Fluid-based biomarker assays offer promising avenues for disease diagnosis and treatment. In cancer diagnosis, for instance, body fluid-based biomarker detection can significantly reduce patient discomfort compared to radiological or invasive diagnostic methods. The enzyme-linked immunosorbent assay (ELISA) is the most widely used tool for detecting biomarkers in body fluids. Relying on an immune response to recognize targets, ELISA demonstrates excellent specificity. However, traditional ELISA relies on colorimetric quantification, a semi-quantitative approach that results in a high limit of detection (LOD). This limitation hinders its suitability for highly sensitive detection of low-concentration biomarkers in early disease screening.

In recent years, various analytical techniques have been explored to improve ELISA’s sensitivity and parallelism. Yet, few methods achieve highly sensitive, simultaneous detection of multiple biomarkers.

Our research group, led by Professor Hai-Chen Wu at the Institute of Chemistry, Chinese Academy of Sciences, has developed Nanopore-based ELISA (NELISA), a novel approach combining nanopore sensing technology with ELISA. This method enables simultaneous, quantitative detection of multiple biomarkers with remarkable sensitivity.

Simple but Powerful

Nanopore sensing is a single-molecule detection technique widely used in DNA/protein sequencing, ion/small-molecule detection, and single-molecule reaction monitoring. The key to ELISA signal output lies in the enzyme-responsive substrate’s signal generation.

In a previous study (Nat. Methods, 21, 102–109 (2024)), we discovered that α-hemolysin nanopores exhibit exceptional discrimination for peptides with FGXD8-like structures. Inspired by this finding, we designed enzyme-responsive probes based on FGXD₈, bridging nanopore sensing and ELISA to provide a new quantitative detection method.

We engineered FGXD₈ probes with specific response structures for alkaline phosphatase, β-galactosidase, glucose oxidase, and horseradish peroxidase, confirming their compatibility with ELISA enzymatic reactions. These probes generate distinct nanopore current signals before and after enzymatic reactions, with signal frequency correlating to target biomarker concentration—enabling precise quantification (Figure 1).

Effective, Even Better

Remarkably, this simple strategy delivered outstanding results:

• Single-plex detection achieved an aM-level LOD.
• Multiplex detection allowed simultaneous quantification of six biomarkers without signal crosstalk.

We applied NELISA to analyze CA19-9, CEA, and AFP in over 100 blood samples. The results aligned closely with clinical chemiluminescent immunoassays. Moreover, NELISA’s LOD was 3–4 orders of magnitude lower than that of chemiluminescent assays, highlighting its potential for ultra-sensitive biomarker detection.

Future Prospects

By expanding enzyme-probe designs, optimizing instrument interfaces, and refining control software and data processing systems, we envision developing a portable nanopore immunoassay analyzer for point-of-care testing, offering a transformative tool for clinical diagnostics. 

The paper is now on line: https://doi.org/10.1038/s41565-025-01918-z.