Applying nanofabrication techniques to diagnostic development

As the dimensions for fabrication technology are pushed lower and lower, fluidic devices can take advantage of the ability to use microscale and nanoscale wafer processing techniques to make nanostructures. Nanofabrication techniques create features that harness the unique effects that occur when structural dimensions are on the same order of magnitude as those of biopolymers and cell structures, typically 1-100 nm. Extended nanofabrication dimensions are in the regime of 10 nm -1000 nm (1 micron).

Nanostructured Surfaces and Biological Assays and Diagnostics

Reactive-ion etching (RIE) of glass wafers can be used to fabricate a nano-roughened glass surface. A nano-roughened surface can be exploited to create an increased surface area for cell and biomolecular docking stations. For example, an increased surface roughness of nanostructured glass, Rq=150 nm, vs. a smooth glass surface, Rq=1 nm, that cancer cells adhere to more readily, was used as a glass/polymer hybrid circulating tumor cell microfluidic flow cell [2]. In another example, the nano-roughened surface was coupled to a molecular recognition element, in this case, an aptamer, for increased selectivity [3].

Recently, Cao et al. reported dielectrophoresis-based protein enrichment to increase immunoassay sensitivity using Ag/SiO2 nano rod arrays deposited on a glass substrate [4]. By using oblique angle deposition (OAD), the growth of the nanorod is governed by the shadowing effect and surface diffusion of adatoms. The silicon oxide nanorods and silicon oxide coated silver nanorods are integrated through OAD on glass wafers that were pre-patterned with microelectrodes. The microelectrodes intensify the electric field strength, while the dielectrophoretic (DEP) force for protein capture is mainly from the high field gradient obtained from the nanorods when applying a 5 V electric potential across the electrodes.

To enhance immunoassay sensitivity, the researchers coupled insulator-based dielectrophoresis (iDEP), possible because of the glass substrate, to enrich protein biomarkers in the vicinity of the immobilized capture antibodies. This is achieved using a 5 V AC electric field at 1 MHz. The fluorescent signals of the detection probes were simultaneously enhanced by the metal enhancement effect of the silver-coated nanorods. This was demonstrated by a rapid fluorescent immunoassay of mouse IgG with a limit of detection of 275.3 fg ml-1 after only 1 minute of settling time [1].

Nanofluidics

Nanofluidics employs submicron channels to confine volumes and harness phenomena at the same scale as the biomolecules themselves. Applications are vast [5], including DNA separation, creating artificial pores for cell studies [1], and commercialized platforms for drug discovery, such as the opto-nanofluidics platform for cell sorting created by Berkeley Lights [6]. In its early days, nanofluidics showed up primarily in patents for nucleic acid analysis, with some applications in particle analysis [7].

There are some interesting electrical field effects best taken advantage of in an insulator material such as glass. If the nanochannel width is on the same order of magnitude of the electrical double layer such that there is overlap of the electrical double layers, a non-uniform velocity profile and transverse electric field is established. These effects result in a viable method to perform size-based separations of small molecules and short strands of dsDNA. biological macromolecules that are the same charge to size ratio. This leads to applications such as probe-free DNA hybridization assays [5].

Another novel physical effect observed when an electric field is applied across a nanofluidic array or channels filled with nanoscale posts or pillars in glass is DNA reptation [5]. In DNA reptation, the nonlinear electric field results in conformational changes in the DNA that lead to a difference in speed of motion across the array based on size. The results are similar to that of the size-based separation that occurs in gel electrophoresis, minus the gel.

Out-of-plane nanopores can be most easily formed as lipid bilayers, therefore the device material selection is often governed by other considerations such as optical transparency, dielectric, mechanical, electrical, chemical or thermal properties required. However, fused silica glass has typically been the material of choice for the fabrication of in-plane nanochannels largely because of its superior optical transparency, thermal stability, chemical and biological inertness, hydrophilic surface, and mechanical stability which make it favorable for chemical and biological applications. Fabrication at the production scale can be cost effective, while at the research scale, many device developers balk at the cost of glass nanofabrication methods which include e-beam lithography with plasma dry etching and focused e-beam lithography.

However, some new methods for fabricating glass nanochannels have emerged. One attractive method that solves both the challenge of nanoscale – accuracy alignment and biological and chemical material-friendly bonding called nano-in-nano integration developed by Xu et al. This combines a plasma treatment surface activation step that allows room temperature bonding with post-bonding molecular self-assembly in the nanochannels. Aligning is assisted by the fabrication of gold reference marks that could be searched manually and recognized using a backscatter detector. Employing the transparency and electrical properties of glass, optofluidics has been used in glass nanochannels for electrokinetic manipulation and resistive-pulse monitoring of the assembly of hepatitis B virus core protein dimers into T = 3 and T = 4 icosahedral capsids under biologically relevant concentrations [8].

Conclusions

While there are significant challenges to using nanofabrication techniques for biological assay and diagnostic development, there are some novel effects that can be harnessed when feature dimensions are on the same order of magnitude as macromolecules, light, and field effects. These unique effects resulting from the length scale of the confined volume and the biopolymer offer rich opportunity for novel separation, identification, and detection methods for in vitro diagnostics. The superior optical, chemical, electrical, thermal, and mechanical properties of glass are an advantage as researchers attempt to harness these forces to probe biological systems and create better, higher performing biological assays and diagnostics.