture and Food Systems"(Waltham MA, 2015), Annual meeting of American Society of Phytopathology (Pasadena CA, 2015), and California Asian Citrus Psyllid and Huanglongbing Research and Extension Summit (Davis CA, 2015). During these meetings, the PIs and students working on this project explained the concept and advantages of the nanosensors, which are well accepted by the audience, especially the growers in major citrus growing states including FL, CA and TX. The co-PI also introduced this research to citrus growers and researchers in China through her invited seminar at the Guangdong Academy of Agricultural Science, where HLB was first identified. In addition, the progress of this project and the performance of the HLB-specific antibody was also reported to California citrus growers through visits of members in the Citrus Research Board (a grower organization) to the co-PI's laboratory.
Changes / Problems
Nothing Reported
Training & Professional Development
Two Ph.D. students, one from Bioengineering and other (female) from Microbiology, received training in antibody generation, purification and characterization of poolyclonal antibodies against specific targets, develop Enzyme-labeled immunosorbent assays using antibodies, synthesis of single layer graphene, making nanogap electrode and applying it for nanogap-based biosensor.
Dissemination Streams
Nothing Reported
Next Reporting Steps
Per the design of our biosensor platform, we will continue to characterize our antibodies and optimize the purification our antibodies to obtain a pair of antibodies for performing a sandwich-assay for the CLAS biomarkers. Furthermore, we will optimize purification to obtain antibodies with minimal cross-reactivity to sample matrix by pre-blocking antisera solutions with healthy citrus phloem extract, followed by affinity purification to harvest non-cross-reactive and biomarker-specific antibodies. Additionally, we will generate CLAS protein markers using a different expression system, via the SUMO RSFDuet vector, to eliminate the His-tag from our expressed CLAS protein markers. We will integrate anti-CLAS pAbs into the nanogap biosensor system. To achieve this goal, we will concurrently specifically immobilize the first antibody to the nanogap region followed by synthesis of gold nanoparticles conjugated with the second antibodies to perform preliminary testing. Meanwhile, per objective 4, we will synthesize magnetic core-gold shell nanoparticles functionalized with the second antibody against the selected HLB biomarker for isolation and concentration. Once we have achieved a working nanogap sensor for detecting purified HLB biomarkers, we will proceed to testing the biosensor system with healthy plant samples artificially spiked with the HLB biomarkers. <br><br>
<br>What was accomplished under these goals? During the second year of this project, research efforts were focused on objectives 1) Evaluation of antibodies that specifically bind to unique proteins produced by the HLB causative agent Candidatus Liberibacter spp. (or CLas) and optimization of antibody purification procedures to increase yield and minimize cross-reactive antibodies, 2) Optimization of graphene nanogap fabrication and testing on a model system using biotin and gold nanoparticle conjugated to avidin. In following up with the previous year's research work, from Specific Aim 1, we have continued to evaluate the titer of antisera generated from another biomarker CLAS-3230 using direct ELISA. ELISA results showed high titers for in antisera generated against CLAS-3230. Thus, we proceeded to affinity purify anti-CLAS-3230 polyclonal antibodies from these antisera for further antibody evaluation and integration into the biosensor development process. Direct ELISA results and Western blot assays showed we obtained purified anti-CLAS-3230 pAbs. After investigating the three methods of focused ion beam (FIB) milling, nanoindentation, and electron-beam lithography (EBL) for fabricating a nanogap in graphene film, we have determined that EBL was the optimal method due to reproducibility and ease-of-fabrication. Using EBL, we have further improved the nanogap width to between 65 nm and 100 nm. We have begun testing biosensing using a model biotin-avidin system. Results showed that increased concentration of gold-conjugated avidin bound to the biotin-functionalized nanogap led to increased conductance in the biosensor. Furthermore, while developing the nanogap sensor system, we have also applied the anti-CLAS-3230 pAbs on our existing nano-FET/chemiresistor platform using single-walled carbon nanotubes and reduced graphene oxide. The project has provided training to two graduate students in antibodies generation, purification and characterization and in the fabrication of nanosensor based on graphene nanopgap electrodes. <br><br><b>Publications</b><br>
Target Audience
Nothing Reported
Changes / Problems
Nothing Reported
Training & Professional Development
The project has provided training to two graduate students in antibodies generation, purification and characerization and in the fabrication of nanosensor based on graphene nanopgap electrodes.
Dissemination Streams
Nothing Reported
Next Reporting Steps
Making a nanogap electrode of a gap size less than 100 nm consistently/reproducibly is paramount for the success of the project. In the coming year we will complete the investigations on the three methods of making the cut in graphene film and decide on the best methid for use to make the nanogap electrode sensor. Subsequently we will work on objectives 3 and 4 of the project: Performing site specific immobilization of one antibody against the selected HLB biomarker in the nanogap between the graphene electrodes. Synthesizing magnetic core-gold shell nanoparticles and functionalizing them with second antibody against the selected HLB biomarker for isolation and concentration. Along with working on these objectives we will continue to work on the characterization of the antibodies for the selected antigen targets. <br><br>
<br>What was accomplished under these goals? The multi-billion dollars citrus industry in U.S. and Worldwide is at grave risk of irrepairable damage from citrus greening disease, also called Huanglongbing (HLB). This deadly disease is caused by the bacterial pathogen Candidatus Liberibacter for which currently there is no cure and management is heavily dependent on early detection. Current methods of HLB detection, which are based on disease symptoms and nucleic acid assay, are not only inaccurate but also unsuitable for field surveys due to variable latent time and sporadic distribution of the pathogen in infected trees. The objective of the proposed research is to develop a point-of-use immunosensor for highly sensitive and selective, facile, rapid, cost-effective and quantitative detection of a HLB biomarker in tree phloem extract. We will target proteins secreted by Ca. Liberibacter as candidate biomarkers as these proteins can systemically distribute in the infected trees, therefore providing direct, selective and reliable detections of HLB. During the first year of this project, research efforts were focused on objectives1) the generation, purification and evaluation of antibodies that specifically bind to unique proteins produced by the HLB causative agent Candidatus Liberibacter spp. (or CLas) and 2) graphene nanogap fabrication. 1. Generation, purification and evaluation of antibodies that specifically bind to unique proteins produced by the HLB causative agent Candidatus Liberibacter spp. (or CLas) We are using CLas secreted proteins as detection markers. The basic idea is that although CLas has sporadic distribution in the phloem of infected trees, proteins secreted from CLas cells into the phloem of citrus can be systematically distributed in the infected trees through the vascular flow. As such, serological detection methods based on CLas-specific secreted proteins will better cope with the large variability in distribution of CLas cells within infected trees and the various degrees of disease progression, and thereby increasing the chances of HLB detection in a direct, sensitive, and highly specific manner. Using a computational prediction pipeline with multiple filtering criteria, we identified 27 CLas proteins that are likely secreted into the phloem by the general protein secretion system called the “Secâ€