Source: ARIZONA STATE UNIVERSITY submitted to NRP
NANOTECHNOLOGY FOR AGRICULTURAL AND FOOD SYSTEMS PARTNERSHIP: PORTABLE AND MULTIPLEXED DETECTION OF AFRICAN SWINE FEVER WITH METAL NANOPARTICLES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1027944
Grant No.
2022-67021-37013
Cumulative Award Amt.
$743,994.00
Proposal No.
2021-08642
Multistate No.
(N/A)
Project Start Date
May 15, 2022
Project End Date
May 14, 2026
Grant Year
2022
Program Code
[A1511]- Agriculture Systems and Technology: Nanotechnology for Agricultural and Food Systems
Recipient Organization
ARIZONA STATE UNIVERSITY
660 S MILL AVE STE 312
TEMPE,AZ 85281-3670
Performing Department
IAFSE-ECEE: Electrical Enginee
Non Technical Summary
African swine fever (ASF) is a highly contagious and often lethal infectious disease of swine. Currently, strict control strategies remain the only effective way of ASF containment, typically replying on early disease detection through rapid field suspicion and laboratory diagnosis followed by slaughtering all affected or exposed swine herds.Therefore, ASF has serious socioeconomic impact, especially in countries that export live pigs, pork and/or products, as well as in countries where these products are important sources of diet. To date, ASF has already spread from sub-Saharan Africa to multiple countries in Africa, Europe, Asia, and Americas. For example, since the introduction of ASFV in 2018 in China, an estimated ~50% of all pigs were wiped out. Since July 2021, ASF has been identifiedin the Dominican Republic and very recently in Haiti, rapidly spreading into the Caribbean's. Because most American countries are not well prepared, including Mexico and several others countries that have strong commercial trade or immigration relationships with the U.S., the U.S. is currently at high risk for ASF introduction. As the world's third largest pig producer, with over 11.5 million tons of pork produced per year, and the world's second largest pork exporter, the U.S. may face far-reaching economic consequences if ASF is introduced.Typical ASF diagnosis requires identification of animals that are or have previously been infected with the ASF virus (ASFV). Given the absence of treatment and vaccines, rapid and accurate ASF diagnostics is crucial to border inspection, surveillance, and outbreak containment. An appropriate diagnosis involves the detection and identification of ASFV-specific nucleic acids, antigens, and/or antibodies. Conventional tests, such as real time polymer chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA), although highly sensitive, are limited to lab use due to required instrument, personnel training, and long turnaround readout time. In comparison, the use of rapid and portable diagnostic tests in the field and next to a pen, where the infected animal populations are, would facilitate earlier and more effective disease control. In addition, reduced cost and simplified operation will facilitate high-frequency deployment of such tests for large-volume and in-time surveillance, which is important to disrupting transmission chain and ASF containment. Currently, there is still a lack of low-cost yet accurate antibody or antigen tests that can be deployed to pen sides for surveillance purposes.To bridge these technological gaps, the Wang lab at Arizona State University (ASU) and the Centro de Investigación en Sanidad Animal (INIA-CISA/CSIC) in Spain, which is also the European Union reference laboratory (EURL) and the Food and Agriculture Organization (FAO) reference Centre (WRC) for ASF, will collaboratively carry out the proposed research. Our long-term goal is to innovate portable electronic sensors that can be deployed worldwide to ASF-prevalent areas for rapid and sensitive detection of ASF-specific antibodies and antigens and to assist ASF containment in future outbreaks. In this project, our objective is to design and validate a new metal nanoparticle (MNP)-based assay platform, namely nanoparticle-supported rapid, electronic detection (NaSRED), and quantify its performance in ASF diagnostics. The NaSRED assay utilizes sets of MNPs of different optical characteristics to interrogate the ASF biomarkers, and generates optically distinguishable signals that serve to detect such markers in a multiplexed fashion. Here, we will simultaneously detect ASF antigens (e.g. p72) and antibodies (e.g. anti-p54, anti-p30, etc.) and evaluate its feasibility to improve the diagnostic accuracy. We will combine structural analysis and optical detection with intuitive physical and mathematical models to comprehensively understand the mechanisms of MNP-based multivalent analyte-binding and subsequent clustering and precipitation at nanometer scale. Further, we will devise a portable (comparable to a U.S. quarter) and inexpensive system (a few dollars in material cost) that can convert the optical signals for electronic readout using simple electronic circuits. Additionally, we will prove the feasibility of rapid ASF detection (expected a few minutes) and assess the assay performance for pen-side field deployment.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113510202040%
3113520202020%
3113599202020%
3113510110120%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal; 3520 - Meat, swine; 3599 - Swine, general/other;

Field Of Science
2020 - Engineering; 1101 - Virology;
Goals / Objectives
African swine fever (ASF) is a highly contagious and often lethal infectious disease of swine. Given the absence of treatment and vaccines, rapid and accurate ASF diagnostics is crucial to border inspection, surveillance, and outbreak containment. Currently, there is still a lack of low-cost yet accurate antibody or antigen tests that can be deployed at the time, simultaneously in the same device to pen sides for surveillance purposes. The Wang lab at ASU and the INIA-CISA/CSIC in Spain propose a multidisciplinary research plan to address the fundamental challenges in low-cost, portable, fast, simple, and quantitative ASF assay format. In this project, our major goal is to design and validate a new metal nanoparticle (MNP)-based assay platform, namely nanoparticle-supported rapid, electronic detection (NaSRED), and quantify its performance in ASF diagnostics. As shown in the management plan, this project has four important tasks.We have four tasks/objectives in this project.In Task 1, we will innovate on a spherical metal nanoparticle (MNP) based NaSRED assay for the detection of ASF antigen based on the p72 ASFV protein and Anti-p72 as antibodies. Expected outcome: we expect a dynamic range of > 3 logs, e.g. from >100 nM to <100 pM in antigen detection. We will also evaluate the NaSRED specificity by using two negative controls (NC), one as PBS buffer and another as a different ASF protein, such as p30. Later we will test at ASU and INIA-CISA/CSIC semipurified soluble ASFV antigen (SAg) /and or P72 antigen and other closely clinically related Classical swine fever virus (CSFV). We should expect minimal signal response for the negative control samples and antigens coming from CSFV. Further, we will combine experimental analysis with physical interpretations and a theoretical model to comprehensively study the mechanisms of MNP-based multivalent analyte-binding.In Task 2, we will design two sets of heterogeneous MNPs, e.g. gold NPs and nanorods (NRs), to display distinct plasmonic wavelengths. Such MNPs will be conjugated with specific ligands towards ASF (SAg) or protein (p72) and antibody (anti-p54, anti-p30) markers, respectively. The simultaneous spectrometric readout of two or more markers at separated wavelengths allows multiplexed profiling of ASF protein markers. The assay performance will be assessed in LoD, cross-reactivity, assay time, etc. Expected outcome: we will mix very low-concentration (e.g. 1 to 100 pM) anti-p30 and high-concentration p72 (e.g. 10 to 100 nM) as the ASF sample to mimic early stage ASF detection, during which antibodies are not released yet by the immune systems. We expect only the SAg/p72 proteins are detectable, triggering AuNR precipitation. Then we will spike both the antigens and antibodies at high concentration (e.g. 10 to 100 nM). This mimics the stage from about two weeks ASF infection and up to at least 1.5 month, during which both antigens and antibodies are expected present in swine serum. We expect the mixing to cause precipitation of both MNPs. Here the exact color would depend on the ratio between the remaining NPs and NRs. Lastly, for very late-stage ASF infection or survivors, only antibodies should be detectable. We will mix low-concentration (e.g. 1 to 100 pM) p72 and high-concentration anti-p30 (e.g. 10 to 100 nM) as the ASF sample. This will cause AuNPs to precipitate. In this task, this multiplexed diagnosis will prove the feasibility of ASF detection across different infection stages at ASU.Towards faster detection for mass screening, particularly considering the need of future pen-side testing, in Task 3 we will develop a rapid detection system to complete the test within a few minutes, assisted by a simple centrifugation followed by mixing. The target ASF protein markers will be quantified in centrifuge tubes with portable electronic readout system, including an integrated circuit board comprising two LEDs for the two selected MNPs, photodetectors, battery connectors, and signal transmitters. Expected outcome: We expect to demonstrate rapid and accurate detection of ASF p72 proteins and anti-p54 (or anti-p30) antibodies. Here we will use p72 as antigen and anti-p72-bound AuNPs as sensor to evaluate different spinning speed from 300 rpm to 2000 rpm, and optimize the centrifugation and incubation processes. The assay limit of detection, dynamic range and linearity will be evaluated using the electronic readout, and compared against the spectroscopic readout. We estimate the total detection time can be reduced to within 5 minutes even including all the sample handling, such as pipetting, centrifugation, vortex, and readout (if by bare eye), or within 30 min if using a 20-min incubation. In addition, we will demonstrate the feasibility of portable readout by integrating low-cost electronic elements on PCB boards with 3D printed low-cost tube holders. This will eliminate the need to extract the assay supernatant to PDMS well plate and also simplify the readout.In Task 4, the NaSRED assay will be evaluated at INIA-CISA/CSIC for antigen detection, antibody detection, and multiplexed detection. Using ASF reference samples, we will evaluate the analytical limit of detection (LoD) and specificity against closely clinically related swine viruses, and estimate diagnostic sensitivity and specificity. During NaSRED validation, we will determine the diagnostic sensitivity and diagnostic specificity. We will further investigate the cross-reactivity with other swine virus diseases, and determine the analytical speci?city of NaSRED by testing experimental samples from closely clinically related Classical swine fever virus (CSFV) as well as and clinical material (EDTA-blood and serum) obtained from a non-infected donor pig. The repeatability will be measured by running 10 replicates of 10 positive pig samples previously experimentally infected with ASFV and 8 replicates of 8 negative samples from non-infected pigs. Lastly, to evaluate the feasibility of deploying the NaSRED assay to the pen-side during future outbreaks, both teams at ASU and INIA-CISA/CSIC will load the NaSRED device, together with needed accessories (pipettes, centrifuge tubes, low-cost centrifuges and vortex mixers, biosamples, batteries, and laptop) on a car, and perform ASF NaSRED assay in the field. We will record the data and compare to the test results obtained in a lab setting. The outside humidity, temperature and the duration of the tests will be recorded as well.
Project Methods
The NaSRED assay proposed here makes uses of nanoscale particles to interrogate ASF-specific antigens (p72 protein) and antibodies (anti-p54 and anti-p30), aiming to achieve improved diagnostic power for early and widely assessable ASF detection.First, we will innovate on a spherical metal nanoparticle (MNP) based NaSRED assay for ASF antigen detection, and assess the assay performance at ASU with colorimetric and spectroscopic readout, using p72 protein purified from the cytoplasm of monkey stable cells (MS) and swine serum samples provided by INIA-CISA/CSIC. We will also evaluate the NaSRED specificity by testing semipurified soluble ASFV antigen and closely clinically related Classical swine fever virus (CSFV). Further, we will combine experimental analysis with physical interpretations and a theoretical model to comprehensively study the mechanisms of MNP-based multivalent analyte-binding.Second, we will design two sets of heterogeneous MNPs, e.g. gold NPs and nanorods (NRs), to display distinct plasmonic wavelengths. Such MNPs will be conjugated with specific ligands towards ASF protein (p72) and antibody (anti-p54, anti-p30) markers, respectively. The simultaneous spectrometric readout of two or more markers at separated wavelengths allows multiplexed profiling of ASF protein markers. We will use this multiplexed diagnosis method to evaluate the feasibility of ASF detection across different infection stages.Thirdly, towards faster detection for mass screening, particularly considering the need of future pen-side testing, we will develop a rapid detection system to complete the test within a few minutes, assisted by a simple centrifugation followed by mixing. The target ASF protein markers will be quantified in centrifuge tubes with portable electronic readout system, including an integrated circuit board comprising two LEDs for the two selected MNPs, photodetectors, battery connectors, and signal transmitters.Lastly, the NaSRED assay will be evaluated at INIA-CISA/CSIC for antigen detection, antibody detection, and multiplexed detection. Using ASF reference samples, we will evaluate the analytical limit of detection and specificity, and determine the diagnostic sensitivity and diagnostic specificity. To evaluate the feasibility of deploying the NaSRED assay to the pen-side during future outbreaks, both teams at ASU and INIA-CISA/CSIC will load the NaSRED device, together with needed accessories on a car, and perform ASF antigen tests in the field.

Progress 05/15/24 to 05/14/25

Outputs
Target Audience:In ASF diagnostics, the INIA-CISA/CSIC, acting as the EURL-ASF and WRC-ASF, continued to play a central role in the preparation and distribution of ASFV reference materials for research and assay validation. During this reporting period, our primary target audiences included: Regulatory and biosafety offices, particularly USDA-APHIS and FADDL, who oversaw the approval, inspection, and safe transfer of ASFV reference samples across international and domestic channels; Our collaborators at INIA-CISA/CSIC, who prepared and shipped a second batch of standardized ASFV serum, blood, and DNA samples representing diverse ASFV genotypes, aiding our platform calibration and validation; Diagnostic assay developers and engineering teams at Arizona State University, who adapted and optimized the NaSRED sensing assay and portable readout system for antibody and antigen detection in ASF reference matrices; And indirectly, veterinary authorities, field surveillance teams, and stakeholders in swine biosecurity, who will benefit from the portable diagnostic tool being developed through this USDA-funded partnership. In this reporting period, a second international shipment of ASFV reference samples was successfully received from our collaborators at INIA-CISA/CSIC (Spain), following coordination with USDA-APHIS and FADDL. These samples are essential for the analytical and diagnostic validation of the NaSRED biosensing system under development. The received materials include: Two sets of 35 ASFV-infected swine serum samples (0.5 ml/vial), collected at various days post-infection from pigs experimentally infected with genotype II and XXIII ASFV strains; Two sets of 34 ASFV-infected EDTA-blood samples (0.5 ml/vial), also from genotype II and XXIII-infected swine; Two panels of 10 ASFV-positive serum samples (1 ml/vial), acquired at staggered infection stages to serve as calibration references; Two sets of 6 ASFV-negative EDTA-blood samples, serving as healthy controls; Two panels of 10 lyophilized URL-ASF reference serum samples, provided for internal verification and validation of ASF antibody detection techniques; Two panels of 21 lyophilized URL-ASF reference DNA samples, representing seven distinct p72 genotypes of ASFV. All materials were received in duplicate to meet the USDA's biosafety requirement for pre-testing at FADDL prior to release. One set was tested and discarded per USDA-FADDL protocol. These reference samples are critical to enable full calibration of NaSRED against real-world diagnostic targets, encompassing both antigen and antibody detection workflows, across multiple ASFV genotypes and infection stages. Changes/Problems:We have not made any major changes in our project objectives or experimental approach during this reporting period. However, we have encountered significant obstacles related to the acquisition and handling of ASFV reference samples, as well as reagent stability and procurement challenges. These issues stemmed from the complex regulatory environment, logistical coordination among multiple institutions, and supply chain limitations. The following highlights the key obstacles we were able to overcome: We initiated the ASU biosafety review in August 2023. The approval process required revisiting and updating internal IBC protocols, conducting additional lab inspections, and providing supplementary personnel training. Externally, since ASFV samples from the INIA-CISA/CSIC lab in Spain needed to be tested by USDA FADDL before being transported to ASU, the permit process had to be renewed and cleared. The entire process was completed by late October 2023. The USDA permit renewal process was initiated in September 2023. To complete the renewal, we had to update the shipment details and submit applications for new international and inter-state permits through USDA APHIS VS Animal Product Import and Export (APIE). After multiple communications with APHIS-APIE, we were able to update the necessary documentation and retain permit privileges. The permit was approved in mid-October 2023. On the collaborator side, we continued close coordination with the INIA-CISA/CSIC lab in Spain for sample preparation. The production of the ASFV reference panel required scaling up virus propagation in swine, which is a time-consuming process. Despite administrative hurdles, our collaborators, Dr. Marisa Arias and Dr. Carmina Gallardo, ensured that the reference samples were prepared even before completing all internal paperwork for project funding. Additionally, institutional account issues with World Courier for international shipment were resolved, and the samples were made ready for shipment in late December 2023. We also worked with USDA FADDL to facilitate sample testing and transport. With the guidance of Dr. Ming Y. Deng, the samples were transported on scheduled dates to JFK airport, received by FADDL, and tested by November 2024. However, due to extended inspection times, we had to renew the USDA permit again before the shipment from FADDL to ASU could proceed. The ASFV reference samples were finally received at ASU in late December 2024. As a consequence of these delays, the project's focus during this reporting period remained on sensor optimization and platform development. The extended timeline also affected the shelf life of key reagents used in the NaSRED assay. Several critical reagents approached expiration, requiring us to validate their stability and, in some cases, repurchase them. Given the lead times associated with reagent manufacturing and delivery, this presented an additional logistical challenge. An important development during this period was the identification of a new reagent source (Bioss) for biotinylated antibodies. These reagents are expected to enhance the assay's performance, particularly for antigen detection, and represent a positive refinement in our experimental approach. What opportunities for training and professional development has the project provided?This project provided valuable training and professional development opportunities for multiple graduate students. Ph.D. candidates Seyedsina Mirjalili and Yeji Choi were extensively trained through their involvement in assay development, device engineering, and analytical validation activities. Throughout the course of the project, Seyedsina Mirjalili successfully completed his Ph.D. degree, with his dissertation work directly supported by this USDA grant. These training experiences have strengthened the students' technical expertise in biosensing technologies, nanoparticle assay development, and portable diagnostic device fabrication, contributing to their academic and professional growth. How have the results been disseminated to communities of interest?The results of this project have been disseminated through multiple scientific conferences and collaborative interactions with key stakeholders. Preliminary findings related to ASFV antigen and antibody detection using the NaSRED platform were presented at international conferences, including µTAS 2024, EIPBN 2024, and the Gordon Research Seminar & Conference 2024, through both oral and poster presentations. These events provided opportunities to engage with experts in biosensing, veterinary diagnostics, and agricultural biosecurity. Additionally, the project team showcased the NaSRED platform for ASFV diagnostics at the Entrepreneurship + Innovation (E+I) event at Arizona State University. This event facilitated engagement with industry representatives, potential commercialization partners, and innovation-focused stakeholders, supporting the translation of project outcomes towards practical deployment. These dissemination activities collectively advance the visibility and potential adoption of the NaSRED platform for ASFV surveillance and outbreak control. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will aim to: Validate the NaSRED assay performance using the ASFV reference samples from INIA-CISA/CSIC, focusing on sensitivity, specificity, and dynamic range for both p72 antigen and anti-p30/anti-p72 antibody detection in swine serum and blood matrices. Further refine the multi-tube reader system and automated assay processing device to ensure reproducible operation and higher throughput for ASFV detection. Benchmark the PED system performance under simulated field conditions to confirm stability, reproducibility, and user-friendliness. Finalize standard operating procedures (SOPs) for NaSRED assay deployment in lab and field settings. Coordinate with INIA-CISA/CSIC and USDA stakeholders on validation progress and plan potential field trial pathways (Task 4). Anticipate at least one manuscript submission summarizing the ASFV NaSRED assay validation results, and one manuscript on NasRED platform.

Impacts
What was accomplished under these goals? 1-1. ASFV Sample Acquisition and Logistics, Task 1 Navigated complex regulatory processes for ASFV reference sample acquisition. Coordinated USDA permit renewals, FADDL testing, and international shipment logistics. Collaborated with INIA-CISA/CSIC to secure reference panels, including serum, EDTA-blood, and DNA samples covering multiple ASFV genotypes. Resolved multi-party institutional hurdles (IBC protocols, APHIS-APIE, World Courier logistics). Received ASFV reference panels at ASU by December 2024. These samples are critical for assay calibration and validation in Task 4. 1-2. ASFV Antigen & Antibody Sensing-Mechanism & Analytical SOPs, Task 1 Established the complete sensing procedure for ASFV p72 and p30 antigen and antibody detection. Optimized key analytical SOPs for biotinylation, AuNP functionalization, and matrix interference mitigation. These standardizations ensure assay consistency and sensitivity, providing a validated foundation for Task 4 validation activities. 2-1. Portable Electronic Detector (PED) Development & Validation, Task 3 Designed and fabricated PED Version 5 (V5) with surface-mount PCB, 3D-printed holders, and portable architecture to minimize noise and enhance usability. Established optical power measurement protocols for LED responsivity and stability . Conducted noise and reproducibility analyses in buffer systems, benchmarking PED performance against ELISA with comparable sensitivity and precision . Optimized PED performance in blood and serum matrices, ensuring analytical reliability in realistic sample conditions . Extended detection capability to inactivated viral particles in saliva . Performed initial calibration with ASFV-spiked swine sera using first shipment samples, confirming PED accuracy . 2-2. Multi-Tube Reader Development & Optimization , Task 3 Developed a multi-tube reader system to enable parallel assay measurements with low-noise electronic readout. Designed constant current LED driver circuits with current mirror and voltage regulation for signal consistency Fabricated dedicated PCB boards with noise-filtering circuits, replacing breadboard setups Validated tube-to-tube consistency and minimized readout variance through hardware-level optimizations 2-3. Optical Path Optimization & Analytical Stability , Task 3 Optimized sensor placement and measurement protocols within microcentrifuge tubes to enhance signal consistency. Implemented measurement logic preventing LED cross-interference, achieving CV <5% across tubes. Addressed matrix gradient effects during incubation/vortexing, refining protocols for sub-fM-level detection. These efforts ensure reliable PED readouts in complex matrices, supporting ASFV assay robustness. 2-4. Automated Device Development for NaSRED Assay Workflow , Task 3 Designed and implemented an automated device integrating centrifugation, incubation, and vortexing steps with programmable control. Developed hardware prototypes with IR sensors, DC motors, LCD interfaces, and user-friendly control systems. Enhanced device functionality with speed measurement (tachometer) and precise time control. Established a preliminary graphical user interface (GUI) for data acquisition and dose-response analysis. This platform reduces user-dependent variability and supports high-throughput ASFV diagnostics. Outcome: During this reporting period, the project has made significant progress transitioning from platform optimization towards validation readiness. A major milestone was the successful acquisition of ASFV reference panels, which was achieved after resolving complex regulatory and logistical challenges involving multiple institutional stakeholders. Concurrently, the team established robust sensing protocols, including comprehensive strategies to mitigate matrix interference in serum/blood-based assays and analytical methods leading to sub-femtomolar detection sensitivity with high accuracy (CV<10%) and Precision (Recovery~%100). In parallel, substantial advancements were made in the development and validation of the portable electronic detector (PED) system. This included the fabrication of a miniaturized, reproducible readout device, as well as the design of supporting hardware platforms such as a multi-tube reader and an automated device for assay processing. These technological improvements have collectively ensured that the NaSRED platform is now fully prepared for ASFV-specific assay validation. The accomplishments directly address the objectives outlined in Tasks 1 and 3, while establishing a solid foundation for the validation activities planned under Task 4.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Maziyar Kalateh Mohammadi, Seyedsina Mirjalili, Md Ashif Ikbal, Hao Xie, and Chao Wang, "Rapid and Sensitive Detection of Thrombospondin-2 Using Nanoparticle Sensors for Cancer Screening and Prognosis," Micromachines, vol. 16, pp. 354, 2025
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Seyedsina Mirjalili, Md Ashif Ikbal, Ching-Wen Hou, Maziyar Kalatehmohammdi, Yeji Choi, Laimonas Kelbauskas, Laura A. VanBlargan, Vel Murugan, Michael S. Diamond, and Chao Wang, " Nanoparticle-Supported, Rapid, Digital Quantification of Neutralizing Antibodies Against SARS-CoV-2 Variants," Biosensors and Bioelectronics, 2025.117549
  • Type: Theses/Dissertations Status: Published Year Published: 2025 Citation: Nanoparticle-Supported Rapid Electronic Detection (NasRED): a Versatile Platform Technology for Reliable, Rapid, Affordable Diagnostics
  • Type: Peer Reviewed Journal Articles Status: Under Review Year Published: 2024 Citation: Yeji Choi, Seyedsina Mirjalili, Md Ashif Ikbal, Sean McClure, Scott Clemens, Jose Solano, John Heggland, Jiawei Zuo, and Chao Wang, "Portable, Rapid, and Digital Detection of SARS-CoV-2 Antibodies and Antigens at Attomolar Level," Under review, bioRxiv 2024.09.04.611305
  • Type: Peer Reviewed Journal Articles Status: Other Year Published: 2025 Citation: Seyedsina Mirjalili, Yeji Choi, Karuppiah Chockalingam, Benjamin Thomas, Xiaohua He, Zhilei Chen, and Chao Wang, "Nanoparticle-Supported, Point-of-Care Detection of Shiga Toxin-Producing E. coli Infection from Food and Human Specimens," Medrxiv, pp. 2025.2004. 2003.25325182, 2025.


Progress 05/15/23 to 05/14/24

Outputs
Target Audience: In ASF diagnostics, the INIA-CISA/CSIC, acting also as the EURL-ASF and WRC-ASF, have the capacity to prepare reference controls and panels, reference standards and other biologicals for the exclusive use of National reference laboratories in the EU and worldwide after a request and contracts. The INIA-CISA/CSIC is responsible of a number of research and development activities with the goal of keeping abreast of developments in ASF surveillance, epizootiology and prevention throughout the world, and retaining expertise to enable rapid ASF diagnosis. They regularly store and supply cell cultures for use in diagnosis, build up and hold an ASF virus collection, organize periodic comparative tests of diagnostic procedures, collect diagnostic data and results of tests, characterize virus isolates, etc.. The existing capacity at INIA-CISA/CSIC will be utilized throughout this research project to ensure proper assessment, evaluation and validation of the proposed NaSRED assay. 1. We completed tests of a batch of ASF samples sent from the INIA-CISA/CSIC. The content of the parcel will include: • 10 vials of the reference panel collection 1 batch 3 (I enclosed the certificate that we always send with the panel description of the serum samples), • 10 vials of 0.5 ml of negative serum samples • 6 vials of 0.5 ml of the purified protein p72 from ASFV ( publication that we have used to get the VP72 (vp73) is attached ) • 6 vials of 0.5 ml of ASF semi-purified Ag (6 vials of 0.5 ml) procedure attached, also it is in the publication and in the WOAH (former OIE) Manual. • 2 vials of ASF positive reference control serum • 2 vials of ASF reference positive-limit control serum standardized sera, conjugate sera and other reference reagents, including antigens and antibodies, will be shipped from INIA-CISA/CSIC to ASU to standardize the tests and reagents. 2. We have met online with our collaborators at the INIA-CISA/CSIC, and decided to include more samples in our tests. The 2nd batch of samples were shipped in Jan 2024, and as of May 15 they are still being tested at the Foreign Animal Disease Diagnostic Laboratory within USDA/APHIS/VS/NVSL. What we expect to receive include: 1.- Panel of serum samples, using amounts of 0.5 ml per serum samples - 10 different serum samples- sending 1-2 ml /each 2.- Panel of DNAs 3.- Several vials of negative blood samples 4.- Several vials, of positive blood samples 5.- Several vial of serum samples from one pig , serum samples obtained at different times post infection. - Serum samples also contains high amount of virus at the beggining of the infection for several weeks ) . Changes/Problems:No major changes. The ASF samples shipped from Spain once again have been under test for more than 4 months. This delay is disappointing but not surprising. We are prepared to perform the tests of these samples once we receive them. What opportunities for training and professional development has the project provided?2 first year Ph.D. students (Sina Mirjalili, and Yeji Choi) were trained on this project. One undergrad student (Sean McClure ) was trained on this project How have the results been disseminated to communities of interest?Not disseminated yet. However, we will present the ASF sensing work at EIPBN 2024 conference on May 31, 2024. It has been accepted as an oral presentation: "Nanoparticle-assisted, Portable Detection of African Swine Fever Infection" What do you plan to do during the next reporting period to accomplish the goals?In the next report period, we will aim to: Further optimize the AuNP functionalization and sensing conditions, and test the sensing of p72 proteins (commercial proteins) spiked in swine serums. Then we will test the assay in the reference samples provided by INIA-CISA/CSIC in Spain. Then later, we will coat the AuNPs with the purified p72 proteins and semi-purified antigens (provided by INIA-CISA/CSIC in Spain) and test the sensing of antibodies spiked in swine serum and then the positive and negative control samples provided by INIA-CISA/CSIC in Spain. We will further optimize the PED device to build a portable system suitable for pen-side use. We will coordinate with INIA-CISA/CSIC in Spain on our progress and decide the next steps. We will anticipate one manuscript on the PED device and one manuscript on the anti-p72 antibody sensing.

Impacts
What was accomplished under these goals? In this year, we focused on prototyped ASF antibody sensing and the sensor development. They are directly related to tasks 1 and 3. First, we have performedassay optimization and readout system development using SARS-CoV-2 antigen and antibody, while we are waiting for our ASF samples to be released from FADDL lab. · We optimized single-tube reader circuit to stabilize the signals and reduce measurement error · We optimized the sensing protocol · We developed co-binder based assay format to detect SARS-COV-2 antibodies and antigens in different biological fluids, including saliva, nasal fluids, sera and blood, and demonstrated aM detection limit, orders of magnitude better than ELISA · We designed multi-tube reader system to allow parallel readout of multiple samples. In addition, we have spiked p72 antibody, p72 antigen, p30 antibody, and p30 antigen in buffer swine sera and performed the tests. We have demonstrated · limit of detection of 2 fM within 10 min reaction time · a dynamic range from 100 pM to 1fM, i.e. about 5 logs · These are already much better than our expected outcome in the proposal: "a dynamic range of > 3 logs,e.g.from >100 nM to <100 pM". This is in part attributed to the sensing protocol optimization and our PED sensor development.

Publications


    Progress 05/15/22 to 05/14/23

    Outputs
    Target Audience: In ASF diagnostics, the INIA-CISA/CSIC, acting also as the EURL-ASF and WRC-ASF, have the capacity to prepare reference controls and panels, reference standards and other biologicals for the exclusive use of National reference laboratories in the EU and worldwide after a request and contracts. The INIA-CISA/CSIC is responsible of a number of research and development activities with the goal of keeping abreast of developments in ASF surveillance, epizootiology and prevention throughout the world, and retaining expertise to enable rapid ASF diagnosis. They regularly store and supply cell cultures for use in diagnosis, build up and hold an ASF virus collection, organize periodic comparative tests of diagnostic procedures, collect diagnostic data and results of tests, characterize virus isolates, etc.. The existing capacity at INIA-CISA/CSIC will be utilized throughout this research project to ensure proper assessment, evaluation and validation of the proposed NaSRED assay. In this year, the INIA-CISA/CSIC has prepared a batch of ASF samples and shipped to the U.S. Changes/Problems:No major changes. We have encountered a lot of obstacles in this first year to obtain the regents and materials. This is partly due to very strict safety rules and our lack of experience dealing with all parties involved on the use of ASF materials. However, we have managed to solve all of them so far. The following highlights the obstacles we were able to overcome: We started ASU biosafety review mid Apr 2022. The approval required internally redoing internal IBC, lab visits, modifying lab protocols, and additional personnel trainings. Externally, because samples from Spain lab need to be tested by FADDL, a permit process has to be cleared. Eventually the whole process was cleared in late Oct 2022. We initiated the USDA permit process in late Apr 2022 for the ASF samples from Spain to be tested by FADDL and then transported to ASU. However, the website was confusing and had errors, and I had to call the USDA APHIS VS Animal Product Import and Export (APIE). Finally, after a number of emails to APHIS-APIE, Vivek G. Kamath was able to point out the right permit to use. Eventually, we were able to get this issue resolved in mid Nov. 2022. We also worked closely with the INIA-CISA/CSIC lab in Spain to initiate the collaboration and the sample preparation. Despite organizational changes from the INIA-CISA/CSIC side that has delayed some paperwork, our collaborator Dr. Marisa Arias has been extremely helpful, and they prepared the purified protein samples even before they were able to complete all the paperwork to receive funds from the project. The purified proteins require growing large number of ASFV to purify and took some time. The protein samples were made ready to ship in late Dec. 2022. We also worked with the USDA FADDL office to test and transport our samples. Dr. Ming Y. Deng was very helpful to guide us through the process. However, due to miscommunication, 10 vials of sample to be used for panel evaluation was discarded, because FADDL required at least 2 identical vials for each sample, but we thought the test will be done by any random vial from the panel. The samples were transported on specific dates to specific airport (JFK) by a designated courier, picked up by FADDL and tested in mid Feb 2023. However, due to miscommunications between ASU financial admins and FADDL, the payment was delayed. The payment was issued and the samples received by ASU in mid Apr 2023. What opportunities for training and professional development has the project provided?3 first year Ph.D. students (Sina Mirjalili, Mohammad Altarfa, and Yeji Choi) were trained on this project. How have the results been disseminated to communities of interest?No, but we are preparing the data. What do you plan to do during the next reporting period to accomplish the goals?In the next report period, we will aim to: Further optimize the AuNP functionalization and sensing conditions, and test the sensing of p72 proteins (commercial proteins) spiked in swine serums. Then we will test the assay in the reference samples provided by INIA-CISA/CSIC in Spain. Then later, we will coat the AuNPs with the purified p72 proteins and semi-purified antigens (provided by INIA-CISA/CSIC in Spain) and test the sensing of antibodies spiked in swine serum and then the positive and negative control samples provided by INIA-CISA/CSIC in Spain. We will further optimize the PED device to build a portable system. We will coordinate with INIA-CISA/CSIC in Spain on our progress and decide the next steps. We will anticipate one manuscript on the PED device and one manuscript on the anti-p72 antibody sensing.

    Impacts
    (N/A)

    Publications