Nanopore Bioelectronics for Next Generation Proteomics: SBIR

OUSD (R&E) critical technology area(s): Advanced Materials, Biotechnology, Microelectronics

Objective: To develop a next-generation single-molecule sensing and sequencing platform that delivers robust, high-accuracy, high-throughput, and scalable reading of biomolecules.

Description: Proteomics has emerged as a crucial field for protecting national security, understanding diseases, developing diagnostics, and designing effective therapeutics. Rapid, agnostic detection of future unknown protein-based biological threats necessitates the development of single-molecule protein sequencing technology that can differentiate the 20 canonical amino acids and beyond. While DNA sequencing has become widely accessible, protein sequencing has lagged due to significant technical complexity, including the 20 primary amino acids, numerous non-canonical and modified amino acids, hundreds of post-translational modifications (PTMs), the inability to amplify samples, a high dynamic range in biological samples, and variable solubility (1). The Defense Advanced Research Project Agency (DARPA) seeks to build a technology that can directly read individual biological polymers (e.g., proteins) using nanopore-based platforms. The technology will enable the identification of unknown biomolecules in real time with reliable devices to address detection gaps for protein-based threats.

The current State of the Art (SOA) in proteomic polymer sequencing is defined by mass spectrometry (MS)-based platforms that allow deep qualitative and quantitative analysis of protein sequence, expression, interactions and post-translational modifications. The predominant “bottom-up” strategy involves enzymatic digestion of proteins into peptides, which are subsequently separated by liquid chromatography prior to identification and quantification using tandem MS. Despite demonstrating great utility, this destructive process often provides incomplete sequence coverage and critical information regarding full-length protein isoforms and the combinatorial arrangement of post-translational modifications on a single protein molecule is lost.

Recent advances in data-independent acquisition approaches, run on SOA instrumentation like Orbitraps and TimsTOFs, now enable the quantification of over 10,000 proteins from bulk samples and can identify over 5,000 proteins at the single-cell level (2). Despite this remarkable capability, the technology is constrained by the need for reference databases to identify proteins of interest. Any novel, single-molecule protein sequencing approach will ultimately be measured against these established technological benchmarks that depend upon inferring protein identities from peptide fragments.

Novel nanopore-based technologies offer a promising path towards direct, real-time, single-molecule sequencing without references, potentially overcoming many of the limitations of current MS-based methods (3,4,5). To meet the need for rapid detection of unknown biological threats, DARPA seeks biological nanopore-based technology to advance our capability to read proteins, including novel sample preparation technologies, engineered protein-based motors and nanopores for molecular transit, and computational models for translating electronic signals into amino acid calls.

Phase I

During Phase I, performers will establish technical feasibility for a novel protein sequencing methodology that can scale to ultrahigh speed, accuracy, and chemical diversity.

Successful methods will develop custom interfaces between sensing/sequencing technologies and microsystems to: (1) Match a wide variety of microsystem interfaces and correct for process variation (2) Remain stable under a wide range of forces (e.g., electrophoretic, electroosmotic) and translocation of molecules (3) Present customizable chemistries that maximize signal differentiation. Performers should address a wide range of challenges, including but not limited to: adaptive sample preparation, nanopore-microsystem interfaces, and high-accuracy sequence calling algorithms. Ultimately, this platform should create a new product category: a microsystem-based, high-throughput, scalable, reconfigurable, real-time single-molecule sensing device and sequencer, that could be developed into a variety of business models (e.g., consumables-plus-instrument, centralized sequencing as a service) analogous to DNA sequencing, enabling commercial sustainability and broad acquisition that enables long term DoW access to technology.

Phase I fixed payable milestones for this program should include:

• Month 1: Kickoff meeting report detailing sample preparation approaches, nanopore and/or motor protein designs, algorithms, and integration plans.
• Month 3: Interim report on performance of prototype system.
• Month 6: Demonstrate feasibility and validation of novel sequencing chemistry. Discrimination of protein analytes with high accuracy and demonstration of a path towards increased throughput and robustness.
• Month 9: Final Phase I report summarizing all key elements of the prototype system including integration of novel chemistries with microsystem interfaces with nanopores and associated motor proteins, sample preparation approaches, prototype stability, and algorithms for discriminating protein analytes.

Phase II

In Phase II, performers will focus on maturing Phase I technology into a scalable and robust solution suitable for transition. The goal is to significantly advance the core technology by enabling more robust sequencing of a wider range of chemical diversity and demonstrating a clear path to high-throughput operation. The project will focus on validating three metrics critical for market entry: operational robustness (extending device and/or reagent shelf-life and stability), systematic reproducibility (across device/reagent batches and complex samples), and the scalability of the sequencing workflow for operationally relevant deployment. The primary task is to quantitatively derisk the transition from prototype to a commercially available technology. The development efforts in this phase should prepare the technology for commercialization, whether as a benchtop prototype system or as the foundation for a sequencing-as-a-service model. This phase should also incorporate advanced data analysis, such as AI/ML-based high-accuracy sequence calling, to manage the increased complexity and throughput.

Phase II fixed payable milestones for the Base Period (12 months) should include:

• Month 1: Report on Phase II Base Period plan, including lessons learned, updated nanopore/motor protein designs, refined sample preparation workflows, updated AI/ML algorithm architectures, and a detailed Phase II integration roadmap. Updated metrics and success criteria for robustness, reproducibility, and scalability.
• Month 3: Interim report demonstrating improved sequencing chemistry capable of discriminating an expanded set of peptides. Quantitative benchmarking of accuracy improvements over Phase I baseline.
• Month 6: Interim report on performance of prototype system including nanopore/motor protein designs and system integration, sample preparation approaches, and AI/ML algorithm development. Demonstrate multi-channel or parallelized nanopore operation.
• Month 9: Demonstrate an initial integrated benchtop prototype system or a functional sequencing-as-a-service workflow, including sample-in to sequence-out operation.
• Report on scalability of the sequencing workflow for operationally relevant deployment scenarios (e.g., throughput targets, time-to-result, operator skill requirements).
• Month 12: Final Phase II base report documenting: (1) final prototype architectures, nanopore/motor protein designs, sample preparation protocols, and all AI/ML algorithms for peptide identification/sequence calling, (2) comprehensive performance metrics (accuracy, throughput, chemical diversity, robustness, reproducibility), (3) quantitative de-risking assessment for transition from lab prototype to commercially viable technology, and (4) updated transition and commercialization plan, including identified strategic partners, target market segments, and business model refinement.

Phase II fixed payable milestones for the Option Period (12 months) should include:

• Month 1: Report on Phase II Option Period plan, including lessons learned, updated nanopore/motor protein designs, refined sample preparation workflows, updated AI/ML algorithm architectures, and a detailed Phase II integration roadmap. Updated metrics and success criteria for robustness, reproducibility, and scalability.
• Month 3: Interim report demonstrating improved sequencing chemistry capable of discriminating an expanded set of peptides. Quantitative benchmarking of accuracy improvements over Phase II Base Period methods.
• Month 6: Interim report on performance of prototype system including nanopore/motor protein designs and system integration, sample preparation approaches, and AI/ML algorithm development.
• Month 9: Demonstrate a final integrated benchtop prototype system or a functional sequencing-as-a-service workflow, including experimental demonstration discriminating operationally relevant analytes (e.g., engineered toxins, antibody medical countermeasures, etc) in near-real-time.
• Month 12: Final report documenting: (1) ) final prototype architectures, nanopore/motor protein designs, sample preparation protocols, and all AI/ML algorithms for peptide identification/sequence calling, (2) finalized commercialization strategy with letters of intent or partnership agreements from strategic partners, and (3) roadmap for Phase III dual-use applications (government and commercial).

Phase III dual use applications

Phase III refers to work that derives from, extends, or completes an effort made under prior SBIR funding agreements, but is funded by sources other than the SBIR program. The performer should pursue both government and commercial applications. For government development, the technology could be transitioned to relevant stakeholders for applications such as real-time field detection of engineered toxins, novel biothreats, and disease signatures to enhance force protection and medical diagnostics. This may involve further ruggedization and integration into existing sensor suites or diagnostic workflows. For commercial development, performers should pursue a strategy to enter markets such as medical diagnostics, pharmaceutical research, and life science tooling. This may involve pursuing business models analogous to DNA sequencing (e.g., consumables-plus-instrument sales, centralized sequencing-as-a-service, or data licensing). The performer should seek strategic partnerships with larger corporations to facilitate market entry and scale manufacturing.

References

1. Lu, C., Bonini, A., Viel, J. H. & Maglia, G. Toward single-molecule protein sequencing using nanopores. Nat. Biotechnol. 43, 312–322 (2025).
2. Guo, T., Steen, J.A. & Mann, M. Mass-spectrometry-based proteomics: from single cells to clinical applications. Nature 638, 901–911 (2025).
3. Motone, K., Kontogiorgos-Heintz, D., Wee, J. et al. Multi-pass, single-molecule nanopore reading of long protein strands. Nature 633, 662–669 (2024).
4. Berhanu, S., Majumder, S., Muntener, T., Whitehouse, J. et al. Sculpting conducting nanopore size and shape through de novo protein design. Science 385,282-288 (2024).
5. Ritmejeris, J., Chen, X. & Dekker, C. Single-molecule protein sequencing with nanopores. Nat Rev Bioeng 3, 303–316 (2025).

Keywords

Biotechnology, Chemical/Biological Defense, Biomedical, Sensors, Microelectronics, Advanced Materials, Single-molecule sequencing, Proteomics

TPOC-1-PoC

DARPA BAA Help Desk

Email

SBIR_BAA@darpa.mil