We would like to congratulate Livio Oliveira de Miranda and his team from Eindhoven University of Technology (Peter Zijlstra’s group), together with their collaborators at KU Leuven (Jeroen Lammertynin’s group) and EPFL (Hatice Altugin’s group), for their recent publication: “A dynamic nanoparticle-on-film biosensor for sub-picomolar continuous monitoring in complex matrices with single-molecule resolution”.
This work is an important step forward in the field of single-molecule detection, a technique that allows researchers to observe and measure molecular interactions at the single molecule level, rather than relying on averages from bulk samples. Detecting individual molecules in real time provides unique insights into fundamental biological processes, molecular mechanisms, and clinical biomarkers that would otherwise remain hidden.
Single-molecule methods are increasingly seen as the future of molecular diagnostics and biosensing. They offer unprecedented sensitivity, resolution, and specificity, enabling applications in healthcare, environmental monitoring, life sciences, and drug development. Compared with traditional ensemble-based approaches, which measure the average behavior of millions of molecules, single-molecule techniques reveal the diversity, kinetics, and dynamics of individual molecular events. This is particularly valuable when working with rare biomarkers, ultra-low concentrations, or complex biological samples such as blood serum.
The article demonstrates a new optical biosensor capable of continuous monitoring at sub-picomolar concentrations with single-molecule sensitivity. By coupling nanoparticles with thin metallic films, the team achieved strong plasmonic field enhancement, making it possible to monitor proteins, DNA, and RNA molecules in real time with minimal background noise. This breakthrough shows how advanced imaging, spectroscopy, and microfluidic fluid handling can work together to push the boundaries of what single-molecule detection can achieve.
In their new article, the researchers introduce a dynamic nanoparticle-on-film (NPoF) biosensor designed to overcome long-standing challenges in single-molecule detection. The innovation lies in combining nanoparticles with a thin metallic film to create a plasmonic nanogap where light and matter interact with extraordinary efficiency. This optical setup produces a strong, localized electromagnetic field that amplifies the signal of an individual molecule, enabling real-time detection with exceptional sensitivity.
Simulations of the plasmonic nanogap Numerical models of the gold nanoparticle–film system, illustrating the plasmonic field enhancement in the nanogap that enables ultrasensitive detection.
Unlike conventional biosensors that provide averaged signals from large populations, this system is able to track the binding and unbinding of individual molecules. Each molecular event is recorded as a measurable change in light scattering, making it possible to follow the natural fluctuations of proteins, DNA, or RNA in complex biological fluids.
Principle of the NPoF biosensor Schematic of the nanoparticle-on-film construct, the optical setup, and microscopy readout, showing how single binding events are detected in real time.
This shift from ensemble measurement to single-molecule resolution opens new possibilities for understanding the kinetics of biomolecular interactions, including binding lifetimes, molecular affinities, and conformational changes.
A key advantage highlighted in the study is the ability of this biosensor to perform continuous monitoring without the need for washing or regeneration steps. Traditional surface plasmon resonance or fluorescence assays often require resetting between measurements. By contrast, the nanoparticle-on-film construct allows reversible binding at the single-molecule level, providing an internal “reset” that ensures uninterrupted operation. This makes the technology ideally suited for long-term studies and dynamic tracking of molecular processes in their native environment.
The publication also emphasizes the importance of operating in complex matrices such as undiluted serum. Biological fluids introduce significant challenges: background noise, non-specific binding, and scattering effects. The authors address this through a concept called kinetic fingerprinting, analyzing the precise duration of individual binding events to distinguish between specific molecular interactions and spurious background signals. This step is crucial for real-world applications, where samples rarely consist of purified targets alone.
By demonstrating sub-picomolar detection limits directly in serum, the study sets a new benchmark for biosensing sensitivity. Applications include monitoring circulating nucleic acids such as microRNAs, detecting low-abundance protein biomarkers, and enabling molecular diagnostics in cases where early detection is critical, such as cancer or inflammatory disease. Beyond healthcare, the ability to achieve such precise single-molecule analysis has potential impact in environmental monitoring, food quality control, and drug development.
To achieve single-molecule detection at such low concentrations, the authors combined advanced nanophotonic design with carefully optimized biochemical assays and fluid handling. Their experimental approach relied on several complementary methods:
The core of the biosensor is a gold nanoparticle placed above a thin gold film, forming a nanoscale gap. This geometry supports resonant coupling between localized and propagating plasmons, creating a highly confined optical field. Any individual molecule binding in this gap produces a measurable change in light scattering, enabling single-molecule resolution imaging without labels or amplification.
The team used objective-type TIRF microscopy to excite the plasmonic film at a steep angle. This illumination enhances the near-field while suppressing background signals from the bulk solution. A CMOS camera captured the scattered light from each single particle binding event, turning nanoscale interactions into clear, analyzable images. This optical setup ensures that the detection is both sensitive and selective, even in complex fluids like serum.
Event detection workflow Optical image processing pipeline that isolates reversible single-particle binding events, converting weak nanoscale signals into clear, analyzable traces.
To achieve continuous operation, the researchers designed a sandwich assay with two types of binders: one attached to the nanoparticle and one on the gold surface. A target molecule (such as DNA, RNA, or protein) bridges the two but then releases after a defined time. These reversible interactions act as an internal reset, avoiding saturation and enabling real-time monitoring of fluctuating concentrations at the single-molecule level.
Since complex samples often produce background signals, the team implemented kinetic fingerprinting. Each binding event has a characteristic lifetime that reveals whether it is specific (true molecular recognition) or non-specific (random adhesion or multivalency). By analyzing the time traces of many individual events, the sensor distinguishes genuine signals from noise, a crucial step to maintain single-molecule sensitivity in diagnostic samples.
Background subtraction in microscopy images Example of how image subtraction removes film roughness and noise, leaving only the point-spread function of true molecular binding events.
For sample delivery and automation, the experiments employed the Laboratory Programmable Syringe Pump, LSPone. This device enabled precise introduction of nanoparticles, buffers, and targets into the flow cell mounted on the microscope. By integrating multiple inlets (12 ports) into a single automated system, the LSPone ensured stable, reproducible conditions and allowed different assays to be conducted on the same chip without manual exchange. This kind of programmable microfluidic control is critical for experiments where even tiny fluctuations in flow or volume can affect single-molecule measurements.
Diffusion and binding simulations Calculated diffusion coefficients and binding frequencies for nanoparticles of different sizes, highlighting why 40 nm particles with optimized fluidics enable femtomolar detection.
Together, these methods, plasmonic nanostructures, wide-field TIRF microscopy, reversible assays, kinetic fingerprinting, and automated fluidics with AMF’s LSPone, created a robust framework for continuous biosensing. The combination of optical sensitivity and controlled fluid handling allowed the team to measure molecular interactions with unprecedented resolution and stability, directly in undiluted serum.
A central aspect of this publication is the ability to deliver precisely controlled flows of nanoparticles, buffers, and biomolecular targets into the sensor chamber without disrupting delicate measurements. For this, the researchers relied on the LSPone.
In the experiments, the LSPone was connected to a custom flow cell mounted on the microscope. Different Eppendorf tubes containing the gold nanoparticles, DNA or RNA targets, and assay buffers were placed on the pump’s ports. By programming the pump, the team could introduce each solution in sequence, all from a single syringe system, and run multiple assays within the same field of view. This minimized manual handling, reduced contamination risks, and ensured consistent fluid delivery.
For single-molecule detection, such precision in fluid handling is critical. Even small fluctuations in flow rate, bubbles, or unstable switching can obscure weak optical signals. The LSPone provides stable and repeatable liquid transport, allowing the sensor to track individual binding events in real time with minimal background interference. Its multiplexing capability also made it possible to test multiple concentrations and targets quickly, an advantage when working at the edge of sensitivity with sub-picomolar analyte levels.
By enabling reproducible sample introduction, cleaning, and switching between solutions, the LSPone was more than just a pump in this study, it acted as the backbone of the experimental workflow. It supported the researchers in reaching the extraordinary achievement of continuous, label-free single-molecule monitoring in undiluted serum.
The LSPone Laboratory Programmable Syringe Pump is AMF’s laboratory-grade programmable syringe pump, designed for researchers who need precision, reproducibility, and flexibility in their microfluidic experiments. With its rotary valve, the LSPone allows seamless switching between multiple liquids in a single setup, an essential feature when working with assays that demand continuous flow and rapid solution changes.
At its core, the LSPone combines:
What makes the LSPone particularly powerful in biosensing and imaging studies is its ability to maintain a stable flow while minimizing carryover and dead volume. This ensures that delicate signals, such as those from individual molecular binding events, are not lost in fluidic noise.
For researchers who need even greater precision at ultra-low flow rates, AMF now also offers the LSPone HD. This new high-definition model integrates AMF’s advanced plunger control system to deliver smoother, more predictable flow at the sub-microliter-per-minute level, making it ideal for applications like single-molecule fluorescence, droplet generation, and cell handling where stability is everything.
Beyond biosensing, the LSPone family is widely used in applications such as:
By combining microfluidic precision with automation, the LSPone and LSPone HD help researchers simplify their setups, reduce errors, and push their experiments into new areas, from fundamental molecular biology to applied diagnostics.
This study marks an important step forward in the future of single-molecule detection, showing how advanced optical techniques and precise fluid handling can work together to push the boundaries of biosensing. For anyone interested in the details of the experiments, the data, and the broader implications, we highly recommend reading the full article.
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