Nanofluidics allows the detection of compounds at the lowest concentration. It studies the behavior and handling of confined fluids in 1-1000 nm scale structures. The scientists revealed that the global detection process has improved with advances in unlabeled detections in nanofluidics, mainly in biological and chemical analyzes. This paper focuses on the unlabeled characterization of biomolecules using nanofluidics.
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Advances in fluid control techniques and nanofabrication have led to the emergence of nanofluidic devices for biomolecule analysis. Over the years, improvements have been reported in the configurations of nanofluidic devices, such as nanoporous membranes, nanopores, nanogaps, nanocavities, and nanopipettes. These advances have allowed scientists to process denatured DNA molecules into nanofluidic channels while designing individual DNA molecules.
What is the need for unlabeled characterization techniques?
One of the challenges scientists face in nanofluidics studies is the extremely low number of molecules detected in a nanofluidic channel. Typically, the detection of biomolecules in nanofluidic-based laser-induced fluorescence microscopy has single-molecule sensitivity. This process has several drawbacks.
One of the key disadvantages involved in the labeling of biomolecules with fluorescent labels includes their separation from unbound dyes. Other limitations include interference from fluorescence signals (photobleaching), changes in electrophoretic mobility, and an inadequate quantitative response. In addition, the low labeling efficiency makes it difficult to detect a single biomolecule or to quantify biomolecules in nanofluidic channels.
Unlabeled characterization of biomolecules in nanofluidics
Label-free detection methods in nanofluidics have been classified into optical and electrical methods. These methods are discussed below:
Optical detection methods:
Typically, detecting molecules in nanofluidic devices using conventional optical methods is a challenge. This is basically due to the short lengths of the optical path. In a nanochannel, the length of the optical path is one millionth of that used in a general optical cell linked to conventional absorbance measurements.
Some strategies used to detect optical signals emitted from a limited number of label-free biomolecules in a nanofluidic channel are diffraction / scattering or differential interference (DIC) contrast techniques and strategies associated with an increase in light-matter interactions (temporal or spatial) using plasmonic and photonic structures.
The scientists used nanofluidic gratings to identify changes in the refractive index and real-time monitoring of DNA amplification. They have designed a device that could also be integrated into a smartphone-based biodetection system to detect and compare 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 unique, unlabeled protein molecules. This technique successfully determined the presence of viruses in a nanochannel. Increased light-matter interactions could overcome the deficiency associated with reduced optical path length in nanofluidic devices. The scientists reported that the integration of plasmonic or photonic structures into nanofluidic channels has substantially improved the detection performance.
Refractive index (IR) sensors can identify small changes in IR due to the presence of analytes on the detection surface. Some nanofluidic devices, based on photonic structures, such as photon crystals based on nanophorate matrices (PhCs), Fabry-Pérot cavities (FP) and plasmonic nanophorates, exploit the simultaneous arrest of photonic energy and molecules present within a nanochannel. .
Raman spectroscopy and infrared absorption (IR) spectroscopy provide essential information associated with molecular bonds and chemical structures in an unlabeled 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 nucleobases of DNA in a single DNA molecule.
Electrical detection methods:
Although electrical detection methods are used as effective detection of unlabeled biomolecules, the small size of the nanochannel poses difficulties due to the high impedance of the liquid in the nanochannel.
One of the standard conduction-based detection methods is the detection of resistive pulses by nanopores, which is associated with the measurement of the change in electric current when biomolecules flow through nanopores.
Scientists have used the method of detecting resistive pulses to differentiate different protein structures due to their conformational changes. In addition, lysozyme was also identified by a nanopore with a diameter of about 21 nm. It is important to note that DNA translocation was detected by a nanopore with a diameter of about 5 nm located in the center of a graphene nanoscape.
In this method, the researchers measured the resistive modulations of the current in the plane generated as a result of DNA translocation. This method was also used to detect aggregate proteins by measuring the current change in proteins as nanopores of specific size transited.
Several biomolecules have been detected measuring nanochannel conductivity changes, for example, bovine serum albumin, cardiac troponin T, microRNA, DNA, and trypsin. Another unlabeled nanofluidic method used to detect biomolecules such as DNA is electroosmotic flux estimation (EOF). One of the electrical detection methods is to measure the transmission current signal in the order of peak-amperes in the nanochannels. Recently, researchers have developed a bionanofluidic sensor using a nanochannel in different reaction schemes.
Future perspectives
In the future, scientists intend to focus on the development of new nanofluidic devices, mainly by formulating unlabeled techniques to detect different chemical and biological molecules. Over the next decade, new nanofluidic-based analytical tools will be developed for biomedical and biochemical research.
Interview: Application of nanofluidics to improve biofabrication
References and future readings
Špačková, B. et al. (2022) Non-labeled nanofluidic dispersion microscopy of size and mass of individual diffuser molecules and nanoparticles. Nature Methods, 19, p. 751–758.
Zhao, Y. et al. (2022) Unlabeled 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 unlabeled detections for nanofluidic analytical devices. Micromachines, 11 (10), 885.
Spackova, B. et al. (2020) Detection of individual biomolecules without nanofluidic label (conference presentation). Proc. SPIE 11254, Nanoscale imaging, detection and operation for biomedical applications XVII, 112540O.
Duan, C. et al. (2013) Review article: Manufacture of nanofluidic devices. Biomicrofluidics, 7, 026501. https://doi.org/10.1063/1.4794973
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