Nanopore-Based Single-Molecule Biosensing Platform Enables High-Sensitivity, Real-Time, Label-Free Detection of All 20 Natural Amino Acids

In the current era, life science research and clinical medicine are advancing rapidly, with precision detection technologies emerging as a key force driving medical progress. As an emerging hotspot in scientific research, single-molecule biosensing technology provides new perspectives and tools for unraveling the mysteries of life and tackling disease challenges. It enables real-time capture of dynamic information at the single-molecule level, breaking through the limitations of traditional macroscopic detection methods and allowing for in-depth exploration of molecular interaction mechanisms. From basic research to clinical applications, single-molecule biosensing technology demonstrates transformative potential, holding promise to reshape paradigms of disease diagnosis and treatment.

The development of single-molecule biosensing technology exemplifies deep interdisciplinary integration. From biophysics to materials science, and from chip fabrication to algorithm design, knowledge from diverse fields has converged to propel this technology from theory into practice. It offers significant advantages over traditional detection methods. For instance, the Coulter counter, which detects cells using micron-scale apertures, brought an automation revolution to medical testing. Single-molecule biosensing technology goes a step further, enabling precise detection of molecular information at the nanoscale, thereby opening new frontiers in life sciences research and clinical diagnostics.

At the Precision Medicine and Molecular Diagnostics Industry Development Forum, part of the 2025 VBEF Future Healthcare and Pharmaceuticals Top 100 Exhibition, Professor Geng Jia, Deputy Director of the Clinical Laboratory Medicine Research Center at West China Hospital, Sichuan University, presented her team’s recent research and achievements. Grounded in real-world clinical pain points and needs, she highlighted the significant clinical opportunities offered by single-molecule biosensing technologies, represented by nanopore sequencing.

 Geng Jia, Deputy Director of the Clinical Laboratory Medicine Research Center, West China Hospital, Sichuan University

Biosensing technology has been widely applied in various fields, including disease diagnosis, drug testing, physiological parameter monitoring, and biopharmaceuticals. However, existing biosensing technologies and devices still face numerous critical challenges that need to be addressed, particularly in the real-time single-molecule sensing of complex samples.

This is because current medical testing predominantly focuses on fixed time points, making it difficult to capture the dynamic changes in physiological and pathological states.However, the onset and progression of disease constitute a dynamic process, making real-time, continuous monitoring crucial for early detection, precision treatment, and prognostic assessment. Furthermore, in intensive care units (ICUs), primary healthcare institutions, remote areas, and during public health emergencies, there is a critical need for convenient, rapid, and real-time point-of-care testing methods to provide timely evidence for clinical decision-making and save patients' lives.

Driven by this demand, nanopore sequencing technology has advanced rapidly. This is because, unlike other traditional sequencing methods, nanopore sequencing achieves true real-time genomic sequencing, outputting results concurrently with the sequencing process. Users can assess sample quality and status in the early stages of sequencing and stop the run once sufficient data have been acquired. Furthermore, nanopore sequencers are portable, enabling personnel to perform sequencing directly at the sampling site and obtain sequence information in real time for taxonomic identification, thereby facilitating rapid identification of target organisms.

In fact, the origins of nanopore sequencing technology can be traced back to the 1990s, when scientists at the University of Washington and the University of California envisioned using nanoscale pores to detect DNA. It took more than two decades of development for this concept to mature into commercial application. The true practical deployment of nanopore sequencing occurred in 2015, during the Ebola outbreak, when European scientists packed nanopore sequencing devices into suitcases and transported them to makeshift local laboratories, completing the entire workflow from sample to report within 24 hours. In the diagnosis and detection of pathogens, the convenience and efficiency of nanopore sequencing have provided substantial support for pathogen typing, source tracing, and the formulation of prevention and control strategies.

Currently, the commercialization of nanopore technology in the field of gene sequencing has gradually matured. However, its applications in areas such as protein sequencing and drug development still face numerous challenges.This is because, during sequencing, a single electrical signal is contributed by multiple consecutive nucleotides or amino acids. The signal complexity generated by combinations of 20 amino acids is far greater than that of the four deoxyribonucleotides; in other words, the structural complexity of proteins is significantly higher than that of DNA, making protein sequencing extremely challenging. Therefore, if individual amino acid molecules produced by peptide hydrolysis can be detected, the difficulty of signal recognition would be substantially reduced.

To achieve highly accurate single-molecule detection of the 20 proteinogenic amino acids, Professor Geng Jia’s team proposed and validated the Nanopore Exopeptidase Real-time Peptide Sequencing (NEPS) method. This approach enables high-sensitivity, label-free, real-time detection of all 20 natural amino acids, providing a viable pathway toward single-molecule protein sequencing.Aided by machine learning algorithms, the signal recognition accuracy reached 99.1%, with a signal recovery rate of 30.9%. The related findings were published in Nature Methods in 2024.

In terms of real-time therapeutic drug monitoring,Nanopore sensing technology demonstrates significant application potential. Excessive drug dosages can easily lead to toxic side effects in patients, while insufficient dosages may compromise therapeutic efficacy. Clinical guidelines and expert consensus specifically recommend regular therapeutic drug monitoring for vulnerable populations, such as the elderly, children, and pregnant women, to provide precise guidance for medication management in these groups.

For organ transplant recipients, the use of immunosuppressants must be precisely controlled to effectively prevent rejection and infection, thereby improving transplant success rates. Furthermore, real-time monitoring of anesthetic drug concentrations during anesthesia and surgery helps ensure procedural safety and reduce postoperative complications. However, currently, while continuous monitoring is achievable for inhaled anesthetic gases, the medical industry has yet to realize continuous monitoring of anesthetic concentrations in the bloodstream.

Currently, common therapeutic drug monitoring requires patients to visit hospitals for registration and blood sampling, with test results typically available only after 1–2 days, resulting in significant time costs. Professor Geng Jia’s team has pioneered the use of nanopore sensing technology to enable direct monitoring of small molecules in whole blood samples. This method eliminates the need for complex sample preparation, requires only 10 microliters of blood, and completes concentration measurements within 5 minutes. Within the therapeutic range, its detection accuracy is comparable to that of liquid chromatography and mass spectrometry, providing a precise basis for clinical medication and holding promise for meeting the urgent need for real-time drug monitoring among hundreds of millions of people.

During the drug development process,Nanopore-based single-molecule sensing technology also holds significant potential. Currently, there is a lack of effective real-time monitoring methods in pharmacokinetic research. By leveraging this technology, continuous monitoring of drug metabolism in animals can be achieved, providing detailed data support for drug development and accelerating the process of new drug discovery.

Looking ahead, single-molecule biosensing technologies, represented by nanopore sequencing, will evolve in increasingly diverse directions. On one hand, researchers will strive to develop full-length protein single-molecule sequencing protocols, providing novel technical approaches for drug development, personalized antibody drug screening, and tumor therapy. On the other hand, chip-based multi-analyte simultaneous monitoring technologies will be progressively refined and, when integrated with artificial intelligence algorithms, will enable intelligent diagnosis and treatment. This will drive the advancement of molecular medicine monitoring and management systems, achieving precise, portable, and rapid detection across proteomics, genomics, and small-molecule metabolomics, thereby offering robust technical support for health management and disease treatment.