Nanofluidic biosensors with sub-100 nm channels represent a cutting-edge advancement in single-molecule detection, enabling unprecedented sensitivity for analyzing biomolecules such as DNA, proteins, and exosomes. These devices exploit the unique physics of nanoscale confinement to achieve precise measurements, making them invaluable for applications ranging from early cancer diagnosis to ultra-rare cell isolation. The underlying principles, fabrication techniques, and emerging applications of these sensors highlight their transformative potential in diagnostics and biomedical research.

At the heart of nanofluidic biosensors is the principle of confinement effects on molecular transport. When biomolecules traverse channels with dimensions comparable to or smaller than their own size, their behavior deviates significantly from bulk solution dynamics. In sub-100 nm channels, electrostatic interactions, steric hindrance, and entropic barriers dominate molecular motion. For instance, DNA molecules elongate and undergo size-dependent migration due to the interplay between confinement and hydrodynamic forces. Proteins experience altered diffusion rates and adsorption kinetics, which can be leveraged for selective detection. These confinement effects enhance signal-to-noise ratios by reducing background interference and enabling single-molecule resolution.

Resistive pulse sensing is a key mechanism employed in nanofluidic biosensors for single-molecule detection. As molecules pass through a constriction in the nanochannel, they temporarily block ionic current flow, generating a transient resistive pulse. The amplitude, duration, and frequency of these pulses provide information about the molecule’s size, charge, and concentration. In sub-100 nm channels, the signal is amplified due to the high surface-to-volume ratio, allowing even small biomolecules like microRNAs or unfolded proteins to be detected. Advanced signal processing algorithms further improve discrimination between closely related species, such as mutant and wild-type DNA sequences.

Fabrication of nanofluidic biosensors requires precise nanoscale engineering to achieve reproducible and robust devices. Nanoimprinting lithography is a widely used technique due to its scalability and high resolution. A master mold with nanoscale features is pressed into a polymer substrate, creating channels with sub-100 nm dimensions. Alternatively, electron beam lithography or focused ion beam milling can directly pattern channels in materials like silicon or glass, though these methods are more time-consuming. Emerging approaches, such as sacrificial layer etching or self-assembly of block copolymers, offer additional routes to create complex nanofluidic networks. Integration with microfluidic systems ensures efficient sample delivery and waste removal, while surface functionalization with recognition elements like antibodies or aptamers enhances specificity.

Optical detection methods are often combined with nanofluidic biosensors to provide complementary information. Fluorescence microscopy can track labeled molecules in real-time, revealing binding kinetics or conformational changes. Plasmonic nanostructures embedded in the channels enhance optical signals through localized surface plasmon resonance, enabling label-free detection of low-abundance analytes. Interferometry and whispering gallery mode sensors further improve sensitivity by measuring refractive index changes induced by molecular interactions. These hybrid approaches maximize the utility of nanofluidic biosensors for diverse applications.

One of the most promising applications of nanofluidic biosensors is early cancer diagnosis through exosome profiling. Exosomes are nanoscale vesicles secreted by tumor cells, carrying molecular signatures of their origin. Traditional detection methods struggle with the heterogeneity and low concentration of exosomes in bodily fluids. Nanofluidic biosensors overcome these challenges by isolating and analyzing individual exosomes based on size, surface markers, or cargo. For example, resistive pulse sensing can distinguish exosomes from similarly sized particles like lipoproteins, while immunoaffinity capture within nanochannels enriches specific subpopulations. Multiplexed detection of exosomal proteins or nucleic acids provides a comprehensive profile for cancer screening and monitoring treatment response.

Ultra-rare cell isolation, such as capturing circulating tumor cells (CTCs) from blood, is another critical application. CTCs are present at concentrations as low as one cell per billion blood cells, necessitating highly sensitive detection. Nanofluidic biosensors achieve this by combining size-based sorting with immunocapture in nanostructured surfaces. The confined geometry increases interactions between target cells and immobilized antibodies, improving capture efficiency. Integrated electrodes or optical sensors then enable phenotypic or genetic analysis of isolated cells, offering insights into metastasis and drug resistance.

The performance of nanofluidic biosensors depends on several factors, including channel dimensions, surface chemistry, and flow control. Smaller channels enhance confinement effects but may increase clogging risks. Surface modifications with antifouling coatings like polyethylene glycol minimize nonspecific adsorption, preserving sensor accuracy. Pressure-driven or electrokinetic flow must be carefully optimized to balance throughput and resolution. Computational modeling aids in predicting molecular behavior under varying conditions, guiding device design for specific applications.

Despite their advantages, challenges remain in scaling up nanofluidic biosensors for clinical use. Batch-to-batch variability in fabrication can affect reproducibility, while sample preprocessing steps may introduce biases. Standardization of protocols and integration with automated systems will be essential for widespread adoption. Future developments may focus on multiplexed arrays for high-throughput screening or portable devices for point-of-care testing. Advances in materials science and nanofabrication will further expand the capabilities of these sensors, solidifying their role in next-generation diagnostics.

In summary, nanofluidic biosensors with sub-100 nm channels offer unparalleled sensitivity for single-molecule detection by leveraging confinement effects and resistive pulse sensing. Their applications in exosome profiling and rare cell isolation hold immense potential for early cancer diagnosis and personalized medicine. Fabrication techniques like nanoimprinting and integration with optical detection methods enhance their functionality, while ongoing research addresses scalability and standardization challenges. As these technologies mature, they are poised to revolutionize biomedical analysis and improve patient outcomes.