Source: The Regents of University of California submitted to NRP
PARTNERSHIP: DEVELOPMENT OF A NANOLITER REACTOR FOR DIGITALIZATION OF NUCLEIC ACID-BASED DETECTION PLATFORMS WITH BROAD APPLICATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1031652
Grant No.
2022-67021-41478
Cumulative Award Amt.
$689,028.47
Proposal No.
2023-04197
Multistate No.
(N/A)
Project Start Date
Nov 1, 2023
Project End Date
Feb 7, 2025
Grant Year
2024
Program Code
[A1511]- Agriculture Systems and Technology: Nanotechnology for Agricultural and Food Systems
Recipient Organization
The Regents of University of California
200 University Office Building
Riverside,CA 92521
Performing Department
(N/A)
Non Technical Summary
Microbial foodborne illness sickens millions and kills thousands in the U.S. annually. One of the major challenges to preventing the transmission of foodborne pathogens is the lack of ideal rapid, portable, and sensitive detection of pathogens from foods samples. Ligand-based detection techniques are portable, rapid but lack the degree of sensitivity needed for detection of pathogens in foods. Nucleic acid amplification-based techniques are highly sensitive, but lack portability and are sensitive to sample-associated inhibitors, thus requiring extensive sample purification. Isothermal amplification techniques like LAMP and RPA show promise because of advantages, one of which is their relative tolerance of inhibitors, allowing for crude extraction/purification in-field. However, these methods can sometimes lack quantitative ability. Similarly, digital polymerase chain reaction (dPCR) also has exhibited tolerance for inhibitors and better quantitative ability, but lacks portability. In this proposal, we seek to develop and evaluate a nanoliter reactor that both digitalizes and detects any nucleic acid amplification-based technique in a portable format. We will explore the degree to which digitalization of RPA and LAMP chemistries in a fully automated, in situ chip can aid rapid detection with the need for only crude sample extraction.
Animal Health Component
30%
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123712202050%
7121499202050%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1499 - Vegetables, general/other; 3712 - Salmon;

Field Of Science
2020 - Engineering;
Goals / Objectives
The aims of the proposal are:Aim 1: Fabrication of a nanoliter digital chip (nano-dChip) for nucleic acid amplification; Aim 2:Development of nano-dChip for the digitalized detection of human noroviruses; Aim 3:Development of nano-dChip for the duplexed digitalized detection of foodborne bacterial pathogens (Salmonella enterica and Listeria monocytogenes); Aim 4:Assessment of the ability of the nano-dChip to detect foodborne bacterial and viral pathogens in crudely processed samples.
Project Methods
In this proposal, we aim to develop and evaluate a novel nanoliter digital chip (nano-dChip) that can readily digitalize any nucleic acid (DNA and RNA) amplification. This dChip provides numerous advantages by dividing nucleic acid amplification reactions into numerous individual reaction wells. Combination with a sensitive isothermal nucleic acid amplification, the nano-dChip will be used to detect representative foodborne pathogens in food samples, including human norovirus (ssRNA), Salmonella enterica (DNA), and Listeria monocytogenes (DNA) in a multiplexed fashion on a small single chip, enabling non-trained operators to rapidly quantify the microbial contaminants in foods.One of the key innovations of our dChip is that the digitalization of amplification product is simple and can significantly enhance the analytical sensitivity, quantitative accuracy, and resistance to sample-associated inhibition, superior to various conventional amplification chemistries. The sensing is performed on a small chip without the need for expertise to operate. In addition, the fabrication of the dChip relies on standard microfabrication thus the costs of each chip is less than $0.50 USD, making it an affordable and disposable unit for wide distribution. A "vacuum lung" design is included without using any external pumps to drive the reagents. Thus, our chip can be used by anyone without special training. Further, a simple and portable commercially available fluorescence microscopeis used to read the fluorescence results, thus completely avoiding bulky and expensive instrumentations.The overall goal of this proposal is to improve food safety by providing an ideal portable digital nanoliter chip that digitalizes nucleic acid amplification signal for the rapid, sensitive, user-friendly, low-cost, and in-field detection of viral and bacterial pathogens in foods. This is important for the long-term goal of preventing the spread of foodborne pathogens in foods and reducing foodborne outbreaks 15.

Progress 11/01/23 to 02/07/25

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through this grant, we have successfully trained multiple Ph.D. and M.S. students, equipping them with valuable research skills and expertise in foodborne pathogen detection. Our students have had the opportunity to present their research findings at prestigious conferences, including the USDA PD Meeting and the ASME 2024 Annual Meeting. Additionally, our research has been disseminated through invited presentations at various academic and industry events, including: "Advances in the Detection and Control of Foodborne Viruses." Future of Food Symposium, McGill University and the Consortium de Recherche et Innovations en Bioprocédés Industriels au Québec (CRiBiQ), Montreal, Quebec, Canada. 5/16/2024. "Moore Lab: Applied and Environmental Virology." UMass Food Science Advisory Board Meeting, Amherst, MA, USA. 4/19/2024. "Moore Lab: Applied and Environmental Virology." Department of Defense Army Combat Feeding Division, Soldier Center, Natick, MA, USA. 2/27/2024. How have the results been disseminated to communities of interest?We have published three journal articles last year: Liu, Li, Stephen J. Dollery, Gregory J. Tobin, Guoyu Lu, and Ke Du. "Cleavable energy transfer labeled oligonucleotide probe for enhanced isothermal amplification detection and nano digital chip-based readout."Nanoscale17, no. 3 (2025): 1381-1391. Liu, Li, Tom Kasputis, Juhong Chen, Matthew D. Moore, and Ke Du. "Fully Integrated Microfluidic Digital Chip for Simple and Highly Quantitative Detection of Norovirus."Analytical Chemistry96, no. 46 (2024): 18408-18415. Kasputis, Tom, Po-Chen Yeh, Li Liu, Jeffrey Marano, James Weger-Lucarelli, Ke Du, Liwei Lin, and Juhong Chen. "Development of a self-powered digital LAMP microfluidic chip (SP-dChip) for the detection of emerging viruses."Lab on a Chip24, no. 14 (2024): 3490-3497. The Analytical Chemistry paper was featured as the supplement cover by the journal. The Nanoscale paper was inlcuded in the Emerging Investigator Special Issue. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, our focus will be on further optimizing the on-chip loop-mediated isothermal amplification (LAMP) process to enhance its sensitivity and specificity for the duplex detection of both viral and bacterial pathogens that are known to cause infectious diseases. By refining reaction conditions and improving assay efficiency, we aim to ensure robust and accurate detection across a wide range of pathogen concentrations. Additionally, we will explore novel and simplified methodologies for nucleic acid extraction, aiming to develop rapid, cost-effective, and user-friendly approaches that streamline the sample preparation process while maintaining high analytical performance. As part of our dissemination efforts, we plan to publish at least two peer-reviewed journal articles detailing our findings and advancements in digital pathogen detection technologies. Furthermore, we will actively participate in international conferences to share our research with the broader scientific community, fostering collaborations and knowledge exchange. Another key objective for the upcoming period is the successful completion of graduate studies for two M.S. students who are currently working on this project. Their research contributions will play a crucial role in advancing our work, and we anticipate that their theses will provide valuable insights into the development of highly sensitive and reliable diagnostic platforms for food safety and public health applications.

Impacts
What was accomplished under these goals? We have successfully developed and demonstrated two distinct types of nanoliter-scale digital chips for the detection of human Norovirus: a silicon microwell-based chip and a polydimethylsiloxane (PDMS) nanoliter reservoir-based chip. Both systems have been designed to enable precise, highly sensitive, and digital detection of foodborne pathogens, even at extremely low concentrations, by utilizing an on-chip loop-mediated isothermal amplification (LAMP) process. Our results confirm that these platforms provide reliable and reproducible pathogen detection, offering a significant advantage in food safety monitoring. Additionally, we demonstrated the capability of duplex detection, allowing for the simultaneous identification and differentiation of multiple Norovirus strains. This capability enhances the robustness of the system, making it a powerful tool for rapid and accurate foodborne pathogen surveillance in various settings, including food processing industries and clinical diagnostics.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Liu, Li, Stephen J. Dollery, Gregory J. Tobin, Guoyu Lu, and Ke Du. "Cleavable energy transfer labeled oligonucleotide probe for enhanced isothermal amplification detection and nano digital chip-based readout." Nanoscale 17, no. 3 (2025): 1381-1391.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Kasputis, Tom, Po-Chen Yeh, Li Liu, Jeffrey Marano, James Weger-Lucarelli, Ke Du, Liwei Lin, and Juhong Chen. "Development of a self-powered digital LAMP microfluidic chip (SP-dChip) for the detection of emerging viruses." Lab on a Chip 24, no. 14 (2024): 3490-3497.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Liu, Li, Tom Kasputis, Juhong Chen, Matthew D. Moore, and Ke Du. "Fully Integrated Microfluidic Digital Chip for Simple and Highly Quantitative Detection of Norovirus." Analytical Chemistry 96, no. 46 (2024): 18408-18415.


Progress 11/01/23 to 10/31/24

Outputs
Target Audience:We have establisheda novel nano digital loop-mediated isothermal amplification (LAMP) assay platform, which employs digital microfluidic chips for absolute quantitative analysis of nucleic acids. This portable chip utilizes LAMP technology to achieve ultra-sensitive detection of target nucleic acids within 30 min and reduces the detection limit to 1 fM without the need for complex instrumentation. By digitizing amplification signals directly from the target sample, our platform offers simplicity, affordability, portability, and quantitative molecular readouts. This innovation represents a crucial step toward on-site detection of foodpathogens, thereby enhancing food safety and mitigating disease outbreaks. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We use this grant to support several graduate and undergraduate students. Our student willpresent this work in the ASME IMECE Conference. How have the results been disseminated to communities of interest?Publications: 1. Tom Kasputis, Po-Chen Yeh, Li Liu, Jeffrey Marano, James Weger-Lucarelli, Ke Du, Liwei Lin, and Juhong Chen. "Development of a self-powered digital LAMP microfluidic chip (SP-dChip) for the detection of emerging viruses."Lab on a Chip24, no. 14 (2024): 3490-3497. 2.Li Liu, Stephen Dollery, Gregory Tobin, Guoyu Lu, and Ke Du. "Digital Nanofluidic Chip for Simple and Highly Quantitative Detection of HPV Target."medRxiv(2024): 2024-03. 3. Li Liu, Tom Kasputis, Juhong Chen, Matthew Moore, and Ke Du. "Fully Integrated Digital Nanofluidic Chip for Simple and Highly Quantitative Detection of Norovirus", under review in Analytical Chemistry. The presentations came out from this grant: "Advances in the Detection and Control of Foodborne Viruses." Future of Food Symposium. McGill University and the Consortium de Recherche et innovations en Bioprocédés industriels au Québec (CRiBiQ). Montreal, Quebec, Canada. 5/16/2024. -"Moore Lab: Applied and Environmental Virology." UMass Food Science Advisory Board Meeting. Amherst, MA, USA. 4/19/2024. -"Moore Lab: Applied and Environmental Virology." Department of Defense Army Combat Feeding Division Soldier Center. Natick, MA, USA. 2/27/2024 -Du Lab: Digital Micro-and Nanofluidic Systems for Infectious Diseases Sensing, scheduled for November, Portland, OR What do you plan to do during the next reporting period to accomplish the goals?We will continue our strong collaobrations of the team to accomplish the goals listedin the proposal.

Impacts
What was accomplished under these goals? A. Fabrication of a nanoliter digital chip (nano-dChip) for nucleic acid amplification: We have successfully fabricated the nanoliter digital chip (nano-dChip) for nucleic acid amplification.We fabricated the Nano-dChip using the standard soft lithography process.The mold of the designed microfluidic chip was fabricated on a silicon wafer using SU-8 photoresist (Kayaku Advanced Materials, Inc.). The main channel and vacuum lungs were 200 µm in width and 100 µm in depth. The dimension of each well was 400*200*100 µm (length*width*depth). To replicate the patterns onto polydimethylsiloxane (PDMS), an elastomer mixture (10:1 weight ratio of Sylgard 184 silicone elastomer base and curing agent) was mixed and degassed in a vacuum chamber. The mixture was then poured onto the silicon wafer and placed into an oven at 80 for 2 hrs. Afterward, the PDMS was peeled off from the silicon wafer. The inlet and outlet of the channel were punched with 1 mm diameter holes to allow the reagent to flow through the channel. The final device was sealed in a pretreated glass coverslip by an oxygen plasma (Electro-Technic Products) system and baked at 125 on a hotplate overnight. After that, hydrophobic modification was made by passivating the microfluidic surface with an anti-biofouling surface treatment to avoid nonspecific absorption of protein/DNA. B.Development of nano-dChip for the digitalized detection of human noroviruses: We have successfully demonstrated the nano-dChip for the digitalized detection of norovirus:This compact, affordable, and disposable device has applicability to a broad array of nucleic acid amplification techniques, catering to both DNA and RNA analysis. It is not only suitable for laboratory environments, but can also be applied in real-world scenarios, especially in the rapid detection of representative foodborne pathogens. In our study, a simple, specific, rapid and sensitive RT-LAMP assay was established, targeting RNA for the detection of the prevalent norovirus GII. Leveraging the LAMP reaction's exceptional specificity with three pairs of six primers and our innovative fluorescence-quencher binding reaction for precise target recognition, this sequence-based detection method we designed significantly reduces background signal intensity compared to traditional LAMP detection methods and offers higher sensitivity and specificity. Our method provides a versatile solution for detecting a wide range of pathogens with shared strains, thus expanding its utility in pathogen detection. C.Development of nano-dChip for the duplexed digitalized detection of foodborne bacterial pathogens:We have demonstrated that the duplex LAMP reaction can be achieved with our probe binding assay. For the next phase, we will demonstrate and optimize the duplex detection offoodborne bacterial and viral pathogens on-chip.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: 1. Tom Kasputis, Po-Chen Yeh, Li Liu, Jeffrey Marano, James Weger-Lucarelli, Ke Du, Liwei Lin, and Juhong Chen. "Development of a self-powered digital LAMP microfluidic chip (SP-dChip) for the detection of emerging viruses." Lab on a Chip 24, no. 14 (2024): 3490-3497.
  • Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Li Liu, Stephen Dollery, Gregory Tobin, Guoyu Lu, and Ke Du. "Digital Nanofluidic Chip for Simple and Highly Quantitative Detection of HPV Target." medRxiv (2024): 2024-03.
  • Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Li Liu, Tom Kasputis, Juhong Chen, Matthew Moore, and Ke Du. "Fully Integrated Digital Nanofluidic Chip for Simple and Highly Quantitative Detection of Norovirus.", Analytical Chemistry, in revision.