Design an at-home test kit to detect and eliminate S. aureus
Overview
Our at-home test kit aims to serve three key functions for detecting and treating S. aureus colonization. First, the kit allows users to assess environmental S. aureus contamination via an in vitro test. The second is to detect the presence of S. aureus directly within the intestine. Upon detection, the final function is to immediately eliminate the bacteria in real-time through lysins delivered by an engineered probiotic. User-friendliness and implementation in everyday life were important design priorities. To achieve this, we focused on creating efficient, convenient detection tools SAeking capsule and SAeking detection cassette that target S. aureus specifically. The kit is intended as a quick, daily use product allowing at-home monitoring and preventive approaches.
Content per unit
10× capsule: An enteric-coated biotherapeutic capsule
5× burette: Transferring the sample solution to the sample pad.
5× detection Cassette: The S. aureus identification Cassette.
1× instruction: Listing operational procedures, principles introduction, and healthy habits in daily lives
Protocol
The SAeking Capsule
We propose an intestinal capsule designed to eliminate S. aureus colonization in vivo through ingestion before or after meals. The capsule aims to reduce infection risks by eradicating S. aureus from the intestinal tract.
The capsule contains two engineered probiotic strains tailored for detection and elimination of S. aureus. B. subtilis is engineered to detect autoinducer peptides (AIPs), quorum-sensing signals emitted by S. aureus. Meanwhile, E. coli is engineered to constantly express endolysins lytic against S. aureus. In the presence of AIPs detected by B. subtilis within the capsule, it will signal the E. coli to induce expression of its self-lysis enzyme. This allows the capsule to directly detect and eliminate S. aureus upon detection of AIPs released from the pathogen.
The capsule utilizes an enteric-coated structure, protecting its contents from the acidic stomach environment and releasing them specifically in the intestines. There, the probiotic strains become active - E. coli begins endolysin production while B. subtilis prepares to detect AIPs. E. coli eventually undergoes regulated self-lysis, releasing endolysins to surround and lyse any S. aureus detected by the coordinated response of the engineered probiotic system.
To facilitate rapid response upon capsule releas, endolysin production in E. coli is initiated during manufacturing. E. coli cells express endolysin before freeze-drying and encapsulation, allowing endolysin accumulation. Upon capsule dissolution in the intestine, rehydrated E. coli can immediately lyse any S. aureus detected by B. subtilis via AIP sensing, without delay for new endolysin production. This coordinated detection and lysis system rapidly eliminates colonized S. aureus in the gut.
The SAeking Detection Cassette
We envision an easy-to-use cassette format for rapid S. aureus detection. The cassette could be stored at room temperature for convenience. To perform a test, the user collects a sample suspected of contamination (e.g. food) and places it in the cassette's sample well. The sample solution then migrates along the strip via capillary action. Present in the conjugation pad is an aptamer (PA#2/8) that is Au-nanoparticle (AuNP) labeled and biotin-conjugated. This aptamer specifically binds to protein A, a characteristic membrane protein of S. aureus.
If S. aureus is in the sample, its protein A will bind to the aptamer-AuNP complex. This complex then binds to a fixed version of the aptamer on the test (T) line. Regardless of protein A presence, unbound aptamer-AuNP continues migrating and biotinylates the control (C) line via streptavidin linkage. Biotin acts as a molecule that allows the aptamer to be immobilized on a streptavidin-coated surface on the C line to serve as a control. The presence of AuNPs can then be visually detected, typically through a color change reaction. The result will be available in 5-20 minutes. Positive results appear as dual lines on the T and C zones, while a single C line indicates a negative sample. This provides a simple, rapid way to detect S. aureus contamination without specialized equipment or skills.
Figure 1. The principle of the S. aureus detection cassette
Figure 2. The detailed illustration of the theoretical S. aureus detection cassette.
T: contains the aptamer that can pair with S.aureus.
C: contains the streptomycin that can bind to the biotin on the AuNP-marked aptamer.
The DNA aptamer-based cassette enables rapid detection of S. aureus within 30 minutest. It is very convenient due to its small size and colorimetric readout eliminate the need for complex procedures. Also, compared to immunological assays, the aptamer-based approach is significantly cheaper to manufacture since aptamers are far easier and less expensive to produce than antibodies. Reducing cost in this way could improve accessibility. Moreover, the test kit features highly efficient detection, revealing results within just 5-20 minutes. This timely detection capability is ideal for applications needing prompt confirmation, such as food contamination monitoring and hospital situations. Additionally, as chemically synthesized molecules, aptamers present no risk of biological contamination (Lakhin et al., 2013). This improves the safety profile for both manufacturers and end-users. These attributes support the potential for broad implementation where low-cost, safe pathogen detection is needed.
While suitable for food safety, expanding the cassette's use to clinical pathogen detection requires further development. Detecting multiple targets would need inclusion of varied aptamers in the conjugation pad and corresponding T lines. Additional validation is still needed to optimize this multi-target format. Multiplexing introduces potential issues like reduced accuracy from interactions between aptamers or complexes. More testing is warranted to refine the implementation approach and its performance when detecting individual targets or mixtures.
Reference
Aptamers vs. Antibodies - Antibody Alternatives - Base Pair Biotechnologies. (2019). Base Pair Biotechnologies. https://www.basepairbio.com/aptamers-vs-antibodies/
Lakhin, A. V., Tarantul, V. Z., & Gening, L. V. (2013). Aptamers: Problems, Solutions and Prospects. Acta Naturae, 5(4), 34-43. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890987/