Enable label-free characterization of biomolecules

Nanofluidics allows the detection of compounds at the smallest concentration. It studies the behavior and manipulation of liquids confined in structures of 1-1000 nm scale. Scientists have revealed that the overall detection process has improved with advances in label-free detections in nanofluidics, mainly 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 biomolecule analysis. Over the years, improvements have been reported in nanofluidic device configurations, such as nanoporous membranes, nanopores, nanogaps, nanocavities, and nanopipettes. These advancements have enabled scientists to process the denatured DNA molecules in nanofluidic channels while developing single DNA molecules.

What is the need for label-free characterization techniques?

One of the challenges facing 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 a single-molecule sensitivity. This process has several drawbacks.

One of the major drawbacks of labeling biomolecules with fluorescent tags is the separation of unbound dyes. Other limitations include interference from fluorescent signals (photobleaching), changes in electrophoretic mobility, and inappropriate quantitative response. In addition, low labeling efficiency hinders 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 are divided into optical and electrical methods. These methods are discussed below:

Optical Detection Methods:

Typically, the detection of molecules in nanofluidic devices via conventional optical methods is challenging. 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 coupled 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 related to an increase in light-matter interactions. (temporary or spatial) using plasmonic and photonic structures.

Scientists used nanofluidic lattices to identify the changes in the refractive index and the real-time monitoring of DNA amplification. They have designed a device that can 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 scattering of the light-based detection system in nanochannels has been used to detect single, label-free protein molecules. This technique has successfully determined the presence of viruses in a nanochannel. Increased light-matter interactions could address the shortcoming associated with reduced optical path lengths in nanofluidic devices. Scientists reported that integrating plasmonic or photonic structures into nanofluidic channels significantly improved detection performance.

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

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

Surface-Enhanced Raman Spectroscopies (SERS) were applied in nanofluidic devices to enhance the mass transport of biomolecules. This nanofluidic technique has been used to detect all four DNA nucleobases in a single DNA molecule.

Electrical Detection Methods:

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

One of the standard conductivity-based detection methods is nanopore resistive pulse detection, which is associated with measuring the change of electrical current when biomolecules flow through nanopores.

Scientists have used the resistive pulse detection method to distinguish different structures of proteins due to their conformational changes. In addition, lysozyme was also identified using a nanopore with a diameter of approximately 21 nm. Importantly, translocation of DNA was detected using a nanopore with a diameter of approximately 5 nm in the center of a graphene nanoribbon.

In this method, researchers measured resistive modulations of in-plane current generated as a result of DNA translocation. This method was also used to detect aggregated proteins by measuring the current change of the proteins when they are transported through nanopores of specific size.

Several biomolecules have been detected by measuring the changes in conductivity in the nanochannel, for example bovine serum albumin, cardiac troponin T, microRNA, DNA and trypsin. Another label-free nanofluidic method used to detect biomolecules such as DNA is electroosmotic flow (EOF) estimation. One of the electricity-based detection methods involves measuring the flowing current signal in the order of pico-amperes in the nanochannels. Recently, researchers have developed a bionanofluidic sensor using a nanochannel in the various reaction schemes.

Future perspectives

In the future, scientists want to focus on developing new nanofluidic devices, mainly by formulating label-free techniques for detecting various chemical and biological molecules. In the next decade, new nanofluidic-based analytical tools will be developed for biomedical and biochemical research.

Interview: Applying nanofluidics to improve bioproduction

References and future lectures

Špačková, B. et al. (2022) Label-free nanofluidic scattering size and mass microscopy of single diffusing molecules and nanoparticles. Nature Methods, 19, pp. 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-molecular protein sequencing. ACS Photonics† 9(3), pp. 730-742. DOI: 10.1021/acsphotonics.1c01825.

Le, T. et al. (2020) Advances in label-free detections for nanofluidic analytical devices. Micromachinery11(10), 885. https://doi.org/10.3390/mi11100885

Spackova, B. et al. (2020) Nanofluidic label-free detection of single biomolecules (conference presentation). proc. KEY 11254Nanoscale imaging, detection and control for biomedical applications XVII, 112540O. https://doi.org/10.1117/12.2544736

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

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