Utilizing Constrained Bicyclic Peptides for In Vitro Diagnostics

Today, we are sharing an important study published in ACS Nanoin 2026 by the research team led by Professor Molly M. Stevens. This study systematically explores, for the first time, the application potential of constrained bicyclic peptides as a novel class of synthetic biorecognition elements in the field of in vitro diagnostics. Targeting the SARS-CoV-2 nucleocapsid protein, the research obtained bicyclic peptides with nanomolar affinity through phage display and successfully applied them to various diagnostic platforms, including enzyme-linked immunosorbent assay (ELISA), nanozyme-linked immunosorbent assay (NLISA), and lateral flow immunoassay (LFIA). The results demonstrate that bicyclic peptides not only exhibit binding performance comparable to antibodies but, leveraging their unique advantages such as controllable chemical synthesis, ease of site-specific modification, and excellent batch-to-batch consistency, also enable the ultra-low concentration detection of target antigens. This provides a new molecular tool for developing the next generation of low-cost, scalable, and highly sensitive biosensors.

01 Research Background

At the core of biosensors and diagnostic technologies are biorecognition elements that can specifically and efficiently bind to disease biomarkers. Currently, the gold standard methods for laboratory testing and point-of-care testing (e.g., ELISA and LFIA) largely rely on antibodies to form "sandwich complexes." Although antibodies offer sufficient sensitivity and specificity, their production depends on complex biological systems, suffering from limitations such as batch-to-batch variation, high cost, and difficulty in performing site-specific chemical modifications. Therefore, developing alternative binding molecules with high affinity, high specificity, and ease of production and engineering is crucial. Constrained bicyclic peptides are a novel class of synthetic affinity agents, typically composed of 10-20 amino acids, forming a rigid bicyclic structure via chemical crosslinking. They were initially developed as drug molecules targeting cancer therapy, garnering attention for their small molecular weight, good tissue penetration, and high affinity. However, their potential as antibody mimics in diagnostic immunoassays remains underexplored. The chemical synthesis nature of bicyclic peptides allows for large-scale, reproducible production and facilitates easy, site-specific modifications away from the antigen-binding site, offering unique advantages for optimizing their performance in diagnostic platforms.

02 Innovative Highlights

First Extension of Therapeutic Bicyclic Peptides to the In Vitro Diagnostics Field

The research team creatively repurposed and validated bicyclic peptide molecules, originally developed for in vivo targeted therapy, as efficient capture or detection probes in in vitro diagnostic assays. This breaks the traditional perception of bicyclic peptides primarily as therapeutic molecules, opening up new applications in the biosensing field.

Comprehensive Validation of Bicyclic Peptide Applicability Across Mainstream Diagnostic Platforms

The study not only proved the effectiveness of bicyclic peptides in traditional ELISA but also further integrated them with emerging NLISA and point-of-care LFIA. This cross-platform validation fully demonstrates the powerful compatibility and flexibility of bicyclic peptides as universal recognition elements.

Achieving Ultra-Low Detection Limits, Rivaling or Even Surpassing Some Antibody-Based Assays

By optimizing bicyclic peptide-antibody sandwich pairs and combining them with the signal amplification of platinum-based nanozymes, the study achieved ultra-low detection limits of 97 pg/mL and 0.04 ng/mL (0.83 pM) in NLISA and LFIA, respectively. This sensitivity surpasses that of most commercially available rapid antigen test strips and approaches the level of PCR detection, proving its potential for highly sensitive diagnostics.

Utilizing Phage Display to Obtain Bicyclic Peptide Libraries Targeting Different Epitopes

Against the full-length, N-terminal domain (NTD), and C-terminal domain (CTD) of the SARS-CoV-2 nucleocapsid protein, the study screened multiple bicyclic peptides with affinities ranging from micromolar to low nanomolar via phage display technology. This demonstrates the ability to rapidly and directionally obtain binding molecules targeting different regions of the same target protein, providing a key resource for constructing non-competitive sandwich detection pairs.

Chemical Synthesis Ensuring High Purity and Site-Specific Labeling

All bicyclic peptides were obtained by chemical synthesis with purity >95% and could be conveniently modified with reporter groups (e.g., biotin) at specific positions (e.g., away from the binding site). This synthetic accessibility, batch-to-batch consistency, and precise chemical modification capability are significant advantages that are difficult to match with traditional antibody production.

03 Results and Discussion

3.1 Screening and Characterization of Bicyclic Peptides

The study used recombinant proteins of different SARS-CoV-2 N protein domains (full-length, NTD, CTD) to perform solution-phase panning on a linear peptide phage library containing three cysteines. After four rounds of selection, several enriched clones were obtained. Biolayer interferometry measurements showed that the screened bicyclic peptides (e.g., B001-B004) had affinities for the target proteins in the micromolar to low nanomolar range. Crystal structure analysis confirmed that bicyclic peptides from the B003/B004 family bound to the C-terminal domain of the N protein, while those from the B001/B002 family bound to the N-terminal domain, providing a structural basis for designing non-competitive sandwich detection pairs.

3.2 Application and Optimization of Bicyclic Peptides in Plate-Based Assays (ELISA/NLISA)

ELISA Screening:Using a mixed sandwich format of "bicyclic peptide capture + antibody detection," the study screened 16 different bicyclic peptide-antibody combinations. Using the signal-to-noise ratio as the criterion, the combination of the CTD-targeting bicyclic peptide B003 and the NTD-targeting antibody 40588-MM124 showed the best performance. Under optimized conditions, this pair achieved detection of the SARS-CoV-2 N protein with a limit of detection (LOD) as low as 250 pg/mL, and showed no cross-reactivity with N proteins from other human coronaviruses.

NLISA Development:To further improve sensitivity, the detection probe was replaced from an enzyme to a platinum-coated nanocatalyst with peroxidase-mimicking activity. After a similar screening process, B004-40143-MM08 was identified as the optimal pair. This NLISA method achieved a LOD of 97 pg/mL, improving sensitivity by approximately 2.5 times compared to traditional ELISA, while maintaining high specificity.

3.3 Application of Bicyclic Peptides in Paper-Based Point-of-Care Testing (LFIA)

The study further applied bicyclic peptides to lateral flow immunoassays to validate their potential in point-of-care diagnostic scenarios.

Preliminary Screening:Using platinum nanozymes as the signal probe, 16 pairs were tested at an antigen concentration of 1 ng/mL. Screening revealed that the B001-40143-MM05 combination produced a clear test line at the target concentration with no non-specific background signal in blank samples, exhibiting the best signal-to-noise ratio.

Performance Evaluation:An LFIA was constructed using the B001-40143-MM05 pair. Prior to signal amplification, the visual LOD was 0.62 ng/mL. After chromogenic substrate-catalyzed amplification, the visual LOD was significantly reduced to 0.04 ng/mL. This sensitivity exceeds that of most commercial rapid antigen test strips. Cross-reactivity experiments confirmed the high specificity of this method for the SARS-CoV-2 N protein.

3.4 Key Discussions and Findings

Platform Performance Does Not Directly Translate:The study found that bicyclic peptide-antibody pairs that performed excellently in ELISA/NLISA did not rank similarly in LFIA. This indicates that the performance of biorecognition elements in different detection platforms needs to be evaluated independently and cannot be simply extrapolated from one platform to another.

Detection Antibody Influences Non-Specific Binding:In LFIA screening, certain detection antibodies (e.g., 40588-MM124) produced significant background signals in blank samples regardless of the capture bicyclic peptide they were paired with, suggesting that the properties of the detection antibody itself are a key factor affecting non-specific binding.

Stability and Specificity Advantages of Bicyclic Peptides:Benefiting from their minimalist architecture and lack of higher-order structure, bicyclic peptides are expected to exhibit superior stability and lower non-specific binding compared to traditional antibodies, as they remove redundant protein domains unrelated to target recognition that may cause undesirable interactions.

04 Conclusion

This study successfully demonstrates that constrained bicyclic peptides are a highly promising new class of biorecognition elements, effectively usable for developing highly sensitive and specific in vitro diagnostic assays. By integrating them with various platforms including ELISA, NLISA, and LFIA, the research achieved ultra-low concentration detection of the SARS-CoV-2 N protein, showcasing the application potential of bicyclic peptides across the complete diagnostic chain from the laboratory to the bedside. Their chemical synthesis nature ensures feasibility for large-scale production and batch-to-batch consistency, while the capability for site-specific modification provides great flexibility for optimizing assay performance.

This work marks an important step in extending bicyclic peptides from the therapeutic to the diagnostic field. Its core value lies in providing a customizable, scalable, and high-performance alternative to antibodies. In the future, this technological platform is expected to have a profound impact in the following directions: ① Multiplex Detection:​ Leveraging bicyclic peptide libraries to rapidly develop assays for various biomarkers of infectious diseases, cancer, or cardiovascular diseases. ② Multiplexed Assays:​ Utilizing the characteristic of different bicyclic peptides being amenable to specific chemical modifications to develop multiplex test strips or chips capable of simultaneously detecting multiple biomarkers. ③ Theranostics:​ Integrating bicyclic peptides used for diagnostics with bicyclic peptide drugs used for therapy to achieve "theranostic" applications. ④ Cost and Accessibility:​ Leveraging the cost advantage of chemical synthesis to promote the adoption of high-performance diagnostic technologies in resource-limited settings. In summary, bicyclic peptides provide a powerful molecular toolbox for developing the next generation of biosensors, promising to advance in vitro diagnostic technologies towards greater sensitivity, speed, and affordability.