Enable label-free characterization of biomolecules

Nanofluidics allows the detection of compounds at the smallest concentration. He studies the behavior and manipulation of fluids confined in structures at the 1-1000 nm scale. The scientists revealed that the overall detection process has improved with advances in label-free detections in nanofluidics, primarily in biological and chemical analysis. This article focuses on the label-free characterization of biomolecules using nanofluidics.

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Advances in fluidic control techniques and nanofabrication have led to the emergence of nanofluidic devices for the analysis of biomolecules. Over the years, improvements in nanofluidic device configurations, such as nanoporous membranes, nanopores, nanogaps, nanocavities, and nanopipettes, have been reported. These advances have allowed scientists to process denatured DNA molecules in nanofluidic channels while designing unique DNA molecules.

What is the need for label-free characterization techniques?

One of the challenges faced by scientists in nanofluidic studies is the extremely low number of molecules detected in a nanofluidic channel. Typically, the detection of biomolecules in nanofluidics-based laser-induced fluorescence microscopy possesses single-molecule sensitivity. This method has several drawbacks.

One of the major drawbacks involved in labeling biomolecules with fluorescent tags includes its separation from unbound dyes. Other limitations involve interference from fluorescence signals (photobleaching), changes in electrophoretic mobility, and inappropriate quantitative response. Additionally, low labeling efficiency hampers the detection of a single biomolecule or the quantification of biomolecules in nanofluidic channels.

Label-free characterization of biomolecules in nanofluidics

Label-free detection methods in nanofluidics have been classified into optical and electrical methods. These methods are described below:

Optical detection methods:

Generally, the detection of molecules in nanofluidic devices via conventional optical methods is difficult. This is mainly due to the short optical path lengths. In a nanochannel, the optical path length is one millionth of that used in a general optical cell related to conventional absorbance measurements.

Some strategies used to detect optical signals emitted by a limited number of label-free biomolecules in a nanofluidic channel are diffraction/scattering or differential interference contrast (DIC) techniques and strategies associated with increased light-matter interactions (temporal or spatial) using plasmonic and photonic structures.

Scientists used a nanofluidic network to identify refractive index changes and real-time monitoring of DNA amplification. They designed a device that could also be integrated into a smartphone-based biosensing system to detect and compare the results with reference nanochannels to directly calculate the refractive index.

Recently, a diffusion of light-based detection system in nanochannels has been used to detect single and unlabeled protein molecules. This technique successfully determined the presence of viruses in a nanochannel. Increasing light-matter interactions could overcome the disadvantage associated with reduced optical path lengths in nanofluidic devices. Scientists have reported that integrating plasmonic or photonic structures into nanofluidic channels has significantly improved detection performance.

Refractive index (IR) sensors can identify small changes in IR due to the presence of analytes on the sensing surface. Some nanofluidic devices, based on photonic structures, such as nanohole-based photonic crystals (PhC), Fabry-Pérot (FP) cavities and plasmonic nanoholes, exploit the simultaneous retention of photon energy and molecules present in a nanochannel.

Raman spectroscopies and infrared (IR) absorption spectroscopies provide essential information associated with molecular bonds and chemical structures in a label-free and non-invasive way.

Surface-enhanced Raman spectroscopies (SERS) have been applied in nanofluidic devices to enhance the mass transport of biomolecules. This nanofluidic technique was used to detect the four DNA nucleobases in a single DNA molecule.

Electrical detection methods:

Although electrical detection methods are used as efficient label-free detection of biomolecules, the small size of the nanochannel poses challenges due to the large impedance of the liquid in the nanochannel.

One of the standard conductive sensing methods is resistive pulse sensing using nanopores, which is associated with measuring the change in electric current when biomolecules pass through nanopores.

Scientists used the resistive pulse detection method to differentiate between different protein structures due to their conformational changes. Additionally, lysozyme was also identified using a nanopore with a diameter of approximately 21 nm. Importantly, DNA translocation was detected using a nanopore with a diameter of approximately 5 nm located in the center of a graphene nanoribbon.

In this method, the researchers measured resistive modulations of the in-plane current generated as a result of DNA translocation. This method has also been used to detect aggregated proteins by measuring the current change of proteins during the transit of nanopores of specific size.

Several biomolecules were detected by measuring conductivity changes in the nanochannel, for example bovine serum albumin, cardiac troponin T, microRNA, DNA and trypsin. Another label-free nanofluidic method used to detect biomolecules like DNA is to estimate electro-osmotic flow (EOF). One of the electricity-based sensing methods is to measure the pico-ampere flux current signal in the nanochannels. Recently, researchers have developed a bionanofluidic sensor using a nanochannel in the different reaction schemes.

Future prospects

Going forward, scientists aim to focus on developing new nanofluidic devices, primarily by formulating label-free techniques to detect different chemical and biological molecules. Over the next decade, new analytical tools based on nanofluidics will be developed for biomedical and biochemical research.

Interview: Application of nanofluidics to improve biomanufacturing

References and further reading

Spačková, B. et al. (2022) Label-free nanofluidic scattering microscopy of the size and mass of single scattering molecules and nanoparticles. natural methods, 19, p. 751–758. https://doi.org/10.1038/s41592-022-01491-6

Zhao, Y. et al. (2022) Label-free optical analysis of biomolecules in solid-state nanopores: towards single-molecule protein sequencing. ACS Photonics. 9(3), p. 730-742. DOI: 10.1021/acsphotonics.1c01825.

Le, T. et al. (2020) Advances in Label-Free Detections for Nanofluidic Analytical Devices. Micromachines, 11(10), 885. https://doi.org/10.3390/mi11100885

Spackova, B. et al. (2020) Single biomolecule detection without a nanofluidic tag (conference presentation). proc. SPIE 11254Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications XVII, 112540O. https://doi.org/10.1117/12.2544736

Duan, C. et al. (2013) Journal article: Fabrication of nanofluidic devices. Biomicrofluidics, 7, 026501. https://doi.org/10.1063/1.4794973

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