A rapid diagnosis of SARS-COV-2 using DNA hydrogel.pptx

The creation of DNA hydrogel on microfluidic pores is a highly quick and precise method for COVID-19 diagnosis. This method is based on the rolling circle amplification of DNA, which produces a DNA hydrogel that obstructs the micropores of the ensuing nylon mesh and blocks the sample fluid's flow channel. A unique design is used for the nylon mesh. A primer is used to immobilize the nylon mesh first, and then the primer is used to attach with a unique padlock probe in the shape of a dumbbell. Because of its unique architecture, the padlock probe may attach to both the genetic material of the pathogen and the primer. Its dumbbell-like form comes from a shared nucleic acid bridge conjugating a pathogen DNA binding site and a primer binding site. Dengue, influenza, and other illnesses can also be diagnosed with this method.

1. A rapid diagnosis of SARS-CoV-2 using DNA hydrogel formation on microfluidic pores Hwang- soo Kim, Naseem Abbas, Sehyun Shin Department of Mechanical Engineering, Korea University, Seoul, 02841, Republic of Korea Nano- Biofluignostic Research Center , Korea University, Seoul, 02841, Republic of Korea Presented By: Ritam Acharya, University Of Kalyani

2. COVID-19 Pandemic Impact GLOBAL HEALTH CRISIS COVID-19 Pandemic has caused significant global health challenges. Over 60 million people have been infected worldwide, and less than a year, over a million people have died from the virus. ECONOMIC IMPACT The pandemic has crippled economies worldwide. TESTING NEEDS Rapid and accurate testing is crucial for disease control.

3. Current Diagnostic Methods RT-qPCR Most widely used method, but requires specialized facilities. It has highest sensitivity and accuracy ANTIBODY-BASED DETECTION Limited by low sensitivity and dependence on specific antibodies ISOTHERMAL AMPLIFICATION Promising alternative, offering rapid analysis and high sensitivity

4. Limitations of the current diagnostic methods Low Sensitivity:   Techniques like LAMP (Loop-mediated Isothermal Amplification of DNA) and RCA (Rolling Circle Amplification) have shown lower sensitivity. Complexity: Requires complex and processes. Specialized facilities: Needs a laboratory environment, skilled operators, and contamination-free operation. Cost and time: Expensive and time-consuming, making it less suitable for rapid, large-scale testing.

5. Objective Of the Study To develop a rapid diagnostic method for SARS-CoV-2. Use DNA hydrogel formation on microfluidic pores. KEY GOALS: High sensitivity to detect low concentrations of the virus. Rapid diagnosis (results within 15 minutes). Cost-effective and accessible in resource-limited settings.

6. Principle A mesh-based RCA (Rolling Circle Amplification) procedure is used in the microfluidic system to detect SARS-CoV-2. It is comprised of a waste chamber with a rubber lid, a sample chamber, a glass tube, and padlock probe nylon mesh. Effective detection is ensured by DNA entanglement blocking micro-holes in the nylon mesh. The sample chamber was filled with a dye-colored fluid to indicate blockage, and a light-density mineral oil was added to prevent evaporation. When a target pathogen was present, DNA binds to the padlock probe, causing a Rolling Circle Amplification (RCA). This process forms DNA hydrogels, blocking flow paths in the mesh. The rubber lid was punctured to monitor fluid motion and travel time. Samples with high pathogen concentrations blocked flow paths, resulting in no flow to the end of the test tube.

7. Experimental Setup Microfluidic System Components: Sample Chamber Glass tube Padlock-probe conjugated nylon mesh Waste chamber with a rubber-lid Nylon Mesh Preparation: Primer Immobilization on nylon mesh with 1-μ m pores. Attachment of padlock probe to the mesh.

8. Steps of the experiment Hybridization: Pathogen DNA hybridizes with immobilized probes on a nylon mesh. Rolling Circle Amplification: Amplify the target pathogen’s DNA, which is bound to the padlock probes immobilized on a nylon mesh. DNA hydrogel formation: Pathogen forms a DNA hydrogel, which blocks the pores of the nylon mesh, preventing fluid flow. Hydrostatic pressure: The system uses hydrostatic pressure to transport fluid from the sample chamber to waste chamber, with flow blockage due to DNA hydrogel formation indicating pathogen presence. Detection: The method enables the highly sensitive detection of SARS-CoV-2 with a Limit Of Detection (LOD) as low as 0.7 aM within 15 minutes, making it ideal for point-of-care diagnostics

9. Rolling Circle Amplification reaction Primer Conjugation: Primer is conjugated with nylon mesh via surface activation and functionalization. Padlock-probe Hybridization: D esigned with primer-binding, pathogen-binding, and self-assembly regions, hybridizes with the primer. Annealing: Padlock probe forms an asymmetric dumbbell shape upon annealing. Target Pathogen Hybridization: Padlock probe hybridizes with the target pathogen . Ligation: Ligase ligates the opened padlock probe to form a closed-loop template. Elongation: DNA polymerase elongates complementary single-stranded DNA, forming a DNA gel due to the dumbbell-shaped template entangling with neighboring DNAs.

10. PAGE analysis of COVID-19 template structures

11. Results

12. Experimental Validation Primer Immobilization Confirmation Fluorescence microscopy confirmed successful immobilization of primers on the nylon mesh surface, demonstrating the specificity of the probe. The mesh immobilized with primers exhibited bright fluorescence, whereas the control mesh without primers showed no fluorescence. Specificity Validation The system's specificity was validated by testing with two different viral templates, SARS-CoV-2 and dengue virus. Only SARS-CoV-2 DNA resulted in hydrogel formation, confirming the target-specific nature of the padlock probe and the RCA process. Rheological Characterization Rheological measurements demonstrated a significant increase in viscosity and yield shear stress with incubation time, confirming the formation of a DNA hydrogel. This robust gel formation effectively blocks fluid flow through the mesh, indicating the presence of the target pathogen.

13. Sensitivity and LOD Determination Incubation Time Limit of Detection (LOD) 5 minutes 30 aM 15 minutes 0.7 aM The system's sensitivity was assessed by varying the concentration of SARS-CoV-2 DNA and incubation time. The LOD was determined by analyzing the flow time and velocity of the dye through the microfluidic system. The LOD values indicate the ability to detect even extremely low concentrations of the virus.

14. Advantages Over Existing Technologies Enhanced Sensitivity The LOD achieved in this study is significantly lower than previously reported methods, demonstrating improved sensitivity. This is attributed to the combination of a densely packed mesh with small pores, minimized driving force, and optimized incubation time. Rapid Detection The system offers a significant reduction in testing time compared to conventional PCR assays, enabling rapid diagnosis and timely intervention. This rapid detection capability is critical for managing the spread of the virus in high-risk settings. Potential for POC Applications The system's simplicity, affordability, and portability make it suitable for deployment at POCs, particularly in resource-limited settings. This accessibility can greatly expand access to testing, facilitating early detection and containment of the virus.

15. Limitations Synthetic Templates: The study was conducted using synthetic nucleic acid templates rather than clinical samples, which limits its real-world applicability without further validation.​ Specificity and Cross-Reactivity: While the padlock probes are designed to be specific, there is always a risk of cross-reactivity with non-target sequences, which could lead to false positives or negatives. Potential for Incomplete Gelation: Incomplete or inconsistent gelation could affect the accuracy and reliability of the detection results, particularly under suboptimal conditions Sensitivity to Environmental Conditions: The performance of the detection system may be affected by variations in environmental conditions such as temperature, humidity, and sample quality. Regulatory and Standardization Challenges : Obtaining regulatory approvals and establishing standardized protocols for widespread use can be a lengthy and complex process. Maintenance and Calibration: Regular maintenance and calibration of the detection system are necessary to ensure accurate and reliable performance, which can be resource-intensive.

16. Further Research and Development Multiplex Detection : Developing the system to simultaneously detect multiple pathogens (e.g., COVID-19, influenza-A, influenza-B) could enhance its utility in diagnosing various infectious diseases at point-of-care (POC) settings. Optimization of Materials : Exploring different materials for the nylon mesh and improving surface activation techniques may further enhance the system's sensitivity, repeatability, and flow resistance. Integration with Portable Devices : Incorporating the system into more compact, user-friendly devices for easy deployment in airports, hospitals, and public health centers could streamline rapid mass testing​​. Automation and Scalability : Developing automated processes for sample preparation, DNA amplification, and data analysis will reduce manual intervention and make the system more suitable for large-scale testing​.

17. Conclusion A microfluidic-based biosensor has been designed for specific COVID-19 detection at point-of-care (POCs). The RCA technique, using a nylon mesh medium, maximizes the detection of the virus. DNA hydrogels form on the mesh, blocking the flow path. This technology has a minimal effective surface area and reduces the amount of DNA hydrogel needed to block the tube. The system can detect SARS-CoV-2 with an excellent low detection limit (LOD) and is highly selective and accurate. The system could be applied for screening tests in airports and other locations where infectious diseases commonly spread. Current research focuses on using a microfluidic platform to detect multiple infectious viruses, including COVID-19, influenza-A, and influenza-B .

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