P113: Silicon Micropillar-Enhanced CRISPR Biosensor for Rapid and Sensitive Detection of Drug-Resistant Bacteria
Poster Presenter
Ruonan Peng
Graduate Research Assistant
University of California, Riverside United States
Objectives
This study presents a silicon micropillar-enhanced CRISPR biosensor for the rapid and sensitive detection of drug-resistant bacteria, leveraging high-density probe immobilization and CRISPR/Cas12a activation to achieve ultrasensitive and efficient pathogen identification.
Method
The silicon chip was fabricated at UCSB Nanofab, and assay testing was conducted at UC Riverside. A high-aspect-ratio silicon micropillar array enhanced fluorescent reporter immobilization. CRISPR/Cas12a-based detection was performed to identify MRSA target with high sensitivity.
Results
We have successfully fabricated silicon micropillar chips with varying heights of 100, 300, and 500 microns to explore their potential for enhancing CRISPR-based bacterial detection. Probe immobilization was achieved using APTES (3-aminopropyltriethoxysilane), glutaraldehyde (GA), and amine-modified (NH2) probes, ensuring stable and efficient surface functionalization. The surface chemistry was optimized to maximize probe loading, and a quantitative fluorescence was analyzed using ImageJ.
The lysis-free detection workflow involves heat lysis of MRSA, followed by Recombinase Polymerase Amplification (RPA) for target DNA amplification and CRISPR/Cas12a for highly specific detection. The off-chip RPA/CRISPR-Cas12a one-pot assay was successfully performed, achieving an off-chip detection limit of 104 CFU/mL. To assess specificity, the assay was further tested on non-target bacteria, including wild-type Staphylococcus aureus (SA), Escherichia coli (E. coli), and kanamycin-resistant E. coli (kanR E. coli). The results confirmed high specificity, with a strong signal detected only for MRSA, demonstrating the assay’s ability to distinguish target bacteria from closely related species.
Additionally, a fluorescent dye without a quencher was used, enabling direct and amplified signal detection with minimal background noise. Given that higher micropillar aspect ratios provide increased probe immobilization, this surface architecture is expected to further enhance sensitivity by improving reaction efficiency and fluorescence output.
On-going work includes integrating the CRISPR reaction directly on-chip to determine the true detection limit and assess the impact of micropillar height on assay sensitivity. Additionally, to improve reagent stability and field applicability, lyoprotectants such as dextran will be incorporated to allow room-temperature storage and easy reconstitution of the reaction components, making the platform more practical for point-of-care diagnostic
Conclusion
This study demonstrates the feasibility of using a silicon micropillar-enhanced CRISPR biosensor for rapid and specific MRSA detection. The successful fabrication of high-aspect-ratio silicon micropillars allowed for enhanced probe immobilization, which is expected to improve reaction efficiency and detection sensitivity. The integration of RPA with CRISPR/Cas12a in a lysis-free detection workflow enables fast, specific bacterial identification without requiring extensive sample processing.
The high specificity of the assay, as demonstrated by its ability to distinguish MRSA from wild-type Staphylococcus aureus (SA), Escherichia coli, and kanamycin-resistant E. coli, suggests that this platform could be adapted for broader applications in antimicrobial resistance screening. The use of a fluorescent dye without a quencher ensures strong signal amplification with minimal background noise, making the detection process more efficient and easier to interpret.
Beyond detection performance, this platform holds promise for practical deployment. Future optimization will focus on integrating the CRISPR reaction directly onto the micropillar chip, refining surface chemistry to enhance on-chip sensitivity, and further improving assay reproducibility. Additionally, incorporating lyoprotectants such as dextran or sucrose will enable room-temperature reagent storage, allowing for easier handling and long-term usability.
These findings highlight the potential of silicon micropillar biosensors as scalable, portable diagnostic tools for point-of-care bacterial detection. By offering a rapid, sensitive, and stable alternative to conventional culture-based methods, this technology could significantly impact clinical diagnostics, public health monitoring, and infectious disease management, providing a practical solution for antibiotic resistance screening in both laboratory and field settings.
